Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Examining the sex- and circadian dependency of a learning phenotype in mice with glycine transporter 1 deletion in two Pavlovian conditioning paradigms

PubMed Central

Dubroqua, Sylvain; Boison, Detlev; Feldon, Joram; Möhler, Hanns; Yee, Benjamin K.

2011-01-01

Behavioural characterisation of transgenic mice has been instrumental in search of therapeutic targets for the modulation of cognitive function. However, little effort has been devoted to phenotypic characterisation across environmental conditions and genomic differences such as sex and strain, which is essential to translational research. The present study is an effort in this direction. It scrutinised the stability and robustness of the phenotype of enhanced Pavlovian conditioning reported in mice with forebrain neuronal deletion of glycine transporter 1 by evaluating the possible presence of sex and circadian dependency, and its consistency across aversive and appetitive conditioning paradigms. The Pavlovian phenotype was essentially unaffected by the time of testing between the two circadian phases, but it was modified by sex in both conditioning paradigms. We observed that the effect size of the phenotype was strongest in female mice tested during the dark phase in the aversive paradigm. Critically, the presence of the phenotype in female mutants was accompanied by an increase in resistance to extinction. Similarly, enhanced conditioned responding once again emerged solely in female mutants in the appetitive conditioning experiment, which was again associated with an increased resistance to extinction across days, but male mutants exhibited an opposite trend towards facilitation of extinction. The present study has thus added hitherto unknown qualifications and specifications of a previously reported memory enhancing phenotype in this mouse line by identifying the determinants of the magnitude and direction of the expressed phenotype. This in-depth comparative approach is of value to the interpretation of behavioural findings in general. PMID:21596148

Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

PubMed

Petit, Fabrice G; Deng, Chuxia; Jamin, Soazik P

2016-01-01

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.

Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

PubMed

Weibel, Raphaël; Reiss, David; Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A J; Wood, John N; Kieffer, Brigitte L; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

PubMed Central

Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice

PubMed Central

Yan, Keqin; Price-O’Dea, Tina

2017-01-01

Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size. PMID:29095838

Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway.

PubMed

Kim, P C; Mo, R; Hui Cc, C

2001-02-01

VACTERL syndrome is a common surgical condition affecting the development of many midaxial organs. The etiology, embryology, and pathogenesis of the VACTERL syndrome are not known. The authors report here new mouse models of VACTERL syndrome involving the Sonic hedgehog (Shh) signaling pathway. Mutant mice involving Shh signaling, the Shh transcription factors Gli2-/- and Gli3-/-, Gli2-/-;Gli3+/- double heterozygotes, and Shh-/- were analyzed. In addition to reported vertebral, anal, tracheoesophageal, and limb anomalies, mutant mice display cardiac, renal, and associated anomalies, namely congenital diaphragmatic hernia and omphalocele, known to be associated in VACTERL syndrome. The Shh transcription factors Gli2 and Gli3 have specific and overlapping roles in the induction of VACTERL phenotypes in a gene-dose dependent manner in these mutants. To the authors' knowledge, these mutant mice represent the first animal model that mimics the human VACTERL syndrome, and suggests that aberrations in Shh signaling might be involved in the VACTERL syndrome.

The Ovary Is an Alternative Site of Origin for High-Grade Serous Ovarian Cancer in Mice

PubMed Central

Coffey, Donna M.; Ma, Lang; Matzuk, Martin M.

2015-01-01

Although named “ovarian cancer,” it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53R172H, into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53R172H-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice. PMID:25815421

The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

PubMed

Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

2015-06-01

Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

PubMed

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-12-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27. Published by Elsevier Inc.

Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

PubMed Central

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-01-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required to ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27. PMID:28964737

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

PubMed Central

Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.

2012-01-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild type mice, whereas Tas1r3 null mice were not different from wild-type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in conditioned taste aversion to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol. PMID:17376151

Abnormalities in brain structure and behavior in GSK-3alpha mutant mice

PubMed Central

2009-01-01

Background Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. Results Similar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. Conclusion Taken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders. PMID:19925672

ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

PubMed Central

2013-01-01

Background The Grin1 (glutamate receptor, ionotropic, NMDA1) gene expresses a subunit of N-methyl-D-aspartate (NMDA) receptors that is considered to play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. Grin1 is a candidate susceptibility gene for neuropsychiatric disorders, including schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder (ADHD). In our previous study, we examined an N-ethyl-N-nitrosourea (ENU)-generated mutant mouse strain (Grin1Rgsc174/Grin1+) that has a non-synonymous mutation in Grin1. These mutant mice showed hyperactivity, increased novelty-seeking to objects, and abnormal social interactions. Therefore, Grin1Rgsc174/Grin1+ mice may serve as a potential animal model of neuropsychiatric disorders. However, other behavioral characteristics related to these disorders, such as working memory function and sensorimotor gating, have not been fully explored in these mutant mice. In this study, to further investigate the behavioral phenotypes of Grin1Rgsc174/Grin1+ mice, we subjected them to a comprehensive battery of behavioral tests. Results There was no significant difference in nociception between Grin1Rgsc174/Grin1+ and wild-type mice. The mutants did not display any abnormalities in the Porsolt forced swim and tail suspension tests. We confirmed the previous observations that the locomotor activity of these mutant mice increased in the open field and home cage activity tests. They displayed abnormal anxiety-like behaviors in the light/dark transition and the elevated plus maze tests. Both contextual and cued fear memory were severely deficient in the fear conditioning test. The mutant mice exhibited slightly impaired working memory in the eight-arm radial maze test. The startle amplitude was markedly decreased in Grin1Rgsc174/Grin1+ mice, whereas no significant differences between genotypes were detected in the prepulse inhibition (PPI) test. The mutant mice showed no obvious deficits in social behaviors in three different social interaction tests. Conclusions This study demonstrated that the Grin1Rgsc174/Grin1+ mutation causes abnormal anxiety-like behaviors, a deficiency in fear memory, and a decreased startle amplitude in mice. Although Grin1Rgsc174/Grin1+ mice only partially recapitulate symptoms of patients with ADHD, schizophrenia, and bipolar disorder, they may serve as a unique animal model of a certain subpopulation of patients with these disorders. PMID:23688147

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

PubMed

Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

2000-12-08

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

PubMed

Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

2017-06-01

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43 Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43 Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Long-term memory deficits in Pavlovian fear conditioning in Ca2+/calmodulin kinase kinase alpha-deficient mice.

PubMed

Blaeser, Frank; Sanders, Matthew J; Truong, Nga; Ko, Shanelle; Wu, Long Jun; Wozniak, David F; Fanselow, Michael S; Zhuo, Min; Chatila, Talal A

2006-12-01

Signaling by the Ca(2+)/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase alpha (CaMKKalpha) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKalpha mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKalpha mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKalpha in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.

Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia.

PubMed

Guo, Xiaochuan; Hamilton, Peter J; Reish, Nicholas J; Sweatt, J David; Miller, Courtney A; Rumbaugh, Gavin

2009-06-01

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

Genes and Alcohol Consumption: Studies with Mutant Mice

PubMed Central

Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

2017-01-01

In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse.

PubMed

Walters, C L; Blendy, J A

2001-12-01

Addiction is a complex process that relies on the ability of an organism to integrate positive and negative properties of drugs of abuse. Therefore, studying the reinforcing as well as aversive components of drugs of abuse in a single model system will enable us to understand the role of final common mediators, such as cAMP response element-binding protein (CREB), in the addiction process. To this end, we analyzed mice with a mutation in the alpha and Delta isoforms of the CREB gene. Previously we have shown that CREB(alphaDelta) mutant mice in a mixed genetic background show attenuated signs of physical dependence, as measured by the classic signs of withdrawal. We have generated a uniform genetically stable F1 hybrid (129SvEv/C57BL/6) mouse line harboring the CREB mutation. We have found the functional activity of CREB in these F1 hybrid mice to be dramatically reduced compared with their wild-type littermates. These mice maintain a reduced withdrawal phenotype after chronic morphine. We are now poised to examine a number of complex behavioral phenotypes related to addiction in a well defined CREB-deficient mouse model. We demonstrate that the aversive properties of morphine are still present in CREB mutant mice despite a reduction of physical withdrawal. On the other hand, these mice do not respond to the reinforcing properties of morphine in a conditioned place preference paradigm. In contrast, CREB mutant mice demonstrate an enhanced response to the reinforcing properties of cocaine compared with their wild-type controls in both conditioned place preference and sensitization behaviors. These data may provide the first paradigm for differential vulnerability to various drugs of abuse.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea

PubMed Central

Kawakami-Schulz, Sharolyn V.; Verdoni, Angela M.; Sattler, Shannon G.; Jessen, Erik; Kao, Winston W.-Y.; Ikeda, Akihiro

2014-01-01

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstncorn1 mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstncorn1 mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstncorn1 cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstncorn1 cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstncorn1 mice. Comparison of gene expression values from WT, Dstncorn1 mutant, and Dstncorn1 rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstncorn1 mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstncorn1 mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstncorn1 mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis. PMID:24550211

Perception of sweet taste is important for voluntary alcohol consumption in mice.

PubMed

Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F

2008-02-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

PubMed

Li, Juan; Prins, Daniel; Park, Hyun Jung; Grinfeld, Jacob; Gonzalez-Arias, Carlos; Loughran, Stephen; Dovey, Oliver M; Klampfl, Thorsten; Bennett, Cavan; Hamilton, Tina L; Pask, Dean C; Sneade, Rachel; Williams, Matthew; Aungier, Juliet; Ghevaert, Cedric; Vassiliou, George S; Kent, David G; Green, Anthony R

2018-02-08

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALR del/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALR del/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALR del/+ HSCs were more proliferative in vitro, but neither CALR del/+ nor CALR del/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF. © 2018 by The American Society of Hematology.

Restricted growth and insulin-like growth factor-1 deficiency in mice lacking presenilin-1 in the neural crest cell lineage

PubMed Central

Nakajima, Mitsunari; Watanabe, Sono; Okuyama, Satoshi; Shen, Jie; Furukawa, Yoshiko

2012-01-01

Presenilin-1 (PS1) is a transmembrane protein that is in many cases responsible for the development of early-onset familial Alzheimer’s disease. PS1 is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. We report here that PS1 is also required for maturation and/or maintenance of the pituitary gland. We generated PS1-conditional knockout (PS1-cKO) mice by crossing floxed PS1 and Wnt1-cre mice, in which PS1 was lacking in the neural crest-derived cell lineage. Although the PS1-cKO mice exhibited no obvious phenotypic abnormalities for several days after birth, reduced body weight in the mutant was evident by the age of 3 to 5 weeks. Pituitary weight and serum insulin-like growth factor (IGF)-1 level were also reduced in the mutant. Histologic analysis revealed severe atrophy of the cytosol in the anterior and intermediate pituitary lobes of the mutant. Immunohistochemistry did not reveal clear differences in the expression levels of thyroid-stimulating hormone, adrenocorticotropic hormone, or prolactin in the mutant pituitary. In contrast, growth hormone expression levels were reduced in the anterior lobe of the mutant. PS1 was defective in the posterior lobe, but not the anterior or intermediate lobes, in the mutant pituitary. These findings suggest that PS1 indirectly mediates the development and/or maintenance of the anterior and intermediate lobes in the pituitary gland via actions in other regions, such as the posterior lobe. PMID:19665542

Increased ethanol preference and serotonin 1A receptor-dependent attenuation of ethanol-induced hypothermia in PACAP-deficient mice.

PubMed

Tanaka, Kazuhiro; Kunishige-Yamamoto, Akiko; Hashimoto, Hitoshi; Shintani, Norihito; Hayata, Atsuko; Baba, Akemichi

2010-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice display remarkable behavioral changes including increased novelty-seeking behavior and reduced hypothermia induced by either serotonin (5-HT)(1A) receptor agonists or ethanol. Because 5-HT(1A) receptors have been implicated in the development of alcohol dependence, we have examined ethanol preference in PACAP-deficient mice using a two-bottle choice and a conditioned place preference test, as well as additive effects of ethanol and 5-HT(1A) receptor agents on hypothermia. PACAP-deficient mice showed an increased preference towards ethanol compared with wild-type mice. However, they showed no preference for the ethanol compartment after conditioning and neither preference nor aversion to sucrose or quinine. The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) restored the attenuated hypothermic response to ethanol in the mutants to similar levels in wild-type mice, with no effect in wild-types. In contrast, the 5-HT(1A) receptor antagonist WAY-100635 attenuated the ethanol-induced hypothermia in wild-type mice, with no effect in the mutants. These results demonstrate increased ethanol preference in PACAP-deficient mice that may be mediated by 5-HT(1A) receptor-dependent attenuation of ethanol-induced central inhibition. Copyright 2009 Elsevier Inc. All rights reserved.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

PubMed Central

Memon, Mushtaq A.; Anway, Matthew D.; Covert, Trevor R.; Uzumcu, Mehmet; Skinner, Michael K.

2008-01-01

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice. PMID:18790002

NMDA Receptors on Dopaminoceptive Neurons Are Essential for Drug-Induced Conditioned Place Preference123

PubMed Central

Tokarski, Krzysztof; Bobula, Bartosz; Zygmunt, Magdalena; Smutek, Magdalena; Kamińska, Katarzyna; Gołembiowska, Krystyna; Hess, Grzegorz; Przewlocki, Ryszard

2016-01-01

Abstract Plasticity of the brain’s dopamine system plays a crucial role in adaptive behavior by regulating appetitive motivation and the control of reinforcement learning. In this study, we investigated drug- and natural-reward conditioned behaviors in a mouse model in which the NMDA receptor-dependent plasticity of dopaminoceptive neurons was disrupted. We generated a transgenic mouse line with inducible selective inactivation of the NR1 subunit in neurons expressing dopamine D1 receptors (the NR1D1CreERT2 mice). Whole-cell recordings of spontaneous EPSCs on neurons in the nucleus accumbens confirmed that a population of neurons lacked the NMDA receptor-dependent component of the current. This effect was accompanied by impaired long-term potentiation in the nucleus accumbens and in the CA1 area of the ventral, but not the dorsal, hippocampus. Mutant mice did not differ from control animals when tested for pavlovian or instrumental conditioning. However, NR1D1CreERT2 mice acquired no preference for a context associated with administration of drugs of abuse. In the conditioned place preference paradigm, mutant mice did not spend more time in the context paired with cocaine, morphine, or ethanol, although these mice acquired a preference for sucrose jelly and an aversion to naloxone injections, as normal. Thus, we observed that the selective inducible ablation of the NMDA receptors specifically blocks drug-associated context memory with no effect on positive reinforcement in general. PMID:27294197

NMDA Receptors on Dopaminoceptive Neurons Are Essential for Drug-Induced Conditioned Place Preference.

PubMed

Sikora, Magdalena; Tokarski, Krzysztof; Bobula, Bartosz; Zajdel, Joanna; Jastrzębska, Kamila; Cieślak, Przemysław Eligiusz; Zygmunt, Magdalena; Sowa, Joanna; Smutek, Magdalena; Kamińska, Katarzyna; Gołembiowska, Krystyna; Engblom, David; Hess, Grzegorz; Przewlocki, Ryszard; Rodriguez Parkitna, Jan

2016-01-01

Plasticity of the brain's dopamine system plays a crucial role in adaptive behavior by regulating appetitive motivation and the control of reinforcement learning. In this study, we investigated drug- and natural-reward conditioned behaviors in a mouse model in which the NMDA receptor-dependent plasticity of dopaminoceptive neurons was disrupted. We generated a transgenic mouse line with inducible selective inactivation of the NR1 subunit in neurons expressing dopamine D1 receptors (the NR1(D1CreERT2) mice). Whole-cell recordings of spontaneous EPSCs on neurons in the nucleus accumbens confirmed that a population of neurons lacked the NMDA receptor-dependent component of the current. This effect was accompanied by impaired long-term potentiation in the nucleus accumbens and in the CA1 area of the ventral, but not the dorsal, hippocampus. Mutant mice did not differ from control animals when tested for pavlovian or instrumental conditioning. However, NR1(D1CreERT2) mice acquired no preference for a context associated with administration of drugs of abuse. In the conditioned place preference paradigm, mutant mice did not spend more time in the context paired with cocaine, morphine, or ethanol, although these mice acquired a preference for sucrose jelly and an aversion to naloxone injections, as normal. Thus, we observed that the selective inducible ablation of the NMDA receptors specifically blocks drug-associated context memory with no effect on positive reinforcement in general.

Smad3 mutant mice develop colon cancer with overexpression of COX-2

PubMed Central

Zhu, Yu-Ping; Liu, Zhuo; Fu, Zhi-Xuan; Li, De-Chuan

2017-01-01

Colon cancer is the second most common cause of cancer-associated mortality in human populations. The aim of the present study was to identify the role of cyclooxygenase-2 (COX-2) in Smad3 mutant mice, which are known to develop colon cancer. Homozygous Smad3 (−/−) mutant mice were generated from inbred and hybrid Smad3 mouse strains by intercrossing the appropriate heterozygotes. Immunohistochemistry with COX-2 antibody was performed throughout this experiment and the data was validated and cross-checked with reverse transcription-polymerase chain reaction (RT-PCR). Homozygous mutant Smad3 mice were generated and the overexpression pattern of COX-2 was identified by immunohistochemistry and validated with RT-PCR. The results of the present study demonstrated a link between the Smad3 mutant mice, colon cancer and COX-2. In addition, the overexpression pattern of COX-2 in Smad3 mutant mice that develop colon cancer was identified. PMID:28454287

Inhaled Anesthetic Responses of Recombinant Receptors and Knockin Mice Harboring α2(S270H/L277A) GABAA Receptor Subunits That Are Resistant to Isoflurane

PubMed Central

Werner, D. F.; Swihart, A.; Rau, V.; Jia, F.; Borghese, C. M.; McCracken, M. L.; Iyer, S.; Fanselow, M. S.; Oh, I.; Sonner, J. M.; Eger, E. I.; Harrison, N. L.; Harris, R. A.

2011-01-01

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABAA receptors (GABAA-Rs) in a manner that makes them plausible targets. We asked whether GABAA-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABAA-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABAA-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC50 for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N2 generation knockins. This effect was not observed at the N4 generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC50) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit. PMID:20807777

Modeling familial British and Danish dementia.

PubMed

Garringer, Holly J; Murrell, Jill; D'Adamio, Luciano; Ghetti, Bernardino; Vidal, Ruben

2010-03-01

Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI ( 2 ) gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles. Transgenic mice expressing wild-type and mutant forms of the BRI(2) protein, Bri ( 2 ) knock-in mutant mice, and Bri ( 2 ) gene knock-out mice have been developed. Transgenic mice expressing a human FDD-mutated form of the BRI ( 2 ) gene have partially reproduced the neuropathological lesions observed in FDD. These mice develop extensive CAA, parenchymal amyloid deposition, and neuroinflammation in the central nervous system. These animal models allow the study of the molecular mechanism(s) underlying the neuronal dysfunction in these diseases and allow the development of potential therapeutic approaches for these and related neurodegenerative conditions. In this review, a comprehensive account of the advances in the development of animal models for FBD and FDD and of their relevance to the study of Alzheimer disease is presented.

Maternal nicotine exposure effects on adolescent learning and memory are abolished in alpha(α)2* nicotinic acetylcholine receptor-null mutant mice.

PubMed

Mojica, Celina; Bai, Yu; Lotfipour, Shahrdad

2018-06-01

The objective of the current study is to test the hypothesis that the deletion of alpha(α)2* nicotinic acetylcholine receptors (nAChRs) (encoded by the Chrna2 gene) ablate maternal nicotine-induced learning and memory deficits in adolescent mice. We use a pre-exposure-dependent contextual fear conditioning behavioral paradigm that is highly hippocampus-dependent. Adolescent wild type and α2-null mutant offspring are exposed to vehicle or maternal nicotine exposure (200 μg/ml, expressed as base) in the drinking water throughout pregnancy until weaning. Adolescent male offspring mice are tested for alterations in growth and development characteristics as well as modifications in locomotion, anxiety, shock-reactivity and learning and memory. As expected, maternal nicotine exposure has no effects on pup number, weight gain and only modestly reduces fluid intake by 19%. Behaviorally, maternal nicotine exposure impedes extinction learning in adolescent wild type mice, a consequence that is abolished in α2-null mutant mice. The effects on learning and memory are not confounded by alternations in stereotypy, locomotion, anxiety or sensory shock reactivity. Overall, the findings highlight that the deletion of α2* nAChRs eliminate the effects of maternal nicotine exposure on learning and memory in adolescent mice. Copyright © 2018 Elsevier Ltd. All rights reserved.

Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice

PubMed Central

Blednov, Y.A.; Benavidez, J.M.; Geil, C.; Perra, S.; Morikawa, H.; Harris, R.A.

2011-01-01

Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., in press), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1 mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57/Bl6 J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electro-physiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion. PMID:21266194

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

PubMed

Lauer, Peter; Metzner, Hubert J; Zettlmeissl, Gerd; Li, Meng; Smith, Austin G; Lathe, Richard; Dickneite, Gerhard

2002-12-01

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation

PubMed Central

Chen, Peng-Chieh; Wakimoto, Hiroko; Conner, David; Araki, Toshiyuki; Yuan, Tao; Roberts, Amy; Seidman, Christine E.; Bronson, Roderick; Neel, Benjamin G.; Seidman, Jonathan G.; Kucherlapati, Raju

2010-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%–15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated phenotypes, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS. PMID:21041952

Deletion of the δ opioid receptor gene impairs place conditioning but preserves morphine reinforcement.

PubMed

Le Merrer, Julie; Plaza-Zabala, Ainhoa; Del Boca, Carolina; Matifas, Audrey; Maldonado, Rafael; Kieffer, Brigitte L

2011-04-01

Converging experimental data indicate that δ opioid receptors contribute to mediate drug reinforcement processes. Whether their contribution reflects a role in the modulation of drug reward or an implication in conditioned learning, however, has not been explored. In the present study, we investigated the impact of δ receptor gene knockout on reinforced conditioned learning under several experimental paradigms. We assessed the ability of δ receptor knockout mice to form drug-context associations with either morphine (appetitive)- or lithium (aversive)-induced Pavlovian place conditioning. We also examined the efficiency of morphine to serve as a positive reinforcer in these mice and their motivation to gain drug injections, with operant intravenous self-administration under fixed and progressive ratio schedules and at two different doses. Mutant mice showed impaired place conditioning in both appetitive and aversive conditions, indicating disrupted context-drug association. In contrast, mutant animals displayed intact acquisition of morphine self-administration and reached breaking-points comparable to control subjects. Thus, reinforcing effects of morphine and motivation to obtain the drug were maintained. Collectively, the data suggest that δ receptor activity is not involved in morphine reinforcement but facilitates place conditioning. This study reveals a novel aspect of δ opioid receptor function in addiction-related behaviors. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

PubMed

Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

2010-12-01

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

Sucrose intake and fasting glucose levels in 5-HT(1A) and 5-HT(1B) receptor mutant mice.

PubMed

Bechtholt, Anita J; Smith, Karen; Gaughan, Stephanie; Lucki, Irwin

2008-03-18

Serotonin (5-HT)(1A) and 5-HT(1B) receptors have been implicated in the incidence and treatment of depression in part through the examination of animals lacking these receptors. Although these receptors have been repeatedly implicated in ingestive behavior there is little information about how 5-HT(1A) and 5-HT(1B) receptor mutant mice react to solutions of varying palatability. In the present experiment male and female 5-HT(1A) and 5-HT(1B) mutant and wild-type mice were presented with increasing concentrations of sucrose using a two-bottle choice procedure. In addition fasting blood glucose levels were assessed. Both male and female 5-HT(1B) mutant mice drank more sucrose than WT mice but also consumed more water. Female, but not male, 5-HT(1A) mutant mice similarly showed increased sucrose consumption, but did not demonstrate increased consumption of water. In addition, the pattern of increased sucrose consumption over genotype and sex was related to fasting blood glucose concentrations such that levels in male 5-HT(1B) mutant mice were reduced relative to wild-type and 5-HT(1A) mutant males, but similar to those of females. The findings in 5-HT(1B) mutant mice emphasize the role of the 5-HT(1B) receptor in regulating ingestive behavior, whereas female sex hormones and 5-HT(1A) receptors may interact to alter sucrose consumption in 5-HT(1A) mutant mice. In addition, these findings may have implications for the role of these receptors in the incidence and treatment of depression since the intake of sucrose has been used as an index of anhedonia in animal models of depression and antidepressant efficacy.

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

PubMed Central

Bortolussi, Giulia; Baj, Gabriele; Vodret, Simone; Viviani, Giulia; Bittolo, Tamara; Muro, Andrés F.

2014-01-01

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1−/− but not in C57BL/6-Ugt1−/− mice. Survival of FVB/NJ-Ugt1−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions. PMID:25062689

Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

PubMed Central

de Alwis, M C; Carter, G R; Chengappa, M M

1980-01-01

A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent. PMID:6778598

Ectodysplasin signalling deficiency in mouse models of hypohidrotic ectodermal dysplasia leads to middle ear and nasal pathology

PubMed Central

Azar, Ali; Piccinelli, Chiara; Brown, Helen; Headon, Denis; Cheeseman, Michael

2016-01-01

Hypohidrotic ectodermal dysplasia (HED) results from mutation of the EDA, EDAR or EDARADD genes and is characterized by reduced or absent eccrine sweat glands, hair follicles and teeth, and defective formation of salivary, mammary and craniofacial glands. Mouse models with HED also carry Eda, Edar or Edaradd mutations and have defects that map to the same structures. Patients with HED have ear, nose and throat disease, but this has not been investigated in mice bearing comparable genetic mutations. We report that otitis media, rhinitis and nasopharyngitis occur at high frequency in Eda and Edar mutant mice and explore the pathogenic mechanisms related to glandular function, microbial and immune parameters in these lines. Nasopharynx auditory tube glands fail to develop in HED mutant mice and the functional implications include loss of lysozyme secretion, reduced mucociliary clearance and overgrowth of nasal commensal bacteria accompanied by neutrophil exudation. Heavy nasopharynx foreign body load and loss of gland protection alters the auditory tube gating function and the auditory tubes can become pathologically dilated. Accumulation of large foreign body particles in the bulla stimulates granuloma formation. Analysis of immune cell populations and myeloid cell function shows no evidence of overt immune deficiency in HED mutant mice. Our findings using HED mutant mice as a model for the human condition support the idea that ear and nose pathology in HED patients arises as a result of nasal and nasopharyngeal gland deficits, reduced mucociliary clearance and impaired auditory tube gating function underlies the pathological sequelae in the bulla. PMID:27378689

Tissue-specific roles of Tbx1 in the development of the outer, middle and inner ear, defective in 22q11DS patients

PubMed Central

Arnold, Jelena S.; Braunstein, Evan M.; Ohyama, Takahiro; Groves, Andrew K.; Adams, Joe C.; Brown, M. Christian; Morrow, Bernice E.

2007-01-01

Most 22q11.2 deletion syndrome (22q11DS) patients have middle and outer ear anomalies, whereas some have inner ear malformations. Tbx1, a gene hemizygously deleted in 22q11DS patients and required for ear development, is expressed in multiple tissues during embryogenesis. To determine the role of Tbx1 in the first pharyngeal pouch (PPI) in forming outer and middle ears, we tissue-specifically inactivated the gene using Foxg1-Cre. In the conditional mutants, PPI failed to outgrow, preventing the middle ear bone condensations from forming. Tbx1 was also inactivated in the otic vesicle (OV), resulting in the failure of inner ear sensory organ formation, and in duplication of the cochleovestibular ganglion (CVG). Consistent with the anatomical defects, the sensory genes, Otx1 and Bmp4 were downregulated, whereas the CVG genes, Fgf3 and NeuroD, were upregulated. To delineate Tbx1 cell-autonomous roles, a more selective ablation, exclusively in the OV, was performed using Pax2-Cre. In contrast to the Foxg1-Cre mutants, Pax2-Cre conditional mutant mice survived to adulthood and had normal outer and middle ears but had the same inner ear defects as the Tbx1 null mice, with the same gene expression changes. These results demonstrate that Tbx1 has non-cell autonomous roles in PPI in the formation of outer and middle ears and cell-autonomous roles in the OV. Periotic mesenchymal markers, Prx2 and Brn4 were normal in both conditional mutants, whereas they were diminished in Tbx1−/− embryos. Thus, Tbx1 in the surrounding mesenchyme in both sets of conditional mutants cannot suppress the defects in the OV that occur in the null mutants. PMID:16600992

Satb2 Ablation Impairs Hippocampus-Based Long-Term Spatial Memory and Short-Term Working Memory and Immediate Early Genes (IEGs)-Mediated Hippocampal Synaptic Plasticity.

PubMed

Li, Ying; You, Qiang-Long; Zhang, Sheng-Rong; Huang, Wei-Yuan; Zou, Wen-Jun; Jie, Wei; Li, Shu-Ji; Liu, Ji-Hong; Lv, Chuang-Ye; Cong, Jin; Hu, Yu-Ying; Gao, Tian-Ming; Li, Jian-Ming

2017-04-18

Special AT-rich sequence-binding protein 2 (Satb2) is a protein binding to the matrix attachment regions of DNA and important for gene regulation. Patients with SATB2 mutation usually suffer moderate to severe mental retardation. However, the mechanisms for the defects of intellectual activities in patients with SATB2 mutation are largely unclear. Here we established the heterozygous Satb2 mutant mice and Satb2 conditional knockout mice to mimic the patients with SATB2 mutation and figured out the role of Satb2 in mental activities. We found that the spatial memory and working memory were significantly damaged in the heterozygous Satb2 mutant mice, early postnatal Satb2-deficient mice (CaMKIIα-Cre + Satb2 fl/fl mice), and adult Satb2 ablation mice (Satb2 fl/fl mice injected with CaMKIIα-Cre virus). Functionally, late phase long-term potentiation (L-LTP) in these Satb2 mutant mice was greatly impaired. Morphologically, in CA1 neurons of CaMKIIα-Cre + Satb2 fl/fl mice, we found decreased spine density of the basal dendrites and less branches of apical dendrites that extended into lacunar molecular layer. Mechanistically, expression levels of immediate early genes (IEGs) including Fos, FosB, and Egr1 were significantly decreased after Satb2 deletion. And, Satb2 could regulate expression of FosB by binding to the promoter of FosB directly. In general, our study uncovers that Satb2 plays an important role in spatial memory and working memory by regulating IEGs-mediated hippocampal synaptic plasticity.

Proteoglycan 4: A Dynamic Regulator of Skeletogenesis and Parathyroid Hormone Skeletal Anabolism

PubMed Central

Novince, Chad M; Michalski, Megan N; Koh, Amy J; Sinder, Benjamin P; Entezami, Payam; Eber, Matthew R; Pettway, Glenda J; Rosol, Thomas J; Wronski, Thomas J; Kozloff, Ken M; McCauley, Laurie K

2014-01-01

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure. PMID:21932346

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Effect of CCS on the Accumulation of FALS SOD1 Mutant-containing Aggregates and on Mitochondrial Translocation of SOD1 Mutants: Implication of a Free Radical Hypothesis

PubMed Central

Kim, Ha Kun; Chung, Youn Wook; Chock, P. Boon; Yim, Moon B.

2011-01-01

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H2O2, mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. PMID:21354101

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome.

PubMed

Hurd, Elizabeth A; Adams, Meredith E; Layman, Wanda S; Swiderski, Donald L; Beyer, Lisa A; Halsey, Karin E; Benson, Jennifer M; Gong, Tzy-Wen; Dolan, David F; Raphael, Yehoash; Martin, Donna M

2011-12-01

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears. Copyright © 2011 Elsevier B.V. All rights reserved.

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome

PubMed Central

Hurd, Elizabeth A.; Adams, Meredith E.; Layman, Wanda S.; Swiderski, Donald L.; Beyer, Lisa A.; Halsey, Karin E.; Benson, Jennifer M.; Gong, Tzy-Wen; Dolan, David F.; Raphael, Yehoash; Martin, Donna M.

2011-01-01

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by Prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7Gt/+ mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7Gt/+ mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7Gt/+ mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7Gt/+ mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears. PMID:21875659

Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95.

PubMed

Zhang, Jingping; Saur, Taixiang; Duke, Angela N; Grant, Seth G N; Platt, Donna M; Rowlett, James K; Isacson, Ole; Yao, Wei-Dong

2014-01-01

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

PubMed

Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

2018-04-11

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

The clock gene Period1 regulates innate routine behaviour in mice

PubMed Central

Bechstein, Philipp; Rehbach, Nils-Jörn; Yuhasingham, Gowzekan; Schürmann, Christoph; Göpfert, Melanie; Kössl, Manfred; Maronde, Erik

2014-01-01

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. We found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour. PMID:24598427

The clock gene Period1 regulates innate routine behaviour in mice.

PubMed

Bechstein, Philipp; Rehbach, Nils-Jörn; Yuhasingham, Gowzekan; Schürmann, Christoph; Göpfert, Melanie; Kössl, Manfred; Maronde, Erik

2014-04-22

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. We found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour.

Pathogenesis of coxsackievirus A9 in mice: role of the viral arginine-glycine-aspartic acid motif.

PubMed

Harvala, Heli; Kalimo, Hannu; Stanway, Glyn; Hyypiä, Timo

2003-09-01

Coxsackievirus A9 (CAV9) contains an arginine-glycine-aspartic acid (RGD) motif which participates in cell entry. Mutants with alterations in the RGD-containing region were utilized to explore the importance of the tripeptide in the pathogenesis of CAV9 in mice. Using in situ hybridization, the parental CAV9 strain was observed to infect skeletal muscle (intercostal, platysma, lingual and thigh muscles) of newborn mice, whereas the RGD-less mutants were detectable only in platysma and lingual muscles. In addition, newborn mice infected with the mutants survived longer than CAV9-infected mice. In adult mice, the parental strain of CAV9, but not the mutants, achieved moderately high titres in the pancreas. These results suggest that the RGD motif has a significant role in the pathogenesis of CAV9 in mice but also that RGD-independent entry routes can be utilized in the infection of murine tissue.

Resistance to collagen-induced arthritis in SHPS-1 mutant mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okuzawa, Chie; Kaneko, Yoriaki; Murata, Yoji

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Here we show that mice expressing a mutant form of SHPS-1 fail to develop type-II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans. Histological examinations of the arthritic paws from immunized wild-type mice revealed that cartilage was destroyed in association with marked mononuclear cell infiltration, while only mild cell infiltration was observed in immunized SHPS-1 mutant mice. Consistently, the serum levels of both IgG and IgG2a specific to CII andmore » of IL-1{beta} in immunized SHPS-1 mutant mice were markedly reduced compared with those apparent for wild-type mice. The CII-induced proliferation of, and production of cytokines by, T cells from immunized SHPS-1 mutant mice were reduced compared to wild-type cells. These results suggest that SHPS-1 is essential for development of CIA.« less

Oxidoreductases that Act as Conditional Virulence Suppressors in Salmonella enterica Serovar Typhimurium

PubMed Central

Anwar, Naeem; Sem, Xiao Hui; Rhen, Mikael

2013-01-01

In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium. PMID:23750221

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

PubMed Central

Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

A Phex Mutation in a Murine Model of X-linked Hypophosphatemia Alters Phosphate Responsiveness of Bone Cells

PubMed Central

Ichikawa, Shoji; Austin, Anthony M.; Gray, Amie K.; Econs, Michael J.

2011-01-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH – high dose phosphate and calcitriol – further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (PhexK496X) and hyperphosphatemic tumoral calcinosis (Galnt3 -/-), and Galnt3/Phex double mutant mice. Phex mutant mice had not only increased Fgf23 expression, but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by up-regulating Fgf23 expression as much as 24 fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for “normal” phosphate levels. PMID:22006791

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Econs, Michael J

2012-02-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

Dental and Cranial Pathologies in Mice Lacking the Cl−/H+-Exchanger ClC-7

PubMed Central

WEN, Xin; LACRUZ, Rodrigo S.; PAINE, Michael L.

2015-01-01

ClC-7 is a 2Cl−/1H+-exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC-7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC-7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC-7, and this study aims to determine the significance of ClC-7 in enamel formation by examining the dentitions of ClC-7 mutant mice. Micro-CT analysis revealed that the molar teeth of 3-week old ClC-7 mutant mice had no roots, and the incisors were smaller than their age-matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild type littermates. Scanning electron microscopy (SEM) showed normal enamel crystallite and prismatic organization in the ClC-7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC-7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro-CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC-7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC-7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis. PMID:25663454

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain

PubMed Central

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A.; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-01-01

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology. PMID:24381309

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

PubMed

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-05-15

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice.

PubMed

Xie, Huirong; Wang, Haibin; Tranguch, Susanne; Iwamoto, Ryo; Mekada, Eisuke; Demayo, Francesco J; Lydon, John P; Das, Sanjoy K; Dey, Sudhansu K

2007-11-13

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

PubMed Central

Farrelly, Dennis; Brown, Karen S.; Tieman, Aaron; Ren, Jianming; Lira, Sergio A.; Hagan, Deborah; Gregg, Richard; Mookhtiar, Kasim A.; Hariharan, Narayanan

1999-01-01

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control. PMID:10588736

Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease

PubMed Central

Olteanu, Dragos; Liu, Xiaofen; Liu, Wen; Roper, Venus C.; Sharma, Neeraj; Yoder, Bradley K.; Satlin, Lisa M.; Schwiebert, Erik M.

2012-01-01

Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88Tg737Rpw) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na+ movement in cilium-deficient (“mutant”) cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent (“rescued”) monolayers. To examine NHE activity, we measured intracellular pH (pHi) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na+-dependent acid-base transporter activity in the nominal absence of CO2/HCO3−. However, only the mutant cells displayed appreciable apical Na+-induced pHi recoveries from NH4+ prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pHi dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pHi 6.23–6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD. PMID:22301060

NCAM deficiency in the mouse forebrain impairs innate and learned avoidance behaviours.

PubMed

Brandewiede, J; Stork, O; Schachner, M

2014-06-01

The neural cell adhesion molecule (NCAM) has been implicated in the development and plasticity of neural circuits and the control of hippocampus- and amygdala-dependent learning and behaviour. Previous studies in constitutive NCAM null mutants identified emotional behaviour deficits related to disturbances of hippocampal and amygdala functions. Here, we studied these behaviours in mice conditionally deficient in NCAM in the postmigratory forebrain neurons. We report deficits in both innate and learned avoidance behaviours, as observed in elevated plus maze and passive avoidance tasks. In contrast, general locomotor activity, trait anxiety or neophobia were unaffected by the mutation. Altered avoidance behaviour of the conditional NCAM mutants was associated with a deficit in serotonergic signalling, as indicated by their reduced responsiveness to (±)-8-hydroxy-2-(dipropylamino)-tetralin-induced hypothermia. Another serotonin-dependent behaviour, namely intermale aggression that is massively increased in constitutively NCAM-deficient mice, was not affected in the forebrain-specific mutants. Our data suggest that genetically or environmentally induced changes of NCAM expression in the late postnatal and mature forebrain determine avoidance behaviour and serotonin (5-HT)1A receptor signalling. © 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

PubMed

Gautam, Dinesh; Jeon, Jongrye; Li, Jian Hua; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Lu, Huiyan; Deng, Chuxia; Gavrilova, Oksana; Wess, Jürgen

2008-01-01

The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Calcium Regulates FGF-23 Expression in Bone

PubMed Central

David, Valentin; Dai, Bing; Martin, Aline; Huang, Jinsong; Han, Xiaobin

2013-01-01

Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D3 [1,25(OH)2D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2−/− and Cyp27b1−/− mutant mice. Gcm2−/− mice exhibited low serum PTH and 1,25(OH)2D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1−/− mice had high PTH, undetectable 1,25(OH)2D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2−/− mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)2D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)2D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1−/− mice in the absence of 1,25(OH)2D and in Gcm2−/− mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)2D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia. PMID:24140714

Calcium regulates FGF-23 expression in bone.

PubMed

David, Valentin; Dai, Bing; Martin, Aline; Huang, Jinsong; Han, Xiaobin; Quarles, L Darryl

2013-12-01

Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D3 [1,25(OH)2D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2(-/-) and Cyp27b1(-/-) mutant mice. Gcm2(-/-) mice exhibited low serum PTH and 1,25(OH)2D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1(-/-) mice had high PTH, undetectable 1,25(OH)2D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2(-/-) mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)2D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)2D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1(-/-) mice in the absence of 1,25(OH)2D and in Gcm2(-/-) mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)2D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia.

AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

PubMed Central

Guo, Wen; Wong, Siu; Bhasin, Shalender

2013-01-01

Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

PubMed

Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

2018-05-01

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.

Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice

PubMed Central

Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y.; DeMayo, Francesco J.; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W.

2005-01-01

Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, ARGAL4DBD, which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators. PMID:15983373

Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice.

PubMed

Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y; DeMayo, Francesco J; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W

2005-07-05

Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, AR(GAL4DBD), which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators.

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

PubMed

Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

2011-05-15

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor.

PubMed

Cannata, David J; Finkelstein, David I; Gantois, Ilse; Teper, Yaroslav; Drago, John; West, Jan M

2009-01-01

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the alpha4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca(2+) and Sr(2+) force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa(50) - pSr(50)) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

Temporal dissection of K-ras(G12D) mutant in vitro and in vivo using a regulatable K-ras(G12D) mouse allele.

PubMed

Wang, Zuoyun; Feng, Yan; Bardeesy, Nabeel; Bardessy, Nabeel; Wong, Kwok-Kin; Liu, Xin-Yuan; Ji, Hongbin

2012-01-01

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(G12D) (ER-K-ras(G12D)) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-ras(G12D) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(G12D) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(G12D) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks

PubMed Central

Pennucci, Roberta; Talpo, Francesca; Astro, Veronica; Montinaro, Valentina; Morè, Lorenzo; Cursi, Marco; Castoldi, Valerio; Chiaretti, Sara; Bianchi, Veronica; Marenna, Silvia; Cambiaghi, Marco; Tonoli, Diletta; Leocani, Letizia; Biella, Gerardo; D'Adamo, Patrizia; de Curtis, Ivan

2016-01-01

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons. PMID:26582364

BEHAVIORAL AND NEUROCHEMICAL CHARACTERIZATION OF THE mlh MUTANT MICE LACKING OTOCONIA.

PubMed

Manes, Marianna; Garcia-Gomes, Mariana de Souza Aranha; Sandini, Thaísa Meira; Zaccarelli-Magalhães, J; Florio, J C; Alexandre-Ribeiro, Sandra Regina; Wadt, Danilo; Bernardi, Maria Martha; Massironi, Silvia Maria Gomes; Mori, Claudia Madalena Cabrera

2018-06-15

Otoconia are crucial for the correct processing of positional information and orientation. Mice lacking otoconia cannot sense the direction of the gravity vector and cannot swim properly. This study aims to characterize the behavior of mergulhador (mlh), otoconia-deficient mutant mice. Additionally, the central catecholamine levels were evaluated to investigate possible correlations between behaviors and central neurotransmitters. A sequence of behavioral tests was used to evaluate the parameters related to the general activity, sensory nervous system, psychomotor system, and autonomous nervous system, in addition to measuring the acquisition of spatial and declarative memory, anxiety-like behavior, motor coordination, and swimming behavior of the mlh mutant mice. As well, the neurotransmitter levels in the cerebellum, striatum, frontal cortex, and hippocampus were measured. Relative to BALB/c mice, the mutant mlh mice showed 1) reduced locomotor and rearing behavior, increased auricular and touch reflexes, decreased motor coordination and increased micturition; 2) decreased responses in the T-maze and aversive wooden beam tests; 3) increased time of immobility in the tail suspension test; 4) no effects in the elevated plus maze or object recognition test; 5) an inability to swim; and 6) reduced turnover of dopaminergic system in the cerebellum, striatum, and frontal cortex. Thus, in our mlh mutant mice, otoconia deficiency reduced the motor, sensory and spatial learning behaviors likely by impairing balance. We did not rule out the role of the dopaminergic system in all behavioral deficits of the mlh mutant mice. Copyright © 2018. Published by Elsevier B.V.

Axonal abnormalities in vanishing white matter.

PubMed

Klok, Melanie D; Bugiani, Marianna; de Vries, Sharon I; Gerritsen, Wouter; Breur, Marjolein; van der Sluis, Sophie; Heine, Vivi M; Kole, Maarten H P; Baron, Wia; van der Knaap, Marjo S

2018-04-01

We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. Axons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed. In the corpus callosum of Eif2b5- mutant mice, myelin sheath thickness, axonal diameter, and G-ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G-ratio in Eif2b5- mutant mice. In more severely affected Eif2b4-Eif2b5 double-mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5 -mutant mice. Co-cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal. In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.

Mutation in the Fas Pathway Impairs CD8+ T Cell Memory1

PubMed Central

Dudani, Renu; Russell, Marsha; van Faassen, Henk; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-γ and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections. PMID:18292515

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis.

PubMed

Tian, Cong; Harris, Belinda S; Johnson, Kenneth R

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets.

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis

PubMed Central

Tian, Cong; Harris, Belinda S.; Johnson, Kenneth R.

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets. PMID:27959908

Performance deficits of mGluR8 knockout mice in learning tasks: the effects of null mutation and the background genotype.

PubMed

Gerlai, R; Adams, B; Fitch, T; Chaney, S; Baez, M

2002-08-01

mGluR8 is a G-protein coupled metabotropic glutamate receptor expressed in the mammalian brain. Members of the mGluR family have been shown to be modulators of neural plasticity and learning and memory. Here we analyze the consequences of a null mutation at the mGluR8 gene locus generated using homologous recombination in embryonic stem cells by comparing the learning performance of the mutants with that of wild type controls in the Morris water maze (MWM) and the context and cue dependent fear conditioning (CFC). Our results revealed robust performance deficits associated with the genetic background, the ICR outbred strain, in both mGluR8 null mutant and the wild type control mice. Mice of this strain origin suffered from impaired vision as compared to CD1 or C57BL/6 mice, a significant impediment in MWM, a visuo-spatial learning task. The CFC task, being less dependent on visual cues, allowed us to reveal subtle performance deficits in the mGluR8 mutants: novelty induced hyperactivity and temporally delayed and blunted responding to shocks and temporally delayed responding to contextual stimuli were detected. The role of mGluR8 as a presynaptic autoreceptor and its contribution to cognitive processes are hypothesized and the utility of gene targeting as compared to pharmacological methods is discussed.

Haemolysis and Perturbations in the Systemic Iron Metabolism of Suckling, Copper-Deficient Mosaic Mutant Mice – An Animal Model of Menkes Disease

PubMed Central

Lenartowicz, Małgorzata; Starzyński, Rafał R.; Krzeptowski, Wojciech; Grzmil, Paweł; Bednarz, Aleksandra; Ogórek, Mateusz; Pierzchała, Olga; Staroń, Robert; Gajowiak, Anna; Lipiński, Paweł

2014-01-01

The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism. PMID:25247420

Altering BDNF expression by genetics and/or environment: impact for emotional and depression-like behaviour in laboratory mice.

PubMed

Chourbaji, Sabine; Brandwein, Christiane; Gass, Peter

2011-01-01

According to the "neurotrophin hypothesis", brain-derived neurotrophic factor (BDNF) is an important candidate gene in depression. Moreover, environmental stress is known to represent a risk factor in the pathophysiology and treatment of this disease. To elucidate, whether changes of BDNF availability signify cause or consequence of depressive-like alterations, it is essential to look for endophenotypes under distinct genetic conditions (e.g. altered BDNF expression). Furthermore it is crucial to examine environment-driven BDNF regulation and its effect on depressive-linked features. Consequently, gene × environment studies investigating prospective genetic mouse models of depression in different environmental contexts become increasingly important. The present review summarizes recent findings in BDNF-mutant mice, which have been controversially discussed as models of depression and anxiety. It furthermore illustrates the potential of environment to serve as naturalistic stressor with the potential to modulate the phenotype in wildtype and mutant mice. Moreover, environment may exert protective effects by regulating BDNF levels as attributed to "environmental enrichment". The effect of this beneficial condition will also be discussed with regard to probable "curative/therapeutic" approaches. Copyright © 2010 Elsevier Ltd. All rights reserved.

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

PubMed Central

Field, H. J.; Wildy, P.

1978-01-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain. PMID:212476

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

PubMed

Field, H J; Wildy, P

1978-10-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.

Mapping and Deciphering Neural Codes of NMDA Receptor-Dependent Fear Memory Engrams in the Hippocampus

PubMed Central

Tsien, Joe Z.

2013-01-01

Mapping and decoding brain activity patterns underlying learning and memory represents both great interest and immense challenge. At present, very little is known regarding many of the very basic questions regarding the neural codes of memory: are fear memories retrieved during the freezing state or non-freezing state of the animals? How do individual memory traces give arise to a holistic, real-time associative memory engram? How are memory codes regulated by synaptic plasticity? Here, by applying high-density electrode arrays and dimensionality-reduction decoding algorithms, we investigate hippocampal CA1 activity patterns of trace fear conditioning memory code in inducible NMDA receptor knockout mice and their control littermates. Our analyses showed that the conditioned tone (CS) and unconditioned foot-shock (US) can evoke hippocampal ensemble responses in control and mutant mice. Yet, temporal formats and contents of CA1 fear memory engrams differ significantly between the genotypes. The mutant mice with disabled NMDA receptor plasticity failed to generate CS-to-US or US-to-CS associative memory traces. Moreover, the mutant CA1 region lacked memory traces for “what at when” information that predicts the timing relationship between the conditioned tone and the foot shock. The degraded associative fear memory engram is further manifested in its lack of intertwined and alternating temporal association between CS and US memory traces that are characteristic to the holistic memory recall in the wild-type animals. Therefore, our study has decoded real-time memory contents, timing relationship between CS and US, and temporal organizing patterns of fear memory engrams and demonstrated how hippocampal memory codes are regulated by NMDA receptor synaptic plasticity. PMID:24302990

Altered Body Weight Regulation in CK1ε Null and tau Mutant Mice on Regular Chow and High Fat Diets

PubMed Central

Zhou, Lili; Summa, Keith C.; Olker, Christopher; Vitaterna, Martha H.; Turek, Fred W.

2016-01-01

Disruption of circadian rhythms results in metabolic dysfunction. Casein kinase 1 epsilon (CK1ε) is a canonical circadian clock gene. Null and tau mutations in CK1ε show distinct effects on circadian period. To investigate the role of CK1ε in body weight regulation under both regular chow (RC) and high fat (HF) diet conditions, we examined body weight on both RC and HF diets in CK1ε −/− and CK1ε tau/tau mice on a standard 24 hr light-dark (LD) cycle. Given the abnormal entrainment of CK1ε tau/tau mice on a 24 hr LD cycle, a separate set of CK1ε tau/tau mice were tested under both diet conditions on a 20 hr LD cycle, which more closely matches their endogenous period length. On the RC diet, both CK1ε −/− and CK1ε tau/tau mutants on a 24 hr LD cycle and CK1ε tau/tau mice on a 20 hr LD cycle exhibited significantly lower body weights, despite similar overall food intake and activity levels. On the HF diet, CK1ε tau/tau mice on a 20 hr LD cycle were protected against the development of HF diet-induced excess weight gain. These results provide additional evidence supporting a link between circadian rhythms and energy regulation at the genetic level, particularly highlighting CK1ε involved in the integration of circadian biology and metabolic physiology. PMID:27144030

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

PubMed

Pendergast, Julie S; Wendroth, Robert H; Stenner, Rio C; Keil, Charles D; Yamazaki, Shin

2017-11-14

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

Working-for-Food Behaviors: A Preclinical Study in Prader-Willi Mutant Mice.

PubMed

Lassi, Glenda; Maggi, Silvia; Balzani, Edoardo; Cosentini, Ilaria; Garcia-Garcia, Celina; Tucci, Valter

2016-11-01

Abnormal feeding behavior is one of the main symptoms of Prader-Willi syndrome (PWS). By studying a PWS mouse mutant line, which carries a paternally inherited deletion of the small nucleolar RNA 116 (Snord116), we observed significant changes in working-for-food behavioral responses at various timescales. In particular, we report that PWS mutant mice show a significant delay compared to wild-type littermate controls in responding to both hour-scale and seconds-to-minutes-scale time intervals. This timing shift in mutant mice is associated with better performance in the working-for-food task, and results in better decision making in these mutant mice. The results of our study reveal a novel aspect of the organization of feeding behavior, and advance the understanding of the interplay between the metabolic functions and cognitive mechanisms of PWS. Copyright © 2016 by the Genetics Society of America.

Deficits in Memory Tasks of Mice with CREB Mutations Depend on Gene Dosage

PubMed Central

Gass, Peter; Wolfer, David P.; Balschun, Detlef; Rudolph, Dorothea; Frey, Uwe; Lipp, Hans-Peter; Schütz, Günther

1998-01-01

Studies in Aplysia, Drosophila, and mice have shown that the transcription factor CREB is involved in formation and retention of long-term memory. To analyze the impact of differential CREB levels on learning and memory, we varied the gene dosage of CREB in two strains of mutant mice: (1) CREBαΔ mice, in which the α and Δ isoforms are disrupted, but a third isoform β is strongly up-regulated; (2) CREBcomp, a compound strain with one αΔ allele and one CREBnull allele in which all CREB isoforms are disrupted. To minimize genetic background effects, CREB mutations were backcrossed into a C57BL/6 and a FVB/N strain, respectively, and studies were performed in F1 hybrids from these lines. CREBcomp but not CREBαΔ F1 hybrids were impaired in water maze learning and fear conditioning, demonstrating a CREB gene dosage effect. However, analysis of the platform searching strategies in the water maze task suggested that CREBcomp mutants are impaired in behavioral flexibility rather than in spatial memory. In contrast to previous experiments using CREBαΔ mice with different genetic background, the F1 hybrid CREBαΔ and CREBcomp mice did not show deficits in a social transmission of food preference task nor in dentate gyrus and CA1 LTP as recorded from slice preparations. These data indicate that the hybrid vigor typical for F1 hybrids may compensate for a reduction in CREB levels in some tests. On the other hand, the persistence of clear behavioral deficits as shown by the F1 hybrid CREBcomp mice in water maze and fear conditioning indicates a robust and repeatable phenomenon that will permit further functional analysis of CREB. PMID:10454354

The Tennessee Mouse Genome Consortium: Identification of ocular mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu

2005-06-01

The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases andmore » disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.« less

Learning-Induced Suboptimal Compensation for PKCι/λ Function in Mutant Mice.

PubMed

Sheng, Tao; Wang, Shaoli; Qian, Dandan; Gao, Jun; Ohno, Shigeo; Lu, Wei

2017-06-01

PKCι/λ has been proposed to be crucial in the early expression of long-term potentiation (LTP). Here, we further investigate the potential role of PKCι/λ in learning and memory by generating PKCι/λ conditional knockout mice specifically lacking PKCι/λ in the hippocampal CA1 pyramidal cells. Surprisingly, PKCι/λ cKO mice show normal hippocampal LTP and memory. Further close-up observation reveals compensation for PKCι/λ expression by PKMζ in PKCι/λ cKO mice. This compensation was not observed under basal conditions, but was detected either after LTP induction or learning-associated behavioral training. Accordingly, in the early stage of LTP expression, a switch from PKCι/λ- to PKMζ-dependent molecular mechanisms was detected in PKCι/λ cKO mice. Notably, when cKO mice were challenged with more difficult hippocampus-dependent learning tasks, moderate learning deficits were detected, suggesting a suboptimal compensation for PKCι/λ's function in PKCι/λ cKO mice. Thus, under physiological conditions, PKCι/λ is essential for hippocampal early-LTP and long-term memory (LTM). © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Sleep apneas are increased in mice lacking monoamine oxidase A.

PubMed

Real, Caroline; Popa, Daniela; Seif, Isabelle; Callebert, Jacques; Launay, Jean-Marie; Adrien, Joëlle; Escourrou, Pierre

2007-10-01

Alterations in the serotonin (5-HT) system have been suggested as a mechanism of sleep apnea in humans and rodents. The objective is to evaluate the contribution of 5-HT to this disorder. We studied sleep and breathing (whole-body plethysmography) in mutant mice that lack monoamine oxidase A (MAOA) and have increased concentrations of monoamines, including 5-HT. Compared to wild-type mice, the mutants showed similar amounts of slow wave sleep (SWS) and rapid eye movement sleep (REMS), but exhibited a 3-fold increase in SWS and REMS apnea indices. Acute administration of the MAOA inhibitor clorgyline decreased REMS amounts and increased the apnea index in wild-type but not mutant mice. Parachlorophenylalanine, a 5-HT synthesis inhibitor, reduced whole brain concentrations of 5-HT in both strains, and induced a decrease in apnea index in mutant but not wild-type mice. Our results show that MAOA deficiency is associated with increased sleep apnea in mice and suggest that an acute or chronic excess of 5-HT contributes to this phenotype.

Enriching the Environment of [alpha]CaMKII[superscript T286A] Mutant Mice Reveals that LTD Occurs in Memory Processing but Must be Subsequently Reversed by LTP

ERIC Educational Resources Information Center

Soto, Florentina; Giese, K. Peter; Edwards, Frances A.; Parsley, Stephanie L.; Pilgram, Sara M.

2007-01-01

[alpha]CaMKII[superscript T286A] mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of [alpha]CaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate…

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice

PubMed Central

Xie, Huirong; Wang, Haibin; Tranguch, Susanne; Iwamoto, Ryo; Mekada, Eisuke; DeMayo, Francesco J.; Lydon, John P.; Das, Sanjoy K.; Dey, Sudhansu K.

2007-01-01

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation. PMID:17986609

Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate.

PubMed

Irie, Fumitoshi; Badie-Mahdavi, Hedieh; Yamaguchi, Yu

2012-03-27

Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism.

Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate

PubMed Central

Irie, Fumitoshi; Badie-Mahdavi, Hedieh; Yamaguchi, Yu

2012-01-01

Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism. PMID:22411800

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.

PubMed

Sasaki, Masato; Knobbe, Christiane B; Munger, Joshua C; Lind, Evan F; Brenner, Dirk; Brüstle, Anne; Harris, Isaac S; Holmes, Roxanne; Wakeham, Andrew; Haight, Jillian; You-Ten, Annick; Li, Wanda Y; Schalm, Stefanie; Su, Shinsan M; Virtanen, Carl; Reifenberger, Guido; Ohashi, Pamela S; Barber, Dwayne L; Figueroa, Maria E; Melnick, Ari; Zúñiga-Pflücker, Juan-Carlos; Mak, Tak W

2012-08-30

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

Fetal alcohol exposure disrupts metabolic signaling in hypothalamic proopiomelanocortin neurons via a circadian mechanism in male mice.

PubMed

Agapito, Maria A; Zhang, Changqing; Murugan, Sengottuvelan; Sarkar, Dipak K

2014-07-01

Early-life ethanol feeding (ELAF) alters the metabolic function of proopiomelanocortin (POMC)-producing neurons and the circadian expression of clock regulatory genes in the hypothalamus. We investigated whether the circadian mechanisms control the action of ELAF on metabolic signaling genes in POMC neurons. Gene expression measurements of Pomc and a selected group of metabolic signaling genes, Stat3, Sirt1, Pgc1-α, and Asb4 in laser-captured microdissected POMC neurons in the hypothalamus of POMC-enhanced green fluorescent protein mice showed circadian oscillations under light/dark and constant darkness conditions. Ethanol programmed these neurons such that the adult expression of Pomc, Stat3, Sirt, and Asb4 gene transcripts became arrhythmic. In addition, ELAF dampened the circadian peak of gene expression of Bmal1, Per1, and Per2 in POMC neurons. We crossed Per2 mutant mice with transgenic POMC-enhanced green fluorescent protein mice to determine the role of circadian mechanism in ELAF-altered metabolic signaling in POMC neurons. We found that ELAF failed to alter arrhythmic expression of most circadian genes, with the exception of the Bmal1 gene and metabolic signaling regulating genes in Per2 mutant mice. Comparison of the ELAF effects on the circadian blood glucose in wild-type and Per2 mutant mice revealed that ELAF dampened the circadian peak of glucose, whereas the Per2 mutation shifted the circadian cycle and prevented the ELAF dampening of the glucose peak. These data suggest the possibility that the Per2 gene mutation may regulate the ethanol actions on Pomc and the metabolic signaling genes in POMC neurons in the hypothalamus by blocking circadian mechanisms.

[Accumulation of the bvg- Bordetella pertussis a virulent mutants in the process of experimental whooping cough in mice].

PubMed

Medkova, A Iu; Siniashina, L N; Rumiantseva, Iu P; Voronina, O L; Kunda, M S; Karataev, G I

2013-01-01

The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

Csf3r mutations in mice confer a strong clonal HSC advantage via activation of Stat5

PubMed Central

Liu, Fulu; Kunter, Ghada; Krem, Maxwell M.; Eades, William C.; Cain, Jennifer A.; Tomasson, Michael H.; Hennighausen, Lothar; Link, Daniel C.

2008-01-01

A fundamental property of leukemic stem cells is clonal dominance of the bone marrow microenvironment. Truncation mutations of CSF3R, which encodes the G-CSF receptor (G-CSFR), are implicated in leukemic progression in patients with severe congenital neutropenia. Here we show that expression of a truncated mutant Csf3r in mice confers a strong clonal advantage at the HSC level that is dependent upon exogenous G-CSF. G-CSF–induced proliferation, phosphorylation of Stat5, and transcription of Stat5 target genes were increased in HSCs isolated from mice expressing the mutant Csf3r. Conversely, the proliferative advantage conferred by the mutant Csf3r was abrogated in myeloid progenitors lacking both Stat5A and Stat5B, and HSC function was reduced in mice expressing a truncated mutant Csf3r engineered to have impaired Stat5 activation. These data indicate that in mice, inappropriate Stat5 activation plays a key role in establishing clonal dominance by stem cells expressing mutant Csf3r. PMID:18292815

Impaired ventilatory acclimatization to hypoxia in mice lacking the immediate early gene fos B.

PubMed

Malik, Mohammad T; Peng, Ying-Jie; Kline, David D; Adhikary, Gautam; Prabhakar, Nanduri R

2005-01-15

Earlier studies on cell culture models suggested that immediate early genes (IEGs) play an important role in cellular adaptations to hypoxia. Whether IEGs are also necessary for hypoxic adaptations in intact animals is not known. In the present study we examined the potential importance of fos B, an IEG in ventilatory acclimatization to hypoxia. Experiments were performed on wild type and mutant mice lacking the fos B gene. Ventilation was monitored by whole body plethysmography in awake animals. Baseline ventilation under normoxia, and ventilatory response to acute hypoxia and hypercapnia were comparable between wild type and mutant mice. Hypobaric hypoxia (0.4 atm; 3 days) resulted in a significant elevation of baseline ventilation in wild type but not in mutant mice. Wild type mice exposed to hypobaric hypoxia manifested an enhanced hypoxic ventilatory response compared to pre-hypobaric hypoxia. In contrast, hypobaric hypoxia had no effect on the hypoxic ventilatory response in mutant mice. Hypercapnic ventilatory responses, however, were unaffected by hypobaric hypoxia in both groups of mice. These results suggest that the fos B, an immediate early gene, plays an important role in ventilatory acclimatization to hypoxia in mice.

Lack of tau proteins rescues neuronal cell death and decreases amyloidogenic processing of APP in APP/PS1 mice.

PubMed

Leroy, Karelle; Ando, Kunie; Laporte, Vincent; Dedecker, Robert; Suain, Valérie; Authelet, Michèle; Héraud, Céline; Pierrot, Nathalie; Yilmaz, Zehra; Octave, Jean-Noël; Brion, Jean-Pierre

2012-12-01

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Shiver me titin! Elucidating titin's role in shivering thermogenesis.

PubMed

Taylor-Burt, Kari R; Monroy, Jenna; Pace, Cinnamon; Lindstedt, Stan; Nishikawa, Kiisa C

2015-03-01

Shivering frequency scales predictably with body mass and is 10 times higher in a mouse than a moose. The link between shivering frequency and body mass may lie in the tuning of muscle elastic properties. Titin functions as a muscle 'spring', so shivering frequency may be linked to titin's structure. The muscular dystrophy with myositis (mdm) mouse is characterized by a deletion in titin's N2A region. Mice that are homozygous for the mdm mutation have a lower body mass, stiffer gait and reduced lifespan compared with their wild-type and heterozygous siblings. We characterized thermoregulation in these mice by measuring metabolic rate and tremor frequency during shivering. Mutants were heterothermic at ambient temperatures of 20-37°C while wild-type and heterozygous mice were homeothermic. Metabolic rate increased at smaller temperature differentials (i.e. the difference between body and ambient temperatures) in mutants than in non-mutants. The difference between observed tremor frequencies and shivering frequencies predicted by body mass was significantly larger for mutant mice than for wild-type or heterozygous mice, even after accounting for differences in body temperature. Together, the heterothermy in mutants, the increase in metabolic rate at low temperature differentials and the decreased tremor frequency demonstrate the thermoregulatory challenges faced by mice with the mdm mutation. Oscillatory frequency is proportional to the square root of stiffness, and we observed that mutants had lower active muscle stiffness in vitro. The lower tremor frequencies in mutants are consistent with reduced active muscle stiffness and suggest that titin affects the tuning of shivering frequency. © 2015. Published by The Company of Biologists Ltd.

A new spontaneous allele at the pink-eyed dilution (p) locus discovered in Mus musculus castaneus.

PubMed

Tsuji, A; Wakayama, T; Ishikawa, A

1995-10-01

Mutant mice characterized by a cream coat and pink eyes were spontaneously discovered among the descendants of Indonesian wild mice (Mus musculus castaneus). This mutant phenotype was controlled by a single autosomal recessive gene that was allelic to the pink-eyed dilution (p) gene. The mutant mouse phenotypically resembled the original p mouse which was the first mutant identified at this locus. Nevertheless, these two alleles differed in origin, a previous report suggesting that the original p allele was derived from Japanese wild mice (M. m. molossinus). Thus the symbol pcas (pink-eyed castaneus) was proposed for the present mutation allele.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon

PubMed Central

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis.

PubMed

Kirdis, Ebru; Jonsson, Ing-Marie; Kubica, Malgorzata; Potempa, Jan; Josefsson, Elisabet; Masalha, Mahmud; Foster, Simon J; Tarkowski, Andrzej

2007-01-01

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.

GH and IGF1: roles in energy metabolism of long-living GH mutant mice.

PubMed

Brown-Borg, Holly M; Bartke, Andrzej

2012-06-01

Of the multiple theories to explain exceptional longevity, the most robust of these has centered on the reduction of three anabolic protein hormones, growth hormone (GH), insulin-like growth factor, and insulin. GH mutant mice live 50% longer and exhibit significant differences in several aspects of energy metabolism as compared with wild-type mice. Mitochondrial metabolism is upregulated in the absence of GH, whereas in GH transgenic mice and dwarf mice treated with GH, multiple aspects of these pathways are suppressed. Core body temperature is markedly lower in dwarf mice, yet whole-body metabolism, as measured by indirect calorimetry, is surprisingly higher in Ames dwarf and Ghr-/- mice compared with normal controls. Elevated adiponectin, a key antiinflammatory cytokine, is also very likely to contribute to longevity in these mice. Thus, several important components related to energy metabolism are altered in GH mutant mice, and these differences are likely critical in aging processes and life-span extension.

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

PubMed Central

Lyerla, Timothy

2010-01-01

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS. PMID:20603711

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

PubMed

van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

PubMed Central

Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

PubMed

Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

PubMed

Gupta, Deepak Prasad; Hwang, Jae-Won; Cho, Eui-Sic; Kim, Won; Song, Chang Ho; Chai, Ok Hee

2017-01-01

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice. © 2017 S. Karger AG, Basel.

EPHRIN-A5 REGULATES INTER-MALE AGGRESSION IN MICE

PubMed Central

Sheleg, Michal; Yochum, Carrie L.; Richardson, Jason R.; Wagner, George C.; Zhou, Renping

2015-01-01

The Eph family of receptor tyrosine kinases play key roles in both the patterning of the developing nervous system and neural plasticity in the mature brain. To determine functions of ephrin-A5, a GPI-linked ligand to the Eph receptors, in animal behavior regulations, we examined effects of its inactivation on male mouse aggression. When tested in the resident-intruder paradigm for offensive aggression, ephrin-A5-mutant animals (ephrin-A5−/−) exhibited severe reduction in conspecific aggression compared to wild-type controls. On the contrary, defensive aggression in the form of target biting was higher in ephrin-A5−/− mice, indicating that the mutant mice are capable of attacking behavior. In addition, given the critical role of olfaction in aggressive behavior, we examined the ability of the ephrin-A5−/− mice to smell and found no differences between the mutant and control animals. Testosterone levels in the mutant mice were also found to be within the normal range. Taken together, our data reveal a new role of ephrin-A5 in the regulation of aggressive behavior in mice. PMID:25746458

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

PubMed Central

Inoue, Isao; Yanai, Kazuhiko; Kitamura, Daisuke; Taniuchi, Ichiro; Kobayashi, Takashi; Niimura, Kaku; Watanabe, Takehiko; Watanabe, Takeshi

1996-01-01

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter. PMID:8917588

Superoxide overproduction and kidney fibrosis: a new animal model

PubMed Central

Guimarães-Souza, Nadia Karina; Yamaleyeva, Liliya Marsovna; Lu, Baisong; Ramos, Ana Claudia Mallet de Souza; Bishop, Colin Edward; Andersson, Karl Erik

2015-01-01

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. PMID:25993073

Candidate genes for panhypopituitarism identified by gene expression profiling

PubMed Central

Mortensen, Amanda H.; MacDonald, James W.; Ghosh, Debashis

2011-01-01

Mutations in the transcription factors PROP1 and PIT1 (POU1F1) lead to pituitary hormone deficiency and hypopituitarism in mice and humans. The dysmorphology of developing Prop1 mutant pituitaries readily distinguishes them from those of Pit1 mutants and normal mice. This and other features suggest that Prop1 controls the expression of genes besides Pit1 that are important for pituitary cell migration, survival, and differentiation. To identify genes involved in these processes we used microarray analysis of gene expression to compare pituitary RNA from newborn Prop1 and Pit1 mutants and wild-type littermates. Significant differences in gene expression were noted between each mutant and their normal littermates, as well as between Prop1 and Pit1 mutants. Otx2, a gene critical for normal eye and pituitary development in humans and mice, exhibited elevated expression specifically in Prop1 mutant pituitaries. We report the spatial and temporal regulation of Otx2 in normal mice and Prop1 mutants, and the results suggest Otx2 could influence pituitary development by affecting signaling from the ventral diencephalon and regulation of gene expression in Rathke's pouch. The discovery that Otx2 expression is affected by Prop1 deficiency provides support for our hypothesis that identifying molecular differences in mutants will contribute to understanding the molecular mechanisms that control pituitary organogenesis and lead to human pituitary disease. PMID:21828248

Functional verification of a porcine myostatin propeptide mutant.

PubMed

Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

Increased Tau Phosphorylation and Tau Truncation, and Decreased Synaptophysin Levels in Mutant BRI2/Tau Transgenic Mice

PubMed Central

Garringer, Holly J.; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI2 gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI2 (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI2 can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration. PMID:23418567

Increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant BRI2/tau transgenic mice.

PubMed

Garringer, Holly J; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI(2) gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI(2) (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI(2) can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration.

Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease.

PubMed

Bogdanik, Laurent P; Sleigh, James N; Tian, Cong; Samuels, Mark E; Bedard, Karen; Seburn, Kevin L; Burgess, Robert W

2013-05-01

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.

A Glycine Betaine Importer Limits Salmonella Stress Resistance and Tissue Colonization by Reducing Trehalose Production

PubMed Central

Pilonieta, M. Carolina; Nagy, Toni A.; Jorgensen, Dana R.; Detweiler, Corrella S.

2012-01-01

SUMMARY Mechanisms by which Salmonella establish chronic infections are not well understood. Microbes respond to stress by importing or producing compatible solutes, small molecules that stabilize proteins and lipids. The Salmonella locus opuABCD (also called OpuC) encodes a predicted importer of the compatible solute glycine betaine. Under stress conditions, if glycine betaine cannot be imported, S. enterica produce the disaccharide trehalose, a highly effective compatible solute. We demonstrate that strains lacking opuABCD accumulate more trehalose under stress conditions than wild-type strains. ΔopuABCD mutant strains are more resistant to high salt, low pH and hydrogen peroxide, conditions that mimic aspects of innate immunity, in a trehalose-dependent manner. In addition, ΔopuABCD mutant strains require the trehalose production genes to out-compete wild-type strains in mice and macrophages. These data suggest that in the absence of opuABCD, trehalose accumulation increases bacterial resistance to stress in broth and mice. Thus, opuABCD reduces bacterial colonization via a mechanism that limits trehalose production. Mechanisms by which microbes limit disease may reveal novel pathways as therapeutic targets. PMID:22375627

Impairment of VGLUT2 but not VGLUT1 signaling reduces neuropathy-induced hypersensitivity.

PubMed

Leo, Sandra; Moechars, Dieder; Callaerts-Vegh, Zsuzsanna; D'Hooge, Rudi; Meert, Theo

2009-11-01

Glutamate is the major excitatory neurotransmitter in the central nervous system with an important role in nociceptive processing. Storage of glutamate into vesicles is controlled by vesicular glutamate transporters (VGLUT). Null mutants for VGLUT1 and VGLUT2 were poorly viable, thus, pain-related behavior was presently compared between heterozygote VGLUT1 and VGLUT2 mice and their respective wild-type littermates using a test battery that included a variety of assays for thermal and mechanical acute nociception, and inflammatory and neuropathic pain syndromes. Behavioral analysis of VGLUT1 mutant mice did not show important behavioral changes in the pain conditions tested. Reduction of VGLUT2 also resulted in unaltered acute nociceptive and inflammatory-induced pain behavior. Interestingly, VGLUT2 heterozygote mice showed an attenuation or absence of some typical neuropathic pain features (e.g., absence of mechanical and cold allodynia after spared nerve injury). Chronic constriction injury in VGLUT2 heterozygote mice showed also reduced levels of cold allodynia, but had no impact on mechanical thresholds. Together, these data suggest that VGLUT2, but not VGLUT1, plays a role in neuropathy-induced allodynia and hypersensitivity, and might be a therapeutic target to prevent and/or treat neuropathic pain.

Drug discrimination and neurochemical studies in alpha7 null mutant mice: tests for the role of nicotinic alpha7 receptors in dopamine release.

PubMed

Quarta, Davide; Naylor, Christopher G; Barik, Jacques; Fernandes, Cathy; Wonnacott, Susan; Stolerman, Ian P

2009-04-01

The nicotine discriminative stimulus has been linked to beta2-containing (beta2*) nicotinic receptors, with little evidence of a role for alpha7 nicotinic receptors, because nicotine discrimination was very weak in beta2 null mutant mice but normal in alpha7 mutants. As both alpha7 and beta2* nicotinic receptors have been implicated in nicotine-stimulated dopamine overflow, this study focused on the dopamine-mediated element in the nicotine stimulus by examining cross-generalisation between amphetamine and nicotine. Male alpha7 nicotinic receptor null mutant mice and wild-type controls were bred in-house and trained to discriminate nicotine (0.8 mg/kg) or (+)-amphetamine (0.6 mg/kg) from saline in a two-lever procedure with a tandem VI-30 FR-10 schedule of food reinforcement. Dopamine release from striatal slices was determined in parallel experiments. An alpha7 nicotinic receptor-mediated component of dopamine release was demonstrated in tissue from wild-type mice using choline as a selective agonist. This response was absent in tissue from null mutant animals. The mutation did not influence acquisition of drug discriminations but subtly affected the results of cross-generalisation tests. In mice trained to discriminate nicotine or amphetamine, there was partial cross-generalisation in wild-type mice and, at certain doses, these effects were attenuated in mutants. Further support for an alpha7 nicotinic receptor-mediated component was provided by the ability of the alpha7 nicotinic receptor antagonist methyllycaconitine to attenuate responses to nicotine and amphetamine in wild-type mice. These findings support the concept of an alpha7 nicotinic receptor-mediated dopaminergic element in nicotine discrimination, warranting further tests with selective dopamine agonists.

Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

PubMed Central

Huang, Ching-Hsun; Pei, Ju-Chun; Luo, Da-Zhong; Chen, Ching; Chen, Yi-Wen; Lai, Wen-Sung

2015-01-01

Accumulating evidence from human genetic studies has suggested several functional candidate genes that might contribute to susceptibility to schizophrenia, including AKT1 and neuregulin 1 (NRG1). Recent findings also revealed that NRG1 stimulates the PI3-kinase/AKT signaling pathway, which might be involved in the functional outcomes of some schizophrenic patients. The aim of this study was to evaluate the effect of Akt1-deficiency and Nrg1-deficiency alone or in combination in the regulation of behavioral phenotypes, cognition, and social functions using genetically modified mice as a model. Male Akt1+/−, Nrg1+/−, and double mutant mice were bred and compared with their wild-type (WT) littermate controls. In Experiment 1, general physical examination revealed that all mutant mice displayed a normal profile of body weight during development and a normal brain activity with microPET scan. In Experiment 2, no significant genotypic differences were found in our basic behavioral phenotyping, including locomotion, anxiety-like behavior, and sensorimotor gating function. However, both Nrg1+/− and double mutant mice exhibited impaired episodic-like memory. Double mutant mice also had impaired sociability. In Experiment 3, a synergistic epistasis between Akt1 and Nrg1 was further confirmed in double mutant mice in that they had impaired social interaction compared to the other 3 groups, especially encountering with a novel male or an ovariectomized female. Double mutant and Nrg1+/− mice also emitted fewer female urine-induced ultrasonic vocalization calls. Collectively, our results indicate that double deficiency of Akt1 and Nrg1 can result in the impairment of social cognitive functions, which might be pertinent to the pathogenesis of schizophrenia-related social cognition. PMID:25688191

Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia.

PubMed

Huang, Ching-Hsun; Pei, Ju-Chun; Luo, Da-Zhong; Chen, Ching; Chen, Yi-Wen; Lai, Wen-Sung

2014-01-01

Accumulating evidence from human genetic studies has suggested several functional candidate genes that might contribute to susceptibility to schizophrenia, including AKT1 and neuregulin 1 (NRG1). Recent findings also revealed that NRG1 stimulates the PI3-kinase/AKT signaling pathway, which might be involved in the functional outcomes of some schizophrenic patients. The aim of this study was to evaluate the effect of Akt1-deficiency and Nrg1-deficiency alone or in combination in the regulation of behavioral phenotypes, cognition, and social functions using genetically modified mice as a model. Male Akt1 (+/-), Nrg1 (+/-), and double mutant mice were bred and compared with their wild-type (WT) littermate controls. In Experiment 1, general physical examination revealed that all mutant mice displayed a normal profile of body weight during development and a normal brain activity with microPET scan. In Experiment 2, no significant genotypic differences were found in our basic behavioral phenotyping, including locomotion, anxiety-like behavior, and sensorimotor gating function. However, both Nrg1 (+/-) and double mutant mice exhibited impaired episodic-like memory. Double mutant mice also had impaired sociability. In Experiment 3, a synergistic epistasis between Akt1 and Nrg1 was further confirmed in double mutant mice in that they had impaired social interaction compared to the other 3 groups, especially encountering with a novel male or an ovariectomized female. Double mutant and Nrg1 (+/-) mice also emitted fewer female urine-induced ultrasonic vocalization calls. Collectively, our results indicate that double deficiency of Akt1 and Nrg1 can result in the impairment of social cognitive functions, which might be pertinent to the pathogenesis of schizophrenia-related social cognition.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

PubMed

Kurosawa, Koreyuki; Inoue, Yui; Kakugawa, Yoichiro; Yamashita, Yoji; Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Momoi, Yuki; Sato, Ikuro; Chiba, Natsuko; Suzuki, Mai; Ogoh, Honami; Yamada, Hidekazu; Miura, Koh; Watanabe, Toshio; Tanuma, Nobuhiro; Tachi, Masahiro; Shima, Hiroshi

2018-05-14

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-ras G12D -expressing and Ppp6c-deficient) mice in which K-ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lip, nipples, external genitalia, anus and palms, and had to be sacrificed by three weeks after induction by tamoxifen, while comparably-treated K-ras G12D -expressing mice did not. HE-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinoma. Immunohistochemical analysis of lip of doubly-mutant versus K-ras G12D mice revealed that cell proliferation and cell size increased approximately two-fold relative to K-ras G12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-ras G12D only mice. Moreover, AKT phosphorylation increased in K-ras G12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6, and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-ras G12D -dependent tumor promotion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

PubMed

Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

2009-01-01

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Reduced Gut Acidity Induces an Obese-Like Phenotype in Drosophila melanogaster and in Mice

PubMed Central

Yen, Jui-Hung; Kuo, Ping-Chang; Yeh, Sheng-Rong; Lin, Hung-Yu; Fu, Tsai-Feng; Wu, Ming-Shiang; Wang, Horng-Dar; Wang, Pei-Yu

2015-01-01

In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients. PMID:26436771

Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

PubMed Central

Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

2001-01-01

Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

PubMed

van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

2014-06-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

PubMed Central

van Lier, Christina J.; Sha, Jian; Kirtley, Michelle L.; Cao, Anthony; Tiner, Bethany L.; Erova, Tatiana E.; Cong, Yingzi; Kozlova, Elena V.; Popov, Vsevolod L.; Baze, Wallace B.

2014-01-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4+ and CD8+ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection. PMID:24686064

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.

PubMed

Ihara, Tatsuya; Mitsui, Takahiko; Nakamura, Yuki; Kira, Satoru; Miyamoto, Tatsuya; Nakagomi, Hiroshi; Sawada, Norifumi; Hirayama, Yuri; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Yoichi; Yoshiyama, Mitsuharu; Andersson, Karl-Erik; Nakao, Atsuhito; Takeda, Masayuki; Koizumi, Schuichi

2017-04-01

The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (Clock Δ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP. Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 Clock Δ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods. No significant differences were observed in behavior patterns between Clock Δ19/Δ19 and WT mice. Clock Δ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in Clock Δ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in Clock Δ19/Δ19 mice than in WT mice. We demonstrated that Clock Δ19/Δ19 mice showed the phenotype of NOC/NP. The Clock Δ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Repeated psychosocial stress at night affects the circadian activity rhythm of male mice.

PubMed

Bartlang, Manuela S; Oster, Henrik; Helfrich-Förster, Charlotte

2015-06-01

We have recently shown that molecular rhythms in the murine suprachiasmatic nucleus (SCN) are affected by repeated social defeat (SD) during the dark/active phase (social defeat dark [SDD]), while repeated SD during the light/inactive phase (social defeat light [SDL]) had no influence on PERIOD2::LUCIFERASE explant rhythms in the SCN. Here we assessed the effects of the same stress paradigm by in vivo biotelemetry on 2 output rhythms of the circadian clock (i.e., activity and core body temperature) in wild-type (WT) and clock-deficient Period (Per)1/2 double-mutant mice during and following repeated SDL and SDD. In general, stress had more pronounced effects on activity compared to body temperature rhythms. Throughout the SD procedure, activity and body temperature were markedly increased during the 2 h of stressor exposure at zeitgeber time (ZT) 1 to ZT3 (SDL mice) and ZT13 to ZT15 (SDD mice), which was compensated by decreased activity during the remaining dark phase (SDL and SDD mice) and light phase (SDL mice) in both genotypes. Considerable differences in the activity between SDL and SDD mice were seen in the poststress period. SDD mice exhibited a reduced first activity bout at ZT13, delayed activity onset, and, consequently, a more narrow activity bandwidth compared with single-housed control (SHC) and SDL mice. Given that this effect was absent in Per1/2 mutant SDD mice and persisted under constant darkness conditions in SDD WT mice, it suggests an involvement of the endogenous clock. Taken together, the present findings demonstrate that SDD has long-lasting consequences for the functional output of the biological clock that, at least in part, appear to depend on the clock genes Per1 and Per2. © 2015 The Author(s).

Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

PubMed

Dang, Mai T; Yokoi, Fumiaki; McNaught, Kevin St P; Jengelley, Toni-Ann; Jackson, Tehone; Li, Jianyong; Li, Yuqing

2005-12-01

A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.

Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response.

PubMed

Larroquette, Frédérique; Seto, Lesley; Gaub, Perrine L; Kamal, Brishna; Wallis, Deeann; Larivière, Roxanne; Vallée, Joanne; Robitaille, Richard; Tsuda, Hiroshi

2015-11-15

Missense mutations (P56S) in Vapb are associated with autosomal dominant motor neuron diseases: amyotrophic lateral sclerosis and lower motor neuron disease. Although transgenic mice overexpressing the mutant vesicle-associated membrane protein-associated protein B (VAPB) protein with neuron-specific promoters have provided some insight into the toxic properties of the mutant proteins, their role in pathogenesis remains unclear. To identify pathological defects in animals expressing the P56S mutant VAPB protein at physiological levels in the appropriate tissues, we have generated Vapb knock-in mice replacing wild-type Vapb gene with P56S mutant Vapb gene and analyzed the resulting pathological phenotypes. Heterozygous P56S Vapb knock-in mice show mild age-dependent defects in motor behaviors as characteristic features of the disease. The homozygous P56S Vapb knock-in mice show more severe defects compared with heterozygous mice reflecting the dominant and dose-dependent effects of P56S mutation. Significantly, the knock-in mice demonstrate accumulation of P56S VAPB protein and ubiquitinated proteins in cytoplasmic inclusions, selectively in motor neurons. The mutant mice demonstrate induction of ER stress and autophagic response in motor neurons before obvious onset of behavioral defects, suggesting that these cellular biological defects might contribute to the initiation of the disease. The P56S Vapb knock-in mice could be a valuable tool to gain a better understanding of the mechanisms by which the disease arises. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice.

PubMed

Beamer, W G; Cunliffe-Beamer, T L; Shultz, K L; Langley, S H; Roderick, T H

1988-05-01

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.

Mouse Drawer System (MDS): An autonomous hardware for supporting mice space research

NASA Astrophysics Data System (ADS)

Liu, Y.; Biticchi, R.; Alberici, G.; Tenconi, C.; Cilli, M.; Fontana, V.; Cancedda, R.; Falcetti, G.

2005-08-01

For the scientific community the ability of flying mice under weightless conditions in space, compared to other rodents, offers many valuable advantages. These include the option of testing a wide range of wild-type and mutant animals, an increased animal number for flight, and a reduced demand on shuttle resources and crew time. In this study, we describe a spaceflight hardware for mice, the Mouse Drawer System (MDS). MDS can interface with Space Shuttle middeck and International Space Station Express Rack. It consists of Mice Chamber, Liquid Handling Subsystem, Food Delivery Subsystem, Air Conditioning Subsystem, Illumination Subsystem, Observation Subsystem and Payload Control Unit. It offers single or paired containment for 6-8 mice with a mean weight of 40 grams/mouse for a period of up to 3 months. Animal tests were conducted in a MDS breadboard to validate the biocompatibility of various subsystems. Mice survived in all tests of short and long duration. Results of blood parameters, histology and air/waste composition analysis showed that MDS subsystems meet the NIH guidelines for temperature, humidity, food and water access, air quality, odour and waste management.

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hsu-Pin; Hsu, Shu-Yuan; Wu, Wen-Ai

Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB{sup +/−} mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species.more » The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.« less

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

2014-01-01

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

Deletion of Ku80 causes early aging independent of chronic inflammation and Rag-1-induced DSBs.

PubMed

Holcomb, Valerie B; Vogel, Hannes; Hasty, Paul

2007-01-01

Animal models of premature aging are often defective for DNA repair. Ku80-mutant mice are disabled for nonhomologous end joining; a pathway that repairs both spontaneous DNA double-strand breaks (DSBs) and induced DNA DSBs generated by the action of a complex composed of Rag-1 and Rag-2 (Rag). Rag is essential for inducing DSBs important for assembling V(D)J segments of antigen receptor genes that are required for lymphocyte development. Thus, deletion of either Rag-1 or Ku80 causes severe combined immunodeficiency (scid) leading to chronic inflammation. In addition, Rag-1 induces breaks at non-B DNA structures. Previously we reported Ku80-mutant mice undergo premature aging, yet we do not know the root cause of this phenotype. Early aging may be caused by either defective repair of spontaneous DNA damage, defective repair of Rag-1-induced breaks or chronic inflammation caused by scid. To address this issue, we analyzed aging in control and Ku80-mutant mice deleted for Rag-1 such that both cohorts are scid and suffer from chronic inflammation. We make two observations: (1) chronic inflammation does not cause premature aging in these mice and (2) Ku80-mutant mice exhibit early aging independent of Rag-1. Therefore, this study supports defective repair of spontaneous DNA damage as the root cause of early aging in Ku80-mutant mice.

Scheduled Feeding Alters the Timing of the Suprachiasmatic Nucleus Circadian Clock in Dexras 1-Deficient Mice

PubMed Central

Bouchard-Cannon, Pascale; Cheng, Hai-Ying M.

2013-01-01

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1−/−) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1−/− mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1−/− mice by ~2 h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. PMID:22928915

Aberrant cognitive phenotypes and altered hippocampal BDNF expression related to epigenetic modifications in mice lacking the post-synaptic scaffolding protein SHANK1: Implications for autism spectrum disorder.

PubMed

Sungur, A Özge; Jochner, Magdalena C E; Harb, Hani; Kılıç, Ayşe; Garn, Holger; Schwarting, Rainer K W; Wöhr, Markus

2017-08-01

Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by persistent deficits in social communication/interaction, together with restricted/repetitive patterns of behavior. ASD is among the most heritable neuropsychiatric conditions, and while available evidence points to a complex set of genetic factors, the SHANK gene family has emerged as one of the most promising candidates. Here, we assessed ASD-related phenotypes with particular emphasis on social behavior and cognition in Shank1 mouse mutants in comparison to heterozygous and wildtype littermate controls across development in both sexes. While social approach behavior was evident in all experimental conditions and social recognition was only mildly affected by genotype, Shank1 -/- null mutant mice were severely impaired in object recognition memory. This effect was particularly prominent in juveniles, not due to impairments in object discrimination, and replicated in independent mouse cohorts. At the neurobiological level, object recognition deficits were paralleled by increased brain-derived neurotrophic factor (BDNF) protein expression in the hippocampus of Shank1 -/- mice; yet BDNF levels did not differ under baseline conditions. We therefore investigated changes in the epigenetic regulation of hippocampal BDNF expression and detected an enrichment of histone H3 acetylation at the Bdnf promoter1 in Shank1 -/- mice, consistent with increased learning-associated BDNF. Together, our findings indicate that Shank1 deletions lead to an aberrant cognitive phenotype characterized by severe impairments in object recognition memory and increased hippocampal BDNF levels, possibly due to epigenetic modifications. This result supports the link between ASD and intellectual disability, and suggests epigenetic regulation as a potential therapeutic target. © 2017 Wiley Periodicals, Inc.

Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides.

PubMed

Yang, Shaobin; Pascual-Guiral, Sònia; Ponce, Rebeca; Giménez-Llort, Lydia; Baltrons, María A; Arancio, Ottavio; Palacio, Jose R; Clos, Victoria M; Yuste, Victor J; Bayascas, Jose R

2017-01-01

The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1) with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G

2007-01-01

We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsivenessmore » to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.« less

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

PubMed

McBrayer, Zofeyah L; Dimova, Jiva; Pisansky, Marc T; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C; O'Connor, Michael B

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration

PubMed Central

McBrayer, Zofeyah L.; Dimova, Jiva; Pisansky, Marc T.; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C.; O’Connor, Michael B.

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors. PMID:26444546

Altered sexual and social behaviors in trp2 mutant mice

PubMed Central

Leypold, Bradley G.; Yu, C. Ron; Leinders-Zufall, Trese; Kim, Michelle M.; Zufall, Frank; Axel, Richard

2002-01-01

We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male–female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners. PMID:11972034

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

PubMed

Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

2016-03-10

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells.

PubMed

Fan, Heng-Yu; Liu, Zhilin; Cahill, Nicola; Richards, JoAnne S

2008-09-01

FSH activates the phosphatidylinositol-3 kinase (PI3K)/acute transforming retrovirus thymoma protein kinase pathway and thereby enhances granulosa cell differentiation in culture. To identify the physiological role of the PI3K pathway in vivo we disrupted the PI3K suppressor, Pten, in developing ovarian follicles. To selectively disrupt Pten expression in granulosa cells, Ptenfl/fl mice were mated with transgenic mice expressing cAMP response element recombinase driven by Cyp19 promoter (Cyp19-Cre). The resultant Pten mutant mice were fertile, ovulated more oocytes, and produced moderately more pups than control mice. These physiological differences in the Pten mutant mice were associated with hyperactivation of the PI3K/acute transforming retrovirus thymoma protein kinase pathway, decreased susceptibility to apoptosis, and increased proliferation of mutant granulosa cells. Strikingly, corpora lutea of the Pten mutant mice persisted longer than those of control mice. Although the follicular and luteal cell steroidogenesis in Ptenfl/fl;Cyp19-Cre mice was similar to controls, viable nonsteroidogenic luteal cells escaped structural luteolysis. These findings provide the novel evidence that Pten impacts the survival/life span of granulosa/luteal cells and that its loss not only results in the facilitated ovulation but also in the persistence of nonsteroidogenic luteal structures in the adult mouse ovary.

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

PubMed Central

Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra

2016-01-01

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

A post-transcriptional compensatory pathway in heterozygous ventricular myosin light chain 2-deficient mice results in lack of gene dosage effect during normal cardiac growth or hypertrophy.

PubMed

Minamisawa, S; Gu, Y; Ross, J; Chien, K R; Chen, J

1999-04-09

Our previous study of homozygous mutants of the ventricular specific isoform of myosin light chain 2 (mlc-2v) demonstrated that mlc-2v plays an essential role in murine heart development (Chen, J., Kubalak, S. W., Minamisawa, S., Price, R. L., Becker, K. D., Hickey, R., Ross, J., Jr., and Chien, K. R. (1998) J. Biol. Chem. 273, 1252-1256). As gene dosage of some myofibrillar proteins can affect muscle function, we have analyzed heterozygous mutants in depth. Ventricles of heterozygous mutants displayed a 50% reduction in mlc-2v mRNA, yet expressed normal levels of protein both under basal conditions and following induction of cardiac hypertrophy by aortic constriction. Heterozygous mutants exhibited cardiac function comparable to that of wild-type littermate controls both prior to and following aortic constriction. There were no significant differences in contractility and responses to calcium between wild-type and heterozygous unloaded cardiomyocytes. We conclude that heterozygous mutants show neither a molecular nor a physiological cardiac phenotype either at base line or following hypertrophic stimuli. These results suggest that post-transcriptional compensatory mechanisms play a major role in maintaining the level of MLC-2v protein in murine hearts. In addition, as our mlc-2v knockout mutants were created by a knock-in of Cre recombinase into the endogenous mlc-2v locus, this study demonstrates that heterozygous mlc-2v cre knock-in mice are appropriate for ventricular specific gene targeting.

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

PubMed

Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

2015-11-09

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

PubMed Central

Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

2015-01-01

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

Bidirectional modulation of fear extinction by mediodorsal thalamic firing in mice.

PubMed

Lee, Sukchan; Ahmed, Touqeer; Lee, Soojung; Kim, Huisu; Choi, Sukwoo; Kim, Duk-Soo; Kim, Sang Jeong; Cho, Jeiwon; Shin, Hee-Sup

2011-12-25

The mediodorsal thalamic nucleus has been implicated in the control of memory processes. However, the underlying neural mechanism remains unclear. Here we provide evidence for bidirectional modulation of fear extinction by the mediodorsal thalamic nucleus. Mice with a knockout or mediodorsal thalamic nucleus-specific knockdown of phospholipase C β4 exhibited impaired fear extinction. Mutant mediodorsal thalamic nucleus neurons in slices showed enhanced burst firing accompanied by increased T-type Ca(2+) currents; blocking of T channels in vivo rescued the fear extinction. Tetrode recordings in freely moving mice revealed that, during extinction, the single-spike (tonic) frequency of mediodorsal thalamic nucleus neurons increased in wild-type mice, but was static in mutant mice. Furthermore, tonic-evoking microstimulations of the mediodorsal thalamic nucleus, contemporaneous with the extinction tones, rescued fear extinction in mutant mice and facilitated it in wild-type mice. In contrast, burst-evoking microstimulation suppressed extinction in wild-type mice, mimicking the mutation. These results suggest that the firing mode of the mediodorsal thalamic nucleus is critical for the modulation of fear extinction.

Bone morphogenetic protein signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior.

PubMed

Caronia, Giuliana; Wilcoxon, Jennifer; Feldman, Polina; Grove, Elizabeth A

2010-05-05

The cortical hem is an embryonic signaling center that generates bone morphogenetic proteins (BMPs) and acts as an organizer for the hippocampus. The role of BMP signaling in hippocampal neurogenesis, however, has not been established. We therefore generated mice that were deficient in Bmpr1b constitutively, and deficient in Bmpr1a conditionally in the dorsal telencephalon. In double mutant male and female mice, the dentate gyrus (DG) was dramatically smaller than in control mice, reflecting decreased production of granule neurons at the peak period of DG neurogenesis. Additionally, the pool of cells that generates new DG neurons throughout life was reduced, commensurate with the smaller size of the DG. Effects of diminished BMP signaling on the cortical hem were at least partly responsible for these defects in DG development. Reduction of the DG and its major extrinsic output to CA3 raised the possibility that the DG was functionally compromised. We therefore looked for behavioral deficits in double mutants and found that the mice were less responsive to fear- or anxiety-provoking stimuli, whether the association of the stimulus with fear or anxiety was learned or innate. Given that no anatomical defects appeared in the double mutant telencephalon outside the DG, our observations support a growing literature that implicates the hippocampus in circuitry mediating fear and anxiety. Our results additionally indicate a requirement for BMP signaling in generating the dorsalmost neuronal lineage of the telencephalon, DG granule neurons, and in the development of the stem cell niche that makes neurons in the adult hippocampus.

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

PubMed Central

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P.

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction. PMID:22312434

Murine GRPR and stathmin control in opposite directions both cued fear extinction and neural activities of the amygdala and prefrontal cortex.

PubMed

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.

Deletion of Core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development

PubMed Central

Wu, Mengrui; Li, Chenguan; Zhu, Guochun; Wang, Yiping; Jules, Joel; Lu, Yun; McConnell, Matthew; Wang, Yong-Jun; Shao, Jian-Zhong; Li, Yi-Ping; Chen, Wei

2015-01-01

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage causing alveolus defects which led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wildtype mice indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors impacted bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx), but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development, but also facilitate the design of novel therapeutic approaches for skeletal diseases. PMID:24798493

Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

PubMed

Wu, Mengrui; Li, Chenguan; Zhu, Guochun; Wang, Yiping; Jules, Joel; Lu, Yun; McConnell, Matthew; Wang, Yong-Jun; Shao, Jian-Zhong; Li, Yi-Ping; Chen, Wei

2014-08-01

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases. Copyright © 2014. Published by Elsevier Inc.

Evaluation of Nitrofurantoin Combination Therapy of Metronidazole-Sensitive and -Resistant Helicobacter pylori Infections in Mice

PubMed Central

Jenks, Peter J.; Ferrero, Richard L.; Tankovic, Jacques; Thiberge, Jean-Michel; Labigne, Agnès

2000-01-01

The main objectives of this study were to determine whether the nitroreductase enzyme encoded by the rdxA gene of Helicobacter pylori was responsible for reductive activation of nitrofurantoin and whether a triple-therapy regimen with nitrofurantoin was able to eradicate metronidazole-sensitive and -resistant H. pylori infections from mice. The susceptibilities to nitrofurantoin of parent and isogenic rdxA mutant strains (three pairs), as well as a series of matched metronidazole-sensitive and -resistant strains isolated from mice (30) and patients (20), were assessed by agar dilution determination of the MIC. Groups of mice colonized with the metronidazole-sensitive H. pylori SS1 strain or a metronidazole-resistant rdxA SS1 mutant were treated with either metronidazole or nitrofurantoin as part of a triple-therapy regimen. One month after the completion of treatment the mice were sacrificed and their stomachs were cultured for H. pylori. The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 μg/ml. There was no significant difference between the susceptibility to nitrofurantoin of the parental strains and those of respective rdxA mutants or between those of matched metronidazole-sensitive and -resistant H. pylori isolates. The regimen with metronidazole eradicated infection from all eight SS1-infected mice and from one of eight mice inoculated with the rdxA mutant (P ≤ 0.001). The regimen with nitrofurantoin failed to eradicate infection from any of the six SS1-infected mice (P ≤ 0.001) and cleared infection from one of seven mice inoculated with the rdxA mutant. These results demonstrate that, despite the good in vitro activity of nitrofurantoin against H. pylori and the lack of cross-resistance between metronidazole and nitrofurantoin, eradication regimens involving nitrofurantoin are unable to eradicate either metronidazole-sensitive or -resistant H. pylori infections from mice. PMID:10991835

Reanalysis of parabiosis of obesity mutants in the age of leptin.

PubMed

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

2015-07-21

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Reanalysis of parabiosis of obesity mutants in the age of leptin

PubMed Central

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M.

2015-01-01

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin−/− double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure. PMID:26150485

Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant

PubMed Central

Infante, E; Aguilar, L D; Gicquel, B; Pando, R Hernandez

2005-01-01

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-γ, interleukin (IL)-4 and tumour necrosis factor (TNF)-α. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette–Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis. PMID:15958066

Contribution of Egr1/zif268 to Activity-Dependent Arc/Arg3.1 Transcription in the Dentate Gyrus and Area CA1 of the Hippocampus

PubMed Central

Penke, Zsuzsa; Chagneau, Carine; Laroche, Serge

2011-01-01

Egr1, a member of the Egr family of transcription factors, and Arc are immediate early genes known to play major roles in synaptic plasticity and memory. Despite evidence that Egr family members can control Arc transcriptional regulation, demonstration of a selective role of Egr1 alone is lacking. We investigated the extent to which activity-dependent Arc expression is dependent on Egr1 by analyzing Arc mRNA expression using fluorescence in situ hybridization in the dorsal dentate gyrus and CA1 of wild-type (WT) and Egr1 knockout mice. Following electroconvulsive shock, we found biphasic expression of Arc in area CA1 in mice, consisting in a rapid (30 min) and transient wave followed by a second late-phase of expression (8 h), and a single but prolonged wave of expression in the dentate gyrus. Egr1 deficiency abolished the latest, but not the early wave of Arc expression in CA1, and curtailed that of the dentate gyrus. Since the early wave of Arc expression was not affected in Egr1 mutant mice, we next analyzed behaviorally induced Arc expression patterns as an index of neural ensemble activation in the dentate gyrus and area CA1 of WT and Egr1 mutant mice. Spatial exploration of novel or familiar environments induced in mice a single early and transient wave of Arc expression in the dentate gyrus and area CA1, which were not affected in Egr1 mutant mice. Analyses of Arc-expressing cells revealed that exploration recruits similar size dentate gyrus and CA1 neural ensembles in WT and Egr1 knockout mice. These findings suggest that hippocampal neural ensembles are normally activated immediately following spatial exploration in Egr1 knockout mice, indicating normal hippocampal encoding of information. They also provide evidence that in condition of strong activation Egr1 alone can control late-phases of activity-dependent Arc transcription in the dentate gyrus and area CA1 of the hippocampus. PMID:21887136

LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

PubMed Central

Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

2012-01-01

GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

PubMed

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

2017-02-15

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK - ) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK - HSV-1 remain elusive. Using three genetically engineered HSV-1 TK - mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK - mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK - HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts. Copyright © 2017 American Society for Microbiology.

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice

PubMed Central

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu

2016-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484–490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. IMPORTANCE Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK−) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK− HSV-1 remain elusive. Using three genetically engineered HSV-1 TK− mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK− mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK− HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts. PMID:27974554

Mutations in Prickle Orthologs Cause Seizures in Flies, Mice, and Humans

PubMed Central

Tao, Hirotaka; Manak, J. Robert; Sowers, Levi; Mei, Xue; Kiyonari, Hiroshi; Abe, Takaya; Dahdaleh, Nader S.; Yang, Tian; Wu, Shu; Chen, Shan; Fox, Mark H.; Gurnett, Christina; Montine, Thomas; Bird, Thomas; Shaffer, Lisa G.; Rosenfeld, Jill A.; McConnell, Juliann; Madan-Khetarpal, Suneeta; Berry-Kravis, Elizabeth; Griesbach, Hilary; Saneto, Russell P.; Scott, Matthew P.; Antic, Dragana; Reed, Jordan; Boland, Riley; Ehaideb, Salleh N.; El-Shanti, Hatem; Mahajan, Vinit B.; Ferguson, Polly J.; Axelrod, Jeffrey D.; Lehesjoki, Anna-Elina; Fritzsch, Bernd; Slusarski, Diane C.; Wemmie, John; Ueno, Naoto; Bassuk, Alexander G.

2011-01-01

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution. PMID:21276947

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

PubMed Central

Giovannini, Marco; Robanus-Maandag, Els; Niwa-Kawakita, Michiko; van der Valk, Martin; Woodruff, James M.; Goutebroze, Laurence; Mérel, Philippe; Berns, Anton; Thomas, Gilles

1999-01-01

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages. PMID:10215625

The Gne M712T mouse as a model for human glomerulopathy.

PubMed

Kakani, Sravan; Yardeni, Tal; Poling, Justin; Ciccone, Carla; Niethamer, Terren; Klootwijk, Enriko D; Manoli, Irini; Darvish, Daniel; Hoogstraten-Miller, Shelley; Zerfas, Patricia; Tian, E; Ten Hagen, Kelly G; Kopp, Jeffrey B; Gahl, William A; Huizing, Marjan

2012-04-01

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs.

PubMed

Feodorova, V A; Pan'kina, L N; Savostina, E P; Sayapina, L V; Motin, V L; Dentovskaya, S V; Shaikhutdinova, R Z; Ivanov, S A; Lindner, B; Kondakova, A N; Bystrova, O V; Kocharova, N A; Senchenkova, S N; Holst, O; Pier, G B; Knirel, Y A; Anisimov, A P

2007-11-01

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice.

PubMed

Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P

1996-09-01

The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

Localization of efferent neurotransmitters in the inner ear of the homozygous Bronx waltzer mutant mouse.

PubMed

Kong, W J; Scholtz, A W; Hussl, B; Kammen-Jolly, K; Schrott-Fischer, A

2002-05-01

Naturally occurring mutant mice provide an excellent model for the study of genetic malformations of the inner ear. Mice homozygous for the Bronx waltzer (bv/bv) mutation are severely hearing impaired or deaf and exhibit a 'waltzing' gait. Functional aspects of cochlear and vestibular efferents in the bv/bv mutant mouse are not well known. The present study was designed to evaluate several candidates of efferent neurotransmitters or neuromodulators including choline acetyltransferase (ChAT), gamma-aminobutyric acid (GABA), and calcitonin gene-related peptide (CGRP) in the inner ear of the bv/bv mutant mouse. Ultrastructural investigations at both light and electron microscopic level were performed. Ultrastructural morphologic evaluations of the cochlea and the vestibular end-organs were also undertaken. It is demonstrated that ChAT, GABA and CGRP immunoreactivities are present in the cochlea and in vestibular end-organs of bv/bv mutant mice. In the organ of Corti, immunoreactivity of ChAT, GABA and CGRP is confined to the inner spiral fibers, tunnel-crossing fibers, and the vesiculated nerve endings synapsing with outer hair cells. Interestingly, immunoreactivity was detectable even where inner hair cells appeared missing. Results also revealed malformations of the outer hair cells with synaptic contacts to efferent nerve endings consistently intact. In the neurosensory epithelia of the vestibular end-organs, the presence of ChAT, GABA, and CGRP immunoreactivity was localized at the vestibular efferents, with the exception of the macula of saccule. In one 8-month-old macula of utricle where the depletion of hair cells appeared highest, ChAT immunostaining was still discernible. Ultrastructural investigation demonstrated that vesiculated efferent nerve endings make synaptic contact with the outer hair cells in the organ of Corti and with type II hair cells in the vestibular end-organs. The present study provides further support that the efferent system in the bv/bv mutant inner ear is morphologically as well as functionally mature. These findings also demonstrate that if and when the onset of efferent degeneration in the bv/bv mutant inner ear occurs, it transpires subsequent to pathological conditions in the hair cells. The present findings give further indication that the efferent systems of the bv/bv mutant inner ear are independent of the afferent systems in many aspects including development, maturation as well as degeneration.

Zinc transporter 3 is involved in learned fear and extinction, but not in innate fear.

PubMed

Martel, Guillaume; Hevi, Charles; Friebely, Olivia; Baybutt, Trevor; Shumyatsky, Gleb P

2010-11-01

Synaptically released Zn²+ is a potential modulator of neurotransmission and synaptic plasticity in fear-conditioning pathways. Zinc transporter 3 (ZnT3) knock-out (KO) mice are well suited to test the role of zinc in learned fear, because ZnT3 is colocalized with synaptic zinc, responsible for its transport to synaptic vesicles, highly enriched in the amygdala-associated neural circuitry, and ZnT3 KO mice lack Zn²+ in synaptic vesicles. However, earlier work reported no deficiency in fear memory in ZnT3 KO mice, which is surprising based on the effects of Zn²+ on amygdala synaptic plasticity. We therefore reexamined ZnT3 KO mice in various tasks for learned and innate fear. The mutants were deficient in a weak fear-conditioning protocol using single tone-shock pairing but showed normal memory when a stronger, five-pairing protocol was used. ZnT3 KO mice were deficient in memory when a tone was presented as complex auditory information in a discontinuous fashion. Moreover, ZnT3 KO mice showed abnormality in trace fear conditioning and in fear extinction. By contrast, ZnT3 KO mice had normal anxiety. Thus, ZnT3 is involved in associative fear memory and extinction, but not in innate fear, consistent with the role of synaptic zinc in amygdala synaptic plasticity.

Peroxisome proliferator-activated receptor gamma agonist troglitazone induces colon tumors in normal C57BL/6J mice and enhances colonic carcinogenesis in Apc1638 N/+ Mlh1+/- double mutant mice.

PubMed

Yang, Kan; Fan, Kun-Hua; Lamprecht, Sergio A; Edelmann, Winfried; Kopelovich, Levy; Kucherlapati, Raju; Lipkin, Martin

2005-09-10

The role of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in colon tumorigenesis remains controversial. Notwithstanding evidence that PPAR-gamma ligands impede murine colorectal carcinogenesis, PPAR-gamma agonists have been shown to enhance in vivo tumor formation in mouse models of human colon cancer. Our study was designed to determine whether troglitazone (TGZ) induces colonic tumor formation in normal C57BL/6J mice and enhances colorectal carcinogenesis in double mutant Apc1638N/+ Mlh1+/- mice fed a standard AIN-76A diet. We report herein that not only does TGZ enhance carcinogenesis in the large intestine of mutant mice predisposed to intestinal carcinogenesis but TGZ also induces colonic tumors in normal mice without gene targeting or carcinogen administration. This observation indicates that preexisting mutational events are not necessary for induction of colonic tumors by activated PPAR-gamma in vivo. (c) 2005 Wiley-Liss, Inc.

Are Sema5a mutant mice a good model of autism? A behavioral analysis of sensory systems, emotionality and cognition

PubMed Central

Gunn, Rhian K.; Huentelman, Matthew J.; Brown, Richard E.

2011-01-01

Semaphorin 5A (Sema5A) expression is reduced in the brain of individuals with autism, thus mice with reduced Sema5A levels may serve as a model of this neurodevelopmental disorder. We tested male and female Sema5a knockout mice (B6.129P2SEMA5A/J) and C57BL/6J controls for emotionality, visual ability, prepulse inhibition, motor learning and cognition. Overall, there were only two genotype differences in emotionality: Sema5a mutant mice had more stretch-attend postures in the elevated plus-maze and more defecations in the open field. All mice could see, but Sema5a mice had better visual ability than C57BL/6J mice. There were no genotype differences in sensory-motor gating. Sema5a mice showed higher levels of activity in the elevated plus-maze and light/dark transition box, and there were sex by genotype differences in the Rotarod, suggesting a sex difference in balance and coordination differentially affected by Sema5a. There were no genotype effects on cognition: Sema5a mice did not differ from C57BL/6J in the Morris water maze, set-shifting or cued and contextual fear conditioning. In the social recognition test, all mice preferred social stimuli, but there was no preference for social novelty, thus the Sema5A mice do not have a deficit in social behavior. Overall, there were a number of sex differences, with females showing greater activity and males performing better in tests of spatial learning and memory, but no deficits in the behavior of Sema5A mice. We conclude that the Sema5a mice do not meet the behavioral criteria for a mouse model of autism. PMID:21777623

An enteric pathogen Salmonella enterica serovar Typhimurium suppresses tumor growth by downregulating CD44high and CD4T regulatory (Treg) cell expression in mice: the critical role of lipopolysaccharide and Braun lipoprotein in modulating tumor growth.

PubMed

Liu, T; Chopra, A K

2010-02-01

An antitumor activity associated with several bacterial pathogens, including Salmonella enterica serovar Typhimurium, has been reported; however, the underlying immunological mechanism(s) that lead to an antitumor effect are currently unclear. Furthermore, such pathogens cannot be used to suppress tumor growth because of their potential for causing sepsis. Recently, we reported the characterization of S. Typhimurium isogenic mutants from which Braun lipoprotein genes (lppA and B) and the multicopy repressor of high temperature requirement (msbB) gene were deleted. In a mouse infection model, two mutants, namely, lppB/msbB and lppAB/msbB, minimally induced proinflammatory cytokine production at high doses and were nonlethal to animals. We showed that immunization of mice with these mutants, followed by challenge with the wild-type S. Typhimurium, could significantly suppress tumor growth, as evidenced by an 88% regression in tumor size in lppB/msbB mutant-immunized animals over a 24-day period. However, the lppAB/msbB mutant alone was not effective in modulating tumor growth in mice, although the lppB/msbB mutant alone caused marginal regression in tumor size. Importantly, we showed that CD44(+) cells grew much faster than CD44(-) cells from human liver tumors in mice, leading us to examine the possibility that S. Typhimurium might downregulate CD44 in tumors and splenocytes of mice. Consequently, we found in S. Typhimurium-infected mice that tumor size regression could indeed be related to the downregulation of CD44(high) and CD4(+)CD25(+) T(reg) cells. Importantly, the role of lipopolysaccharide and Braun lipoprotein was critical in S. Typhimurium-induced antitumor immune responses. Taken together, we have defined new immune mechanisms leading to tumor suppression in mice by S. Typhimurium.

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

PubMed Central

Yamamoto, N; Naraparaju, V R

1996-01-01

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice. PMID:8881764

Mitochondrial dysfunction precedes neurodegeneration in mahogunin (Mgrn1) mutant mice

PubMed Central

Sun, Kaihua; Johnson, Brian S.; Gunn, Teresa M.

2007-01-01

Oxidative stress, ubiquitination defects and mitochondrial dysfunction are commonly associated with neurodegeneration. Mice lacking mahogunin ring finger-1 (MGRN1) or attractin (ATRN) develop age-dependent spongiform neurodegeneration through an unknown mechanism. It has been suggested that they act in a common pathway. As MGRN1 is an E3 ubiquitin ligase, proteomic analysis of Mgrn1 mutant and control brains was performed to explore the hypothesis that loss of MGRN1 causes neurodegeneration via accumulation of its substrates. Many mitochondrial proteins were reduced in Mgrn1 mutants. Subsequent assays confirmed significantly reduced mitochondrial complex IV expression and activity as well as increased oxidative stress in mutant brains. Mitochondrial dysfunction was obvious many months before onset of vacuolation, implicating this as a causative factor. Compatible with the hypothesis that ATRN and MGRN1 act in the same pathway, mitochondrial dysfunction and increased oxidative stress were also observed in the brains of Atrn mutants. Our results suggest that the study of Mgrn1 and Atrn mutant mice will provide insight into a causative molecular mechanism common to many neurodegenerative disorders. PMID:17720281

Role of immune cells in animal models for inherited neuropathies: facts and visions.

PubMed

Mäurer, Mathias; Kobsar, Igor; Berghoff, Martin; Schmid, Christoph D; Carenini, Stefano; Martini, Rudolf

2002-04-01

Mice heterozygously deficient in the peripheral myelin adhesion molecule P0 (P0+/- mice) are models for some forms of Charcot-Marie-Tooth (CMT) neuropathies. In addition to the characteristic hallmarks of demyelination, elevated numbers of CD8-positive T-lymphocytes and F4/80-positive macrophages are striking features in the nerves of these mice. These immune cells increase in number with age and progress of demyelination, suggesting that they might be functionally related to myelin damage. In order to investigate the pathogenetic role of lymphocytes, the myelin mutants were cross-bred with recombination activating gene 1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. The immunodeficient myelin mutants showed a less severe myelin degeneration. The beneficial effect of lymphocyte-deficiency was reversible, since demyelination worsened in immunodeficient myelin-mutants when reconstituted with bone marrow from wild-type mice. Ultrastructural analysis revealed macrophages in close apposition to myelin and demyelinated axons. We therefore cross-bred the P0+/- mice with spontaneous osteopetrotic (op) mutants deficient in the macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the corresponding double mutants the numbers of macrophages were not elevated in the peripheral nerves, and the demyelinating phenotype was less severe than in the genuine P0+/- mice, demonstrating that macrophages are also functionally involved in the pathogenesis of genetically mediated demyelination. We also examined other models for inherited neuropathies for a possible involvement of immune cells. We chose mice deficient in the gap junction component connexin 32, a model for the X-linked form of CMT. Similar to P0-deficient mice, T-lymphocytes and macrophages were elevated and macrophages showed a close apposition to degenerating myelin. We conclude that the involvement of T-lymphocytes and macrophages is a common pathogenetic feature in various forms of slowly progressive inherited neuropathies.

Rho GTPase protein Cdc42 is critical for postnatal cartilage development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagahama, Ryo; Department of Orthodontics, School of Dentistry, Showa University, Tokyo; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp

2016-02-19

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 {sup fl/fl}; Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 {sup fl/fl}) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system.more » The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages. - Highlights: • Tamoxifen-induced cartilage specific inactivated Cdc42 mutant mice were generated. • Cdc42 mutant mice were shorter limbs and body. • Severe defects were found in growth plate chondrocytes.« less

Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice.

PubMed

Nakamura, Kazufumi; Miura, Daiji; Saito, Yukihiro; Yunoki, Kei; Koyama, Yasushi; Satoh, Minoru; Kondo, Megumi; Osawa, Kazuhiro; Hatipoglu, Omer F; Miyoshi, Toru; Yoshida, Masashi; Morita, Hiroshi; Ito, Hiroshi

2017-01-01

The klotho gene was identified as an "aging-suppressor" gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in klotho mutant mice. Four-week-old klotho mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, klotho and WT: n = 12, each) or not containing EPA (control food, klotho and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in klotho mice after 4 weeks of control food, but they were not elevated in klotho mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (NOX4), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of klotho mice. Activity of NOX was also significantly higher in SMCs of klotho mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of klotho mice. EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in klotho mutant mice.

Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABA(A) receptors.

PubMed

Blednov, Y A; Borghese, C M; McCracken, M L; Benavidez, J M; Geil, C R; Osterndorff-Kahanek, E; Werner, D F; Iyer, S; Swihart, A; Harrison, N L; Homanics, G E; Harris, R A

2011-01-01

GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.

Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAA Receptors

PubMed Central

Borghese, C. M.; McCracken, M. L.; Benavidez, J. M.; Geil, C. R.; Osterndorff-Kahanek, E.; Werner, D. F.; Iyer, S.; Swihart, A.; Harrison, N. L.; Homanics, G. E.; Harris, R. A.

2011-01-01

GABA type A receptors (GABAA-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABAA-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705–714, 2004; Pharmacol Biochem Behav 90:95–104, 2008; J Psychiatr Res 42:184–191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism. PMID:20876231

Heparan Sulfate Biosynthesis Enzyme, Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling.

PubMed

Zhang, Rui; Cao, Peijuan; Yang, Zhongzhou; Wang, Zhenzhen; Wu, Jiu-Lin; Chen, Yan; Pan, Yi

2015-01-01

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Diet-induced obesity increases the frequency of Pig-a mutant erythrocytes in male C57BL/6J mice.

PubMed

Wickliffe, Jeffrey K; Dertinger, Stephen D; Torous, Dorothea K; Avlasevich, Svetlana L; Simon-Friedt, Bridget R; Wilson, Mark J

2016-12-01

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p < 0.02) in erythrocytes as compared to non-obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

PubMed Central

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-01-01

Objective Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. Methods FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. Results FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. Conclusion These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. PMID:24022275

A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

PubMed

Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

2007-06-15

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

GATA-3 is required for early T lineage progenitor development

PubMed Central

Hosoya, Tomonori; Kuroha, Takashi; Moriguchi, Takashi; Cummings, Dustin; Maillard, Ivan; Lim, Kim-Chew

2009-01-01

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors. PMID:19934022

Chronic Desipramine Treatment Rescues Depression-Related, Social and Cognitive Deficits in Engrailed-2 Knockout Mice

PubMed Central

Brielmaier, Jennifer; Senerth, Julia M.; Silverman, Jill L.; Matteson, Paul G.; Millonig, James H.; DiCicco-Bloom, Emanuel; Crawley, Jacqueline N.

2014-01-01

Engrailed-2 (En2) is a homeobox transcription factor that regulates neurodevelopmental processes including neuronal connectivity and elaboration of monoaminergic neurons in the ventral hindbrain. We previously reported abnormalities in brain noradrenergic concentrations in En2 null mutant mice that were accompanied by increased immobility in the forced swim test, relevant to depression. An EN2 genetic polymorphism has been associated with autism spectrum disorders (ASD), and mice with a deletion in En2 display social abnormalities and cognitive deficits that may be relevant to multiple neuropsychiatric conditions. The present study evaluated the ability of chronic treatment with desipramine (DMI), a selective norepinephrine reuptake inhibitor and classical antidepressant, to reverse behavioral abnormalities in En2 −/− mice. DMI treatment significantly reduced immobility in the tail suspension and forced swim tests, restored sociability in the three-chambered social approach task, and reversed impairments in contextual fear conditioning in En2 −/− mice. Our findings indicate that modulation of brain noradrenergic systems rescues the depression-related phenotype in En2 −/− mice and suggest new roles for norepinephrine in the pathophysiology of the social and cognitive deficits seen in neuropsychiatric disorders such as autism or schizophrenia. PMID:24730055

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

PubMed

Horiuchi, Keiko; Perez-Cerezales, Serafín; Papasaikas, Panagiotis; Ramos-Ibeas, Priscila; López-Cardona, Angela Patricia; Laguna-Barraza, Ricardo; Fonseca Balvís, Noelia; Pericuesta, Eva; Fernández-González, Raul; Planells, Benjamín; Viera, Alberto; Suja, Jose Angel; Ross, Pablo Juan; Alén, Francisco; Orio, Laura; Rodriguez de Fonseca, Fernando; Pintado, Belén; Valcárcel, Juan; Gutiérrez-Adán, Alfonso

2018-04-03

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Vertebrate intersectin1 is repurposed to facilitate cortical midline connectivity and higher order cognition.

PubMed

Sengar, Ameet S; Ellegood, Jacob; Yiu, Adelaide P; Wang, Hua; Wang, Wei; Juneja, Subhash C; Lerch, Jason P; Josselyn, Sheena A; Henkelman, R Mark; Salter, Michael W; Egan, Sean E

2013-02-27

Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.

Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling.

PubMed

Mishina, Yuji; Starbuck, Michael W; Gentile, Michael A; Fukuda, Tomokazu; Kasparcova, Viera; Seedor, J Gregory; Hanks, Mark C; Amling, Michael; Pinero, Gerald J; Harada, Shun-ichi; Behringer, Richard R

2004-06-25

Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.

Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko

2012-09-07

Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively activemore » mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.« less

THE REELIN RECEPTORS VLDLR AND ApoER2 REGULATE SENSORIMOTOR GATING IN MICE

PubMed Central

Barr, Alasdair M.; Fish, Kenneth N.; Markou, Athina

2007-01-01

Summary Postmortem brain loss of reelin is noted in schizophrenia patients. Accordingly, heterozygous reeler mutant mice have been proposed as a putative model of this disorder. Little is known, however, about the involvement of the two receptors for reelin, Very-Low-Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E Receptor 2 (ApoER2), on pre-cognitive processes of relevance to deficits seen in schizophrenia. Thus, we evaluated sensorimotor gating in mutant mice heterozygous or homozygous for the two reelin receptors. Mutant mice lacking one of these reelin receptors were tested for prepulse inhibition (PPI) of the acoustic startle reflex prior to and following puberty, and on a crossmodal PPI task, involving the presentation of acoustic and tactile stimuli. Furthermore, because schizophrenia patients show increased sensitivity to N-methyl-D-aspartate (NMDA) receptor blockade, we assessed the sensitivity of these mice to the PPI-disruptive effects of the NMDA receptor antagonist phencyclidine. The results demonstrated that acoustic PPI did not differ between mutant and wildtype mice. However, VLDLR homozygous mice displayed significant deficits in crossmodal PPI, while ApoER2 heterozygous and homozygous mice displayed significantly increased crossmodal PPI. Both ApoER2 and VLDLR heterozygous and homozygous mice exhibited greater sensitivity to the PPI-disruptive effects of phencyclidine than wildtype mice. These results indicate that partial or complete loss of either one of the reelin receptors results in a complex pattern of alterations in PPI function that include alterations in crossmodal, but not acoustic, PPI and increased sensitivity to NMDA receptor blockade. Thus, reelin receptor function appears to be critically involved in crossmodal PPI and the modulation of the PPI response by NMDA receptors. These findings have relevance to a range of neuropsychiatric disorders that involve sensorimotor gating deficits, including schizophrenia.. PMID:17261317

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

PubMed Central

Misra, Chaitali; Sachan, Nita; McNally, Caryn Rothrock; Koenig, Sara N.; Nichols, Haley A.; Guggilam, Anuradha; Lucchesi, Pamela A.; Pu, William T.; Srivastava, Deepak; Garg, Vidu

2012-01-01

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans. PMID:22589735

Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice.

PubMed

Nonogaki, Katsunori; Ohba, Yukie; Sumii, Makiko; Oka, Yoshitomo

2008-07-18

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of beta-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

PubMed

Tiner, Bethany L; Sha, Jian; Ponnusamy, Duraisamy; Baze, Wallace B; Fitts, Eric C; Popov, Vsevolod L; van Lier, Christina J; Erova, Tatiana E; Chopra, Ashok K

2015-12-01

Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but with a much diminished virulence potential. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Corticostriatal circuit defects in Hoxb8 mutant mice

PubMed Central

Nagarajan, Naveen; Jones, Bryan W.; West, Peter J.; Marc, Robert; Capecchi, Mario R.

2018-01-01

Hoxb8 mutant mice exhibit compulsive grooming and hair removal dysfunction similar to humans with the OCD-spectrum disorder, trichotillomania. Since, in the mouse brain, the only detectable cells that label with Hoxb8 cell lineage appear to be microglia, we suggested that defective microglia cause the neuropsychiatric disorder. Does the Hoxb8 mutation in microglia lead to neural circuit dysfunctions? We demonstrate that Hoxb8 mutants contain corticostriatal circuit defects. Golgi staining, ultra-structural, and electrophysiological studies of mutants reveal excess dendritic spines, pre- and post-synaptic structural defects, long-term potentiation and miniature postsynaptic current defects. Hoxb8 mutants also exhibit hyperanxiety and social behavioral deficits similar to mice with neuronal mutations in Sapap3, Slitrk5 and Shank3, reported models of OCD and autism spectrum disorders (ASD’s). Long-term treatment of Hoxb8 mutants with fluoxetine, a serotonin reuptake inhibitor (SSRI), reduces excessive grooming, hyperanxiety and social behavioral impairments. These studies provide linkage between the neuronal defects induced by defective Hoxb8-microglia, and neuronal dysfunctions directly generated by mutations in synaptic components that result in mice that display similar pathological grooming, hyperanxiety and social impairment deficits. Our results shed light on Hoxb8 microglia driven circuit-specific defects and therapeutic approaches that will become essential to developing novel therapies for neuropsychiatric diseases such as OCD and ASD’s with Hoxb8-microglia being the central target. PMID:28948967

Embryonic epithelial Pten deletion through Nkx2.1-cre leads to thyroid tumorigenesis in a strain-dependent manner

PubMed Central

Tiozzo, Caterina; Danopoulos, Soula; Lavarreda-Pearce, Maria; Baptista, Sheryl; Varimezova, Radka; Al Alam, Denise; Warburton, David; Virender, Rehan; De Langhe, Stijn; Di Cristofano, Antonio

2014-01-01

Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Ptenflox/flox with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T4) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background. PMID:22167068

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice.

PubMed

Huang, Tzyy-Nan; Hsueh, Yi-Ping

2017-01-01

Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase ( CASK ) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability. Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein-protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N -methyl-D-aspartate receptor subunit 2B ( Grin2b ) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein-protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice. The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal. The functions of other CASK protein interactions cannot be addressed using CASK T740A mice. Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility.

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

PubMed Central

ISHIKAWA, Akira; SUGIYAMA, Makoto; HONDO, Eiichi; KINOSHITA, Keiji; YAMAGISHI, Yuki

2015-01-01

Oca2p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging. PMID:25739360

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging.

PubMed

Ishikawa, Akira; Sugiyama, Makoto; Hondo, Eiichi; Kinoshita, Keiji; Yamagishi, Yuki

2015-01-01

Oca2(p-cas) (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2(p-cas) revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

PubMed

Dluzen, D E; Gao, X; Story, G M; Anderson, L I; Kucera, J; Walro, J M

2001-07-01

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice. Copyright 2001 Academic Press.

Contribution of NMDA receptor hypofunction in prefrontal and cortical excitatory neurons to schizophrenia-like phenotypes.

PubMed

Rompala, Gregory R; Zsiros, Veronika; Zhang, Shuqin; Kolata, Stefan M; Nakazawa, Kazu

2013-01-01

Pharmacological and genetic studies support a role for NMDA receptor (NMDAR) hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1) deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice), in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in in situ hybridization immunocytochemistry as well as the lack of NMDA currents with in vitro recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (e.g. spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior). Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.

Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice.

PubMed

Razzaque, Mohammed S; Lanske, Beate

2006-07-01

The essential role of low levels of vitamin D during aging is well documented. However, possible effects of high levels of vitamin D on the aging process are not yet clear. Recent in vivo genetic-manipulation studies have shown increased serum level of vitamin D and altered mineral-ion homeostasis in mice that lack either fibroblast growth factor 23 (Fgf23) or klotho (Kl) genes. These mice develop identical phenotypes consistent with premature aging. Elimination or reduction of vitamin-D activity from Fgf23 and Kl mutant mice, either by dietary restriction or genetic manipulation could rescue premature aging-like features and ectopic calcifications, resulting in prolonged survival of both mutants. Such in vivo experimental studies indicated that excessive vitamin-D activity and altered mineral-ion homeostasis could accelerate the aging process.

A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection

PubMed Central

Spera, Juan Manuel; Ugalde, Juan Esteban; Mucci, Juan; Comerci, Diego J.; Ugalde, Rodolfo Augusto

2006-01-01

Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella. PMID:17053080

Ablation of RIC8A function in mouse neurons leads to a severe neuromuscular phenotype and postnatal death.

PubMed

Ruisu, Katrin; Kask, Keiu; Meier, Riho; Saare, Merly; Raid, Raivo; Veraksitš, Alar; Karis, Alar; Tõnissoo, Tambet; Pooga, Margus

2013-01-01

Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed Ric8a conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (SynCre) into the floxed Ric8a (Ric8a (F/F) ) background ablated RIC8A function in most differentiated neuron populations. Mutant SynCre (+/-) Ric8 (lacZ/F) mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of SynCre (+/-) Ric8a (lacZ/F) mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

PubMed Central

Ruisu, Katrin; Kask, Keiu; Meier, Riho; Saare, Merly; Raid, Raivo; Veraksitš, Alar; Karis, Alar; Tõnissoo, Tambet; Pooga, Margus

2013-01-01

Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed Ric8a conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (SynCre) into the floxed Ric8a (Ric8a F/F) background ablated RIC8A function in most differentiated neuron populations. Mutant SynCre +/- Ric8 lacZ/F mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of SynCre +/- Ric8a lacZ/F mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype. PMID:23977396

A 1-bp deletion in the gammaC-crystallin leads to dominant cataracts in mice.

PubMed

Zhao, Liya; Li, Kai; Bao, Shimin; Zhou, Yuxun; Liang, Yinming; Zhao, Guoji; Chen, Ye; Xiao, Junhua

2010-08-01

To date around 140 genetic alleles have been identified as being responsible for mouse cataract pathology, including Crya, Cryb, Cryg, Maf, Pax6, Pitx3, Sox, Connexins, MIP, and Lim-2. We obtained a dominant cataract mouse model from a spontaneous mutation in the F1 hybrids of outbred strain ICR mice crossed to the inbred strain BALB/cJ mice. Heterozygous and homozygous mutants expressed a nuclear cataract in both eyes. In 8-day-old mice, histological analysis showed that polygon epithelial cells were in the equatorial region and cortex underneath, and vacuole and sponge-like degeneration were in the cortical area underneath the posterior lens capsule. The nucleus of the lens was a deeply stained pink, with the shorter fibers losing their normal arrangement. For the entire eye, there was a blank zone in the equatorial region in 8-day-old mice; however, there was a certain degree of atrophy in cornea tension and retina in the lens in 3-month-old mice. The lens had been serious damaged in the homozygous mutants. For mutation mapping, heterozygous carriers were mated to wild-type C3H/HeJ mice, and offspring (F1 generation) with cataracts were backcrossed to the wild-type C3H/HeJ mice again. N2 mice with cataracts were used for genotyping. Using genome-wide linkage analysis, the mutation was mapped to chromosome 1 and the Cryg gene cluster between two markers was confirmed as the candidate gene. After direct sequencing the cDNA of the Cryg gene cluster, a 1-bp deletion was found in exon 3 of the Crygc gene, leading to a stop codon at the 76th amino acid of exon 3 which results in production of a truncated protein in mutant mice (Leu160Stop). Bioinformatic analysis of the mutant gammaC-crystallin reveals that the COOH-terminal of the mutant protein deletes a beta-sheet, which affects the function of the lens proteins and leads to the development of cataracts.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

PubMed

Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Shi, Yun-Liang; Wan, Xiao-Ling; Yang, Yi-Chao

2017-08-07

Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5)  = 2.595, P = 0.049), 60 (t (5)  = 7.838, P = 0.001) and 90 dpi (t (5)  = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5)  = 5.139, P = 0.004), 60 (t (5)  = 6.138, P = 0.002) and 90 dpi (t (5)  = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t (5)  = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t (5)  = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t (5)  = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi. These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

PubMed

Monteiro, Gaby E R; Jansen van Vuren, Petrus; Wichgers Schreur, Paul J; Odendaal, Lieza; Clift, Sarah J; Kortekaas, Jeroen; Paweska, Janusz T

2018-04-02

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). Copyright © 2018. Published by Elsevier B.V.

p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice

NASA Technical Reports Server (NTRS)

Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R. A.

2001-01-01

Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/- mice and 2.1-fold increase in p53-/- mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.

The roles of Eph receptors in contextual fear conditioning memory formation.

PubMed

Dines, Monica; Grinberg, Svetlana; Vassiliev, Maria; Ram, Alon; Tamir, Tal; Lamprecht, Raphael

2015-10-01

Eph receptors regulate glutamate receptors functions, neuronal morphology and synaptic plasticity, cellular events believed to be involved in memory formation. In this study we aim to explore the roles of Eph receptors in learning and memory. Toward that end, we examined the roles of EphB2 and EphA4 receptors, key regulators of synaptic functions, in fear conditioning memory formation. We show that mice lacking EphB2 (EphB2(-/-)) are impaired in short- and long-term contextual fear conditioning memory. Mice that express a carboxy-terminally truncated form of EphB2 that lacks forward signaling, instead of the full EphB2, are impaired in long-term, but not short-term, contextual fear conditioning memory. Long-term contextual fear conditioning memory is attenuated in CaMKII-cre;EphA4(lx/-) mice where EphA4 is removed from all pyramidal neurons of the forebrain. Mutant mice with targeted kinase-dead EphA4 (EphA4(KD)) exhibit intact long-term contextual fear conditioning memory showing that EphA4 kinase-mediated forward signaling is not needed for contextual fear memory formation. The ability to form long-term conditioned taste aversion (CTA) memory is not impaired in the EphB2(-/-) and CaMKII-cre;EphA4(lx/-) mice. We conclude that EphB2 forward signaling is required for long-term contextual fear conditioning memory formation. In contrast, EphB2 mediates short-term contextual fear conditioning memory formation in a forward signaling-independent manner. EphA4 mediates long-term contextual fear conditioning memory formation in a kinase-independent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

PubMed Central

Perng, Guey-Chuen; Esmaili, Daniel; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Osorio, Nelson; Mott, Kevin R.; Maguen, Barak; Jin, Ling; Nesburn, Anthony B.; Wechsler, Steven L.

2001-01-01

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex. PMID:11533165

Masking responses to light in period mutant mice.

PubMed

Pendergast, Julie S; Yamazaki, Shin

2011-10-01

Masking is an acute effect of an external signal on an overt rhythm and is distinct from the process of entrainment. In the current study, we investigated the phase dependence and molecular mechanisms regulating masking effects of light pulses on spontaneous locomotor activity in mice. The circadian genes, Period1 (Per1) and Per2, are necessary components of the timekeeping machinery and entrainment by light appears to involve the induction of the expression of Per1 and Per2 mRNAs in the suprachiasmatic nuclei (SCN). We assessed the roles of the Per genes in regulating masking by assessing the effects of light pulses on nocturnal locomotor activity in C57BL/6J Per mutant mice. We found that Per1(-/-) and Per2(-/-) mice had robust negative masking responses to light. In addition, the locomotor activity of Per1(-/-)/Per2(-/-) mice appeared to be rhythmic in the light-dark (LD) cycle, and the phase of activity onset was advanced (but varied among individual mice) relative to lights off. This rhythm persisted for 1 to 2 days in constant darkness in some Per1(-/-)/Per2(-/-) mice. Furthermore, Per1(-/-)/Per2(-/-) mice exhibited robust negative masking responses to light. Negative masking was phase dependent in wild-type mice such that maximal suppression was induced by light pulses at zeitgeber time 14 (ZT14) and gradually weaker suppression occurred during light pulses at ZT16 and ZT18. By measuring the phase shifts induced by the masking protocol (light pulses were administered to mice maintained in the LD cycle), we found that the phase responsiveness of Per mutant mice was altered compared to wild-types. Together, our data suggest that negative masking responses to light are robust in Per mutant mice and that the Per1(-/-)/Per2(-/-) SCN may be a light-driven, weak/damping oscillator.

VGF ablation blocks the development of hyperinsulinemia and hyperglycemia in several mouse models of obesity.

PubMed

Watson, Elizabeth; Hahm, Seung; Mizuno, Tooru M; Windsor, Joan; Montgomery, Carla; Scherer, Philipp E; Mobbs, Charles V; Salton, Stephen R J

2005-12-01

Targeted deletion of the gene encoding the neuronal and endocrine secreted peptide precursor called VGF (nonacronymic) produces a lean, hypermetabolic, hyperactive mouse. Because VGF mutant mice are resistant to specific forms of diet-, lesion-, and genetically induced obesity, we investigated the role that this polypeptide plays in glucose homeostasis. We report that VGF mutant mice have increased insulin sensitivity by hyperinsulinemic euglycemic clamp analysis, and by insulin and glucose tolerance testing. Blunted counterregulatory responses in VGF-deficient mice were likely influenced by their significantly lower liver glycogen levels. VGF deficiency lowered circulating glucose and insulin levels in several murine models of obesity that are also susceptible to adult onset diabetes mellitus, including A(y)/a agouti, ob/ob, and MC4R(-)/MC4R(-) mice. Interestingly, ablation of Vgf in ob/ob mice decreased circulating glucose and insulin levels but did not affect adiposity, whereas MC4R(-)/MC4R(-) mice that are additionally deficient in VGF have improved insulin responsiveness at 7-8 wk of age, when lean MC4R(-)/MC4R(-) mice already have impaired insulin tolerance but are not yet obese. VGF mutant mice also resisted developing obesity and hyperglycemia in response to a high-fat/high-carbohydrate diet, and after gold thioglucose treatment, which is toxic to hypothalamic glucose-sensitive neurons. Lastly, circulating adiponectin, an adipose-synthesized protein the levels of which are correlated with improved insulin sensitivity, increased in VGF mutant compared with wild-type mice. Modulation of VGF levels and/or VGF signaling may consequently represent an alternative means to regulate circulating glucose levels and insulin sensitivity.

Stromal deletion of the APC tumor suppressor in mice triggers development of endometrial cancer

PubMed Central

Tanwar, Pradeep S.; Zhang, LiHua; Roberts, Drucilla J.; Teixeira, Jose M.

2011-01-01

The contribution of the stromal microenvironment to the progression of endometrial cancer (EC) has not been well explored. We have conditionally expressed a mutant allele of adenomatous polyposis coli (APCcKO) in murine uterine stroma cells to study its effect on uterine development and function. In addition to metrorrhagia, the mice develop complex atypical endometrial gland hyperplasia that progresses to endometrial carcinoma in situ and endometrial adenocarcinoma as evidenced by myometrial invasion. Stromal cells subjacent to the carcinoma cells express αSMA with fewer cells expressing PDGFR-α compared to normal stromal cells suggesting that the mutant stromal cells have acquired a more myofibroblastic phenotype, which have been described as cancer-associated fibroblasts and have been shown to induce carcinogenesis in other organ systems. Analyses of human EC specimens showed substantial αSMA expression in the stroma compared with normal endometrial stroma cells. We also show that APCcKO mutant uteri and human EC have decreased stromal levels of TGFβ and BMP activities and that the mutant uteri failed to respond to exogenous estradiol stimulation. The mutant stroma cells also had higher levels of VEGF and SDF signaling components and diminished expression of ERα and PR which is common in advanced stages of human EC and is an indicator of poor prognosis. Our results indicate that de novo mutation or loss of heterozygosity in stromal APC is sufficient to induce endometrial hyperplasia and endometrial carcinogenesis by mechanisms that are consistent with unopposed estrogen signaling in the endometrial epithelium. PMID:21363919

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

PubMed

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Effect of pituitary hollow fiber units and thyroid supplementation on growth in the little mouse (41949)

NASA Technical Reports Server (NTRS)

Harkness, John E.; Hymer, W. C.; Rosenberger, James L.; Grindeland, Richard E.

1984-01-01

It is shown that the implantation of encapsulated pituitary cells into heterozygous lit/+ mice inhibited the average percentage change in weight gain as compared to controls. However, homozygous lit/lit mice receiving cell-filled capsules consistently had higher percentage weight gains than their control counterparts. It was also found that thyroid-supplemented mutant mice with pituitary cell implants had significantly higher organ and carcass weights than other mutant groups.

Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line

PubMed Central

Kumagai, Ayako; Fujita, Akira; Yokoyama, Tomoki; Nonobe, Yuki; Hasaba, Yasuhiro; Sasaki, Tsutomu; Itoh, Yumi; Koura, Minako; Suzuki, Osamu; Adachi, Shigeki; Ryo, Haruko; Kohara, Arihiro; Tripathi, Lokesh P.; Sanosaka, Masato; Fukushima, Toshiki; Takahashi, Hiroyuki; Kitagawa, Kazuo; Nagaoka, Yasuo; Kawahara, Hidehisa; Mizuguchi, Kenji; Nomura, Taisei; Matsuda, Junichiro; Tabata, Toshihide; Takemori, Hiroshi

2014-01-01

Memantine is a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer’s disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds. PMID:25513882

Roles of HAUSP-mediated p53 regulation in central nervous system development.

PubMed

Kon, N; Zhong, J; Kobayashi, Y; Li, M; Szabolcs, M; Ludwig, T; Canoll, P D; Gu, W

2011-08-01

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

PubMed

Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

2016-09-28

Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.

Mecp2 truncation in male mice promotes affiliative social behavior

PubMed Central

Pearson, B.L.; Defensor, E.B.; Pobbe, R.L.H.; Yamamoto, L.H.L.; Bolivar, V.J.; Blanchard, D.C.; Blanchard, R.J.

2018-01-01

Mouse models of Rett syndrome, with targeted mutations in the Mecp2 gene, show a high degree of phenotypic consistency with the clinical syndrome. In addition to severe and age-specific regression in motor and cognitive abilities, a variety of studies have demonstrated that Mecp2 mutant mice display impaired social behavior. Conversely, other studies indicate complex enhancements of social behavior in Mecp2 mutant mice. Since social behavior is a complicated accumulation of constructs, we performed a series of classic and refined social behavior tasks and revealed a relatively consistent pattern of enhanced pro-social behavior in hypomorphic Mecp2308/Y mutant mice. Analyses of repetitive motor acts, and cognitive stereotypy did not reveal any profound differences due to genotype. Taken together, these results suggest that the mutations associated with Rett syndrome are not necessarily associated with autism-relevant social impairment in mice. However, this gene may be a valuable candidate for revealing basic mechanisms of affiliative behavior. PMID:21909962

The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation.

PubMed

Kawabe, T; Naka, T; Yoshida, K; Tanaka, T; Fujiwara, H; Suematsu, S; Yoshida, N; Kishimoto, T; Kikutani, H

1994-06-01

An engagement of CD40 with CD40 ligand (CD40L) expressed on activated T cells is known to provide an essential costimulatory signal to B cells in vitro. To investigate the role of CD40 in in vivo immune responses, CD40-deficient mice were generated by gene targeting. The significant reduction of CD23 expression on mature B cells and relatively decreased number of IgM bright and IgD dull B cells were observed in the mutant mice. The mutant mice mounted IgM responses but no IgG, IgA, and IgE responses to thymus-dependent (TD) antigens. However, IgG as well as IgM responses to thymus-independent (TI) antigens were normal. Furthermore, the germinal center formation was defective in the mutant mice. These results suggest that CD40 is essential for T cell-dependent immunoglobulin class switching and germinal center formation, but not for in vivo T cell-dependent IgM responses and T cell-independent antibody responses.

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma

PubMed Central

Schneeberger, Valentina E.; Ren, Yuan; Luetteke, Noreen; Huang, Qingling; Chen, Liwei; Lawrence, Harshani R.; Lawrence, Nicholas J.; Haura, Eric B.; Koomen, John M.; Coppola, Domenico; Wu, Jie

2015-01-01

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma. PMID:25730908

Changes in the striatal proteome of YAC128Q mice exhibit gene-environment interactions between mutant huntingtin and manganese.

PubMed

Wegrzynowicz, Michal; Holt, Hunter K; Friedman, David B; Bowman, Aaron B

2012-02-03

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat within the Huntingtin (HTT) gene, though the clinical presentation of disease and age-of-onset are strongly influenced by ill-defined environmental factors. We recently reported a gene-environment interaction wherein expression of mutant HTT is associated with neuroprotection against manganese (Mn) toxicity. Here, we are testing the hypothesis that this interaction may be manifested by altered protein expression patterns in striatum, a primary target of both neurodegeneration in HD and neurotoxicity of Mn. To this end, we compared striatal proteomes of wild-type and HD (YAC128Q) mice exposed to vehicle or Mn. Principal component analysis of proteomic data revealed that Mn exposure disrupted a segregation of WT versus mutant proteomes by the major principal component observed in vehicle-exposed mice. Identification of altered proteins revealed novel markers of Mn toxicity, particularly proteins involved in glycolysis, excitotoxicity, and cytoskeletal dynamics. In addition, YAC128Q-dependent changes suggest that axonal pathology may be an early feature in HD pathogenesis. Finally, for several proteins, genotype-specific responses to Mn were observed. These differences include increased sensitivity to exposure in YAC128Q mice (UBQLN1) and amelioration of some mutant HTT-induced alterations (SAE1, ENO1). We conclude that the interaction of Mn and mutant HTT may suppress proteomic phenotypes of YAC128Q mice, which could reveal potential targets in novel treatment strategies for HD.

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

PubMed Central

Young, Douglas; Mayer, Franziska; Vidotto, Nella; Schweizer, Tatjana; Berth, Ramon; Abramowski, Dorothee; Shimshek, Derya R.; van der Putten, P. Herman; Schmid, Peter

2013-01-01

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model. PMID:24086450

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

PubMed

Nakayama, Takafumi; Sawai, Tomoko; Masuda, Ikuko; Kaneko, Shinya; Yamauchi, Kazumi; Blyth, Benjamin J; Shimada, Yoshiya; Tachibana, Akira; Kakinuma, Shizuko

2017-10-01

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Thyroid hormone is essential for pituitary somatotropes and lactotropes.

PubMed

Stahl, J H; Kendall, S K; Brinkmeier, M L; Greco, T L; Watkins-Chow, D E; Campos-Barros, A; Lloyd, R V; Camper, S A

1999-04-01

Mice homozygous for a disruption in the alpha-subunit essential for TSH, LH, and FSH activity (alphaGsu-/-) exhibit hypothyroidism and hypogonadism similar to that observed in TSH receptor-deficient hypothyroid mice (hyt) and GnRH-deficient hypogonadal mutants (hpg). Although the five major hormone-producing cells of the anterior pituitary are present in alphaGsu-/- mice, the relative proportions of each cell type are altered dramatically. Thyrotropes exhibit hypertrophy and hyperplasia, and somatotropes and lactotropes are underrepresented. The size and number of gonadotropes in alphaGsu mutants are not remarkable in contrast to the hypertrophy characteristic of gonadectomized animals. The reduction in lactotropes is more severe in alphaGsu mutants (13-fold relative to wild-type) than in hyt or hpg mutants (4.5- and 1.5-fold, respectively). In addition, T4 replacement therapy of alphaGsu mutants restores lactotropes to near-normal levels, illustrating the importance of T4, but not alpha-subunit, for lactotrope proliferation and function. T4 replacement is permissive for gonadotrope hypertrophy in alphaGsu mutants, consistent with the role for T4 in the function of gonadotropes. This study reveals the importance of thyroid hormone in developing the appropriate proportions of anterior pituitary cell types.

Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity

PubMed Central

Yang, Chunxing; Danielson, Eric W.; Qiao, Tao; Metterville, Jake; Brown, Robert H.; Landers, John E.; Xu, Zuoshang

2016-01-01

Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments. PMID:27681617

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

PubMed

Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

2014-06-04

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models.

PubMed

Shannon, Harlan E; Yang, Lijuan

2004-01-01

Neuropeptide Y (NPY) administered exogenously is anticonvulsant, and, NPY null mutant mice are more susceptible to kainate-induced seizures. In order to better understand the potential role of NPY in epileptogenesis, the present studies investigated the development of amygdala kindling, post-kindling seizure thresholds, and anticonvulsant effects of carbamazepine and levetiracetam in 129S6/SvEv NPY(+/+) and NPY(-/-) mice. In addition, susceptibility to pilocarpine- and kainate-induced seizures was compared in NPY(+/+) and (-/-) mice. The rate of amygdala kindling development did not differ in the NPY(-/-) and NPY(+/+) mice either when kindling stimuli were presented once daily for at least 20 days, or, 12 times daily for 2 days. However, during kindling development, the NPY(-/-) mice had higher seizure severity scores and longer afterdischarge durations than the NPY(+/+) mice. Post-kindling, the NPY(-/-) mice had markedly lower afterdischarge thresholds and longer afterdischarge durations than NPY (+/+) mice. Carbamazepine and levetiracetam increased the seizure thresholds of both NPY (-/-) and (+/+) mice. In addition, NPY (-/-) mice had lower thresholds for both kainate- and pilocarpine-induced seizures. The present results in amygdala kindling and chemical seizure models suggest that NPY may play a more prominent role in determining seizure thresholds and severity of seizures than in events leading to epileptogenesis. In addition, a lack of NPY does not appear to confer drug-resistance in that carbamazepine and levetiracetam were anticonvulsant in both wild type (WT) and NPY null mutant mice.

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.

PubMed

Won, Hyejung; Lee, Hye-Ryeon; Gee, Heon Yung; Mah, Won; Kim, Jae-Ick; Lee, Jiseok; Ha, Seungmin; Chung, Changuk; Jung, Eun Suk; Cho, Yi Sul; Park, Sae-Geun; Lee, Jung-Soo; Lee, Kyungmin; Kim, Daesoo; Bae, Yong Chul; Kaang, Bong-Kiun; Lee, Min Goo; Kim, Eunjoon

2012-06-13

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Alterations of arcuate nucleus neuropeptidergic development in contactin-deficient mice: comparison with anorexia and food-deprived mice.

PubMed

Fetissov, Sergueï O; Bergström, Ulrika; Johansen, Jeanette E; Hökfelt, Tomas; Schalling, Martin; Ranscht, Barbara

2005-12-01

A mutation in the Contactin-1 gene results in an ataxic and anorectic phenotype that is apparent by postnatal day 10 and lethal by postnatal day 19 [Berglund et al. (1999) Neuron 24, 739-750]. The resemblance of this phenotype with the anorexia (anx/anx) mouse mutation prompted us to investigate the hypothalamic neurochemistry of Contactin knock-out (KO) mice. Contactin was expressed in the hypothalamic neuropil of wild-type (WT) but not Contactin KO mice. In the KO condition, neuropeptide Y (NPY) and agouti-related protein (AgRP) immunoreactivity (IR) accumulated in the somata of arcuate nucleus neurons, whereas IR for these neuropeptides as well as for alpha-melanocyte-stimulating hormone (alpha-MSH) decreased in the corresponding axon projections. These changes in the pattern of neuropeptide expression in the Contactin-deficient hypothalamus were similar but more pronounced than those found in anx/anx mice. Increased levels of NPY and AgRP and decreased concentrations of pro-opiomelanocortin mRNA in arcuate neurons accompanied these changes. In relating these alterations a 24-h food deprivation period, we observed in 3-week-old WT mice an elevation of NPY- and AgRP-IR in the perikarya of arcuate neurons without notable reduction of NPY- or AgRP-IR in nerve fibers, suggesting that the decrease of arcuate projections can be associated with postnatal anorectic phenotype. Our data implicate Contactin in the postnatal development of the NPY/AgRP and alpha-MSH arcuate neurons and suggest that similar to anx/anx mutant mice, compromised orexigenic signaling via NPY/AgRP neurons may contribute to reduced food intake by the Contactin-mutant animals.

ProSAAS-derived peptides are regulated by cocaine and are required for sensitization to the locomotor effects of cocaine.

PubMed

Berezniuk, Iryna; Rodriguiz, Ramona M; Zee, Michael L; Marcus, David J; Pintar, John; Morgan, Daniel J; Wetsel, William C; Fricker, Lloyd D

2017-11-01

To identify neuropeptides that are regulated by cocaine, we used a quantitative peptidomic technique to examine the relative levels of neuropeptides in several regions of mouse brain following daily intraperitoneal administration of 10 mg/kg cocaine or saline for 7 days. A total of 102 distinct peptides were identified in one or more of the following brain regions: nucleus accumbens, caudate putamen, frontal cortex, and ventral tegmental area. None of the peptides detected in the caudate putamen or frontal cortex were altered by cocaine administration. Three peptides in the nucleus accumbens and seven peptides in the ventral tegmental area were significantly decreased in cocaine-treated mice. Five of these ten peptides are derived from proSAAS, a secretory pathway protein and neuropeptide precursor. To investigate whether proSAAS peptides contribute to the physiological effects of psychostimulants, we examined acute responses to cocaine and amphetamine in the open field with wild-type (WT) and proSAAS knockout (KO) mice. Locomotion was stimulated more robustly in the WT compared to mutant mice for both psychostimulants. Behavioral sensitization to amphetamine was not maintained in proSAAS KO mice and these mutants failed to sensitize to cocaine. To determine whether the rewarding effects of cocaine were altered, mice were tested in conditioned place preference (CPP). Both WT and proSAAS KO mice showed dose-dependent CPP to cocaine that was not distinguished by genotype. Taken together, these results suggest that proSAAS-derived peptides contribute differentially to the behavioral sensitization to psychostimulants, while the rewarding effects of cocaine appear intact in mice lacking proSAAS. © 2017 International Society for Neurochemistry.

Insertion mutation at the C-terminus of the serotonin transporter disrupts brain serotonin function and emotion-related behaviors in mice.

PubMed

Zhao, S; Edwards, J; Carroll, J; Wiedholz, L; Millstein, R A; Jaing, C; Murphy, D L; Lanthorn, T H; Holmes, A

2006-06-19

The 5-hydroxytryptamine transporter (5-HTT) regulates 5-hydroxytryptamine (5-HT) neurotransmission by removing 5-HT from the synaptic cleft. Emerging evidence from clinical and genetic studies implicates the 5-HTT in various neuropsychiatric conditions, including anxiety and depression. Here we report that a 5-HTT null mutant mouse line was generated by gene trapping that disrupted the sequence encoding the C-terminus of 5-HTT. This mutation resulted in significant reduction of 5-HTT mRNA and loss of 5-HTT protein. Brain levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid, were markedly decreased in C-terminus 5-HTT -/- mice, while 5-HT uptake or 5-HT content in platelets was absent. Behavioral phenotyping showed that C-terminus 5-HTT -/- mice were normal on a screen for gross behavioral, neurological, and sensory functions. In the tail suspension test for depression-related behavior, C-terminus 5-HTT -/- mice showed increased immobility relative to their +/+ controls. By comparison, a previously generated line of 5-HTT -/- mice lacking exon 2, encoding the N-terminus of the 5-HTT, showed abnormally high immobility in response to repeated, but not acute, exposure to the tail suspension test. In a novel, brightly-lit open field, both C-terminus 5-HTT -/- mice and N-terminus 5-HTT -/- mice displayed decreased center time and reduced locomotor activity compared with their +/+ controls. Both mutant lines buried significantly fewer marbles than their +/+ controls in the marble burying test. These findings further demonstrate the neurobiological functions of the 5-HTT and add to a growing literature linking genetic variation in 5-HTT function with emotional abnormalities.

A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis

PubMed Central

Yang, Yong; Kulka, Kathleen; Montelaro, Ronald C.; Reinhart, Todd A.; Sissons, James; Aderem, Alan; Ojha, Anil K.

2014-01-01

Summary Chronic tuberculosis in an immunocompetent host is a consequence of the delicately balanced growth of Mycobacterium tuberculosis (Mtb) in the face of host defense mechanisms. We identify an Mtb enzyme (TdmhMtb) that hydrolyzes the mycobacterial glycolipid trehalose dimycolate and plays a critical role in balancing the intracellular growth of the pathogen. TdmhMtb is induced under nutrient limiting conditions and remodels the Mtb envelope to increase nutrient influx, but concomitantly sensitizes Mtb to stresses encountered in the host. Consistent with this, a ΔtdmhMtb mutant is more resilient to stress and grows to higher levels than wild-type in immunocompetent mice. By contrast, mutant growth is retarded in MyD88−/− mice indicating that TdmhMtb provides a growth advantage to intracellular Mtb in an immunocompromised host. Thus, the effects and counter-effects of TdmhMtb play an important role in balancing intracellular growth of Mtb in a manner that is directly responsive to host innate immunity. PMID:24528862

Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida

PubMed Central

Schmerk, Crystal L.; Duplantis, Barry N.; Wang, Diana; Burke, Robert D.; Chou, Alicia Y.; Elkins, Karen L.; Ludu, Jagjit S.; Nano, Francis E.

2009-01-01

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice. PMID:19372153

Targeted Deletion of ERK5 MAP Kinase in the Developing Nervous System Impairs Development of GABAergic Interneurons in the Main Olfactory Bulb and Behavioral Discrimination between Structurally Similar Odorants

PubMed Central

Zou, Junhui; Pan, Yung-Wei; Wang, Zhenshan; Chang, Shih-Yu; Wang, Wenbin; Wang, Xin; Tournier, Cathy; Storm, Daniel R.; Xia, Zhengui

2012-01-01

ERK5 MAP kinase is highly expressed in the developing nervous system and has been implicated in promoting the survival of immature neurons in culture. However, its role in the development and function of the mammalian nervous system has not been established in vivo. Here, we report that conditional deletion of the erk5 gene in mouse neural stem cells during development reduces the number of GABAergic interneurons in the main olfactory bulb (OB). Our data suggest that this is due to a decrease in proliferation and an increase in apoptosis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of ERK5 mutant mice. Interestingly, ERK5 mutant mice have smaller OB and are impaired in odor discrimination between structurally similar odorants. We conclude that ERK5 is a novel signaling pathway regulating developmental OB neurogenesis and olfactory behavior. PMID:22442076

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene.

PubMed

Nagaraj, R Y; Nosek, C M; Brotto, M A; Nishi, M; Takeshima, H; Nosek, T M; Ma, J

2000-11-09

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

Neural cell adhesion molecule, NCAM, regulates thalamocortical axon pathfinding and the organization of the cortical somatosensory representation in mouse

PubMed Central

Enriquez-Barreto, Lilian; Palazzetti, Cecilia; Brennaman, Leann H.; Maness, Patricia F.; Fairén, Alfonso

2012-01-01

To study the potential role of neural cell adhesion molecule (NCAM) in the development of thalamocortical (TC) axon topography, wild type, and NCAM null mutant mice were analyzed for NCAM expression, projection, and targeting of TC afferents within the somatosensory area of the neocortex. Here we report that NCAM and its α-2,8-linked polysialic acid (PSA) are expressed in developing TC axons during projection to the neocortex. Pathfinding of TC axons in wild type and null mutant mice was mapped using anterograde DiI labeling. At embryonic day E16.5, null mutant mice displayed misguided TC axons in the dorsal telencephalon, but not in the ventral telencephalon, an intermediate target that initially sorts TC axons toward correct neocortical areas. During the early postnatal period, rostrolateral TC axons within the internal capsule along the ventral telencephalon adopted distorted trajectories in the ventral telencephalon and failed to reach the neocortex in NCAM null mutant animals. NCAM null mutants showed abnormal segregation of layer IV barrels in a restricted portion of the somatosensory cortex. As shown by Nissl and cytochrome oxidase staining, barrels of the anterolateral barrel subfield (ALBSF) and the most distal barrels of the posteromedial barrel subfield (PMBSF) did not segregate properly in null mutant mice. These results indicate a novel role for NCAM in axonal pathfinding and topographic sorting of TC axons, which may be important for the function of specific territories of sensory representation in the somatosensory cortex. PMID:22723769

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism.

PubMed

Dhamne, Sameer C; Silverman, Jill L; Super, Chloe E; Lammers, Stephen H T; Hameed, Mustafa Q; Modi, Meera E; Copping, Nycole A; Pride, Michael C; Smith, Daniel G; Rotenberg, Alexander; Crawley, Jacqueline N; Sahin, Mustafa

2017-01-01

Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant ( Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery.

High-fat hyperphagia in neurotrophin-4 deficient mice reveals potential role of vagal intestinal sensory innervation in long-term controls of food intake.

PubMed

Byerly, Mardi S; Fox, Edward A

2006-06-12

Neurotrophin-4 (NT-4) deficient mice exhibit substantial loss of intestinal vagal afferent innervation and short-term deficits in feeding behavior, suggesting reduced satiation. However, they do not show long-term changes in feeding or body weight because of compensatory behaviors. The present study examined whether high-fat hyperphagia induction would overcome compensation and reveal long-term effects associated with the reduced vagal sensory innervation of NT-4 mutants. First, modifications of a feeding schedule previously developed in rats were examined in wild-type mice to identify the regimen most effective at producing hyperphagia. The most successful schedule, which was run for 26 days, included access to a 43%-fat diet and pelleted chow every other day and access to only powdered chow on the alternate days. On high-fat access days mice consumed 25% more calories than mice with continuous daily access to the same high-fat diet and pelleted chow. This feeding regimen also induced hyperphagia in NT-4 deficient mice and their wild-type controls: on high-fat exposure days mutants consumed 35% more calories relative to continuous-access mutants, and wild types ate 25% more than continuous-access wild types. Moreover, on high-fat access days the alternating NT-4 mutants significantly increased caloric intake by 9% compared to alternating wild types. Thus, high-fat hyperphagia appeared to override compensation, permitting short-term changes in meal consumption by mutants that accrued into long-term changes in total daily food intake. This raises the possibility that intestinal vagal sensory innervation contributes to long-term, as well as to short-term regulation of food intake.

The Hajdu Cheney Mutation Is a Determinant of B-Cell Allocation of the Splenic Marginal Zone.

PubMed

Yu, Jungeun; Zanotti, Stefano; Walia, Bhavita; Jellison, Evan; Sanjay, Archana; Canalis, Ernesto

2018-01-01

The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35 high CD23 - splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35 int CD23 + ) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19 + B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The Effects of GATA-1 and NF-E2 Deficiency on Bone Biomechanical, Biochemical, and Mineral Properties

PubMed Central

Kacena, Melissa A.; Gundberg, Caren M.; Kacena, William J.; Landis, William J.; Boskey, Adele L.; Bouxsein, Mary L.; Horowitz, Mark C.

2014-01-01

Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone. PMID:23359245

Motor coordination in mice with hotfoot, Lurcher, and double mutations of the Grid2 gene encoding the delta-2 excitatory amino acid receptor.

PubMed

Lalonde, R; Hayzoun, K; Selimi, F; Mariani, J; Strazielle, C

2003-11-01

Grid2(ho/ho) is a loss of function gene mutation resulting in abnormal dendritic arborizations of Purkinje cells. These mutants were compared in a series of motor coordination tests requiring balance and equilibrium to nonataxic controls (Grid2(ho/+)) and to a double mutant (Grid2(ho/Lc)) with an inserted Lc mutation. The performance of Grid2(ho/ho) mutant mice was poorer than that of controls on stationary beam, coat hanger, unsteady platform, and rotorod tests. Grid2(ho/Lc) did not differ from Grid2(Lc/+) mice. However, the insertion of the Lc mutation in Grid2(ho/Lc) potentiated the deficits found in Grid2(ho/ho) in stationary beam, unsteady platform, and rotorod tests. These results indicate a deleterious effect of the Lc mutation on Grid2-deficient mice.

Effect of inactivation of the global oxidative stress regulator oxyR on the colonization ability of Escherichia coli O1:K1:H7 in a mouse model of ascending urinary tract infection.

PubMed

Johnson, James R; Clabots, Connie; Rosen, Henry

2006-01-01

To survive within the host urinary tract, Escherichia coli strains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of Escherichia coli as a possible virulence factor in UTI by determining the impact of oxyR inactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47(phox-/-)) mice. The oxyR and oxyS genes of wild-type E. coli strain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an oxyRS mutant. During in vitro growth in broth or human urine, the oxyRS mutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and oxyRS mutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H(2)O(2), which, like the altered growth kinetics observed with oxyRS inactivation, were reversed by restoration of oxyR on a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its oxyRS mutant (by >1 log(10)) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an oxyR-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47(phox-/-) C57BL mice were similarly susceptible, and the oxyR mutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47(phox) genotype, as in CBA/J mice. Within the E. coli Reference collection, 94% of strains were positive for oxyR. These findings fulfill the second and third of Koch's molecular postulates for oxyR as a candidate virulence-facilitating factor in E. coli and indicate that oxyR is a broadly prevalent potential target for future preventive interventions against UTI due to E. coli. They also suggest that neutrophil phagocyte oxidase is not critical for defense against E. coli UTI and that the major oxidative stresses against which OxyR protects E. coli within the host milieu are not phagocyte derived.

HoxB2 binds mutant SOD1 and is altered in transgenic model of ALS.

PubMed

Zhai, Jinbin; Lin, Hong; Canete-Soler, Rafaela; Schlaepfer, William W

2005-09-15

Mutations in Cu/Zn superoxide dismutase (SOD1) cause approximately 20% of familial amyotrophic lateral sclerosis by a toxic gain of function; however, the precise mechanisms remain unclear. Here, we report the identification of HoxB2, a homeodomain-containing transcription factor, as a G93A mutant SOD1 interactive protein in a yeast two-hybrid screen. We show that HoxB2 co-precipitates and co-localizes with mutant SOD1 in neuronal cell lines, as well as in brain and spinal cord of G93A mutant SOD1 transgenic mice. Mutagenesis further shows that this interaction is mediated by the central homeodomain of HoxB2. In motor neuron-like NSC-34 cells, overexpression of HoxB2 or its homeodomain decreases the insolubility of mutant SOD1 and inhibits G93A or G86R mutant SOD1-induced neuronal cell death. In human and mouse tissues, we show that expression of HoxB2 persists in adult spinal cord and is primarily localized in nuclei of motor neurons. In G93A transgenic mice, HoxB2 co-localizes with mutant SOD1 and is redistributed to perikarya and proximal neurites of motor neurons. In addition, there is progressive accumulation of HoxB2 and mutant SOD1 as punctate inclusions in the neuropil surrounding motor neurons. Taken together, our findings demonstrate that interaction of HoxB2 with mutant SOD1 occurs in motor neurons of G93A mutant SOD1 transgenic mice and suggest that this interaction may modulate the neurotoxicity of mutant SOD1.

Increased ethanol consumption despite taste aversion in mice with a human tryptophan hydroxylase 2 loss of function mutation.

PubMed

Lemay, Francis; Doré, François Y; Beaulieu, Jean-Martin

2015-11-16

Polymorphisms in the gene encoding the brain serotonin synthesis enzyme Tph2 have been identified in mental illnesses, with co-morbidity of substance use disorder. However, little is known about the impact of Tph2 gene variants on addiction. Mice expressing a human Tph2 loss of function variant were used to investigate consequences of aversive conditions on ethanol intake. Mice were familiarized either with ethanol or a solution containing both ethanol and the bittering agent quinine. Effect of familiarization to ethanol or an ethanol-quinine solution was then evaluated using a two-bottles preference test in Tph2-KI and control littermates. Mice from both genotypes displayed similar levels of ethanol consumption and quinine avoidance when habituated to ethanol alone. In contrast, addition of quinine to ethanol during the familiarization period resulted in a reduction of avoidance for the quinine-ethanol solution only in mutant mice. These results indicate that loss of function mutation in Tph2 results in greater motivation for ethanol consumption under aversive conditions and may confer enhanced sensitivity to alcohol use disorder. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Nature vs. nurture: can enrichment rescue the behavioural phenotype of BDNF heterozygous mice?

PubMed

Chourbaji, Sabine; Brandwein, Christiane; Vogt, Miriam A; Dormann, Christof; Hellweg, Rainer; Gass, Peter

2008-10-10

In earlier experiments we have demonstrated that group-housing in a rather impoverished "standard" environment can be a crucial stress factor in male C57Bl/6 mice. The present study aimed at investigating the effect of combining a probable genetic vulnerability--postulated by the "Neurotrophin Hypothesis of Depression"--with the potentially modulating influence of a stressful environment such as "impoverished" standard housing conditions. For that purpose mice with a partial deletion of brain-derived neurotrophic factor (BDNF) were group-housed under standard and enriched housing conditions and analysed in a well-established test battery for emotional behaviours. Standard group-housing affected emotional behaviour in male and female BDNF heterozygous mice, causing an increase in anxiety, changes in exploration as well as nociception. Providing the animals' cages with supplementary enrichment, however, led to a rescue of emotional alterations, which emphasises the significance of external factors and their relevance for a valid investigation of genetic aspects in these mutants as well as others, which may be examined in terms of stress-responsiveness or emotionality.

A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

ERIC Educational Resources Information Center

Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

2006-01-01

Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

WNTLESS IS REQUIRED FOR PERIPHERAL LUNG DIFFERENTIATION AND PULMONARY VASCULAR DEVELOPMENT

PubMed Central

Cornett, Bridget; Snowball, John; Varisco, Brian M.; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-01-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. PMID:23523683

Wntless is required for peripheral lung differentiation and pulmonary vascular development.

PubMed

Cornett, Bridget; Snowball, John; Varisco, Brian M; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-07-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. Copyright © 2013 Elsevier Inc. All rights reserved.

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis.

PubMed

Lu, Fangli; Huang, Shiguang; Kasper, Lloyd H

2009-01-22

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8(+), CD4(+), B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4(+), CD8(+), B cells (microMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8(+) T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except microMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4(+), CD8(+), B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8(+) T cells are the most important component.

Overactivation of hedgehog signaling alters development of the ovarian vasculature in mice.

PubMed

Ren, Yi; Cowan, Robert G; Migone, Fernando F; Quirk, Susan M

2012-06-01

The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2(cre/+)SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2(cre/+)SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2(cre/+)SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately.

Overactivation of Hedgehog Signaling Alters Development of the Ovarian Vasculature in Mice1

PubMed Central

Ren, Yi; Cowan, Robert G.; Migone, Fernando F.; Quirk, Susan M.

2012-01-01

ABSTRACT The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2cre/+SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2cre/+SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2cre/+SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately. PMID:22402963

Period 2 gene deletion abolishes β-endorphin neuronal response to ethanol

PubMed Central

Agapito, Maria; Mian, Nadia; Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of β-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β-endorphin neurons in mice. Methods Per2 mutant (mPer2Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated β-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β-endorphin neuronal activity following acute and chronic ethanol treatments, was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Results Per2 mutant mice showed a higher basal level of β-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison to control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β-endorphin secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β-endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions These results suggest for the first time that the Per2 gene may be critically involved in regulating β-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β-endorphin neuronal responses to ethanol. PMID:20586752

Bone marrow-specific knock-in of a non-activatable Ikkα kinase mutant influences haematopoiesis but not atherosclerosis in Apoe-deficient mice.

PubMed

Tilstam, Pathricia V; Gijbels, Marion J; Habbeddine, Mohamed; Cudejko, Céline; Asare, Yaw; Theelen, Wendy; Zhou, Baixue; Döring, Yvonne; Drechsler, Maik; Pawig, Lukas; Simsekyilmaz, Sakine; Koenen, Rory R; de Winther, Menno P J; Lawrence, Toby; Bernhagen, Jürgen; Zernecke, Alma; Weber, Christian; Noels, Heidi

2014-01-01

The Ikkα kinase, a subunit of the NF-κB-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikkα mutant knock-in on haematopoiesis and atherosclerosis in mice. Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA)Apoe(-/-) ) or with Ikkα(+/+)Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA)Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα(AA) mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach.

Maintenance of basal levels of autophagy in Huntington's disease mouse models displaying metabolic dysfunction.

PubMed

Baldo, Barbara; Soylu, Rana; Petersén, Asa

2013-01-01

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin protein. Neuropathology in the basal ganglia and in the cerebral cortex has been linked to the motor and cognitive symptoms whereas recent work has suggested that the hypothalamus might be involved in the metabolic dysfunction. Several mouse models of HD that display metabolic dysfunction have hypothalamic pathology, and expression of mutant huntingtin in the hypothalamus has been causally linked to the development of metabolic dysfunction in mice. Although the pathogenic mechanisms by which mutant huntingtin exerts its toxic functions in the HD brain are not fully known, several studies have implicated a role for the lysososomal degradation pathway of autophagy. Interestingly, changes in autophagy in the hypothalamus have been associated with the development of metabolic dysfunction in wild-type mice. We hypothesized that expression of mutant huntingtin might lead to changes in the autophagy pathway in the hypothalamus in mice with metabolic dysfunction. We therefore investigated whether there were changes in basal levels of autophagy in a mouse model expressing a fragment of 853 amino acids of mutant huntingtin selectively in the hypothalamus using a recombinant adeno-associate viral vector approach as well as in the transgenic BACHD mice. We performed qRT-PCR and Western blot to investigate the mRNA and protein expression levels of selected autophagy markers. Our results show that basal levels of autophagy are maintained in the hypothalamus despite the presence of metabolic dysfunction in both mouse models. Furthermore, although there were no major changes in autophagy in the striatum and cortex of BACHD mice, we detected modest, but significant differences in levels of some markers in mice at 12 months of age. Taken together, our results indicate that overexpression of mutant huntingtin in mice do not significantly perturb basal levels of autophagy.

Interferon β induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

PubMed

Chort, Alice; Alves, Sandro; Marinello, Martina; Dufresnois, Béatrice; Dornbierer, Jean-Gabriel; Tesson, Christelle; Latouche, Morwena; Baker, Darren P; Barkats, Martine; El Hachimi, Khalid H; Ruberg, Merle; Janer, Alexandre; Stevanin, Giovanni; Brice, Alexis; Sittler, Annie

2013-06-01

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu.

PubMed

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-12-01

Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell-deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)-null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1-null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. © 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

Amyloid Precursor Protein Haploinsufficiency Preferentially Mediates Brain Iron Accumulation in Mice Transgenic for The Huntington's Disease Mutation.

PubMed

Berggren, Kiersten; Agrawal, Sonal; Fox, Julia A; Hildenbrand, Justin; Nelson, Ryan; Bush, Ashley I; Fox, Jonathan H

2017-01-01

Huntington's disease (HD) is an autosomal dominant disorder caused by a CAG expansion in the huntingtin gene that results in expression of mutant huntingtin protein. Iron accumulates in HD brain neurons. Amyloid precursor protein (APP) promotes neuronal iron export. However, the role of APP in brain iron accumulation in HD is unclear. To determine the effects of APP insufficiency on HD in YAC128 mice. We crossed APP hemizygous mice (APP+/-) with YAC128 mice that are transgenic (Tg) for human mutant huntingtin (hmHTT) to generate APP+/+ hmHTT-/-, APP+/- hmHTT-/-, APP+/+ hmHTT+/- and APP+/- hmHTT+/- progeny. Mice were evaluated for behavioral, biochemical and neuropathology HD outcomes at 2-12 months of age. APP heterozygosity decreased cortical APP 25% and 60% in non-Tg and Tg mice, respectively. Cerebral and striatal iron levels were increased by APP knockdown in Tg mice only. Nest-building behavior was decreased in Tg mice; APP knockdown decreased nest building in non-Tg but not Tg mice. Rota-rod endurance was decreased in Tg mice. APP+/- hHTT+/- mice demonstrated additional decreases in rota-rod endurance from 4-10 months of age. Tg mice had smaller striatal volumes and fewer striatal neurons but were not affected by APP knockdown. APP heterozygosity results in greater decreases of cortical APP in Tg versus non-Tg mice. Mutant huntingtin transgenic mice develop brain iron accumulation as a result of greater suppression of APP levels. Elevated brain iron in Tg mice was associated with a decline in motor endurance consistent with a disease promoting effect of iron in the YAC128 model of human HD.

High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect.

PubMed

Pardue, Machelle T; Faulkner, Amanda E; Fernandes, Alcides; Yin, Hang; Schaeffel, Frank; Williams, Robert W; Pozdeyev, Nikita; Iuvone, P Michael

2008-02-01

Nob mice share the same mutation in the Nyx gene that is found in humans with complete congenital stationary night blindness (CSNB1). Nob mutant mice were studied to determine whether this defect resulted in myopia, as it does in humans. Refractive development was measured in unmanipulated wild-type C57BL/6J (WT) and nob mice from 4 to 12 weeks of age by using an infrared photorefractor. The right eye was form deprived by means of a skull-mounted goggling apparatus at 4 weeks of age. Refractive errors were recorded every 2 weeks after goggling. The content of dopamine and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection (HPLC-ECD) in retinas of nob and WT mice under light- and dark-adapted conditions. The nob mice had greater hyperopic refractive errors than did the WT mice under normal visual conditions, until 12 weeks of age when both strains had similar refractions. At 6 weeks of age, refractions became less hyperopic in the nob mice but continued to become more hyperopic in the WT mice. After 2 weeks of form deprivation (6 weeks of age), the nob mice displayed a significant myopic shift (~4 D) in refractive error relative to the opposite and control eyes, whereas WT mice required 6 weeks of goggling to elicit a similar response. As expected with loss of ON pathway transmission, light exposure did not alter DOPAC levels in the nob mice. However, dopamine and DOPAC levels were significantly lower in the nob mice compared with WT. Under normal laboratory visual conditions, only minor differences in refractive development were observed between the nob and WT mice. The largest myopic shift in the nob mice resulted after form deprivation, suggesting that visual pathways dependent on nyctalopin and/or abnormally low dopaminergic activity play a role in regulating refractive development. These findings demonstrate an interaction of genetics and environment in refractive development.

High susceptibility to experimental myopia in a mouse model with a retinal ON pathway defect

PubMed Central

Pardue, Machelle T.; Faulkner, Amanda E.; Fernandes, Alcides; Yin, Hang; Schaeffel, Frank; Williams, Robert W.; Pozdeyev, Nikita; Iuvone, P. Michael

2009-01-01

Purpose Nob mice share the same mutation in the Nyx gene that is found in humans with complete congenital stationary night blindness (CSNB1). We studied nob mutant mice to determine whether this defect resulted in myopia as it does in humans. Methods Refractive development was measured in unmanipulated wildtype C57BL/6J (WT) and nob mice from 4 to 12 weeks of age using an infrared photorefractor. The right eye was form-deprived by means of a skull-mounted goggling apparatus at 4 weeks of age. Refractive errors were recorded every 2 weeks after goggling. The content of dopamine and the dopamine metabolite, DOPAC, were measured using HPLC-ECD in retinas of nob and WT mice under light- and dark-adapted conditions. Results Nob mice had greater hyperopic refractive errors than WT mice under normal visual conditions until 12 weeks of age, when both strains had similar refractions. At 6 weeks of age, refractions became less hyperopic in nob mice but continued to become more hyperopic in WT mice. Following two weeks of form deprivation (6 weeks of age), nob mice displayed a significant myopic shift (~4 D) in refractive error relative to the opposite and control eyes, while WT mice required 6 weeks of goggling to elicit a similar response. As expected with loss of ON pathway transmission, light exposure did not alter DOPAC levels in nob mice. However, dopamine and DOPAC levels were significantly lower in nob mice compared to WT. Conclusions Under normal laboratory visual conditions, only minor differences in refractive development were observed between nob and WT mice. The largest myopic shift in nob mice resulted after form deprivation, suggesting that visual pathways dependent on nyctalopin and/or abnormally low dopaminergic activity play a role in regulating refractive development. These findings demonstrate an interaction of genetics and environment in refractive development. PMID:18235018

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection.

PubMed

Gupta, P; Gupta, R K; Harjai, K

2013-01-01

Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.

PTB deficiency causes the loss of adherens junctions in the dorsal telencephalon and leads to lethal hydrocephalus.

PubMed

Shibasaki, Takayuki; Tokunaga, Akinori; Sakamoto, Reiko; Sagara, Hiroshi; Noguchi, Shigeru; Sasaoka, Toshikuni; Yoshida, Nobuaki

2013-08-01

Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.

The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

PubMed

Glasco, Derrick M; Pike, Whitney; Qu, Yibo; Reustle, Lindsay; Misra, Kamana; Di Bonito, Maria; Studer, Michele; Fritzsch, Bernd; Goffinet, André M; Tissir, Fadel; Chandrasekhar, Anand

2016-09-01

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

EphA4 is Involved in Sleep Regulation but Not in the Electrophysiological Response to Sleep Deprivation.

PubMed

Freyburger, Marlène; Pierre, Audrey; Paquette, Gabrielle; Bélanger-Nelson, Erika; Bedont, Joseph; Gaudreault, Pierre-Olivier; Drolet, Guy; Laforest, Sylvie; Blackshaw, Seth; Cermakian, Nicolas; Doucet, Guy; Mongrain, Valérie

2016-03-01

Optimal sleep is ensured by the interaction of circadian and homeostatic processes. Although synaptic plasticity seems to contribute to both processes, the specific players involved are not well understood. The EphA4 tyrosine kinase receptor is a cell adhesion protein regulating synaptic plasticity. We investigated the role of EphA4 in sleep regulation using electrocorticography in mice lacking EphA4 and gene expression measurements. EphA4 knockout (KO) mice, Clock(Δ19/Δ19) mutant mice and littermates, C57BL/6J and CD-1 mice, and Sprague-Dawley rats were studied under a 12 h light: 12 h dark cycle, under undisturbed conditions or 6 h sleep deprivation (SLD), and submitted to a 48 h electrophysiological recording and/or brain sampling at different time of day. EphA4 KO mice showed less rapid eye movement sleep (REMS), enhanced duration of individual bouts of wakefulness and nonrapid eye movement sleep (NREMS) during the light period, and a blunted daily rhythm of NREMS sigma activity. The NREMS delta activity response to SLD was unchanged in EphA4 KO mice. However, SLD increased EphA4 expression in the thalamic/hypothalamic region in C57BL/6J mice. We further show the presence of E-boxes in the promoter region of EphA4, a lower expression of EphA4 in Clock mutant mice, a rhythmic expression of EphA4 ligands in several brain areas, expression of EphA4 in the suprachiasmatic nuclei of the hypothalamus (SCN), and finally an unchanged number of cells expressing Vip, Grp and Avp in the SCN of EphA4 KO mice. Our results suggest that EphA4 is involved in circadian sleep regulation. © 2016 Associated Professional Sleep Societies, LLC.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice.

PubMed

Quinzii, Catarina M; Garone, Caterina; Emmanuele, Valentina; Tadesse, Saba; Krishna, Sindu; Dorado, Beatriz; Hirano, Michio

2013-02-01

Primary human CoQ(10) deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ(10) biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ(10) of control cells, revealed reduced activity of CoQ(10)-dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30-50% CoQ(10) of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ(10) deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2(kd/kd) mice at presymptomatic, phenotypic-onset, and end-stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ(9) deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney-specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2(kd/kd) mice.

Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

PubMed

Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

2013-04-01

GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Ubiquitin Ligase Component Siah1a Is Required for Completion of Meiosis I in Male Mice

PubMed Central

Dickins, Ross A.; Frew, Ian J.; House, Colin M.; O'Bryan, Moira K.; Holloway, Andrew J.; Haviv, Izhak; Traficante, Nadia; de Kretser, David M.; Bowtell, David D. L.

2002-01-01

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including β-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division. PMID:11884614

Hypolipidemic effects of starch and γ-oryzanol from wx/ae double-mutant rice on BALB/c.KOR-Apoe(shl) mice.

PubMed

Nakaya, Makoto; Shojo, Aiko; Hirai, Hiroaki; Matsumoto, Kenji; Kitamura, Shinichi

2013-01-01

waxy/amylose-extender (wx/ae) double-mutant japonica rice (Oryza sativa L.) produces resistant starch (RS) and a large amount of γ-oryzanol. Our previous study has shown the hypolipidemic effect of wx/ae brown rice on mice. To identify the functional constituents of the hypolipidemic activity in wx/ae rice, we prepared pure wx/ae starch and γ-oryzanol from wx/ae rice and investigated their effect on the lipid metabolism in BALB/c.KOR/Stm Slc-Apoe(shl) mice. The mice were fed for 3 weeks a diet containing non-mutant rice starch, non-mutant rice starch plus γ-oryzanol, wx/ae starch, or wx/ae starch plus γ-oryzanol. γ-Oryzanol by itself had no effect on the lipid metabolism, and wx/ae starch prevented an accumulation of triacylglycerol (TAG) in the liver. Interestingly, the combination of wx/ae starch plus γ-oryzanol not only prevented a TAG accumulation in the liver, but also partially suppressed the rise in plasma TAG concentration, indicating that wx/ae starch and γ-oryzanol could have a synergistic effect on the lipid metabolism.

Role for the epidermal growth factor receptor in chemotherapy-induced alopecia.

PubMed

Bichsel, Kyle J; Gogia, Navdeep; Malouff, Timothy; Pena, Zachary; Forney, Eric; Hammiller, Brianna; Watson, Patrice; Hansen, Laura A

2013-01-01

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.

The effects of a skeletal muscle titin mutation on walking in mice.

PubMed

Pace, Cinnamon M; Mortimer, Sarah; Monroy, Jenna A; Nishikawa, Kiisa C

2017-01-01

Titin contributes to sarcomere assembly, muscle signaling, and mechanical properties of muscle. The mdm mouse exhibits a small deletion in the titin gene resulting in dystrophic mutants and phenotypically normal heterozygotes. We examined the effects of this mutation on locomotion to assess how, and if, changes to muscle phenotype explain observed locomotor differences. Mutant mice are much smaller in size than their siblings and gait abnormalities may be driven by differences in limb proportions and/or by changes to muscle phenotype caused by the titin mutation. We quantified differences in walking gait among mdm genotypes and also determined whether genotypes vary in limb morphometrics. Mice were filmed walking, and kinematic and morphological variables were measured. Mutant mice had a smaller range of motion at the ankle, shorter stride lengths, and shorter stance duration, but walked at the same relative speeds as the other genotypes. Although phenotypically similar to wildtype mice, heterozygous mice frequently exhibited intermediate gait mechanics. Morphological differences among genotypes in hindlimb proportions were small and do not explain the locomotor differences. We suggest that differences in locomotion among mdm genotypes are due to changes in muscle phenotype caused by the titin mutation.

Seizure phenotypes, periodicity, and sleep-wake pattern of seizures in Kcna-1 null mice.

PubMed

Wright, Samantha; Wallace, Eli; Hwang, Youngdeok; Maganti, Rama

2016-02-01

This study was undertaken to describe seizure phenotypes, natural progression, sleep-wake patterns, as well as periodicity of seizures in Kcna-1 null mutant mice. These mice were implanted with epidural electroencephalography (EEG) and electromyography (EMG) electrodes, and simultaneous video-EEG recordings were obtained while animals were individually housed under either diurnal (LD) condition or constant darkness (DD) over ten days of recording. The video-EEG data were analyzed to identify electrographic and behavioral phenotypes and natural progression and to examine the periodicity of seizures. Sleep-wake patterns were analyzed to understand the distribution and onset of seizures across the sleep-wake cycle. Four electrographically and behaviorally distinct seizure types were observed. Regardless of lighting condition that animals were housed in, Kcna-1 null mice initially expressed only a few of the most severe seizure types that progressively increased in frequency and decreased in seizure severity. In addition, a circadian periodicity was noted, with seizures peaking in the first 12h of the Zeitgeber time (ZT) cycle, regardless of lighting conditions. Interestingly, seizure onset differed between lighting conditions where more seizures arose out of sleep in LD conditions, whereas under DD conditions, the majority occurred out of the wakeful state. We suggest that this model be used to understand the circadian pattern of seizures as well as the pathophysiological implications of sleep and circadian disturbances in limbic epilepsies. Copyright © 2015 Elsevier Inc. All rights reserved.

Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice

PubMed Central

Barathi, Veluchamy A.; Kwan, Jia Lin; Tan, Queenie S. W.; Weon, Sung Rhan; Seet, Li Fong; Goh, Liang Kee; Vithana, Eranga N.; Beuerman, Roger W.

2013-01-01

SUMMARY Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error. PMID:23649821

Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

PubMed

Lebel, Michel

2002-01-01

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

The Absence of Sensory Axon Bifurcation Affects Nociception and Termination Fields of Afferents in the Spinal Cord

PubMed Central

Tröster, Philip; Haseleu, Julia; Petersen, Jonas; Drees, Oliver; Schmidtko, Achim; Schwaller, Frederick; Lewin, Gary R.; Ter-Avetisyan, Gohar; Winter, York; Peters, Stefanie; Feil, Susanne; Feil, Robert; Rathjen, Fritz G.; Schmidt, Hannes

2018-01-01

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli. PMID:29472841

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

PubMed

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N; Toole, Bryan P; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A

2009-08-01

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice*

PubMed Central

Simhadri, Srilatha; Peterson, Shaun; Patel, Dharm S.; Huo, Yanying; Cai, Hong; Bowman-Colin, Christian; Miller, Shoreh; Ludwig, Thomas; Ganesan, Shridar; Bhaumik, Mantu; Bunting, Samuel F.; Jasin, Maria; Xia, Bing

2014-01-01

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis. PMID:25016020

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

PubMed Central

Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

2013-01-01

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID:23596295

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

PubMed

Bossi, Simone; Musante, Ilaria; Bonfiglio, Tommaso; Bonifacino, Tiziana; Emionite, Laura; Cerminara, Maria; Cervetto, Chiara; Marcoli, Manuela; Bonanno, Giambattista; Ravazzolo, Roberto; Pittaluga, Anna; Puliti, Aldamaria

2018-01-01

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1 crv4/crv4 ) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1 crv4 and Grm5 ko mice to generate double mutants (Grm1 crv4/crv4 Grm5 ko/ko ) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1 crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1 crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1 crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1 crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

PubMed

Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

2013-01-23

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

Mutation of the inhibitory ethanol site in GABAA ρ1 receptors promotes tolerance to ethanol-induced motor incoordination.

PubMed

Blednov, Yuri A; Borghese, Cecilia M; Ruiz, Carlos I; Cullins, Madeline A; Da Costa, Adriana; Osterndorff-Kahanek, Elizabeth A; Homanics, Gregg E; Harris, R Adron

2017-09-01

Genes encoding the ρ1/2 subunits of GABA A receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABA A ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Learning from a paradox: recent insights into Fanconi anaemia through studying mouse models.

PubMed

Bakker, Sietske T; de Winter, Johan P; te Riele, Hein

2013-01-01

Fanconi anaemia (FA) is a rare autosomal recessive or X-linked inherited disease characterised by an increased incidence of bone marrow failure (BMF), haematological malignancies and solid tumours. Cells from individuals with FA show a pronounced sensitivity to DNA interstrand crosslink (ICL)-inducing agents, which manifests as G2-M arrest, chromosomal aberrations and reduced cellular survival. To date, mutations in at least 15 different genes have been identified that cause FA; the products of all of these genes are thought to function together in the FA pathway, which is essential for ICL repair. Rapidly following the discovery of FA genes, mutant mice were generated to study the disease and the affected pathway. These mutant mice all show the characteristic cellular ICL-inducing agent sensitivity, but only partially recapitulate the developmental abnormalities, anaemia and cancer predisposition seen in individuals with FA. Therefore, the usefulness of modelling FA in mice has been questioned. In this Review, we argue that such scepticism is unjustified. We outline that haematopoietic defects and cancer predisposition are manifestations of FA gene defects in mice, albeit only in certain genetic backgrounds and under certain conditions. Most importantly, recent work has shown that developmental defects in FA mice also arise with concomitant inactivation of acetaldehyde metabolism, giving a strong clue about the nature of the endogenous lesion that must be repaired by the functional FA pathway. This body of work provides an excellent example of a paradox in FA research: that the dissimilarity, rather than the similarity, between mice and humans can provide insight into human disease. We expect that further study of mouse models of FA will help to uncover the mechanistic background of FA, ultimately leading to better treatment options for the disease.

Glycine Receptors Containing α2 or α3 Subunits Regulate Specific Ethanol-Mediated Behaviors

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth

2015-01-01

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRα1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRα2 and α3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyRα2 or α3 subunits were distinct from effects previously observed in mice with knock-in mutations in the α1 subunit. We provide evidence that GlyRα2 and α3 subunits may regulate ethanol consumption and the aversive response to ethanol. PMID:25678534

Anterior segment dysgenesis correlation with epithelial-mesenchymal transition in Smad4 knockout mice.

PubMed

Li, Jing; Qin, Yu; Zhao, Fang-Kun; Wu, Di; He, Xue-Fei; Liu, Jia; Zhao, Jiang-Yue; Zhang, Jin-Song

2016-01-01

To explore the molecular mechanisms in lens development and the pathogenesis of Peters anomaly in Smad4 defective mice. Le-Cre transgenic mouse line was employed to inactivate Smad4 in the surface ectoderm selectively. Pathological techniques were used to reveal the morphological changes of the anterior segment in Smad4 defective eye. Immunohistochemical staining was employed to observe the expression of E-cadherin, N-cadherin and α-SMA in anterior segment of Smad4 defective mice and control mice at embryonic (E) day 16.5. Real-time quantitative polymerase chain reaction (qPCR) was performed to detect the expression of Snail, Zeb1, Zeb2 and Twist2 in lens of Smad4 defective mice and control mice at E16.5. Statistical evaluations were performed using the unpaired Student's t-test (two-tailed) by SPSS 11.0 software. Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Loss of Smad4 function inhibited E-cadherin expression in the lens epithelium cells and corneal epithelium cells in Smad4 defective eye. Expression of N-cadherin was up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and N-cadherin were down-regulated at the future trabecular meshwork region in mutant eye. The qPCR results showed that the expression of Twist2 was increased significantly in the mutant lens (P<0.01). Smad4 is essential to eye development and likely a candidate pathogenic gene to Peters anomaly by regulating epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays an essential role in lens development.

Deficits of learning and memory in Hemojuvelin knockout mice.

PubMed

Li, Jinglong; Zhang, Peng; Liu, Hongju; Ren, Wei; Song, Jinjing; Rao, Elizabeth; Takahashi, Eiki; Zhou, Ying; Li, Weidong; Chen, Xiaoping

2015-10-01

Iron is involved in various physiological processes of the human body to maintain normal functions. Abnormal iron accumulation in brain has been reported as a pathogenesis of several neurodegenerative disorders and cognitive impairments. Hemojuvelin (HVJ) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis. Although iron accumulation in brain has been related to neurodegenerative diseases, it remains unknown the effect of mutation of HVJ gene on cognitive performance. In our studies, HJV(-/-) mice showed deficits in novel object recognition and Morris water maze tests. Furthermore, the expression ration of apoptotic marker Bax and anti-apoptotic marker Bcl-2 in the hippocampus and prefrontal cortex showed higher levels in HJV(-/-) mice. Our results suggested that deletion of HJV gene could increase apoptosis in brain which might contribute to learning and memory deficits in mutant mice. These results indicated that HJV(-/-) mice would be a useful model to study cognitive impairment induced by iron overload in brain.

VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting.

PubMed

Hahm, Seung; Fekete, Csaba; Mizuno, Tooru M; Windsor, Joan; Yan, Hai; Boozer, Carol N; Lee, Charlotte; Elmquist, Joel K; Lechan, Ronald M; Mobbs, Charles V; Salton, Stephen R J

2002-08-15

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

Mutant characterization and in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for Aspergillus fumigatus virulence

PubMed Central

Sasse, Anna; Hamer, Stefanie N; Amich, Jorge; Binder, Jasmin; Krappmann, Sven

2016-01-01

Pathogenicity of the saprobe Aspergillus fumigatus strictly depends on nutrient acquisition during infection, as fungal growth determines colonisation and invasion of a susceptible host. Primary metabolism has to be considered as a valid target for antimycotic therapy, based on the fact that several fungal anabolic pathways are not conserved in higher eukaryotes. To test whether fungal proliferation during invasive aspergillosis relies on endogenous biosynthesis of aromatic amino acids, defined auxotrophic mutants of A. fumigatus were generated and assessed for their infectious capacities in neutropenic mice and found to be strongly attenuated in virulence. Moreover, essentiality of the complete biosynthetic pathway could be demonstrated, corroborated by conditional gene expression in infected animals and inhibitor studies. This brief report not only validates the aromatic amino acid biosynthesis pathway of A. fumigatus to be a promising antifungal target but furthermore demonstrates feasibility of conditional gene expression in a murine infection model of aspergillosis. PMID:26605426

An autism-associated serotonin transporter variant disrupts multisensory processing.

PubMed

Siemann, J K; Muller, C L; Forsberg, C G; Blakely, R D; Veenstra-VanderWeele, J; Wallace, M T

2017-03-21

Altered sensory processing is observed in many children with autism spectrum disorder (ASD), with growing evidence that these impairments extend to the integration of information across the different senses (that is, multisensory function). The serotonin system has an important role in sensory development and function, and alterations of serotonergic signaling have been suggested to have a role in ASD. A gain-of-function coding variant in the serotonin transporter (SERT) associates with sensory aversion in humans, and when expressed in mice produces traits associated with ASD, including disruptions in social and communicative function and repetitive behaviors. The current study set out to test whether these mice also exhibit changes in multisensory function when compared with wild-type (WT) animals on the same genetic background. Mice were trained to respond to auditory and visual stimuli independently before being tested under visual, auditory and paired audiovisual (multisensory) conditions. WT mice exhibited significant gains in response accuracy under audiovisual conditions. In contrast, although the SERT mutant animals learned the auditory and visual tasks comparably to WT littermates, they failed to show behavioral gains under multisensory conditions. We believe these results provide the first behavioral evidence of multisensory deficits in a genetic mouse model related to ASD and implicate the serotonin system in multisensory processing and in the multisensory changes seen in ASD.

AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy

PubMed Central

Spindler, Matthew J.; Burmeister, Brian T.; Huang, Yu; Hsiao, Edward C.; Salomonis, Nathan; Scott, Mark J.; Srivastava, Deepak; Carnegie, Graeme K.; Conklin, Bruce R.

2013-01-01

Background A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown. Methodology/Principal Findings To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction. Conclusions These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy. PMID:23658642

Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

PubMed Central

Ding, Yuetong; Qu, Yibo; Feng, Jia; Wang, Meizhi; Han, Qi; So, Kwok-Fai; Wu, Wutian; Zhou, Libing

2014-01-01

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI. PMID:25003601

Interleukin-6 is an essential determinant of on-time parturition in the mouse.

PubMed

Robertson, Sarah A; Christiaens, Inge; Dorian, Camilla L; Zaragoza, Dean B; Care, Alison S; Banks, Anke M; Olson, David M

2010-08-01

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

Dopamine-Dependent Compensation Maintains Motor Behavior in Mice with Developmental Ablation of Dopaminergic Neurons

PubMed Central

DeMaro, Joseph A.; Knoten, Amanda; Hoshi, Masato; Pehek, Elizabeth; Johnson, Eugene M.; Gereau, Robert W.

2013-01-01

The loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and consequent depletion of striatal dopamine are known to underlie the motor deficits observed in Parkinson's disease (PD). Adaptive changes in dopaminergic terminals and in postsynaptic striatal neurons can compensate for significant losses of striatal dopamine, resulting in preservation of motor behavior. In addition, compensatory changes independent of striatal dopamine have been proposed based on PD therapies that modulate nondopaminergic circuits within the basal ganglia. We used a genetic strategy to selectively destroy dopaminergic neurons in mice during development to determine the necessity of these neurons for the maintenance of normal motor behavior in adult and aged mice. We find that loss of 90% of SNc dopaminergic neurons and consequent depletion of >95% of striatal dopamine does not result in changes in motor behavior in young-adult or aged mice as evaluated by an extensive array of motor behavior tests. Treatment of aged mutant mice with the dopamine receptor antagonist haloperidol precipitated motor behavior deficits in aged mutant mice, indicating that <5% of striatal dopamine is sufficient to maintain motor function in these mice. We also found that mutant mice exhibit an exaggerated response to l-DOPA compared with control mice, suggesting that preservation of motor function involves sensitization of striatal dopamine receptors. Our results indicate that congenital loss of dopaminergic neurons induces remarkable adaptions in the nigrostriatal system where limited amounts of dopamine in the dorsal striatum can maintain normal motor function. PMID:24155314

β-Myosin heavy chain variant Val606Met causes very mild hypertrophic cardiomyopathy in mice, but exacerbates HCM phenotypes in mice carrying other HCM mutations.

PubMed

Blankenburg, Robert; Hackert, Katarzyna; Wurster, Sebastian; Deenen, René; Seidman, J G; Seidman, Christine E; Lohse, Martin J; Schmitt, Joachim P

2014-07-07

Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in β-cardiac myosin heavy chain (β-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. To characterize disease caused by β-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations. © 2014 American Heart Association, Inc.

Hip1-related Mutant Mice Grow and Develop Normally but Have Accelerated Spinal Abnormalities and Dwarfism in the Absence of HIP1†

PubMed Central

Hyun, Teresa S.; Li, Lina; Oravecz-Wilson, Katherine I.; Bradley, Sarah V.; Provot, Melissa M.; Munaco, Anthony J.; Mizukami, Ikuko F.; Sun, Hanshi; Ross, Theodora S.

2004-01-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo. PMID:15121852

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1.

PubMed

Hyun, Teresa S; Li, Lina; Oravecz-Wilson, Katherine I; Bradley, Sarah V; Provot, Melissa M; Munaco, Anthony J; Mizukami, Ikuko F; Sun, Hanshi; Ross, Theodora S

2004-05-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.

Etiology of a genetically complex seizure disorder in Celf4 mutant mice

PubMed Central

Wagnon, Jacy L.; Mahaffey, Connie L.; Sun, Wenzhi; Yang, Yan; Chao, Hsiao-Tuan; Frankel, Wayne N.

2011-01-01

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adult. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at seven weeks of age. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice. PMID:21745337

Unbound (bioavailable) IGF1 enhances somatic growth

PubMed Central

Elis, Sebastien; Wu, Yingjie; Courtland, Hayden-William; Cannata, Dara; Sun, Hui; Beth-On, Mordechay; Liu, Chengyu; Jasper, Hector; Domené, Horacio; Karabatas, Liliana; Guida, Clara; Basta-Pljakic, Jelena; Cardoso, Luis; Rosen, Clifford J.; Frystyk, Jan; Yakar, Shoshana

2011-01-01

SUMMARY Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or ‘free IGF1’. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions. PMID:21628395

Loss of Magel2 impairs the development of hypothalamic Anorexigenic circuits

PubMed Central

Maillard, Julien; Park, Soyoung; Croizier, Sophie; Vanacker, Charlotte; Cook, Joshua H.; Prevot, Vincent; Tauber, Maithe; Bouret, Sebastien G.

2016-01-01

Prader–Willi syndrome (PWS) is a genetic disorder characterized by a variety of physiological and behavioral dysregulations, including hyperphagia, a condition that can lead to life-threatening obesity. Feeding behavior is a highly complex process with multiple feedback loops that involve both peripheral and central systems. The arcuate nucleus of the hypothalamus (ARH) is critical for the regulation of homeostatic processes including feeding, and this nucleus develops during neonatal life under of the influence of both environmental and genetic factors. Although much attention has focused on the metabolic and behavioral outcomes of PWS, an understanding of its effects on the development of hypothalamic circuits remains elusive. Here, we show that mice lacking Magel2, one of the genes responsible for the etiology of PWS, display an abnormal development of ARH axonal projections. Notably, the density of anorexigenic α-melanocyte-stimulating hormone axons was reduced in adult Magel2-null mice, while the density of orexigenic agouti-related peptide fibers in the mutant mice appeared identical to that in control mice. On the basis of previous findings showing a pivotal role for metabolic hormones in hypothalamic development, we also measured leptin and ghrelin levels in Magel2-null and control neonates and found that mutant mice have normal leptin and ghrelin levels. In vitro experiments show that Magel2 directly promotes axon growth. Together, these findings suggest that a loss of Magel2 leads to the disruption of hypothalamic feeding circuits, an effect that appears to be independent of the neurodevelopmental effects of leptin and ghrelin and likely involves a direct neurotrophic effect of Magel2. PMID:27288456

The neurotrophin-inducible gene Vgf regulates hippocampal function and behavior through a brain-derived neurotrophic factor-dependent mechanism.

PubMed

Bozdagi, Ozlem; Rich, Erin; Tronel, Sophie; Sadahiro, Masato; Patterson, Kamara; Shapiro, Matthew L; Alberini, Cristina M; Huntley, George W; Salton, Stephen R J

2008-09-24

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, in which it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knock-out mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nm), and tPA STOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75(NTR) function-blocking antiserum, or previous tetanic stimulation. Although LTP was normal in slices from VGF knock-out mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior.

The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior Through a BDNF-Dependent Mechanism

PubMed Central

Bozdagi, Ozlem; Rich, Erin; Tronel, Sophie; Sadahiro, Masato; Patterson, Kamara; Shapiro, Matthew L.; Alberini, Cristina M.; Huntley, George W.; Salton, Stephen R. J.

2009-01-01

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, where it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knockout mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nM), and by tPASTOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75NTR function-blocking antiserum, nor by prior tetanic stimulation. Although LTP was normal in slices from VGF knockout mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism, and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior. PMID:18815270

A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic diabetes insipidus

PubMed Central

Li, Jian Hua; Chou, Chung-Lin; Li, Bo; Gavrilova, Oksana; Eisner, Christoph; Schnermann, Jürgen; Anderson, Stasia A.; Deng, Chu-Xia; Knepper, Mark A.; Wess, Jürgen

2009-01-01

X-linked nephrogenic diabetes insipidus (XNDI) is a severe kidney disease caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene that result in the loss of renal urine-concentrating ability. At present, no specific pharmacological therapy has been developed for XNDI, primarily due to the lack of suitable animal models. To develop what we believe to be the first viable animal model of XNDI, we generated mice in which the V2R gene could be conditionally deleted during adulthood by administration of 4-OH-tamoxifen. Radioligand-binding studies confirmed the lack of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, and further analysis indicated that upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, including polyuria, polydipsia, and resistance to the antidiuretic actions of vasopressin. Gene expression analysis suggested that activation of renal EP4 PGE2 receptors might compensate for the lack of renal V2R activity in XNDI mice. Strikingly, both acute and chronic treatment of the mutant mice with a selective EP4 receptor agonist greatly reduced all major manifestations of XNDI, including changes in renal morphology. These physiological improvements were most likely due to a direct action on EP4 receptors expressed on collecting duct cells. These findings illustrate the usefulness of the newly generated V2R mutant mice for elucidating and testing new strategies for the potential treatment of humans with XNDI. PMID:19729836

Unbound (bioavailable) IGF1 enhances somatic growth.

PubMed

Elis, Sebastien; Wu, Yingjie; Courtland, Hayden-William; Cannata, Dara; Sun, Hui; Beth-On, Mordechay; Liu, Chengyu; Jasper, Hector; Domené, Horacio; Karabatas, Liliana; Guida, Clara; Basta-Pljakic, Jelena; Cardoso, Luis; Rosen, Clifford J; Frystyk, Jan; Yakar, Shoshana

2011-09-01

Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or 'free IGF1'. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions.

Cortico-striatal synaptic defects and OCD-like behaviors in SAPAP3 mutant mice

PubMed Central

Welch, Jeffrey M.; Lu, Jing; Rodriguiz, Ramona M.; Trotta, Nicholas C.; Peca, Joao; Ding, Jin-Dong; Feliciano, Catia; Chen, Meng; Adams, J. Paige; Luo, Jianhong; Dudek, Serena M.; Weinberg, Richard J.; Calakos, Nicole; Wetsel, William C.; Feng, Guoping

2008-01-01

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, though the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of SAPAP3 exhibit increased anxiety and compulsive grooming behavior leading to facial hair loss and skin lesions; both behaviors are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural, and biochemical studies of SAPAP3 mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of SAPAP3 in the striatum rescues the synaptic and behavioral defects of SAPAP3 mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviors. PMID:17713528

Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

PubMed Central

Salido, Eduardo C.; Li, Xiao M.; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J.; Roy-Chowdhury, Jayanta

2006-01-01

Mutations in the alanine–glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt−/− mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

Anti-H-Y responses of H-2b mutant mice.

PubMed

Simpson, E; Gordon, R D; Chandler, P R; Bailey, D

1978-10-01

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

Longitudinal brain MRI study in a mouse model of Rett Syndrome and the effects of choline.

PubMed

Ward, B C; Agarwal, S; Wang, K; Berger-Sweeney, J; Kolodny, N H

2008-07-01

Rett Syndrome (RTT), the second most common cause of mental retardation in girls, is associated with mutations of an X-linked gene encoding the transcriptional repressor protein MeCP2. Mecp2(1lox) mutant mice express no functional MeCP2 protein and exhibit behavioral abnormalities similar to those seen in RTT patients. Here we monitor the development of both whole brain and regional volumes between 21 and 42 days of age in this model of RTT using MRI. We see decreases in whole brain volumes in both male and female mutant mice. Cerebellar and ventricular volumes are also decreased in RTT males. Previous work has suggested that perinatal choline supplementation alleviates some of the behavioral deficits in both male and female Mecp2(1lox) mutant mice. Here we show that perinatal choline supplementation also positively affects whole brain volume in heterozygous females, and cerebellar volume in male RTT mice.

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

PubMed

Peachey, Neal S; Hasan, Nazarul; FitzMaurice, Bernard; Burrill, Samantha; Pangeni, Gobinda; Karst, Son Yong; Reinholdt, Laura; Berry, Melissa L; Strobel, Marge; Gregg, Ronald G; McCall, Maureen A; Chang, Bo

2017-08-01

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6 nob8 mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6 nob8 retina were comparable to control. The Grm6 nob8 phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6 nob8 mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease. NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models.

Behavioral Actions of Alcohol: Phenotypic Relations from Multivariate Analysis of Mutant Mouse Data

PubMed Central

Blednov, Yuri A.; Mayfield, R. Dayne; Belknap, John; Harris, R. Adron

2012-01-01

Behavioral studies of genetically diverse mice have proven powerful for determining relationships between phenotypes and have been widely used in alcohol research. Most of these studies rely on naturally occurring genetic polymorphisms among inbred strains and selected lines. Another approach is to introduce variation by engineering single gene mutations in mice. We have tested 37 different mutant mice and their wild type controls for a variety (31) of behaviors and have mined this dataset by K-means clustering and analysis of correlations. We found a correlation between a stress-related response (activity in a novel environment) and alcohol consumption and preference for saccharin. We confirmed several relationships detected in earlier genetic studies including positive correlation of alcohol consumption with saccharin consumption, and negative correlations with conditioned taste aversion and alcohol withdrawal severity. Introduction of single gene mutations either eliminated or greatly diminished these correlations. The three tests of alcohol consumption used (continuous two bottle choice, and two limited access tests: Drinking In the Dark and Sustained High Alcohol Consumption) share a relationship with saccharin consumption, but differ from each other in their correlation networks. We suggest that alcohol consumption is controlled by multiple physiological systems where single gene mutations can disrupt the networks of such systems. PMID:22405477

Microglia ablation alleviates myelin-associated catatonic signs in mice

PubMed Central

Janova, Hana; Arinrad, Sahab; Balmuth, Evan; Mitjans, Marina; Bittner, Robert A.; Pan, Hong; Goebbels, Sandra; Begemann, Martin; Gerwig, Ulrike C.; Langner, Sönke; Werner, Hauke B.; Davatzikos, Christos; Völzke, Henry; West, Brian L.; Reif, Andreas; Grabe, Hans Jörgen; Nave, Klaus-Armin

2017-01-01

The underlying cellular mechanisms of catatonia, an executive “psychomotor” syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling PLX5622 depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities. PMID:29252214

c-Kit mutation reduce intestinal epithelial cell proliferation and migration, but not influence intestinal permeability stimulated by lipopolysaccharide.

PubMed

Xue, Hong; Wang, Feng Yun; Kang, Qian; Tang, Xu Dong

2018-06-20

The proto-oncogene c-kit, as a marker of interstitial cells of Cajal (ICCs) in the gastrointestinal tract, plays an important role in the ICCs. Although limited evidences showed c-kit is present in the colonic epithelium but its roles remain unclear. In the present study, we aimed to investigate the expression, location and function of c-kit in the intestinal epithelium. Immunofluorescence, western blotting, and RT-PCR were performed to detect the expression and location of c-kit in the intestinal mucosa of WT mice. We investigated intestinal epithelial proliferation and migration in vivo by performing 5-Bromodeoxyuridine (BrdU) incorporation and Ki-67 staining in WT and Wads m/m mice. An Ussing chamber with fluorescein-isothiocyanate dextran 4000 was used to detect the transepithelial electric resistance (TER), short circuit current (ISC) and permeability across ex vivo colon segments under control and endotoxaemia conditions. We demonstrated that c-kit was located and expressed in the gut crypt compartment in WT mice, which was demonstrated in the c-kit mutant mice (Wads m/m ). In addition, both the number of proliferating cells and the percentage of the distance migrated were lower in the Wads m/m mice than those in the WT mice. Moreover, the intestinal permeability, TER and tight junction were unaltered in the Wads m/m mice under endotoxic conditions compared with those in both the control condition and the WT mice. Altogether, these observations imply that the expression of c-kit in the colonic epithelium is involved in the proliferation and permeability of the colonic epithelium. Copyright © 2018. Published by Elsevier GmbH.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice

PubMed Central

Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena

2017-01-01

Abstract CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60–80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. PMID:28369421

Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheep, goats, and mice.

PubMed

Garcia, J P; Adams, V; Beingesser, J; Hughes, M L; Poon, R; Lyras, D; Hill, A; McClane, B A; Rood, J I; Uzal, F A

2013-07-01

Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

Structural abnormalities of corpus callosum and cortical axonal tracts accompanied by decreased anxiety-like behavior and lowered sociability in spock3- mutant mice.

PubMed

Yamamoto, Ayako; Uchiyama, Koji; Nara, Tomoka; Nishimura, Naomichi; Hayasaka, Michiko; Hanaoka, Kazunori; Yamamoto, Tatsuro

2014-01-01

Spock3/Testican-3 is a nervous system-expressed heparan sulfate proteoglycan belonging to a subgroup of the BM-40/SPARC/osteonectin family, the role of which in brain development is unclear. Because Spock1, a member of the Spock family, inhibits their attachment to substrates and the neurite outgrowth of cultured neuronal cells, Spock3 is also thought to be similarly involved in the neuronal development. In the present study, we established a Spock3-mutant mouse harboring a deletion extending from the presumptive upstream regulatory region to exon 4 of the Spock3 locus and performed histological and behavioral studies on these mutant mice. In wild-type (WT) mice, all Spock members were clearly expressed during brain development. In adults, intense Spock1 and Spock2 expressions were observed throughout the entire brain; whereas, Spock3 expression was no longer visible except in the thalamic nuclei. Thus, Spock3 expression is mostly confined to the developmental stage of the brain. In adult mutant mice, the cells of all cortical layers were swollen. The corpus callosum was narrowed around the central region along the rostral-caudal axis and many small spaces were observed without myelin sheaths throughout the entire corpus callosum. In addition, the cortical input and output fibers did not form into thick bundled fibers as well as the WT counterparts did. Moreover, a subpopulation of corticospinal axonal fibers penetrated into the dorsal striatum with moderately altered orientations. Consistent with these modifications of brain structures, the mutant mice exhibited decreased anxiety-like behavior and lowered sociability. Together, these results demonstrate that Spock3 plays an important role in the formation or maintenance of major neuronal structures in the brain. © 2014 S. Karger AG, Basel.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

PubMed Central

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-01-01

Background Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. Methods C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1–specific basophil degranulation, and Cyp c 1–induced allergic symptoms in the mouse model. Results A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1–induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Conclusions Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. PMID:27876628

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy.

PubMed

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-06-01

Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Circadian Clock Gene Csnk1e Regulates Rapid Eye Movement Sleep Amount, and Nonrapid Eye Movement Sleep Architecture in Mice

PubMed Central

Zhou, Lili; Bryant, Camron D.; Loudon, Andrew; Palmer, Abraham A.; Vitaterna, Martha Hotz; Turek, Fred W.

2014-01-01

Study Objectives: Efforts to identify the genetic basis of mammalian sleep have included quantitative trait locus (QTL) mapping and gene targeting of known core circadian clock genes. We combined three different genetic approaches to identify and test a positional candidate sleep gene — the circadian gene casein kinase 1 epsilon (Csnk1e), which is located in a QTL we identified for rapid eye movement (REM) sleep on chromosome 15. Measurements and Results: Using electroencephalographic (EEG) and electromyographic (EMG) recordings, baseline sleep was examined in a 12-h light:12-h dark (LD 12:12) cycle in mice of seven genotypes, including Csnk1etau/tau and Csnk1e-/- mutant mice, Csnk1eB6.D2 and Csnk1eD2.B6 congenic mice, and their respective wild-type littermate control mice. Additionally, Csnk1etau/tau and wild-type mice were examined in constant darkness (DD). Csnk1etau/tau mutant mice and both Csnk1eB6.D2 and Csnk1eD2.B6 congenic mice showed significantly higher proportion of sleep time spent in REM sleep during the dark period than wild-type controls — the original phenotype for which the QTL on chromosome 15 was identified. This phenotype persisted in Csnk1etau/tau mice while under free-running DD conditions. Other sleep phenotypes observed in Csnk1etau/tau mice and congenics included a decreased number of bouts of nonrapid eye movement (NREM) sleep and an increased average NREM sleep bout duration. Conclusions: These results demonstrate a role for Csnk1e in regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture, and support Csnk1e as a causal gene in the sleep QTL on chromosome 15. Citation: Zhou L; Bryant CD; Loudon A; Palmer AA; Vitaterna MH; Turek FW. The circadian clock gene Csnk1e regulates rapid eye movement sleep amount, and nonrapid eye movement sleep architecture in mice. SLEEP 2014;37(4):785-793. PMID:24744456

Treadmill performance of mice with cerebellar lesions: 1. Purkinje cell degeneration mutant mice.

PubMed

Le Marec, N; Lalonde, R

1998-02-01

The purpose of this study was to evaluate the sensorimotor skills of a spontaneous mouse mutant, Purkinje cell degeneration (PCD), marked by selective cerebellar cortical atrophy on a treadmill activated at 1 of 2 speeds and at 1 of 3 slopes, requiring forward movements to avoid footshocks. There was no difference in latencies before falling from the belt between PCD mutants and controls during acquisition. However, PCD mutants were impaired on the fast treadmill during retention, implicating the cerebellum in the memory of a motor skill. During acquisition of the slow treadmill task at the 2 lowest slopes of inclination, PCD mutants spent more time walking than controls, an indication of a decreased ability of coordinating whole body movements. The same pattern of higher walking time on the slow treadmill in PCD mutants was evident during retention. These results indicate that the cerebellar cortex is involved in the acquisition and the retention of a task requiring equilibrium.

Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

PubMed Central

Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Goldstein, Lawrence S. B.

2008-01-01

Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment containing variable numbers of non-mutant, non-motor neurons. Despite high-level mutant expression within 100% of motor neurons and oligodendrocytes, in most of these chimeras, the presence of WT non-motor neurons substantially delayed onset of motor neuron degeneration, increasing disease-free life by 50%. Disease onset is therefore non-cell autonomous, and mutant SOD1 damage within cell types other than motor neurons and oligodendrocytes is a central contributor to initiation of motor neuron degeneration. PMID:18492803

Synergy between Prkdc and Trp53 regulates stem cell proliferation and GI-ARS after irradiation.

PubMed

Gurley, Kay E; Ashley, Amanda K; Moser, Russell D; Kemp, Christopher J

2017-11-01

Ionizing radiation (IR) is one of the most widely used treatments for cancer. However, acute damage to the gastrointestinal tract or gastrointestinal acute radiation syndrome (GI-ARS) is a major dose-limiting side effect, and the mechanisms that underlie this remain unclear. Here we use mouse models to explore the relative roles of DNA repair, apoptosis, and cell cycle arrest in radiation response. IR induces DNA double strand breaks and DNA-PK mutant Prkdc scid/scid mice are sensitive to GI-ARS due to an inability to repair these breaks. IR also activates the tumor suppressor p53 to trigger apoptotic cell death within intestinal crypt cells and p53 deficient mice are resistant to apoptosis. To determine if DNA-PK and p53 interact to govern radiosensitivity, we compared the response of single and compound mutant mice to 8 Gy IR. Compound mutant Prkdc scid/scid /Trp53 -/- mice died earliest due to severe GI-ARS. While both Prkdc scid/scid and Prkdc scid/scid /Trp53 -/- mutant mice had higher levels of IR-induced DNA damage, particularly within the stem cell compartment of the intestinal crypt, in Prkdc scid/scid /Trp53 -/- mice these damaged cells abnormally progressed through the cell cycle resulting in mitotic cell death. This led to a loss of Paneth cells and a failure to regenerate the differentiated epithelial cells required for intestinal function. IR-induced apoptosis did not correlate with radiosensitivity. Overall, these data reveal that DNA repair, mediated by DNA-PK, and cell cycle arrest, mediated by p53, cooperate to protect the stem cell niche after DNA damage, suggesting combination approaches to modulate both pathways may be beneficial to reduce GI-ARS. As many cancers harbor p53 mutations, this also suggests targeting DNA-PK may be effective to enhance sensitivity of p53 mutant tumors to radiation.

CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: A new mammalian circadian oscillator model including the cAMP module

NASA Astrophysics Data System (ADS)

Wang, Jun-Wei; Zhou, Tian-Shou

2009-12-01

In this paper, we develop a new mathematical model for the mammalian circadian clock, which incorporates both transcriptional/translational feedback loops (TTFLs) and a cAMP-mediated feedback loop. The model shows that TTFLs and cAMP signalling cooperatively drive the circadian rhythms. It reproduces typical experimental observations with qualitative similarities, e.g. circadian oscillations in constant darkness and entrainment to light-dark cycles. In addition, it can explain the phenotypes of cAMP-mutant and Rev-erbα-/--mutant mice, and help us make an experimentally-testable prediction: oscillations may be rescued when arrhythmic mice with constitutively low concentrations of cAMP are crossed with Rev-erbα-/- mutant mice. The model enhances our understanding of the mammalian circadian clockwork from the viewpoint of the entire cell.

A Novel Intergenic ETnII-β Insertion Mutation Causes Multiple Malformations in Polypodia Mice

PubMed Central

Lehoczky, Jessica A.; Thomas, Peedikayil E.; Patrie, Kevin M.; Owens, Kailey M.; Villarreal, Lisa M.; Galbraith, Kenneth; Washburn, Joe; Johnson, Craig N.; Gavino, Bryant; Borowsky, Alexander D.; Millen, Kathleen J.; Wakenight, Paul; Law, William; Van Keuren, Margaret L.; Gavrilina, Galina; Hughes, Elizabeth D.; Saunders, Thomas L.; Brihn, Lesil; Nadeau, Joseph H.; Innis, Jeffrey W.

2013-01-01

Mouse early transposon insertions are responsible for ∼10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5′ LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development. PMID:24339789

Targeted gene deletion of miRNAs in mice by TALEN system.

PubMed

Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

2013-01-01

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Interleukin-17 promotes development of castration-resistant prostate cancer potentially through creating an immunotolerant and pro-angiogenic tumor microenvironment

PubMed Central

Zhang, Qiuyang; Liu, Sen; Zhang, Qingsong; Xiong, Zhenggang; Wang, Alun R.; Myers, Leann; Melamed, Jonathan; Tang, Wendell W.; You, Zongbing

2014-01-01

BACKGROUND Interleukin-17 (IL-17) has been demonstrated to promote formation and growth of hormone-naïve prostate adenocarcinoma in mice. IL-17’s role in development of castration-resistant prostate cancer is unknown. In the present study, we investigated IL-17’s role in castration-resistant prostate cancer in a mouse model. METHODS IL-17 receptor C (IL-17RC) deficient mice were interbred with Pten conditional mutant mice to produce RC+ mice that maintained IL-17RC expression and RC− mice that were IL-17RC deficient. Male RC+ and RC− mice were Pten-null and were castrated at 16 weeks of age when invasive prostate cancer had already formed. At 30 weeks of age, all male mice were analyzed for the prostate phenotypes. RESULTS RC− mice displayed prostates that were smaller than RC+ mice. Approximately 23% of prostatic glands in RC− mice, in contrast to 65% of prostatic glands in RC+ mice, developed invasive adenocarcinomas. Compared to castrate RC+ mice, castrate RC− mouse prostate had lower rates of cellular proliferation and higher rates of apoptosis as well as lower levels of MMP7, YBX1, MTA1, and UBE2C proteins. In addition, castrate RC− mouse prostate had less angiogenesis, which was associated with decreased levels of COX-2 and VEGF. Moreover, castrate RC− mouse prostate had fewer inflammatory cells including lymphocytes, myeloid-derived suppressor cells, and macrophages. CONCLUSIONS Taken together, our findings suggest that IL-17 promotes development of invasive prostate adenocarcinomas under castrate conditions, potentially through creating an immunotolerant and pro-angiogenic tumor microenvironment. PMID:24691769

Conditional ablation of the Notch2 receptor in the ocular lens

PubMed Central

Saravanamuthu, Senthil S.; Le, Tien T.; Gao, Chun Y.; Cojocaru, Radu I.; Pandiyan, Pushpa; Liu, Chunqiao; Zhang, Jun; Zelenka, Peggy S.; Brown, Nadean L.

2011-01-01

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. PMID:22173065

Suppression of calbindin-D28k expression exacerbates SCA1 phenotype in a disease mouse model.

PubMed

Vig, Parminder J S; Wei, Jinrong; Shao, Qingmei; Lopez, Maripar E; Halperin, Rebecca; Gerber, Jill

2012-09-01

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder caused by the expansion of a polyglutamine tract in the mutant protein ataxin-1. The cerebellar Purkinje cells (PCs) are the major targets of mutant ataxin-1. The mechanism of PC death in SCA1 is not known; however, previous work indicates that downregulation of specific proteins involved in calcium homeostasis and signaling by mutant ataxin-1 is the probable cause of PC degeneration in SCA1. In this study, we explored if targeted deprivation of PC specific calcium-binding protein calbindin-D28k (CaB) exacerbates ataxin-1 mediated toxicity in SCA1 transgenic (Tg) mice. Using behavioral tests, we found that though both SCA1/+ and SCA1/+: CaB null (-/+) double mutants exhibited progressive impaired performance on the rotating rod, a simultaneous enhancement of exploratory activity, and absence of deficits in coordination, the double mutants were more severely impaired than SCA1/+ mice. With increasing age, SCA1/+ mice showed a progressive loss in the expression and localization of CaB and other PC specific calcium-binding and signaling proteins. In double mutants, these changes were more pronounced and had an earlier onset. Gene expression profiling of young mice exhibiting no behavior or biochemical deficits revealed a differential expression of many genes common to SCA1/+ and CaB-/+ lines, and unique to SCA1/+: CaB-/+ phenotype. Our study provides further evidence for a critical role of CaB in SCA1 pathogenesis, which may help identify new therapeutic targets to treat SCA1 or other cerebellar ataxias.

Activation of dopamine D3 receptors inhibits reward-related learning induced by cocaine.

PubMed

Kong, H; Kuang, W; Li, S; Xu, M

2011-03-10

Memories of learned associations between the rewarding properties of drugs and environmental cues contribute to craving and relapse in humans. The mesocorticolimbic dopamine (DA) system is involved in reward-related learning induced by drugs of abuse. DA D3 receptors are preferentially expressed in mesocorticolimbic DA projection areas. Genetic and pharmacological studies have shown that DA D3 receptors suppress locomotor-stimulant effects of cocaine and reinstatement of cocaine-seeking behaviors. Activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is also inhibited by D3 receptors. How D3 receptors modulate cocaine-induced reward-related learning and associated changes in cell signaling in reward circuits in the brain, however, have not been fully investigated. In the present study, we show that D3 receptor mutant mice exhibit potentiated acquisition of conditioned place preference (CPP) at low doses of cocaine compared to wild-type mice. Activation of ERK and CaMKIIα, but not the c-Jun N-terminal kinase and p38, in the nucleus accumbens, amygdala and prefrontal cortex is also potentiated in D3 receptor mutant mice compared to that in wild-type mice following CPP expression. These results support a model in which D3 receptors modulate reward-related learning induced by low doses of cocaine by inhibiting activation of ERK and CaMKIIα in reward circuits in the brain. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

β-1,2-Mannosyltransferases 1 and 3 Participate in Yeast and Hyphae O- and N-Linked Mannosylation and Alter Candida albicans Fitness During Infection.

PubMed

Courjol, Flavie; Jouault, Thierry; Mille, Céline; Hall, Rebecca; Maes, Emmanuel; Sendid, Boualem; Mallet, Jean Maurice; Guerardel, Yann; Gow, Neil A R; Poulain, Daniel; Fradin, Chantal

2015-09-01

β-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of β-1,2-oligomannosides (β-Mans) to phosphopeptidomannan and phospholipomannan. β-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor β-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with β-Mans but only at the nonreducing end of α-1,2-mannotriose. β-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 β-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for β-1,2-mannosylation of CWMPs and expression of β-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no β-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the β-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

Rac1 Dosage Is Crucial for Normal Endochondral Bone Growth.

PubMed

Suzuki, Dai; Bush, Jason R; Bryce, Dawn-Marie; Kamijo, Ryutaro; Beier, Frank

2017-10-01

Rac1, a member of the small Rho GTPase family, plays multiple cellular roles. Studies of mice conditionally lacking Rac1 have revealed essential roles for Rac1 in various tissues, including cartilage and limb mesenchyme, where Rac1 loss produces dwarfism and long bone shortening. To gain further insight into the role of Rac1 in skeletal development, we have used transgenic mouse lines to express a constitutively active (ca) Rac1 mutant protein in a Cre recombinase-dependent manner. Overexpression of caRac1 in limb bud mesenchyme or chondrocytes leads to reduced body weight and shorter bones compared with control mice. Histological analysis of growth plates showed that caRac1;Col2-Cre mice displayed ectopic hypertrophic chondrocytes in the proliferative zone and enlarged hypertrophic zones. These mice also displayed a reduced proportion of proliferating cell nuclear antigen-positive cells in the proliferative zone and nuclear β-catenin localization in the ectopic hypertrophic chondrocytes. Importantly, overexpression of caRac1 partially rescued the phenotypes of Rac1fl/fl;Col2-Cre and Rac1fl/fl;Prx1-Cre conditional knockout mice, including body weight, bone length, and growth plate disorganization. These results suggest that tight regulation of Rac1 activity is necessary for normal cartilage development. Copyright © 2017 Endocrine Society.

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice.

PubMed

Tseng, Hsiu-Ting; Park, Young Joo; Lee, Yoon Kwang; Moore, David D

2015-05-08

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp(-/-)) double mutant mice. Both ob/ob and double mutant ob/ob;Shp(-/-) mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp(-/-) mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp(-/-) liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp(-/-) mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp(-/-) mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes. Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.

Distinct Neurobehavioural Effects of Cannabidiol in Transmembrane Domain Neuregulin 1 Mutant Mice

PubMed Central

Long, Leonora E.; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S.; Arnold, Jonathon C.; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT2A receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABAA receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT2A binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes. PMID:22509273

Distinct neurobehavioural effects of cannabidiol in transmembrane domain neuregulin 1 mutant mice.

PubMed

Long, Leonora E; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S; Arnold, Jonathon C; Karl, Tim

2012-01-01

The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ(9)-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT(2A) receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABA(A) receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT(2A) binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes.

Neuronal-specific overexpression of a mutant valosin-containing protein associated with IBMPFD promotes aberrant ubiquitin and TDP-43 accumulation and cognitive dysfunction in transgenic mice.

PubMed

Rodriguez-Ortiz, Carlos J; Hoshino, Hitomi; Cheng, David; Liu-Yescevitz, Liqun; Blurton-Jones, Mathew; Wolozin, Benjamin; LaFerla, Frank M; Kitazawa, Masashi

2013-08-01

Mutations in valosin-containing protein (VCP) cause a rare, autosomal dominant disease called inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). One-third of patients with IBMPFD develop frontotemporal dementia, characterized by an extensive neurodegeneration in the frontal and temporal lobes. Neuropathologic hallmarks include nuclear and cytosolic inclusions positive to ubiquitin and transactive response DNA-binding protein 43 (TDP-43) in neurons and glial activation in affected regions. However, the pathogenic mechanisms by which mutant VCP triggers neurodegeneration remain unknown. Herein, we generated a mouse model selectively overexpressing a human mutant VCP in neurons to study pathogenic mechanisms of mutant VCP-mediated neurodegeneration and cognitive impairment. The overexpression of VCPA232E mutation in forebrain regions produced significant progressive impairments of cognitive function, including deficits in spatial memory, object recognition, and fear conditioning. Although overexpressed or endogenous VCP did not seem to focally aggregate inside neurons, TDP-43 and ubiquitin accumulated with age in transgenic mouse brains. TDP-43 was also found to co-localize with stress granules in the cytosolic compartment. Together with the appearance of high-molecular-weight TDP-43 in cytosolic fractions, these findings demonstrate the mislocalization and accumulation of abnormal TDP-43 in the cytosol of transgenic mice, which likely lead to an increase in cellular stress and cognitive impairment. Taken together, these results highlight an important pathologic link between VCP and cognition. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Genetic dissection of the role of cannabinoid type-1 receptors in the emotional consequences of repeated social stress in mice.

PubMed

Dubreucq, Sarah; Matias, Isabelle; Cardinal, Pierre; Häring, Martin; Lutz, Beat; Marsicano, Giovanni; Chaouloff, Francis

2012-07-01

The endocannabinoid system (ECS) tightly controls emotional responses to acute aversive stimuli. Repeated stress alters ECS activity but the role played by the ECS in the emotional consequences of repeated stress has not been investigated in detail. This study used social defeat stress, together with pharmacology and genetics to examine the role of cannabinoid type-1 (CB(1)) receptors on repeated stress-induced emotional alterations. Seven daily social defeat sessions increased water (but not food) intake, sucrose preference, anxiety, cued fear expression, and adrenal weight in C57BL/6N mice. The first and the last social stress sessions triggered immediate brain region-dependent changes in the concentrations of the principal endocannabinoids anandamide and 2-arachidonoylglycerol. Pretreatment before each of the seven stress sessions with the CB(1) receptor antagonist rimonabant prolonged freezing responses of stressed mice during cued fear recall tests. Repeated social stress abolished the increased fear expression displayed by constitutive CB(1) receptor-deficient mice. The use of mutant mice lacking CB(1) receptors from cortical glutamatergic neurons or from GABAergic neurons indicated that it is the absence of the former CB(1) receptor population that is responsible for the fear responses in socially stressed CB(1) mutant mice. In addition, stress-induced hypolocomotor reactivity was amplified by the absence of CB(1) receptors from GABAergic neurons. Mutant mice lacking CB(1) receptors from serotonergic neurons displayed a higher anxiety but decreased cued fear expression than their wild-type controls. These mutant mice failed to show social stress-elicited increased sucrose preference. This study shows that (i) release of endocannabinoids during stress exposure impedes stress-elicited amplification of cued fear behavior, (ii) social stress opposes the increased fear expression and delayed between-session extinction because of the absence of CB(1) receptors from cortical glutamatergic neurons, and (iii) CB(1) receptors on central serotonergic neurons are involved in the sweet consumption response to repeated stress.

Genetic Dissection of the Role of Cannabinoid Type-1 Receptors in the Emotional Consequences of Repeated Social Stress in Mice

PubMed Central

Dubreucq, Sarah; Matias, Isabelle; Cardinal, Pierre; Häring, Martin; Lutz, Beat; Marsicano, Giovanni; Chaouloff, Francis

2012-01-01

The endocannabinoid system (ECS) tightly controls emotional responses to acute aversive stimuli. Repeated stress alters ECS activity but the role played by the ECS in the emotional consequences of repeated stress has not been investigated in detail. This study used social defeat stress, together with pharmacology and genetics to examine the role of cannabinoid type-1 (CB1) receptors on repeated stress-induced emotional alterations. Seven daily social defeat sessions increased water (but not food) intake, sucrose preference, anxiety, cued fear expression, and adrenal weight in C57BL/6N mice. The first and the last social stress sessions triggered immediate brain region-dependent changes in the concentrations of the principal endocannabinoids anandamide and 2-arachidonoylglycerol. Pretreatment before each of the seven stress sessions with the CB1 receptor antagonist rimonabant prolonged freezing responses of stressed mice during cued fear recall tests. Repeated social stress abolished the increased fear expression displayed by constitutive CB1 receptor-deficient mice. The use of mutant mice lacking CB1 receptors from cortical glutamatergic neurons or from GABAergic neurons indicated that it is the absence of the former CB1 receptor population that is responsible for the fear responses in socially stressed CB1 mutant mice. In addition, stress-induced hypolocomotor reactivity was amplified by the absence of CB1 receptors from GABAergic neurons. Mutant mice lacking CB1 receptors from serotonergic neurons displayed a higher anxiety but decreased cued fear expression than their wild-type controls. These mutant mice failed to show social stress-elicited increased sucrose preference. This study shows that (i) release of endocannabinoids during stress exposure impedes stress-elicited amplification of cued fear behavior, (ii) social stress opposes the increased fear expression and delayed between-session extinction because of the absence of CB1 receptors from cortical glutamatergic neurons, and (iii) CB1 receptors on central serotonergic neurons are involved in the sweet consumption response to repeated stress. PMID:22434220

GRK5 deficiency leads to susceptibility to intermittent hypoxia-induced cognitive impairment.

PubMed

Singh, Prabhakar; Peng, Wei; Zhang, Qiang; Ding, XueFeng; Suo, William Z

2016-04-01

Obstructive sleep apnea (OSA) leads to cognitive impairment in about 25% patients, though it remains elusive what makes one more susceptible than the other to be cognitively impaired. G protein-coupled receptor kinase-5 (GRK5) deficiency is recently found to render subjects more susceptible to cognitive impairment triggered by over-expression of Swedish mutant ß-amyloid precursor protein. This study is to determine whether GRK5 deficiency also renders subjects more susceptible to the OSA-triggered cognitive impairment. Both wild type (WT) and GRK5 knockout (KO) mice were placed in conditions absence and presence of intermittent hypoxia (IH) with 8%/21% O2 90-s cycle for 8h a day for a month, and then followed by behavioral assessments with battery of tasks. We found that the selected IH condition only induced marginally abnormal behavior (slightly elevated anxiety with most others unchanged) in the WT mice but it caused significantly more behavioral deficits in the KO mice, ranging from elevated anxiety, impaired balancing coordination, and impaired short-term spatial memory. These results suggest that GRK5 deficiency indeed makes the mice more susceptible to wide range of behavioral impairments, including cognitive impairments. Published by Elsevier B.V.

The Lambda Select cII Mutation Detection System.

PubMed

Besaratinia, Ahmad; Tommasi, Stella

2018-04-26

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

CD18 deficiency improves liver injury in the MCD model of steatohepatitis

PubMed Central

Pierce, Andrew A.; Siao, Kevin; Mattis, Aras N.; Goodsell, Amanda; Baron, Jody L.; Maher, Jacquelyn J.

2017-01-01

Neutrophils and macrophages are important constituents of the hepatic inflammatory infiltrate in non-alcoholic steatohepatitis. These innate immune cells express CD18, an adhesion molecule that facilitates leukocyte activation. In the context of fatty liver, activation of infiltrated leukocytes is believed to enhance hepatocellular injury. The objective of this study was to determine the degree to which activated innate immune cells promote steatohepatitis by comparing hepatic outcomes in wild-type and CD18-mutant mice fed a methionine-choline-deficient (MCD) diet. After 3 weeks of MCD feeding, hepatocyte injury, based on serum ALT elevation, was 40% lower in CD18-mutant than wild-type mice. Leukocyte infiltration into the liver was not impaired in CD18-mutant mice, but leukocyte activation was markedly reduced, as shown by the lack of evidence of oxidant production. Despite having reduced hepatocellular injury, CD18-mutant mice developed significantly more hepatic steatosis than wild-type mice after MCD feeding. This coincided with greater hepatic induction of pro-inflammatory and lipogenic genes as well as a modest reduction in hepatic expression of adipose triglyceride lipase. Overall, the data indicate that CD18 deficiency curbs MCD-mediated liver injury by limiting the activation of innate immune cells in the liver without compromising intrahepatic cytokine activation. Reduced liver injury occurs at the expense of increased hepatic steatosis, which suggests that in addition to damaging hepatocytes, infiltrating leukocytes may influence lipid homeostasis in the liver. PMID:28873429

CD18 deficiency improves liver injury in the MCD model of steatohepatitis.

PubMed

Pierce, Andrew A; Duwaerts, Caroline C; Siao, Kevin; Mattis, Aras N; Goodsell, Amanda; Baron, Jody L; Maher, Jacquelyn J

2017-01-01

Neutrophils and macrophages are important constituents of the hepatic inflammatory infiltrate in non-alcoholic steatohepatitis. These innate immune cells express CD18, an adhesion molecule that facilitates leukocyte activation. In the context of fatty liver, activation of infiltrated leukocytes is believed to enhance hepatocellular injury. The objective of this study was to determine the degree to which activated innate immune cells promote steatohepatitis by comparing hepatic outcomes in wild-type and CD18-mutant mice fed a methionine-choline-deficient (MCD) diet. After 3 weeks of MCD feeding, hepatocyte injury, based on serum ALT elevation, was 40% lower in CD18-mutant than wild-type mice. Leukocyte infiltration into the liver was not impaired in CD18-mutant mice, but leukocyte activation was markedly reduced, as shown by the lack of evidence of oxidant production. Despite having reduced hepatocellular injury, CD18-mutant mice developed significantly more hepatic steatosis than wild-type mice after MCD feeding. This coincided with greater hepatic induction of pro-inflammatory and lipogenic genes as well as a modest reduction in hepatic expression of adipose triglyceride lipase. Overall, the data indicate that CD18 deficiency curbs MCD-mediated liver injury by limiting the activation of innate immune cells in the liver without compromising intrahepatic cytokine activation. Reduced liver injury occurs at the expense of increased hepatic steatosis, which suggests that in addition to damaging hepatocytes, infiltrating leukocytes may influence lipid homeostasis in the liver.

Sequence analysis of laci mutations obtained from lung cells of radon-exposed big blue{trademark} transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Layton, A.D.; Cross, F.T.; Steigler, G.L.

1994-12-31

We have exposed Big Blue{trademark} transgenic mice by inhalation to 320, 640 and 960 Working Level Months (WLM) of radon progeny. Mice were sacrificed after 3, 6 and 9 days; the time periods required to obtain the exposures. Control mice were also sacrificed at each time interval. In each case all tissues were excised, flash frozen in liquid nitrogen, and stored at -80{degrees}C for further analysis. Twelve lacI mutations have been isolated from the lung tissue of a mouse from the 960-WLM exposure group; the lacI genes from these mutants have been sequenced. Sequence data indicate that three of themore » mutants have a C;G deletion at BP 978 and are possibly clonal in origin. Two mutants have multiple events within the gene: one has a an A:T to C:G transversion and a C:G insertion separated by 291 BPs; the second has a G:C to A:T transition as well as an A:T deletion followed by 6 base pairs downstream by a T:A insertion. Other mutations include a single G:C to A:T transition, a two base pair deletion, and a C:G to T:A transition. Mutant plaques are being evaluated from individual mice at other dose levels. Time course experiments are also planned. These studies will help define the molecular fine structure of mutations induced by high-LET radiation exposure.« less

Further studies on cortical tangential migration in wild type and Pax-6 mutant mice.

PubMed

Jiménez, D; López-Mascaraque, L; de Carlos, J A; Valverde, F

2002-01-01

In this study we present new data concerning the tangential migration from the medial and lateral ganglionic eminences (MGE and LGE) to the cerebral cortex during development. We have used Calbindin as a useful marker to follow the itinerary of tangential migratory cells during early developmental stages in wild-type and Pax-6 homozygous mutant mice. In the wild-type mice, at early developmental stages, migrating cells advance through the intermediate zone (IZ) and preplate (PP). At more advanced stages, migrating cells were present in the subplate (SP) and cortical plate (CP) to reach the entire developing cerebral cortex. We found that, in the homozygous mutant mice (Pax-6(Sey-Neu)/Pax-6(Sey-Neu)), this tangential migration is severely affected at early developmental stages: migrating cells were absent in the IZ, which were only found some days later, suggesting that in the mutant mice, there is a temporal delay in tangential migration. We have also defined some possible mechanisms to explain certain migratory routes from the basal telencephalon to the cerebral cortex. We describe the existence of two factors, which we consider to be essential for the normal migration; the first one is the cell adhesion molecule PSA-NCAM, whose role in other migratory systems is well known. The second factor is Robo-2, whose expression delimits a channel for the passage of migratory cells from the basal telencephalon to the cerebral cortex.

Uterine Msx-1 and Wnt4 signaling becomes aberrant in mice with the loss of leukemia inhibitory factor or Hoxa-10: evidence for a novel cytokine-homeobox-Wnt signaling in implantation.

PubMed

Daikoku, Takiko; Song, Haengseok; Guo, Yong; Riesewijk, Anne; Mosselman, Sietse; Das, Sanjoy K; Dey, Sudhansu K

2004-05-01

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

PubMed

Sha, Jian; Agar, Stacy L; Baze, Wallace B; Olano, Juan P; Fadl, Amin A; Erova, Tatiana E; Wang, Shaofei; Foltz, Sheri M; Suarez, Giovanni; Motin, Vladimir L; Chauhan, Sadhana; Klimpel, Gary R; Peterson, Johnny W; Chopra, Ashok K

2008-04-01

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Natural human apoA-I mutations L141RPisa and L159RFIN alter HDL structure and functionality and promote atherosclerosis development in mice.

PubMed

Tiniakou, Ioanna; Kanaki, Zoi; Georgopoulos, Spiros; Chroni, Angeliki; Van Eck, Miranda; Fotakis, Panagiotis; Zannis, Vassilis I; Kardassis, Dimitris

2015-11-01

Mutations in human apolipoprotein A-I (apoA-I) are associated with low high-density lipoprotein (HDL) cholesterol levels and pathological conditions such as premature atherosclerosis and amyloidosis. In this study we functionally characterized two natural human apoA-I mutations, L141RPisa and L159RFIN, in vivo. We generated transgenic mice expressing either wild-type (WT) or the two mutant forms of human apoA-I on a mouse apoA-I(-/-) background and analyzed for abnormalities in their lipid and lipoprotein profiles. HDL structure and functionality, as well as atherosclerosis development following a 14-week high-fat diet were assessed in these mice. The expression of either apoA-I mutant was associated with markedly reduced serum apoA-I (<10% of WT apoA-I), total and HDL-cholesterol levels (∼20% and ∼7% of WT apoA-I, respectively) and the formation of few small size HDL particles with preβ2 and α3, α4 electrophoretic mobility. HDL particles containing either of the two apoA-I mutants exhibited attenuated anti-oxidative properties as indicated by their inability to prevent low-density lipoprotein oxidation, and by decreased activities of paraoxonase-1 and platelet-activating factor acetylhydrolase. However, the apoA-I(L141R)Pisa or apoA-I(L159R)FIN-containing HDL particles demonstrated increased capacity to promote ATP-Binding Cassette Transporter A1-mediated cholesterol efflux from macrophages. Expression of apoA-I(L141R)Pisa or apoA-I(L159R)FIN mutations in mice was associated with increased diet-induced atherosclerosis compared to either WT apoA-I transgenic or apoA-I(-/-) mice. These findings suggest that natural apoA-I mutations L141RPisa and L159RFIN affect the biogenesis and the functionality of HDL in vivo and predispose to diet-induced atherosclerosis in the absence of any other genetic defect. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Modeled changes of cerebellar activity in mutant mice are predictive of their learning impairments

NASA Astrophysics Data System (ADS)

Badura, Aleksandra; Clopath, Claudia; Schonewille, Martijn; de Zeeuw, Chris I.

2016-11-01

Translating neuronal activity to measurable behavioral changes has been a long-standing goal of systems neuroscience. Recently, we have developed a model of phase-reversal learning of the vestibulo-ocular reflex, a well-established, cerebellar-dependent task. The model, comprising both the cerebellar cortex and vestibular nuclei, reproduces behavioral data and accounts for the changes in neural activity during learning in wild type mice. Here, we used our model to predict Purkinje cell spiking as well as behavior before and after learning of five different lines of mutant mice with distinct cell-specific alterations of the cerebellar cortical circuitry. We tested these predictions by obtaining electrophysiological data depicting changes in neuronal spiking. We show that our data is largely consistent with the model predictions for simple spike modulation of Purkinje cells and concomitant behavioral learning in four of the mutants. In addition, our model accurately predicts a shift in simple spike activity in a mutant mouse with a brainstem specific mutation. This combination of electrophysiological and computational techniques opens a possibility of predicting behavioral impairments from neural activity.

Modeled changes of cerebellar activity in mutant mice are predictive of their learning impairments

PubMed Central

Badura, Aleksandra; Clopath, Claudia; Schonewille, Martijn; De Zeeuw, Chris I.

2016-01-01

Translating neuronal activity to measurable behavioral changes has been a long-standing goal of systems neuroscience. Recently, we have developed a model of phase-reversal learning of the vestibulo-ocular reflex, a well-established, cerebellar-dependent task. The model, comprising both the cerebellar cortex and vestibular nuclei, reproduces behavioral data and accounts for the changes in neural activity during learning in wild type mice. Here, we used our model to predict Purkinje cell spiking as well as behavior before and after learning of five different lines of mutant mice with distinct cell-specific alterations of the cerebellar cortical circuitry. We tested these predictions by obtaining electrophysiological data depicting changes in neuronal spiking. We show that our data is largely consistent with the model predictions for simple spike modulation of Purkinje cells and concomitant behavioral learning in four of the mutants. In addition, our model accurately predicts a shift in simple spike activity in a mutant mouse with a brainstem specific mutation. This combination of electrophysiological and computational techniques opens a possibility of predicting behavioral impairments from neural activity. PMID:27805050

Laser Interferometer Measurements of the Viscoelastic Properties of Tectorial Membrane Mutants

NASA Astrophysics Data System (ADS)

Jones, Gareth; Russell, Ian; Lukashkin, Andrei

2011-11-01

The visco-elastic properties of the tectorial membrane (TM) can be determined by measuring the propagation velocity of travelling waves over a range of frequencies. This study presents a new method using laser interferometry and compares the TM's material properties (sheer storage modulus, G' and viscosity, η) at basal and apical locations in wild-type mice and basal locations of three mutant groups (TectaY1870C/+, Tectb-/- and Otoa-/-). The G' and η values calculated for the wild-type mice are similar to estimates derived using other methods whereas the mutant groups all exhibit slower wave propagation velocities and reduced longitudinal coupling.

Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.

PubMed Central

Zuckermann, F A; Zsak, L; Mettenleiter, T C; Ben-Porat, T

1990-01-01

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to stimulate the secondary anti-PrV CTL response. Cultures of both murine or swine lymphocytes that had been stimulated with gIII- virus contained only approximately half as many lytic units as did those stimulated with either wild-type virus, a gX- virus mutant, or a gI-/gp63- virus mutant. Thus, a large proportion of the PrV-specific CTLs that are induced by immunization with PrV of both mice and pigs are directed against gIII. Furthermore, glycoproteins gI, gp63, and gX play at most a minor role in the CTL response of these animals to PrV. PMID:2153244

In vivo contribution of amino acid sulfur to cartilage proteoglycan sulfation

PubMed Central

Pecora, Fabio; Gualeni, Benedetta; Forlino, Antonella; Superti-Furga, Andrea; Tenni, Ruggero; Cetta, Giuseppe; Rossi, Antonio

2006-01-01

Cytoplasmic sulfate for sulfation reactions may be derived either from extracellular fluids or from catabolism of sulfur-containing amino acids and other thiols. In vitro studies have pointed out the potential relevance of sulfur-containing amino acids as sources for sulfation when extracellular sulfate concentration is low or when its transport is impaired such as in DTDST [DTD (diastrophic dysplasia) sulfate transporter] chondrodysplasias. In the present study, we have considered the contribution of cysteine and cysteine derivatives to in vivo macromolecular sulfation of cartilage by using the mouse model of DTD we have recently generated [Forlino, Piazza, Tiveron, Della Torre, Tatangelo, Bonafe, Gualeni, Romano, Pecora, Superti-Furga et al. (2005) Hum. Mol. Genet. 14, 859–871]. By intraperitoneal injection of [35S]cysteine in wild-type and mutant mice and determination of the specific activity of the chondroitin 4-sulfated disaccharide in cartilage, we demonstrated that the pathway by which sulfate is recruited from the intracellular oxidation of thiols is active in vivo. To check whether cysteine derivatives play a role, sulfation of cartilage proteoglycans was measured after treatment for 1 week of newborn mutant and wild-type mice with hypodermic NAC (N-acetyl-L-cysteine). The relative amount of sulfated disaccharides increased in mutant mice treated with NAC compared with the placebo group, indicating an increase in proteoglycan sulfation due to NAC catabolism, although pharmacokinetic studies demonstrated that the drug was rapidly removed from the bloodstream. In conclusion, cysteine contribution to cartilage proteoglycan sulfation in vivo is minimal under physiological conditions even if extracellular sulfate availability is low; however, the contribution of thiols to sulfation becomes significant by increasing their plasma concentration. PMID:16719839

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional β-Cell Mass in Mice.

PubMed

Fontés, Ghislaine; Ghislain, Julien; Benterki, Isma; Zarrouki, Bader; Trudel, Dominique; Berthiaume, Yves; Poitout, Vincent

2015-12-01

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal β-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced β-cell function. β-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic β-cell secretory defect but is associated with insulin resistance and a β-cell mass deficit in aging mutants. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Transcription Factor FoxO1 Is Essential for Enamel Biomineralization

PubMed Central

Poché, Ross A.; Sharma, Ramaswamy; Garcia, Monica D.; Wada, Aya M.; Nolte, Mark J.; Udan, Ryan S.; Paik, Ji-Hye; DePinho, Ronald A.; Bartlett, John D.; Dickinson, Mary E.

2012-01-01

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta. PMID:22291941

Competitive advantage of Borrelia burgdorferi with outer surface protein BBA03 during tick-mediated infection of the mammalian host.

PubMed

Bestor, Aaron; Rego, Ryan O M; Tilly, Kit; Rosa, Patricia A

2012-10-01

Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

Competitive Advantage of Borrelia burgdorferi with Outer Surface Protein BBA03 during Tick-Mediated Infection of the Mammalian Host

PubMed Central

Rego, Ryan O. M.; Tilly, Kit; Rosa, Patricia A.

2012-01-01

Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle. PMID:22851744

Chemical rescue of cleft palate and midline defects in conditional GSK-3beta mice.

PubMed

Liu, Karen J; Arron, Joseph R; Stankunas, Kryn; Crabtree, Gerald R; Longaker, Michael T

2007-03-01

Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

PubMed Central

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N.; Toole, Bryan P.; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A.

2009-01-01

Summary The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb. PMID:19633173

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

PubMed Central

Kajiwara, Chiaki; Kondo, Shiho; Uda, Shizuha; Dai, Lei; Ichiyanagi, Tomoko; Chiba, Tomoki; Ishido, Satoshi; Koji, Takehiko; Udono, Heiichiro

2012-01-01

Summary It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty. PMID:23213375

Plasmodium yoelii: induction of attenuated mutants by irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waki, S.; Yonome, I.; Suzuki, M.

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number ofmore » blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.« less

Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone

PubMed Central

Zhao, Liangliang; Hadziahmetovic, Majda; Wang, Chenguang; Xu, Xueying; Song, Ying; Jinnah, H.A.; Wodzinska, Jolanta; Iacovelli, Jared; Wolkow, Natalie; Krajacic, Predrag; Weissberger, Alyssa Cwanger; Connelly, John; Spino, Michael; Lee, Michael K.; Connor, James; Giasson, Benoit; Harris, Z. Leah; Dunaief, Joshua L.

2016-01-01

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. PMID:26303407

Induction of the Hajdu-Cheney Syndrome Mutation in CD19 B Cells in Mice Alters B-Cell Allocation but Not Skeletal Homeostasis.

PubMed

Yu, Jungeun; Zanotti, Stefano; Schilling, Lauren; Schoenherr, Chris; Economides, Aris N; Sanjay, Archana; Canalis, Ernesto

2018-06-01

Mice harboring Notch2 mutations replicating Hajdu-Cheney syndrome (Notch2 tm1.1ECan ) have osteopenia and exhibit an increase in splenic marginal zone B cells with a decrease in follicular B cells. Whether the altered B-cell allocation is responsible for the osteopenia of Notch2 tm1.1ECan mutants is unknown. To determine the effect of NOTCH2 activation in B cells on splenic B-cell allocation and skeletal phenotype, a conditional-by-inversion (COIN) Hajdu-Cheney syndrome allele of Notch2 (Notch2 [ΔPEST]COIN ) was used. Cre recombination generates a permanent Notch2 ΔPEST allele expressing a transcript for which sequences coding for the proline, glutamic acid, serine, and threonine-rich (PEST) domain are replaced by a stop codon. CD19-Cre drivers were backcrossed into Notch2 [ΔPEST]COIN/[ΔPEST]COIN to generate CD19-specific Notch2 ΔPEST/ΔPEST mutants and control Notch2 [ΔPEST]COIN/[ΔPEST]COIN littermates. There was an increase in marginal zone B cells and a decrease in follicular B cells in the spleen of CD19 Cre/WT ;Notch2 ΔPEST/ΔPEST mice, recapitulating the splenic phenotype of Notch2 tm1.1ECan mice. The effect was reproduced when the NOTCH1 intracellular domain was induced in CD19-expressing cells (CD19 Cre/WT ;Rosa Notch1/WT mice). However, neither CD19 Cre/WT ;Notch2 ΔPEST/ΔPEST nor CD19 Cre/WT ;Rosa Notch1/WT mice had a skeletal phenotype. Moreover, splenectomies in Notch2 tm1.1ECan mice did not reverse their osteopenic phenotype. In conclusion, Notch2 activation in CD19-expressing cells determines B-cell allocation in the spleen but has no skeletal consequences. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice.

PubMed

Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena; Pizzorusso, Tommaso

2017-06-15

CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60-80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. © The Author 2017. Published by Oxford University Press.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

PubMed

Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

2015-08-24

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Evidence for an Intrinsic Renal Tubular Defect in Mice with Genetic Hypophosphatemic Rickets

PubMed Central

Cowgill, Larry D.; Goldfarb, Stanley; Lau, Kai; Slatopolsky, Eduardo; Agus, Zalman S.

1979-01-01

To investigate the role of parathyroid hormone (PTH) and(or) an intrinsic renal tubular reabsorptive defect for phosphate in mice with hereditary hypophosphatemic rickets, we performed clearance and micropuncture studies in hypophosphatemic mutants and nonaffected littermate controls. Increased fractional excretion of phosphate in mutants (47.2±4 vs. 30.8±2% in controls) was associated with reduced fractional and absolute reabsorption in the proximal convoluted tubule and more distal sites. Acute thyropara-thyroidectomy (TPTX) increased phosphate reabsorption in both mutants and controls with a fall in fractional phosphate excretion to ≅7.5% in both groups indicating that PTH modified the degree of phosphaturia in the intact mutants. Absolute reabsorption in the proximal tubule and beyond remained reduced in the mutants, however, possibly because of the reduced filtered load. Serum PTH levels were the same in intact mutants and normals as was renal cortical adenylate cyclase activity both before and after PTH stimulation. To evaluate the possibility that the phosphate wasting was caused by an intrinsic tubular defect that was masked by TPTX, glomerular fluid phosphate concentration was raised by phosphate infusion in TPTX mutants to levels approaching those of control mice. Phosphate excretion rose markedly and fractional reabsorption fell, but there was no change in absolute phosphate reabsorption in either the proximal tubule or beyond, indicating a persistent reabsorptive defect in the absence of PTH. We conclude that hereditary hypophosphatemia in the mouse is associated with a renal tubular defect in phosphate reabsorption, which is independent of PTH and therefore represents a specific intrinsic abnormality of phosphate transport. PMID:221535

Moderate Light-Induced Degeneration of Rod Photoreceptors with Delayed Transducin Translocation in shaker1 Mice

PubMed Central

Zallocchi, Marisa; Wang, Wei-Min; Delimont, Duane; Cosgrove, Dominic

2011-01-01

Purpose. Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice. Methods. Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated. Results. In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months. Conclusions. These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light. PMID:21447681

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

DOE PAGES

Freudenberger Catanzaro, Kelly C.; Champion, Anna E.; Mohapatra, Nrusingh; ...

2017-05-30

Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. But, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locusmore » in F. tularensis. Following daily subculture of F. novicida in Chamberlain’s defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ11212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ11212–1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Thus, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.« less

Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freudenberger Catanzaro, Kelly C.; Champion, Anna E.; Mohapatra, Nrusingh

Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. But, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locusmore » in F. tularensis. Following daily subculture of F. novicida in Chamberlain’s defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ11212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ11212–1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Thus, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.« less

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

PubMed Central

Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

Dominant negative DISC1 mutant mice display specific social behaviour deficits and aberration in BDNF and cannabinoid receptor expression.

PubMed

Kaminitz, Ayelet; Barzilay, Ran; Segal, Hadar; Taler, Michal; Offen, Daniel; Gil-Ad, Irit; Mechoulam, Raphael; Weizman, Abraham

2014-01-01

OBJECTIVES. Disrupted in schizophrenia 1 (DISC1) is considered the most prominent candidate gene for schizophrenia. In this study, we aimed to characterize behavioural and brain biochemical traits in a mouse expressing a dominant negative DISC1mutant (DN-DISC1). DN-DISC1 mice underwent behavioural tests to evaluate object recognition, social preference and social novelty seeking. ELISA was conducted on brain tissue to evaluate BDNF levels. Western blot was employed to measure BDNF receptor (TrkB) and cannabinoid receptor CB1. The mutant DISC1 mice displayed deficits in preference to social novelty while both social preference and object recognition were intact. Biochemical analysis of prefrontal cortex and hippocampus revealed a modest reduction in cortical TrkB protein levels of male mice while no differences in BDNF levels were observed. We found sex dependent differences in the expression of cannabinoid-1 receptors. We describe novel behavioural and biochemical abnormalities in the DN-DISC1 mouse model of schizophrenia. The data shows for the first time a possible link between DISC1 mutation and the cannabinoid system.

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

PubMed Central

Pauper, Eva; Gregoire, Elodie P.; Klopfenstein, Muriel; de Rooij, Dirk G.; Mark, Manuel; Schedl, Andreas; Ghyselinck, Norbert B.; Chaboissier, Marie-Christine

2012-01-01

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1KOSox9cKO ), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1KOSox9cKO mice. Moreover, since testis development occurs in XY Rspo1KOSox9cKO mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9cKO mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised. PMID:23300469

Loss of Magel2 impairs the development of hypothalamic Anorexigenic circuits.

PubMed

Maillard, Julien; Park, Soyoung; Croizier, Sophie; Vanacker, Charlotte; Cook, Joshua H; Prevot, Vincent; Tauber, Maithe; Bouret, Sebastien G

2016-08-01

Prader-Willi syndrome (PWS) is a genetic disorder characterized by a variety of physiological and behavioral dysregulations, including hyperphagia, a condition that can lead to life-threatening obesity. Feeding behavior is a highly complex process with multiple feedback loops that involve both peripheral and central systems. The arcuate nucleus of the hypothalamus (ARH) is critical for the regulation of homeostatic processes including feeding, and this nucleus develops during neonatal life under of the influence of both environmental and genetic factors. Although much attention has focused on the metabolic and behavioral outcomes of PWS, an understanding of its effects on the development of hypothalamic circuits remains elusive. Here, we show that mice lacking Magel2, one of the genes responsible for the etiology of PWS, display an abnormal development of ARH axonal projections. Notably, the density of anorexigenic α-melanocyte-stimulating hormone axons was reduced in adult Magel2-null mice, while the density of orexigenic agouti-related peptide fibers in the mutant mice appeared identical to that in control mice. On the basis of previous findings showing a pivotal role for metabolic hormones in hypothalamic development, we also measured leptin and ghrelin levels in Magel2-null and control neonates and found that mutant mice have normal leptin and ghrelin levels. In vitro experiments show that Magel2 directly promotes axon growth. Together, these findings suggest that a loss of Magel2 leads to the disruption of hypothalamic feeding circuits, an effect that appears to be independent of the neurodevelopmental effects of leptin and ghrelin and likely involves a direct neurotrophic effect of Magel2. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

lbtA and lbtB Are Required for Production of the Legionella pneumophila Siderophore Legiobactin

PubMed Central

Allard, Kimberly A.; Viswanathan, V. K.; Cianciotto, Nicholas P.

2006-01-01

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2′,2′-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression. PMID:16452417

The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

PubMed

Orlow, S J; Brilliant, M H

1999-02-01

The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle. Copyright 1999 Academic Press.

Yersinia pestis with regulated delayed attenuation as a vaccine candidate to induce protective immunity against plague.

PubMed

Sun, Wei; Roland, Kenneth L; Kuang, Xiaoying; Branger, Christine G; Curtiss, Roy

2010-03-01

Two mutant strains of Yersinia pestis KIM5+, a Deltacrp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P(BAD) crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD(50)s) of the Deltacrp and araC P(BAD) crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5+, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 x 10(7) CFU of the Deltacrp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 x 10(4) CFU of the araC P(BAD) crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.

EphA4 is Involved in Sleep Regulation but Not in the Electrophysiological Response to Sleep Deprivation

PubMed Central

Freyburger, Marlène; Pierre, Audrey; Paquette, Gabrielle; Bélanger-Nelson, Erika; Bedont, Joseph; Gaudreault, Pierre-Olivier; Drolet, Guy; Laforest, Sylvie; Blackshaw, Seth; Cermakian, Nicolas; Doucet, Guy; Mongrain, Valérie

2016-01-01

Study Objectives: Optimal sleep is ensured by the interaction of circadian and homeostatic processes. Although synaptic plasticity seems to contribute to both processes, the specific players involved are not well understood. The EphA4 tyrosine kinase receptor is a cell adhesion protein regulating synaptic plasticity. We investigated the role of EphA4 in sleep regulation using electrocorticography in mice lacking EphA4 and gene expression measurements. Methods: EphA4 knockout (KO) mice, ClockΔ19/Δ19 mutant mice and littermates, C57BL/6J and CD-1 mice, and Sprague-Dawley rats were studied under a 12 h light: 12 h dark cycle, under undisturbed conditions or 6 h sleep deprivation (SLD), and submitted to a 48 h electrophysiological recording and/or brain sampling at different time of day. Results: EphA4 KO mice showed less rapid eye movement sleep (REMS), enhanced duration of individual bouts of wakefulness and nonrapid eye movement sleep (NREMS) during the light period, and a blunted daily rhythm of NREMS sigma activity. The NREMS delta activity response to SLD was unchanged in EphA4 KO mice. However, SLD increased EphA4 expression in the thalamic/hypothalamic region in C57BL/6J mice. We further show the presence of E-boxes in the promoter region of EphA4, a lower expression of EphA4 in Clock mutant mice, a rhythmic expression of EphA4 ligands in several brain areas, expression of EphA4 in the suprachiasmatic nuclei of the hypothalamus (SCN), and finally an unchanged number of cells expressing Vip, Grp and Avp in the SCN of EphA4 KO mice. Conclusions: Our results suggest that EphA4 is involved in circadian sleep regulation. Citation: Freyburger M, Pierre A, Paquette G, Bélanger-Nelson E, Bedont J, Gaudreault PO, Drolet G, Laforest S, Blackshaw S, Cermakian N, Doucet G, Mongrain V. EphA4 is involved in sleep regulation but not in the electrophysiological response to sleep deprivation. SLEEP 2016;39(3):613–624. PMID:26612390

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

PubMed

Carrión, Javier; Folgueira, Cristina; Soto, Manuel; Fresno, Manuel; Requena, Jose M

2011-07-27

Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

Minocycline reduces neuroinflammation but does not ameliorate neuron loss in a mouse model of neurodegeneration

PubMed Central

Cheng, Shanshan; Hou, Jinxing; Zhang, Chen; Xu, Congyu; Wang, Long; Zou, Xiaoxia; Yu, Huahong; Shi, Yun; Yin, Zhenyu; Chen, Guiquan

2015-01-01

Minocycline is a broad-spectrum tetracycline antibiotic. A number of preclinical studies have shown that minocycline exhibits neuroprotective effects in various animal models of neurological diseases. However, it remained unknown whether minocycline is effective to prevent neuron loss. To systematically evaluate its effects, minocycline was used to treat Dicer conditional knockout (cKO) mice which display age-related neuron loss. The drug was given to mutant mice prior to the occurrence of neuroinflammation and neurodegeneration, and the treatment had lasted 2 months. Levels of inflammation markers, including glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule1 (Iba1) and interleukin6 (IL6), were significantly reduced in minocycline-treated Dicer cKO mice. In contrast, levels of neuronal markers and the total number of apoptotic cells in Dicer cKO mice were not affected by the drug. In summary, inhibition of neuroinflammation by minocycline is insufficient to prevent neuron loss and apoptosis. PMID:26000566

Cognitive assessment of mice strains heterozygous for cell-adhesion genes reveals strain-specific alterations in timing.

PubMed

Gallistel, C R; Tucci, Valter; Nolan, Patrick M; Schachner, Melitta; Jakovcevski, Igor; Kheifets, Aaron; Barboza, Luendro

2014-03-05

We used a fully automated system for the behavioural measurement of physiologically meaningful properties of basic mechanisms of cognition to test two strains of heterozygous mutant mice, Bfc (batface) and L1, and their wild-type littermate controls. Both of the target genes are involved in the establishment and maintenance of synapses. We find that the Bfc heterozygotes show reduced precision in their representation of interval duration, whereas the L1 heterozygotes show increased precision. These effects are functionally specific, because many other measures made on the same mice are unaffected, namely: the accuracy of matching temporal investment ratios to income ratios in a matching protocol, the rate of instrumental and classical conditioning, the latency to initiate a cued instrumental response, the trials on task and the impulsivity in a switch paradigm, the accuracy with which mice adjust timed switches to changes in the temporal constraints, the days to acquisition, and mean onset time and onset variability in the circadian anticipation of food availability.

Cognitive assessment of mice strains heterozygous for cell-adhesion genes reveals strain-specific alterations in timing

PubMed Central

Gallistel, C. R.; Tucci, Valter; Nolan, Patrick M.; Schachner, Melitta; Jakovcevski, Igor; Kheifets, Aaron; Barboza, Luendro

2014-01-01

We used a fully automated system for the behavioural measurement of physiologically meaningful properties of basic mechanisms of cognition to test two strains of heterozygous mutant mice, Bfc (batface) and L1, and their wild-type littermate controls. Both of the target genes are involved in the establishment and maintenance of synapses. We find that the Bfc heterozygotes show reduced precision in their representation of interval duration, whereas the L1 heterozygotes show increased precision. These effects are functionally specific, because many other measures made on the same mice are unaffected, namely: the accuracy of matching temporal investment ratios to income ratios in a matching protocol, the rate of instrumental and classical conditioning, the latency to initiate a cued instrumental response, the trials on task and the impulsivity in a switch paradigm, the accuracy with which mice adjust timed switches to changes in the temporal constraints, the days to acquisition, and mean onset time and onset variability in the circadian anticipation of food availability. PMID:24446498

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model.

PubMed

Lin, Yi-Jiun; Huang, Li-Rung; Yang, Hung-Chih; Tzeng, Horng-Tay; Hsu, Ping-Ning; Wu, Hui-Lin; Chen, Pei-Jer; Chen, Ding-Shinn

2010-05-18

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20-30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176-185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-gamma response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.

Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice.

PubMed

Sakakibara, Yasufumi; Kasahara, Yoshiyuki; Hall, F Scott; Lesch, Klaus-Peter; Murphy, Dennis L; Uhl, George R; Sora, Ichiro

2014-10-01

A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the "short" allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT(+/-)) and homozygous (SERT(-/-)) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated. We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions. Adult SERT(-/-) mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT(-/-) mice. Conversely, SERT(+/-) and SERT(-/-) mice did display higher spontaneous alternation during early adolescence and adulthood. SERT(+/-) and SERT(-/-) also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT(-/-) mice also showed an increased spine density in the basolateral amygdala. Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence.

K-RasV14I recapitulates Noonan syndrome in mice

PubMed Central

Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

FUS/TLS acts as an aggregation-dependent modifier of polyglutamine disease model mice.

PubMed

Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Yamada, Mizuki; Doi, Hiroshi; Takumi, Toru; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Hicks, Geoffrey G; Hattori, Nobutaka; Shimogori, Tomomi; Nukina, Nobuyuki

2016-10-14

FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington's disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of (HD, but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS +/- double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

PubMed

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Genetic and pharmacological intervention of the p75NTR pathway alters morphological and behavioural recovery following traumatic brain injury in mice.

PubMed

Alder, Janet; Fujioka, Wendy; Giarratana, Anna; Wissocki, Jenna; Thakkar, Keya; Vuong, Phung; Patel, Bijal; Chakraborty, Trisha; Elsabeh, Rami; Parikh, Ankit; Girn, Hartaj S; Crockett, David; Thakker-Varia, Smita

2016-01-01

Neurotrophin levels are elevated after TBI, yet there is minimal regeneration. It was hypothesized that the pro-neurotrophin/p75NTR pathway is induced more than the mature neurotrophin/Trk pathway and that interfering with p75 signalling improves recovery following TBI. Lateral Fluid Percussion (LFP) injury was performed on wildtype and p75 mutant mice. In addition, TrkB agonist 7,8 Dihydroxyflavone or p75 antagonist TAT-Pep5 were tested. Western blot and immunohistochemistry revealed biochemical and cellular changes. Morris Water Maze and Rotarod tests demonstrated cognitive and vestibulomotor function. p75 was up-regulated and TrkB was down-regulated 1 day post-LFP. p75 mutant mice as well as mice treated with the p75 antagonist or the TrkB agonist exhibited reduced neuronal death and degeneration and less astrocytosis. The cells undergoing apoptosis appear to be neurons rather than glia. There was improved motor function and spatial learning in p75 mutant mice and mice treated with the p75 antagonist. Many of the pathological and behavioural consequences of TBI might be due to activation of the pro-neurotrophin/p75 toxic pathway overriding the protective mechanisms of the mature neurotrophin/Trk pathway. Targeting p75 can be a novel strategy to counteract the damaging effects of TBI.

IGFBP4 Is Required for Adipogenesis and Influences the Distribution of Adipose Depots.

PubMed

Maridas, David E; DeMambro, Victoria E; Le, Phuong T; Mohan, Subburaman; Rosen, Clifford J

2017-10-01

Insulinlike growth factor (IGF) I induces adipogenesis in vitro. IGF-binding protein 4 (IGFBP4) is highly expressed in adipocytes and osteoblasts and is inhibitory of IGFs in vitro. We previously reported that Igfbp4 null mice (Igfbp4-/-) had decreased fat proportions at 8 and 16 weeks of age. However, the mechanism leading to the reduced adiposity remains unknown. The purpose of this study was to elucidate how IGFBP4 mediates adipose tissue development in vivo. Our results showed that inguinal and gonadal white adipose tissue (gWAT) from Igfbp4-/- mice had decreased weights and Pparγ expression. Cultures of primary bone marrow stromal cells (BMSCs) and ear mesenchymal stem cells (eMSCs) from mutant mice showed reduced adipogenesis. Both BMSCs and eMSC had a strong induction of Igfbp4 expression during adipogenesis. Furthermore, the increase in phosphorylated Akt (p-Akt), a downstream target of IGF-I signaling, in wild-type cells, was blunted in mutant eMSCs. On a high-fat diet (HFD) there were sexual differences in adipocyte expansion of Igfbp4-/- mice. Mutant males gained weight by expanding their white fat depots. However, Igfbp4-/- female mice were protected against diet-induced obesity. Ovariectomized Igfbp4-/- female mice gained weight in a manner similar to that seen in ovariectomized controls. Thus, Igfbp4 is required for inguinal fat expansion in female mice but not in male mice. However, gWAT expansion, which is prevented by estrogen during HFD, does not require Igfbp4. Copyright © 2017 Endocrine Society.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

PubMed

Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Rajderkar, Sudha; Ray, Manas K; Mochida, Yoshiyuki; Allen, Benjamin; Lefebvre, Veronique; Hung, Irene H; Ornitz, David M; Kunieda, Tetsuo; Mishina, Yuji

2016-12-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

PubMed Central

Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

2015-01-01

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

Milk composition and lactation of beta-casein-deficient mice.

PubMed Central

Kumar, S; Clarke, A R; Hooper, M L; Horne, D S; Law, A J; Leaver, J; Springbett, A; Stevenson, E; Simons, J P

1994-01-01

beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition. Images PMID:8016126

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice

PubMed Central

Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Ray, Manas K.; Mochida, Yoshiyuki; Lefebvre, Veronique; Hung, Irene H.; Kunieda, Tetsuo; Mishina, Yuji

2016-01-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome. PMID:28027321

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

PubMed Central

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-01-01

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration. PMID:25950469

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice.

PubMed

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-05-07

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.

Interval timing in genetically modified mice: a simple paradigm

PubMed Central

Balci, F.; Papachristos, E. B.; Gallistel, C. R.; Brunner, D.; Gibson, J.; Shumyatsky, G. P.

2009-01-01

We describe a behavioral screen for the quantitative study of interval timing and interval memory in mice. Mice learn to switch from a short-latency feeding station to a long-latency station when the short latency has passed without a feeding. The psychometric function is the cumulative distribution of switch latencies. Its median measures timing accuracy and its interquartile interval measures timing precision. Next, using this behavioral paradigm, we have examined mice with a gene knockout of the receptor for gastrin-releasing peptide that show enhanced (i.e. prolonged) freezing in fear conditioning. We have tested the hypothesis that the mutants freeze longer because they are more uncertain than wild types about when to expect the electric shock. The knockouts however show normal accuracy and precision in timing, so we have rejected this alternative hypothesis. Last, we conduct the pharmacological validation of our behavioral screen using D-amphetamine and methamphetamine. We suggest including the analysis of interval timing and temporal memory in tests of genetically modified mice for learning and memory and argue that our paradigm allows this to be done simply and efficiently. PMID:17696995

Interval timing in genetically modified mice: a simple paradigm.

PubMed

Balci, F; Papachristos, E B; Gallistel, C R; Brunner, D; Gibson, J; Shumyatsky, G P

2008-04-01

We describe a behavioral screen for the quantitative study of interval timing and interval memory in mice. Mice learn to switch from a short-latency feeding station to a long-latency station when the short latency has passed without a feeding. The psychometric function is the cumulative distribution of switch latencies. Its median measures timing accuracy and its interquartile interval measures timing precision. Next, using this behavioral paradigm, we have examined mice with a gene knockout of the receptor for gastrin-releasing peptide that show enhanced (i.e. prolonged) freezing in fear conditioning. We have tested the hypothesis that the mutants freeze longer because they are more uncertain than wild types about when to expect the electric shock. The knockouts however show normal accuracy and precision in timing, so we have rejected this alternative hypothesis. Last, we conduct the pharmacological validation of our behavioral screen using d-amphetamine and methamphetamine. We suggest including the analysis of interval timing and temporal memory in tests of genetically modified mice for learning and memory and argue that our paradigm allows this to be done simply and efficiently.

Deficiency of Sbds in the mouse pancreas leads to features of Shwachman-Diamond syndrome, with loss of zymogen granules.

PubMed

Tourlakis, Marina E; Zhong, Jian; Gandhi, Rikesh; Zhang, Siyi; Chen, Lingling; Durie, Peter R; Rommens, Johanna M

2012-08-01

Shwachman-Diamond syndrome (SDS) is the second leading cause of hereditary exocrine pancreatic dysfunction. More than 90% of patients with SDS have biallelic loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene, which encodes a factor involved in ribosome function. We investigated whether mutations in Sbds lead to similar pancreatic defects in mice. Pancreas-specific knock-out mice were generated using a floxed Sbds allele and bred with mice carrying a null or disease-associated missense Sbds allele. Cre recombinase, regulated by the pancreatic transcription factor 1a promoter, was used to disrupt Sbds specifically in the pancreas. Models were assessed for pancreatic dysfunction and growth impairment. Disruption of Sbds in the mouse pancreas was sufficient to recapitulate SDS phenotypes. Pancreata of mice with Sbds mutations had decreased mass, fat infiltration, but general preservation of ductal and endocrine compartments. Pancreatic extracts from mutant mice had defects in formation of the 80S ribosomal complex. The exocrine compartment of mutant mice was hypoplastic and individual acini produced few zymogen granules. The null Sbds allele resulted in an earlier onset of phenotypes as well as endocrine impairment. Mutant mice had reduced serum levels of digestive enzymes and overall growth impairment. We developed a mouse model of SDS with pancreatic phenotypes similar to those of the human disease. This model could be used to investigate organ-specific consequences of Sbds-associated ribosomopathy. Sbds genotypes correlated with phenotypes. Defects developed specifically in the pancreata of mice, reducing growth of mice and production of digestive enzymes. SBDS therefore appears to be required for normal pancreatic development and function. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

PubMed Central

Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

2016-01-01

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

Comparative analysis of charged particle-induced autosomal mutations in murine cells and tissues

NASA Astrophysics Data System (ADS)

Kronenberg, Amy; Gauny, Stacey; Turker, Mitchell; Dan, Cristian; Kwoh, Ely

Carcinogenesis requires the accumulation of mutations and most of these mutations of occur on autosomes. This study seeks to determine the effect of the tissue microenvironment on the frequency and types of autosomal mutations in epithelial cells exposed to the types of charged particles in space radiation environments. Epithelial cells are the principal cells at risk for the development of solid cancers in humans. Aprt heterozygous mice from a cross between C57BL/6 and DBA/2 mice (B6D2F1) are used for these studies. The tissue of interest here is the kidney. We evaluated the effects of Fe ion on cytotoxicity, mutant frequency, and mutant spectra in kidney epithelium exposed in vivo. In vitro studies use primary kidney clones from B6D2F1 mice. Animals or cells were exposed to graded doses (0-2 Gy) of 1 GeV/amu Fe ions at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. Animals were given whole body exposure in plexiglas holders. Cells were irradiated in T-75 flasks as monolayers. Cytotoxicity for cells exposed as monolayers were performed immediately post-irradiation. In vitro mutation assays were performed after a 5-6 day expression period post-irradiation, at which time cells were seeded in standard medium supplemented with 2,6 diaminopurine to screen for Aprt mutants. Colony formation was assessed in parallel in standard medium. In contrast, mice were euthanized after 2-4 months post-irradiation (early) or 8-10 months post-irradiation (late) to determine the cytotoxic and mutagenic response to Fe ion irradiation. Once the kidneys were digested, the cytotoxicity and mutation assays were performed using the same methodology employed for cells in vitro. Individual Apr t mutant colonies were collected from separate flasks exposed in vitro to 2 Gy of Fe ions. A similar group of Aprt mutants were collected from separate, un-irradiated flasks Aprt mutant colonies were also collected from individual kidneys for un-irradiated mice and for mice exposed to 2 Gy of Fe ions. Mutant spectra were analyzed via PCR using a series of heterozygous markers along mouse chromosome 8. Cytotoxicity assays were performed immediately after Fe ion exposure of cells from two primary clones. Cells irradiated in vitro demonstrated a dose-dependent decrement in cloning efficiency with no evidence of a shoulder. The results demonstrate the two clones behave similarly (unpaired t-test, p>0.3) with a D0 of 84.3 cGy for the combined data set. Mutation data were obtained using cells from one of the primary clones. In three experiments, we observed a linear dose-response for Aprt mutation with an induced mutant frzction of 1.06 x 10-3 /Gy. Kidney epithelial cells irradiated in vivo and incubated for 2-4 months in situ prior to harvest also showed an exponential reduction of cloning efficiency. Cells harvested 8-10 months postirradiation showed evidence of recovery for doses up to 1.5 Gy, but there was no improvement in cloning efficiency for kidney cells exposed to 2 Gy Fe ions in vivo evaluated at the late time point. Results for Aprt mutation induction in vivo indicated considerable inter-animal variation within each dose group (0, 1.0, 1.5 and 2 Gy). Fe ion exposures were mutagenic to the kidney, even at the lowest dose (p<0.01). A comparison of the mutant frequency results at the two harvest times indicates that the dose response did not vary with incubation time in vivo. Analysis of the pooled data from the 2-4 months harvests and the 8-10 month harvests indicated an increase in mutant frequency of 1.49 fold per Gy (p=0.01, CI 1.11-2.01). Molecular analysis of Aprtdeficient cells collected after a 2 Gy exposure to Fe ions in vitro showed an increased proportion of mutants arising via interstitial deletion or mitotic recombination, with an indication of an increase in chromosome loss. Similar results have been obtained for Aprt mutants isolated from mice exposed to 2 Gy of Fe ions, as compared with mutants collected from sham-irradiated mice. Taken together, the results to date demonstrate that Fe ions are mutagenic to mouse kidney epithelium exposed in vitro assayed at short times post-irradiation and they are also mutagenic to kidney epithelium exposed in vivo. While cytotoxicity is somewhat ameliorated in vivo, toxicity was evident at the highest dose up to 8-10 months post-exposure. A comparison of the Aprt mutant frequency analyses (in vitro vs. in vivo) and mutation spectra analyses (in vitro vs. in vivo) reflect similar trends. Supported by NASA grant T-403X to A. Kronenberg.

Intact interval timing in circadian CLOCK mutants.

PubMed

Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

Mutant KRAS promotes malignant pleural effusion formation

PubMed Central

Αgalioti, Theodora; Giannou, Anastasios D.; Krontira, Anthi C.; Kanellakis, Nikolaos I.; Kati, Danai; Vreka, Malamati; Pepe, Mario; Spella, Μagda; Lilis, Ioannis; Zazara, Dimitra E.; Nikolouli, Eirini; Spiropoulou, Nikolitsa; Papadakis, Andreas; Papadia, Konstantina; Voulgaridis, Apostolos; Harokopos, Vaggelis; Stamou, Panagiota; Meiners, Silke; Eickelberg, Oliver; Snyder, Linda A.; Antimisiaris, Sophia G.; Kardamakis, Dimitrios; Psallidas, Ioannis; Μarazioti, Antonia; Stathopoulos, Georgios T.

2017-01-01

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition. PMID:28508873

Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress.

PubMed

Bale, T L; Contarino, A; Smith, G W; Chan, R; Gold, L H; Sawchenko, P E; Koob, G F; Vale, W W; Lee, K F

2000-04-01

Corticotropin-releasing hormone (Crh) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, Crh released from the paraventricular nucleus (PVN) of the hypothalamus activates Crh receptors on anterior pituitary corticotropes, resulting in release of adrenocorticotropic hormone (Acth) into the bloodstream. Acth in turn activates Acth receptors in the adrenal cortex to increase synthesis and release of glucocorticoids. The receptors for Crh, Crhr1 and Crhr2, are found throughout the central nervous system and periphery. Crh has a higher affinity for Crhr1 than for Crhr2, and urocortin (Ucn), a Crh-related peptide, is thought to be the endogenous ligand for Crhr2 because it binds with almost 40-fold higher affinity than does Crh. Crhr1 and Crhr2 share approximately 71% amino acid sequence similarity and are distinct in their localization within the brain and peripheral tissues. We generated mice deficient for Crhr2 to determine the physiological role of this receptor. Crhr2-mutant mice are hypersensitive to stress and display increased anxiety-like behaviour. Mutant mice have normal basal feeding and weight gain, but decreased food intake following food deprivation. Intravenous Ucn produces no effect on mean arterial pressure in the mutant mice.

The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice.

PubMed

Zaraket, Hassan; Bridges, Olga A; Russell, Charles J

2013-05-01

After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures

PubMed Central

Spieker, Janine; Mudersbach, Thomas; Vogel-Höpker, Astrid; Layer, Paul G.

2017-01-01

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation. PMID:28118357

Altered striatal function in a mutant mouse lacking D1A dopamine receptors.

PubMed Central

Drago, J; Gerfen, C R; Lachowicz, J E; Steiner, H; Hollon, T R; Love, P E; Ooi, G T; Grinberg, A; Lee, E J; Huang, S P

1994-01-01

Of the five known dopamine receptors, D1A and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei of the basal ganglia. Movement disorders, including Parkinson disease and various dystonias, are thought to result from imbalanced activity in these pathways. Dopamine regulates movement through its differential effects on D1A receptors expressed by direct output neurons and D2 receptors expressed by indirect output neurons. To further examine the interaction of D1A and D2 neuronal pathways in the striatum, we used homologous recombination to generate mutant mice lacking functional D1A receptors (D1A-/-). D1A-/- mutants are growth retarded and die shortly after weaning age unless their diet is supplemented with hydrated food. With such treatment the mice gain weight and survive to adulthood. Neurologically, D1A-/- mice exhibit normal coordination and locomotion, although they display a significant decrease in rearing behavior. Examination of the striatum revealed changes associated with the altered phenotype of these mutants. D1A receptor binding was absent in striatal sections from D1A-/- mice. Striatal neurons normally expressing functional D1A receptors are formed and persist in adult homozygous mutants. Moreover, substance P mRNA, which is colocalized specifically in striatal neurons with D1A receptors, is expressed at a reduced level. In contrast, levels of enkephalin mRNA, which is expressed in striatal neurons with D2 receptors, are unaffected. These findings show that D1A-/- mice exhibit selective functional alterations in the striatal neurons giving rise to the direct striatal output pathway. Images Fig. 2 Fig. 4 PMID:7809078

Activity of Gemifloxacin against Quinolone-Resistant Streptococcus pneumoniae Strains In Vitro and in a Mouse Pneumonia Model

PubMed Central

Azoulay-Dupuis, E.; Bédos, J. P.; Mohler, J.; Moine, P.; Cherbuliez, C.; Peytavin, G.; Fantin, B.; Köhler, T.

2005-01-01

Gemifloxacin is a novel fluoronaphthyridone quinolone with enhanced in vitro activity against Streptococcus pneumoniae. We investigated the activities of gemifloxacin and trovafloxacin, their abilities to select for resistance in vitro and in vivo, and their efficacies in a mouse model of acute pneumonia. Immunocompetent Swiss mice were infected with 105 CFU of a virulent, encapsulated S. pneumoniae strain, P-4241, or its isogenic parC, gyrA, parC gyrA, and efflux mutant derivatives (serotype 3); and leukopenic mice were infected with 107 CFU of two poorly virulent clinical strains (serotype 11A) carrying either a parE mutation or a parC, gyrA, and parE triple mutation. The drugs were administered six times every 12 h, starting at either 3 or 18 h postinfection. In vitro, gemifloxacin was the most potent agent against strains with and without acquired resistance to fluoroquinolones. While control mice died within 6 days, gemifloxacin at doses of 25 and 50 mg/kg of body weight was highly effective (survival rates, 90 to 100%) against the wild-type strain and against mutants harboring a single mutation, corresponding to area under the time-versus-serum concentration curve at 24 h (AUC24)/MIC ratios of 56.5 to 113, and provided a 40% survival rate against a mutant with a double mutation (parC and gyrA). A total AUC24/MIC ratio of 28.5 was associated with poor efficacy and the emergence of resistant mutants. Trovafloxacin was as effective as gemifloxacin against mutants with single mutations but did not provide any protection against the mutant with double mutations, despite treatment with a high dose of 200 mg/kg. Gemifloxacin preferentially selected for parC mutants both in vitro and in vivo. PMID:15728901

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides.

PubMed

Stokin, Gorazd B; Almenar-Queralt, Angels; Gunawardena, Shermali; Rodrigues, Elizabeth M; Falzone, Tomás; Kim, Jungsu; Lillo, Concepción; Mount, Stephanie L; Roberts, Elizabeth A; McGowan, Eileen; Williams, David S; Goldstein, Lawrence S B

2008-11-15

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.

Autism-Relevant Social Abnormalities and Cognitive Deficits in Engrailed-2 Knockout Mice

PubMed Central

Brielmaier, Jennifer; Matteson, Paul G.; Silverman, Jill L.; Senerth, Julia M.; Kelly, Samantha; Genestine, Matthieu; Millonig, James H.

2012-01-01

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders. PMID:22829897

Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.

PubMed

MacGrogan, Donal; D'Amato, Gaetano; Travisano, Stanislao; Martinez-Poveda, Beatriz; Luxán, Guillermo; Del Monte-Nieto, Gonzalo; Papoutsi, Tania; Sbroggio, Mauro; Bou, Vanesa; Gomez-Del Arco, Pablo; Gómez, Manuel Jose; Zhou, Bin; Redondo, Juan Miguel; Jiménez-Borreguero, Luis J; de la Pompa, José Luis

2016-05-13

The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. The aim of this study is to determine the functional specificity of Notch in valve development. Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function. © 2016 American Heart Association, Inc.

A Critical Period for Postnatal Adaptive Plasticity in a Model of Motor Axon Miswiring

PubMed Central

Castiblanco-Urbina, Maria A.; Winzeck, Stefan; Sundermeier, Julia; Theis, Fabian J.; Fouad, Karim; Huber, Andrea B.

2015-01-01

The correct wiring of neuronal circuits is of crucial importance for precise neuromuscular functionality. Therefore, guidance cues provide tight spatiotemporal control of axon growth and guidance. Mice lacking the guidance cue Semaphorin 3F (Sema3F) display very specific axon wiring deficits of motor neurons in the medial aspect of the lateral motor column (LMCm). While these deficits have been investigated extensively during embryonic development, it remained unclear how Sema3F mutant mice cope with these errors postnatally. We therefore investigated whether these animals provide a suitable model for the exploration of adaptive plasticity in a system of miswired neuronal circuitry. We show that the embryonically developed wiring deficits in Sema3F mutants persist until adulthood. As a consequence, these mutants display impairments in motor coordination that improve during normal postnatal development, but never reach wildtype levels. These improvements in motor coordination were boosted to wildtype levels by housing the animals in an enriched environment starting at birth. In contrast, a delayed start of enriched environment housing, at 4 weeks after birth, did not similarly affect motor performance of Sema3F mutants. These results, which are corroborated by neuroanatomical analyses, suggest a critical period for adaptive plasticity in neuromuscular circuitry. Interestingly, the formation of perineuronal nets, which are known to close the critical period for plastic changes in other systems, was not altered between the different housing groups. However, we found significant changes in the number of excitatory synapses on limb innervating motor neurons. Thus, we propose that during the early postnatal phase, when perineuronal nets have not yet been formed around spinal motor neurons, housing in enriched environment conditions induces adaptive plasticity in the motor system by the formation of additional synaptic contacts, in order to compensate for coordination deficits. PMID:25874621

CLOCKΔ19 mutation modifies the manner of synchrony among oscillation neurons in the suprachiasmatic nucleus.

PubMed

Sujino, Mitsugu; Asakawa, Takeshi; Nagano, Mamoru; Koinuma, Satoshi; Masumoto, Koh-Hei; Shigeyoshi, Yasufumi

2018-01-16

In mammals, the principal circadian oscillator exists in the hypothalamic suprachiasmatic nucleus (SCN). In the SCN, CLOCK works as an essential component of molecular circadian oscillation, and ClockΔ19 mutant mice show unique characteristics of circadian rhythms such as extended free running periods, amplitude attenuation, and high-magnitude phase-resetting responses. Here we investigated what modifications occur in the spatiotemporal organization of clock gene expression in the SCN of ClockΔ19 mutants. The cultured SCN, sampled from neonatal homozygous ClockΔ19 mice on an ICR strain comprising PERIOD2::LUCIFERASE, demonstrated that the Clock gene mutation not only extends the circadian period, but also affects the spatial phase and period distribution of circadian oscillations in the SCN. In addition, disruption of the synchronization among neurons markedly attenuated the amplitude of the circadian rhythm of individual oscillating neurons in the mutant SCN. Further, with numerical simulations based on the present studies, the findings suggested that, in the SCN of the ClockΔ19 mutant mice, stable oscillation was preserved by the interaction among oscillating neurons, and that the orderly phase and period distribution that makes a phase wave are dependent on the functionality of CLOCK.

The Role of Neuropeptide Y (NPY) in Uncontrolled Alcohol Drinking and Relapse Behavior Resulting from Exposure to Stressful Events

DTIC Science & Technology

2011-01-01

effects of stressors on excessive and uncontrolled drinking and relapse-like drinking. We proposed to use foot-shock as the stressor to elicit increase...did significantly increase deprivation-induced relapse-like drinking, and that this effect was more robust in mutant mice lacking the NPY Y1R. Thus...ethanol consumption using a models of excessive uncontrolled drinking, and mutant mice lacking NPY or the Y1R were more sensitive to the effects of

A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

PubMed

Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

2012-07-01

Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague. Published 2012. This article is a US Government work and is in the public domain in the USA.

Enhancement of DEN-induced liver tumorigenesis in heme oxygenase-1 G143H mutant transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Jianfeng; Wang, Dayong; Xiao, Haifeng

Heme oxygenase (HO) is the rate-limiting enzyme in heme metabolism. HO-1 exhibits anti-oxidative and anti-inflammatory function via the actions of its metabolite, respectively. A growing body of evidence demonstrates that HO-1 is implicated in the pathogenesis and progression of several types of cancer. However, whether HO-1 takes part in healthy-premalignant-malignant transformation is still undefined. In this study, we took advantage of transgenic mice which over-expressed HO-1 dominant negative mutant (HO-1 G143H) and observed its susceptibility to DEN-induced hepatocarcinogenesis. Our results indicate that HO-1 G143H mutant accelerates the progression of tumorigenesis and tumor growth. The mechanism is closely related to enhancementmore » of ROS production which induce more hepatocytes death and secretion of inflammatory cytokines, proliferation of surviving hepatocytes. Our result provides the direct evidence that HO-1 plays an important protective role in liver carcinogenesis. Alternatively, we suggest the possible explanation on effect of HO-1 promoter polymorphism which involved in tumorigenesis. - Highlights: • Enhancement of DEN-induced hepatocarcinogenesis in HO-1 G143H Tg mice. • HO-1G143H mutant enhanced DEN-induced ROS production and liver injury. • HO-1G143H mutant aggravated DEN-induced changes of inflammatory factors and cell proliferation.« less

A Mutant Receptor Tyrosine Phosphatase, CD148, Causes Defects in Vascular Development

PubMed Central

Takahashi, Takamune; Takahashi, Keiko; St. John, Patricia L.; Fleming, Paul A.; Tomemori, Takuya; Watanabe, Toshio; Abrahamson, Dale R.; Drake, Christopher J.; Shirasawa, Takuji; Daniel, Thomas O.

2003-01-01

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPη), participates in developmental vascularization. A mutant allele, CD148ΔCyGFP, was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions. PMID:12588999

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins

PubMed Central

Prasad, Tuhina; Weiner, Joshua A.

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γdel/del null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs. PMID:22275881

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins.

PubMed

Prasad, Tuhina; Weiner, Joshua A

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γ(del/del) null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs.

Mechanisms of motor recovery after subtotal spinal cord injury: insights from the study of mice carrying a mutation (WldS) that delays cellular responses to injury.

PubMed

Zhang, Z; Guth, L; Steward, O

1998-01-01

Partial lesions of the mammalian spinal cord result in an immediate motor impairment that recovers gradually over time; however, the cellular mechanisms responsible for the transient nature of this paralysis have not been defined. A unique opportunity to identify those injury-induced cellular responses that mediate the recovery of function has arisen from the discovery of a unique mutant strain of mice in which the onset of Wallerian degeneration is dramatically delayed. In this strain of mice (designated WldS for Wallerian degeneration, slow), many of the cellular responses to spinal cord injury are also delayed. We have used this experimental animal model to evaluate possible causal relationships between these delayed cellular responses and the onset of functional recovery. For this purpose, we have compared the time course of locomotor recovery in C57BL/6 (control) mice and in WldS (mutant) mice by hemisecting the spinal cord at T8 and evaluating locomotor function at daily postoperative intervals. The time course of locomotor recovery (as determined by the Tarlov open-field walking procedure) was substantially delayed in mice carrying the WldS mutation: C57BL/6 control mice began to stand and walk within 6 days (mean Tarlov score of 4), whereas mutant mice did not exhibit comparable locomotor function until 16 days postoperatively. (a) The rapid return of locomotor function in the C57BL/6 mice suggests that the recovery resulted from processes of functional plasticity rather than from regeneration or collateral sprouting of nerve fibers. (b) The marked delay in the return of locomotor function in WldS mice indicates that the processes of neuroplasticity are induced by degenerative changes in the damaged neurons. (c) These strains of mice can be effectively used in future studies to elucidate the specific biochemical and physiological alterations responsible for inducing functional plasticity and restoring locomotor function after spinal cord injury.

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

PubMed

Tajan, Mylène; Pernin-Grandjean, Julie; Beton, Nicolas; Gennero, Isabelle; Capilla, Florence; Neel, Benjamin G; Araki, Toshiyuki; Valet, Philippe; Tauber, Maithé; Salles, Jean-Pierre; Yart, Armelle; Edouard, Thomas

2018-04-12

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins.In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

PubMed Central

Perera, Nirma D.; Sheean, Rebecca K.; Lau, Chew L.; Shin, Yea Seul; Beart, Philip M.; Horne, Malcolm K.; Turner, Bradley J.

2018-01-01

ABSTRACT Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS. PMID:28980850

Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

PubMed

Chien, Li-Ting; Hartzell, H Criss

2008-11-01

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Test systems for measuring ocular parameters and visual function in mice.

PubMed

Schaeffel, Frank

2008-05-01

New techniques are described to measure refractive state, pupil responses, corneal curvature, ocular dimensions and spatial vision in mice. These variables are important for studies on myopia development in mice, but they are also valuable for phenotyping mouse mutants and for pharmacological studies.

Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABAA receptor

PubMed Central

Oh, I; Rau, V; Lor, C; Laha, KT; Jurd, R; Rudolph, U; Eger, EI; Pearce, RA

2015-01-01

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 [.proportional]M and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons). PMID:25680234

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

PubMed

Bandara, Aloka B; DeShazer, David; Inzana, Thomas J; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

2008-09-01

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

Strong sonic hedgehog signaling in the mouse ventral spinal cord is not required for oligodendrocyte precursor cell (OPC) generation but is necessary for correct timing of its generation.

PubMed

Hashimoto, Hirokazu; Jiang, Wen; Yoshimura, Takeshi; Moon, Kyeong-Hye; Bok, Jinwoong; Ikenaka, Kazuhiro

2017-11-06

In the mouse neural tube, sonic hedgehog (Shh) secreted from the floor plate (FP) and the notochord (NC) regulates ventral patterning of the neural tube, and later is essential for the generation of oligodendrocyte precursor cells (OPCs). During early development, the NC is adjacent to the neural tube and induces ventral domains in it, including the FP. In the later stage of development, during gliogenesis in the spinal cord, the pMN domain receives strong Shh signaling input. While this is considered to be essential for the generation of OPCs, the actual role of this strong input in OPC generation remains unclear. Here we studied OPC generation in bromi mutant mice which show abnormal ciliary structure. Shh signaling occurs within cilia and has been reported to be weak in bromi mutants. At E11.5, accumulation of Patched1 mRNA, a Shh signaling reporter, is observed in the pMN domain of wild type but not bromi mutants, whereas expression of Gli1 mRNA, another Shh reporter, disappeared. Thus, Shh signaling input to the pMN domain at E12.5 was reduced in bromi mutant mice. In these mutants, induction of the FP structure was delayed and its size was reduced compared to wild type mice. Furthermore, while the p3 and pMN domains were induced, the length of the Nkx2.2-positive region and the number of Olig2-positive cells decreased. The number of OPCs was also significantly decreased in the E12.5 and E14.5 bromi mutant spinal cord. In contrast, motor neuron (MN) production, detected by HB9 expression, significantly increased. It is likely that the transition from MN production to OPC generation in the pMN domain is impaired in bromi mutant mice. These results suggest that strong Shh input to the pMN domain is not required for OPC generation but is essential for producing a sufficient number of OPCs. Copyright © 2017 Elsevier Ltd. All rights reserved.

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

PubMed

Hirobe, Tomohisa; Ishikawa, Akira

2015-12-01

The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A mouse model for Costello syndrome reveals an Ang II–mediated hypertensive condition

PubMed Central

Schuhmacher, Alberto J.; Guerra, Carmen; Sauzeau, Vincent; Cañamero, Marta; Bustelo, Xosé R.; Barbacid, Mariano

2008-01-01

Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin–Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies. PMID:18483625

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid β-protein precursor abolishes its anti-thrombotic properties in vivo.

PubMed

Xu, Feng; Davis, Judianne; Hoos, Michael; Van Nostrand, William E

2017-07-01

Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid β-protein precursor (AβPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AβPP are abundant in brain. Regions of AβPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AβPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. To determine the extent of KPI function of AβPP in regulating cerebral thrombosis we generated a recombinant reactive center KPI R13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AβPP knock out and AβPP/KPI R13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. Recombinant mutant KPI R13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AβPP/KPI R13I mutant mice were similarly deficient as AβPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. We demonstrate that the anti-thrombotic function of AβPP primarily resides in the KPI activity of the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Disruption of insulin-like growth factor-II imprinting during embryonic development rescues the dwarf phenotype of mice null for pregnancy-associated plasma protein-A.

PubMed

Bale, Laurie K; Conover, Cheryl A

2005-08-01

Pregnancy-associated plasma protein-A (PAPP-A), an insulin-like growth factor-binding protein (IGFBP) protease, increases insulin-like growth factor (IGF) activity through cleavage of inhibitory IGFBP-4 and the consequent release of IGF peptide for receptor activation. Mice homozygous for targeted disruption of the PAPP-A gene are born as proportional dwarfs and exhibit retarded bone ossification during fetal development. Phenotype and in vitro data support a model in which decreased IGF-II bioavailability during embryogenesis results in growth retardation and reduction in overall body size. To test the hypothesis that an increase in IGF-II during embryogenesis would overcome the growth deficiencies, PAPP-A-null mice were crossed with DeltaH19 mutant mice, which have increased IGF-II expression and fetal overgrowth due to disruption of IgfII imprinting. DeltaH19 mutant mice were 126% and PAPP-A-null mice were 74% the size of controls at birth. These size differences were evident at embryonic day 16.5. Importantly, double mutants were indistinguishable from controls both in terms of size and skeletal development. Body size programmed during embryo development persisted post-natally. Thus, disruption of IgfII imprinting and consequent elevation in IGF-II during fetal development was associated with rescue of the dwarf phenotype and ossification defects of PAPP-A-null mice. These data provide strong genetic evidence that PAPP-A plays an essential role in determining IGF-II bioavailability for optimal fetal growth and development.

Late-onset of spinal neurodegeneration in knock-in mice expressing a mutant BiP.

PubMed

Jin, Hisayo; Mimura, Naoya; Kashio, Makiko; Koseki, Haruhiko; Aoe, Tomohiko

2014-01-01

Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

PubMed

Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

2016-08-18

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

PubMed Central

Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

2016-01-01

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

PubMed Central

Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

2014-01-01

Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

Atypical patterns of neural infection produced in mice by drug-resistant strains of herpes simplex virus.

PubMed

Field, H J; Anderson, J R; Wildy, P

1982-03-01

Mice inoculated intracerebrally (i.c.) with a mutant strain of HSV were found to develop cataracts 1 to 2 months after inoculation. Cataract formation was subsequently shown to follow an acute retinitis which commenced within 1 week of inoculation. The mutant had been selected for high resistance to the nucleoside analogue acyclovir and has been shown previously to be defective in the induction of thymidine kinase and also to express an altered DNA polymerase. The LD50 for mice inoculated i.c. was greater than 10(5) p.f.u. compared with approx 7 p.f.u. for the parental strain. Studies of virus replication following i.c. inoculation with a sublethal dose of the mutant revealed that only small amounts of infectious virus were produced in the brain, but during a period from 6 to 12 days after inoculation vigorous replication occurred in retinal tissue, producing very high titres of virus.

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice.

PubMed

Ulrich, Ricky L; Amemiya, Kei; Waag, David M; Roy, Chad J; DeShazer, David

2005-03-14

Burkholderia mallei is an obligate mammalian pathogen that causes the zoonotic disease glanders. Two live attenuated B. mallei strains, a capsule mutant and a branched-chain amino acid auxotroph, were evaluated for use as vaccines against aerosol-initiated glanders in mice. Animals were aerogenically vaccinated and serum samples were obtained before aerosol challenge with a high-dose (>300 times the LD50) of B. mallei ATCC 23344. Mice vaccinated with the capsule mutant developed a Th2-like Ig subclass antibody response and none survived beyond 5 days. In comparison, the auxotrophic mutant elicited a Th1-like Ig subclass antibody response and 25% of the animals survived for 1 month postchallenge. After a low-dose (5 times the LD50) aerosol challenge, the survival rates of auxotroph-vaccinated and unvaccinated animals were 50 and 0%, respectively. Thus, live attenuated strains that promote a Th1-like Ig response may serve as promising vaccine candidates against aerosol infection with B. mallei.

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging.

PubMed

Hall, Mary P; Woodroofe, Carolyn C; Wood, Monika G; Que, Ivo; Van't Root, Moniek; Ridwan, Yanto; Shi, Ce; Kirkland, Thomas A; Encell, Lance P; Wood, Keith V; Löwik, Clemens; Mezzanotte, Laura

2018-01-09

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

An Early Postnatal Oxytocin Treatment Prevents Social and Learning Deficits in Adult Mice Deficient for Magel2, a Gene Involved in Prader-Willi Syndrome and Autism.

PubMed

Meziane, Hamid; Schaller, Fabienne; Bauer, Sylvian; Villard, Claude; Matarazzo, Valery; Riet, Fabrice; Guillon, Gilles; Lafitte, Daniel; Desarmenien, Michel G; Tauber, Maithé; Muscatelli, Françoise

2015-07-15

Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. This study aimed to determine the behavioral phenotype of Magel2-deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. We assessed the social and cognitive behavior of Magel2-deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2-deficient pups has a curative effect. Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Hippocampal mutant APP and amyloid beta-induced cognitive decline, dendritic spine loss, defective autophagy, mitophagy and mitochondrial abnormalities in a mouse model of Alzheimer's disease.

PubMed

Manczak, Maria; Kandimalla, Ramesh; Yin, Xiangling; Reddy, P Hemachandra

2018-04-15

The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (Aβ) in 12-month-old APP transgenic mice. Using rotarod and Morris water maze tests, immunoblotting and immunofluorescence, Golgi-cox staining and transmission electron microscopy, we assessed cognitive behavior, protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and quantified dendritic spines and mitochondrial number and length in 12-month-old APP mice that express Swedish mutation. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial ATP. Morris water maze and rotarod tests revealed that hippocampal learning and memory and motor learning and coordination were impaired in APP mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Drp1 and Fis1 and decreased levels of fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 and TFAM), autophagy (ATG5 and LC3BI, LC3BII), mitophagy (PINK1 and TERT), synaptic (synaptophysin and PSD95) and dendritic (MAP2) proteins were found in 12-month-old APP mice relative to age-matched non-transgenic WT mice. Golgi-cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in APP mice. These findings suggest that hippocampal accumulation of mutant APP and Aβ is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins and reduced dendritic spines and hippocampal-based learning and memory impairments, and mitochondrial structural and functional changes in 12-month-old APP mice.

Glycosylation on Hemagglutinin Affects the Virulence and Pathogenicity of Pandemic H1N1/2009 Influenza A Virus in Mice

PubMed Central

Li, Yongtao; Bradley, Konrad C.; Cao, Jiyue; Chen, Huanchun; Jin, Meilin; Zhou, Hongbo

2013-01-01

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor. PMID:23637827

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R4496C/+) mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca(2+) handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca(2+) release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

Phenotype detection in morphological mutant mice using deformation features.

PubMed

Roy, Sharmili; Liang, Xi; Kitamoto, Asanobu; Tamura, Masaru; Shiroishi, Toshihiko; Brown, Michael S

2013-01-01

Large-scale global efforts are underway to knockout each of the approximately 25,000 mouse genes and interpret their roles in shaping the mammalian embryo. Given the tremendous amount of data generated by imaging mutated prenatal mice, high-throughput image analysis systems are inevitable to characterize mammalian development and diseases. Current state-of-the-art computational systems offer only differential volumetric analysis of pre-defined anatomical structures between various gene-knockout mice strains. For subtle anatomical phenotypes, embryo phenotyping still relies on the laborious histological techniques that are clearly unsuitable in such big data environment. This paper presents a system that automatically detects known phenotypes and assists in discovering novel phenotypes in muCT images of mutant mice. Deformation features obtained from non-linear registration of mutant embryo to a normal consensus average image are extracted and analyzed to compute phenotypic and candidate phenotypic areas. The presented system is evaluated using C57BL/10 embryo images. All cases of ventricular septum defect and polydactyly, well-known to be present in this strain, are successfully detected. The system predicts potential phenotypic areas in the liver that are under active histological evaluation for possible phenotype of this mouse line.

Behavioral Characterization of Knockin Mice with Mutations M287L and Q266I in the Glycine Receptor α1 Subunit

PubMed Central

Blednov, Yuri A.; Benavidez, Jill M.; Homanics, Gregg E.

2012-01-01

We used behavioral pharmacology to characterize heterozygous knockin mice with mutations (Q266I or M287L) in the α1 subunit of the glycine receptor (GlyR) (J Pharmacol Exp Ther 340:304–316, 2012). These mutations were designed to reduce (M287L) or eliminate (Q266I) ethanol potentiation of GlyR function. We asked which behavioral effects of ethanol would be reduced more in the Q266I mutant than the M287L and found rotarod ataxia to be the behavior that fulfilled this criterion. Compared with controls, the mutant mice also differed in ethanol consumption, ethanol-stimulated startle response, signs of acute physical dependence, and duration of loss of righting response produced by ethanol, butanol, ketamine, pentobarbital, and flurazepam. Some of these behavioral changes were mimicked in wild-type mice by acute injections of low, subconvulsive doses of strychnine. Both mutants showed increased acoustic startle response and increased sensitivity to strychnine seizures. Thus, in addition to reducing ethanol action on the GlyRs, these mutations reduced glycinergic inhibition, which may also alter sensitivity to GABAergic drugs. PMID:22037202

Improvement in motor performance of Weaver mutant mice following lesions of the cerebellum.

PubMed

Grüsser, C; Grüsser-Cornehls, U

1998-12-01

In Weaver mutants (B6CBA wv/wv) cerebellar granule cells degenerate almost completely postnatally. A partial loss of Purkinje cells (PC) and a degeneration of dopaminergic cells in the substantia nigra have also been found. Weaver mice suffer from striking motor symptoms, including difficulty in walking without toppling over. In an attempt to influence the poor motor performance, the cerebellum in young animals was removed, thus eliminating the faulty output of surviving PCs, demonstrated electrophysiologically. Unoperated Weaver, lesioned wildtypes and one sham mouse were used as controls. Before and after operation, a battery of behavioural tests was performed. In Weaver mice, tumbling to the side (t) and the relation of t to the motor activity (k) while traversing an open-field matrix, (t/k), improved considerably, as did manoeuvring on a slanted wire mesh, but keeping balance on a wooden bench did not to the same degree. Locomotor activity alone improved in some animals. In wildtypes no significant changes occurred after operation, with the exception of a strong reduction in locomotor activity. The experiments demonstrate that the motor performance in Weaver mutant mice benefits from removal of their cerebellum.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

PubMed Central

van Bueren, Kelly Lammerts; Papangeli, Irinna; Rochais, Francesca; Pearce, Kerra; Roberts, Catherine; Calmont, Amelie; Szumska, Dorota; Kelly, Robert G.; Bhattacharya, Shoumo; Scambler, Peter J.

2010-01-01

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures. PMID:20122914

Vezatin, an integral membrane protein of adherens junctions, is required for the sound resilience of cochlear hair cells

PubMed Central

Bahloul, Amel; Simmler, Marie-Christine; Michel, Vincent; Leibovici, Michel; Perfettini, Isabelle; Roux, Isabelle; Weil, Dominique; Nouaille, Sylvie; Zuo, Jian; Zadro, Cristina; Licastro, Danilo; Gasparini, Paolo; Avan, Paul; Hardelin, Jean-Pierre; Petit, Christine

2009-01-01

Loud sound exposure is a significant cause of hearing loss worldwide. We asked whether a lack of vezatin, an ubiquitous adherens junction protein, could result in noise-induced hearing loss. Conditional mutant mice bearing non-functional vezatin alleles only in the sensory cells of the inner ear (hair cells) indeed exhibited irreversible hearing loss after only one minute exposure to a 105 dB broadband sound. In addition, mutant mice spontaneously underwent late onset progressive hearing loss and vestibular dysfunction related to substantial hair cell death. We establish that vezatin is an integral membrane protein with two adjacent transmembrane domains, and cytoplasmic N- and C-terminal regions. Late recruitment of vezatin at junctions between MDCKII cells indicates that the protein does not play a role in the formation of junctions, but rather participates in their stability. Moreover, we show that vezatin directly interacts with radixin in its actin-binding conformation. Accordingly, we provide evidence that vezatin associates with actin filaments at cell–cell junctions. Our results emphasize the overlooked role of the junctions between hair cells and their supporting cells in the auditory epithelium resilience to sound trauma. PMID:20049712

Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

PubMed

Kimura, Wataru; Machii, Masashi; Xue, XiaoDong; Sultana, Nishat; Hikosaka, Keisuke; Sharkar, Mohammad T K; Uezato, Tadayoshi; Matsuda, Masashi; Koseki, Haruhiko; Miura, Naoyuki

2011-01-01

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development. Copyright © 2010 Wiley-Liss, Inc.

Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

PubMed Central

McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD. PMID:10454355

Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone beta1 receptor gene.

PubMed

McDonald, M P; Wong, R; Goldstein, G; Weintraub, B; Cheng, S Y; Crawley, J N

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor beta (TRbeta) gene on chromosome 3, representing a mutation of the ligand-binding domain of the TRbeta gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRbeta gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRbeta gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.

Overexpression of mutant HSP27 causes axonal neuropathy in mice.

PubMed

Lee, Jinho; Jung, Sung-Chul; Joo, Jaesoon; Choi, Yu-Ri; Moon, Hyo Won; Kwak, Geon; Yeo, Ha Kyung; Lee, Ji-Su; Ahn, Hye-Jee; Jung, Namhee; Hwang, Sunhee; Rheey, Jingeun; Woo, So-Youn; Kim, Ji Yon; Hong, Young Bin; Choi, Byung-Ok

2015-06-19

Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM.

PubMed

Lemieux, Maxime; Laflamme, Olivier D; Thiry, Louise; Boulanger-Piette, Antoine; Frenette, Jérôme; Bretzner, Frédéric

2016-03-01

Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM(2J) mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM(2J) mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM(2J) mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits. Copyright © 2016 the American Physiological Society.

Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection.

PubMed

Jyot, Jeevan; Balloy, Viviane; Jouvion, Gregory; Verma, Amrisha; Touqui, Lhousseine; Huerre, Michel; Chignard, Michel; Ramphal, Reuben

2011-05-15

The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research. Using a model of pneumonia in Toll-like receptor (TLR) 2,4(-/-) mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology. Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS(+) strains killed mice within a day, and the T2SS(+) strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS(+) strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria. The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.

Intact Interval Timing in Circadian CLOCK Mutants

PubMed Central

Cordes, Sara; Gallistel, C. R.

2008-01-01

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion–Induced Kidney Growth

PubMed Central

Wu, Huijuan; Chen, Jianchun; Xu, Jinxian; Dong, Zheng; Meyuhas, Oded

2016-01-01

The molecular mechanisms underlying renal growth and renal growth–induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys. PMID:26296742

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

PubMed

Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

2014-05-21

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

Brain state-dependent abnormal LFP activity in the auditory cortex of a schizophrenia mouse model

PubMed Central

Nakao, Kazuhito; Nakazawa, Kazu

2014-01-01

In schizophrenia, evoked 40-Hz auditory steady-state responses (ASSRs) are impaired, which reflects the sensory deficits in this disorder, and baseline spontaneous oscillatory activity also appears to be abnormal. It has been debated whether the evoked ASSR impairments are due to the possible increase in baseline power. GABAergic interneuron-specific NMDA receptor (NMDAR) hypofunction mutant mice mimic some behavioral and pathophysiological aspects of schizophrenia. To determine the presence and extent of sensory deficits in these mutant mice, we recorded spontaneous local field potential (LFP) activity and its click-train evoked ASSRs from primary auditory cortex of awake, head-restrained mice. Baseline spontaneous LFP power in the pre-stimulus period before application of the first click trains was augmented at a wide range of frequencies. However, when repetitive ASSR stimuli were presented every 20 s, averaged spontaneous LFP power amplitudes during the inter-ASSR stimulus intervals in the mutant mice became indistinguishable from the levels of control mice. Nonetheless, the evoked 40-Hz ASSR power and their phase locking to click trains were robustly impaired in the mutants, although the evoked 20-Hz ASSRs were also somewhat diminished. These results suggested that NMDAR hypofunction in cortical GABAergic neurons confers two brain state-dependent LFP abnormalities in the auditory cortex; (1) a broadband increase in spontaneous LFP power in the absence of external inputs, and (2) a robust deficit in the evoked ASSR power and its phase-locking despite of normal baseline LFP power magnitude during the repetitive auditory stimuli. The “paradoxically” high spontaneous LFP activity of the primary auditory cortex in the absence of external stimuli may possibly contribute to the emergence of schizophrenia-related aberrant auditory perception. PMID:25018691

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C.

PubMed

Peng, Chaohua; Zhu, Gaochun; Liu, Xiangqian; Li, He

2018-04-30

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1.

PubMed

Gopal, Pooja; Tasneen, Rokeya; Yee, Michelle; Lanoix, Jean-Philippe; Sarathy, Jansy; Rasic, George; Li, Liping; Dartois, Véronique; Nuermberger, Eric; Dick, Thomas

2017-07-14

Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

The cleft lip and palate defects in Dancer mutant mice result from gain of function of the Tbx10 gene

PubMed Central

Bush, Jeffrey O.; Lan, Yu; Jiang, Rulang

2004-01-01

Cleft lip and palate (CL/P) is a common disfiguring birth defect with complex, poorly understood etiology. Mice carrying a spontaneous mutation, Dancer (Dc), exhibit CL/P in homozygotes and show significantly increased susceptibility to CL/P in heterozygotes [Deol, M. S. & Lane, P. W. (1966) J. Embryol. Exp. Morphol. 16, 543–558 and Trasler, D. G., Kemp, D. & Trasler, T. A. (1984) Teratology 29, 101–104], providing an animal model for understanding the molecular pathogenesis of CL/P. We genetically mapped Dc to within a 1-cM region near the centromere of chromosome 19. In situ hybridization analysis showed that one positional candidate gene, Tbx10, is ectopically expressed in Dc mutant embryos. Positional cloning of the Dc locus revealed an insertion of a 3.3-kb sequence containing the 5′ region of the p23 gene into the first intron of Tbx10, which causes ectopic expression of a p23-Tbx10 chimeric transcript encoding a protein product identical to a normal variant of the Tbx10 protein. Furthermore, we show that ectopic expression of Tbx10 in transgenic mice recapitulates the Dc mutant phenotype, indicating that CL/Pin Dc mutant mice results from the p23 insertion-induced ectopic Tbx10 expression. These results identify gain of function of a T-box transcription factor gene as a mechanism underlying CL/P pathogenesis. PMID:15118109

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

PubMed Central

2011-01-01

Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis. PMID:21794145

Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model.

PubMed

Hollister, Kristin; Kusam, Saritha; Wu, Hao; Clegg, Ninah; Mondal, Arpita; Sawant, Deepali V; Dent, Alexander L

2013-10-01

The transcriptional repressor Bcl6 controls development of the follicular Th cell (T(FH)) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring T(FH) cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) T(FH) cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the T(FH) cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting T(FH) cell survival.

Zn II(atsm) is protective in amyotrophic lateral sclerosis model mice via a copper delivery mechanism.

PubMed

McAllum, Erin J; Roberts, Blaine R; Hickey, James L; Dang, Theresa N; Grubman, Alexandra; Donnelly, Paul S; Liddell, Jeffrey R; White, Anthony R; Crouch, Peter J

2015-09-01

Mutations in the metalloprotein Cu,Zn-superoxide dismutase (SOD1) cause approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease for which effective therapeutics do not yet exist. Transgenic rodent models based on over-expression of mutant SOD1 have been developed and these have provided opportunity to test new therapeutic strategies and to study the mechanisms of mutant SOD1 toxicity. Although the mechanisms of mutant SOD1 toxicity are yet to be fully elucidated, incorrect or incomplete metallation of SOD1 confers abnormal folding, aggregation and biochemical properties, and improving the metallation state of SOD1 provides a viable therapeutic option. The therapeutic effects of delivering copper (Cu) to mutant SOD1 have been demonstrated recently. The aim of the current study was to determine if delivery of zinc (Zn) to SOD1 was also therapeutic. To investigate this, SOD1G37R mice were treated with the metal complex diacetyl-bis(4-methylthiosemicarbazonato)zinc(II) [Zn(II)(atsm)]. Treatment resulted in an improvement in locomotor function and survival of the mice. However, biochemical analysis of spinal cord tissue collected from the mice revealed that the treatment did not increase overall Zn levels in the spinal cord nor the Zn content of SOD1. In contrast, overall levels of Cu in the spinal cord were elevated in the Zn(II)(atsm)-treated SOD1G37R mice and the Cu content of SOD1 was also elevated. Further experiments demonstrated transmetallation of Zn(II)(atsm) in the presence of Cu to form the Cu-analogue Cu(II)(atsm), indicating that the observed therapeutic effects for Zn(II)(atsm) in SOD1G37R mice may in fact be due to in vivo transmetallation and subsequent delivery of Cu. Copyright © 2015. Published by Elsevier Inc.

Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

PubMed Central

Murakami, Shunichi; Balmes, Gener; McKinney, Sandra; Zhang, Zhaoping; Givol, David; de Crombrugghe, Benoit

2004-01-01

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1. PMID:14871928

GSK-3β Function in Bone Regulates Skeletal Development, Whole-Body Metabolism, and Male Life Span

PubMed Central

Gillespie, J. R.; Bush, J. R.; Bell, G. I.; Aubrey, L. A.; Dupuis, H.; Ferron, M.; Kream, B.; DiMattia, G.; Patel, S.; Woodgett, J. R.; Karsenty, G.; Hess, D. A.; Beier, F.

2016-01-01

Glycogen synthase kinase 3 β (GSK-3β) is an essential negative regulator or “brake” on many anabolic-signaling pathways including Wnt and insulin. Global deletion of GSK-3β results in peri-natal lethality and various skeletal defects. The goal of our research was to determine GSK-3β cell-autonomous effects and postnatal roles in the skeleton. We used the 3.6-kb Col1a1 promoter to inactivate the Gsk3b gene (Col1a1-Gsk3b knockout) in skeletal cells. Mutant mice exhibit decreased body fat and postnatal bone growth, as well as delayed development of several skeletal elements. Surprisingly, the mutant mice display decreased circulating glucose and insulin levels despite normal expression of GSK-3β in metabolic tissues. We showed that these effects are due to an increase in global insulin sensitivity. Most of the male mutant mice died after weaning. Prior to death, blood glucose changed from low to high, suggesting a possible switch from insulin sensitivity to resistance. These male mice die with extremely large bladders that are preceded by damage to the urogenital tract, defects that are also seen type 2 diabetes. Our data suggest that skeletal-specific deletion of GSK-3β affects global metabolism and sensitizes male mice to developing type 2 diabetes. PMID:23904355

A Novel Interaction Between Aging and ER Overload in a Protein Conformational Dementia

PubMed Central

Schipanski, Angela; Lange, Sascha; Segref, Alexandra; Gutschmidt, Aljona; Lomas, David A.; Miranda, Elena; Schweizer, Michaela; Hoppe, Thorsten; Glatzel, Markus

2013-01-01

Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention. PMID:23335331

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

PubMed

Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

2014-04-01

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Vitamin C restores healthy aging in a mouse model for Werner syndrome

PubMed Central

Massip, Laurent; Garand, Chantal; Paquet, Eric R.; Cogger, Victoria C.; O’Reilly, Jennifer N.; Tworek, Leslee; Hatherell, Avril; Taylor, Carla G.; Thorin, Eric; Zahradka, Peter; Le Couteur, David G.; Lebel, Michel

2013-01-01

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-κB, as well as protein kinase Cδ and Hif-1α transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARα. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS. PMID:19741171

Deletion of Numb/Numblike in glutamatergic neurons leads to anxiety-like behavior in mice.

PubMed

Qian, Wenyu; Hong, Yang; Zhu, Minyan; Zhou, Liang; Li, Hongchang; Li, Huashun

2017-06-15

Endocytic adaptor protein Numb is the first identified cell fate determinant in Drosophila melanogaster. It has been implicated in Notch signaling pathway and regulation of neural stem cells proliferation in the central nervous system. Numb is also expressed in postmitotic neurons, in vitro studies showed that Numb is involved in neuronal morphologic development, such as neurite growth, axonal growth and spine development. However, in vivo functions of Numb in the postmitotic neurons are largely unknown. Here we show that deletion of Numb/Numblike in glutamatergic neurons causes anxiety-like behavior in mouse. In this study, we conditionally deleted Numb and its homologous gene Numblike in the glutamatergic neurons in dorsal forebrain, and thoroughly characterized the behavioral phenotypes of mutant mice. On a battery of tests for anxiety-like behavior, the conditional double knockout mice showed increased anxiety-like behavior on light/dark exploration and novel open field tests, but not on elevated zero maze tests. The conditional double knockout mice also displayed novelty induced hyperactivity in novel open field test. Control measures of general health, motor functions, startle response, sensorimotor gating, depression-related behaviors did not show differences between genotypes. Our present findings provide new insight into the indispensable functions of Numb/Numblike in the brain and behavior, and suggest that Numb/Numblike may play a role in mediating neuronal functions that underlie behaviors related to anxiety. Copyright © 2017. Published by Elsevier B.V.

Muscle spindle feedback directs locomotor recovery and circuit reorganization after spinal cord injury.

PubMed

Takeoka, Aya; Vollenweider, Isabel; Courtine, Grégoire; Arber, Silvia

2014-12-18

Spinal cord injuries alter motor function by disconnecting neural circuits above and below the lesion, rendering sensory inputs a primary source of direct external drive to neuronal networks caudal to the injury. Here, we studied mice lacking functional muscle spindle feedback to determine the role of this sensory channel in gait control and locomotor recovery after spinal cord injury. High-resolution kinematic analysis of intact mutant mice revealed proficient execution in basic locomotor tasks but poor performance in a precision task. After injury, wild-type mice spontaneously recovered basic locomotor function, whereas mice with deficient muscle spindle feedback failed to regain control over the hindlimb on the lesioned side. Virus-mediated tracing demonstrated that mutant mice exhibit defective rearrangements of descending circuits projecting to deprived spinal segments during recovery. Our findings reveal an essential role for muscle spindle feedback in directing basic locomotor recovery and facilitating circuit reorganization after spinal cord injury. Copyright © 2014 Elsevier Inc. All rights reserved.