Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini
In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging.
Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini
In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738
McLaughlin, Precious J; Bakall, Benjamin; Choi, Jiwon; Liu, Zhonglin; Sasaki, Takako; Davis, Elaine C; Marmorstein, Alan D; Marmorstein, Lihua Y
A mutation in the EFEMP1 gene causes Malattia Leventinese, an inherited macular degenerative disease with strong similarities to age-related macular degeneration. EFEMP1 encodes fibulin-3, an extracellular matrix protein of unknown function. To investigate its biological role, the murine Efemp1 gene was inactivated through targeted disruption. Efemp1(-/-) mice exhibited reduced reproductivity, and displayed an early onset of aging-associated phenotypes including reduced lifespan, decreased body mass, lordokyphosis, reduced hair growth, and generalized fat, muscle and organ atrophy. However, these mice appeared to have normal wound healing ability. Efemp1(-/-) mice on a C57BL/6 genetic background developed multiple large hernias including inguinal hernias, pelvic prolapse and protrusions of the xiphoid process. In contrast, Efemp1(-/-) mice on a BALB/c background rarely had any forms of hernias, indicating the presence of modifiers for fibulin-3's function in different mouse strains. Histological analysis revealed a marked reduction of elastic fibers in fascia, a thin layer of connective tissue maintaining and protecting structures throughout the body. No apparent macular degeneration associated defects were found in Efemp1(-/-) mice, suggesting that loss of fibulin-3 function is not the mechanism by which the mutation in EFEMP1 causes macular degeneration. These data demonstrate that fibulin-3 plays an important role in maintaining the integrity of fascia connective tissues and regulates aging.
Kim, Youngsoo; Kim, Seong Hun; Kim, Kook Hwan; Chae, Sujin; Kim, Chanki; Kim, Jeongjin; Shin, Hee-Sup; Lee, Myung-Shik; Kim, Daesoo
Really interesting new gene (RING) finger protein 170 (RNF170) is an E3 ubiquitin ligase known to mediate ubiquitination-dependent degradation of type-I inositol 1,4,5-trisphosphate receptors (ITPR1). It has recently been demonstrated that a point mutation of RNF170 gene is linked with autosomal-dominant sensory ataxia (ADSA), which is characterized by an age-dependent increase of walking abnormalities, a rare genetic disorder reported in only two families. Although this mutant allele is known to be dominant, the functional identity thereof has not been clearly established. Here, we generated mice lacking Rnf170 (Rnf170(-/-)) to evaluate the effect of its loss of function in vivo. Remarkably, Rnf170(-/-) mice began to develop gait abnormalities in old age (12 months) in the form of asynchronous stepping between diagonal limb pairs with a fixed step sequence during locomotion, while age-matched wild-type mice showed stable gait patterns using several step sequence repertoires. As reported in ADSA patients, they also showed a reduced sensitivity for proprioception and thermal nociception. Protein blot analysis revealed that the amount of Itpr1 protein was significantly elevated in the cerebellum and spinal cord but intact in the cerebral cortex in Rnf170(-/-) mice. These results suggest that the loss of Rnf170 gene function mediates ADSA-associated phenotypes and this gives insights on the cure of patients with ADSA and other age-dependent walking abnormalities.
Gu, Changgui; Coomans, Claudia P.; Hu, Kun; Scheer, Frank A. J. L.; Stanley, H. Eugene; Meijer, Johanna H.
In healthy humans and other animals, behavioral activity exhibits scale invariance over multiple timescales from minutes to 24 h, whereas in aging or diseased conditions, scale invariance is usually reduced significantly. Accordingly, scale invariance can be a potential marker for health. Given compelling indications that exercise is beneficial for mental and physical health, we tested to what extent a lack of exercise affects scale invariance in young and aged animals. We studied six or more mice in each of four age groups (0.5, 1, 1.5, and 2 y) and observed an age-related deterioration of scale invariance in activity fluctuations. We found that limiting the amount of exercise, by removing the running wheels, leads to loss of scale-invariant properties in all age groups. Remarkably, in both young and old animals a lack of exercise reduced the scale invariance in activity fluctuations to the same level. We next showed that scale invariance can be restored by returning the running wheels. Exercise during the active period also improved scale invariance during the resting period, suggesting that activity during the active phase may also be beneficial for the resting phase. Finally, our data showed that exercise had a stronger influence on scale invariance than the effect of age. The data suggest that exercise is beneficial as revealed by scale-invariant parameters and that, even in young animals, a lack of exercise leads to strong deterioration in these parameters. PMID:25675516
Jha, S; Dong, B E; Xue, Y; Delotterie, D F; Vail, M G; Sakata, K
Reduced promoter IV-driven expression of brain-derived neurotrophic factor (BDNF) is implicated in stress and major depression. We previously reported that defective promoter IV (KIV) caused depression-like behavior in young adult mice, which was reversed more effectively by enriched environment treatment (EET) than antidepressants. The effects of promoter IV-BDNF deficiency and EET over the life stages remain unknown. Since early-life development (ED) involves dynamic epigenetic processes, we hypothesized that EET during ED would provide maximum antidepressive effects that would persist later in life due to enhanced, long-lasting BDNF induction. We tested this hypothesis by determining EET effects across three life stages: ED (0–2 months), young adult (2–4 months), and old adult (12–14 months). KIV mice at all life stages showed depression-like behavior in the open-field and tail-suspension tests compared with wild-type mice. Two months of EET reduced depression-like behavior in ED and young adult, but not old adult mice, with the largest effect in ED KIV mice. This effect lasted for 1 month after discontinuance of EET only in ED mice. BDNF protein induction by EET in the hippocampus and frontal cortex was also the largest in ED mice and persisted only in the hippocampus of ED KIV mice after discontinuance of EET. No gender-specific effects were observed. The results suggest that defective promoter IV causes depression-like behavior, regardless of age and gender, and that EET during ED is particularly beneficial to individuals with promoter IV-BDNF deficiency, while additional treatment may be needed for older adults. PMID:27648918
Ng, Lily; Cordas, Emily; Wu, Xuefeng; Vella, Kristen R.; Hollenberg, Anthony N.
A key function of the thyroid hormone receptor β (Thrb) gene is in the development of auditory function. However, the roles of the 2 receptor isoforms, TRβ1 and TRβ2, expressed by the Thrb gene are unclear, and it is unknown whether these isoforms promote the maintenance as well as development of hearing. We investigated the function of TRβ1 in mice with a Thrbb1 reporter allele that expresses β-galactosidase instead of TRβ1. In the immature cochlea, β-galactosidase was detected in the greater epithelial ridge, sensory hair cells, spiral ligament, and spiral ganglion and in adulthood, at low levels in the hair cells, support cells and root cells of the outer sulcus. Although deletion of all TRβ isoforms causes severe, early-onset deafness, deletion of TRβ1 or TRβ2 individually caused no obvious hearing loss in juvenile mice. However, over subsequent months, TRβ1 deficiency resulted in progressive loss of hearing and loss of hair cells. TRβ1-deficient mice had minimal changes in serum thyroid hormone and thyrotropin levels, indicating that hormonal imbalances were unlikely to cause hearing loss. The results suggest mutually shared roles for TRβ1 and TRβ2 in cochlear development and an unexpected requirement for TRβ1 in the maintenance of hearing in adulthood. PMID:26241124
Feliszek, Monika; Bindila, Laura; Lutz, Beat; Zimmer, Andreas; Bilkei-Gorzo, Andras; Schlicker, Eberhard
Activation of cannabinoid CB1 receptors may offer new therapeutic strategies, but the efficiency of CB1 receptor agonists may be impaired by tolerance development upon prolonged administration. We compared the influence of repeated administration of Δ(9)-tetrahydrocannabinol (THC) 10 mg/kg on the motility and on basal and CB1 receptor-stimulated (35)S-GTPγS binding of adolescent and aged mice. Moreover, we determined the influence of JZL 184 (which inhibits the 2-arachidonoylglycerol, 2-AG, degrading enzyme monoacylglycerol lipase, MAGL) on (35)S-GTPγS binding and 2-AG levels of young adult mice. Mouse motility was tested in the open field. (35)S-GTPγS binding was studied in hippocampal membranes. THC and CP 55,940 were used as cannabinoid agonists in the behavioural and biochemical studies, respectively. 2-AG levels were quantified by liquid chromatography-multiple reaction monitoring. The THC (10 mg/kg)-induced hypomotility was stronger in untreated than in THC-pretreated adolescent mice but similar in both treatment groups of aged mice. Basal and stimulated (35)S-GTPγS binding was decreased in membranes from THC-pretreated adolescent but not affected in membranes from aged mice. Treatment of young adult mice with JZL 184 (4, 10 and 40 mg/kg) for 14 days did not affect basal binding. Stimulated binding tended to be decreased by 25 % only in mice treated with JZL 184 (40 mg/kg). Hippocampal 2-AG level was increased by JZL 184 at 40 and 10 but not affected at 4 mg/kg. In conclusion, CB1 receptor tolerance does not occur in aged mice pretreated with THC and in young adult mice treated with a low dose of the MAGL inhibitor JZL 184.
Lind, Thomas; Gustafson, Ann-Marie; Calounova, Gabriela; Hu, Lijuan; Rasmusson, Annica; Jonsson, Kenneth B.; Wernersson, Sara; Åbrink, Magnus; Andersson, Göran; Larsson, Sune; Melhus, Håkan; Pejler, Gunnar
Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted. PMID:27936149
Godwin, A R; Capecchi, M R
Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development.
Miyado, Mami; Miyado, Kenji; Katsumi, Momori; Saito, Kazuki; Nakamura, Akihiro; Shihara, Daizou; Ogata, Tsutomu; Fukami, Maki
In mice, the onset of parturition is triggered by a rapid decline in circulating progesterone. Progesterone withdrawal occurs as a result of functional luteolysis, which is characterized by an increase in the enzymatic activity of 20α-hydroxysteroid dehydrogenase (20α-HSD) in the corpus luteum and is mediated by the prostaglandin F2α (PGF2α) signaling. Here, we report that the genetic knockout (KO) of Mamld1, which encodes a putative non-DNA-binding regulator of testicular steroidogenesis, caused defective functional luteolysis and subsequent parturition failure and neonatal deaths. Progesterone receptor inhibition induced the onset of parturition in pregnant KO mice, and MAMLD1 regulated the expression of Akr1c18, the gene encoding 20α-HSD, in cultured cells. Ovaries of KO mice at late gestation were morphologically unremarkable; however, Akr1c18 expression was reduced and expression of its suppressor Stat5b was markedly increased. Several other genes including Prlr, Cyp19a1, Oxtr, and Lgals3 were also dysregulated in the KO ovaries, whereas PGF2α signaling genes remained unaffected. These results highlight the role of MAMLD1 in labour initiation. MAMLD1 likely participates in functional luteolysis by regulating Stat5b and other genes, independent of the PGF2α signaling pathway.
Wiedmer, Petra; Schwarz, Franziska; Große, Birgit; Schindler, Nancy; Tuchscherer, Armin; Russo, Vincenzo C; Tschöp, Matthias H; Hoeflich, Andreas
IGFBP-2 affects growth and metabolism and is thought to impact on energy homeostasis and the accretion of body fat via its heparin binding domains (HBD). In order to assess the function of the HBD present in the linker domain (HBD1) we have generated transgenic mice overexpressing mutant human IGFBP-2 lacking the PKKLRP sequence and carrying a PNNLAP sequence instead. Transgenic mice expressed high amounts of human IGFBP-2, while endogenous IGFBP-2 or IGF-I serum concentrations were not affected. In both genders we performed a longitudinal analysis of growth and metabolism including at least 4 separate time points between the age of 10 and 52 weeks. Body composition was assessed by nuclear magnetic resonance (NMR) analysis. Food intake was recorded by an automated online-monitoring. We describe negative effects of mutant human IGFBP-2 on body weight, longitudinal growth and lean body mass (p < 0.05). Very clearly, negative effects of mutant IGFBP-2 were not observed for fat mass accretion throughout life. Instead, relative fat mass was increased in transgenic mice of both genders (p < 0.05). In male mice transgene expression significantly increased absolute mass of total body fat over all age groups (p < 0.05). Food intake was increased in female but decreased in male transgenic mice at an age of 11 weeks. Thus our study clearly provides gender- and time-specific effects of HBD1-deficient hIGFBP-2 (H1d-BP-2) on fat mass accretion and food intake. While our data are in principal agreement with current knowledge on the role of HB-domains for fat accretion we now may also speculate on a role of HBD1 for the control of eating behavior.
Farkas, Jozsef; Sandor, Balazs; Tamas, Andrea; Kiss, Peter; Hashimoto, Hitoshi; Nagy, Andras D; Fulop, Balazs D; Juhasz, Tamas; Manavalan, Sridharan; Reglodi, Dora
Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.
Kirkland, James L; Stout, Michael B; Sierra, Felipe
Recently discovered interventions that target fundamental aging mechanisms have been shown to increase life span in mice and other species, and in some cases, these same manipulations have been shown to enhance health span and alleviate multiple age-related diseases and conditions. Aging is generally associated with decreases in resilience, the capacity to respond to or recover from clinically relevant stresses such as surgery, infections, or vascular events. We hypothesize that the age-related increase in susceptibility to those diseases and conditions is driven by or associated with the decrease in resilience. Thus, a test for resilience at middle age or even earlier could represent a surrogate approach to test the hypothesis that an intervention delays the process of aging itself. For this, animal models to test resilience accurately and predictably are needed. In addition, interventions that increase resilience might lead to treatments aimed at enhancing recovery following acute illnesses, or preventing poor outcomes from medical interventions in older, prefrail subjects. At a meeting of basic researchers and clinicians engaged in research on mechanisms of aging and care of the elderly, the merits and drawbacks of investigating effects of interventions on resilience in mice were considered. Available and potential stressors for assessing physiological resilience as well as the notion of developing a limited battery of such stressors and how to rank them were discussed. Relevant ranking parameters included value in assessing general health (as opposed to focusing on a single physiological system), ease of use, cost, reproducibility, clinical relevance, and feasibility of being repeated in the same animal longitudinally. During the discussions it became clear that, while this is an important area, very little is known or established. Much more research is needed in the near future to develop appropriate tests of resilience in animal models within an aging context
Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S
The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.
Esposito, Fabienne; Rochat, Lucien; Juillerat Van der Linden, Anne-Claude; Lekeu, Françoise; Charnallet, Annik; Van der Linden, Martial
Apathy is common in aging and generally defined on the basis of three dimensions: lack of initiative, lack of interest and emotional blunting. Curiously, no study until now has examined the associations and dissociations between these dimensions in elderly people (with or without dementia). These questions were addressed in two studies. In the first study, we explored the distribution of scores and the relationships between the three dimensions of apathy in 56 patients with dementia, focusing mainly on lack of initiative and lack of interest. Apathy was hetero-evaluated with the Apathy Inventory (AI), a scale widely used to assess the apathy dimensions in aging. In the second study, given the AI's limitations, we investigated in more detail the relationship between lack of initiative and interest in 115 elderly people using a new questionnaire specifically designed to assess these two dimensions. Results showed that lack of initiative was closely related to lack of interest (Study 1). Although we used a more specific questionnaire, these facets of apathy did not constitute two separable dimensions, but reflected a common main factor of apathy in aging (Study 2). Thus, the distinction between lack of initiative and lack of interest seems questionable. Only a multifactorial approach that includes the various psychological factors involved in apathy would enable one to gain a better understanding of the different manifestations of apathy and to highlight possible dissociations between them.
Al-Ajmi, Nouf; Saretzki, Gabriele; Miles, Colin; Spyridopoulos, Ioakim
Ageing is associated with an overall decline in the functional capacity of tissues and stem cells, including haematopoietic stem and progenitor cells (HSPCs), as well as telomere dysfunction. Dietary restriction (DR) is a recognised anti-ageing intervention that extends lifespan and improves health in several organisms. To investigate the role of telomeres and telomerase in haematopoietic ageing, we compared the HSPC profile and clonogenic capacity of bone marrow cells from wild type with telomerase-deficient mice and the effect of DR on these parameters. Compared with young mice, aged wild type mice demonstrated a significant accumulation of HSPCs (1.3% vs 0.2%, P=0.002) and elevated numbers of granulocyte/macrophage colony forming units (CFU-GM, 26.4 vs 17.3, P=0.0037) consistent with myeloid "skewing" of haematopoiesis. DR was able to restrict the increase in HSPC number as well as the myeloid "skewing" in aged wild type mice. In order to analyse the influence of short telomeres on the ageing phenotype we examined mice lacking the RNA template for telomerase, TERC(-/-). Telomere shortening resulted in a similar bone marrow phenotype to that seen in aged mice, with significantly increased HSPC numbers and an increased formation of all myeloid colony types but at a younger age than wild type mice. However, an additional increase in erythroid colonies (BFU-E) was also evident. Mice lacking telomerase reverse transcriptase without shortened telomeres, TERT(-/-), also presented with augmented haematopoietic ageing which was ameliorated by DR, demonstrating that the effect of DR was not dependent on the presence of telomerase in HSPCs. We conclude that whilst shortened telomeres mimic some aspects of haematopoietic ageing, both shortened telomeres and the lack of telomerase produce specific phenotypes, some of which can be prevented by dietary restriction.
Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming; Gupta, Sapna; Slifker, Michael J; Li, Yue-Sheng; Andrews, Andrew J; Kruger, Warren D
Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.
Pettan-Brewer, Christina; Treuting, Piper M.
Old mice will have a subset of lesions as part of the progressive decline in organ function that defines aging. External and palpable lesions will be noted by the research, husbandry, or veterinary staff during testing, cage changing, or physical exams. While these readily observable lesions may cause alarm, not all cause undue distress or are life-threatening. In aging research, mice are maintained until near end of life that, depending on strain and genetic manipulation, can be upwards of 33 months. Aging research has unique welfare issues related to age-related decline, debilitation, fragility, and associated pain of chronic diseases. An effective aging research program includes the collaboration and education of the research, husbandry, and veterinary staff, and of the members of the institution animal care and use committee. This collaborative effort is critical to humanely maintaining older mice and preventing excessive censorship due to non-lethal diseases. Part of the educational process is becoming familiar with how old mice appear clinically, at necropsy and histopathologically. This baseline knowledge is important in making the determination of humane end points, defining health span, contributing causes of death and effects of interventions. The goal of this paper is to introduce investigators to age-associated diseases and lesion patterns in mice from clinical presentation to pathologic assessment. To do so, we present and illustrate the common clinical appearances, necropsy and histopathological lesions seen in subsets of the aging colonies maintained at the University of Washington. PMID:22953032
Silvani, Alessandro; Berteotti, Chiara; Bastianini, Stefano; Lo Martire, Viviana; Mazza, Roberta; Pagotto, Uberto; Quarta, Carmelo; Zoccoli, Giovanna
Cannabinoid type 1 (CB1) receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD). We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD) and HFD. CB1 cannabinoid receptor knock-out (KO) and wild-type (WT) mice were fed SD or HFD for 4 months (n = 9-10 per group). Mice were instrumented with electroencephalographic (EEG) and electromyographic electrodes. Recordings were performed during baseline (48 hours), sleep deprivation (gentle handling, 6 hours), sleep recovery (18 hours), and after cage switch (insomnia model paradigm, 6 hours). We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) than WT during the dark (active) period but not during the light (rest) period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice.
Xu, Wenjing; Barrientos, Tomasa; Mao, Lan; Rockman, Howard A; Sauve, Anthony A; Andrews, Nancy C
Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.
Suh, Eul; Izzo Matic, Agnella; Otting, Margarete; Walsh, Joseph T., Jr.; Richter, Claus-Peter
Lasers can be used to stimulate neural tissue, including the sciatic nerve or auditory neurons. Wells and coworkers suggested that neural tissue is likely stimulated by heat.[1,2] Ion channels that can be activated by heat are the TRPV channels, a subfamily of the Transient Receptor Potential (TRP) ion channels. TRPV channels are nonselective cation channels found in sensory neurons involved in nociception. In addition to various chemicals, TRPV channels can also be thermally stimulated. The activation temperature for the different TRPV channels varies and is 43°C for TRPV1 and 39°C for TRPV3. By performing an immunohistochemical staining procedure on frozen 20 μm cochlear slices using a primary TRPV1 antibody, we observed specific immunostaining of the spiral ganglion cells. Here we show that in mice that lack the gene for the TRPV1 channel optical radiation cannot evoke action potentials on the auditory nerve.
Wilhelm, Dagmar; Jans, David A.; Bowles, Josephine; Koopman, Peter
Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. PMID:28107452
Maleki, Nasim; Barmettler, Gabi; Moulton, Eric A.; Scrivani, Steven; Veggeberg, Rosanna; Spierings, Egilius L.H.; Burstein, Rami; Becerra, Lino; Borsook, David
Gray matter loss in cortical regions is a normal ageing process for the healthy brain. There have been few studies on the process of ageing of the brain in chronic neurological disorders. In this study, we evaluated changes in the cortical thickness by age in 92 female subjects (46 migraine patients, and 46 healthy controls) using high field MRI. The results indicate that in contrast to healthy subjects migraineurs show lack of thinning in the insula by age. The functional significance of the lack of thinning is unknown, but may contribute to the overall cortical hyperexcitability of the migraine brain since the region is tightly involved in a number of majo brain networks involved in interoception, salience, nociception, and autonomic function, including the default mode network. PMID:25775358
Background and purpose Cyclooxygenase-2 (COX-2) promotes inflammation by synthesizing pro-inflammatory prostaglandins from arachidonic acid. Inflammation is an early response to bone fracture, and ablation of COX-2 activity impairs fracture healing. Arachidonic acid is also converted into leukotrienes by 5-lipoxygenase (5-LO). We hypothesized that 5-LO is a negative regulator of fracture healing and that in the absence of COX-2, excess leukotrienes synthesized by 5-LO will impair fracture healing. Methods Fracture healing was assessed in mice with a targeted 5-LO mutation (5-LOKO mice) and control mice by radiographic and histological observations, and measured by histomorphometry and torsional mechanical testing. To assess effects on arachidonic acid metabolism, prostaglandin E2, F2α, and leukotriene B4 levels were measured in the fracture calluses of control, 5-LOKO COX-1KO, and COX-2KO mice by enzyme linked immunoassays. Results Femur fractures in 5-LOKO mice rapidly developed a cartilaginous callus that was replaced with bone to heal fractures faster than in control mice. Femurs from 5-LOKO mice had substantially better mechanical properties after 1 month of healing than did control mice. Callus leukotriene levels were 4-fold higher in mice homozygous for a targeted mutation in the COX-2 gene (COX-2KO), which indicated that arachidonic acid was shunted into the 5-LO pathway in the absence of COX-2. Interpretation These experiments show that 5-LO negatively regulates fracture healing and that shunting of arachidonic acid into the 5-LO pathway may account, at least in part, for the impaired fracture healing response observed in COX-2KO mice. PMID:21067431
The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain. PMID:26107521
Murovets, Vladimir O; Bachmanov, Alexander A; Zolotarev, Vasiliy A
The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.
Magner, William J.; Chang, Andrew C.; Owens, Jennie; Hong, M-J. P.; Brooks, Andrew
In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice. PMID:11097211
Tonniges, Jeffrey R; Albert, Benjamin; Calomeni, Edward P; Roy, Shuvro; Lee, Joan; Mo, Xiaokui; Cole, Susan E; Agarwal, Gunjan
The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.
Tonniges, Jeffrey R.; Albert, Benjamin; Calomeni, Edward P.; Roy, Shuvro; Lee, Joan; Mo, Xiaokui; Cole, Susan E.; Agarwal, Gunjan
The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson’s trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM. PMID:27329311
d'Anglemont de Tassigny, Xavier; Fagg, Lisa A.; Dixon, John P. C.; Day, Kate; Leitch, Harry G.; Hendrick, Alan G.; Zahn, Dirk; Franceschini, Isabelle; Caraty, Alain; Carlton, Mark B. L.; Aparicio, Samuel A. J. R.; Colledge, William H.
The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (β-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic–pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54- and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic–pituitary–gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms. PMID:17563351
Zhou, Yong-Xing; Zhao, Mingrui; Li, Dan; Shimazu, Kazuhiro; Sakata, Kazuko; Deng, Chu-Xia; Lu, Bai
Smad4 is a central mediator of TGF-beta signals, which are known to play essential roles in many biological processes. Using a Cre-loxP approach to overcome early embryonic lethality, we have studied functions of TGF-beta/Smad4 signals in the central nervous system (CNS). No obvious deficits were detected in mice carrying the targeted disruption of Smad4 in the CNS. The overall morphology of the hippocampus appeared normal. There was no change in the proliferation of neuronal precursor cells, nor in several forms of synaptic plasticity. In contrast, deletion of Smad4 resulted in a marked decrease in the number of cerebellar Purkinje cells and parvalbumin-positive interneurons. Accompanied by the abnormality in the cerebellum, mutant mice also exhibited significantly increased vertical activity. Thus, our study reveals an unexpected role for Smad4 in cerebellar development and in the control of motor function.
Renné, Thomas; Pozgajová, Miroslava; Grüner, Sabine; Schuh, Kai; Pauer, Hans-Ulrich; Burfeind, Peter; Gailani, David; Nieswandt, Bernhard
Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)–mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy. PMID:16009717
Domingo, J.S.; Mercedes Gomez, M.A.B.; Corbella, J.
The embryotoxic and teratogenic potential of aluminum hydroxide, a therapeutic drug used as an antacid and phosphate binder, was investigated in Swiss mice. Mated female mice were given by gavage daily doses of 0, 66.5, 133 or 266 mg/kg of A1(OH)/sub 3/ on gestation days 6 through 15 and killed on gestation day 18. Females were evaluated for body weight gain, food consumption appearance and behavior, survival rates, and reproduction data. No significant effects attributable to A1(OH)/sub 3/ were noted in comparisons of maternal body weight and food consumption values, appearance and behavior. No treatment-related changes were recorded in the number of total implants, resorptions, the number of live and dead fetuses, fetal size parameters or fetal sex distribution data. Gross external, soft tissue and skeletal examination of the A1-treated fetuses did not reveal differences at any dose in comparison with the controls. Thus, no evidence of maternal toxicity, embryo/fetal toxicity or teratogenicity was observed with A1(OH)/sub 3/ in mice.
Ojeda-Fernández, Luisa; Recio-Poveda, Lucía; Aristorena, Mikel; Lastres, Pedro; Blanco, Francisco J.; Sanz-Rodríguez, Francisco; Gallardo-Vara, Eunate; de las Casas-Engel, Mateo; Corbí, Ángel; Arthur, Helen M.; Bernabeu, Carmelo; Botella, Luisa M.
Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Engfl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Engfl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients. PMID:27010826
Zamakhchari, Mai F.; Sima, Corneliu; Sama, Kishore; Fine, Noah; Glogauer, Michael; Van Dyke, Thomas E.; Gyurko, Robert
Excess reactive oxygen species production is central to the development of diabetic complications. The contribution of leukocyte reactive oxygen species produced by the NADPH oxidase to altered inflammatory responses associated with uncontrolled hyperglycemia is poorly understood. To get insight into the role of phagocytic superoxide in the onset of diabetic complications, we used a model of periodontitis in mice with chronic hyperglycemia and lack of leukocyte p47phox (Akita/Ncf1) bred from C57BL/6-Ins2Akita/J (Akita) and neutrophil cytosolic factor 1 knockout (Ncf1) mice. Akita/Nfc1 mice showed progressive cachexia starting at early age and increased mortality by six months. Their lungs developed infiltrative interstitial lesions that obliterated air spaces as early as 12 weeks when fungal colonization of lungs also was observed. Neutrophils of Akita/Ncf1 mice had normal degranulation and phagocytic efficiency when compared with wild-type mice. Although Akita/Ncf1 mice had increased prevalence of oral infections and more severe periodontitis compared with wild-type mice, bone loss was only marginally higher compared with Akita and Ncf1 null mice. Altogether these results indicate that lack of leukocyte superoxide production in mice with chronic hyperglycemia results in interstitial pneumonia and increased susceptibility to infections. PMID:26747235
Keller, Kirsten; Maass, Martina; Dizayee, Sara; Leiss, Veronika; Annala, Suvi; Köth, Jessica; Seemann, Wiebke K.; Müller-Ehmsen, Jochen; Mohr, Klaus; Nürnberg, Bernd; Engelhardt, Stefan; Herzig, Stefan; Birnbaumer, Lutz; Matthes, Jan
Aims Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression. Methods and results β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2−/−) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2−/−). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or β1-tg, but similar to β1-tg/Gαi2−/−, mice. Beyond 300 days, mortality of β1-tg/Gαi2−/− mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β1-tg, and Gαi2−/− mice, but significant impairment for β1-tg/Gαi2−/− mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2−/− and 394 ± 80% in β1-tg/Gαi2−/−, respectively). Conclusions Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or
Porras-García, M. Elena; Ruiz, Rocío; Pérez-Villegas, Eva M.; Armengol, José Á.
The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum. PMID:23630472
Porras-García, M Elena; Ruiz, Rocío; Pérez-Villegas, Eva M; Armengol, José Á
The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input-output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.
Kronenberg, Golo; Mosienko, Valentina; Gertz, Karen; Alenina, Natalia; Hellweg, Rainer; Klempin, Friederike
The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.
Melville, Katherine M; Kelly, Natalie H; Khan, Sohaib A; Schimenti, John C; Ross, F Patrick; Main, Russell P; van der Meulen, Marjolein C H
Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERα), mice with global deletion of ERα (ERαKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin-like growth factor 1 (IGF-1) serum levels, which may independently affect bone. Mice with tissue-specific ERα deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERα is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast-specific ERαKO mice (pOC-ERαKO) by crossing mice expressing a floxed ERα gene (ERα(fl/fl)) with mice transgenic for the osteocalcin-Cre promoter (OC-Cre). Having confirmed that serum levels of estrogen and IGF-1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC-ERαKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERα in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This p
Lao, Taotao; Jiang, Zhiqiang; Yun, Jeong; Qiu, Weiliang; Guo, Feng; Huang, Chunfang; Mancini, John Dominic; Gupta, Kushagra; Laucho-Contreras, Maria E; Naing, Zun Zar Chi; Zhang, Li; Perrella, Mark A; Owen, Caroline A; Silverman, Edwin K; Zhou, Xiaobo
Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.
Eleftheriou, B E; Zolovick, A J; Elias, M F
Electroencephalographic (EEG) changes, as measured by the awake state, slow-wave sleep (SWS), rapid-eye movement (REM) patterns and ratio of REM/total sleep, were recorded in aging male mice of DBA/2J and C57BL/6J strains. Results indicate that there is a significant increase in the awake state accompanied by significant decrease in SWS with advancing age for both strains, although these changes appear more pronounced in DBA/2J mice than C57BL/6J mice. Of considerable significance is the finding that REM sleep is absent in mice of DBA/2J strain at 23.5 months of age. Based on these findings, the conclusion was reached that strain DBA/2J ages significantly faster than C57BL/6J. The difference in aging between the two strains emphasizes the need for additional studies dealing with genetic aspects of aging.
Benice, Ted S.; Raber, Jacob
Compared with age-matched male mice, female mice experience a more severe age-related cognitive decline (ACD). Since androgens are less abundant in aged female mice compared with aged male mice, androgen supplementation may enhance cognition in aged female mice. To test this, we assessed behavioral performance on a variety of tasks in 22- to…
Hamrick, Mark W; Pennington, Catherine; Byron, Craig D
GDF-8, also known as myostatin, is a member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared to normal mice. We describe here morphology of the lumbar spine in myostatin knockout (Mstn(-/-)) mice using histological and densitometric techniques. The Mstn(-/-) mice examined in this study weigh approximately 10% more than controls (p<0.001) but the iliopsoas muscle is over 50% larger in the knockout mice than in wild-type mice (p<0.001). Peripheral quantitative computed tomography (pQCT) data from the fifth lumbar vertebra show that mice lacking myostatin have approximately 50% greater trabecular bone mineral density (p=0.001) and significantly greater cortical bone mineral content than normal mice. Toluidine blue staining of the intervertebral disc between L4-L5 reveals loss of proteoglycan staining in the hyaline end plates and inner annulus fibrosus of the knockout mice. Loss of cartilage staining in the caudal end plate of L4 is due to ossification of the end plate in the myostatin-deficient animals. Results from this study suggest that increased muscle mass in mice lacking myostatin is associated with increased bone mass as well as degenerative changes in the intervertebral disc.
Newton, P. M.; Kim, J. A.; McGeehan, A. J.; Paredes, J. P.; Chu, K.; Wallace, M. J.; Roberts, A. J.; Hodge, C. W.; Messing, R. O.
The protein kinase C (PKC) family of serine–threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCε have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCε null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCε null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of μ-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCε as a key regulator of opiate sensitivity in mice. PMID:16899053
Cheung, L Y M; Rizzoti, K; Lovell-Badge, R; Tissier, P R
The Notch signalling pathway ligand delta-like 1 homologue (Dlk1, also named Pref1) is expressed throughout the developing pituitary and becomes restricted to mostly growth hormone (GH) cells within the adult gland. We have investigated the role of Dlk1 in pituitary development and function from late embryogenesis to adulthood using a mouse model completely lacking the expression of Dlk1. We confirm that Dlk1-null mice are shorter and weigh less than wild-type littermates from late gestation, at parturition and in adulthood. A loss of Dlk1 leads to significant reduction in GH content throughout life, whereas other pituitary hormones are reduced to varying degrees depending on sex and age. Both the size of the pituitary and the proportion of hormone-producing cell populations are unchanged, suggesting that there is a reduction in hormone content per cell. In vivo challenge of mutant and wild-type littermates with growth hormone-releasing hormone and growth hormone-releasing hexapeptide shows that reduced GH secretion is unlikely to account for the reduced growth of Dlk1 knockout animals. These data suggest that loss of Dlk1 gives rise to minor pituitary defects manifesting as an age- and sex-dependent reduction in pituitary hormone contents. However, Dlk1 expression in other tissue is most likely responsible for the weight and length differences observed in mutant animals. PMID:23279263
Laubach, V E; Shesely, E G; Smithies, O; Sherman, P A
Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon gamma, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock. Images Fig. 2 Fig. 3 PMID:7479866
Liu, Xingkai; Zhang, Ping; Martin, Robert C; Cui, Guozhen; Wang, Guangyi; Tan, Yi; Cai, Lu; Lv, Guoyue; Li, Yan
Fibroblast growth factor 21 (FGF21) concentrations are increased in human subjects who either have type 2 diabetes or nonalcoholic fatty liver disease (NAFLD). While excessive fat in the liver promotes the release of pro-inflammatory cytokines, NAFLD progresses from steatosis to non alcoholic steatohepatitis (NASH), a more aggressive form of hepatic damage, and lastly toward cirrhosis and HCC. In our previous study, loss of FGF21 is associated with hyper-proliferation, aberrant p53, and HCC development in diabetes mice. In this study, we proposed to investigate the liver metabolic disorders by diabetes and the potential roles of FGF21 played in NASH and potential carcinogenetic transformation of HCC. NASH was induced in FGF21 knockout (FGF21KO) mice by streptozotocin administration or fed with high fat diet (HFD). The pathological transformation of steatohepatities as well as parameters of inflammation, lipid metabolism, cellular events, mesenchymal-epithelial transition (MET) and Wnt/β-catenin signaling was determined in the FGF21 KO diabetic mice and HFD fed mice. We found that mice lacking the FGF21 gene are more prone to develop NASH. A compromised microenvironment of NASH, which could facilitate the HCC carcinogenetic transformation, was found in FGF21 KO mice under metabolic disorders by diabetes and HFD feeding. This study provided further evidence that lack of FGF21 worsened the metabolic disorders in NASH and could render a tumor microenvironment for HCC initiation and progression in the liver of diabetes mice. PMID:27293995
Castagnaro, Silvia; Gregorio, Ilaria; Bonaldo, Paolo
Collagen VI is an extracellular matrix (ECM) protein with a broad distribution in different tissues and mostly deposited at the close periphery of the cell surface. Previous studies revealed that collagen VI protects neurons from the toxicity of amyloid-βpeptides and from UV-induced damage. However, the physiological role of this protein in the central nervous system (CNS) remains unknown. Here, we established primary neural cultures from murine cortex and hippocampus, and carried out in vitro and in vivo studies in wild-type and collagen VI null (Col6a1−/−) mice. Col6a1−/− neural cultures displayed an increased incidence of spontaneous apoptosis and higher vulnerability to oxidative stress, accompanied by altered regulation of autophagy with increased p62 protein levels and decreased LC3 lipidation. Analysis of brain sections confirmed increased apoptosis and abnormal regulation of autophagy in the CNS of collagen VI-deficient animals. To investigate the in vivo physiological consequences of these CNS defects, we carried out functional studies and found that motor and memory task performances were impaired in aged Col6a1−/− mice. These findings indicate that lack of collagen VI leads to spontaneous apoptosis and defective autophagy in neural cells, and point at a protective role for this ECM protein in the CNS during physiological aging. PMID:27060109
Tung, Yu-Tang; Chen, Ying-Ju; Chuang, Hsiao-Li; Huang, Wen-Ching; Lo, Chun-Tsung; Liao, Chen-Chung; Huang, Chi-Chang
Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help
Derbré, Frederic; Gomez-Cabrera, Mari Carmen; Nascimento, Ana Lucia; Sanchis-Gomar, Fabian; Martinez-Bello, Vladimir Essau; Tresguerres, Jesus A F; Fuentes, Teresa; Gratas-Delamarche, Arlette; Monsalve, Maria; Viña, Jose
Low mitochondriogenesis is critical to explain loss of muscle function in aging and in the development of frailty. The aim of this work was to explain the mechanism by which mitochondriogenesis is decreased in aging and to determine to which extent it may be prevented by exercise training. We used aged rats and compared them with peroxisome proliferator-activated receptor-γ coactivator-1α deleted mice (PGC-1α KO). PGC-1α KO mice showed a significant decrease in the mitochondriogenic pathway in muscle. In aged rats, we found a loss of exercise-induced expression of PGC-1α, nuclear respiratory factor-1 (NRF-1), and of cytochrome C. Thus muscle mitochondriogenesis, which is activated by exercise training in young animals, is not in aged or PGC-1α KO ones. Other stimuli to increase PGC-1α synthesis apart from exercise training, namely cold induction or thyroid hormone treatment, were effective in young rats but not in aged ones. To sum up, the low mitochondrial biogenesis associated with aging may be due to the lack of response of PGC-1α to different stimuli. Aged rats behave as PGC-1α KO mice. Results reported here highlight the role of PGC-1α in the loss of mitochondriogenesis associated with aging and point to this important transcriptional coactivator as a target for pharmacological interventions to prevent age-associated sarcopenia.
Walker, L. C.; Parker, C. A.; Lipinski, W. J.; Callahan, M. J.; Carroll, R. T.; Gandy, S. E.; Smith, J. D.; Jucker, M.; Bisgaier, C. L.
To assess the influence of age and diet on cerebral pathology in mice lacking apolipoprotein E (apoE), four male apoE knockout mice (epsilon -/-), and five male wild-type (epsilon +/+) littermate controls were placed on a high-fat/high-cholesterol diet for 7 weeks beginning at 17 months of age. All four aged knockout mice developed xanthomatous lesions in the brain consisting mostly of crystalline cholesterol clefts, lipid globules, and foam cells. Smaller xanthomas were confined mainly to the choroid plexus and ventral fornix in the roof of the third ventricle, occasionally extending subpially along the choroidal fissure and into the adjacent parenchyma. More advanced xanthomas disrupted adjoining neural tissue in the fornix, hippocampus, and dorsal diencephalon; in one case, over 60% of one telencephalic hemisphere, including nearly the entire neocortex, was obliterated by the lesion. No xanthomas were observed in aged wild-type controls fed the high-fat/high-cholesterol diet. Brains from 42 additional animals, fed only conventional chow, were examined; 3 of 15 aged (15- to 23-month-old) apoE knockout mice developed small choroidal xanthomas. In contrast, no lesions were observed in five young (2- to 4-month-old) apoE knockout mice or in any wild-type controls between the ages of 2 and 23 months. Our findings indicate that disorders of lipid metabolism can induce significant pathological changes in the central nervous system of aged apoE knockout mice, particularly those on a high-fat/high-cholesterol diet. It may be fruitful to seek potential interactions between genetic factors and diet in modulating the risk of Alzheimer's disease and other neurodegenerative disorders in aged humans. Images Figure 1 Figure 2 PMID:9358763
Kohnken, Rebecca A; Schwahn, Denise J
FVB/N mice with ‘space cadet’ syndrome are prone to audiogenic seizures and are considered excitotoxic ‘sensitive’ mice due to the neuronal damage that accompanies seizures. FVB/N mice found dead demonstrate acute neuronal cell death—attributed to a massive seizure episode—within the hippocampus and cerebrocortical laminae. However, the behavioral features of FVB/N mice and numerous studies using excitotoxins to induce seizure activity indicate that this strain experiences multiple sublethal seizures. To assess whether FVB/N mice develop histologically detectable lesions, we evaluated the brains of 86 aged (154-847 d) FVB/N mice without a history of seizures. The hippocampus and cerebrocortical laminae were evaluated histologically for neuronal atrophy and gliosis. Neuronal atrophy was quantified by counting neurons in the hippocampus (CA3 and dentate gyrus) and cerebral cortex. Gliosis was quantified by using immunohistochemistry for glial fibrillary acidic protein and glial counting in the cerebral cortex. In addition, ventricular area was calculated. Our study revealed no changes in brain weight with age, no neuronal loss or gliosis, no correlation between neuronal or glial cell profile densities and brain weight or age, and no differences in ventricular size between FVB/N and control mice. Neuronal densities in the cerebral cortex and granule cells of the dentate gyrus were lower in FVB/N mice than in control Swiss Webster mice. We conclude that although acute lesions of seizure activity are a previous feature of the FVB/N strain, chronic seizure activity in these mice either is negligible or does not cause morphologic or phenotypic changes. PMID:27053564
Perez-Alcazar, Marta; Daborg, Jonny; Stokowska, Anna; Wasling, Pontus; Björefeldt, Andreas; Kalm, Marie; Zetterberg, Henrik; Carlström, Karl E; Blomgren, Klas; Ekdahl, Christine T; Hanse, Eric; Pekna, Marcela
Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.
SIVILS, JEFFREY C.; GONZALEZ, IVEN; BAIN, LISA J.
The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ABC active transporter family that can transport several steroid hormone conjugates, including 17β-estradiol glucuronide, dehydroepiandrosterone sulfate (DHEAS), and estrone 3-sulfate. The present study investigated the role that MRP1 plays in maintaining proper hormone levels in the serum and testes. Serum and testicular steroid hormone levels were examined in both wild-type mice and Mrp1 null mice. Serum testosterone levels were reduced 5-fold in mice lacking Mrp1, while testicular androstenedione, testosterone, estradiol, and dehydroepiandrosterone (DHEA) were significantly reduced by 1.7- to 4.5-fold in Mrp1 knockout mice. Investigating the mechanisms responsible for the reduction in steroid hormones in Mrp1-/- mice revealed no differences in the expression or activity of enzymes that inactivate steroids, the sulfotransferases or glucuronosyltransferases. However, steroid biosynthetic enzyme levels in the testes were altered. Cyp17 protein levels were increased by 1.6-fold, while Cyp17 activity using progesterone as a substrate was also increased by 1.4-2.0-fold in mice lacking Mrp1. Additionally, the ratio of 17β-hydroxysteroid dehydrogenase to 3β-hydroxysteroid dehydrogenase, and steroidogenic factor 1 to 3βhydroxysteroid dehydrogenase were significantly increased in the testes of Mrp1-/- mice. These results indicate that Mrp1-/- mice have lowered steroid hormones levels, and suggests that upregulation of steroid biosynthetic enzymes may be an attempt to maintain proper steroid hormone homeostasis. PMID:20178799
Bulwa, Zachary B; Sharlin, Jordan A; Clark, Peter J; Bhattacharya, Tushar K; Kilby, Chessa N; Wang, Yanyan; Rhodes, Justin S
Individual differences in dopamine D2 receptor (D2R) expression in the brain are thought to influence motivation and reinforcement for ethanol and other rewards. D2R exists in two isoforms, D2 long (D2LR) and D2 short (D2SR), produced by alternative splicing of the same gene. The relative contributions of D2LR versus D2SR to ethanol and sugar water drinking are not known. Genetic engineering was used to produce a line of knockout (KO) mice that lack D2LR and consequently have increased expression of D2SR. KO and wild-type (WT) mice of both sexes were tested for intake of 20% ethanol, 10% sugar water and plain tap water using established drinking-in-the-dark procedures. Mice were also tested for effects of the D2 antagonist eticlopride on intake of ethanol to determine whether KO responses were caused by lack of D2LR or overrepresentation of D2SR. Locomotor activity on running wheels and in cages without wheels was also measured for comparison. D2L KO mice drank significantly more ethanol than WT in both sexes. KO mice drank more sugar water than WT in females but not in males. Eticlopride dose dependently decreased ethanol intake in all groups except male KO. KO mice were less physically active than WT in cages with or without running wheels. Results suggest that overrepresentation of D2SR contributes to increased intake of ethanol in the KO mice. Decreasing wheel running and general levels of physical activity in the KO mice rules out the possibility that higher intake results from higher motor activity. Results extend the literature implicating altered expression of D2R in risk for addiction by delineating the contribution of individual D2R isoforms. These findings suggest that D2LR and D2SR play differential roles in consumption of alcohol and sugar rewards.
Rolán, Hortensia García; Tsolis, Renée M
The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.
Malik, Mohammad T; Peng, Ying-Jie; Kline, David D; Adhikary, Gautam; Prabhakar, Nanduri R
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.
Kovács-Valasek, Andrea; Szabadfi, Krisztina; Dénes, Viktória; Szalontai, Bálint; Tamás, Andrea; Kiss, Péter; Szabó, Aliz; Setalo, Gyorgy; Reglődi, Dóra; Gábriel, Robert
Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.
Thorn, Stephanie R; Giesy, Sarah L; Myers, Martin G; Boisclair, Yves R
Mice lacking leptin (ob/ob) or its full-length receptor (db/db) are obese and reproductively incompetent. Fertility, pregnancy, and lactation are restored, respectively, in ob/ob mice treated with leptin through mating, d 6.5 post coitum, and pregnancy. Therefore, leptin signaling is needed for lactation, but the timing of its action and the affected mammary process remain unknown. To address this issue, we used s/s mice lacking only leptin-dependent signal transducer and activator of transcription (STAT)3 signaling. These mice share many features with db/db mice, including obesity, but differ by retaining sufficient activity of the hypothalamic-pituitary-ovarian axis to support reproduction. The s/s mammary epithelium was normal at 3 wk of age but failed to expand through the mammary fat pad (MFP) during the subsequent pubertal period. Ductal growth failure was not corrected by estrogen therapy and did not relate to inadequate IGF-I production by the MFP or to the need for epithelial or stromal leptin-STAT3 signaling. Ductal growth failure coincided with adipocyte hypertrophy and increased MFP production of leptin, TNFalpha, and IL6. These cytokines, however, were unable to inhibit the proliferation of a collection of mouse mammary epithelial cell lines. In conclusion, the very first step of postnatal mammary development fails in s/s mice despite sufficient estrogen IGF-I and an hypothalamic-pituitary-ovarian axis capable of supporting reproduction. This failure is not caused by mammary loss of leptin-dependent STAT3 signaling or by the development of inflammation. These data imply the existence of an unknown mechanism whereby leptin-dependent STAT3 signaling and obesity alter mammary ductal development.
Neugebauer, Nichole M; Henehan, Robert M; Hales, Claire A; Picciotto, Marina R
Previous work has indicated that the neuropeptide galanin decreases sensitivity to the rewarding effects of morphine and cocaine, but increases alcohol drinking. The aim of the current study was to examine the role of galanin signaling in nicotine reward by testing the effects of nicotine in mice lacking galanin peptide (GAL-/-) as compared to wild-type (GAL+/+) controls. Using an unbiased, three-chamber conditioned place preference (CPP) paradigm the dose-response function for nicotine CPP was tested in GAL-/- and GAL+/+ mice. Since activation of extracellular signal-related kinase (ERK2) is involved in the rewarding effects of several classes of drugs of abuse, we then measured the level of ERK2 phosphorylation in the nucleus accumbens shell (NACsh) and core (NACco) of GAL-/- and GAL+/+ mice following re-exposure to the CPP chamber previously paired with nicotine as a marker of mesolimbic system activation. Finally, we examined whether acute nicotine administration affects ERK2 activity in GAL-/- and GAL+/+ mice. GAL-/- mice required a higher dose of nicotine to induce a significant CPP compared to GAL+/+ mice. In the conditioning groups showing significant expression of nicotine CPP, only GAL+/+ mice showed ERK2 activation in the NACsh. This suggests that the nicotine CPP observed in GAL+/+ mice resulted in differential recruitment of ERK signaling in the NACsh compared to GAL-/- mice. In addition, no activation of ERK2 was observed following acute nicotine administration in either genotype. These data, along with prior results, suggest that galanin alters sensitivity to drugs of abuse differentially, with morphine, cocaine and amphetamine place preference suppressed, and nicotine and alcohol preference increased, by galanin signaling.
Yellon, Steven M; Oshiro, Bryan T; Chhaya, Tejas Y; Lechuga, Thomas J; Dias, Rejane M; Burns, Alexandra E; Force, Lindsey; Apostolakis, Ede M
Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition.
Bonasera, Stephen J.; Schenk, A. Katrin; Luxenberg, Evan J.; Wang, Xidao; Basbaum, Allan; Tecott, Laurence H.
Although central serotonergic systems are known to influence responses to noxious stimuli, mechanisms underlying serotonergic modulation of pain responses are unclear. We proposed that serotonin 2C receptors (5-HT2CRs), which are expressed within brain regions implicated in sensory and affective responses to pain, contribute to the serotonergic modulation of pain responses. In mice constitutively lacking 5-HT2CRs (2CKO mice) we found normal baseline sensory responses to noxious thermal, mechanical and chemical stimuli. In contrast, 2CKO mice exhibited a selective enhancement of affect-related ultrasonic afterdischarge vocalizations in response to footshock. Enhanced affect-related responses to noxious stimuli were also exhibited by 2CKO mice in a fear-sensitized startle assay. The extent to which a brief series of unconditioned footshocks produced enhancement of acoustic startle responses was markedly increased in 2CKO mice. As mesolimbic dopamine pathways influence affective responses to noxious stimuli, and these pathways are disinhibited in 2CKO mice, we examined the sensitivity of footshock-induced enhancement of startle to dopamine receptor blockade. Systemic administration of the dopamine D2/D3 receptor antagonist raclopride selectively reduced footshock-induced enhancement of startle without influencing baseline acoustic startle responses. We propose that 5-HT2CRs regulate affective behavioral responses to unconditioned aversive stimuli through mechanisms involving the disinhibition of ascending dopaminergic pathways. PMID:26630489
Hammerschmidt, Kurt; Whelan, Gabriela; Eichele, Gregor; Fischer, Julia
Mouse models play an increasingly important role in the identification and functional assessment of speech-associated genes, with a focus on genes involved in vocal production, and possibly vocal learning. Moreover, mice reportedly show direct projections from the cortex to brainstem vocal motor neurons, implying a degree of volitional control over vocal output. Yet, deaf mice did not reveal differences in call structures compared to their littermates, suggesting that auditory input is not a prerequisite for the development of species-specific sounds. To elucidate the importance of cortical structures for the development of mouse ultrasonic vocalizations (USVs) in more detail, we studied Emx1-CRE;Esco2(fl/fl) mice, which lack the hippocampus and large parts of the cortex. We conducted acoustic analyses of the USVs of 28 pups during short-term isolation and 23 adult males during courtship encounters. We found no significant differences in the vocalizations of Emx1-CRE;Esco2(fl/fl) mice, and only minor differences in call type usage in adult mice, compared to control littermates. Our findings question the notion that cortical structures are necessary for the production of mouse USVs. Thus, mice might be less suitable to study the mechanisms supporting vocal learning than previously assumed, despite their value for studying the genetic foundations of neurodevelopment more generally.
Reines, Brandon; Cheng, Lily I; Matzinger, Polly
Complete regeneration of damaged extremities, including both the epithelium and the underlying tissues, is thought to occur mainly in embryos, fetuses, and juvenile mammals, but only very rarely in adult mammals. Surprisingly, we found that common strains of mice are able to regenerate all of the tissues necessary to completely fill experimentally punched ear holes, but only if punched at middle age. Although young postweaning mice regrew the epithelium without typical pre-scar granulation tissue, they showed only minimal regeneration of connective tissues. In contrast, mice punched at 5-11 months of age showed true amphibian-like blastema formation and regrowth of cartilage, fat, and dermis, with blood vessels, sebaceous glands, hair follicles, and, in black mice, melanocytes. These data suggest that at least partial appendage regeneration may be more common in adult mammals than previously thought and call into question the common view that regenerative ability is lost with age. The data suggest that the age at which various inbred mouse strains become capable of epimorphic regeneration may be correlated with adult body weight.
Cheng, Lily I.; Matzinger, Polly
Abstract Complete regeneration of damaged extremities, including both the epithelium and the underlying tissues, is thought to occur mainly in embryos, fetuses, and juvenile mammals, but only very rarely in adult mammals. Surprisingly, we found that common strains of mice are able to regenerate all of the tissues necessary to completely fill experimentally punched ear holes, but only if punched at middle age. Although young postweaning mice regrew the epithelium without typical pre-scar granulation tissue, they showed only minimal regeneration of connective tissues. In contrast, mice punched at 5–11 months of age showed true amphibian-like blastema formation and regrowth of cartilage, fat, and dermis, with blood vessels, sebaceous glands, hair follicles, and, in black mice, melanocytes. These data suggest that at least partial appendage regeneration may be more common in adult mammals than previously thought and call into question the common view that regenerative ability is lost with age. The data suggest that the age at which various inbred mouse strains become capable of epimorphic regeneration may be correlated with adult body weight. PMID:19226206
Shirato, Suguru; Maeda, Hidetaka; Miura, Gen; Frishman, Laura J
The purpose of this study was to determine the contributions of postreceptoral neurons to the light-adapted ERG of the Nob mouse, a model for complete-type congenital stationary night blindness (CSNB1) that lacks a b-wave from depolarizing bipolar cells. Ganzfeld ERGs were recorded from anesthetized adult control mice, control mice injected intravitreally with L-2-amino-4-phosphonobutyric acid (Control APB mice) to remove On pathway activity, and Nob mice. ERGs also were recorded after PDA (cis-2,3-piperidine-dicarboxylic acid, 3-5mM) was injected to block transmission to hyperpolarizing (Off) bipolar and horizontal cells, and all third-order neurons. Stimuli were brief (<4ms, 0.4-2.5log sc td s) and long (200ms, 2.5-4.6log sc td) LED flashes (lambda(max)=513nm, on a rod suppressing background (2.6log sc td). Sinusoidal modulation of the LEDs (mean, 2.6log sc td; contrast, 100%; 3-36Hz) was used to study flicker ERGs. Brief-flash ERGs of Nob mice presented as long-lasting negative waves with a positive-going intrusion that started about 50ms after the flash and peaked around 120ms. Control APB mice had similar responses, and in both cases, PDA removed the positive-going intrusion. For long flashes, PDA removed a small, slow "d-wave" after light offset. With sinusoidal stimulation, the fundamental (F1) amplitude of control mice ERG peaked at 8Hz ( approximately 70microV). For Nob mice the peak was approximately 20microV at 6Hz before PDA and approximately 10muV at 3Hz or lower after PDA. F1 responses were present up to 21Hz in control and Nob eyes and 15Hz in Nob eyes after PDA. Between 3 and 6Hz, F1 phase was 170-210 degrees more delayed in Nob than control mice; phase was hardly altered by PDA. With vector analysis, a substantial postreceptoral input to the Nob flicker ERG was revealed. In control mice, the second harmonic (F2) response showed peaks of approximately 10mocrpV at 3Hz and 13Hz. Nob mice showed almost no F2. In summary, in this study it was found that
Seal, Rebecca P; Akil, Omar; Yi, Eunyoung; Weber, Christopher M; Grant, Lisa; Yoo, Jong; Clause, Amanda; Kandler, Karl; Noebels, Jeffrey L; Glowatzki, Elisabeth; Lustig, Lawrence R; Edwards, Robert H
The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.
Yun, Nadezhda E; Seregin, Alexey V; Walker, David H; Popov, Vsevolod L; Walker, Aida G; Smith, Jeanon N; Miller, Milagros; de la Torre, Juan C; Smith, Jennifer K; Borisevich, Viktoriya; Fair, Joseph N; Wauquier, Nadia; Grant, Donald S; Bockarie, Bayon; Bente, Dennis; Paessler, Slobodan
Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.
Holm, Anne Trommelholt; Wulf-Johansson, Helle; Hvidsten, Svend; Jorgensen, Patricia Troest; Schlosser, Anders; Pilecki, Bartosz; Ormhøj, Maria; Moeller, Jesper Bonnet; Johannsen, Claus; Baun, Christina; Andersen, Thomas; Schneider, Jan Philipp; Hegermann, Jan; Ochs, Matthias; Götz, Alexander A; Schulz, Holger; de Angelis, Martin Hrabě; Vestbo, Jørgen; Holmskov, Uffe; Sorensen, Grith Lykke
Microfibrillar-associated protein 4 (MFAP4) is localized to elastic fibers in blood vessels and the interalveolar septa of the lungs and is further present in bronchoalveolar lavage. Mfap4 has been previously suggested to be involved in elastogenesis in the lung. We tested this prediction and aimed to characterize the pulmonary function changes and emphysematous changes that occur in Mfap4-deficient (Mfap4(-/-)) mice. Significant changes included increases in total lung capacity and compliance, which were evident in Mfap4(-/-) mice at 6 and 8 mo but not at 3 mo of age. Using in vivo breath-hold gated microcomputed tomography (micro-CT) in 8-mo-old Mfap4(-/-) mice, we found that the mean density of the lung parenchyma was decreased, and the low-attenuation area (LAA) was significantly increased by 14% compared with Mfap4(+/+) mice. Transmission electron microscopy (TEM) did not reveal differences in the organization of elastic fibers, and there was no difference in elastin content, but a borderline significant increase in elastin mRNA expression in 3-mo-old mice. Stereological analysis showed that alveolar surface density in relation to the lung parenchyma and total alveolar surface area inside of the lung were both significantly decreased in Mfap4(-/-) mice by 25 and 15%, respectively. The data did not support an essential role of MFAP4 in pulmonary elastic fiber organization or content but indicated increased turnover in young Mfap4(-/-) mice. However, Mfap4(-/-) mice developed a spontaneous loss of lung function, which was evident at 6 mo of age, and moderate air space enlargement, with emphysema-like changes.
Lucas, Elizabeth K.; Dougherty, Sarah E.; McMeekin, Laura J.; Trinh, Alisa T.; Reid, Courtney S.; Cowell, Rita M.
Accumulating evidence implicates the transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) in the pathophysiology of Huntington Disease (HD). Adult PGC-1α −/− mice exhibit striatal neurodegeneration, and reductions in the expression of PGC-1α have been observed in striatum and muscle of HD patients as well as in animal models of the disease. However, it is unknown whether decreased expression of PGC-1α alone is sufficient to lead to the motor phenotype and striatal pathology characteristic of HD. For the first time, we show that young PGC-1α −/− mice exhibit severe rotarod deficits, decreased rearing behavior, and increased occurrence of tremor in addition to the previously described hindlimb clasping. Motor impairment and striatal vacuolation are apparent in PGC-1α −/− mice by four weeks of age and do not improve or decline by twelve weeks of age. The behavioral and pathological phenotype of PGC-1α −/− mice can be completely recapitulated by conditional nervous system deletion of PGC-1α, indicating that peripheral effects are not responsible for the observed abnormalities. Evaluation of the transcriptional profile of PGC-1α −/− striatal neuron populations and comparison to striatal neuron profiles of R6/2 HD mice revealed that PGC-1α deficiency alone is not sufficient to cause the transcriptional changes observed in this HD mouse model. In contrast to R6/2 HD mice, PGC-1α −/− mice show increases in the expression of medium spiny neuron (MSN) markers with age, suggesting that the observed behavioral and structural abnormalities are not primarily due to MSN loss, the defining pathological feature of HD. These results indicate that PGC-1α is required for the proper development of motor circuitry and transcriptional homeostasis in MSNs and that developmental disruption of PGC-1α leads to long-term alterations in motor functioning. PMID:22916173
Powell, David R; DaCosta, Christopher M; Gay, Jason; Ding, Zhi-Ming; Smith, Melinda; Greer, Jennifer; Doree, Deon; Jeter-Jones, Sabrina; Mseeh, Faika; Rodriguez, Lawrence A; Harris, Angela; Buhring, Lindsey; Platt, Kenneth A; Vogel, Peter; Brommage, Robert; Shadoan, Melanie K; Sands, Arthur T; Zambrowicz, Brian
Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs <1% of the UGE of DKOs. Consistent with their increased UGE, DKOs had lower fasting blood glucose and improved intraperitoneal glucose tolerance than Sglt2 KOs. In conclusion, 1) Sglt2 is the major renal glucose transporter, but Sglt1 reabsorbs 70% of filtered glucose if Sglt2 is absent; 2) mice lacking Sglt2 display improved glucose tolerance despite UGE that is 30% of maximum; 3) Sglt1 KO mice respond to oral glucose with increased circulating GLP-1; and 4) DKO mice have improved glycemic control over mice lacking Sglt2 alone. These data suggest that, in patients with type 2 diabetes, combining pharmacological SGLT2 inhibition with complete renal and/or partial intestinal SGLT1 inhibition may improve glycemic control over that achieved by SGLT2 inhibition alone.
Naulé, Lydie; Robert, Vincent; Parmentier, Caroline; Martini, Mariangela; Keller, Matthieu; Cohen-Solal, Martine; Hardin-Pouzet, Hélène; Grange-Messent, Valérie; Franceschini, Isabelle; Mhaouty-Kodja, Sakina
Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and β. Here we investigate the role of ERβ on central effects of oestradiol, by generating a mouse line specifically lacking the ERβ gene in neuronal and glial cells. Central ERβ deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERβ in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.
Esther, C R; Howard, T E; Marino, E M; Goddard, J M; Capecchi, M R; Bernstein, K E
Mammals produce two isozymes of angiotensin-converting enzyme (ACE). Somatic ACE plays an important role in the control of blood pressure. The function of testis ACE, produced by male and germ cells, is not known. To examine the roles of these isozymes, we used targeted homologous recombination to introduce a modified ACE allele into a mouse line. Mice homozygous for this mutant allele lack both ACE isozymes and have markedly reduced blood pressures. Contrary to a previous report, we found heterozygous male mice to have normal blood pressures. Homozygous mutant mice also have severe renal disease. The renal papilla is markedly reduced, and the intrarenal arteries exhibit vascular hyperplasia associated with a perivascular inflammatory infiltrate. These animals cannot effectively concentrate urine. They also have an abnormally low urinary sodium to potassium ratio despite reduced levels of aldosterone. Homozygous mutant male mice sire significantly smaller litters than wild-type male mice; however, no defect in sperm number, morphology, or motility was detected. ACE-deficient animals demonstrate the role of this enzyme in systemic blood pressure, renal development and function, and male fertility.
Ruzza, C; Pulga, A; Rizzi, A; Marzola, G; Guerrini, R; Calo', G
Neuropeptide S (NPS) is the endogenous ligand of a previously orphan receptor now named NPSR. In the brain NPS regulates several biological functions including anxiety, arousal, locomotion, food intake, learning and memory, pain and drug abuse. Mice lacking the NPSR gene (NPSR(-/-)) represent an useful tool to investigate the neurobiology of the NPS/NPSR system. NPSR(-/-) mice have been generated in a 129S6/SvEv genetic background. In the present study we generated CD-1 congenic NPSR(+/+) and NPSR(-/-) mice and investigated their phenotype and sensitivity to NPS in various behavioural assays. The phenotype analysis revealed no locomotor differences between NPSR(+/+) and NPSR(-/-) mice. The behaviour of NPSR(+/+) and NPSR(-/-) mice in the righting reflex test was superimposable. No differences were recorded between the two genotypes in the elevated plus maze, open field and stress-induced hyperthermia tests, with the exception of rearing behaviour that was reduced in knockout animals. Moreover the behaviour of NPSR(+/+) and NPSR(-/-) mice in the forced swimming, novel object recognition and formalin assays was similar. The stimulatory effects of NPS in the locomotor activity test and its anxiolytic-like actions in the elevated plus maze and open field assays were evident in NPSR(+/+) but not NPSR(-/-) animals. In conclusion, the present study indicates that the NPS/NPSR system does not tonically control locomotion, sensitivity to diazepam, anxiety, depressive-like behaviours, memory and pain transmission in mice. Furthermore our results clearly show that the product of the NPSR gene represents the mandatory protein for all the NPS biological effects so far described.
Valenza, Marta; DiLeo, Alyssa; Steardo, Luca; Cottone, Pietro; Sabino, Valentina
Rationale The Sigma-1 receptor (Sig-1R) is a chaperone protein that has been implicated in drug abuse and addiction. Multiple studies have characterized the role the Sig-1R plays in psychostimulants addiction, but fewer studies have specifically investigated its role in alcohol addiction. We have previously shown that antagonism of the Sig-1R reduces excessive drinking and motivation to drink, whereas agonism induces binge-like drinking in rodents. Objectives The objectives of these studies were to investigate the impact of Sig-1R gene deletion in C57Bl/6J mice on ethanol drinking and other ethanol-related behaviors. Methods We used an extensive panel of behavioral tests to examine ethanol actions in male, adult mice lacking Oprs1, the gene encoding the Sig-1R. To compare ethanol drinking behavior, Sig-1 knockout (KO) and wild type (WT) mice were subject to a two-bottle choice, continuous access paradigm with different concentrations of ethanol (3%–20% v/v) vs. water. Consumption of sweet and bitter solutions was also assessed in Sig-1R KO and WT mice. Finally, motor stimulant sensitivity, taste aversion and ataxic effects of ethanol were assessed. Results Sig-1R KO mice displayed higher ethanol intake compared to WT mice; the two genotypes did not differ in their sweet or bitter taste perception. Sig-1R KO mice showed lower sensitivity to ethanol stimulant effects, but greater sensitivity to its taste aversive effects. Ethanol-induced sedation was unaltered in the mutants. Conclusions Our results suggest that the deletion of the Sig-1R increases ethanol consumption, likely by decreasing its rewarding effects, and therefore indicating that the Sig-1R is involved in modulation of the reinforcing effects of alcohol. PMID:26462569
Luria, Ayala; Morisseau, Christophe; Tsai, Hsing-Ju; Yang, Jun; Inceoglu, Bora; De Taeye, Bart; Watkins, Steven M; Wiest, Michelle M; German, J Bruce; Hammock, Bruce D
Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.
Hanner, Fiona; Chambrey, Régine; Bourgeois, Soline; Meer, Elliott; Mucsi, István; Rosivall, László; Shull, Gary E; Lorenz, John N; Eladari, Dominique; Peti-Peterdi, János
Macula densa (MD) cells express the Na(+)/H(+) exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2(+/+)) and NHE2 knockout (NHE2(-/-)) mice. Immunofluorescence studies indicated that NHE2(-/-) mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2(-/-) mice was increased approximately 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2(-/-) mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2(+/+) mice, but the response was blunted in NHE2(-/-) mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2(-/-) mice compared with wild type. NHE2(-/-) mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2(-/-) kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE(2)-renin release pathway.
WEN, Xin; LACRUZ, Rodrigo S.; PAINE, Michael L.
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
Wen, Xin; Lacruz, Rodrigo S; Paine, Michael L
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 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.
Trigo, José M.; Zimmer, Andreas; Maldonado, Rafael
The endogenous opioid system plays an important role in the behavioral effects of nicotine. Thus, μ-opioid receptor and the endogenous opioids derived from proenkephalin are involved in the central effects of nicotine. However, the role played by the different endogenous opioid peptides in the acute and chronic effects of nicotine remains to be fully established. Mice lacking β-endorphin were acutely injected with nicotine at different doses to evaluate locomotor, anxiogenic and antinociceptive responses. The rewarding properties of nicotine were evaluated by using the conditioned place-preference paradigm. Mice chronically treated with nicotine were acutely injected with mecamylamine to study the behavioral expression of nicotine withdrawal. Mice lacking β-endorphin exhibited a spontaneous hypoalgesia and hyperlocomotion and a reduction on the anxiogenic and rewarding effects induced by nicotine. Nicotine induced similar antinociception and hypolocomotion in both genotypes and no differences were found in the development of physical dependence. The dissociation between nicotine rewarding properties and physical dependence suggests a differential implication of β-endorphin in these addictive related responses. PMID:19376143
Trigo, José M; Zimmer, Andreas; Maldonado, Rafael
The endogenous opioid system plays an important role in the behavioral effects of nicotine. Thus, micro-opioid receptor and the endogenous opioids derived from proenkephalin are involved in the central effects of nicotine. However, the role played by the different endogenous opioid peptides in the acute and chronic effects of nicotine remains to be fully established. Mice lacking beta-endorphin were acutely injected with nicotine at different doses to evaluate locomotor, anxiogenic and antinociceptive responses. The rewarding properties of nicotine were evaluated by using the conditioned place-preference paradigm. Mice chronically treated with nicotine were acutely injected with mecamylamine to study the behavioral expression of nicotine withdrawal. Mice lacking beta-endorphin exhibited a spontaneous hypoalgesia and hyperlocomotion and a reduction on the anxiogenic and rewarding effects induced by nicotine. Nicotine induced similar antinociception and hypolocomotion in both genotypes and no differences were found in the development of physical dependence. The dissociation between nicotine rewarding properties and physical dependence suggests a differential implication of beta-endorphin in these addictive related responses.
Fuller, Charles; Ringgold, Kristyn
The circadian pacemaker can be phase shifted and entrained by appropriately timed locomotor activity, however the mechanism(s) involved remain poorly understood. Recent work in our lab has suggested the involvement of the vestibular otolith organs in activity-induced changes within the circadian timing system (CTS). For example, we have shown that changes in circa-dian period and phase in response to locomotion (wheel running) require functional macular gravity receptors. We believe the neurovestibular system is responsible for the transduction of gravitoinertial input associated with the types of locomotor activity that are known to af-fect the pacemaker. This study investigated the hypothesis that daily, timed gravitoinertial stimuli, as applied by centrifugation. would induce entrainment of circadian rhythms in only those animals with functional afferent vestibular input. To test this hypothesis, , chemically labyrinthectomized (Labx) mice, mice lacking macular vestibular input (head tilt or hets) and wildtype (WT) littermates were implanted i.p. with biotelemetry and individually housed in a 4-meter diameter centrifuge in constant darkness (DD). After 2 weeks in DD, the mice were exposed daily to 2G via centrifugation from 1000-1200 for 9 weeks. Only WT mice showed entrainment to the daily 2G pulse. The 2G pulse was then re-set to occur at 1200-1400 for 4 weeks. Only WT mice demonstrated a phase shift in response to the re-setting of the 2G pulse and subsequent re-entrainment to the new centrifugation schedule. These results provide further evidence that gravitoinertial stimuli require a functional vestibular system to both en-train and phase shift the CTS. Entrainment among only WT mice supports the role of macular gravity receptive cells in modulation of the CTS while also providing a functional mechanism by which gravitoinertial stimuli, including locomotor activity, may affect the pacemaker.
Bello, Estefanía P; Mateo, Yolanda; Gelman, Diego M; Noaín, Daniela; Shin, Jung H; Low, Malcolm J; Alvarez, Verónica A; Lovinger, David M; Rubinstein, Marcelo
Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2loxP/loxP; Dat+/IRES-cre, referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine. PMID:21743470
Contreras-Jurado, Constanza; García-Serrano, Laura; Martínez-Fernández, Mónica; Ruiz-Llorente, Lidia; Paramio, Jesus M; Aranda, Ana
Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies.
Li, Nan; Liu, Zhenghui; Zhang, Yue; Chen, Qiaoyuan; Liu, Peng; Cheng, C Yan; Lee, Will M; Chen, Yongmei; Han, Daishu
The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double-knockout (Axl(-/-)Mer(-/-)) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freund's adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testes, impaired permeability of the blood-testis barrier and generation of autoantibodies against germ cell antigens in Axl(-/-)Mer(-/-) mice. Immunization also induced mild EAO in Axl or Mer single-gene-knockout mice. By contrast, a single immunization failed to induce EAO in wild-type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.
Martínez-Fernández, Mónica; Ruiz-Llorente, Lidia; Paramio, Jesus M.; Aranda, Ana
Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies. PMID:25254665
Espinoza, Stefano; Ghisi, Valentina; Emanuele, Marco; Leo, Damiana; Sukhanov, Ilya; Sotnikova, Tatiana D; Chieregatti, Evelina; Gainetdinov, Raul R
Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3β. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences.
Hsu, Jong-Wei; Hsu, Iawen; Xu, Defeng; Miyamoto, Hiroshi; Liang, Liang; Wu, Xue-Ru; Shyr, Chih-Rong; Chang, Chawnshang
Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.
Background Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease. Results Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10−/−) to study its functional role. Moreover, mice devoid of exon 10 (E10+/−) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/− and wild-type mice, and their muscular grip strength was less than that of E10+/− mice. The performance of E10+/− mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found. Conclusion Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions. PMID:24261309
Schneider, Jan Philipp; Arkenau, Martina; Knudsen, Lars; Wedekind, Dirk; Ochs, Matthias
Pulmonary surfactant, a mixture of lipids and proteins at the air-liquid interface of alveoli, prevents the lungs from collapsing due to surface tension. One constituent is surfactant-associated protein-D (SP-D), a protein involved in surfactant homeostasis and innate immunity. Mice deficient in SP-D (SP-D (-/-)) has been described as developing a characteristic phenotype which affects the surfactant system (including changes in the intra-cellular and intra-alveolar surfactant pool, alveolar epithelial type II cells and alveolar macrophages), lung architecture and its inflammatory state (development of an emphysema-like pathology, inflammatory cell infiltration). Furthermore, it has been described that these mice develop sub-pleural fibrosis and a thickening of alveolar septal walls. The aim of the present study was to systematically investigate the long term progression of this phenotype with special focus on parenchymal remodeling, whether there are progressive emphysematous changes and whether there is progressive septal wall thickening which might indicate the development of pulmonary fibrosis. By means of design-based stereology and light microscopy, lungs of wild type (wt) and SP-D (-/-) mice of four age groups (3, 6, 12 and ∼18 months) were investigated. The data do not suggest a relevant spontaneous pro-fibrotic remodeling or a destructive process in the aging SP-D (-/-) mice. We demonstrated neither a significant destructive emphysema nor significant thickening of alveolar septal walls, but the data suggest an increase in the number weighted mean alveolar volume in aging SP-D (-/-) mice without loss of alveoli or alveolar epithelial surface area per lung. This increase may reflect over-distension due to altered mechanical properties of alveoli. In the light of our findings and data from the literature, the question arises as to whether a lack of SP-D promotes structural changes in the lung which have been described as being associated with aging lungs
Kim, Susie; Hill, Adele; Warman, Matthew L.; Smits, Patrick
Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development. PMID:23185636
Luck, Chloe; Vitaterna, Martha H; Wevrick, Rachel
The etiology of abnormal eating behaviors, including binge-eating disorder, is poorly understood. The neural circuits modulating the activities of the neurotransmitters dopamine and serotonin are proposed to be dysfunctional in individuals suffering from eating disorders. Prader-Willi syndrome is a neurodevelopmental disorder that causes extreme food seeking and binge-eating behaviors together with reduced satiety. One of the genes implicated in Prader-Willi syndrome, Magel2, is highly expressed in the regions of the brain that control appetite. Our objective was to examine behaviors relevant to feeding and the neural circuits controlling feeding in a mouse model of Prader-Willi syndrome that lacks expression of the Magel2 gene. We performed behavioral tests related to dopaminergic function, measuring cocaine-induced hyperlocomotion, binge eating, and saccharin-induced anhedonia in Magel2-deficient mice. Next, we analyzed dopaminergic neurons in various brain regions and compared these findings between genotypes. Finally, we examined biochemical markers in the brain under standard diet, high-fat diet, and withdrawal from a high-fat diet conditions. We identified abnormal behaviors and biomarkers reflecting dopaminergic dysfunction in mice lacking Magel2. Our results provide a biological framework for clinical studies of dopaminergic function in children with Prader-Willi syndrome, and may also provide insight into binge-eating disorders that occur in the general population. (PsycINFO Database Record
Chan, Wilson; Singh, Sanmeet; Keshav, Taj; Dewan, Ramita; Eberly, Christian; Maurer, Robert; Nunez-Parra, Alexia; Araneda, Ricardo C.
The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1−/− and the M1/M3−/− knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing. PMID:28210219
McHugh, Kevin P.; Hodivala-Dilke, Kairbaan; Zheng, Ming-Hao; Namba, Noriyuki; Lam, Jonathan; Novack, Deborah; Feng, Xu; Ross, F. Patrick; Hynes, Richard O.; Teitelbaum, Steven L.
Osteoclasts express the αvβ3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the β3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that αvβ3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, αvβ5, does not substitute for αvβ3 during cytokine stimulation or authentic osteoclastogenesis. β3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, β3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in β3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin αvβ3 is essential for normal osteoclast function. PMID:10683372
Kronenberg, Golo; Harms, Christoph; Sobol, Robert W; Cardozo-Pelaez, Fernando; Linhart, Heinz; Winter, Benjamin; Balkaya, Mustafa; Gertz, Karen; Gay, Shanna B; Cox, David; Eckart, Sarah; Ahmadi, Michael; Juckel, Georg; Kempermann, Gerd; Hellweg, Rainer; Sohr, Reinhard; Hörtnagl, Heide; Wilson, Samuel H; Jaenisch, Rudolf; Endres, Matthias
Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung-/- embryonic fibroblasts, and conferred death of cultured Ung-/- hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/- but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung-/- mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.
Gamu, Daniel; Trinh, Anton; Bombardier, Eric; Tupling, A Russell
Several rodent models of obesity have been shown to develop excessive adiposity only when voluntary cage ambulation is restricted. We have previously shown that mice lacking the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump regulatory protein sarcolipin (Sln(-/-)), an uncoupler of Ca(2+) uptake, develop excessive diet-induced obesity under standard housing conditions. However, it is unclear whether this phenotype is due, in part, to the sedentary housing environment in which these animals are kept. To address this, we allowed wild-type and Sln(-/-) animals ad libitum access to voluntary wheel running while consuming a standard chow or high-fat diet for 8 weeks. During this period, wheel revolutions were monitored along with weekly mass gain. Postdiet glucose tolerance and visceral adiposity were also taken. The volume of wheel running completed was similar between genotype, regardless of diet. Although voluntary activity reduced mass gain relative to sedentary controls within each diet (P < 0.05), visceral adiposity was surprisingly unaltered with activity. However, Sln(-/-) mice developed excessive obesity (P < 0.05) and glucose intolerance (P < 0.05) with high-fat feeding relative to wild-type controls. These findings indicate that the excessive diet-induced obese phenotype previously observed in Sln(-/-) mice is not the result of severely restricted daily ambulation, but in fact the inability to recruit uncoupling of the Ca(2+)-ATPase pump.
Pifferi, Simone; Dibattista, Michele; Sagheddu, Claudia; Boccaccio, Anna; Al Qteishat, Ahmed; Ghirardi, Filippo; Tirindelli, Roberto; Menini, Anna
Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. Ca(2+) activates a Cl(-) current that produces an efflux of Cl(-) ions and amplifies the depolarization. The molecular identity of Ca(2+)-activated Cl(-) channels is still elusive, although some bestrophins have been shown to function as Ca(2+)-activated Cl(-) channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca(2+)-activated Cl(-) channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca(2+)-activated Cl(-) currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca(2+)-activated Cl(-) currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.
Pifferi, Simone; Dibattista, Michele; Sagheddu, Claudia; Boccaccio, Anna; Al Qteishat, Ahmed; Ghirardi, Filippo; Tirindelli, Roberto; Menini, Anna
Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca2+ into the cilia. Ca2+ activates a Cl− current that produces an efflux of Cl− ions and amplifies the depolarization. The molecular identity of Ca2+-activated Cl− channels is still elusive, although some bestrophins have been shown to function as Ca2+-activated Cl− channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca2+-activated Cl− currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca2+-activated Cl− currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons. PMID:19622610
Yang, Lin; Kirby, James E.; Sunwoo, Hongjae; Lee, Jeannie T.
X-chromosome inactivation (XCI) compensates for differences in X-chromosome number between male and female mammals. XCI is orchestrated by Xist RNA, whose expression in early development leads to transcriptional silencing of one X chromosome in the female. Knockout studies have established a requirement for Xist with inviability of female embryos that inherit an Xist deletion from the father. Here, we report that female mice lacking Xist RNA can, surprisingly, develop and survive to term. Xist-null females are born at lower frequency and are smaller at birth, but organogenesis is mostly normal. Transcriptomic analysis indicates significant overexpression of hundreds of X-linked genes across multiple tissues. Therefore, Xist-null mice can develop to term in spite of a deficiency of dosage compensation. However, the degree of X-autosomal dosage imbalance was less than anticipated (1.14-fold to 1.36-fold). Thus, partial dosage compensation can be achieved without Xist, supporting the idea of inherent genome balance. Nevertheless, to date, none of the mutant mice has survived beyond weaning stage. Sudden death is associated with failure of postnatal organ maturation. Our data suggest Xist-independent mechanisms of dosage compensation and demonstrate that small deviations from X-autosomal balance can have profound effects on overall fitness. PMID:27542829
Allen, Annette E; Cameron, Morven A; Brown, Timothy M; Vugler, Anthony A; Lucas, Robert J
The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-);Cnga3(-/-);Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod α transducin (Gnat1); the cone-specific α3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone α transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction.
Zhang, Yu; Kwon, Sohee; Yamaguchi, Teppei; Cubizolles, Fabien; Rousseaux, Sophie; Kneissel, Michaela; Cao, Chun; Li, Na; Cheng, Hwei-Ling; Chua, Katrin; Lombard, David; Mizeracki, Adam; Matthias, Gabriele; Alt, Frederick W; Khochbin, Saadi; Matthias, Patrick
Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.
Malakauskas, Sandra M.; Kourany, Wissam M.; Zhang, Xiao Yin; Lu, Danhong; Stevens, Robert D.; Koves, Timothy R.; Hohmeier, Hans E.; Muoio, Deborah M.; Newgard, Christopher B.; Le, Thu H.
Collectrin is a downstream target of the transcription factor hepatocyte nuclear factor-1α (HNF-1α), which is mutated in maturity-onset diabetes of the young subtype 3 (MODY3). Evidence from transgenic mouse models with collectrin overexpression in pancreatic islets suggests divergent roles for collectrin in influencing β-cell mass and insulin exocytosis. To clarify the function of collectrin in the pancreas, we used a mouse line with targeted deletion of the gene. We examined pancreas morphology, glucose homeostasis by ip glucose tolerance testing (IPGTT) and insulin tolerance testing (IPITT), and pancreas function by in vivo acute-phase insulin response determination and glucose-stimulated insulin secretion from isolated islets. We find no difference in either pancreas morphology or function between wild-type and collectrin-deficient animals (Tmem27−/y). However, we note that by 6 months of age, Tmem27−/y mice exhibit increased insulin sensitivity by IPITT and decreased adiposity by dual-energy x-ray absorptiometry scanning compared with wild-type. We have previously reported that Tmem27−/y mice exhibit profound aminoaciduria due to failed renal recovery. We now demonstrate that Tmem27−/y animals also display inappropriate excretion of some short-chain acylcarnitines derived from amino acid and fatty acid oxidation. We provide further evidence for compensatory up-regulation of oxidative metabolism in Tmem27−/y mice, along with enhanced protein turnover associated with preserved lean mass even out to 1.5 yr of age. Our studies suggest that collectrin-deficient mice activate a number of adaptive mechanisms to defend energy homeostasis in the setting of ongoing nutrient losses. PMID:19246514
Fontán-Lozano, Angela; Romero-Granados, Rocío; del-Pozo-Martín, Yaiza; Suárez-Pereira, Irene; Delgado-García, José María; Penninger, Josef M; Carrión, Angel Manuel
Memory deficits in aging affect millions of people and are often disturbing to those concerned. Dissection of the molecular control of learning and memory is paramount to understand and possibly enhance cognitive functions. Old-age memory loss also has been recently linked to altered Ca(2+) homeostasis. We have previously identified DREAM (downstream regulatory element antagonistic modulator), a member of the neuronal Ca(2+) sensor superfamily of EF-hand proteins, with specific roles in different cell compartments. In the nucleus, DREAM is a Ca(2+)-dependent transcriptional repressor, binding to specific DNA signatures, or interacting with nucleoproteins regulating their transcriptional properties. Also, we and others have shown that dream mutant (dream(-/-)) mice exhibit marked analgesia. Here we report that dream(-/-) mice exhibit markedly enhanced learning and synaptic plasticity related to improved cognition. Mechanistically, DREAM functions as a negative regulator of the key memory factor CREB in a Ca(2+)-dependent manner, and loss of DREAM facilitates CREB-dependent transcription during learning. Intriguingly, 18-month-old dream(-/-) mice display learning and memory capacities similar to young mice. Moreover, loss of DREAM protects from brain degeneration in aging. These data identify the Ca(2+)-regulated "pain gene" DREAM as a novel key regulator of memory and brain aging.
Kashiwazaki, Hiromi; Kakizaki, Masatoshi; Ikehara, Yuzuru; Togayachi, Akira; Narimatsu, Hisashi; Watanabe, Rihito
Carbohydrate structures, including Lewis X (Le(x)), which is not synthesized in mutant mice that lack α1,3-fucosyltransferase 9 (Fut9(-/-)), are involved in cell-cell recognition and inflammation. However, immunological alteration in Fut9(-/-) mice has not been studied. Thus, the inflammatory response of Fut9(-/-) mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9(-/-) mice after infection was more extensive compared with that of wild-type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild-type mice. Furthermore, the reduction in cell numbers in the spleens of wild-type mice after infection was not observed in the infected Fut9(-/-) mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9(-/-) and wild-type mice except for interferon-β expression, some of those in the spleens, including interferon-γ, interleukin-6, and monocyte chemoattractant protein 1, showed higher levels in Fut9(-/-) than in wild-type mice. Furthermore, Fut9(-/-) mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild-type mice. These results indicate that Le(x) structures are involved in host responses against viral or bacterial challenges.
Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma; Cornett, Ben; Mackie, Ken; Bradshaw, Heather
Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic
Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma; Cornett, Ben; Mackie, Ken; Bradshaw, Heather; Hohmann, Andrea G
Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic
Barakat, Waleed; Elshazly, Shimaa M; Mahmoud, Amr A A
Spirulina is a blue-green alga used as a dietary supplement. It has been shown to possess anti-inflammatory, antioxidant, and hepatoprotective properties. This study was designed to evaluate the antitumor effect of spirulina (200 and 800 mg/kg) against a murine model of solid Ehrlich carcinoma compared to a standard chemotherapeutic drug, 5-fluorouracil (20 mg/kg). Untreated mice developed a palpable solid tumor after 13 days. Unlike fluorouracil, spirulina at the investigated two dose levels failed to exert any protective effect. In addition, spirulina did not potentiate the antitumor effect of fluorouracil when they were administered concurrently. Interestingly, their combined administration resulted in a dose-dependent increase in mortality. The present study demonstrates that spirulina lacks antitumor effect against this model of solid Ehrlich carcinoma and increased mortality when combined with fluorouracil. However, the implicated mechanism is still elusive.
Barakat, Waleed; Elshazly, Shimaa M.; Mahmoud, Amr A. A.
Spirulina is a blue-green alga used as a dietary supplement. It has been shown to possess anti-inflammatory, antioxidant, and hepatoprotective properties. This study was designed to evaluate the antitumor effect of spirulina (200 and 800 mg/kg) against a murine model of solid Ehrlich carcinoma compared to a standard chemotherapeutic drug, 5-fluorouracil (20 mg/kg). Untreated mice developed a palpable solid tumor after 13 days. Unlike fluorouracil, spirulina at the investigated two dose levels failed to exert any protective effect. In addition, spirulina did not potentiate the antitumor effect of fluorouracil when they were administered concurrently. Interestingly, their combined administration resulted in a dose-dependent increase in mortality. The present study demonstrates that spirulina lacks antitumor effect against this model of solid Ehrlich carcinoma and increased mortality when combined with fluorouracil. However, the implicated mechanism is still elusive. PMID:26366170
Ushiki, Takashi; Huntington, Nicholas D.; Glaser, Stefan P.; Kiu, Hiu; Georgiou, Angela; Zhang, Jian-Guo; Nicola, Nicos A.; Roberts, Andrew W.; Alexander, Warren S.
The Suppressors of Cytokine Signalling (SOCS) proteins are negative regulators of cytokine signalling required to prevent excess cellular responses. SOCS1 and SOCS3 are essential to prevent inflammatory disease, SOCS1 by attenuating responses to IFNγ and gamma-common (γc) cytokines, and SOCS3 via regulation of G-CSF and IL-6 signalling. SOCS1 and SOCS3 show significant sequence homology and are the only SOCS proteins to possess a KIR domain. The possibility of overlapping or redundant functions was investigated in inflammatory disease via generation of mice lacking both SOCS1 and SOCS3 in hematopoietic cells. Loss of SOCS3 significantly accelerated the pathology and inflammatory disease characteristic of SOCS1 deficiency. We propose a model in which SOCS1 and SOCS3 operate independently to control specific cytokine responses and together modulate the proliferation and activation of lymphoid and myeloid cells to prevent rapid inflammatory disease. PMID:27583437
Wey, Margaret Chia-Ying; Fernandez, Elizabeth; Martinez, Paul Anthony; Sullivan, Patricia; Goldstein, David S; Strong, Randy
Previous studies have reported elevated levels of biogenic aldehydes in the brains of patients with Parkinson's disease (PD). In the brain, aldehydes are primarily detoxified by aldehyde dehydrogenases (ALDH). Reduced ALDH1 expression in surviving midbrain dopamine neurons has been reported in brains of patients who died with PD. In addition, impaired complex I activity, which is well documented in PD, reduces the availability of the NAD(+) co-factor required by multiple ALDH isoforms to catalyze the removal of biogenic aldehydes. We hypothesized that chronically decreased function of multiple aldehyde dehydrogenases consequent to exposure to environmental toxins and/or reduced ALDH expression, plays an important role in the pathophysiology of PD. To address this hypothesis, we generated mice null for Aldh1a1 and Aldh2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Aldh1a1(-/-)×Aldh2(-/-) mice exhibited age-dependent deficits in motor performance assessed by gait analysis and by performance on an accelerating rotarod. Intraperitoneal administration of L-DOPA plus benserazide alleviated the deficits in motor performance. We observed a significant loss of neurons immunoreactive for tyrosine hydroxylase (TH) in the substantia nigra and a reduction of dopamine and metabolites in the striatum of Aldh1a1(-/-)×Aldh2(-/-) mice. We also observed significant increases in biogenic aldehydes reported to be neurotoxic, including 4-hydroxynonenal (4-HNE) and the aldehyde intermediate of dopamine metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL). These results support the hypothesis that impaired detoxification of biogenic aldehydes may be important in the pathophysiology of PD and suggest that Aldh1a1(-/-)×Aldh2(-/-) mice may be a useful animal model of PD.
Verwey, Michael; Grant, Alanna; Meti, Nicholas; Adye-White, Lauren; Torres-Berrío, Angelica; Rioux, Veronique; Lévesque, Martin; Charron, Frederic; Flores, Cecilia
Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon(-/-) embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon(-/-) mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon(-/-) mice at either age. Moreover, adult Cdon(-/-) mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon(-/-) mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.
Grant, Alanna; Meti, Nicholas; Adye-White, Lauren; Rioux, Veronique
Abstract Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon−/− embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon−/− mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon−/− mice at either age. Moreover, adult Cdon−/− mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon−/− mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry. PMID:27419218
Liu, Su; Lee, Yi-Fen; Chou, Samuel; Uno, Hideo; Li, Gonghui; Brookes, Paul; Massett, Michael P; Wu, Qiao; Chen, Lu-Min; Chang, Chawnshang
The estimated incidence of mitochondrial diseases in humans is approximately 1:5000 to 1:10,000, whereas the molecular mechanisms for more than 50% of human mitochondrial disease cases still remain unclear. Here we report that mice lacking testicular nuclear receptor 4 (TR4(-/-)) suffered mitochondrial myopathy, and histological examination of TR4(-/-) soleus muscle revealed abnormal mitochondrial accumulation. In addition, increased serum lactate levels, decreased mitochondrial ATP production, and decreased electron transport chain complex I activity were found in TR4(-/-) mice. Restoration of TR4 into TR4(-/-) myoblasts rescued mitochondrial ATP generation capacity and complex I activity. Further real-time PCR quantification and promoter studies found TR4 could modulate complex I activity via transcriptionally regulating the complex I assembly factor NDUFAF1, and restoration of NDUFAF1 level in TR4(-/-) myoblasts increased mitochondrial ATP generation capacity and complex I activity. Together, these results suggest that TR4 plays vital roles in mitochondrial function, which may help us to better understand the pathogenesis of mitochondrial myopathy, and targeting TR4 via its ligands/activators may allow us to develop better therapeutic approaches.
Kahan, V; Ribeiro, D A; Egydio, F; Barros, L A; Tomimori, J; Tufik, S; Andersen, M L
Skin naturally changes with age, becoming more fragile. Various stimuli can alter skin integrity. The aim of this study was to evaluate whether sleep deprivation affects the integrity of DNA in skin and exacerbates the effects of aging. Fifteen-month old female Hairless mice underwent 72 h of paradoxical sleep deprivation or 15 days of chronic sleep restriction. Punch biopsies of the skin were taken to evaluate DNA damage by single cell gel (comet) assay. Neither paradoxical sleep deprivation nor sleep restriction increased genetic damage, measured by tail movement and tail intensity values. Taken together, the findings are consistent with the notion that aging overrides the effect of sleep loss on the genetic damage in elderly mice.
Chassaing, Benoit; Miles-Brown, Jennifer; Pellizzon, Michael; Ulman, Edward; Ricci, Matthew; Zhang, Limin; Patterson, Andrew D.; Vijay-Kumar, Matam
Diet-induced obesity is often modeled by comparing mice fed high-fat diet (HFD), which is made from purified ingredients, vs. normal chow diet (NCD), which is a low-fat assemblage of relatively unrefined plant and animal products. The mechanism by which HFD promotes adiposity is complex but thought to involve low-grade inflammation and altered gut microbiota. The goal of this study was to investigate the extent to which HFD-induced adiposity is driven by fat content vs. other factors that differentiate HFD vs. NCD. Mice were fed NCD, HFD, or other compositionally defined diets (CDD), designed to mimic NCD and/or explore the role of HFD components. A range of metabolic parameters reflecting low-grade inflammation and adiposity were assayed. Relative to NCD, HFD, and to a lesser, but, nonetheless, significant extent, CDD induced increased adiposity, indicating both lipid content and other aspects of HFD are obesogenic. Moreover, HFD and CDD induced a rapid and marked loss of cecal and colonic mass. Such CDD-induced effects were not affected by adjusting dietary protein levels/types but could be largely eliminated by exchanging insoluble fiber (cellulose) for soluble fiber (inulin). Replacing cellulose with inulin in HFD also protected mice against decreased intestinal mass, hyperphagia, and increased adiposity. Such beneficial effects of inulin were microbiota dependent, correlated with elevated fecal short-chain fatty acid levels analyzed via 1H-NMR-based metabolomics and were partially recapitulated by administration of short-chain fatty acid. HFD-induced obesity is strongly promoted by its lack of soluble fiber, which supports microbiota-mediated intestinal tissue homeostasis that prevents inflammation driving obesity and metabolic syndrome. PMID:26185332
Anwar, Sabina; Peters, Owen; Millership, Steven; Ninkina, Natalia; Doig, Natalie; Connor-Robson, Natalie; Threlfell, Sarah; Kooner, Gurdeep; Deacon, Robert M.; Bannerman, David M.; Bolam, J. Paul; Chandra, Sreeganga S.; Cragg, Stephanie J.; Wade-Martins, Richard; Buchman, Vladimir L.
The synucleins (α, β and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signalling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4-14 month old) animals using a combination of behavioural, biochemical, histological and electrochemical techniques. Adult triple synuclein null (TKO) mice displayed no overt phenotype, and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms which differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson’s disease (PD) may ultimately inform on the selectivity of the disease process. PMID:21593311
Montoya, S.E.; Thiels, E.; Card, J.P.; Lazo, J.S.
Bleomycin hydrolase is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer’s disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the central nervous system were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/J X B6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the central nervous system. PMID:17391860
Clancy, J. S.; Takeshima, H.; Hamilton, S. L.; Reid, M. B.
Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37 degrees C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.
Gomes, Daniel Cláudio de Oliveira; Pinto, Eduardo Fonseca; de Melo, Luiz Dione Barbosa; Lima, Wallace Pacienza; Larraga, Vicente; Lopes, Ulisses Gazos; Rossi-Bergmann, Bartira
We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.
Justesen, Jeannette; Mosekilde, Lis; Holmes, Megan; Stenderup, Karin; Gasser, Jürg; Mullins, John J; Seckl, Jonathan R; Kassem, Moustapha
Glucocorticoids (GCs) exert potent, but poorly characterized, effects on the skeleton. The cellular activity of GCs is regulated at a prereceptor level by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). The type 1 isoform, which predominates in bone, functions as a reductase in intact cells and regenerates active cortisol (corticosterone) from circulating inert 11-keto forms. The aim of the present study was to investigate the role of this intracrine activation of GCs on normal bone physiology in vivo using mice deficient in 11betaHSD1 (HSD1(-/-)). The HSD1(-/-) mice exhibited no significant changes in cortical or trabecular bone mass compared with wild-type (Wt) mice. Aged HSD1(-/-) mice showed age-related bone loss similar to that observed in Wt mice. Histomorphometric analysis showed similar bone formation and bone resorption parameters in HSD1(-/-) and Wt mice. However, examination of bone marrow composition revealed a total absence of marrow adipocytes in HSD1(-/-) mice. Cells from Wt and HSD1(-/-) mice exhibited similar growth rates as well as similar levels of production of osteoblastic markers. The adipocyte-forming capacity of in vitro cultured bone marrow stromal cells and trabecular osteoblasts was similar in HSD1(-/-) and Wt mice. In conclusion, our results suggest that 11betaHSD1 amplification of intracellular GC actions in mice may be required for bone marrow adipocyte formation, but not for bone formation. The clinical relevance of this observation remains to be determined.
Ribeiro-Resende, Victor Túlio; Araújo Gomes, Tiago; de Lima, Silmara; Nascimento-Lima, Maiara; Bargas-Rega, Michele; Santiago, Marcelo Felipe; Reis, Ricardo Augusto de Melo; de Mello, Fernando Garcia
The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration.
Ribeiro-Resende, Victor Túlio; Gomes, Tiago Araújo; de Lima, Silmara; Nascimento-Lima, Maiara; Bargas-Rega, Michele; Santiago, Marcelo Felipe; Reis, Ricardo Augusto de Melo; de Mello, Fernando Garcia
The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration. PMID:25330147
Buran, Bradley N; Strenzke, Nicola; Neef, Andreas; Gundelfinger, Eckart D; Moser, Tobias; Liberman, M Charles
Synaptic ribbons, found at the presynaptic membrane of sensory cells in both ear and eye, have been implicated in the vesicle-pool dynamics of synaptic transmission. To elucidate ribbon function, we characterized the response properties of single auditory nerve fibers in mice lacking Bassoon, a scaffolding protein involved in anchoring ribbons to the membrane. In bassoon mutants, immunohistochemistry showed that fewer than 3% of the hair cells' afferent synapses retained anchored ribbons. Auditory nerve fibers from mutants had normal threshold, dynamic range, and postonset adaptation in response to tone bursts, and they were able to phase lock with normal precision to amplitude-modulated tones. However, spontaneous and sound-evoked discharge rates were reduced, and the reliability of spikes, particularly at stimulus onset, was significantly degraded as shown by an increased variance of first-spike latencies. Modeling based on in vitro studies of normal and mutant hair cells links these findings to reduced release rates at the synapse. The degradation of response reliability in these mutants suggests that the ribbon and/or Bassoon normally facilitate high rates of exocytosis and that its absence significantly compromises the temporal resolving power of the auditory system.
Beeler, Jeff A; Cao, Zhen Fang Huang; Kheirbek, Mazen A; Zhuang, Xiaoxi
Both the dorsal and ventral striatum have been demonstrated to have a critical role in reinforcement learning and addiction. Dissecting the specific function of these striatal compartments and their associated nigrostriatal and mesoaccumbens dopamine pathways, however, has proved difficult. Previous studies using lesions to isolate the contribution of nigrostriatal and mesoaccumbens dopamine in mediating the locomotor and reinforcing effects of psychostimulant drugs have yielded inconsistent and inconclusive results. Using a naturally occurring mutant mouse line, aphakia, that lacks a nigrostriatal dopamine pathway but retains an intact mesoaccumbens pathway, we show that the locomotor activating effects of cocaine, including locomotor sensitization, are dependent on an intact nigrostriatal dopamine projection. In contrast, cocaine reinforcement, as measured by conditioned place preference and cocaine sensitization of sucrose preference, is intact in these mice. In light of the well-established role of the nucleus accumbens in mediating the effects of psychostimulants, these data suggest that the nigrostriatal pathway can act as a critical effector mechanism for the nucleus accumbens highlighting the importance of intrastriatal connectivity and providing insight into the functional architecture of the striatum.
Tateda, K; Matsumoto, T; Miyazaki, S; Yamaguchi, K
This study was designed to define the lipopolysaccharide (LPS) sensitivity of aged mice in terms of lethality and cytokine production and to determine down-regulating responses of corticosterone and interleukin 10 (IL-10). The 50% lethal doses of LPS in young (6- to 7-week-old) and aged (98- to 102-week-old) mice were 601 and 93 microg per mouse (25.6 and 1.6 mg per kg of body weight), respectively. Aged mice were approximately 6.5-fold more sensitive to the lethal toxicity of LPS in micrograms per mouse (16-fold more sensitive in milligrams per kilogram) than young mice. Levels in sera of tumor necrosis factor-alpha (TNF-alpha) IL-1alpha, and IL-6 after intraperitoneal injection of 100 microg of LPS peaked at 1.5, 3, and 3 h, respectively, and declined thereafter in both groups of mice. However, the peak values of these cytokines were significantly higher in aged than in young mice (P < 0.05). Gamma interferon (IFN-gamma) was detectable at 3 h, and sustained high levels were still detected after 12 h in both age groups. Although there were no significant differences in levels of IFN-gamma in sera from both groups, aged mice showed higher IFN-gamma levels throughout the 3- to 12-h study period. Administration of increasing doses of LPS revealed that aged mice had a lower threshold to IL-1alpha production than young mice. In addition, aged mice were approximately 4-fold more sensitive to the lethal toxicity of exogenous TNF in units per mouse (10-fold more sensitive in units per kilogram) than young mice. With regard to down-regulating factors, corticosterone amounts were similar at basal levels and no differences in kinetics after the LPS challenge were observed, whereas IL-10 levels in sera were significantly higher in aged mice at 1.5 and 3 h than in young mice (P < 0.01). These results indicate that aged mice are more sensitive to the lethal toxicities of LPS and TNF than young mice. We conclude that a relatively activated, or primed, state for LPS
Sher, Roger B.; Cox, Gregory A.; Mills, Kevin D.; Sundberg, John P.
Rhabdomyosarcomas (RSCs) are skeletal muscle neoplasms found in humans and domestic mammals. The A/J inbred strain developed a high frequency (between 70–80%) of adult pleomorphic type (APT) RSC at >20 months of age while BALB/cByJ also develop RSC but less frequently. These neoplasms invaded skeletal muscle surrounding either the axial or proximal appendicular skeleton and were characterized by pleomorphic cells with abundant eosinophilic cytoplasm, multiple nuclei, and cross striations. The diagnosis was confirmed by detection of alpha-sarcomeric actin and myogenin in the neoplastic cells using immunocytochemistry. The A/J strain, but not the related BALB/c substrains, is also characterised by a progressive muscular dystrophy homologous to limb-girdle muscular dystrophy type 2B. The association between the development of RSC in similar muscle groups to those most severely affected by the progressive muscular dystrophy suggested that these neoplasms developed from abnormal regeneration of the skeletal muscle exacerbated by the dysferlin mutation. Transcriptome analyses of RSCs revealed marked downregulation of genes in muscular development and function signaling networks. Non-synonymous coding SNPs were found in Myl1, Abra, Sgca, Ttn, and Kcnj12 suggesting these may be important in the pathogenesis of RSC. These studies suggest that A strains of mice can be useful models for dissecting the molecular genetic basis for development, progression, and ultimately for testing novel anticancer therapeutic agents dealing with rhabdomyosarcoma. PMID:21853140
Duvoisin, Robert M; Zhang, Connie; Pfankuch, Timothy F; O'Connor, Heather; Gayet-Primo, Jacqueline; Quraishi, Salma; Raber, Jacob
To study the role of the metabotropic glutamate receptor 8 (mGluR8), mice lacking this receptor were generated by homologous recombination. Homozygous mGluR8-deficient mice are about 8% heavier than their wild-type age-matched controls after reaching 4 weeks of age. This weight difference is not caused by an altered food intake and is not exacerbated by feeding the animals a high-fat diet. Moreover, mGluR8-/- mice are mildly insulin resistant, possibly as a result of being overweight. Behavioral testing revealed a reduced locomotor activity of mGluR8-/- mice compared with wild-type mice during the first 3 days in a novel enclosed environment. However after 3 days, the locomotor activities of wild-type and mGluR8-/- mice were similar, suggesting a reduced exploratory behavior of mGluR8-/- mice in a novel enclosed environment. By contrast, there were no genotype differences in locomotor activity in the open field, plus maze, or in total time spent exploring objects during object recognition tests, indicating that there is a dissociation between effects of mGluR8 deficiency in exploratory activity in a novel safe enclosed environment vs. a more anxiogenic novel open environment. The absence of mGluR8 also leads to increased measures of anxiety in the open field and elevated plus maze. Whether the diverse phenotypic differences observed in mGluR8-/- mice result from the misregulation of a unique neural pathway, possibly in the thalamus or hypothalamus, or whether they are the consequence of multiple developmental and functional alterations in synaptic transmission, remains to be determined.
Mishima, Yuriko; Shinoda, Yo; Sadakata, Tetsushi; Kojima, Masami; Wakana, Shigeharu; Furuichi, Teiichi
Chronic stress is associated with anxiety and depressive disorders, and can cause weight gain. Ca(2+)-dependent activator protein for secretion 2 (CAPS2) is involved in insulin release. Caps2 knockout (KO) mice exhibit decreased body weight, reduced glucose-induced insulin release, and abnormal psychiatric behaviors. We chronically administered the stress hormone corticosterone (CORT), which induces anxiety/depressive-like behavior and normally increases plasma insulin levels, via the drinking water for 10 weeks, and we examined the stress response in KO mice. Chronic CORT exposure inhibited stress-induced serum CORT elevation in wild-type (WT) mice, but not in KO mice. Poor weight gain in CORT-treated animals was observed until week 6 in WT mice, but persisted for the entire duration of the experiment in KO mice, although there is no difference in drug*genotype interaction. Among KO mice, food consumption was unchanged, while water consumption was higher, over the duration of the experiment in CORT-treated animals, compared with untreated animals. Moreover, serum insulin and leptin levels were increased in CORT-treated WT mice, but not in KO mice. Lastly, both WT and KO mice displayed anxiety/depressive-like behavior after CORT administration. These results suggest that Caps2 KO mice have altered endocrine responses to CORT administration, while maintaining CORT-induced anxiety/depressive-like behavior.
Roschger, Paul; Zillikens, M. Carola; Waarsing, Jan H.; van der Kemp, Annemiete; Schreuders-Koedam, Marijke; Fratzl-Zelman, Nadja; Leenen, Pieter J.M.; Hoenderop, Joost G.J.; Klaushofer, Klaus; Bindels, René J.M.; van Leeuwen, Johannes P.T.M.
Trpv5 plays an important role in calcium (Ca2+) homeostasis, among others by mediating renal calcium reabsorption. Accordingly, Trpv5 deficiency strongly stresses Ca2+ homeostasis in order to maintain stable serum Ca2+. We addressed the impact of lifelong challenge of calcium homeostasis on the bone phenotype of these mice. Aging significantly increased serum 1,25(OH)2D3 and PTH levels in both genotypes but they were more elevated in Trpv5−/− mice, whereas serum Ca2+ was not affected by age or genotype. Age-related changes in trabecular and cortical bone mass were accelerated in Trpv5−/− mice, including reduced trabecular and cortical bone thickness as well as reduced bone mineralization. No effect of Trpv5 deficiency on bone strength was observed. In 78-week-old mice no differences were observed between the genotypes regarding urinary deoxypyridinoline, osteoclast number, differentiation and activity as well as osteoclast precursor numbers, as assessed by flow cytometry. In conclusion, life-long challenge of Ca2+ homeostasis present in Trpv5−/− mice causes accelerated bone aging and a low cortical and trabecular bone mass phenotype. The phenotype of the Trpv5−/− mice suggests that maintenance of adequate circulatory Ca2+ levels in patients with disturbances in Ca2+ homeostasis should be a priority in order to prevent bone loss at older age. PMID:27102152
Praag, Henriette van; Shubert, Tiffany; Zhao, Chunmei; Gage, Fred H.
Aging causes changes in the hippocampus that may lead to cognitive decline in older adults. In young animals, exercise increases hippocampal neurogenesis and improves learning. We investigated whether voluntary wheel running would benefit mice that were sedentary until 19 months of age. Specifically, young and aged mice were housed with or without a running wheel and injected with bromodeoxyuridine or retrovirus to label newborn cells. After 1 month, learning was tested in the Morris water maze. Aged runners showed faster acquisition and better retention of the maze than age-matched controls. The decline in neurogenesis in aged mice was reversed to 50% of young control levels by running. Moreover, fine morphology of new neurons did not differ between young and aged runners, indicating that the initial maturation of newborn neurons was not affected by aging. Thus, voluntary exercise ameliorates some of the deleterious morphological and behavioral consequences of aging. PMID:16177036
Nishio, Naomi; Okawa, Yayoi; Sakurai, Hidetoshi
One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice. PMID:19424869
Hayashi, Y.; Kurashima, C.; Utsuyama, M.; Hirokawa, K.
This study reports that spontaneous autoimmune sialadenitis developed in aging female, rather than male, BDF1 mice. The lesions first appeared in 6-month-old female BDF1 mice and were aggravated with advancing age, especially in 24-month-old and 30-month-old senescent mice. In contrast, significant inflammatory changes did not develop in aging male BDF1 mice. The presence of antisalivary duct antibody was found in sera from mice with sialadenitis. The infiltrating cells in the lesions of submandibular salivary glands were mainly composed of T cells, especially Lyt 1+ and L3T4+ cells. Moreover, mild inflammatory lesions were observed in parotid, sublingual salivary glands, pancreas, or kidneys in some mice that developed spontaneously occurring sialadenitis. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3260751
Jansen, Hailey J.; Moghtadaei, Motahareh; Mackasey, Martin; Rafferty, Sara A.; Bogachev, Oleg; Sapp, John L.; Howlett, Susan E.; Rose, Robert A.
Atrial fibrillation (AF) is prevalent in aging populations; however not all individuals age at the same rate. Instead, individuals of the same chronological age can vary in health status from fit to frail. Our objective was to determine the impacts of age and frailty on atrial function and arrhythmogenesis in mice using a frailty index (FI). Aged mice were more frail and demonstrated longer lasting AF compared to young mice. Consistent with this, aged mice showed longer P wave duration and PR intervals; however, both parameters showed substantial variability suggesting differences in health status among mice of similar chronological age. In agreement with this, P wave duration and PR interval were highly correlated with FI score. High resolution optical mapping of the atria demonstrated reduced conduction velocity and action potential duration in aged hearts that were also graded by FI score. Furthermore, aged mice had increased interstitial fibrosis along with changes in regulators of extracellular matrix remodelling, which also correlated with frailty. These experiments demonstrate that aging results in changes in atrial structure and function that create a substrate for atrial arrhythmias. Importantly, these changes were heterogeneous due to differences in health status, which could be identified using an FI. PMID:28290548
Chauhan, Anil Kumar; Moretti, Federico Andrea; Iaconcig, Alessandra; Baralle, Francisco Ernesto; Muro, Andrés Fernando
The extracellular matrix (ECM) plays an important role in the central nervous system (CNS) by modulating the migration of cells, axons and dendrites of neurons. Fibronectin (FN) is a major component of the ECM in the CNS and plays essential roles in development, cell adhesion and cell migration. Specific FN-isoforms, generated by alternative splicing at three conserved regions, the extra domain B (EDB), extra domain A (EDA) and type III homologies connecting segment (IIICS), have been shown to modulate these processes in vitro and in vivo. The inclusion of the EDA exon in the brain is highly regulated during development and aging, suggesting an important role of this exon in brain function. However, the direct role of FN-isoforms in brain function and behaviour is still obscure. Therefore, to directly assess the role of the FN-EDA exon in vivo, we have generated two mouse strains devoid of EDA exon regulated splicing in the FN gene that constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the EDA exon in all tissues. Here, we show the behavioural consequences of the absence of regulated splicing of the EDA exon in the FN gene. Deletion of the EDA domain in the FN protein results in reduced motor-coordination abilities and vertical exploratory capacity, whereas mice that constitutively include the EDA domain displayed a decrease in locomotory activity in the open field (OF) test. These results strongly suggest that regulated splicing of the EDA exon is necessary for a normal function of the brain.
TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...
Vidovic, Diana; Harris, Lachlan; Harvey, Tracey J; Evelyn Heng, Yee Hsieh; Smith, Aaron G; Osinski, Jason; Hughes, James; Thomas, Paul; Gronostajski, Richard M; Bailey, Timothy L; Piper, Michael
Nuclear factor one X (NFIX) has been shown to play a pivotal role during the development of many regions of the brain, including the neocortex, the hippocampus and the cerebellum. Mechanistically, NFIX has been shown to promote neural stem cell differentiation through the activation of astrocyte-specific genes and via the repression of genes central to progenitor cell self-renewal. Interestingly, mice lacking Nfix also exhibit other phenotypes with respect to development of the central nervous system, and whose underlying causes have yet to be determined. Here we examine one of the phenotypes displayed by Nfix(-/-) mice, namely hydrocephalus. Through the examination of embryonic and postnatal Nfix(-/-) mice we reveal that hydrocephalus is first seen at around postnatal day (P) 10 in mice lacking Nfix, and is fully penetrant by P20. Furthermore, we examined the subcommissural organ (SCO), the Sylvian aqueduct and the ependymal layer of the lateral ventricles, regions that when malformed and functionally perturbed have previously been implicated in the development of hydrocephalus. SOX3 is a factor known to regulate SCO development. Although we revealed that NFIX could repress Sox3-promoter-driven transcriptional activity in vitro, SOX3 expression within the SCO was normal within Nfix(-/-) mice, and Nfix mutant mice showed no abnormalities in the structure or function of the SCO. Moreover, these mutant mice exhibited no overt blockage of the Sylvian aqueduct. However, the ependymal layer of the lateral ventricles was frequently absent in Nfix(-/-) mice, suggesting that this phenotype may underlie the development of hydrocephalus within these knockout mice.
Porter, A J; Pillidge, K; Tsai, Y C; Dudley, J A; Hunt, S P; Peirson, S N; Brown, L A; Stanford, S C
Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: ‘Hom’) wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents (‘Het’, derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors. PMID:25558794
Jia, Yun-Fang; Song, Ning-Ning; Mao, Rong-Rong; Li, Jin-Nan; Zhang, Qiong; Huang, Ying; Zhang, Lei; Han, Hui-Li; Ding, Yu-Qiang; Xu, Lin
Dysfunction of central serotonin (5-HT) system has been proposed to be one of the underlying mechanisms for anxiety and depression, and the association of diabetes mellitus and psychiatric disorders has been noticed by the high prevalence of anxiety/depression in patients with diabetes mellitus. This promoted us to examine these behaviors in central 5-HT-deficient mice and those also suffering with diabetes mellitus. Mice lacking either 5-HT or central serotonergic neurons were generated by conditional deletion of Tph2 or Lmx1b respectively. Simultaneous depletion of both central serotonergic neurons and pancreatic islet cells was achieved by administration of diphtheria toxin (DT) in Pet1-Cre;Rosa26-DT receptor (DTR) mice. The central 5-HT-deficient mice showed reduced anxiety-like behaviors as they spent more time in and entered more often into the light box in the light/dark box test compared with controls; similar results were observed in the elevated plus maze test. However, they displayed no differences in the immobility time of the forced swimming and tail suspension tests suggesting normal depression-like behaviors in central 5-HT-deficient mice. As expected, DT-treated Pet1-Cre;Rosa26-DTR mice lacking both central serotonergic neurons and pancreatic islet endocrine cells exhibited several classic diabetic symptoms. Interestingly, they displayed increased anxiety-like behaviors but reduced immobility time in the forced swimming and tail suspension tests. Furthermore, the hippocampal neurogenesis was dramatically enhanced in these mice. These results suggest that the deficiency of central 5-HT may not be sufficient to induce anxiety/depression-like behaviors in mice, and the enhanced hippocampal neurogenesis may contribute to the altered depression-like behaviors in the 5-HT-deficient mice with diabetes. Our current investigation provides understanding the relationship between diabetes mellitus and psychiatric disorders. PMID:25294992
Swärd, Karl; Sadegh, Mardjaneh K; Mori, Michiko; Erjefält, Jonas S; Rippe, Catarina
Caveolae are invaginations in the plasma membrane that depend on caveolins and cavins for maturation. Here, we investigated the pulmonary phenotype in mice lacking cavin-1. Bright field and electron-microscopy showed that the cavin-1-deficient mice lacked caveolae in the lung, had an increased lung tissue density, and exhibited hypertrophic remodeling of pulmonary arteries. The right ventricle of the heart moreover had an increased mass and the right ventricular pressure was elevated. A microarray analysis revealed upregulation of Arg1 and downregulation of Ddah1, molecules whose altered expression has previously been associated with pulmonary arterial hypertension. Taken together, this work demonstrates vascular remodeling and increased pulmonary blood pressure in cavin-1 deficient mice and associates this phenotype with altered expression of Arg1 and Ddah1. PMID:24303100
Chae, T; Kwon, Y T; Bronson, R; Dikkes, P; Li, E; Tsai, L H
The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.
Yamada, Y.; Fausto, N.
Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver. Images Figure 2 Figure 4 Figure 6 Figure 8 Figure 9 Figure 10 Figure 12 PMID:9626061
Berkowicz, Susan R.; Featherby, Travis J.; Whisstock, James C.; Bird, Phillip I.
Background: Brinps 1–3, and Astrotactins (Astn) 1 and 2, are members of the Membrane Attack Complex/Perforin (MACPF) superfamily that are predominantly expressed in the mammalian brain during development. Genetic variation at the human BRINP2/ASTN1 and BRINP1/ASTN2 loci has been implicated in neurodevelopmental disorders. We, and others, have previously shown that Brinp1−/− mice exhibit behavior reminiscent of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Method: We created Brinp2−/− mice and Brinp3−/− mice via the Cre-mediated LoxP system to investigate the effect of gene deletion on anatomy and behavior. Additionally, Brinp2−/−Brinp3−/− double knock-out mice were generated by interbreeding Brinp2−/− and Brinp3−/− mice. Genomic validation was carried out for each knock-out line, followed by histological, weight and behavioral examination. Brinp1−/−Brinp2−/−Brinp3−/− triple knock-out mice were also generated by crossing Brinp2/3 double knock-out mice with previously generated Brinp1−/− mice, and examined by weight and histological analysis. Results: Brinp2−/− and Brinp3−/− mice differ in their behavior: Brinp2−/− mice are hyperactive, whereas Brinp3−/− mice exhibit marked changes in anxiety-response on the elevated plus maze. Brinp3−/− mice also show evidence of altered sociability. Both Brinp2−/− and Brinp3−/− mice have normal short-term memory, olfactory responses, pre-pulse inhibition, and motor learning. The double knock-out mice show behaviors of Brinp2−/− and Brinp3−/− mice, without evidence of new or exacerbated phenotypes. Conclusion: Brinp3 is important in moderation of anxiety, with potential relevance to anxiety disorders. Brinp2 dysfunction resulting in hyperactivity may be relevant to the association of ADHD with chromosome locus 1q25.2. Brinp2−/− and Brinp3−/− genes do not compensate in the mammalian brain and likely have
Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J
A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.
Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J
A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. Images Fig. 1 Fig. 2 PMID:8633004
Thomas, D W; Mannon, R B; Mannon, P J; Latour, A; Oliver, J A; Hoffman, M; Smithies, O; Koller, B H; Coffman, T M
Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus. PMID:9835625
Mironova, Elena; Peti-Peterdi, Janos; Bugaj, Vladislav; Stockand, James D.
We tested whether ATP release through Connexin 30 (Cx30) is part of a local purinergic regulatory system intrinsic to the aldosterone-sensitive distal nephron (ASDN) important for proper control of sodium excretion; if changes in sodium intake influence ATP release via Cx30; and if this allows a normal ENaC response to changes in systemic sodium levels. In addition, we define the consequences of disrupting ATP regulation of ENaC in Cx30−/− mice. Urinary ATP levels in wild-type mice increase with sodium intake, being lower and less dependent on sodium intake in Cx30−/− mice. Loss of inhibitory ATP regulation causes ENaC activity to be greater in Cx30−/− versus wild-type mice, particularly with high sodium intake. This results from compromised ATP release rather than end-organ resistance: ENaC in Cx30−/− mice responds to exogenous ATP. Thus, loss of paracrine ATP feedback regulation of ENaC in Cx30−/− mice disrupts normal responses to changes in sodium intake. Consequently, ENaC is hyperactive in Cx30−/− mice lowering sodium excretion particularly during increases in sodium intake. Clamping mineralocorticoids high in Cx30−/− mice fed a high sodium diet causes a marked decline in renal sodium excretion. This is not the case in wild-type mice, which are capable of undergoing aldosterone-escape. This loss of the ability of ENaC to respond to changes in sodium levels contributes to salt-sensitive hypertension in Cx30−/− mice. PMID:21075848
Mironova, Elena; Peti-Peterdi, Janos; Bugaj, Vladislav; Stockand, James D
We tested whether ATP release through Connexin 30 (Cx30) is part of a local purinergic regulatory system intrinsic to the aldosterone-sensitive distal nephron (ASDN) important for proper control of sodium excretion; if changes in sodium intake influence ATP release via Cx30; and if this allows a normal ENaC response to changes in systemic sodium levels. In addition, we define the consequences of disrupting ATP regulation of ENaC in Cx30(-/-) mice. Urinary ATP levels in wild-type mice increase with sodium intake, being lower and less dependent on sodium intake in Cx30(-/-) mice. Loss of inhibitory ATP regulation causes ENaC activity to be greater in Cx30(-/-) versus wild-type mice, particularly with high sodium intake. This results from compromised ATP release rather than end-organ resistance: ENaC in Cx30(-/-) mice responds to exogenous ATP. Thus, loss of paracrine ATP feedback regulation of ENaC in Cx30(-/-) mice disrupts normal responses to changes in sodium intake. Consequently, ENaC is hyperactive in Cx30(-/-) mice lowering sodium excretion particularly during increases in sodium intake. Clamping mineralocorticoids high in Cx30(-/-) mice fed a high sodium diet causes a marked decline in renal sodium excretion. This is not the case in wild-type mice, which are capable of undergoing aldosterone-escape. This loss of the ability of ENaC to respond to changes in sodium levels contributes to salt-sensitive hypertension in Cx30(-/-) mice.
Sakai, Kaori; Shoji, Hirotaka; Kohno, Takao; Miyakawa, Tsuyoshi; Hattori, Mitsuharu
The secreted glycoprotein Reelin is believed to play critical roles in the pathogenesis of several neuropsychiatric disorders. The highly basic C-terminal region (CTR) of Reelin is necessary for efficient activation of its downstream signaling, and the brain structure of knock-in mice that lack the CTR (ΔC-KI mice) is impaired. Here, we performed a comprehensive behavioral test battery on ΔC-KI mice, in order to evaluate the effects of partial loss-of-function of Reelin on brain functions. The ΔC-KI mice were hyperactive and exhibited reduced anxiety-like and social behaviors. The working memory in ΔC-KI mice was impaired in a T-maze test. There was little difference in spatial reference memory, depression-like behavior, prepulse inhibition, or fear memory between ΔC-KI and wild-type mice. These results suggest that CTR-dependent Reelin functions are required for some specific normal brain functions and that ΔC-KI mice recapitulate some aspects of neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder. PMID:27346785
Lassance-Soares, Roberta M; Sood, Subeena; Chakraborty, Nabarun; Jhamnani, Sunny; Aghili, Nima; Nashin, Hajra; Hammamieh, Rasha; Jett, Marti; Epstein, Stephen E; Burnett, Mary Susan
Chronic stress is associated with increased risk of cardiovascular diseases. Aging is also associated with vascular dysfunction. We hypothesize that chronic stress accelerates collateral dysfunction in old mice. Mice were subjected to either chronic social defeat (CSD) or chronic cold stress (CCS). The CSD mice were housed in a box inside an aggressor's cage and exposed to the aggressor. The CCS group was placed in iced water. After chronic stress, mice underwent femoral artery ligation (FAL) and flow recovery was measured. For the CSD group, appearance and use scores of the foot and a behavioral test were performed. CSD impaired collateral flow recovery after FAL. Further, stressed mice had greater ischemic damage, impaired foot function, and altered behavior. The CCS mice also showed impaired collateral flow recovery. Chronic stress causes hind limb collateral dysfunction in old mice, a conclusion reinforced by the fact that two types of stress produced similar changes.
Diderich, Karin E M; Nicolaije, Claudia; Priemel, Matthias; Waarsing, Jan H; Day, Judd S; Brandt, Renata M C; Schilling, Arndt F; Botter, Sander M; Weinans, Harrie; van der Horst, Gijsbertus T J; Hoeijmakers, Jan H J; van Leeuwen, Johannes P T M
Trichothiodystrophy (TTD) is a rare, autosomal recessive nucleotide excision repair (NER) disorder caused by mutations in components of the dual functional NER/basal transcription factor TFIIH. TTD mice, carrying a patient-based point mutation in the Xpd gene, strikingly resemble many features of the human syndrome and exhibit signs of premature aging. To examine to which extent TTD mice resemble the normal process of aging, we thoroughly investigated the bone phenotype. Here, we show that female TTD mice exhibit accelerated bone aging from 39 weeks onwards as well as lack of periosteal apposition leading to reduced bone strength. Before 39 weeks have passed, bones of wild-type and TTD mice are identical excluding a developmental defect. Albeit that bone formation is decreased, osteoblasts in TTD mice retain bone-forming capacity as in vivo PTH treatment leads to increased cortical thickness. In vitro bone marrow cell cultures showed that TTD osteoprogenitors retain the capacity to differentiate into osteoblasts. However, after 13 weeks of age TTD females show decreased bone nodule formation. No increase in bone resorption or the number of osteoclasts was detected. In conclusion, TTD mice show premature bone aging, which is preceded by a decrease in mesenchymal stem cells/osteoprogenitors and a change in systemic factors, identifying DNA damage and repair as key determinants for bone fragility by influencing osteogenesis and bone metabolism.
Ytterberg, S R; Mahowald, M L; Messner, R P
Chronic inflammatory myositis similar to human polymyositis occurs in mice after infection with a strain of Coxsackievirus B 1 (CVB 1). To investigate the role of T cells in the pathogenesis of this disorder, we compared disease expression in T cell-deficient athymic nude (nu/nu) mice and heterozygotes (nu/+) with normal T cell function. Acute infectious myositis occurred in nu/nu and nu/+ mice. Chronic (greater than 21 d postinfection) weakness and myositis, however, developed only in nu/+. Resistance to disease in nu/nu mice was not explained by insusceptibility to infection; the amount of virus lethal for 50% of mice and virus replication were comparable in both groups. Additionally, anti-CVB 1 antibody production was similar in both groups. Reconstitution of infected nu/nu mice with spleen cells from normal mice resulted in disease. These results demonstrate that chronic weakness after infection with this virus is not simply a sequela of acute myonecrosis and suggest that T cells play a pivotal role in the pathogenesis of chronic myositis. Images PMID:3038960
Ma, Ke; Cabrero, Agatha; Saha, Pradip K; Kojima, Hideto; Li, Lan; Chang, Benny Hung-Junn; Paul, Antoni; Chan, Lawrence
Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generated Adipo(-/-) mice by gene targeting. Adipo(-/-) mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels of Adipo(-/-) and Adipo(+/+) mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed that Adipo(-/-) and Adipo(+/+) mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured beta-oxidation and found it to be significantly increased in muscle and liver of Adipo(-/-) mice. In conclusion, our study indicates that absence of adipo causes increased beta-oxidation but does not cause glucose intolerance or insulin resistance in mice.
Andres-Mateos, Eva; Mejias, Rebeca; Sasaki, Masayuki; Li, Xiaojie; Lin, Brian M; Biskup, Saskia; Zhang, Li; Banerjee, Rebecca; Thomas, Bobby; Yang, Lichuan; Liu, Guosheng; Beal, M Flint; Huso, David L; Dawson, Ted M; Dawson, Valina L
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinson's disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knockout (KO) mice lacking the kinase domain of LRRK2. Here we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild type (WT) mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP. PMID:20016100
van der Eerden, Bram C J; Fratzl-Zelman, Nadja; Nijenhuis, Tom; Roschger, Paul; Zügel, Ulrich; Steinmeyer, Andreas; Hoenderop, Joost G J; Bindels, René J M; Klaushofer, Klaus; van Leeuwen, Johannes P T M
Mice lacking the renal epithelial Ca(2+) channel TRPV5 (TRPV5(-/-)) display impaired renal Ca(2+) reabsorption, hypercalciuria, and intestinal Ca(2+) hyperabsorption, due to secondary hypervitaminosis D. Using these mice, we previously demonstrated that ZK191784 acts as an intestine-specific 1,25(OH)(2) D(3) antagonist without affecting serum calcium levels. On the other hand, it acted as an agonist in the kidney and the effects of ZK191784 on bone were ambiguous. The present study was undertaken to further evaluate the effect of the vitamin D receptor antagonist on murine bone in mice lacking TRPV5. Eight-week-old female Trpv5(+/+) and Trpv5(-/-) mice were treated for 4 weeks with or without 50 µg/kg/day ZK191784. Quantitative backscattered electron imaging showed that the reduced bone matrix mineralization found in femoral bones of Trpv5(-/-) mice was partially but significantly restored upon ZK191784 treatment, just as we observed for trabecular bone thickness. This supports the significance of 1,25(OH)(2) D(3) and optimal control of Ca(2+) homeostasis for bone formation and matrix mineralization. Restoration also took place at the bone gene expression level, where 1α-hydroxylase (Cyp27b1) mRNA in femurs from ZK-treated Trpv5(-/-) mice was upregulated compared to control levels. The downregulated 24-hydroxylase (Cyp24a1) gene expression in femoral bone indicated local vitamin D resistance in the mice treated with ZK191784. Phosphate homeostasis was unaffected between the groups as shown by unaltered serum PO(4)(3-) and fibroblast growth factor (FGF) 23 as well as Fgf23 mRNA expression in bone. In conclusion, circulating 1,25(OH)(2) D(3) is important for optimal control of Ca(2+) homeostasis but also for controlled bone formation and matrix mineralization.
Rivard, Germain F; Moser, Emily G; D'Ambrose, Steven P; Lin, David M
A goal of the Guide for the Care and Use of Laboratory Animals is to improve animal welfare by minimizing sources of fear, anxiety, and stress. As a result, it includes recommendations on overcrowding, frequency of cage changes, enrichment, and group housing. However, human odorants are a potential but unexplored source of fear, anxiety, and stress. Although mice have been maintained for decades for animal research, whether mice perceive humans as predators is unknown. If so, this would necessitate changes in animal care and use procedures to minimize this source of chronic fear, anxiety, and stress. Odorants from predator urine are well known to elicit strong fear responses in mice, leading to modification of animal behavior and elevated levels of stress. To begin asking whether human odors influence mouse behavior, we tested the effect of human urine odor on fear response in mice. We assessed mouse behavior by using a modified shuttle cage to record various parameters of mouse exposure to odorants. We found that mice displayed fear responses to 2,4,5-trimethylthiazoline, a synthetic analog of red fox feces, but no fear response to DMSO, the diluent for 2,4,5-trimethylthiazoline. In contrast, mice exposed to human urine samples showed no significant fear response.
Zeitz, Ute; Weber, Karin; Soegiarto, Desi W; Wolf, Eckhard; Balling, Rudi; Erben, Reinhold G
It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild-type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by approximately 60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic beta cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the vitamin D-responsive elements in pancreatic insulin synthesis and secretion in vivo.
Nagashima, Shuichi; Yagyu, Hiroaki; Tozawa, Ryuichi; Tazoe, Fumiko; Takahashi, Manabu; Kitamine, Tetsuya; Yamamuro, Daisuke; Sakai, Kent; Sekiya, Motohiro; Okazaki, Hiroaki; Osuga, Jun-ichi; Honda, Akira; Ishibashi, Shun
Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.
Rivard, Germain F; Moser, Emily G; D'Ambrose, Steven P; Lin, David M
A goal of the Guide for the Care and Use of Laboratory Animals is to improve animal welfare by minimizing sources of fear, anxiety, and stress. As a result, it includes recommendations on overcrowding, frequency of cage changes, enrichment, and group housing. However, human odorants are a potential but unexplored source of fear, anxiety, and stress. Although mice have been maintained for decades for animal research, whether mice perceive humans as predators is unknown. If so, this would necessitate changes in animal care and use procedures to minimize this source of chronic fear, anxiety, and stress. Odorants from predator urine are well known to elicit strong fear responses in mice, leading to modification of animal behavior and elevated levels of stress. To begin asking whether human odors influence mouse behavior, we tested the effect of human urine odor on fear response in mice. We assessed mouse behavior by using a modified shuttle cage to record various parameters of mouse exposure to odorants. We found that mice displayed fear responses to 2,4,5-trimethylthiazoline, a synthetic analog of red fox feces, but no fear response to DMSO, the diluent for 2,4,5-trimethylthiazoline. In contrast, mice exposed to human urine samples showed no significant fear response. PMID:24602539
Gowen, Brian B.; Bailey, Kevin W.; Scharton, Dionna; Vest, Zachery; Westover, Jonna B.; Skirpstunas, Ramona; Ikegami, Tetsuro
Rift Valley fever virus (RVFV) causes severe disease in humans and livestock. There are currently no approved antivirals or vaccines for the treatment or prevention of RVF disease in humans. A major virulence factor of RVFV is the NSs protein, which inhibits host transcription including the interferon (IFN)-β gene and promotes the degradation of dsRNA-dependent protein kinase, PKR. We analyzed the efficacy of the live-attenuated MP-12 vaccine strain and MP-12 variants that lack the NSs protein as post-exposure vaccinations. Although parental MP-12 failed to elicit a protective effect in mice challenged with wild-type (wt) RVFV by the intranasal route, significant protection was demonstrated by vaccination with MP-12 strains lacking NSs when they were administered at 20 to 30 min post-exposure. Viremia and virus replication in liver, spleen and brain were also inhibited by post-exposure vaccination with MP-12 lacking NSs. The protective effect was mostly lost when vaccination was delayed 6 or 24 h after intranasal RVFV challenge. When mice were challenged subcutaneously, efficacy of MP-12 lacking NSs was diminished, most likely due to more rapid dissemination of wt RVFV. Our findings suggest that post-exposure vaccination with MP-12 lacking NSs may be developed as a novel post-exposure treatment to prevent RVF. PMID:23523764
Gowen, Brian B; Bailey, Kevin W; Scharton, Dionna; Vest, Zachery; Westover, Jonna B; Skirpstunas, Ramona; Ikegami, Tetsuro
Rift Valley fever virus (RVFV) causes severe disease in humans and livestock. There are currently no approved antivirals or vaccines for the treatment or prevention of RVF disease in humans. A major virulence factor of RVFV is the NSs protein, which inhibits host transcription including the interferon (IFN)-β gene and promotes the degradation of dsRNA-dependent protein kinase, PKR. We analyzed the efficacy of the live-attenuated MP-12 vaccine strain and MP-12 variants that lack the NSs protein as post-exposure vaccinations. Although parental MP-12 failed to elicit a protective effect in mice challenged with wild-type (wt) RVFV by the intranasal route, significant protection was demonstrated by vaccination with MP-12 strains lacking NSs when they were administered at 20-30 min post-exposure. Viremia and virus replication in liver, spleen and brain were also inhibited by post-exposure vaccination with MP-12 lacking NSs. The protective effect was mostly lost when vaccination was delayed 6 or 24 h after intranasal RVFV challenge. When mice were challenged subcutaneously, efficacy of MP-12 lacking NSs was diminished, most likely due to more rapid dissemination of wt RVFV. Our findings suggest that post-exposure vaccination with MP-12 lacking NSs may be developed as a novel post-exposure treatment to prevent RVF.
Wesson, Daniel W.; Varga-Wesson, Adrienn G.; Borkowski, Anne H.; Wilson, Donald A.
Orienting responses are physiological and active behavioral reactions evoked by novel stimulus perception and are critical for survival. We explored whether odor orienting responses are impacted throughout both adulthood and normal and pathological aging in mice. Novel odor investigation (including duration and bout numbers) and its subsequent habituation as assayed in the odor habituation task were preserved in adult C57BL/6J mice up to 12mo of age with <6% variability between age groups in investigation time. Separately, using whole-body plethysmography we found that both spontaneous respiration and odor-evoked sniffing behaviors were strikingly preserved in wildtype (WT) mice up to 26mo of age. In contrast, mice accumulating amyloid-β protein in the brain by means of overexpressing mutations in the human amyloid precursor protein gene (APP) showed preserved spontaneous respiration up to 12mo, but starting at 14mo showed significant differences from WT. Similar to WTs, odor-evoked sniffing was not impacted in APP mice up to 26mo. These results show that odor-orienting responses are minimally impacted throughout aging in mice, and suggest that the olfactomotor network is mostly spared of insults due to aging. PMID:21524667
Uchida, Kunitoshi; Dezaki, Katsuya; Damdindorj, Boldbaatar; Inada, Hitoshi; Shiuchi, Tetsuya; Mori, Yasuo; Yada, Toshihiko; Minokoshi, Yasuhiko; Tominaga, Makoto
OBJECTIVE TRPM2 is a Ca2+-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5′-diphosphoribose, which has been suggested to be involved in intracellular Ca2+ increase in immunocytes and pancreatic β-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. RESEARCH DESIGN AND METHODS Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca2+ in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets. RESULTS Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated β-cells, smaller intracellular Ca2+ increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups. CONCLUSIONS These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy. PMID:20921208
WANG, KASPER S.; MA, TONGHUI; FILIZ, FERDA; VERKMAN, A. S.; BASTIDAS, J. AUGUSTO
Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (−/−) mice. The transepithelial osmotic water permeability coefficient (Pf) of in vivo perfused colon of +/+ mice, measured using the volume marker 14C-labeled polyethylene glycol, was 0.016 ± 0.002 cm/s. Pf of proximal colon was greater than that of distal colon (0.020 ± 0.004 vs. 0.009 ± 0.003 cm/s, P < 0.01). Pf was significantly lower in −/− mice when measured in full-length colon (0.009 ± 0.002 cm/s, P < 0.05) and proximal colon (0.013 ± 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. −/− mice (0.80 ± 0.01 vs. 0.81 ± 0.01), but there was a slightly higher water content in defecated stool from +/+ mice (0.68 ± 0.01 vs. 0.65 ± 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 ± 9 vs. 51 ± 8 μl · min−1 · g−1). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration. PMID:10915657
Roh, Danny S; Du, Yiqin; Gabriele, Michelle L; Robinson, Andria R; Niedernhofer, Laura J; Funderburgh, James L
The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age-related spontaneous loss of CE, we characterized CE in Ercc1(-/Δ) mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5- to 6-month-old Ercc1(-/Δ) mice, age-matched wild-type (WT) littermates, and old WT mice (24- to 34-month-old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole-mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1(-/Δ) mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair-deficient mice. CD45(+) immune cells were associated with the posterior surface of CE from Ercc1(-/Δ) mice and the tissue expressed increased IL-1α, Cxcl2, and TNFα, pro-inflammatory proteins associated with senescence-associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1(-/Δ) mice offer a rapid and accurate model to study CE pathogenesis and therapy.
Roh, Danny S.; Du, Yiqin; Gabriele, Michelle L.; Robinson, Andria R.; Niedernhofer, Laura J.; Funderburgh, James L.
Summary The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age-related spontaneous loss of CE, we characterized CE in Ercc1−/Δ mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5- to 6-month-old Ercc1−/Δ mice, age-matched wild-type (WT) litter-mates, and old WT mice (24- to 34-month-old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole-mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet’s thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1−/Δ mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair–deficient mice. CD45+ immune cells were associated with the posterior surface of CE from Ercc1−/Δ mice and the tissue expressed increased IL-1α, Cxcl2, and TNFα, proinflammatory proteins associated with senescence-associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1−/Δ mice offer a rapid and accurate model to study CE pathogenesis and therapy. PMID:23927039
Koenig, Claire M; Walker, Cheryl K; Qi, Lihong; Pessah, Isaac N; Berman, Robert F
Misoprostol is a synthetic analogue of prostaglandin E1 that is administered to women at high doses to induce uterine contractions for early pregnancy termination and at low doses to aid in cervical priming during labor. Because of the known teratogenic effects of misoprostol when given during gestation and its effects on axonal growth in vitro, we examined misoprostol for its potential as a neurodevelopmental toxicant when administered to neonatal C57BL6/J mice. Mice were injected subcutaneously (s.c.) with 0.4, 4 or 40 µg/kg misoprostol on postnatal day 7, the approximate developmental stage in mice of human birth, after which neonatal somatic growth, and sensory and motor system development were assessed. These doses were selected to span the range of human exposure used to induce labor. In addition, adult mice underwent a battery of behavioral tests relevant to neurodevelopmental disorders such as autism including tests for anxiety, stereotyped behaviors, social communication and interactions, and learning and memory. No significant effects of exposure were found for any measure of development or behavioral endpoints. In conclusion, the results of the present study in C57BL/6J mice do not provide support for neurodevelopmental toxicity after misoprostol administration approximating human doses and timed to coincide with the developmental stage of human birth.
Shi, Hao; Gatzke, Florian; Molle, Julia M; Lee, Han Bin; Helm, Emma T; Oldham, Jessie J; Zhang, Lei; Gerrard, David E; Bennett, Anton M
The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways.
Muhl, Lars; Ehnman, Monika; Tannenberg, Philip; Lawrence, Anna-Lisa; Betsholtz, Christer; Eriksson, Ulf
Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor β, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd-/-), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in subpopulations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd-/- mice. Furthermore, Pdgfd-/- mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis. PMID:27032083
Choi, Su-Yeon; Han, Kihoon; Cutforth, Tyler; Chung, Woosuk; Park, Haram; Lee, Dongsoo; Kim, Ryunhee; Kim, Myeong-Heui; Choi, Yeeun; Shen, Kang; Kim, Eunjoon
Synaptic adhesion molecules regulate diverse aspects of neuronal synapse development, including synapse specificity, formation, and maturation. Neph2, also known as Kirrel3, is an immunoglobulin superfamily adhesion molecule implicated in intellectual disability, neurocognitive delay associated with Jacobsen syndrome, and autism spectrum disorders. We here report mice lacking Neph2 (Neph2-/- mice) display moderate hyperactivity in a familiar, but not novel, environment and defective novel object recognition with normal performances in Morris water maze spatial learning and memory, contextual fear conditioning and extinction, and pattern separation tests. These mice also show normal levels of anxiety-like behaviors, social interaction, and repetitive behaviors. At the synapse level, Neph2-/- dentate gyrus granule cells exhibit unaltered dendritic spine density and spontaneous excitatory synaptic transmission. These results suggest that Neph2 is important for normal locomotor activity and object recognition memory. PMID:26283919
Corsetti, Patrícia P; de Almeida, Leonardo A; Carvalho, Natália B; Azevedo, Vasco; Silva, Teane M A; Teixeira, Henrique C; Faria, Ana C; Oliveira, Sergio C
IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.
Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John
Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.
Henninger, Nils; Bouley, James; Sikoglu, Elif M; An, Jiyan; Moore, Constance M; King, Jean A; Bowser, Robert; Freeman, Marc R; Brown, Robert H
Axonal degeneration is a critical, early event in many acute and chronic neurological disorders. It has been consistently observed after traumatic brain injury, but whether axon degeneration is a driver of traumatic brain injury remains unclear. Molecular pathways underlying the pathology of traumatic brain injury have not been defined, and there is no efficacious treatment for traumatic brain injury. Here we show that mice lacking the mouse Toll receptor adaptor Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) gene, a key mediator of Wallerian degeneration, demonstrate multiple improved traumatic brain injury-associated phenotypes after injury in a closed-head mild traumatic brain injury model. Sarm1(-/-) mice developed fewer β-amyloid precursor protein aggregates in axons of the corpus callosum after traumatic brain injury as compared to Sarm1(+/+) mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phophorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after traumatic brain injury. Strikingly, whereas wild-type mice exibited a number of behavioural deficits after traumatic brain injury, we observed a strong, early preservation of neurological function in Sarm1(-/-) animals. Finally, using in vivo proton magnetic resonance spectroscopy we found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1(-/-) mice compared to controls immediately following traumatic brain injury. Our results indicate that the SARM1-mediated prodegenerative pathway promotes pathogenesis in traumatic brain injury and suggest that anti-SARM1 therapeutics are a viable approach for preserving neurological function after traumatic brain injury.
Van der Zee, Catharina E E M
The neural substrate of adaptive thermoregulation in mice lacking both brain-type creatine kinase isoforms is further investigated. The cytosolic brain-type creatine kinase (CK-B) and mitochondrial ubiquitous creatine kinase (UbCKmit) are expressed in neural cells throughout the central and peripheral nervous system, where they have an important role in cellular energy homeostasis. Several integral functions appear altered when creatine kinases are absent in the brain (Jost et al., 2002; Streijger et al., 2004, 2005), which has been explained by inefficient neuronal transmission. The CK--/-- double knockout mice demonstrate every morning a body temperature drop of ~1.0 °C, and they have impaired thermogenesis, as revealed by severe hypothermia upon cold exposure. This defective thermoregulation is not associated with abnormal food intake, decreased locomotive activity, or increased torpor sensitivity. Although white and brown adipose tissue fat pads are diminished in CK--/-- mice, intravenous norepinephrine infusion results in a normal brown adipose tissue response with increasing core body temperatures, indicating that the sympathetic innervation functions correctly (Streijger et al., 2009). This study revealed c-fos changes following a cold challenge, and that neuropeptide Y levels were decreased in the paraventricular nucleus of wildtype, but not CK--/--, mice. A reduction in hypothalamic neuropeptide Y is coupled to increased uncoupling protein 1 expression in brown adipose tissue, resulting in thermogenesis. In CK--/-- mice the neuropeptide Y levels did not change. This lack of hypothalamic plasticity of neuropeptide Y might be the result of inefficient neuronal transmission or can be explained by the previous observation of reduced circulating levels of leptin in CK--/-- mice.
Kim, Hyoung-Mi; Wangemann, Philine
Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4(-/-), results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4(+/-) and Slc26a4(-/-) mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4(-/-) compared to Slc26a4(+/-) mice. In Slc26a4(+/-) and Slc26a4(-/-) mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4(+/-) mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4(-/-) mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.
Yotsumoto, Naoki; Takeoka, Michiko; Yokoyama, Minesuke
Calponin h1 (CNh1) is an actin-binding protein originally isolated from vascular smooth muscle and has been reported to suppress bone formation. We are therefore curious how CNh1 is involved in bone loss that is caused by space flight in microgravity. We assessed the effects of tail suspension (TS) in C57BL/6J wild (CN+/+) and CNh1-deleted (CN-/-) mice to elucidate the role of CNh1 in bone loss under weightless conditions. Bone mineral density (BMD) of tibiae was measured by single energy X-ray absorptiometry, and bone volume fraction (BV/TV), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were measured by bone histomorphometry. BMD, BV/TV, MAR, and BFR/BS were lower in CN+/+ mice with TS than in those without. In the CN-/- group, however, the decrease in each of these parameters by TS was ameliorated. Decreases in serum osteocalcin levels by TS in CN+/+ mice were attenuated in CN-/- mice. Furthermore, urinary deoxypyridinolin (DPD), an indicator of bone resorption, was increased in CN+/+ mice following TS, but not in CN-/- mice. In transfection experiments, the degree of induction of bone formation markers, alkaline phosphatase (ALP) activity and bone morphogenetic protein (BMP)-4 mRNA expression, under stimulation with BMP-2, was lower in MC3T3-E1 mouse osteoblast-like cells expressing CNh1 than that in mock transfected cells. Notably, the BMP-2-induced ALP activity was decreased by CNh1 expression, which was partially rescued by treatment with the Rho kinase inhibitor Y27632. Taken together, these results indicate that CNh1 is responsible for weightlessness-induced bone loss in part through Rho signaling pathway.
Varney, Seth; Polston, Keith F.; Jessen, Tammy; Carneiro, Ana M.D.
Recent studies indicate multiple roles for integrin αvβ3 in adult neurons, including response to pharmacological agents such as cocaine and selective serotonin reuptake inhibitors. In this study, we examined the role of the integrin β3 gene (Itgb3) in the response to environmental stimuli by subjecting Itgb3+/+ and Itgb3−/− mice to unpredictable chronic mild stressors. We found that genetic abrogation of integrin β3 expression elicits an exaggerated vulnerability to chronic unpredictable stress in the open field test. In this test, chronic stress elicited significant decreases in stereotypic behavior and horizontal locomotor activity, including increases in anxiety behaviors. Mild chronic stress led to reductions in dopamine turnover in midbrains of Itgb3+/+, but not Itgb3−/− mice, suggesting a disruption of stress-dependent regulation of DA homeostasis. Chronic stress elicited altered synaptic expression of syntaxin and synaptophysin in midbrains of Itgb3−/− mice, when compared to Itgb3+/+. Semi-quantitative Western blot studies revealed that the synaptic expression, but not total tissue expression, of multiple signaling proteins is correlated with integrin αv levels in the midbrain. Moreover, loss of integrin β3 expression modifies this correlation network. Together, these findings demonstrate that Itgb3−/− mice display a pattern of changes indicating disrupted regulation of midbrain synaptic systems involved in conferring resilience to mild stressors. PMID:26634222
Chidgey, M; Brakebusch, C; Gustafsson, E; Cruchley, A; Hail, C; Kirk, S; Merritt, A; North, A; Tselepis, C; Hewitt, J; Byrne, C; Fassler, R; Garrod, D
The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.
Espinosa, F; Marks, G; Heintz, N; Joho, R H
The voltage-gated potassium channels Kv3.1 and Kv3.3 are widely expressed in the brain, including areas implicated in the control of motor activity and in areas thought to regulate arousal states. Although Kv3.1 and Kv3.3-single mutants show some physiological changes, previous studies revealed relatively subtle behavioral alterations suggesting that Kv3.1 and Kv3.3 channel subunits may be encoded by a pair of redundant genes. In agreement with this hypothesis, Kv3.1/Kv3.3-deficient mice display a 'strong' mutant phenotype that includes motor dysfunction (ataxia, myoclonus, tremor) and hyperactivity when exposed to a novel environment. In this paper we report that Kv3.1/Kv3.3-deficient mice are also constitutively hyperactive. Compared to wildtype mice, double mutants display 'restlessness' that is particularly prominent during the light period, when mice are normally at rest, characterized by more than a doubling of ambulatory and stereotypic activity, and accompanied by a 40% sleep reduction. When we reinvestigated both single mutants, we observed constitutive increases of ambulatory and stereotypic activity in conjunction with sleep loss in Kv3.1-single mutants but not in Kv3.3-single mutants. These findings indicate that the absence of Kv3.1-channel subunits is primarily responsible for the increased motor drive and the reduction in sleep time.
Namiranian, Khodadad; Brink, Christa D; Goodman, Jerry Clay; Robertson, Claudia S; Bryan, Robert M
The purpose of this study was to determine whether the potassium channel, TREK-1, was neuroprotective after traumatic brain injury (TBI). Since there are no selective blockers, we used TREK-1 knockout (KO) mice for our study. Wild-type (WT) and TREK-1 KO mice were anesthetized and subjected to controlled-cortical impact injury (deformation of the brain by 1.5 mm by a 3-mm diameter rod traveling at a 3 m/s). Laser Doppler perfusion (LDP) decreased by ∼80% in the injured cortex and remained at that level in both WT and TREK-1 KO mice (n=10 and 11, respectively). Laser Doppler perfusion decreased by 50% to 60% in cortical areas directly adjacent to the site of injury. There were no statistical differences in LDP between genotype. The contusion volume, determined 15 days after the TBI using hematoxylin and eosin-stained coronal brain sections, was 4.1±0.8 (n=10) and 5.1±0.5 (n=11) mm3 for WT and TREK-1 KO, respectively (not significant, P=0.34). Cell counts of viable neurons in the CA1 and CA3 regions of the hippocampus were similar between WT and TREK-1 KO mice (P=0.51 and 0.84 for CA1 and CA3, respectively). We conclude that TREK-1 expression does not provide brain protection after TBI. PMID:21157470
Bär, Janina; Kumar, Vinod; Roth, Wera; Schwarz, Nicole; Richter, Miriam; Leube, Rudolf E; Magin, Thomas M
Keratins perform major structural and regulatory functions in epithelia. Owing to redundancy, their respective contribution to epidermal integrity, adhesion, and cell junction formation has not been addressed in full. Unexpectedly, the constitutive deletion of type II keratins in mice was embryonic lethal ∼ E9.5 without extensive tissue damage. This prompted us to analyze keratin functions in skin where keratins are best characterized. Here, we compare the mosaic and complete deletion of all type II keratins in mouse skin, with distinct consequences on epidermal integrity, adhesion, and organismal survival. Mosaic knockout (KO) mice survived ∼ 12 days while global KO mice died perinatally because of extensive epidermal damage. Coinciding with absence of keratins, epidermal fragility, inflammation, increased epidermal thickness, and increased proliferation were noted in both strains of mice, accompanied by significantly smaller desmosomes. Decreased desmosome size was due to accumulation of desmosomal proteins in the cytoplasm, causing intercellular adhesion defects resulting in intercellular splits. Mixing different ratios of wild-type and KO keratinocytes revealed that ∼ 60% of keratin-expressing cells were sufficient to maintain epithelial sheets under stress. Our data reveal a major contribution of keratins to the maintenance of desmosomal adhesion and epidermal integrity with relevance for the treatment of epidermolysis bullosa simplex and other keratinopathies.
Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew
N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…
Adinolfi, Elena; Capece, Marina; Franceschini, Alessia; Falzoni, Simonetta; Giuliani, Anna L; Rotondo, Alessandra; Sarti, Alba C; Bonora, Massimo; Syberg, Susanne; Corigliano, Domenica; Pinton, Paolo; Jorgensen, Niklas R; Abelli, Luigi; Emionite, Laura; Raffaghello, Lizzia; Pistoia, Vito; Di Virgilio, Francesco
The ATP receptor P2X7 (P2X7R or P2RX7) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study, we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with postmortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intratumoral IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an antitumor immune response, and to restrict tumor growth and metastatic diffusion.
Kotelevtsev, Yuri; Brown, Roger W.; Fleming, Stewart; Kenyon, Christopher; Edwards, Christopher R.W.; Seckl, Jonathan R.; Mullins, John J.
Deficiency of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in humans leads to the syndrome of apparent mineralocorticoid excess (SAME), in which cortisol illicitly occupies mineralocorticoid receptors, causing sodium retention, hypokalemia, and hypertension. However, the disorder is usually incompletely corrected by suppression of cortisol, suggesting additional and irreversible changes, perhaps in the kidney. To examine this further, we produced mice with targeted disruption of the 11β-HSD2 gene. Homozygous mutant mice (11β-HSD2–/–) appear normal at birth, but ∼50% show motor weakness and die within 48 hours. Both male and female survivors are fertile but exhibit hypokalemia, hypotonic polyuria, and apparent mineralocorticoid activity of corticosterone. Young adult 11β-HSD2–/– mice are markedly hypertensive, with a mean arterial blood pressure of 146 ± 2 mmHg, compared with 121 ± 2 mmHg in wild-type controls and 114 ± 4 mmHg in heterozygotes. The epithelium of the distal tubule of the nephron shows striking hypertrophy and hyperplasia. These histological changes do not readily reverse with mineralocorticoid receptor antagonism in adulthood. Thus, 11β-HSD2–/– mice demonstrate the major features of SAME, providing a unique rodent model to study the molecular mechanisms of kidney resetting leading to hypertension. J. Clin. Invest. 103:683–689 (1999) PMID:10074485
Namiranian, Khodadad; Brink, Christa D; Goodman, Jerry Clay; Robertson, Claudia S; Bryan, Robert M
The purpose of this study was to determine whether the potassium channel, TREK-1, was neuroprotective after traumatic brain injury (TBI). Since there are no selective blockers, we used TREK-1 knockout (KO) mice for our study. Wild-type (WT) and TREK-1 KO mice were anesthetized and subjected to controlled-cortical impact injury (deformation of the brain by 1.5 mm by a 3-mm diameter rod traveling at a 3 m/s). Laser Doppler perfusion (LDP) decreased by ∼80% in the injured cortex and remained at that level in both WT and TREK-1 KO mice (n=10 and 11, respectively). Laser Doppler perfusion decreased by 50% to 60% in cortical areas directly adjacent to the site of injury. There were no statistical differences in LDP between genotype. The contusion volume, determined 15 days after the TBI using hematoxylin and eosin-stained coronal brain sections, was 4.1±0.8 (n=10) and 5.1±0.5 (n=11) mm(3) for WT and TREK-1 KO, respectively (not significant, P=0.34). Cell counts of viable neurons in the CA1 and CA3 regions of the hippocampus were similar between WT and TREK-1 KO mice (P=0.51 and 0.84 for CA1 and CA3, respectively). We conclude that TREK-1 expression does not provide brain protection after TBI.
Saxena, Ankita; Kesari, V P
Pesticides, spinosad, imidacloprid and neem oil are widely used both in residential and agricultural environments because of its broad spectrum insecticidal activity and effectiveness. The present study was undertaken to estimate genotoxicity of formulations of some pesticides in mice. Three pesticides of diverse group studied were spinosad (45% w/v), imidacloprid (17.8%, w/v) and neem oil. Animals were exposed 37, 4.5 and 50 mg kg⁻¹ b.wt. for spinosad, imidacloprid and neem oil, respectively, through oral gavage for 5 consecutive days. A vehicle control group and one positive control (cyclophosphamide; 20 mg kg⁻¹ b. wt.) were also selected. The results showed that cyclophosphamide produced 1.12% micronuclei in mice, as against 0.18 in vehicle control, 0.30 in spinosad, 0.28 in imidacloprid and 0.22% in neem oil, respectively. The gross percentage of chromosomal aberration in mice were 28.5% in cyclophosphamide against 6.5% in vehicle control, 8.0% in spinosad, 9.5% in imidacloprid and 7.0% in neem oil, respectively. The overall findings of the present study revealed that all the three pesticide formulations, imidacloprid, spinosad and neem oil at tested dose did not show any genotoxic effect in mice.
Trajkovic, Marija; Visser, Theo J; Mittag, Jens; Horn, Sigrun; Lukas, Jan; Darras, Veerle M; Raivich, Genadij; Bauer, Karl; Heuer, Heike
In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum levels. The MCT8-null mice described here, however, developed without overt deficits but also exhibited distorted 3,5,3'-triiodothyronine (T3) and thyroxine (T4) serum levels, resulting in increased hepatic activity of type 1 deiodinase (D1). In the mutants' brains, entry of T4 was not affected, but uptake of T3 was diminished. Moreover, the T4 and T3 content in the brain of MCT8-null mice was decreased, the activity of D2 was increased, and D3 activity was decreased, indicating the hypothyroid state of this tissue. In the CNS, analysis of T3 target genes revealed that in the mutants, the neuronal T3 uptake was impaired in an area-specific manner, with strongly elevated thyrotropin-releasing hormone transcript levels in the hypothalamic paraventricular nucleus and slightly decreased RC3 mRNA expression in striatal neurons; however, cerebellar Purkinje cells appeared unaffected, since they did not exhibit dendritic outgrowth defects and responded normally to T3 treatment in vitro. In conclusion, the circulating thyroid hormone levels of MCT8-null mice closely resemble those of humans with MCT8 mutations, yet in the mice, CNS development is only partially affected.
Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a resu...
Schachner, Melitta; Morellini, Fabio; Fellini, Laetitia
Geometry, e.g., the shape of the environment, can be used by numerous animal species to orientate, but data concerning the mouse are lacking. We addressed the question of whether mice are capable of using geometry for navigating. To test whether aging could affect searching strategies, we compared adult (3- to 5-mo old) and aged (20- to 21-mo old)…
Dies, Kira A; Bodell, Adria; Hisama, Fuki M; Guo, Chao-Yu; Barry, Brenda; Chang, Bernard S; Barkovich, A James; Walsh, Christopher A
Schizencephaly is a rare malformation of cortical development characterized by congenital clefts extending from the pial surface to the lateral ventricle that are lined by heterotopic gray matter. The clinical presentation is variable and can include motor or cognitive impairment and epilepsy. The causes of schizencephaly are heterogeneous and can include teratogens, prenatal infection, or maternal trauma. Reported genetic causes include chromosomal aneuploidy, EMX2 mutations, and possible autosomal recessive familial cases based on recurrence in siblings. In an effort to identify risk factors for schizencephaly, we conducted a survey of 48 parents or primary caretakers of patients with schizencephaly born between 1983 and 2004. We discovered that young maternal age, lack of prenatal care, and alcohol use were all significantly associated with risk of schizencephaly. Our results suggest that there are important nongenetic, intrauterine events that predispose to schizencephaly.
Tsujimura, Atsushi; Matsuki, Masato; Takao, Keizo; Yamanishi, Kiyofumi; Miyakawa, Tsuyoshi; Hashimoto-Gotoh, Tamotsu
KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimer's disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds. PMID:18958194
Pacheco-Costa, Rafael; Hassan, Iraj; Reginato, Rejane D; Davis, Hannah M; Bruzzaniti, Angela; Allen, Matthew R; Plotkin, Lilian I
Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.
Clark, Daniel D; Gorman, Michael R; Hatori, Megumi; Meadows, Jason D; Panda, Satchidananda; Mellon, Pamela L
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and β-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.
Martínez-Herrero, Sonia; Larrayoz, Ignacio M.; Narro-Íñiguez, Judit; Villanueva-Millán, María J.; Recio-Fernández, Emma; Pérez-Matute, Patricia; Oteo, José A.; Martínez, Alfredo
The link between intestinal inflammation, microbiota, and colorectal cancer is intriguing and the potential underlying mechanisms remain unknown. Here we evaluate the influence of adrenomedullin (AM) in microbiota composition and its impact on colitis with an inducible knockout (KO) mouse model for AM. Microbiota composition was analyzed in KO and wild type (WT) mice by massive sequencing. Colitis was induced in mice by administration of azoxymethane (AOM) followed by dextran sulfate sodium (DSS) in the drinking water. Colitis was evaluated using a clinical symptoms index, histopathological analyses, and qRT-PCR. Abrogation of the adm gene in the whole body was confirmed by PCR and qRT-PCR. KO mice exhibit significant changes in colonic microbiota: higher proportion of δ-Proteobacteria class; of Coriobacteriales order; and of other families and genera was observed in KO feces. Meanwhile these mice had a lower proportion of beneficial bacteria, such as Lactobacillus gasseri and Bifidobacterium choerinum. TLR4 gene expression was higher (p < 0.05) in KO animals. AM deficient mice treated with DSS exhibited a significantly worse colitis with profound weight loss, severe diarrhea, rectal bleeding, colonic inflammation, edema, infiltration, crypt destruction, and higher levels of pro-inflammatory cytokines. No changes were observed in the expression levels of adhesion molecules. In conclusion, we have shown that lack of AM leads to changes in gut microbiota population and in a worsening of colitis conditions, suggesting that endogenous AM is a protective mediator in this pathology. PMID:27965594
Clark, Daniel D.; Gorman, Michael R.; Hatori, Megumi; Meadows, Jason D.; Panda, Satchidananda; Mellon, Pamela L.
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and β-galactosidase, we demonstrated the lack of these SCN markers in all Six6- null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN. PMID:23382588
Punia, Sohan; Rumery, Kyle K; Yu, Elizabeth A; Lambert, Christopher M; Notkins, Abner L; Weaver, David R
Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.
Tung, Victoria W. K.; Burton, Thomas J.; Quail, Stephanie L.; Mathews, Miranda A.; Camp, Aaron J.
Balance and maintaining postural equilibrium are important during stationary and dynamic movements to prevent falls, particularly in older adults. While our sense of balance is influenced by vestibular, proprioceptive, and visual information, this study focuses primarily on the vestibular component and its age-related effects on balance. C57Bl/6J mice of ages 1, 5–6, 8–9 and 27–28 months were tested using a combination of standard (such as grip strength and rotarod) and newly-developed behavioral tests (including balance beam and walking trajectory tests with a vestibular stimulus). In the current study, we confirm a decline in fore-limb grip strength and gross motor coordination as age increases. We also show that a vestibular stimulus of low frequency (2–3 Hz) and duration can lead to age-dependent changes in balance beam performance, which was evident by increases in latency to begin walking on the beam as well as the number of times hind-feet slip (FS) from the beam. Furthermore, aged mice (27–28 months) that received continuous access to a running wheel for 4 weeks did not improve when retested. Mice of ages 1, 10, 13 and 27–28 months were also tested for changes in walking trajectory as a result of the vestibular stimulus. While no linear relationship was observed between the changes in trajectory and age, 1-month-old mice were considerably less affected than mice of ages 10, 13 and 27–28 months. Conclusion: this study confirms there are age-related declines in grip strength and gross motor coordination. We also demonstrate age-dependent changes to finer motor abilities as a result of a low frequency and duration vestibular stimulus. These changes showed that while the ability to perform the balance beam task remained intact across all ages tested, behavioral changes in task performance were observed. PMID:26869921
Liu, Richard R.; Brown, Craig E.; Murphy, Timothy H.
The cystine/glutamate exchanger (xCT) supplies intracellular cyst(e)ine for the production of glutathione, a major cellular anti-oxidant. xCT is enriched in brain regions associated with neurogenesis. Previous studies have shown that the malfunction of this protein greatly attenuates cell proliferation in vitro and is associated with brain atrophy in vivo. Using mice that are homozygous for a function-blocking deletion in xCT (Sut mice), we examined in vivo the role of xCT in cell proliferation in neurogenic regions of the subventricular zone (SVZ) and denate gyrus (DG) in the adult brain. Our results indicate that a high level of cellular proliferation in the adult brain persists even in the absence of functional xCT. Furthermore, in both young adult and middle-aged mice (3 and 11 months old), rates of SVZ cell proliferation were comparable between Sut and wild-type controls, although there was trend towards reduced proliferation in Sut mice (12% and 9% reduction, respectively). To our surprise, rates of cell proliferation in the DG were elevated in both 3- and 11-month-old Sut mice relative to controls (22% and 28% increase, respectively). These results demonstrate that xCT expression plays a role in regulating cellular proliferation in the DG, but not the SVZ of adult mice. Furthermore, unlike previous in vitro studies, our in vivo observations clearly indicate that xCT is not essential for ongoing cellular proliferation.
Kuraji, Manatsu; Matsuno, Tomonori; Satoh, Tazuko
Oral dryness, a serious problem for the aging Japanese society, is induced by aging-related hyposalivation and causes dysphagia, dysgeusia, inadaptation of dentures, and growth of oral Candida albicans. Oxidative stress clearly plays a role in decreasing saliva secretion and treatment with antioxidants such astaxanthin supplements may be beneficial. Therefore, we evaluated the effects of astaxanthin on the oral saliva secretory function of aging mice. The saliva flow increased in astaxanthin-treated mice 72 weeks after administration while that of the control decreased by half. The plasma d-ROMs values of the control but not astaxanthin-treated group measured before and 72 weeks after treatment increased. The diacron-reactive oxygen metabolites (d-ROMs) value of astaxanthin-treated mice 72 weeks after treatment was significantly lower than that of the control group was. The plasma biological antioxidative potential (BAP) values of the control but not astaxanthin-treated mice before and 72 weeks after treatment decreased. Moreover, the BAP value of the astaxanthin-treated group 72 weeks after treatment was significantly higher than that of the control was. Furthermore, the submandibular glands of astaxanthin-treated mice had fewer inflammatory cells than the control did. Specifically, immunofluorescence revealed a significantly large aquaporin-5 positive cells in astaxanthin-treated mice. Our results suggest that astaxanthin treatment may prevent age-related decreased saliva secretion. PMID:27698533
Thuy, Le Thi Thanh; Van Thuy, Tuong Thi; Matsumoto, Yoshinari; Hai, Hoang; Ikura, Yoshihiro; Yoshizato, Katsutoshi; Kawada, Norifumi
Cytoglobin (Cygb) was identified in hepatic stellate cells (HSCs) and pericytes of all organs; however, the effects of Cygb on cellular functions remain unclear. Here, we report spontaneous and age-dependent malformations in multiple organs of Cygb−/− mice. Twenty-six percent of young Cygb−/− mice (<1 year old) showed heart hypertrophy, cystic disease in the kidney or ovary, loss of balance, liver fibrosis and lymphoma. Furthermore, 71.3% (82/115) of aged Cygb−/− mice (1–2 years old) exhibited abnormalities, such as heart hypertrophy and cancer development in multiple organs; by contrast, 5.8% (4/68) of aged wild-type (WT) mice had abnormalities (p < 0.0001). Interestingly, serum and urine analysis demonstrated that the concentration of nitric oxide metabolites increased significantly in Cygb−/− mice, resulting in an imbalance in the oxidative stress and antioxidant defence system that was reversed by NG-monomethyl-L-arginine treatment. A senescent phenotype and evidence of DNA damage were found in primary HSCs and the liver of aged Cygb−/− mice. Moreover, compared with HSC+/+, HSC−/− showed high expression of Il-6 and chemokine mRNA when cocultured with mouse Hepa 1–6 cells. Thus, the absence of Cygb in pericytes provokes organ abnormalities, possibly via derangement of the nitric oxide and antioxidant defence system and through accelerated cellular senescence. PMID:27146058
Gut, Philipp; Huber, Katrin; Lohr, Jennifer; Brühl, Barbara; Oberle, Stephan; Treier, Mathias; Ernsberger, Uwe; Kalcheim, Chaya; Unsicker, Klaus
The diversification of neural-crest-derived sympathoadrenal (SA) progenitor cells into sympathetic neurons and neuroendocrine adrenal chromaffin cells was thought to be largely understood. In-vitro studies with isolated SA progenitor cells had suggested that chromaffin cell differentiation depends crucially on glucocorticoids provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor gene had revealed that adrenal chromaffin cells develop mostly normally in these mice. Alternative cues from the adrenal cortex that may promote chromaffin cell determination and differentiation have not been identified. We therefore investigated whether the chromaffin cell phenotype can develop in the absence of an adrenal cortex, using mice deficient for the nuclear orphan receptor steroidogenic factor-1 (SF1), which lack adrenal cortical cells and gonads. We show that in Sf1-/- mice typical chromaffin cells assemble correctly in the suprarenal region adjacent to the suprarenal sympathetic ganglion. The cells display most features of chromaffin cells, including the typical large chromaffin granules. Sf1-/- chromaffin cells are numerically reduced by about 50% compared with the wild type at embryonic day (E) 13.5 and E17.5. This phenotype is not accounted for by reduced survival or cell proliferation beyond E12.5. However, already at E12.5 the 'adrenal' region in Sf1-/- mice is occupied by fewer PHOX2B+ and TH+ SA cells as well as SOX10+ neural crest cells. Our results suggest that cortical cues are not essential for determining chromaffin cell fate, but may be required for proper migration of SA progenitors to and/or colonization of the adrenal anlage.
London, Barry; Wang, Dao W; Hill, Joseph A; Bennett, Paul B
The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito.We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild-type (Kv1.4+/+), heterozygous (Kv1.4+/-) and homozygous ‘knockout’ (Kv1.4−/−) mice. Kv1.4 RNA was truncated in Kv1.4−/− mice and protein expression was absent.Adult myocytes isolated from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF−1; means ± s.e.m.) were 53.8 ± 5.3, 45.3 ± 2.2 and 44.4 ± 2.8 in cells from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4−/−). The steady-state values (800 ms after the voltage clamp step) were 30.9 ± 2.9, 26.9 ± 3.8 and 23.5 ± 2.2, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4−/−). The inactivating portion of the current was unchanged in the targeted mice.The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best-fitting V½ (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/− and Kv1.4−/− mice were -53.5 ± 3.7, -51.1 ± 2.6 and -54.2 ± 2.4 mV, respectively. The slope factors (k) were -10.1 ± 1.4, -8.8 ± 1.4 and -9.5 ± 1.2 mV, respectively. The fast time constants for development of inactivation at -30 mV were 27.8 ± 2.2, 26.2 ± 5.1 and 19.6 ± 2.1 ms in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes, respectively. At +30 mV, they were 35.5 ± 2.6, 30.0 ± 2.1 and 28.7 ± 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at -80 mV were 32.5 ± 8.2, 23.3 ± 1.8 and 39.0 ± 3.7 ms, respectively
Morris, Jay; Nakata, Paul A; McConn, Michele; Brock, Amanda; Hirschi, Kendal D
Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.
Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.
Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035
Sun, X Z; Harada, Y N; Takahashi, S; Shiomi, N; Shiomi, T
Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.
Anderson, Catherine B.; Kinnamon, Sue C.
Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. PMID:26136251
Fitzgerald, Megan L.; Lupica, Carl R.; Pickel, Virginia M.
Cortical and striatal regions of the brain contain high levels of the cannabinoid-1 (CB1) receptor, the central neuronal mediator of activity-dependent synaptic plasticity evoked by endocannabinoids. The expression levels of parvalbumin, a calcium-binding protein found in fast-spiking interneurons of both regions, may be controlled in part by synaptic activity during critical periods of development. However, there is presently no evidence that CB1 receptor expression affects parvalbumin levels in either cortical or striatal interneurons. To assess this possibility, we examined parvalbumin immunoreactivity in the dorsolateral striatum, primary motor cortex (M1), and prefrontal cortex (PFC) of CB1 knockout and wild-type C57/BL6 mice. Quantitative densitometry showed a significant decrease in parvalbumin immunoreactivity within individual neurons in each of these regions of CB1 knockout mice relative to the controls. A significantly lower density (number of cells per unit area) of parvalbumin-labeled neurons was observed in the striatum, but not the cortical regions of CB1 knockout mice. These findings suggest that CB1 receptor deletion may elicit a compensatory mechanism for network homeostasis affecting parvalbumin-containing cortical and striatal interneurons. PMID:21445945
Lei, Lei; Spradling, Allan C
Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.
Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C
Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli.
Zigman, Jeffrey M.; Nakano, Yoshihide; Coppari, Roberto; Balthasar, Nina; Marcus, Jacob N.; Lee, Charlotte E.; Jones, Juli E.; Deysher, Amy E.; Waxman, Amanda R.; White, Ryan D.; Williams, Todd D.; Lachey, Jennifer L.; Seeley, Randy J.; Lowell, Bradford B.; Elmquist, Joel K.
Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Since its discovery, accumulating evidence has suggested that ghrelin may play a role in signaling and reversing states of energy insufficiency. For example, ghrelin levels rise following food deprivation, and ghrelin administration stimulates feeding and increases body weight and adiposity. However, recent loss-of-function studies have raised questions regarding the physiological significance of ghrelin in regulating these processes. Here, we present results of a study using a novel GHSR-null mouse model, in which ghrelin administration fails to acutely stimulate food intake or activate arcuate nucleus neurons. We show that when fed a high-fat diet, both female and male GHSR-null mice eat less food, store less of their consumed calories, preferentially utilize fat as an energy substrate, and accumulate less body weight and adiposity than control mice. Similar effects on body weight and adiposity were also observed in female, but not male, GHSR-null mice fed standard chow. GHSR deletion also affected locomotor activity and levels of glycemia. These findings support the hypothesis that ghrelin-responsive pathways are an important component of coordinated body weight control. Moreover, our data suggest that ghrelin signaling is required for development of the full phenotype of diet-induced obesity. PMID:16322794
Torrentera, Fabiola Aguilar; Glaichenhaus, Nicolas; Laman, Jon D.; Carlier, Yves
Although BALB/c mice develop lesions when infected with Leishmania mexicana, the mechanisms which are responsible for susceptibility to this parasite have not been elucidated. In contrast, susceptibility of BALB/c mice to Leishmania major has been shown to depend on the early production of interleukin-4 (IL-4) by T cells which react to the parasitic LACK antigen. Here, we demonstrate that the lesions induced by L. mexicana are delayed compared to those induced by L. major but rapidly develop at later time points. Interestingly, while LACK-tolerant BALB/c-derived IE-LACK transgenic mice were resistant to L. major, they were susceptible to L. mexicana and developed lesions similar to those observed in wild-type BALB/c mice. The latter result was observed despite the fact that (i) LACK was expressed by L. mexicana, (ii) splenocytes from BALB/c mice were able to stimulate LACK-specific T-cell hybridoma cells when incubated with live L. mexicana promastigotes, and (iii) LACK-specific T cells contributed to IL-4 production in L. mexicana-infected BALB/c mice. Thus, in contrast to what was observed for L. major-infected mice, LACK-specific T cells do not play a critical role in determining susceptibility to L. mexicana. Although BALB/c mice are susceptible to both L. major and L. mexicana, the mechanisms which are responsible for susceptibility to these parasites are likely to be different. PMID:11119565
Maulik, Mahua; Thinakaran, Gopal; Kar, Satyabrata
Niemann-Pick type C (NPC) disease, a rare autosomal recessive disorder caused mostly by mutation in NPC1 gene, is pathologically characterized by the accumulation of free cholesterol in brain and other tissues. This is accompanied by gliosis and loss of neurons in selected brain regions, including the cerebellum. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer’s disease, including the presence of neurofibrillary tangles and increased levels of amyloid precursor protein (APP)-derived β-amyloid (Aβ) peptides in vulnerable brain neurons. To evaluate the role of Aβ in NPC disease, we determined the gene expression profile in selected brain regions of our recently developed bigenic ANPC mice, generated by crossing APP transgenic (Tg) mice with heterozygous Npc1-deficient mice. The ANPC mice exhibited exacerbated neuronal and glial pathology compared to other genotypes [i.e., APP-Tg, double heterozygous (Dhet), Npc1-null and wild-type mice]. Analysis of expression profiles of 86 selected genes using real-time RT-PCR arrays showed a wide-spectrum of alterations in the four genotypes compared to wild-type controls. The changes observed in APP-Tg and Dhet mice are limited to only few genes involved mostly in the regulation of cholesterol metabolism, whereas Npc1-null and ANPC mice showed alterations in the expression profiles of a number of genes regulating cholesterol homeostasis, APP metabolism, vesicular trafficking and cell death mechanism in both hippocampus and cerebellum compared to wild-type mice. Intriguingly, ANPC and Npc1-null mice, with some exceptions, exhibited similar changes, although more genes were differentially expressed in the affected cerebellum than the relatively spared hippocampus. The altered gene profiles were found to match with the corresponding protein levels. These results suggest that lack of Npc1 protein can alter the expression profile of selected transcripts as well as proteins, and APP
Assi, Roland; Brownson, Kirstyn E.; Hall, Michael R.; Kuwahara, Go; Vasilas, Penny; Dardik, Alan
Background: Endovascular angioplasty and stent placement is currently the most frequent treatment for iliac artery occlusive disease. However, despite a successful endovascular procedure, some patients do not experience symptomatic improvement and satisfaction with their care. This study seeks to identify patient-related factors associated with lack of symptomatic improvement after endovascular iliac artery treatment in male veterans. Methods: Retrospective review of patients treated with endovascular methods for iliac artery occlusive disease between January 2008 and July 2012 at VA Connecticut Healthcare System. Symptomatic improvement on the first post-operative visit was evaluated, with bilateral treatments counted separately. Results: Sixty-two patients had 91 iliac arteries treated with angioplasty and stent placement. Forty-seven (52 percent) legs had critical limb ischemia, and 77 (85 percent) had at least two-vessel distal runoff. Angiographic success was 100 percent. Patient-reported symptomatic improvement at the first post-operative visit was 55 percent (50/91). Lack of symptomatic improvement correlated with older age (OR 1.09 [1.03-1.17], p = 0.008), presence of critical limb ischemia (OR 3.03 [1.09-8.65], p = 0.034), and need for additional surgical intervention (OR 5.61 [1.65-17.36], p = 0.006). Survival, primary and secondary patency, and freedom from restenosis were comparable between patients who reported symptomatic improvement and those who did not. Conclusions: Despite angiographically successful revascularization, patients who are older or have critical limb ischemia who are treated with isolated endovascular iliac artery intervention are more likely to require additional interventions and less likely to experience symptomatic improvement. These patients may need more extensive infra-inguinal revascularization than isolated iliac angioplasty and stent placement, despite a preserved ankle-brachial index. Quality of life needs to be measured
Besmer, Dahlia M; Curry, Jennifer M; Roy, Lopamudra D; Tinder, Teresa L; Sahraei, Mahnaz; Schettini, Jorge; Hwang, Sun-Il; Lee, Yong Y; Gendler, Sandra J; Mukherjee, Pinku
MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.
Coleman, Mitchell C.; Olivier, Alicia K.; Jacobus, James A.; Mapuskar, Kranti A.; Mao, Gaowei; Martin, Sean M.; Riley, Dennis P.; Gius, David
Abstract Aims: This study determined whether acute radiation-induced liver injury seen in Sirtuin3−/− mice after exposure to Cs-137 γ-rays was mediated by superoxide anion (O2•−). Results: Male wild-type (WT) and SIRT3−/− mice were given 2×2 Gy whole-body radiation doses separated by 24 h and livers were harvested 20 h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3−/− animals, relative to WT animals, before irradiation, but this increase was not detected 20 h after radiation exposure. In addition, irradiated livers from SIRT3−/− animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2 mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30 min before each fraction. Innovation: Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2•− in the liver injury process initiated by whole-body irradiation in SIRT3−/− mice. Conclusion: These results support the hypothesis that O2•− mediates acute liver injury in SIRT3−/− animals exposed to whole-body γ-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo. Antioxid. Redox Signal. 20, 1423–1435. PMID:23919724
Trebino, Catherine E.; Stock, Jeffrey L.; Gibbons, Colleen P.; Naiman, Brian M.; Wachtmann, Timothy S.; Umland, John P.; Pandher, Karamjeet; Lapointe, Jean-Martin; Saha, Sipra; Roach, Marsha L.; Carter, Demetrius; Thomas, Nathalie A.; Durtschi, Becky A.; McNeish, John D.; Hambor, John E.; Jakobsson, Per-Johan; Carty, Thomas J.; Perez, Jose R.; Audoly, Laurent P.
Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states. PMID:12835414
Streijger, Femke; Pluk, Helma; Oerlemans, Frank; Beckers, Gaby; Bianco, Antonio C.; Ribeiro, Miriam O.; Wieringa, Bé; Van der Zee, Catharina E.E.M.
The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0°C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK−−/−− mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK−−/−− mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect. PMID:19419668
Pesquero, J B; Araujo, R C; Heppenstall, P A; Stucky, C L; Silva, J A; Walther, T; Oliveira, S M; Pesquero, J L; Paiva, A C; Calixto, J B; Lewin, G R; Bader, M
Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using an in vitro preparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.
Moles, Anna; Kieffer, Brigitte L; D'Amato, Francesca R
Endogenous opioid binding to micro receptors is hypothesized to mediate natural rewards and has been proposed to be the basis of infant attachment behavior. Here, we report that micro-opioid receptor knockout mouse pups emit fewer ultrasonic vocalizations when removed from their mothers but not when exposed to cold or male mice odors. Moreover these knockout pups do not show a preference toward their mothers' cues and do not show ultrasonic calls potentiation after brief maternal exposure. Results from this study may indicate a molecular mechanism for diseases characterized by deficits in attachment behavior, such as autism or reactive attachment disorder.
Enns, Linda C.; Morton, John F.; Treuting, Piper R.; Emond, Mary J.; Wolf, Norman S.; McKnight, G. S.; Rabinovitch, Peter S.; Ladiges, Warren C.
Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIβ, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ−/−) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIβ−/− male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age
Zhang, J; Henrion, D; Ebrahimian, T; Benessiano, J; Colucci-Guyon, E; Langa, F; Lévy, B I; Boulanger, C M
Experiments were designed to investigate endothelial function in the aorta of mice lacking the gene for the cytoskeleton protein vimentin (vim -/- ). Rings with and without endothelium from wild-type (vim +/+ ), heterozygous (vim +/- ), and homozygous (vim -/- ) mice were suspended in organ chambers to record of changes in isometric tension. During phenylephrine contraction, acetylcholine evoked comparable endothelium-dependent relaxations in the three groups. In the presence of Nomega-nitro-L-arginine, acetylcholine caused endothelium-dependent contractions, which were greater in vim -/- than in vim +/+ and vim +/- aortas. Indomethacin did not affect relaxation to acetylcholine in vim +/+ or in vim +/-, but it significantly increased the maximal response in vim -/- (67 +/- 7 vs. 102 +/- 4%). Response to acetylcholine in vim -/- aortas was not affected by cyclooxygenase type 2 inhibitor NS-398, the thromboxane receptor antagonist SQ-29,548, or superoxide dismutase. Relaxations to sodium nitroprusside were not different between vim +/+ and vim -/- mice and were not affected by cyclooxygenase inhibition. Cyclic guanosine monophosphate levels, which were increased to a comparable level by acetylcholine in vim +/+ and vim -/-, were augmented by indomethacin in vim -/- aortas but not in vim +/+ aortas. Expression of endothelial nitric oxide synthase was not different between vim +/+ and vim -/- preparations. These results suggest that despite comparable endothelium-dependent responses to acetylcholine, endothelial cells from vim -/- mice release a cyclooxygenase product that compensates the augmented contribution of nitric oxide.
Thomas, Susan N.; Rutkowski, Joseph M.; Pasquier, Miriella; Kuan, Emma L.; Alitalo, Kari; Randolph, Gwendalyn J.; Swartz, Melody A.
Lymphatic vessels transport interstitial fluid, soluble antigen, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower antibody titers. In contrast, although delayed, T cell responses were robust after 21 days, including high levels of antigen-specific CD8+ T cells and production of IFN-γ, IL-4 and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. Additionally, one-year-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity. PMID:22844119
Thimraj, Tania A; Birru, Rahel L; Mitra, Ankita; Schulz, Holger; Leikauf, George D; Ganguly, Koustav
Superoxide dismutase 3, extracellular (SOD3) polymorphisms have been implicated in reduced pulmonary function development and altered risk for chronic obstructive pulmonary disease. We previously reported that gene-targeted Sod3-/- mice have impaired lung function and human SOD3 variants are associated with reduced pulmonary function in children. Reduced lung SOD3 levels were reported in mice with lower lung function with the greatest difference occurring during alveogenesis phase [postnatal (P) days 14-28]. Interactions between homeobox (HOX), wingless-type MMTV integration site member (WNT), and fibroblast growth factor (FGF) signaling govern complex developmental processes in several organs. A subset of HOX family members, HOXA5 and HOXB5, is expressed in the developing lung. Therefore, in this study we assessed the transcript expression of these family members and their downstream targets in Sod3-/- mice during alveogenesis (P14). In the lung of Sod3-/- mice, Hoxa5 and Hoxb5 increased. These transcription factors regulate WNT gene expression and were accompanied by increases in their downstream targets Wnt2 and Wnt5A, canonical and noncanonical WNT members, respectively. The WNT signaling target, lymphoid enhancer binding factor 1 (Lef1), also increased along with its downstream targets Fgf2, Fgf7, and Fgf10 in the lungs of Sod3-/- mice. Due to limited knowledge on the role of FGF2 in lung development, we further examined FGF2 protein and found increased levels in the bronchial and alveolar type II epithelial cells of Sod3-/- mice compared to age-matched controls. Thus, our findings suggest that deficient management of extracellular superoxide can lead to altered lung developmental signaling during alveogenesis in mice.
Kannangara, Timal S; Lucero, Melanie J; Gil-Mohapel, Joana; Drapala, Robert J; Simpson, Jessica M; Christie, Brian R; van Praag, Henriette
Cell proliferation and neurogenesis are diminished in the aging mouse dentate gyrus. However, it is not known whether isolated or social living affects cell genesis and stress levels in old animals. To address this question, aged (17-18 months old) female C57Bl/6 mice were single or group housed, under sedentary or running conditions. We demonstrate that both individual and socially housed aged C57Bl/6 mice have comparable basal cell proliferation levels and demonstrate increased running-induced cell genesis. To assess stress levels in young and aged mice, corticosterone (CORT) was measured at the onset of the active/dark cycle and 4h later. In young mice, no differences in CORT levels were observed as a result of physical activity or housing conditions. However, a significant increase in stress in socially housed, aged sedentary animals was observed at the onset of the dark cycle; CORT returned to basal levels 4h later. Together, these results indicate that voluntary exercise reduces stress in group housed aged animals and enhances hippocampal cell proliferation.
Schiffmann, Serge N.; Cheron, Guy; Lohof, Ann; d’Alcantara, Pablo; Meyer, Michael; Parmentier, Marc; Schurmans, Stéphane
In the cerebellum, the parallel fiber-Purkinje cell synapse can undergo long-term synaptic plasticity suggested to underlie motor learning and resulting from variations in intracellular calcium concentration ([Ca2+]i). Ca2+ binding proteins are enriched in the cerebellum, but their role in information processing is not clear. Here, we show that mice deficient in calretinin (Cr−/−) are impaired in tests of motor coordination. An impairment in Ca2+ homeostasis in Cr−/− Purkinje cells was supported by the high Ca2+-saturation of calbindin-D28k in these cells. The firing behavior of Purkinje cells is severely affected in Cr−/− alert mice, with alterations of simple spike firing rate, complex spike duration, and simple spike pause. In contrast, in slices, transmission at parallel fiber- or climbing fiber-Purkinje cell synapses is unaltered, indicating that marked modifications of the firing behavior in vivo can be undetectable in slice. Thus, these results show that calretinin plays a major role at the network level in cerebellar physiology. PMID:10220453
Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier
The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523
Cenik, Bercin K.; Garg, Ankit; McAnally, John R.; Shelton, John M.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.; Liu, Ning
Maintenance of skeletal muscle structure and function requires a precise stoichiometry of sarcomeric proteins for proper assembly of the contractile apparatus. Absence of components of the sarcomeric thin filaments causes nemaline myopathy, a lethal congenital muscle disorder associated with aberrant myofiber structure and contractility. Previously, we reported that deficiency of the kelch-like family member 40 (KLHL40) in mice results in nemaline myopathy and destabilization of leiomodin-3 (LMOD3). LMOD3 belongs to a family of tropomodulin-related proteins that promote actin nucleation. Here, we show that deficiency of LMOD3 in mice causes nemaline myopathy. In skeletal muscle, transcription of Lmod3 was controlled by the transcription factors SRF and MEF2. Myocardin-related transcription factors (MRTFs), which function as SRF coactivators, serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. Conversely, conditions that favor actin polymerization de-repress MRTFs and activate SRF-dependent genes. We demonstrated that the actin nucleator LMOD3, together with its stabilizing partner KLHL40, enhances MRTF-SRF activity. In turn, SRF cooperated with MEF2 to sustain the expression of LMOD3 and other components of the contractile apparatus, thereby establishing a regulatory circuit to maintain skeletal muscle function. These findings provide insight into the molecular basis of the sarcomere assembly and muscle dysfunction associated with nemaline myopathy. PMID:25774500
Cruz-Aguado, Reyniel; Winkler, Daniella; Shaw, Christopher A
Beta-methylamino-L-alanine (BMAA) is an excitotoxin allegedly involved in ALS-parkinsonism-dementia complex (ALS-PDC), a neurological disorder found in Guam and its surrounding islands, in which motor neuron disease symptoms can present alone or can co-occur with parkinsonism and dementia. Although in vitro experiments have shown BMAA's neurotoxic properties, studies using adult animals and systemic administration which better model the case of environmentally-induced human neurodegenerative diseases have not supported the involvement of BMAA in these disorders. In order to better test the hypothesized role of BMAA in neurodegeneration, we fed adult mice BMAA at a dose (28 mg/kg body weight, daily for 30 days) that reproduces the natural levels and tested the animals with a battery of behavioural tests, the latter including the evaluation of motor coordination, motor neuron-mediated reflexes, locomotion, muscular strength and memory. We also assessed whether BMAA exposure triggers cell death in the central nervous system (CNS) of mice by examining neuronal numbers and glial response in the spinal cord and the brain. No motor, cognitive or neuropathological outcome resulted from this feeding paradigm. Our findings support neither the causal role of BMAA in neurodegeneration nor the specific involvement of this amino acid in ALS-PDC.
Suidan, Georgette L.; Vanderhorst, Veronique; Hampton, Thomas G.; Wong, Siu Ling; Voorhees, Jaymie R.; Wagner, Denisa D.
The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system. PMID:23516593
Kenner, Lukas; Hoebertz, Astrid; Beil, F Timo; Beil, Timo; Keon, Niamh; Karreth, Florian; Eferl, Robert; Scheuch, Harald; Szremska, Agnieszka; Amling, Michael; Schorpp-Kistner, Marina; Angel, Peter; Wagner, Erwin F
Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.
Johnson, Linda K; Widi, Antin Yn; Rowarth, Serrin; Baxter, Alan G
Here we describe the gross and microscopic findings of naturally occurring, β-hemolytic Escherichia coli peritonitis in B6.129-Myd88(tm1Aki) male and female mice. Over approximately 5 mo, 10 homozygous mutant mice deficient in myeloid differentiation factor 88 (C57BL/6 strain; male and female) that had not been used in research protocols developed rapid-onset abdominal swelling associated with copious viscous ascites. Each mouse developed an anterior peritonitis, primarily involving the parietal peritoneum and the visceral surface of the spleen, liver, diaphragm, and stomach. Inflammation was confined to the organ surfaces, with no indication of septicemia or grossly apparent gastrointestinal perforation or other tissue compromise that would initiate peritonitis. Peritonitis was likely attributable to compromised antibacterial innate immunity; cohoused, similarly immunodeficient littermates did not develop similar clinical signs. An unusual finding in all cases was mesothelial cell hyperplasia and hypertrophy. Although the underlying innate immune deficiency accounts for much of the observed pathology, the remarkable mesothelial cell morphology and the episodic nature of the peritonitis in some littermates and not others remain unexplained.
Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F
The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.
Liu, Ning; Bezprozvannaya, Svetlana; Shelton, John M.; Frisard, Madlyn I.; Hulver, Matthew W.; McMillan, Ryan P.; Wu, Yaru; Voelker, Kevin A.; Grange, Robert W.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.
MicroRNAs modulate cellular phenotypes by inhibiting expression of mRNA targets. In this study, we have shown that the muscle-specific microRNAs miR-133a-1 and miR-133a-2 are essential for multiple facets of skeletal muscle function and homeostasis in mice. Mice with genetic deletions of miR-133a-1 and miR-133a-2 developed adult-onset centronuclear myopathy in type II (fast-twitch) myofibers, accompanied by impaired mitochondrial function, fast-to-slow myofiber conversion, and disarray of muscle triads (sites of excitation-contraction coupling). These abnormalities mimicked human centronuclear myopathies and could be ascribed, at least in part, to dysregulation of the miR-133a target mRNA that encodes dynamin 2, a GTPase implicated in human centronuclear myopathy. Our findings reveal an essential role for miR-133a in the maintenance of adult skeletal muscle structure, function, bioenergetics, and myofiber identity; they also identify a potential modulator of centronuclear myopathies. PMID:21737882
Mito, Takayuki; Ishizaki, Hikari; Suzuki, Michiko; Morishima, Hitomi; Ota, Azusa; Ishikawa, Kaori; Nakada, Kazuto; Maeno, Akiteru; Shiroishi, Toshihiko; Hayashi, Jun-Ichi
The spectra of phenotypes associated with aging and mitochondrial diseases sometimes appear to overlap with each other. We used aged mice and a mouse model of mitochondrial diseases (transmitochondrial mito-miceΔ with deleted mtDNA) to study whether premature aging phenotypes observed in mtDNA mutator mice are associated with aging or mitochondrial diseases. Here, we provide convincing evidence that all the mice examined had musculoskeletal disorders of osteoporosis and muscle atrophy, which correspond to phenotypes prevalently observed in the elderly. However, precise investigation of musculoskeletal disorders revealed that the spectra of osteoporosis and muscle atrophy phenotypes in mtDNA mutator mice were very close to those in mito-miceΔ, but different from those of aged mice. Therefore, mtDNA mutator mice and mito-miceΔ, but not aged mice, share the spectra of musculoskeletal disorders.
Giza, Joanna; Urbanski, Michael J.; Prestori, Francesca; Bandyopadhyay, Bhaswati; Yam, Annie; Friedrich, Victor; Kelley, Kevin; D'Angelo, Egidio; Goldfarb, Mitchell
Deletion of the human SHANK3 gene near the terminus of chromosome 22q is associated with Phelan-McDermid syndrome and autism spectrum disorders. Nearly all such deletions also span the tightly linked IB2 gene. We show here that IB2 protein is broadly expressed in the brain and is highly enriched within postsynaptic densities. Experimental disruption of the IB2 gene in mice reduces AMPA and enhances NMDA receptor-mediated glutamatergic transmission in cerebellum, changes the morphology of Purkinje cell dendritic arbors, and induces motor and cognitive deficits suggesting an autism phenotype. These findings support a role for human IB2 mutation as a contributing genetic factor in Chr22qter-associated cognitive disorders. PMID:21048139
Shimshek, Derya R; Schweizer, Tatjana; Schmid, Peter; van der Putten, P Herman
Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species.
Hirsch, Emilio; Pozzato, Michela; Vercelli, Alessandro; Barberis, Laura; Azzolino, Ornella; Russo, Chiara; Vanni, Cristina; Silengo, Lorenzo; Eva, Alessandra; Altruda, Fiorella
Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation. PMID:11940671
Chen, Yei-Tsung; Collins, Loretta L.; Uno, Hideo; Chou, Samuel M.; Meshul, Charles K.; Chang, Shu-Shi; Chang, Chawnshang
Since Testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological functions remain largely unknown. In this study, the TR4 knockout (TR4−/−) mouse model was used to investigate the role of TR4 in the adult cerebellum. Behaviorally, these null mice exhibit unsteady gait, as well as involuntary postural and kinetic movements, indicating a disturbance of cerebellar function. In the TR4−/− brain, cerebellar restricted hypoplasia is severe and cerebellar vermal lobules VI and VII are underdeveloped, while no structural alterations in the cerebral cortex are observed. Histological analysis of the TR4−/− cerebellar cortex reveals reductions in granule cell density, as well as a decreased number of parallel fiber boutons that are enlarged in size. Further analyses reveal that the levels of GABA and GAD are decreased in both Purkinje cells and interneurons of the TR4−/− cerebellum, suggesting that the inhibitory circuits signaling within and from the cerebellum may be perturbed. In addition, in the TR4−/− cerebellum, immunoreactivity of GluR2/3 was reduced in Purkinje cells, but increased in the deep cerebellar nuclei. Together, these results suggest that the behavioral phenotype of TR4−/− mice may result from disrupted inhibitory pathways in the cerebellum. No progressive atrophy was observed at various adult stages in the TR4−/− brain, therefore the disturbances most likely originate from a failure to establish proper connections between principal neurons in the cerebellum during development. PMID:17706948
Akil, Omar; Chang, Jolie; Hiel, Hakim; Kong, Jee-Hyun; Yi, Eunyoung; Glowatzki, Elisabeth; Lustig, Lawrence R
After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.
Sarnyai, Zoltán; Sibille, Etienne L.; Pavlides, Constantine; Fenster, Robert J.; McEwen, Bruce S.; Tóth, Miklós
The hippocampus is a major limbic target of the brainstem serotonergic neurons that modulate fear, anxiety, and learning through postsynaptic serotonin1A receptors (5-HT1A receptors). Because chronic stress selectively down-regulates the 5-HT1A receptors in the hippocampus, we hypothesized that mice lacking these receptors may exhibit abnormalities reminiscent of symptoms of stress-related psychiatric disorders. In particular, a hippocampal deficit in the 5-HT1A receptor could contribute to the cognitive abnormalities often seen in these disorders. To test whether a deficit in 5-HT1A receptors impairs hippocampus-related functions, we studied hippocampal-dependent learning and memory, synaptic plasticity in the hippocampus, and limbic neuronal excitability in 5-HT1A-knockout (KO) mice. 5-HT1A-KO animals showed a deficit in hippocampal-dependent learning and memory tests, such as the hidden platform (spatial) version of the Morris water maze and the delayed version of the Y maze. The performance of KO mice was not impaired in nonhippocampal memory tasks such as the visible platform (nonspatial) version of the Morris water maze, the immediate version of the Y maze, and the spontaneous-alternation test of working memory. Furthermore, paired-pulse facilitation in the dentate gyrus of the hippocampus was impaired in 5-HT1A-KO mice. Finally, 5-HT1A-KO mice, as compared with wild-type animals, displayed higher limbic excitability manifested as lower seizure threshold and higher lethality in response to kainic acid administration. These results demonstrate that 5-HT1A receptors are required for maintaining normal hippocampal functions and implicate a role for the 5-HT1A receptor in hippocampal-related symptoms, such as cognitive disturbances, in stress-related disorders. PMID:11121072
Sun, Chao; Warland, David K; Ballesteros, Jose M; van der List, Deborah; Chalupa, Leo M
The structural and functional properties of the visual system are disrupted in mutant animals lacking the beta2 subunit of the nicotinic acetylcholine receptor. In particular, eye-specific retinogeniculate projections do not develop normally in these mutants. It is widely thought that the developing retinas of beta2(-/-) mutants do not manifest correlated activity, leading to the notion that retinal waves play an instructional role in the formation of eye-specific retinogeniculate projections. By multielectrode array recordings, we show here that the beta2(-/-) mutants have robust retinal waves during the formation of eye-specific projections. Unlike in WT animals, however, the mutant retinal waves are propagated by gap junctions rather than cholinergic circuitry. These results indicate that lack of retinal waves cannot account for the abnormalities that have been documented in the retinogeniculate pathway of the beta2(-/-) mutants and suggest that other factors must contribute to the deficits in the visual system that have been noted in these animals.
Shelkovnikova, Tatyana A.; Peters, Owen M.; Deykin, Alexey V.; Connor-Robson, Natalie; Robinson, Hannah; Ustyugov, Alexey A.; Bachurin, Sergey O.; Ermolkevich, Tatyana G.; Goldman, Igor L.; Sadchikova, Elena R.; Kovrazhkina, Elena A.; Skvortsova, Veronica I.; Ling, Shuo-Chien; Da Cruz, Sandrine; Parone, Philippe A.; Buchman, Vladimir L.; Ninkina, Natalia N.
Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice. PMID:23867462
Rico, Laura; Del Rio, Marcela; Bravo, Ana; Ramirez, Angel; Jorcano, José L; Page, M Angustias; Larcher, Fernando
The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopic epidermal protein overexpression need to be carefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratin K5 gene regulatory sequences. Histopathological analysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocyte-derived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3-4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance.
Zhang, Tiantian; Suryawanshi, Yogesh R; Kordish, Dennis H; Woyczesczyk, Helene M; Jeng, David; Essani, Karim
Neuregulin (NRG), an epidermal growth factor is known to promote the growth of various cell types, including human melanoma cells through ErbB family of tyrosine kinases receptors. Tanapoxvirus (TPV)-encoded protein TPV-15L, a functional mimic of NRG, also acts through ErbB receptors. Here, we show that the TPV-15L protein promotes melanoma proliferation. TPV recombinant generated by deleting the 15L gene (TPVΔ15L) showed replication ability similar to that of wild-type TPV (wtTPV) in owl monkey kidney cells, human lung fibroblast (WI-38) cells, and human melanoma (SK-MEL-3) cells. However, a TPV recombinant with both 15L and the thymidine kinase (TK) gene 66R ablated (TPVΔ15LΔ66R) replicated less efficiently compared to TPVΔ15L and the parental virus. TPVΔ15L exhibited more robust tumor regression in the melanoma-bearing nude mice compared to other TPV recombinants. Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma.
Slaga, T J; Bracken, W M; Viaje, A; Berry, D L; Fischer, S M; Miller, D R
The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.
Fay, Matthew J; Nguyen, My Trang; Snouwaert, John N; Dye, Rebecca; Grant, Delores J; Bodnar, Wanda M; Koller, Beverly H
UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s.
Fay, Matthew J.; Nguyen, My Trang; Snouwaert, John N.; Dye, Rebecca; Grant, Delores J.; Bodnar, Wanda M.
UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s. PMID:26354949
Guillén, Natalia; Acín, Sergio; Navarro, María A; Surra, Joaquín Carlos; Arnal, Carmen; Lou-Bonafonte, José Manuel; Muniesa, Pedro; Martínez-Gracia, María Victoria; Osada, Jesús
The low incidence of cardiovascular disease in countries bordering the Mediterranean basin, where olive oil is the main source of dietary fat, has stimulated interest in the chemical composition of olive oil and in the production of other oils enriched with its minor components. This review summarizes what has been learned about the effects of different olive oil preparations on the development of atherosclerosis and about the prognostic value of associated plasma variables in the disease from experiments on genetically modified mice that spontaneously develop atherosclerosis. The limitations of this animal model associated with its morphological and physiological differences with humans are minimized by the similarity of the two genomes and by the potential for increased understanding attainable, given that the dietary interventions reported here would have taken 400 years to achieve in humans. As observed in traditional Mediterranean populations, it has been confirmed that extra virgin olive oil is beneficial when consumed judiciously and in a diet that is low in cholesterol due to the relative scarcity of animal products. Furthermore, the use of genomic techniques has led to the identification of new markers of response to olive oil. In conclusion, multidisciplinary research into extra virgin olive oil is expanding our knowledge of the substance's biological properties.
Ghilan, M; Hryciw, B N; Brocardo, P S; Bostrom, C A; Gil-Mohapel, J; Christie, B R
The fragile X mental retardation protein (FMRP) is an important regulator of protein translation, and a lack of FMRP expression leads to a cognitive disorder known as fragile X syndrome (FXS). Clinical symptoms characterizing FXS include learning impairments and heightened anxiety in response to stressful situations. Here, we report that, in response to acute stress, mice lacking FMRP show a faster elevation of corticosterone and a more immediate impairment in N-methyl-d-aspartate receptor (NMDAR) dependent long-term potentiation (LTP) in the dentate gyrus (DG). These stress-induced LTP impairments were rescued by administering the glucocorticoid receptor (GR) antagonist RU38486. Administration of RU38486 also enhanced LTP in Fmr1(-/y) mice in the absence of acute stress to wild-type levels, and this enhancement was blocked by application of the NMDAR antagonist 2-amino-5-phosphonopentanoic acid. These results suggest that a loss of FMPR results in enhanced GR signaling that may adversely affect NMDAR dependent synaptic plasticity in the DG.
Kolesnikov, Alexander V; Fan, Jie; Crouch, Rosalie K; Kefalov, Vladimir J
Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization.
Heredia, Dante J; Gershon, Michael D; Koh, Sang Don; Corrigan, Robert D; Okamoto, Takanubu; Smith, Terence K
Although there is general agreement that mucosal 5-hydroxytryptamine (5-HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5-HT is critical. We therefore tested the hypothesis that the secretion of 5-HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1(-/-) mice), which is the rate-limiting enzyme in the biosynthesis of mucosal but not neuronal 5-HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1(+/+)) mice, Tph1(-/-) animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1(+/+) colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1(+/+)); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1(-/-) colon; and (9) the 5-HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1(+/+) mice, failed to alter CMMCs in Tph1(-/-) animals. Our observations suggest that mucosal 5-HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.
Chugh, Deepti; Ali, Idrish; Bakochi, Anahita; Bahonjic, Elma; Etholm, Lars; Ekdahl, Christine T.
Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and 2 months of age) and tonic-clonic (3.5-4 months) phase of synapsin 2 knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis factor-α, and a decrease in chemokine keratinocyte chemoattractant/ growth-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-2 and neurofascin and potassium chloride co-transporter KCC2. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin 2 knockout mice may be initiated or spread. PMID:26177381
Benderro, Girriso F; LaManna, Joseph C
In order to maintain normal cellular function, mammalian tissue oxygen concentrations must be tightly regulated within a narrow physiological range. The hormone erythropoietin (EPO) is essential for maintenance of tissue oxygen supply by stimulating red blood cell production and promoting their survival. In this study we compared the effects of 290 Torr atmospheric pressure on the kidney EPO protein levels in young (4-month-old) and aged (24-month-old) C57BL/6 mice. The mice were sacrificed after being anesthetized, and kidney samples were collected and processed by Western blot analysis. Relatively low basal expression of EPO during normoxia in young mice showed significant upregulation in hypoxia and stayed upregulated throughout the hypoxic period (threefold compared to normoxic control), showing a slight decline toward the third week. Whereas, a relatively higher normoxic basal EPO protein level in aged mice did not show significant increase until seventh day of hypoxia, but showed significant upregulation in prolonged hypoxia. Hence, we confirmed that there is a progressively increased accumulation of EPO during chronic hypoxia in young and aged mouse kidney, and the EPO upregulation during hypoxia showed a similarity with the pattern of increase in hematocrit, which we have reported previously.
El Idrissi, Abdeslem
Aging of the brain is characterized by several neurochemical modifications involving structural proteins, neurotransmitters, neuropeptides and related receptors. Alterations of neurochemical indices of synaptic function have been considered as indicators of age-related impairment of central functions, such as locomotion, memory and sensory performances. Several studies demonstrated that GABA receptors, glutamic acid decarboxylase (GAD65&67), and different subpopulations of GABAergic neurons are markedly decreased in experimental animal brains during aging. Thus, the age-related decline in cognitive functions could be attributable, at least in part, to decrements in GABA inhibitory neurotransmission. In this study, using a passive avoidance test, we show that chronic supplementation of taurine to aged mice significantly ameliorates the age-dependent decline in memory acquisition and retention. We have previously shown that taurine supplementation caused changes in the GABAergic system. These changes include increased levels of the neurotransmitters GABA and glutamate, increased expression of glutamic acid decarboxylase and the neuropeptide somatostatin and increase in the number of somatostatin-positive neurons. These specific alterations of the inhibitory system caused by taurine treatment oppose those naturally occurring in aging, and suggest a protective role of taurine against the normal aging process. Increased understanding of age-related neurochemical changes in the GABAergic system will be important in elucidating the underpinnings of the functional changes of aging. Taurine might help forestall the age-related decline in cognitive functions through alterations of the GABAergic system.
Gong, Tzy-Wen L.; Karolyi, I. Jill; MacDonald, James; Beyer, Lisa; Raphael, Yehoash; Kohrman, David C.; Camper, Sally A.
The vertebrate cochlea is a complex organ optimized for sound transduction. Auditory hair cells, with their precisely arranged stereocilia bundles, transduce sound waves to electrical signals that are transmitted to the brain. Mutations in the unconventional myosin XV cause deafness in both human DFNB3 families and in shaker 2 (sh2) mice as a result of defects in stereocilia. In these mutant mice, hair cells have relatively normal spatial organization of stereocilia bundles but lack the graded, stair-step organization. We used sh2 mice as an experimental model to investigate the molecular consequences of the sh2 mutation in the Myo15 gene. Gene expression profiling with Affymetrix GeneChips in deaf homozygous (sh2/sh2) mice at 3 weeks and 3 months of age, and in age-matched, normal-hearing heterozygotes (+/sh2) identified only a few genes whose expression was affected by genotype, but a large number with age-associated changes in expression in both normal mice and sh2/sh2 homozygotes. Microarray data analyzed using Robust Multiarray Average identified Aim1, Dbi, and Tm4sf3 as genes with increased expression in sh2/sh2 homozygotes. These increases were confirmed by quantitative reverse transcription-polymerase chain reaction. Genes exhibiting altered expression with age encoded collagens and proteins involved in collagen maturation, extracellular matrix, and bone mineralization. These results identified potential cellular pathways associated with myosin XV defects, and age-associated molecular events that are likely to be involved in maturation of the cochlea and auditory function. PMID:16794912
Auffret, Julien; Viengchareun, Say; Carré, Nadège; Denis, Raphaël G P; Magnan, Christophe; Marie, Pierre-Yves; Muscat, Adeline; Fève, Bruno; Lombès, Marc; Binart, Nadine
Stimulating conversion of white fat to metabolically active adipocytes (beige fat) constitutes a promising strategy against weight gain and its deleterious associated-disorders. We provide direct evidence that prolactin (PRL), best known for its actions on the mammary gland, plays a pivotal role in energy balance through the control of adipocyte differentiation and fate. Here we show that lack of prolactin receptor (PRLR) causes resistance to high-fat-diet-induced obesity due to enhanced energy expenditure and increased metabolic rate. Mutant mice displayed reduced fat mass associated with appearance of massive brown-like adipocyte foci in perirenal and subcutaneous but not in gonadal fat depots under a high-fat diet. Positron emission tomography imaging further demonstrated the occurrence of these thermogenic brown fat depots in adult mice, providing additional support for recruitable brown-like adipocytes (beigeing) in white fat depots. Consistent with the activation of brown adipose tissue, PRLR inactivation increases expression of master genes controlling brown adipocyte fate (PRDM16) and mitochondrial function (PGC1α, UCP1). Altered pRb/Foxc2 expression suggests that this PRL-regulated pathway may contribute to beige cell commitment. Together, these results provide direct genetic evidence that PRLR affects energy balance and metabolic adaptation in rodents via effects on brown adipose tissue differentiation and function.
Sliwa, Leopold; Macura, Barbara; Majewska-Szczepanik, Monika; Szczepanik, Marian
The inflammation of the reproductive system can affect reproduction causing partial or complete infertility. It is well known that lipopolysaccharide (LPS) triggers an inflammatory response in the whole organism, including immunologically privileged organs, e.g. the testicles. Adult male TCRalpha-/-, TCRdelta-/-, CD1d-/- and beta2m-/- on B10.PL (H-2(u)) and B10.PL control mice were intraperitonealy (i.p.) injected with lipopolysaccharide (LPS). The animals were killed 24h and 10 days post LPS treatment and their gonads were prepared for microscopic examination. Histological changes in the testes after LPS injection were found only in control B10PL and CD1d-/- mice. The experiments revealed disturbances in Leydig's glands structure, blood vessel dilatation in the interstitial tissue as well as degeneration of seminal tubule epithelium, disruption ofspermatogenesis and subsequent decrease of sperm cell number in the tubule lumen. These changes were noticed mainly 10 days after LPS treatment. Lack of either TCRalphabeta+ CD8+ or TCRgammadelta+ lymphocytes diminishes the response of testicular macrophages to LPS whereas the absence of CD1d-dependent NKT cells does not affect macrophage reactivity.
Rithidech, Kanokporn Noy; Udomtanakunchai, Chatchanok; Honikel, Louise; Whorton, Elbert
It is clear that high-dose radiation is harmful. However, despite extensive research, assessment of potential health-risks associated with exposure to low-dose radiation (at doses below or equal to 0.1 Gy) is still challenging. Recently, we reported that 0.05 Gy of 137Cs gamma rays (the existing limit for radiation-exposure in the workplace) was incapable of inducing significant in vivo genomic instability (measured by the presence of late-occurring chromosomal damage at 6 months post-irradiation) in bone marrow (BM) cells of two mouse strains, one with constitutively high and one with intermediate levels of the repair enzyme DNA-dependent protein-kinase catalytic-subunit (DNA-PKcs). In this study, we present evidence for a lack of genomic instability in BM cells of the severely combined-immunodeficiency (SCID/J) mouse (which has an extremely low-level of DNA-PKcs activity) exposed whole-body to low-dose radiation (0.05 Gy). Together with our previous report, the data indicate that low-dose radiation (0.05 Gy) is incapable of inducing genomic instability in vivo (regardless of the levels of DNA-PKcs activity of the exposed mice), yet higher doses of radiation (0.1 and 1 Gy) do induce genomic instability in mice with intermediate and extremely low-levels of DNA-PKcs activity (indicating an important role of DNA-PKcs in DNA repair). PMID:23549227
Boettger, Thomas; Hübner, Christian A; Maier, Hannes; Rust, Marco B; Beck, Franz X; Jentsch, Thomas J
Hearing depends on a high K(+) concentration bathing the apical membranes of sensory hair cells. K(+) that has entered hair cells through apical mechanosensitive channels is transported to the stria vascularis for re-secretion into the scala media(). K(+) probably exits outer hair cells by KCNQ4 K(+) channels(), and is then transported by means of a gap junction system connecting supporting Deiters' cells and fibrocytes() back to the stria vascularis. We show here that mice lacking the K(+)/Cl(-) (K-Cl) co-transporter Kcc4 (coded for by Slc12a7) are deaf because their hair cells degenerate rapidly after the beginning of hearing. In the mature organ of Corti, Kcc4 is restricted to supporting cells of outer and inner hair cells. Our data suggest that Kcc4 is important for K(+) recycling() by siphoning K(+) ions after their exit from outer hair cells into supporting Deiters' cells, where K(+) enters the gap junction pathway. Similar to some human genetic syndromes(), deafness in Kcc4-deficient mice is associated with renal tubular acidosis. It probably results from an impairment of Cl(-) recycling across the basolateral membrane of acid-secreting alpha-intercalated cells of the distal nephron.
Kowalik, Agnes S; Johnson, Charis L; Chadi, Sami A; Weston, Jacqueline Y; Fazio, Elena N; Pin, Christopher L
Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2alpha phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.
Haverfield, Jenna; Nakagawa, Shoma; Love, Daniel; Tsichlaki, Elina; Nomikos, Michail; Lai, F. Anthony; Swann, Karl; FitzHarris, Greg
The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca2+ concentration ([Ca2+]i) in the ooplasm as a result of sperm-egg fusion. We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca2+ responses at fertilisation. To test this hypothesis we performed the first examination of Ca2+ dynamics in eggs from young and naturally-aged mice. Strikingly, we find that Ca2+ stores and resting [Ca2+]i are unchanged with age. Although eggs from aged mice feature a reduced ability to replenish intracellular Ca2+ stores following depletion, this difference had no effect on the duration, number, or amplitude of Ca2+ oscillations following intracytoplasmic sperm injection or expression of phospholipase C zeta. In contrast, we describe a substantial reduction in the frequency and duration of oscillations in aged eggs upon parthenogenetic activation with SrCl2. We conclude that the ability to mount and respond to an appropriate Ca2+ signal at fertilisation is largely unchanged by advancing maternal age, but subtle changes in Ca2+ handling occur that may have more substantial impacts upon commonly used means of parthenogenetic activation. PMID:26785810
Vecsey, Christopher G.; Park, Alan J.; Khatib, Nora; Abel, Ted
Sleep deprivation (SD) following hippocampus-dependent learning in young mice impairs memory when tested the following day. Here, we examined the effects of SD on remote memory in both young and aged mice. In young mice, we found that memory is still impaired 1 mo after training. SD also impaired memory in aged mice 1 d after training, but, by a…
Massie, H R; Baird, M B; Williams, T R
Adult Drosophila melanogaster fruit flies ranging in age from 2 to 7.5 weeks with a median colony survival time of 6.4 weeks at 25 degrees C showed no increase in DNA crosslinking with age. The purified denatured DNA used for crosslink determinations varied in molecular weight from 2.02 to 3.84 times 10(5) daltons and was crosslinked to the extent of 6.2-8.8% with no age-related trend.
Wu, Xiang; Chen, Huixin; Huang, Chunhui; Gu, Xinmei; Wang, Jialing; Xu, Dilin; Yu, Xin; Shuai, Chu; Chen, Liping; Li, Shun; Xu, Yiguo; Gao, Tao; Ye, Mingrui; Su, Wei; Liu, Haixiong; Zhang, Jinrong; Wang, Chuang; Chen, Junping; Wang, Qinwen; Cui, Wei
Post-operative cognitive dysfunction (POCD) is associated with elderly patients undergoing surgery. However, pharmacological treatments for POCD are limited. In this study, we found that curcumin, an active compound derived from Curcuma longa, ameliorated the cognitive dysfunction following abdominal surgery in aged mice. Further, curcumin prevented surgery-induced anti-oxidant enzyme activity. Curcumin also increased brain-derived neurotrophic factor (BDNF)-positive area and expression of pAkt in the brain, suggesting that curcumin activated BDNF signaling in aged mice. Furthermore, curcumin neutralized cholinergic dysfunction involving choline acetyltransferase expression induced by surgery. These results strongly suggested that curcumin prevented cognitive impairments via multiple targets, possibly by increasing the activity of anti-oxidant enzymes, activation of BDNF signaling, and neutralization of cholinergic dysfunction, concurrently. Based on these novel findings, curcumin might be a potential agent in POCD prophylaxis and treatment.
Zhao, Yun-peng; Tian, Qing-yun; Liu, Ben; Cuellar, Jason; Richbourgh, Brendon; Jia, Tang-hong; Liu, Chuan-ju
Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases. PMID:25777988
Popik, Piotr; Kamysz, Elzbieta; Kreczko, Joanna; Wróbel, Małgorzata
Like other endogenous enkephalinase inhibitors, human opiorphin peptide (QRFSR) attenuates catabolism of enkephalins and appears to be a promising therapeutic, displaying antinociceptive action in several pain models. However, its opioid-like side-effect profile is insufficiently characterized. In the present set of experiments, acute intraperitoneal administration of opiorphin produced an antinociceptive effect in the tail-flick test in mice (0.3mg/kg) and this action was inhibited by opioid receptor antagonist naloxone (3mg/kg). Repeated treatment with opiorphin changed the antinociceptive response neither to itself nor to morphine, suggesting the lack of tolerance and morphine cross-tolerance, respectively. Repeated treatment with opiorphin (3mg/kg) also failed to produce opioid dependence. Opiorphin (0.3 or 1mg/kg) produced no rewarding effects in the conditioned place preference test. However, at the dose of 3mg/kg, the peptide produced antidepressant-like effect in the forced swim test, and it did not affect locomotor activity. The present set of results confirms the beneficial effects of opiorphin in pain management, and suggests a lack of opioid-like side effects as well as the presence of antidepressant-like actions of this peptide.
Finkel, Deborah; Reynolds, Chandra A.; Berg, Stig; Pedersen, Nancy L.
Sex differences in the etiology of normal cognitive functioning in aging remain largely unexplored. We conducted an investigation of genetic and environmental contributions to sex differences in level of cognitive performance and rate of decline in the Swedish Adoption/Twin Study of Aging (SATSA) (Finkel & Pedersen, 2004) data set. Behavioral…
2003 AAR PM Meeting
Particulate Matter: Atmospheric Sciences,
Exposure and the Fourth Colloquium on PM and Human Health
LACK OF EFFECT OF AGE AND ANTIOXIDANT DEPLETION ON RESPIRATORY RESPONSES TO CONCENTRATED AMBIENT PARTICULATES (CAPs) IN RATS. JA Dye, LC Walsh, C...
Thanos, P.K.; Thanos, P.K.; Bermeo, C.; Rubinstein, M.; Suchland, K.L.; Wang, G.-J.; Grandy, D.K.; Volkow, N.D.
Methylphenidate (MP) and amphetamine (AMPH) are the most frequently prescribed medications for the treatment of attention-deficit/hyperactivity disorder (ADHD). Both drugs are believed to derive their therapeutic benefit by virtue of their dopamine (DA)-enhancing effects, yet an explanation for the observation that some patients with ADHD respond well to one medication but not to the other remains elusive. The dopaminergic effects of MP and AMPH are also thought to underlie their reinforcing properties and ultimately their abuse. Polymorphisms in the human gene that codes for the DA D4 receptor (D4R) have been repeatedly associated with ADHD and may correlate with the therapeutic as well as the reinforcing effects of responses to these psychostimulant medications. Conditioned place preference (CPP) for MP, AMPH and cocaine were evaluated in wild-type (WT) mice and their genetically engineered littermates, congenic on the C57Bl/6J background, that completely lack D4Rs (knockout or KO). In addition, the locomotor activity in these mice during the conditioning phase of CPP was tested in the CPP chambers. D4 receptor KO and WT mice showed CPP and increased locomotor activity in response to each of the three psychostimulants tested. D4R differentially modulates the CPP responses to MP, AMPH and cocaine. While the D4R genotype affected CPP responses to MP (high dose only) and AMPH (low dose only) it had no effects on cocaine. Inasmuch as CPP is considered an indicator of sensitivity to reinforcing responses to drugs these data suggest a significant but limited role of D4Rs in modulating conditioning responses to MP and AMPH. In the locomotor test, D4 receptor KO mice displayed attenuated increases in AMPH-induced locomotor activity whereas responses to cocaine and MP did not differ. These results suggest distinct mechanisms for D4 receptor modulation of the reinforcing (perhaps via attenuating dopaminergic signalling) and locomotor properties of these stimulant drugs
García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa
There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction. - Highlights: • Naloxone-precipitated morphine withdrawal increases sympathetic activity in the PVN and heart. • Co-localization of TH phosphorylated at serine 40/c-Fos in the VLM after morphine withdrawal • Naloxone
Gamella-Pozuelo, Luis; Grande, María T; Clemente-Lorenzo, Milagros; Murillo-Gómez, Cayetana; De Pablo, Flora; López-Novoa, José M; Hernández-Sánchez, Catalina
Catecholamines are essential for the maintenance of physiological homeostasis under basal and stress conditions. We aim to determine the impact of deletion of a single allele of the tyrosine hydroxylase (Th) gene might have on aging arterial pressure and life-span. We found that Th haploinsufficiency prevents age-associated increase of arterial pressure (AP) in mature adult mice, and it results in the extension of the half-life of Th-heterozygous (TH-HET) mice respect to their wild-type (WT) littermates. Heart performance was similar in both genotypes. To further investigate the lack of increase in AP with age in TH-HET mice, we measured the AP response to intra-peritoneal administration of substances involved in AP regulation. The response to acetylcholine and the basal sympathetic tone were similar in both genotypes, while norepinephrine had a greater pressor effect in TH-HET mice, which correlated with altered adrenoreceptor expression in blood vessels and the heart. Furthermore, sympatho-adrenomedular response to stress was attenuated in TH-HET mice. Plasma catecholamine levels and urine glucose increased markedly in WT but not in TH-HET mice after stress. Our results showed that TH-HET mice are resistant to age-associated hypertension, present a reduction in the sympathetic response to stress and display an extended half-life.
Nakamura, Brooke N.; Fielder, Thomas J.; Hoang, Yvonne D.; Lim, Jinhwan; McConnachie, Lisa A.; Kavanagh, Terrance J.
Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm−/− mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm−/− females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm−/− females. Prepubertal Gclm−/− and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm−/− females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/− females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm−/− females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm−/− females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development. PMID:21558310
Carlson, Bradley A; Xu, Xue-Ming; Gladyshev, Vadim N; Hatfield, Dolph L
Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2'-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N(6)-isopentenyladenosine at position 37 (i(6)A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i(6)A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium.
Bay-Richter, C; O'Callaghan, M J; Mathur, N; O'Tuathaigh, C M P; Heery, D M; Fone, K C F; Waddington, J L; Moran, P M
Drugs that induce psychosis, such as D-amphetamine (AMP), and those that alleviate it, such as antipsychotics, are suggested to exert behavioral effects via dopamine receptor D2 (D2). All antipsychotic drugs are D2 antagonists, but D2 antagonism underlies the severe and debilitating side effects of these drugs; it is therefore important to know whether D2 is necessary for their behavioral effects. Using D2-null mice (Drd2-/-), we first investigated whether D2 is required for AMP disruption of latent inhibition (LI). LI is a process of learning to ignore irrelevant stimuli. Disruption of LI by AMP models impaired attention and abnormal salience allocation consequent to dysregulated dopamine relevant to schizophrenia. AMP disruption of LI was seen in both wild-type (WT) and Drd2-/-. This was in contrast to AMP-induced locomotor hyperactivity, which was reduced in Drd2-/-. AMP disruption of LI was attenuated in mice lacking dopamine receptor D1 (Drd1-/-), suggesting that D1 may play a role in AMP disruption of LI. Further supporting this possibility, we found that D1 antagonist SKF83566 attenuated AMP disruption of LI in WT. Remarkably, both haloperidol and clozapine attenuated AMP disruption of LI in Drd2-/-. This demonstrates that antipsychotic drugs can attenuate AMP disruption of learning to ignore irrelevant stimuli in the absence of D2 receptors. Data suggest that D2 is not essential either for AMP to disrupt or for antipsychotic drugs to reverse AMP disruption of learning to ignore irrelevant stimuli and further that D1 merits investigation in the mediation of AMP disruption of these processes.
Kennard, John A.; Woodruff-Pak, Diana S.
Knowledge of age sensitivity, the capacity of a behavioral test to reliably detect age-related changes, has utility in the design of experiments to elucidate processes of normal aging. We review the application of these tests in studies of normal aging and compare and contrast the age sensitivity of the Barnes maze, eyeblink classical conditioning, fear conditioning, Morris water maze, and rotorod. These tests have all been implemented to assess normal age-related changes in learning and memory in rodents, which generalize in many cases to age-related changes in learning and memory in all mammals, including humans. Behavioral assessments are a valuable means to measure functional outcomes of neuroscientific studies of aging. Highlighted in this review are the attributes and limitations of these measures in mice in the context of age sensitivity and processes of brain aging. Attributes of these tests include reliability and validity as assessments of learning and memory, well-defined neural substrates, and sensitivity to neural and pharmacological manipulations and disruptions. These tests engage the hippocampus and/or the cerebellum, two structures centrally involved in learning and memory that undergo functional and anatomical changes in normal aging. A test that is less well represented in studies of normal aging, the context pre-exposure facilitation effect (CPFE) in fear conditioning, is described as a method to increase sensitivity of contextual fear conditioning to changes in the hippocampus. Recommendations for increasing the age sensitivity of all measures of normal aging in mice are included, as well as a discussion of the potential of the under-studied CPFE to advance understanding of subtle hippocampus-mediated phenomena. PMID:21647305
Vecsey, Christopher G.; Park, Alan J.; Khatib, Nora
Sleep deprivation (SD) following hippocampus-dependent learning in young mice impairs memory when tested the following day. Here, we examined the effects of SD on remote memory in both young and aged mice. In young mice, we found that memory is still impaired 1 mo after training. SD also impaired memory in aged mice 1 d after training, but, by a month after training, sleep-deprived and control aged animals performed similarly, primarily due to remote memory decay in the control aged animals. Gene expression analysis supported the finding that SD has similar effects on the hippocampus in young and aged mice. PMID:25776037
Lei, Lei; Chen, Jianlin; Hou, Shuping; Ding, Yiling; Yang, Zhangsheng; Zeng, Hao; Baseman, Joel
Plasmid-free Chlamydia trachomatis and Chlamydia muridarum fail to induce severe pathology. To evaluate whether the attenuated pathogenicity is due to insufficient infection or inability of the plasmidless chlamydial organisms to trigger pathological responses, we compared plasmid-competent and plasmid-free C. muridarum infections in 5 different strains of mice. All 5 strains developed hydrosalpinx following intravaginal inoculation with plasmid-competent, but not inoculation with plasmid-free, C. muridarum. The lack of hydrosalpinx induction by plasmid-free C. muridarum correlated with significantly reduced live organism recovery from the lower genital tract and shortened infection in the upper genital tract. The plasmid-free C. muridarum organisms failed to induce hydrosalpinx even when the organisms were directly inoculated into the oviduct via an intrabursal injection, which was accompanied by significantly reduced survival of the plasmidless organisms in the genital tracts. Furthermore, plasmid-competent C. muridarum organisms after UV inactivation were no longer able to induce hydrosalpinx even when directly delivered into the oviduct at a high dose. Together, these observations suggest that decreased survival of and shortened infection with plasmid-free C. muridarum may contribute significantly to its attenuated pathogenicity. We conclude that adequate live chlamydial infection in the oviduct may be necessary to induce hydrosalpinx. PMID:24343644
George, Sunil K; Jiao, Yan; Bishop, Colin E; Lu, Baisong
Mitochondrial reactive oxygen species (ROS) have been implicated in spermatogenic damage, although direct in vivo evidence is lacking. We recently generated a mouse in which the inner mitochondrial membrane peptidase 2-like (Immp2l) gene is mutated. This Immp2l mutation impairs the processing of signal peptide sequences from mitochondrial cytochrome c₁ and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion, which causes age-dependent spermatogenic damage. Here we confirm age-dependent spermatogenic damage in a new cohort of mutants, which started at the age of 10.5 months. Compared with age-matched controls, protein carbonyl content was normal in testes of 2- to 5-month-old mutants, but significantly elevated in testes of 13-month-old mutants, indicating elevated oxidative stress in the testes at the time of impaired spermatogenesis. Testicular expression of superoxide dismutases was not different between control and mutant mice, whereas that of catalase was increased in young and old mutants. The expression of cytosolic glutathione peroxidase 4 (phospholipid hydroperoxidase) in testes was significantly reduced in 13-month-old mutants, concomitant with impaired spermatogenesis. Apoptosis of all testicular populations was increased in mutant mice with spermatogenic damage. The mitochondrial DNA (mtDNA) mutation rate in germ cells of mutant mice with impaired spermatogenesis was unchanged, excluding a major role of mtDNA mutation in ROS-mediated spermatogenic damage. Our data show that increased mitochondrial ROS are one of the driving forces for spermatogenic impairment.
Dumont, F.; Robert, F.
The cellular organization of the thymus was investigated in 3- and 12-month-old NZB × SJL F1 hybrid (NS) mice. Age-dependent alterations were demonstrated which differed strikingly according to the sex of the animals. In female mice, marked abnormalities of the thymus developed during ageing. They consisted of a more or less pronounced hypertrophy accompanied by histological changes and modifications in the nature of the lymphocyte populations. Three types of qualitative changes were found at 12 months of age: (1) depletion of cortical thymocytes as evidenced by histology, by the evaluation of peanut-agglutinin (PNA) binding and by cell electrophoresis; (2) hyperplasia of the medullary lymphoid tissue, probably reflecting the expansion of a population of mature T lymphocytes. This was further suggested by a rise (up to 60%) in the frequency of lymphocytes lacking both PNA receptor and B cell markers, by an increased proportion (57%) of high electrophoretic mobility (EPM) lymphocytes and by an augmentation of in vitro reactivities to phytohaemagglutinin (PHA) and, although to a lesser extent, to concanavalin A (Con A). (3) The appearance of significant numbers of B lymphocytes (up to 20%) as assessed by surface immunoglobulin (sIg) and complement receptor (CR) detection which was accompanied by a vigorous responsiveness of thymus cells to lipopolysaccharide (LPS). None of these abnormalities was seen in the male mice. Instead, the thymus of NS males displayed a nearly normal age-related involution without major change in the proportions of its lymphocyte subpopulations. NS mice thus provide an interesting model of thymic disease influenced by sex-linked factors. ImagesFig. 3 PMID:7438550
Lin, Lejun; Cao, Bofeng; Xu, Zhiying; Sui, Yanbin; Chen, Jiao; Luan, Qiang; Yang, Ruifang; Li, Shanchun; Li, Ke Feng
Aging is characterized by various cellular changes in the brain. Hippocampus is important for systemic aging and lifespan control. There is still a lack of comprehensive overview of metabolic changes in hippocampus during aging. In this study, we first created an accelerated brain aging mice model through the chronic administration of d-galactose. We then performed a multiplatform metabolomic profiling of mice hippocampus using the combination of in vivo 9.4 T HMRS and in vitro LC-MS/MS based lipidomics. We found N-acetylaspartic acid (NAA), gama-aminobutyric acid (GABA), glutamate/glutamine, taurine, choline, sphingolipids (SMs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs) and phosphatidylserines (PSs), all of them decreasing with the aging process in mice hippocampus. The changes of sphingolipids and phospholipids were not limited to one single class or molecular species. In contrast, we found the significant accumulation of lactate, myoinositol and phosphatidylcholines (PCs) along with aging in hippocampus. SM (d18:1/20:2), PE (36:2), PG (34:1), PI (36:4), PS (18:0/20:4) and PC (36:0) have the most significant changes along with aging. Network analysis revealed the striking loss of biochemical connectivity and interactions between hippocampal metabolites with aging. The correlation pattern between metabolites in hippocampus could function as biomarkers for aging or diagnosis of aging-related diseases.
Rice, Kelly A; Albacarys, Lauren K; Metcalf Pate, Kelly A; Perkins, Cheryl; Henderson, Kenneth S; Watson, Julie
Detecting and controlling murine fur mites continues to be challenging. Here we compared the efficacy of fur-pluck, cage PCR, and fur PCR testing of mice naturally infested with Myocoptes musculinus and make recommendations regarding the application of these diagnostic strategies in aged or treated mice. We compared all 3 diagnostic methods in groups of infested and noninfested control mice over time. For fur plucks, we used a scoring system to quantitatively compare mite infestations across ages. Mice that were 4 wk old had higher egg and mite scores than did older mice, with average scores at 4 wk corresponding to 40 to 100 individual fur mites and eggs per sample. Furthermore, 15% and 20% of samples from infested mice at 24 and 28 wk of age, respectively, lacked all fur mites and eggs. Cage PCR results varied as mice grew older. Fur PCR testing was the most sensitive and specific assay in untreated infested mice, particularly when mite densities were low. In addition, we compared fur-pluck and fur PCR tests for evaluating the efficacy of selamectin treatment. Two treatments with selamectin eliminated Myocoptes fur-mite infestations. At 8 wk after treatment, all fur-pluck samples were negative, but one-third of treated infested cages remained positive by fur PCR assay; at 16 wk after treatment, all cages were negative by fur PCR assay. Because offspring of infested mice were invariably heavily infested, breeding of suspected infested mice with subsequent testing of offspring was the definitive testing strategy when fur-pluck and PCR results conflicted.
Mendoza, Mirian; Qiu, Qi; Casares, Sofia
Background. Plasmodium yoelii 17XNL is a nonlethal malaria strain in mice of different genetic backgrounds including the C57BL/6 mice (I-Ab/I-Enull) used in this study as a control strain. We have compared the trends of blood stage infection with the nonlethal murine strain of P. yoelii 17XNL malaria protozoan in immunocompetent Nonobese Diabetic (NOD) mice prone to type 1 diabetes (T1D) and C57BL/6 mice (control mice) that are not prone to T1D and self-cure the P. yoelii 17XNL infection. Prediabetic NOD mice could not mount a protective antibody response to the P. yoelii 17XNL-infected red blood cells (iRBCs), and they all succumbed shortly after infection. Our data suggest that the lack of anti-P. yoelii 17XNL-iRBCs protective antibodies in NOD mice is a result of parasite-induced, Foxp3+ T regulatory (Treg) cells able to suppress the parasite-specific antibody secretion. Conclusions. The NOD mouse model may help in identifying new mechanisms of B-cell evasion by malaria parasites. It may also serve as a more accurate tool for testing antimalaria therapeutics due to the lack of interference with a preexistent self-curing mechanism present in other mouse strains. PMID:28074170
Wang, Chelsia Qiuxia; Motoda, Lena; Satake, Masanobu; Ito, Yoshiaki; Taniuchi, Ichiro; Tergaonkar, Vinay; Osato, Motomi
The RUNX family genes encode transcription factors that are involved in development and human diseases. RUNX1 is one of the most frequently mutated genes in human hematological malignancies and is a critical factor for the generation and maintenance of hematopoietic stem cells. Another Runx family gene, Runx3, is known to be expressed in hematopoietic cells. However, its involvement in hematopoiesis remains unclear. Here we show the hematopoietic phenotypes in Runx3 conditional knockout (KO) mice (Runx3(fl/fl);Mx1-Cre(+)): whereas young Runx3 KO mice did not exhibit any significant hematopoietic defects, aged Runx3 KO mice developed a myeloproliferative disorder characterized by myeloid-dominant leukocytosis, splenomegaly, and an increase of hematopoietic stem/progenitor cells (HSPCs). Notably, Runx3-deficient cells showed hypersensitivity to granulocyte-colony stimulating factor, suggesting enhanced proliferative and mobilization capability of Runx3-deficient HSPCs when stimulated. These results suggest that, besides Runx1, Runx3 also plays a role in hematopoiesis.
Kaczorowski, Catherine C.; Disterhoft, John F.
Normal aging disrupts hippocampal neuroplasticity and learning and memory. Aging deficits were exposed in a subset (30%) of middle-aged mice that performed below criterion on a hippocampal-dependent contextual fear conditioning task. Basal neuronal excitability was comparable in middle-aged and young mice, but learning-related modulation of the…
Carter, Todd A; Greenhall, Jennifer A; Yoshida, Shigeo; Fuchs, Sebastian; Helton, Robert; Swaroop, Anand; Lockhart, David J; Barlow, Carrolee
Background Progressive neurological dysfunction is a key aspect of human aging. Because of underlying differences in the aging of mice and humans, useful mouse models have been difficult to obtain and study. We have used gene-expression analysis and polymorphism screening to study molecular senescence of the retina and hippocampus in two rare inbred mouse models of accelerated neurological senescence (SAMP8 and SAMP10) that closely mimic human neurological aging, and in a related normal strain (SAMR1) and an unrelated normal strain (C57BL/6J). Results The majority of age-related gene expression changes were strain-specific, with only a few common pathways found for normal and accelerated neurological aging. Polymorphism screening led to the identification of mutations that could have a direct impact on important disease processes, including a mutation in a fibroblast growth factor gene, Fgf1, and a mutation in and ectopic expression of the gene for the chemokine CCL19, which is involved in the inflammatory response. Conclusion We show that combining the study of inbred mouse strains with interesting traits and gene-expression profiling can lead to the discovery of genes important for complex phenotypes. Furthermore, full-genome polymorphism detection, sequencing and gene-expression profiling of inbred mouse strains with interesting phenotypic differences may provide unique insights into the molecular genetics of late-manifesting complex diseases. PMID:15960800
Huang, Teng-Wei; Kochukov, Mikhail Y.; Ward, Christopher S.; Merritt, Jonathan; Thomas, Kaitlin; Nguyen, Tiffani; Arenkiel, Benjamin R.
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Severe breathing abnormalities are common in RTT and are reproduced in mouse models of RTT. Previously, we found that removing MeCP2 from the brainstem and spinal cord in mice caused early lethality and abnormal breathing. To determine whether loss of MeCP2 in functional components of the respiratory network causes specific breathing disorders, we used the Cre/LoxP system to differentially manipulate MeCP2 expression throughout the brainstem respiratory network, specifically within HoxA4-derived tissues, which include breathing control circuitry within the nucleus tractus solitarius and the caudal part of ventral respiratory column but do not include more rostral parts of the breathing control circuitry. To determine whether respiratory phenotypes manifested in animals with MeCP2 removed from specific pons medullary respiratory circuits, we performed whole-body plethysmography and electrophysiological recordings from in vitro brainstem slices from mice lacking MeCP2 in different circuits. Our results indicate that MeCP2 expression in the medullary respiratory network is sufficient for normal respiratory rhythm and preventing apnea. However, MeCP2 expression within components of the breathing circuitry rostral to the HoxA4 domain are neither sufficient to prevent the hyperventilation nor abnormal hypoxic ventilatory response. Surprisingly, we found that MeCP2 expression in the HoxA4 domain alone is critical for survival. Our study reveals that MeCP2 is differentially required in select respiratory components for different aspects of respiratory functions, and collectively for the integrity of this network functions to maintain proper respiration. SIGNIFICANCE STATEMENT Breathing abnormalities are a significant clinical feature in Rett syndrome and are robustly reproduced in the mouse models of this disease. Previous work has established that alterations
Wahl, Elizabeth C; Aronson, James; Liu, Lichu; Fowlkes, John L; Thrailkill, Kathryn M; Bunn, Robert C; Skinner, Robert A; Miller, Mike J; Cockrell, Gael E; Clark, Lindsey M; Ou, Yang; Isales, Carlos M; Badger, Thomas M; Ronis, Martin J; Sims, John; Lumpkin, Charles K
Skeletal changes accompanying aging are associated with both increased risk of fractures and impaired fracture healing, which, in turn, is due to compromised bone regeneration potential. These changes are associated with increased serum levels of selected proinflammatory cytokines, e.g., tumor necrosis factor α (TNF-α). We have used a unique model of bone regeneration to demonstrate (1) that aged-related deficits in direct bone formation can be restored to young mice by treatment with TNF blockers and (2) that the cyclin-dependent kinase inhibitor p21 is a candidate for mediation of the osteoinhibitory effects of TNF. It has been hypothesized recently that TNF antagonists may represent novel anabolic agents, and we believe that the data presented here represent a successful test of this hypothesis. © 2010 American Society for Bone and Mineral Research PMID:19580462
Matsukawa, Hiroshi; Wolf, Alexander M; Matsushita, Shinichi; Joho, Rolf H; Knöpfel, Thomas
Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.
Yu, Carol; Luo, Xiaoyan; Farhat, Nada; Daneault, Caroline; Duquette, Natacha; Martel, Cécile; Lambert, Jean; Thorin‐Trescases, Nathalie; Rosiers, Christine Des; Thorin, Éric
Background Angiopoietin‐like‐2 (angptl2) is produced by several cell types including endothelial cells, adipocytes and macrophages, and contributes to the inflammatory process in cardiovascular diseases. We hypothesized that angptl2 impairs endothelial function, and that lowering angptl2 levels protects the endothelium against high‐fat diet (HFD)‐induced fat accumulation and hypercholesterolemia. Methods and Results Acute recombinant angptl2 reduced (P<0.05) acetylcholine‐mediated vasodilation of isolated wild‐type (WT) mouse femoral artery, an effect reversed (P<0.05) by the antioxidant N‐acetylcysteine. Accordingly, in angptl2 knockdown (KD) mice, ACh‐mediated endothelium‐dependent vasodilation was greater (P<0.05) than in WT mice. In arteries from KD mice, prostacyclin contributed to the overall dilation unlike in WT mice. After a 3‐month HFD, overall vasodilation was not altered, but dissecting out the endothelial intrinsic pathways revealed that NO production was reduced in arteries isolated from HFD‐fed WT mice (P<0.05), while NO release was maintained in KD mice. Similarly, endothelium‐derived hyperpolarizing factor (EDHF) was preserved in mesenteric arteries from HFD‐fed KD mice but not in those from WT mice. Finally, the HFD increased (P<0.05) total cholesterol–to–high‐density lipoprotein ratios, low‐density lipoprotein–to–high‐density lipoprotein ratios, and leptin levels in WT mice only, while glycemia remained similar in the 2 strains. KD mice displayed less triglyceride accumulation in the liver (P<0.05 versus WT), and adipocyte diameters in mesenteric and epididymal white adipose tissues were smaller (P<0.05) in KD than in WT fed an HFD, while inflammatory gene expression increased (P<0.05) in the fat of WT mice only. Conclusions Lack of angptl2 expression limits the metabolic stress induced by an HFD and maintains endothelial function in mice. PMID:25128474
Bourdenx, Mathieu; Dovero, Sandra; Thiolat, Marie-Laure; Bezard, Erwan; Dehay, Benjamin
Neurodegenerative diseases are characterized by the degeneration of specific brain areas associated with accumulation of disease-related protein in extra- or intra-cellular deposits. Their preclinical investigations are mostly based on genetically-engineered animals. Despite their interest, these models are often based on high level of disease-related protein expression, thus questioning their relevance to human pathology and calling for the alternate use of ecological models. In the past few years, Octodon degus has emerged as a promising animal model displaying age-dependent Alzheimer’s disease-related pathology. As neurodegenerative-related proteins often co-deposit in the brain of patients, we assessed the occurrence of α-synuclein-related pathology in this model using state-of-the-art immunohistochemistry and biochemistry. Despite our efforts and in contrast with previously published results, our study argues against the use of Octodon degus as a suitable natural model of neurodegenerative disorder as we failed to identify either Parkinson’s disease- or Alzheimer’s disease-related brain pathologies. PMID:28374864
Wakabayashi, Chisato; Numakawa, Tadahiro; Odaka, Haruki; Ooshima, Yoshiko; Kiyama, Yuji; Manabe, Toshiya; Kunugi, Hiroshi; Iwakura, Yoichiro
Interleukin 1 (IL-1) plays a critical role in stress responses, and its mRNA is induced in the brain by restraint stress. Previously, we reported that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lacked IL-1Ra molecules that antagonize the IL-1 receptor, showed anti-depression-like behavior via adrenergic modulation at the age of 8 weeks. Here, we report that IL-1Ra KO mice display an anxiety-like phenotype that is induced spontaneously by aging in the elevated plus-maze (EPM) test. This anxiety-like phenotype was improved by the administration of diazepam. The expression of the anxiety-related molecule glucocorticoid receptor (GR) was significantly reduced in 20-week-old but not in 11-week-old IL-1Ra KO mice compared to wild-type (WT) littermates. The expression of the mineralocorticoid receptor (MR) was not altered between IL-1Ra KO mice and WT littermates at either 11 or 20 weeks old. Analysis of monoamine concentration in the hippocampus revealed that tryptophan, the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA), and the dopamine metabolite homovanillic acid (HVA) were significantly increased in 20-week-old IL-1Ra KO mice compared to littermate WT mice. These findings strongly suggest that the anxiety-like behavior observed in older mice was caused by the complicated alteration of monoamine metabolism and/or GR expression in the hippocampus.
Reguera, Rosa M.; Balaña-Fouce, Rafael; Showalter, Melissa; Hickerson, Suzanne; Beverley, Stephen M.
Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from L-ornithine has been well studied in protozoa, the metabolic origin(s) of L-ornithine have received less attention. Arginase (EC 188.8.131.52) catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg− knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or L-ornithine for growth. A complemented line (arg−/+ARG) expressing arginase from a multicopy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors α-difluoromethylornithine (DFMO) and Nω-hydroxy-L-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg− parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of L-arginine metabolism in both pro- and anti-parasitic responses involving host nitric
Timaru-Kast, Ralph; Luh, Clara; Gotthardt, Philipp; Huang, Changsheng; Schäfer, Michael K; Engelhard, Kristin; Thal, Serge C
After traumatic brain injury (TBI) elderly patients suffer from higher mortality rate and worse functional outcome compared to young patients. However, experimental TBI research is primarily performed in young animals. Aim of the present study was to clarify whether age affects functional outcome, neuroinflammation and secondary brain damage after brain trauma in mice. Young (2 months) and old (21 months) male C57Bl6N mice were anesthetized and subjected to a controlled cortical impact injury (CCI) on the right parietal cortex. Animals of both ages were randomly assigned to 15 min, 24 h, and 72 h survival. At the end of the observation periods, contusion volume, brain water content, neurologic function, cerebral and systemic inflammation (CD3+ T cell migration, inflammatory cytokine expression in brain and lung, blood differential cell count) were determined. Old animals showed worse neurological function 72 h after CCI and a high mortality rate (19.2%) compared to young (0%). This did not correlate with histopathological damage, as contusion volumes were equal in both age groups. Although a more pronounced brain edema formation was detected in old mice 24 hours after TBI, lack of correlation between brain water content and neurological deficit indicated that brain edema formation is not solely responsible for age-dependent differences in neurological outcome. Brains of old naïve mice were about 8% smaller compared to young naïve brains, suggesting age-related brain atrophy with possible decline in plasticity. Onset of cerebral inflammation started earlier and primarily ipsilateral to damage in old mice, whereas in young mice inflammation was delayed and present in both hemispheres with a characteristic T cell migration pattern. Pulmonary interleukin 1β expression was up-regulated after cerebral injury only in young, not aged mice. The results therefore indicate that old animals are prone to functional deficits and strong ipsilateral cerebral inflammation
Truksa, Jaroslav; Gelbart, Terri; Peng, Hongfan; Beutler, Ernest; Beutler, Bruce; Lee, Pauline
Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo, we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.
Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S.; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M. S. E.; Ackermann, Teresa F.; Föller, Michael; Schleicher, Erwin; Sheffield, William P.; Arthur, J. Simon C.; Lang, Florian; Qadri, Syed M.
The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice. PMID:26611568
Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M S E; Ackermann, Teresa F; Föller, Michael; Schleicher, Erwin; Sheffield, William P; Arthur, J Simon C; Lang, Florian; Qadri, Syed M
The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.
Rb-Ai.62 483 DIFFERENTIAL RESPONSES TO ADJUVANTS OF MACROPHAGES FROM i/i YOUNG VIRGIN AGIN (U) MINNESOTA UNIV DULUTH DEPT OF MEDICAL MICROBIOLOGY RN...ADDRESS (City, State. an ZI 0EC 18 198E- Dept. of Medical Microbiology & Immunology 800 N. Quincy Street E1 8 Duluth, MN 55812-2487 Arlington, VA 22217-5...Aging Breeder Mice by Pamela R. Petrequin and Arthur G. Johnson Dept. of Medical Microbiology /Immunology University of Minnesota-Duluth School of
Blank, Carrine E
Most microbial taxa lack a conventional microfossil or biomarker record, and so we currently have little information regarding how old most microbial clades and their associated traits are. Building on the previously published oxygen age constraint, two new age constraints are proposed based on the ability of microbial clades to metabolize chitin and aromatic compounds derived from lignin. Using the archaeal domain of life as a test case, phylogenetic analyses, along with published metabolic and genetic data, showed that members of the Halobacteriales and Thermococcales are able to metabolize chitin. Ancestral state reconstruction combined with phylogenetic analysis of the genes underlying chitin degradation predicted that the ancestors of these two groups were also likely able to metabolize chitin or chitin-related compounds. These two clades were therefore assigned a maximum age of 1.0 Ga (when chitin likely first appeared). Similar analyses also predicted that the ancestor to the Sulfolobus solfataricus-Sulfolobus islandicus clade was able to metabolize phenol using catechol dioxygenase, so this clade was assigned a maximum age of 475 Ma. Inferred ages of archaeal clades using relaxed molecular clocks with the new age constraints were consistent with those inferred with the oxygen age constraints. This work expands our current toolkit to include Paleoproterozoic, Neoproterozoic, and Paleozoic age constraints, and should aid in our ability to phylogenetically reconstruct the antiquity of a wide array of microbial clades and their associated morphological and biogeochemical traits, spanning deep geologic time. Such hypotheses-although built upon evolutionary inferences-are fundamentally testable.
Blank, Carrine E.
A phylogenomic dating approach was used to identify potential age constraints for multiple archaeal groups, many of which have no fossil, isotopic, or biomarker record. First, well-resolved phylogenetic trees were inferred with the use of multiple gene sequences obtained from whole genome sequences. Next, the ability to use oxygen as a terminal electron acceptor was coded into characters, and ancestral state reconstruction was used to identify clades with taxa that metabolize oxygen and likely had an aerobic ancestor. Next, the habitat of the ancestor was inferred. If the local presence of Cyanobacteria could be excluded from the putative ancestral habitat, then these clades would have originated after the rise in atmospheric oxygen 2.32 Ga. With this method, an upper age of 2.32 Ga (an "oxygen age constraint") is proposed for four major archaeal clades: the Sulfolobales, Thermoplasmatales, Thermoproteus neutrophilus/Pyrobaculum spp., and the Thermoproteales. It was also shown that the halophilic archaea likely had an aerobic common ancestor, yet the possibility of local oxygen oases before oxygenation of the atmosphere could not be formally rejected. Thus, an oxygen age constraint was not assessed for this group. This work suggests that many archaeal groups are not as ancient as many in the research community have previously assumed, and it provides a new method for establishing upper age constraints for major microbial groups that lack a conventional fossil record.
Solon-Biet, Samantha M.; Walters, Kirsty A.; Simanainen, Ulla K.; McMahon, Aisling C.; Ruohonen, Kari; Ballard, John William O.; Raubenheimer, David; Le Couteur, David G.; Simpson, Stephen J.
In invertebrates, reproductive output and lifespan are profoundly impacted by dietary macronutrient balance, with these traits achieving their maxima on different diet compositions, giving the appearance of a resource-based tradeoff between reproduction and longevity. For the first time in a mammal, to our knowledge, we evaluate the effects of dietary protein (P), carbohydrate (C), fat (F), and energy (E) on lifespan and reproductive function in aging male and female mice. We show that, as in invertebrates, the balance of macronutrients has marked and largely opposing effects on reproductive and longevity outcomes. Mice were provided ad libitum access to one of 25 diets differing in P, C, F, and E content, with reproductive outcomes assessed at 15 months. An optimal balance of macronutrients exists for reproductive function, which, for most measures, differs from the diets that optimize lifespan, and this response differs with sex. Maximal longevity was achieved on diets containing a P:C ratio of 1:13 in males and 1:11 for females. Diets that optimized testes mass and epididymal sperm counts (indicators of gamete production) contained a higher P:C ratio (1:1) than those that maximized lifespan. In females, uterine mass (an indicator of estrogenic activity) was also greatest on high P:C diets (1:1) whereas ovarian follicle number was greatest on P:C 3:1 associated with high-F intakes. By contrast, estrous cycling was more likely in mice on lower P:C (1:8), and the number of corpora lutea, indicative of recent ovulations, was greatest on P:C similar to those supporting greatest longevity (1:11). PMID:25733862
Qian, Xiao-Lan; Zhang, Wei; Liu, Ming-Zheng; Zhou, Yu-Bing; Zhang, Jing-Min; Han, Li; Peng, You-Mei; Jiang, Jin-hua; Wang, Qing-Duan
Postoperative cognitive dysfunction (POCD) is a frequent complication following major surgery in the elderly. However, the exact pathogenic mechanisms are still unknown. Dexmedetomidine, a selective alpha 2 adrenal receptor agonist, was revealed anesthesia and brain protective role. The present study aimed to examine whether dexmedetomdine protects against POCD induced by major surgical trauma under general anesthesia in aged mice. In the present study, cognitive function was assessed by Y-maze. Proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α), apoptosis-related factor caspase-3 and Bax were detected by real-time PCR, Western blot or immunohistochemistry. The results showed that anesthesia alone caused weak cognitive dysfunction on the first day after general anesthesia. Cognitive function in mice with splenectomy under general anesthesia was significantly exacerbated at the first and third days after surgery, and was significantly improved by dexmedetomidine administration. Splenectomy increased the expression of IL-1β, TNF-α, Bax and caspase-3 in hippocampus. These changes were significantly inversed by dexmedetomidine. These results suggest that hippocampal inflammatory response and neuronal apoptosis may contribute to POCD, and selective alpha 2 adrenal receptor excitation play a protective role.
Ding, Hua; Nedrud, John G; Blanchard, Thomas G; Zagorski, Brandon M; Li, Guanghui; Shiu, Jessica; Xu, Jinghua; Czinn, Steven J
Helicobacter pylori (H. pylori) infection can be significantly reduced by immunization in mice. Th17 cells play an essential role in the protective immune response. Th1 immunity has also been demonstrated to play a role in the protective immune response and can compensate in the absence of IL-17. To further address the potential of Th1 immunity, we investigated the efficacy of immunization in mice deficient in IL-23p19, a cytokine that promotes Th17 cell development. We also examined the course of Helicobacter infection in unimmunized mice treated with Th1 promoting cytokine IL-12. C57BL/6, IL-12 p35 KO, and IL-23 p19 KO mice were immunized and challenged with H. pylori. Protective immunity was evaluated by CFU determination and QPCR on gastric biopsies. Gastric and splenic IL-17 and IFNγ levels were determined by PCR or by ELISA. Balb/c mice were infected with H. felis and treated with IL-12 therapy and the resulting gastric bacterial load and inflammatory response were assessed by histologic evaluation. Vaccine induced reductions in bacterial load that were comparable to wild type mice were observed in both IL-12 p35 and IL-23 p19 KO mice. In the absence of IL-23 p19, IL-17 levels remained low but IFNγ levels increased significantly in both immunized challenged and unimmunized/challenged mice. Additionally, treatment of H. felis-infected Balb/c mice with IL-12 resulted in increased gastric inflammation and the eradication of bacteria in most mice. These data suggest that Th1 immunity can compensate for the lack of IL-23 mediated Th17 responses, and that protective Th1 immunity can be induced in the absence of immunization through cytokine therapy of the infected host.
Patel, Bhavik Anil; Fidalgo, Sara; Wang, Chunfang; Parmar, Leena; Mandona, Kasonde; Panossian, Annabelle; Flint, Melanie S; Ranson, Richard N; Saffrey, M Jill; Yeoman, Mark S
Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age-related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg(-1)). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to "anti-TNFα" drugs as potential treatments for age-related chronic constipation.
Gabbi, Chiara; Kong, Xiaomu; Suzuki, Hitoshi; Kim, Hyun-Jin; Gao, Min; Jia, Xiao; Ohnishi, Hideo; Ueta, Yoichi; Warner, Margaret; Guan, Youfei; Gustafsson, Jan-Åke
The present study demonstrates a key role for the oxysterol receptor liver X receptor β (LXRβ) in the etiology of diabetes insipidus (DI). Given free access to water, LXRβ(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRβ(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRβ(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRβ was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRβ(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRβ(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRβ is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance.
Shahroudi, Mahdieh Jafari; Mehri, Soghra; Hosseinzadeh, Hossein
Objectives: Aging is an unconscious and gradual process that can lead to changes in biological systems. Induction of oxidative stress and apoptosis, hepatotoxicity and neurotoxicity are involved in the aging process. Regarding the antioxidant property of black seed oil, the aim of this study was to evaluate the anti-aging effect of Nigella sativa (N. sativa) oil on d-galactose-induced aging in mice. Methods: For induction of aging, D-galactose (500 mg/kg, subcoutaneously SC) was administrated to male mice for 42 days. Animals were treated with D-galactose alone or with b lack seed oil (0.1, 0.2, 0.5 mL/kg, intraperitoneally (ip)). Additionally, vitamin E (200 mg/kg) was used as a positive control. At the end of treatment, the malondialdehyde (MDA) and the glutathione (GSH) contents in brain and liver tissues were measured. Also, enzymes in serum, including aspartate aminotransferase (AST) and alanine amino transferase (ALT), were determined. The levels of the proteins Bax, Bcl2, caspase-3 (pro and cleaved) in brain and liver tissues were evaluated. Results: Administration of D-galactose (500 mg/kg, SC) for 42 days increased serum levels of ALT and AST, as well as the MDA content, in brain and liver tissues, but decreased the GSH content. Additionally, the levels of apoptotic proteins, including Bax, procaspase-3 and caspase-3 cleaved, were markedly increased. N. sativa oil (0.1 and 0.2 mL/kg) diminished the levels of the biochemical markers ALT and AST. Administration of black seed oil (0.1, 0.2 and 0.5 mL/kg) reduced lipid peroxidation and at doses 0.1 and 0.2 mL/kg significantly recovered the GSH content. The oil decreased Bax/Bcl2 levels and at 0.1 mL/kg down-regulated the expressions of caspase-3 (pro and cleaved) proteins in brain and liver tissues. Conclusion: Through its antioxidant and anti-apoptosis properties, black seed oil exhibited an anti-aging effect in a model of aging induced with D-galactose. PMID:28392960
Liu, X; Huh, JY; Gong, H; Chamberland, JP; Brinkoetter, MT; Hamnvik, O-PR; Mantzoros, CS
BACKGROUND/OBJECTIVES Obesity is characterized by chronic inflammation and immune dysregulation, as well as insulin resistance, but the link between obesity and adaptive immunity remains to be fully studied. METHODS To elucidate the role of adaptive immunity on body composition, glucose homeostasis and inflammation, recombination-activating gene 1 knockout (Rag1 − / −) mice, without mature T-lymphocytes or B-lymphocytes, were maintained on a low- or high-fat diet (LFD and HFD, respectively) for 11 weeks. RESULTS Rag1 − / − mice fed HFD gained significantly more weight and had increased body fat compared with wild type. Downregulation of energy expenditure as well as brown fat uncoupling protein UCP-1 and UCP-3 gene expression were noticed in HFD-fed Rag1 − / − mice compared with LFD. HFD mice had significantly decreased energy intake compared with LFD mice, consistent with decreased agouti-related protein and increased pro-opiomelanocortin gene expression levels in the hypothalamus. Moreover, compared with wild type, Rag1 − / − mice had lower interleukin (IL)-4 levels, a cytokine recently found to induce browning in white adipocytes, and higher IL-12 levels in HFD-fed Rag1 − / − mice. Despite that HFD Rag1 − / − mice were more obese, they had similar glucose, insulin and adiponectin levels, while leptin was marginally increased. CONCLUSIONS Mice with deficiency in adaptive immunity are obese, partly owing to decreased energy expenditure, but are metabolically normal, suggesting that mature lymphocytes have necessary roles in the development of obesity-related metabolic dysregulation. PMID:25994806
Elias, P K; Elias, M F; Eleftheriou, B E
Two inbred strains of mice, C57BL/6J and DBA/2J, ranging in age from 2 to 38 months, were tested in an open field using the free exploration method. Scores were obtained for locomotor activity, exploratory behavior and emotionality. Strain differences were observed for all three variables. Beginning at late maturity (12 months), locomotor activity decreased with increasing age. Exploratory behavior was at a low level for DBA/2J mice at all ages. For C57BL/6J mice, exploratory behavior decreased significantly between 2 and 6 months and remained stable thereafter. Emotionality remained unchanged with advancing age for both strains of mice.
Buchanan, J.B.; Sparkman, N.L.; Chen, J.; Johnson, R.W.
Summary Peripheral immune stimulation as well as certain types of psychological stress increases brain levels of inflammatory cytokines such as interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNFα). We have demonstrated that aged mice show greater increases in central inflammatory cytokines, as well as greater cognitive deficits, compared to adults in response to peripheral lipopolysaccharide (LPS) administration. Because aged mice are typically more sensitive to systemic stressors such as LPS, and certain psychological stressors induce physiological responses similar to those that follow LPS, we hypothesized that aged mice would be more sensitive to the physiological and cognitive effects of mild stress than adult mice. Here, adult (3–5 mo) and aged (22–23 mo) male BALB/c mice were trained in the Morris water maze for 5 days. Mice were then exposed to a mild restraint stress of 30 minutes before being tested in a working memory version of the water maze over a 3 day period. On day 4 mice were stressed and then killed for collection of blood and brain. In a separate group of animals, mice were killed immediately after one, two or three 30 min restraint sessions and blood for peripheral corticosterone and cytokine protein measurement, and brains were dissected for central cytokine mRNA measurement. Stress disrupted spatial working memory in both adult and aged mice but to a much greater extent in the aged mice. In addition, aged mice showed an increase in stress-induced expression of hippocampal IL-1β mRNA and MHC class II protein compared to non-stressed controls while expression in adult mice was unaffected by stress. These data show that aged mice are more sensitive to both the cognitive and inflammatory effects of mild stress than are adult mice and suggest a possible a role for IL-1β. PMID:18407425
Heimesaat, Markus M.; Grundmann, Ursula; Alutis, Marie E.; Fischer, André; Bereswill, Stefan
Host immune responses are pivotal for combating enteropathogenic infections. We here assessed the impact of the innate receptor nucleotide oligomerization domain protein 2 (NOD2) in murine Campylobacter jejuni-infection. Conventionally colonized IL-10–/– mice lacking NOD2 and IL-10–/– controls were perorally challenged with C. jejuni strain 81-176 and displayed comparable pathogenic colonization of intestines until day 14 postinfection (p.i.). Whereas overall intestinal microbiota compositions were comparable in naive mice, NOD2–/– IL-10–/– mice exhibited less fecal bifidobacteria and lactobacilli than IL-10–/– counterparts after infection. Interestingly, NOD2–/– IL-10–/– mice were clinically more compromised during the early phase of infection, whereas, conversely, IL-10–/– animals exhibited more frequently bloody feces lateron. While colonic apoptotic cell and T lymphocyte numbers were comparable in either C. jejuni-infected mice, B lymphocytes were lower in the colon of infected NOD2–/– IL-10–/– mice versus controls. At day 14 p.i., colonic TNF and IL-23p19 mRNA levels were upregulated in NOD2–/– IL-10–/– mice only. Translocation rates of intestinal commensals to mesenteric lymphnodes and extra-intestinal compartments including liver and kidney were comparable, whereas viable bacteria were more frequently detected in spleens derived from IL-10–/– as compared to NOD2–/– IL-10–/– mice. In conclusion, NOD2 is involved during C. jejuni infection in conventionally colonized IL-10–/– mice in a time-dependent manner. PMID:28386467
Agah, Azin; Kyriakides, Themis R.; Lawler, Jack; Bornstein, Paul
Thrombospondin (TSP) 1 and 2, share the same overall structure and interact with a number of the same cell-surface receptors. In an attempt to elucidate their biological roles more clearly, we generated double-TSP1/TSP2-null animals and compared their phenotype to those of TSP1- and TSP2-null mice. Double-null mice exhibited an apparent phenotype that primarily represented the sum of the abnormalities observed in the single-null mice. However, surprisingly, the wound-healing response in double-null mice resembled that in TSP1-null animals and differed from that in TSP2-nulls. Thus, although the excisional wounds of TSP2-null mice are characterized by increased neovascularization and heal at an accelerated rate, TSP1-null and double-null animals demonstrated delayed healing, as indicated by the prolonged persistence of inflammation and delayed scab loss. Immunohistochemical analysis showed that, similar to TSP1-null mice, the granulation tissue of double-null mice was not excessively vascularized. Furthermore as in TSP1-nulls, decreases in macrophage recruitment and in the levels of monocyte chemoattractant protein-1 indicated that the inflammatory phase of the wound-healing response was impaired in double-null mice. Our data demonstrate that the consequences of a lack of TSP1 predominate in the response of double-null mice, and dictate the course of wound healing. These findings reflect distinct temporal and spatial expressions of TSP1 and TSP2 in the healing wound. PMID:12213711
do Carmo, Jussara M; da Silva, Alexandre A; Cai, Zhengwei; Lin, Shuying; Dubinion, John H; Hall, John E
Although the central nervous system melanocortin system is an important regulator of energy balance, the role of proopiomelanocortin (POMC) neurons in mediating the chronic effects of leptin on appetite, blood pressure, and glucose regulation is unknown. Using Cre/loxP technology we tested whether leptin receptor deletion in POMC neurons (LepR(flox/flox)/POMC-Cre mice) attenuates the chronic effects of leptin to increase mean arterial pressure (MAP), enhance glucose use and oxygen consumption, and reduce appetite. LepR(flox/flox)/POMC-Cre, wild-type, LepR(flox/flox), and POMC-Cre mice were instrumented for MAP and heart rate measurement by telemetry and venous catheters for infusions. LepR(flox/flox)/POMC-Cre mice were heavier, hyperglycemic, hyperinsulinemic, and hyperleptinemic compared with wild-type, LepR(flox/flox), and POMC-Cre mice. Despite exhibiting features of metabolic syndrome, LepR(flox/flox)/POMC-Cre mice had normal MAP and heart rate compared with LepR(flox/flox) but lower MAP and heart rate compared with wild-type mice. After a 5-day control period, leptin was infused (2 μg/kg per minute, IV) for 7 days. In control mice, leptin increased MAP by ≈5 mm Hg despite decreasing food intake by ≈35%. In contrast, leptin infusion in LepR(flox/flox)/POMC-Cre mice reduced MAP by ≈3 mm Hg and food intake by ≈28%. Leptin significantly decreased insulin and glucose levels in control mice but not in LepR(flox/flox)/POMC-Cre mice. Leptin increased oxygen consumption in LepR(flox/flox)/POMC-Cre and wild-type mice. Activation of POMC neurons is necessary for the chronic effects of leptin to raise MAP and reduce insulin and glucose levels, whereas leptin receptors in other areas of the brain other than POMC neurons appear to play a key role in mediating the chronic effects of leptin on appetite and oxygen consumption.
Kim, Hea-Jin; Koh, Hae-Young
Hallucinations and delusions are the most prominent symptoms of schizophrenia and characterized by impaired reality testing. Representation-mediated taste aversion (RMTA) has been proposed as a potential behavioral assessment of reality testing and has been applied to a neurodevelopmental rat model of schizophrenia. However, the theory underlying this approach has not been generalized yet with any demonstration of impaired reality testing in other animal models of schizophrenia, such as genetically-modified mice. We devised a RMTA procedure for mice that combines a Pavlovian association protocol pairing odor conditioned stimulus (CS) with sugar reward unconditioned stimulus (US), and a conditioned taste aversion (CTA) method. In this RMTA paradigm, we compared performances of wild-type (PLCβ1+/+) mice and phospholipase C β1 knock-out (PLCβ1-/-) mice which are known as one of the genetic models for schizophrenia. With a minimal amount of initial odor-sugar associative training, both PLCβ1+/+ and PLCβ1-/- mice were able to form an aversion to the sugar reward when the odor CS predicting sugar was paired with nausea. With an extended initial training, however, only PLCβ1-/- mice could form a RMTA. This persistent RMTA displayed by PLCβ1-/- mice shows their inability to distinguish real sugar from the CS-evoked representation of sugar at a stage in associative learning where wild-type mice normally could differentiate the two. These results demonstrate an impaired reality testing first observed in a genetic mouse model of schizophrenia, and suggest that RMTA paradigm may, with general applicability, allow diverse biological approaches to impaired reality testing.
Kobayashi, Yusuke; Hirawa, Nobuhito; Tabara, Yasuharu; Muraoka, Hidenori; Fujita, Megumi; Miyazaki, Nobuko; Fujiwara, Akira; Ichikawa, Yasuhiro; Yamamoto, Yuichiro; Ichihara, Naoaki; Saka, Sanae; Wakui, Hiromichi; Yoshida, Shin-ichiro; Yatsu, Keisuke; Toya, Yoshiyuki; Yasuda, Gen; Kohara, Katsuhiko; Kita, Yoshikuni; Takei, Kohtaro; Goshima, Yoshio; Ishikawa, Yoshihiro; Ueshima, Hirotsugu; Miki, Tetsuro; Umemura, Satoshi
We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na(+)-Ca(2+) exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.
Kim, Hea-jin; Koh, Hae-Young
Hallucinations and delusions are the most prominent symptoms of schizophrenia and characterized by impaired reality testing. Representation-mediated taste aversion (RMTA) has been proposed as a potential behavioral assessment of reality testing and has been applied to a neurodevelopmental rat model of schizophrenia. However, the theory underlying this approach has not been generalized yet with any demonstration of impaired reality testing in other animal models of schizophrenia, such as genetically-modified mice. We devised a RMTA procedure for mice that combines a Pavlovian association protocol pairing odor conditioned stimulus (CS) with sugar reward unconditioned stimulus (US), and a conditioned taste aversion (CTA) method. In this RMTA paradigm, we compared performances of wild-type (PLCβ1+/+) mice and phospholipase C β1 knock-out (PLCβ1-/-) mice which are known as one of the genetic models for schizophrenia. With a minimal amount of initial odor-sugar associative training, both PLCβ1+/+ and PLCβ1-/- mice were able to form an aversion to the sugar reward when the odor CS predicting sugar was paired with nausea. With an extended initial training, however, only PLCβ1-/- mice could form a RMTA. This persistent RMTA displayed by PLCβ1-/- mice shows their inability to distinguish real sugar from the CS-evoked representation of sugar at a stage in associative learning where wild-type mice normally could differentiate the two. These results demonstrate an impaired reality testing first observed in a genetic mouse model of schizophrenia, and suggest that RMTA paradigm may, with general applicability, allow diverse biological approaches to impaired reality testing. PMID:26731530
Becerril, Sara; Rodríguez, Amaia; Catalán, Victoria; Sáinz, Neira; Ramírez, Beatriz; Gómez-Ambrosi, Javier; Frühbeck, Gema
Leptin and nitric oxide (NO) are implicated in the control of energy homeostasis. The aim of the present study was to examine the impact of the absence of the inducible NO synthase (iNOS) gene on the regulation of energy balance in ob/ob mice analyzing the changes in gene expression levels in brown adipose tissue (BAT). Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated and the expression of genes involved in energy balance including fatty acid and glucose metabolism as well as mitochondrial genes were analyzed by microarrays. DBKO mice exhibited an improvement in energy balance with a decrease in body weight (P < 0.001), total fat pads (P < 0.05), and food intake (P < 0.05), as well as an enhancement in BAT function compared with ob/ob mice. To better understand the molecular events associated with this improvement, BAT gene expression was analyzed. Of particular interest, gene expression levels of the key subunit of the Mediator complex Med1 was upregulated (P < 0.05) in DBKO mice. Real-time PCR and immunohistochemistry further confirmed this data. Med1 is implicated in adipogenesis, lipid metabolic and biosynthetic processes, glucose metabolism, and mitochondrial metabolic pathways. Med1 plays an important role in the transcriptional control of genes implicated in energy homeostasis, suggesting that the improvement in energy balance and BAT function of the DBKO mice is mediated, at least in part, through the transcription coactivator Med1.
Maccarrone, Mauro; Valverde, Olga; Barbaccia, Maria L; Castañé, Anna; Maldonado, Rafael; Ledent, Catherine; Parmentier, Marc; Finazzi-Agrò, Alessandro
Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.
Garrison, Sheldon R.; Stucky, Cheryl L.
Objective Investigate age-related differences in mechanical sensitivity and determine the contribution of transient receptor potential ankyrin 1 (TRPA1) to mechanical hypersensitivity during chronic inflammation in young and aged animals. Methods Mechanical sensitivity in young (3-month) and aged (24-month) wild-type (TRPA1+/+) and TRPA1-deficient (TRPA1-/-) mice was measured behaviorally for 8-weeks following injection of Complete Freund's Adjuvant (CFA) into the plantar hindpaw. Histological analysis and hindpaw measurements evaluated inflammation. Ex-vivo skin-saphenous nerve preparations quantified C-fiber sensitivity. Results In naïve wild-type mice, aged animals were less sensitive to mechanical stimuli than young. Afferent recordings from TRPA1-/- mice indicate that TRPA1 contributes to the normal mechanical sensitivity in both age groups. Following CFA injection, both young and aged TRPA1+/+ mice exhibited mechanical hypersensitivity. Development of mechanical hypersensitivity was delayed until week 4 in young TRPA1-/- mice, when they exhibited a sharp decrease (9-fold) in mechanical thresholds. In contrast, CFA-injected aged TRPA1-/- mice did not exhibit mechanical hypersensitivity at any time during the entire 8-weeks. Recordings of C-fibers supported these findings and showed that action potential firing increased in both young (25%) and aged (60%) TRPA1+/+ mice 8 weeks after CFA. Interestingly, mechanical firing increased markedly in C-fibers from young TRPA1-/- mice (80%) but not in C-fibers from aged TRPA1-/- mice after CFA. Conclusions These data reveal marked differences in long-term mechanical behavioral sensitivity of aged and young mice, and suggest that TRPA1 may be a key contributor to the transition from acute to chronic inflammatory mechanical pain and nociceptor sensitization selectively in aged mice. PMID:24891324
Patel, Bhavik Anil; Fidalgo, Sara; Wang, Chunfang; Parmar, Leena; Mandona, Kasonde; Panossian, Annabelle; Flint, Melanie S.; Ranson, Richard N.; Saffrey, M. Jill; Yeoman, Mark S.
Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age–related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg−1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to “anti-TNFα” drugs as potential treatments for age-related chronic constipation. PMID:28198447
De Stefano, M Egle; Leone, Lucia; Lombardi, Loredana; Paggi, Paola
Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.
Ting, Stephen B; Wilanowski, Tomasz; Auden, Alana; Hall, Mark; Voss, Anne K; Thomas, Tim; Parekh, Vishwas; Cunningham, John M; Jane, Stephen M
The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. Here we show by gene targeting, mapping and genetic complementation studies that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, as the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3-/- embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs.
Hinton, David J; Lee, Moonnoh R; Jacobson, Taylor L; Mishra, Prasanna K; Frye, Mark A; Mrazek, David A; Macura, Slobodan I; Choi, Doo-Sup
Acamprosate is clinically used to treat alcohol-dependent patients. While the molecular and pharmacological mechanisms of acamprosate remain unclear, it has been shown to regulate γ-aminobutyric acid (GABA) or glutamate levels in the cortex and striatum. To investigate the effect of acamprosate on brain metabolites in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), we employed in vivo 16.4 T proton magnetic resonance spectroscopy. We utilized type 1 equilibrative nucleoside transporter (ENT1) null mice since acamprosate attenuates ethanol drinking in these mice. Our findings demonstrated that ethanol withdrawal reduced GABA levels and increased phosphorylated choline compounds in the mPFC of both wild-type and ENT1 null mice. Notably, acamprosate normalized these withdrawal-induced changes only in ENT1 null mice. In the NAc, ethanol withdrawal increased glutamate and glutamine (Glx) levels only in wild-type mice. Interestingly, acamprosate reduced Glx levels in the NAc compared to the withdrawal state in both genotypes. These results provide a molecular basis for the pharmacological effect of acamprosate in the cortical-striatal circuit.
Zhu, Hongyan; Mingler, Melissa K; McBride, Melissa L; Murphy, Andrew J; Valenzuela, David M; Yancopoulos, George D; Williams, Michael T; Vorhees, Charles V; Rothenberg, Marc E
NPSR1 is a G protein coupled receptor expressed in multiple brain regions involved in modulation of stress. Central administration of NPS, the putative endogenous ligand of NPSR1, can induce hyperlocomotion, anxiolytic effects and activation of the HPA axis. The role of NPSR1 in the brain remains unsettled. Here we used NPSR1 gene-targeted mice to define the functional role of NPSR1 under basal conditions on locomotion, anxiety- and/or depression-like behavior, corticosterone levels, acoustic startle with prepulse inhibition, learning and memory, and under NPS-induced locomotor activation, anxiolysis, and corticosterone release. Male, but not female, NPSR1-deficient mice exhibited enhanced depression-like behavior in a forced swim test, reduced acoustic startle response, and minor changes in the Morris water maze. Neither male nor female NPSR1-deficient mice showed alterations of baseline locomotion, anxiety-like behavior, or corticosterone release after exposure to a forced swim test or methamphetamine challenge in an open-field. After intracerebroventricular (ICV) administration of NPS, NPSR1-deficient mice failed to show normal NPS-induced increases in locomotion, anxiolysis, or corticosterone release compared with WT NPS-treated mice. These findings demonstrate that NPSR1 is essential in mediating NPS effects on behavior.
Bhandaru, Madhuri; Kempe, Daniela S; Rotte, Anand; Capuano, Paola; Pathare, Ganesh; Sopjani, Mentor; Alesutan, Ioana; Tyan, Leonid; Huang, Dan Yang; Siraskar, Balasaheb; Judenhofer, Martin S; Stange, Gerti; Pichler, Bernd J; Biber, Jürg; Quintanilla-Martinez, Leticia; Wagner, Carsten A; Pearce, David; Föller, Michael; Lang, Florian
Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.
Wijnholds, J; Evers, R; van Leusden, M R; Mol, C A; Zaman, G J; Mayer, U; Beijnen, J H; van der Valk, M; Krimpenfort, P; Borst, P
The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.
Nanobashvili, Avtandil; Airaksinen, Matti S.; Kokaia, Merab; Rossi, Jari; Asztély, Fredrik; Olofsdotter, Klara; Mohapel, Paul; Saarma, Mart; Lindvall, Olle; Kokaia, Zaal
Seizure activity regulates gene expression for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and their receptor components, the transmembrane c-Ret tyrosine kinase and the glycosylphosphatidylinositol-anchored GDNF family receptor (GFR) α1 and α2 in limbic structures. We demonstrate here that epileptogenesis, as assessed in the hippocampal kindling model, is markedly suppressed in mice lacking GFRα2. Moreover, at 6 to 8 wk after having reached the epileptic state, the hyperexcitability is lower in GFRα2 knock-out mice as compared with wild-type mice. These results provide evidence that signaling through GFRα2 is involved in mechanisms regulating the development and persistence of kindling epilepsy. Our data suggest that GDNF and NRTN may modulate seizure susceptibility by altering the function of hilar neuropeptide Y-containing interneurons and entorhinal cortical afferents at dentate granule cell synapses. PMID:11050250
Bouleftour, Wafa; Boudiffa, Maya; Wade-Gueye, Ndeye Marième; Bouët, Guénaëlle; Cardelli, Marco; Laroche, Norbert; Vanden-Bossche, Arnaud; Thomas, Mireille; Bonnelye, Edith; Aubin, Jane E; Vico, Laurence; Lafage-Proust, Marie Hélène; Malaval, Luc
Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing
Choi, Soonwook; Yu, Eunah; Lee, Seongwon; Llinás, Rodolfo R.
In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca2+ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states. PMID:26056284
Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco; Nielsen, Morten Frost; Kassem, Moustapha; Kousteni, Stavroula
Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5(A214V)) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5 mutation, we show here that Lrp5 GOF mutations in both humans and mice do not activate β-catenin signaling in osteoblasts. Consistent with a lack of β-catenin activation in their osteoblasts, Lrp5(A214V) mice have normal trilinear hematopoiesis. In contrast to leukemic mice with constitutive activation of β-catenin in osteoblasts (Ctnnb1(CAosb)), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5(A214V) mice. Likewise, peripheral blood count analysis in HBM patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.
Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco; Nielsen, Morten Frost; Kassem, Moustapha; Kousteni, Stavroula
Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5A214V) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5 mutation, we show here that Lrp5 GOF mutations in both humans and mice do not activate β-catenin signaling in osteoblasts. Consistent with a lack of β-catenin activation in their osteoblasts, Lrp5A214V mice have normal trilinear hematopoiesis. In contrast to leukemic mice with constitutive activation of β-catenin in osteoblasts (Ctnnb1CAosb), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5A214V mice. Likewise, peripheral blood count analysis in HBM patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza. PMID:26681532
Hillian, Antoinette D; McMullen, Megan R; Sebastian, Becky M; Roychowdhury, Sanjoy; Rowchowdhury, Sanjoy; Kashyap, Sangeeta R; Schauer, Philip R; Kirwan, John P; Feldstein, Ariel E; Nagy, Laura E
Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa(-/-) mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.
Meusel, Andrej; Teupser, Daniel; Ricken, Albert; Thiery, Joachim; Schiller, Jürgen; Huster, Daniel; Schöneberg, Torsten
Dysfunction of the melanocortin system can result in severe obesity accompanied with dyslipidemia and symptoms of the metabolic syndrome but the effect on vascular atherogenesis is not known. To study the impact of obesity and dyslipidemia on the cardiovascular system, we generated mice double-deficient for the melanocortin type 4 receptor (Mc4rmut mice) and the LDL receptor (Ldlr-/- mice). Mc4rmut mice develop obesity due to hyperphagia. Double-mutant mice (Mc4rmut;Ldlr-/-) exhibited massive increases in body weight, plasma cholesterol and triacylglycerol levels and developed atherosclerosis. Atherosclerotic lesion size was affected throughout the aortic root and brachiocephalic artery not only under semisynthetic, cholesterol-containing diet but also under cholesterol-free standard chow. The Mc4rmut mice developed a hepatic steatosis which contributes to increased plasma cholesterol levels even under cholesterol-free standard chow. Transcripts of cholesterol biosynthesis components and liver cholesterol levels did not significantly differ between wild-type and all mutant mouse strains but RNA sequencing data and biochemical measurements point to an altered bile acid elimination in Mc4rmut;Ldlr-/-. Therefore, the unchanged endogenous cholesterol biosynthesis together with a reduced hepatic VLDL and LDL-cholesterol clearance most likely led to increased plasma lipid levels and consequently to atherosclerosis in this animal model. Our data indicate that dysfunction of the melanocortin-regulated food intake and the resulting obesity significantly add to the proatherogenic lipoprotein profile caused by LDL receptor deficiency and, therefore, can be regarded as relevant risk factor for atherosclerosis. PMID:28030540
Mirsoian, Annie; Bouchlaka, Myriam N.; Sckisel, Gail D.; Chen, Mingyi; Pai, Chien-Chun Steven; Maverakis, Emanuel; Spencer, Richard G.; Fishbein, Kenneth W.; Siddiqui, Sana; Monjazeb, Arta M.; Martin, Bronwen; Maudsley, Stuart; Hesdorffer, Charles; Ferrucci, Luigi; Longo, Dan L.; Blazar, Bruce R.; Wiltrout, Robert H.; Taub, Dennis D.
Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF+ macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT. PMID:25366964
Ryan, Michael J; Jackson, Janna R; Hao, Yanlei; Williamson, Courtney L; Dabkowski, Erinne R; Hollander, John M; Alway, Stephen E
This study tested the hypothesis that resveratrol supplementation would lower oxidative stress in exercised muscles of aged mice. Young (3 months) and aged (27 months) C57BL/6 mice received a control or a 0.05% trans-resveratrol-supplemented diet for 10 days. After 7 days of dietary intervention, 20 maximal electrically evoked isometric contractions were obtained from the plantar flexors of one limb in anesthetized mice. Exercise was conducted for three consecutive days. Resveratrol supplementation blunted the exercise-induced increase in xanthine oxidase activity in muscles from young (25%) and aged (53%) mice. Resveratrol lowered H(2)O(2) levels in control (13%) and exercised (38%) muscles from aged animals, reduced Nox4 protein in both control and exercised muscles of young (30%) and aged mice (40%), and increased the ratio of reduced glutathione to oxidized glutathione in exercised muscles from young (38%) and aged (135%) mice. Resveratrol prevented the increase in lipid oxidation, increased catalase activity, and increased MnSOD activity in exercised muscles from aged mice. These data show that dietary resveratrol suppresses muscle indicators of oxidative stress in response to isometric contractions in aged mice.
Currais, Antonio; Prior, Marguerite; Lo, David; Jolivalt, Corinne; Schubert, David; Maher, Pamela
Mounting evidence supports a link between diabetes, cognitive dysfunction, and aging. However, the physiological mechanisms by which diabetes impacts brain function and cognition are not fully understood. To determine how diabetes contributes to cognitive dysfunction and age-associated pathology, we used streptozotocin to induce type 1 diabetes (T1D) in senescence-accelerated prone 8 (SAMP8) and senescence-resistant 1 (SAMR1) mice. Contextual fear conditioning demonstrated that T1D resulted in the development of cognitive deficits in SAMR1 mice similar to those seen in age-matched, nondiabetic SAMP8 mice. No further cognitive deficits were observed when the SAMP8 mice were made diabetic. T1D dramatically increased Aβ and glial fibrillary acidic protein immunoreactivity in the hippocampus of SAMP8 mice and to a lesser extent in age-matched SAMR1 mice. Further analysis revealed aggregated Aβ within astrocyte processes surrounding vessels. Western blot analyses from T1D SAMP8 mice showed elevated amyloid precursor protein processing and protein glycation along with increased inflammation. T1D elevated tau phosphorylation in the SAMR1 mice but did not further increase it in the SAMP8 mice where it was already significantly higher. These data suggest that aberrant glucose metabolism potentiates the aging phenotype in old mice and contributes to early stage central nervous system pathology in younger animals.
Higuchi, Hiroshi; Niki, Takeshi; Shiiya, Tomohiro
Neuropeptide Y (NPY) is a potent neurotransmitter for feeding. Besides NPY, orexigenic neuropeptides such as agouti-related protein (AgRP), and anorexigenic neuropeptides such as alpha-melatonin stimulating hormone (MSH) and cocaine-amphetamine-regulated transcript (CART) are also involved in central feeding regulation. During fasting, NPY and AgRP gene expressions are up-regulated and POMC and CART gene expressions are down-regulated in hypothalamus. Based on the network of peptidergic neurons, the former are involved in positive feeding regulation, and the latter are involved in negative feeding, which exert these feeding-regulated peptides especially in paraventricular nucleus (PVN). To clarify the compensatory mechanism of knock-out of NPY system on feeding, change in gene expressions of appetite-related neuropeptides and the feeding behavior was studied in NPY Y5-KO mice. Food intake was increased in Y5-KO mice. Fasting increased the amounts of food and water intake in the KO mice more profoundly. These data indicated the compensatory phenomenon of feeding behavior in Y5-KO mice. RT-PCR and ISH suggested that the compensation of feeding is due to change in gene expressions of AgRP, CART and POMC in hypothalamus. Thus, these findings indicated that the compensatory mechanism involves change in POMC/CART gene expression in arcuate nucleus (ARC). The POMC/CART gene expression is important for central compensatory regulation in feeding behavior.
Wöhr, Markus; Silverman, Jill L.; Scattoni, Maria L.; Turner, Sarah M.; Harris, Mark J.; Saxena, Roheeni; Crawley, Jacqueline N.
Mutations in neurexin and neuroligin genes have been associated with neurodevelopmental disabilities including autism. Autism spectrum disorder is diagnosed by aberrant reciprocal social interactions, deficits in social communication, and repetitive, stereotyped patterns of behaviors, along with narrow restricted interests. Mouse models have been successfully used to study physiological and behavioral outcomes of mutations in the trans-synaptic neurexin-neuroligin complex. To further understand the behavioral consequences of NEUROLIGIN2 (NLGN2) mutations, we assessed several behavioral phenotypes relevant to autism in neuroligin2 null (Nlgn2-/-), heterozygote (Nlgn2+/-), and wildtype (Nlgn2+/+) littermate control mice. Reduced breeding efficiency and high reactivity to handling was observed in Nlgn2-/- mice, resulting in low numbers of adult mice available for behavioral assessment. Consistent with previous findings, Nlgn2-/- mice displayed normal social behaviors, concomitant with reduced exploratory activity, impaired rotarod performance, and delays on several developmental milestones. No spontaneous stereotypies or repetitive behaviors were detected. Acoustic, tactile, and olfactory sensory information processing as well as sensorimotor gating were not affected. Nlgn2-/- pups isolated from mother and littermates emitted fewer ultrasonic vocalizations and spent less time calling than Nlgn2+/+ littermate controls. The present findings add to the growing literature on the role of neurexins and neuroligins in physiology and behavior relevant to neurodevelopmental disorders. PMID:22820233
Fodstad, O; Hansen, C T; Cannon, G B; Statham, C N; Lichtenstein, G R; Boyd, M R
To elucidate the in vivo role of natural killer (NK) cells, the growth of several murine and human tumors was studied in four variants of athymic, nude mice with different levels of NK activity. Beige-nude mice, homozygous for both the beige and the nude genes, had very low levels of NK activity, and their response to the B-cell mitogen, bacterial lipopolysaccharide, was lower than that of high-NK, adult NIH nude mice. Young and adult NIH nudes had different NK levels and showed different response in assays for K-cell, T-cell, and B-cell activity. The B-cell-defective NIH-II mice had slightly lower NK levels than adult NIH animals, but much lower response in the antibody-dependent cell-mediated cytotoxicity assay. No correlation was found between host NK activity and the s.c. growth of various human (LOX, CEM, K562) and murine (YAC-1) tumor cells. Low NK activity was not associated with increased lung colony formation in a metastasis model using i.v.-injected human (LOX) and murine (B16F10) melanoma cells. No relationship was found between host NK activity and the rate of elimination of i.v.-injected 5-iodo-2'-deoxyuridine-labeled LOX, B16F10, and YAC-1 cells from lungs, liver, or spleen. The results fail to support the view that NK cells exert significant direct effects on tumor cells in vivo.
Sakoori, Kazuto; Akagi, Takumi; Hashikawa, Tsutomu; Morimura, Naoko; Yamada, Kazuyuki; Aruga, Jun
Recent genetic linkage analysis has shown that LRRTM1 (Leucine rich repeat transmembrane neuronal 1) is associated with schizophrenia. Here, we characterized Lrrtm1 knockout mice behaviorally and morphologically. Systematic behavioral analysis revealed reduced locomotor activity in the early dark phase, altered behavioral responses to novel environments (open-field box, light-dark box, elevated plus maze, and hole board), avoidance of approach to large inanimate objects, social discrimination deficit, and spatial memory deficit. Upon administration of the NMDA receptor antagonist MK-801, Lrrtm1 knockout mice showed both locomotive activities in the open-field box and responses to the inanimate object that were distinct from those of wild-type mice, suggesting that altered glutamatergic transmission underlay the behavioral abnormalities. Furthermore, administration of a selective serotonin reuptake inhibitor (fluoxetine) rescued the abnormality in the elevated plus maze. Morphologically, the brains of Lrrtm1 knockout mice showed reduction in total hippocampus size and reduced synaptic density. The hippocampal synapses were characterized by elongated spines and diffusely distributed synaptic vesicles, indicating the role of Lrrtm1 in maintaining synaptic integrity. Although the pharmacobehavioral phenotype was not entirely characteristic of those of schizophrenia model animals, the impaired cognitive function may warrant the further study of LRRTM1 in relevance to schizophrenia. PMID:21818371
Solano, Rosa M; Casarejos, Maria J; Menéndez-Cuervo, Jamie; Rodriguez-Navarro, Jose A; García de Yébenes, Justo; Mena, Maria A
Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals, and abnormal neurotransmitter release. The role of glia in parkin deficiency is little known. We cultured midbrain glia from wild-type (WT) and parkin knock-out (PK-KO) mice. After 18-20 d in vitro, PK-KO glial cultures had less astrocytes, more microglia, reduced proliferation, and increased proapoptotic protein expression. PK-KO glia had greater levels of intracellular glutathione (GSH), increased mRNA expression of the GSH-synthesizing enzyme gamma-glutamylcysteine synthetase, and greater glutathione S-transferase and lower glutathione peroxidase activities than WT. The reverse happened in glia cultured in serum-free defined medium (EF12) or in old cultures. PK-KO glia was more susceptible than WT to transference to EF12 or neurotoxins (1-methyl-4-phenylpyridinium, blockers of GSH synthesis or catalase, inhibitors of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3 kinases), aging of the culture, or combination of these insults. PK-KO glia was less susceptible than WT to Fe2+ plus H2O2 and less responsive to protection by deferoxamine. Old WT glia increased the expression of heat shock protein 70, but PK-KO did not. Glia conditioned medium (GCM) from PK-KO was less neuroprotective and had lower levels of GSH than WT. GCM from WT increased the levels of dopamine markers in midbrain neuronal cultures transferred to EF12 more efficiently than GCM from PK-KO, and the difference was corrected by supplementation with GSH. PK-KO-GCM was a less powerful suppressor of apoptosis and microglia in neuronal cultures. Our data prove that abnormal glial function is critical in parkin mutations, and its role increases with aging.
Schneider, Augusto; Matkovich, Scot J.; Victoria, Berta; Spinel, Lina; Bartke, Andrzej; Golusinski, Pawel; Masternak, Michal M.
The Ames dwarf (df/df) mice have extended longevity and can preserve the ovarian reserve longer than Normal (N) mice. Based on this, the aim of our study was to evaluate the ovarian microRNA (miRNA) profile in young and aged df/df and N mice. Ovarian tissue was collected at 5–6 months and at 21–22 months of age for miRNA sequencing. We detected a total of 404 miRNAs in the ovarian samples, from which the abundance of 22 and 33 miRNAs changed with age in N and df/df mice, respectively. Of these, only three miRNAs were commonly regulated with age between N and df/df mice, indicating a very divergent miRNA profile between genotypes. We also detected that 46 miRNAs were regulated between N and df/df mice, of which 23 were regulated exclusively in young mice, 12 exclusively in old mice and 12 commonly regulated at young and old ages. Many genes likely to be targeted by these miRNAs are involved in the FoxO, mTOR, PI3k/Akt and insulin signaling pathways. These results suggest that the aging process has a differential impact on the ovarian miRNA profile in df/df mice, and suggest that these miRNAs can be central players in the maintenance of a younger ovarian phenotype. PMID:28046124
Pitychoutis, Pothitos M; Belmer, Arnauld; Moutkine, Imane; Adrien, Joëlle; Maroteaux, Luc
Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. Recently, a population-specific serotonin 2B (5-HT2B) receptor stop codon (ie, HTR2B Q20*) was reported to segregate with severely impulsive individuals, whereas 5-HT2B mutant (Htr2B(-/-)) mice also showed high impulsivity. Interestingly, in the same cohort, early-onset schizophrenia was more prevalent in HTR2B Q*20 carriers. However, the putative role of 5-HT2B receptor in the neurobiology of schizophrenia has never been investigated. We assessed the effects of the genetic and the pharmacological ablation of 5-HT2B receptors in mice subjected to a comprehensive series of behavioral test screenings for schizophrenic-like symptoms and investigated relevant dopaminergic and glutamatergic neurochemical alterations in the cortex and the striatum. Domains related to the positive, negative, and cognitive symptom clusters of schizophrenia were affected in Htr2B(-/-) mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr2B(-/-) mice presented with enhanced locomotor response to the psychostimulants dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment. We report herein that 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provide first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders.
Peterson, Rhett L.
Previously, transplantation of ovaries from young cycling mice into old postreproductive-age mice increased life span. We anticipated that the same factors that increased life span could also influence health span. Female CBA/J mice received new (60 d) ovaries at 12 and 17 months of age and were evaluated at 16 and 25 months of age, respectively. There were no significant differences in body weight among any age or treatment group. The percentage of fat mass was significantly increased at 13 and 16 months of age but was reduced by ovarian transplantation in 16-month-old mice. The percentages of lean body mass and total body water were significantly reduced in 13-month-old control mice but were restored in 16- and 25-month-old recipient mice by ovarian transplantation to the levels found in six-month-old control mice. In summary, we have shown that skeletal muscle mass, which is negatively influenced by aging, can be positively influenced or restored by reestablishment of active ovarian function in aged female mice. These findings provide strong incentive for further investigation of the positive influence of young ovaries on restoration of health in postreproductive females. PMID:27747096
Onaolapo, Olakunle J.; Onaolapo, Adejoke Y.; Omololu, Tope A.; Oludimu, Adedunke T.; Segun-Busari, Toluwalase; Omoleke, Taofeeq
This study tested the hypothesis that aging significantly affects the influence of exogenous testosterone on neurobehavior in gonadally intact male mice. Groups of prepubertal and aged male mice received daily vehicle or testosterone propionate (TP; 2.5 or 5.0 mg/kg intraperitoneal [i.p.]) for 21 days. Behaviors were assessed on days 1 and 21. Weight gain was significant in prepubertal mice. Locomotion and rearing increased in prepubertal mice after first dose and decreased after last dose of TP. Rearing was suppressed in aged mice throughout. Suppression of grooming occurred in both age groups at day 21. Significant increase in working memory in both age groups was seen in the radial-arm maze (at specific doses) and in prepubertal mice in the Y-maze. Elevated plus maze test showed mixed anxiolytic/anxiogenic effects. Aged mice had higher serum testosterone. In conclusion, age is an important determinant for the influence of exogenous testosterone on behavior in gonadally intact male mice. PMID:27158222
Atorrasagasti, Catalina; Kippes, Néstor; Malvicini, Mariana; Alaniz, Laura; Garcia, Mariana; Piccioni, Flavia; Fiore, Esteban J.; Bayo, Juan; Bataller, Ramón; Guruceaga, Elizabeth; Corrales, Fernando; Podhajcer, Osvaldo; Mazzolini, Guillermo
Introduction Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. Methods Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC+/+) and knock-out (SPARC−/−) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC−/− and SPARC+/+ mice using Affymetrix Mouse Gene ST 1.0 array. Results SPARC expression was found induced in fibrotic livers of mouse and human. SPARC−/− mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC−/− mice when compared to SPARC+/+ mice; in addition, MMP-2 expression was increased in SPARC−/− mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. Conclusions Overall our data suggest that SPARC plays
Hu, Weilian; Dai, Dehui; Li, Wei
Blakeslea trispora powder that contains 1.9 % lycopene was tested for its anti-aging effect on adult mice. 48 adult mice were administered with the powder at 0, 267, 534, 1,068 mg lycopene kg(-1) body daily for 30 days. The body weight, hematology, clinical chemical and antioxidant activities in major organs of adult mice were measured. The powder had no effect on the body weight, hematology, clinical chemical parameters of adult mice but improved the antioxidant activities in major organs of adult mice. Increased activities of superoxide dismutase, catalase and glutathione peroxidase and a decreased amount of malondialdehyde in liver, brain, kidney and skin of adult mice when a high-dose of the B. trispora powder was administered, suggests that it has the ability to enhance the antioxidation system and improve the anti-aging abilities of adult mice.
Reynders, Ana; Gaillard, Stéphane; Moqrich, Aziz
The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation. PMID:24925072
Roberds, S L; Anderson, J; Basi, G; Bienkowski, M J; Branstetter, D G; Chen, K S; Freedman, S B; Frigon, N L; Games, D; Hu, K; Johnson-Wood, K; Kappenman, K E; Kawabe, T T; Kola, I; Kuehn, R; Lee, M; Liu, W; Motter, R; Nichols, N F; Power, M; Robertson, D W; Schenk, D; Schoor, M; Shopp, G M; Shuck, M E; Sinha, S; Svensson, K A; Tatsuno, G; Tintrup, H; Wijsman, J; Wright, S; McConlogue, L
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.
Marics, Irène; Malapert, Pascale; Reynders, Ana; Gaillard, Stéphane; Moqrich, Aziz
The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.
Takamatsu, Y.; Hagino, Y.; Sato, A.; Takahashi, T.; Nagasawa, S.Y.; Kubo, Y.; Mizuguchi, M.; Uhl, G.R.; Sora, I.; Ikeda, K.
The symptoms of attention-deficit/hyperactivity disorder (ADHD) are characterized by inattention and hyperactivity-impulsivity. It is a common childhood neurodevelopmental disorder that often persists into adulthood. Improvements in ADHD symptoms using psychostimulants have been recognized as a paradoxical calming effect. The psychostimulant methylphenidate (MPH) is currently used as the first-line medication for the management of ADHD. Recent studies have drawn attention to altered dopamine-mediated neurotransmission in ADHD, particularly reuptake by the dopamine transporter (DAT). This hypothesis is supported by the observation that DAT knockout mice exhibit marked hyperactivity that is responsive to acute MPH treatment. However, other behaviors relevant to ADHD have not been fully clarified. In the present study, we observed learning impairment in shuttle-box avoidance behavior together with hyperactivity in a novel environment in DAT knockout mice. Methylphenidate normalized these behaviors and enhanced escape activity in the tail suspension test. Interestingly, the effective dose of MPH increased extracellular dopamine in the prefrontal cortex but not striatum, suggesting an important role for changes in prefrontal dopamine in ADHD. Research that uses rodent models such as DAT knockout mice may be useful for elucidating the pathophysiology of ADHD. PMID:25817856
Minett, Michael S.; Pereira, Vanessa; Sikandar, Shafaq; Matsuyama, Ayako; Lolignier, Stéphane; Kanellopoulos, Alexandros H.; Mancini, Flavia; Iannetti, Gian D.; Bogdanov, Yury D.; Santana-Varela, Sonia; Millet, Queensta; Baskozos, Giorgios; MacAllister, Raymond; Cox, James J.; Zhao, Jing; Wood, John N.
Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids. PMID:26634308
Minett, Michael S; Pereira, Vanessa; Sikandar, Shafaq; Matsuyama, Ayako; Lolignier, Stéphane; Kanellopoulos, Alexandros H; Mancini, Flavia; Iannetti, Gian D; Bogdanov, Yury D; Santana-Varela, Sonia; Millet, Queensta; Baskozos, Giorgios; MacAllister, Raymond; Cox, James J; Zhao, Jing; Wood, John N
Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.
Shahmoradi, Ali; Reinecke, Lisa; Kroos, Christina; Wichert, Sven P.; Oster, Henrik; Wehr, Michael C.; Taneja, Reshma; Hirrlinger, Johannes; Rossner, Moritz J.
Increasing evidence suggests that clock genes may be implicated in a spectrum of psychiatric diseases, including sleep and mood related disorders as well as schizophrenia. The bHLH transcription factors SHARP1/DEC2/BHLHE41 and SHARP2/DEC1/BHLHE40 are modulators of the circadian system and SHARP1/DEC2/BHLHE40 has been shown to regulate homeostatic sleep drive in humans. In this study, we characterized Sharp1 and Sharp2 double mutant mice (S1/2-/-) using online EEG recordings in living animals, behavioral assays and global gene expression profiling. EEG recordings revealed attenuated sleep/wake amplitudes and alterations of theta oscillations. Increased sleep in the dark phase is paralleled by reduced voluntary activity and cortical gene expression signatures reveal associations with psychiatric diseases. S1/2-/- mice display alterations in novelty induced activity, anxiety and curiosity. Moreover, mutant mice exhibit impaired working memory and deficits in prepulse inhibition resembling symptoms of psychiatric diseases. Network modeling indicates a connection between neural plasticity and clock genes, particularly for SHARP1 and PER1. Our findings support the hypothesis that abnormal sleep and certain (endo)phenotypes of psychiatric diseases may be caused by common mechanisms involving components of the molecular clock including SHARP1 and SHARP2. PMID:25340473
Allen, Justin M; Zamurs, Laura; Brachvogel, Bent; Schlötzer-Schrehardt, Ursula; Hansen, Uwe; Lamandé, Shireen R; Rowley, Lynn; Fitzgerald, Jamie; Bateman, John F
WARP is a recently identified extracellular matrix molecule with restricted expression in permanent cartilages and a distinct subset of basement membranes in peripheral nerves, muscle, and the central nervous system vasculature. WARP interacts with perlecan, and we also demonstrate here that WARP binds type VI collagen, suggesting a function in bridging connective tissue structures. To understand the in vivo function of WARP, we generated a WARP-deficient mouse strain. WARP-null mice were healthy, viable, and fertile with no overt abnormalities. Motor function and behavioral testing demonstrated that WARP-null mice exhibited a significantly delayed response to acute painful stimulus and impaired fine motor coordination, although general motor function was not affected, suggesting compromised peripheral nerve function. Immunostaining of WARP-interacting ligands demonstrated that the collagen VI microfibrillar matrix was severely reduced and mislocalized in peripheral nerves of WARP-null mice. Further ultrastructural analysis revealed reduced fibrillar collagen deposition within the peripheral nerve extracellular matrix and abnormal partial fusing of adjacent Schwann cell basement membranes, suggesting an important function for WARP in stabilizing the association of the collagenous interstitial matrix with the Schwann cell basement membrane. In contrast, other WARP-deficient tissues such as articular cartilage, intervertebral discs, and skeletal muscle showed no detectable abnormalities, and basement membranes formed normally. Our data demonstrate that although WARP is not essential for basement membrane formation or musculoskeletal development, it has critical roles in the structure and function of peripheral nerves.
Taguchi, K; Tokuno, M; Yamasaki, K; Kadowaki, D; Seo, H; Otagiri, M
Acetaminophen (APAP), a widely used analgesic and antipyretic drug, has the potential to cause lethal hepatotoxicity. Mice are widely used for developing murine models of APAP-induced hepatotoxicity, and many researchers have used these models for APAP-related studies including the fields of biology, pharmacology and toxicology. Although drug-induced hepatotoxicity is dependent on a number of factors (species, gender and age), very few studies have investigated the effect of aging on APAP hepatotoxicity. In this study, we evaluated the effect of age on APAP-induced hepatotoxicity in different weekly-aged mice to establish a model of APAP-induced hepatotoxicity that is an accurate reflection of general experimental conditions. Male ICR mice 4, 6, 8, 10 and 12 weeks old were given APAP intraperitoneally, and mortality, hepatic damage and the plasma concentration of APAP metabolites were evaluated. It was found that younger male ICR mice were relatively resistant to hepatotoxicity induced by intraperitoneal APAP administration. In addition, the APAP-glucuronide concentration in plasma remained essentially the same among the differently-aged mice, while APAP-sulfate levels were dramatically decreased in an age-dependent manner. Thus, it is recommended that mice of the same ages be used in studies related to APAP-induced hepatotoxixity. These results provide evidence in support of not only the age-related changes in susceptibility to APAP-derived hepatotoxicity in mice but also in developing mouse models for APAP-related studies.
Khodr, C E; Hurley, D L; Phelps, C J
The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid-stimulating hormone. Prolactin therapy initiated before 21 days maintains normal-sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 microg of ovine prolactin (oPRL), 50 microg of oPRL, or 50 microg of recombinant mouse prolactin (rmPRL) beginning at 3 days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P
Dupont, Aline; Sommer, Felix; Zhang, Kaiyi; Repnik, Urska; Basic, Marijana; Bleich, André; Kühnel, Mark; Bäckhed, Fredrik; Litvak, Yael; Fulde, Marcus; Rosenshine, Ilan; Hornef, Mathias W.
Enteropathogenic Escherichia coli (EPEC) represents a major causative agent of infant diarrhea associated with significant morbidity and mortality in developing countries. Although studied extensively in vitro, the investigation of the host-pathogen interaction in vivo has been hampered by the lack of a suitable small animal model. Using RT-PCR and global transcriptome analysis, high throughput 16S rDNA sequencing as well as immunofluorescence and electron microscopy, we characterize the EPEC-host interaction following oral challenge of newborn mice. Spontaneous colonization of the small intestine and colon of neonate mice that lasted until weaning was observed. Intimate attachment to the epithelial plasma membrane and microcolony formation were visualized only in the presence of a functional bundle forming pili (BFP) and type III secretion system (T3SS). Similarly, a T3SS-dependent EPEC-induced innate immune response, mediated via MyD88, TLR5 and TLR9 led to the induction of a distinct set of genes in infected intestinal epithelial cells. Infection-induced alterations of the microbiota composition remained restricted to the postnatal period. Although EPEC colonized the adult intestine in the absence of a competing microbiota, no microcolonies were observed at the small intestinal epithelium. Here, we introduce the first suitable mouse infection model and describe an age-dependent, virulence factor-dependent attachment of EPEC to enterocytes in vivo. PMID:27159323
Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F; Ehlen, Harald W A; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate
Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.
Verbitsky, Miguel; Yonan, Amanda L.; Malleret, Gael; Kandel, Eric R.; Gilliam, T. Conrad; Pavlidis, Paul
We have carried out a global survey of age-related changes in mRNA levels in the 57BL/6NIA mouse hippocampus and found a difference in the hippocampal gene expression profile between 2-month-old young mice and 15-month-old middle-aged mice correlated with an age-related cognitive deficit in hippocampal-based explicit memory formation. Middle-aged…
Maekawa, Akiko; Kanaoka, Yoshihide; Xing, Wei; Austen, K Frank
The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C(4), which then undergoes extracellular metabolism to LTD(4) and LTE(4). LTE(4), the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT(1)R and CysLT(2)R, respectively). We had recognized a greater potency for LTE(4) than LTC(4) or LTD(4) in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE(4) in mice lacking both the CysLT(1)R and CysLT(2)R could establish the existence of a separate LTE(4) receptor. We now report that the intradermal injection of LTE(4) into the ear of mice deficient in both CysLT(1)R and CysLT(2)R elicits a vascular leak that exceeds the response to intradermal injection of LTC(4) or LTD(4), and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE(4) is approximately 64-fold more potent in the CysLT(1)R/CysLT(2)R double-deficient mice than in sufficient mice. The administration of a CysLT(1)R antagonist augmented the permeability response of the CysLT(1)R/CysLT(2)R double-deficient mice to LTC(4), LTD(4), and LTE(4). Our findings establish the existence of a third receptor, CysLT(E)R, that responds preferentially to LTE(4), the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.
Sahu, Ravi P.; DaSilva, Sonia C.; Rashid, Badri; Martel, Kellie Clay; Jernigan, Danielle; Mehta, Shama R.; Mohamed, Deena R.; Rezania, Samin; Bradish, Joshua R.; Armstrong, Andrew B.; Warren, Simon; Konger, Raymond L.
Recent studies suggest that peroxisome proliferator-activated receptor gamma (PPARγ) agonists may have cancer chemopreventive activity. Other studies have shown that loss of epidermal PPARγ results in enhanced chemical carcinogenesis in mice via unknown mechanisms. However, ultraviolet B (UVB) exposure represents the primary etiological agent for skin cancer formation and the role of PPARγ in photobiology and photocarcinogenesis is unknown. In previous studies, we demonstrated that UVB irradiation of cells results in the formation of oxidized glycerophosphocholines that exhibit PPARγ ligand activity. We therefore hypothesized that PPARγ would prove to be a chemopreventive target in photocarcinogenesis. We first showed that UVB irradiation of mouse skin causes generation of PPARγ agonist species in vivo. We then generated SKH-1 hairless, albino mice deficient in epidermal Pparg (Pparg−/−epi) using a cytokeratin 14 driven Cre-LoxP strategy. Using a chronic model of UVB photocarcinogenesis, we next showed that Pparg−/−epi mice exhibit an earlier onset of tumor formation, increased tumor burden, and tumor progression. Increased tumor burden in Pparg−/−epi mice was accompanied by a significant increase in epidermal hyperplasia and p53 positive epidermal cells in surrounding skin lacking tumors. Following acute UVB irradiation, Pparg−/−epi mice exhibited an augmentation of both UVB-induced caspase 3/7 activity and inflammation. Increased apoptosis and inflammation was also observed following treatment with the PPARγ antagonist GW9662. With chronic UVB irradiation, Pparg−/−epi mice exhibited a sustained increase in erythema and transepidermal water loss relative to wildtype littermates. This suggests that PPARγ agonists could have possible chemopreventive activity in non-melanoma skin cancer. PMID:22467332
Besmer, Dahlia M.; Curry, Jennifer M.; Roy, Lopamudra D.; Tinder, Teresa L.; Sahraei, Mahnaz; Schettini, Jorge; Hwang, Sun-Il; Lee, Yong Y.; Gendler, Sandra J.; Mukherjee, Pinku
MUC1 is over expressed and aberrantly glycosolated in >60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In the present study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared to both KC and KCM. Cell lines derived from KCKO tumors have significantly lower tumorigenic capacity compared to cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared to mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or matrix metalloproteinase-9 (MMP9). Further, significantly fewer KCKO cells entered the G2M phase of the cell cycle compared to the KCM cells. Proteomics and western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of MAPK, as well as a significant decrease in Nestin and Tubulin α-2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse in order to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro. PMID:21558393
Ceccarelli, Manuela; Micheli, Laura; D'Andrea, Giorgio; De Bardi, Marco; Scheijen, Blanca; Ciotti, MariaTeresa; Leonardi, Luca; Luvisetto, Siro; Tirone, Felice
Cerebellar granule neurons develop postnatally from cerebellar granule precursors (GCPs), which are located in the external granule layer (EGL) where they massively proliferate. Thereafter, GCPs become postmitotic, migrate inward to form the internal granule layer (IGL), further differentiate and form synapses with Purkinje cell dendrites. We previously showed that the Btg family gene, Tis21/Btg2, is required for normal GCP migration. Here we investigated the role in cerebellar development of the related gene, Btg1, which regulates stem cell quiescence in adult neurogenic niches, and is expressed in the cerebellum. Knockout of Btg1 in mice caused a major increase of the proliferation of the GCPs in the EGL, whose thickness increased, remaining hyperplastic even after postnatal day 14, when the EGL is normally reduced to a few GCP layers. This was accompanied by a slight decrease of differentiation and migration of the GCPs and increase of apoptosis. The GCPs of double Btg1/Tis21-null mice presented combined major defects of proliferation and migration outside the EGL, indicating that each gene plays unique and crucial roles in cerebellar development. Remarkably, these developmental defects lead to a permanent increase of the adult cerebellar volume in Btg1-null and double mutant mice, and to impairment in all mutants, including Tis21-null, of the cerebellum-dependent motor coordination. Gain- and loss-of-function strategies in a GCP cell line revealed that Btg1 regulates the proliferation of GCPs selectively through cyclin D1. Thus, Btg1 plays a critical role for cerebellar maturation and function.
Zhao, L; Ding, T; Cyrus, T; Cheng, Y; Tian, H; Ma, M; Falotico, R; Praticò, D
Background and purpose: Chronic proliferative responses of different vascular cell types have been involved in the pathogenesis of atherosclerosis. However, their functional role remains to be established. Sirolimus reduces neointimal proliferation after balloon angioplasty and chronic graft vessel disease. These studies were undertaken to investigate the effects of this anti-proliferative drug on atherogenesis. Experimental approach: Low-density lipoprotein receptor-deficient (LDL r-KO) mice on a cholesterol-rich diet were randomized to receive placebo or sirolimus (0.1; 0.3; or 1 mg·kg−1) in their diet for 8 or 16 weeks. Results: In both studies, plasma levels of the drug increased in a dose-dependent fashion, animals gained weight normally and, among groups, plasma lipids levels did not differ significantly. Compared with placebo, plasma levels of interleukin-6, monocyte chemoattractant protein-1, interferon γ, tumour necrosis factor α and CD40, and their mRNA levels in aortic tissue were significantly reduced in sirolimus-treated mice. This effect resulted in a significant and dose-dependent reduction in atherosclerotic lesions, in both the root and aortic tree. Also these lesions contained less monocyte/macrophages and smooth muscle cells, but more collagen. Conclusions and implications: The present results demonstrated that at low doses, sirolimus was an effective and safe anti-atherogenic agent in the LDL r-KO mice. It attenuated the progression of atherosclerosis and modulated the plaque phenotype by reducing the pro-inflammatory vascular responses typical of the disease. British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00080.x PMID:19220291
Walsh, Michael E.; Liu, Yuhong; Zhang, Yiqiang; Jaramillo, Carlos A.; Macleod, Gregory T.; Van Remmen, Holly
Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to –a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1−/−), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1−/− mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1−/− mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1−/− mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1−/− and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor
Littrell, Ofelia M.; Pomerleau, Francois; Huettl, Peter; Surgener, Stewart; McGinty, Jacqueline F.; Middaugh, Lawrence D.; Granholm, Ann-Charlotte; Gerhardt, Greg A.; Boger, Heather A.
Glial cell line-derived neurotrophic factor (GDNF) supports the viability of midbrain dopamine (DA) neurons that degenerate in Parkinson’s disease. Middle aged, 12-month-old, Gdnf heterozygous (Gdnf+/−) mice have diminished spontaneous locomotor activity and enhanced synaptosomal DA uptake compared to wildtype mice. In this study, dopamine transporter (DAT) function in middle-aged, 12-month-old Gdnf+/− mice was more thoroughly investigated using in vivo electrochemistry. Gdnf+/− mice injected with the DAT inhibitor, nomifensine, exhibited significantly more locomotor activity than wildtype mice. In vivo electrochemistry with carbon fiber microelectrodes demonstrated enhanced clearance of DA in the striatum of Gdnf+/− mice, suggesting greater surface expression of DAT than in wildtype littermates. Additionally, 12 month old Gdnf+/− mice expressed greater D2 receptor mRNA and protein in the striatum than wildtype mice. Neurochemical analyses of striatal tissue samples indicated significant reductions in DA and a faster DA metabolic rate in Gdnf+/− mice than in wildtype mice. Altogether, these data support an important role for GDNF in the regulation of uptake, synthesis, and metabolism of DA during aging. PMID:21144620
Rehg, Jerold E.; Rahija, Richard; Bush, Dorothy; Bradley, Alys; Ward, Jerrold M.
A few reports indicated the incidence of hematolymphoid neoplasms in old CD-1 mice, but the cellular lineage of CD-1 mouse neoplasms has not be published. In this study, immunohistochemistry (IHC) was used to characterize the cellular lineage of spontaneous hematolymphoid neoplasms arising in young female CD-1 mice used as health monitoring sentinels and aging female CD-1 mice used as controls in 80 wk carcinogenesis studies. Lymphoblastic lymphomas of T-cell and B-cell lineage were common in mice 12 mo or less of age, whereas a wide range of non-lymphoblastic B-cell lymphomas and lymphoblastic T-cell lymphomas were common in mice > 12 mo old. Renal hyaline droplets positive for lysozyme were observed in aged mice with a histiocytic-associated large B-cell lymphoma (HA-BCL) and a myeloid leukemia. Endogenous ecotropic MuLV genes have been recovered from CD-1 mice, but MuLV protein expression has not been previously demonstrated. We reported for the first time the expression of MuLV protein by IHC in lymphomas and some normal tissues of both young and aging CD-1 mice. This report should help to differentiate spontaneous lymphomas and leukemias in CD-1 mice from those induced by chemicals and other methods. PMID:26224701
Ramaraj, P; Kessler, S P; Colmenares, C; Sen, G C
Although angiotensin-converting enzyme (ACE) has been studied primarily in the context of its role in blood pressure regulation, this widely distributed enzyme has many other physiological functions. The ACE gene encodes two isozymes. The somatic isozyme is expressed in many tissues, including vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, whereas the testicular or germinal angiotensin-converting enzyme is expressed only in sperm. The ACE gene knockout mice lack both isozymes and they exhibit low blood pressure, kidney dysfunctions, and male infertility. Here, we report the use of a sperm-specific promoter and interbreeding of transgenic and gene knockout mice for generating a mouse strain that expressed ACE only in sperm. The experimental mice maintained the kidney defects of ACE-/- mice, but unlike the knockout strain, the males were fertile. Thus, we established that the role of ACE in male fertility is completely dependent on its exclusive expression in sperm. Our study clearly demonstrated how transgenic and knockout techniques can be combined for ascribing a specific physiological function to the expression of a multifunctional protein in a given tissue. PMID:9664078
Flegal, Matthew; Blimkie, Melinda; Roch-Lefevre, Sandrine; Gregoire, Eric; Klokov, Dmitry
Health effects of tritium, a β-emitter and a by-product of the nuclear industry, is a subject of significant controversy. This mouse in vivo study was undertaken to monitor biological effects of low level tritium exposure. Mice were exposed to tritiated drinking water (HTO) at 10 KBq/L, 1 MBq/L and 20 MBq/L concentrations for one month. The treatment did not result in a significant increase of apoptosis in splenocytes. To examine if this low level tritium exposure alters radiosensitivity, the extracted splenocytes were challenged in vitro with 2 Gy γ-radiation, and apoptotic responses at 1 and 24 h were measured. No alterations in the radiosensitivity were detected in cells from mice exposed to tritium compared to sham-treated mice. In contrast, low dose γ-irradiation at 20 or 100 mGy, resulted in a significant increase in resistance to apoptotic cell death after 2 Gy irradiation; an indication of the radioadaptive response. Overall, our data suggest that low concentrations of tritium given to mice as HTO in drinking water do not exert cytotoxic effect in splenocytes, nor do they change cellular sensitivity to additional high dose γ-radiation. The latter may be considered as the lack of a radioadaptive response, typically observed after low dose γ-irradiation.
He, Qiuming; Morillon, Y Maurice; Spidale, Nicholas A; Kroger, Charles J; Liu, Bo; Sartor, R Balfour; Wang, Bo; Tisch, Roland
Inefficient thymic negative selection of self-specific T cells is associated with several autoimmune diseases, including type 1 diabetes. The factors that influence the efficacy of thymic negative selection, as well as the kinetics of thymic output of autoreactive T cells remain ill-defined. We investigated thymic production of β cell-specific T cells using a thymus-transplantation model. Thymi from different aged NOD mice, representing distinct stages of type 1 diabetes, were implanted into NOD.scid recipients, and the diabetogenicity of the resulting T cell pool was examined. Strikingly, the development of diabetes-inducing β cell-specific CD4(+) and CD8(+) T cells was regulated in an age-dependent manner. NOD.scid recipients of newborn NOD thymi developed diabetes. However, recipients of thymi from 7- and 10-d-old NOD donor mice remained diabetes-free and exhibited a progressive decline in islet infiltration and β cell-specific CD4(+) and CD8(+) T cells. A similar temporal decrease in autoimmune infiltration was detected in some, but not all, tissues of recipient mice implanted with thymi from NOD mice lacking expression of the autoimmune regulator transcription factor, which develop multiorgan T cell-mediated autoimmunity. In contrast, recipients of 10 d or older thymi lacked diabetogenic T cells but developed severe colitis marked by increased effector T cells reactive to intestinal microbiota. These results demonstrate that thymic development of autoreactive T cells is limited to a narrow time window and occurs in a reciprocal manner compared with colonic microbiota-responsive T cells in NOD mice.
Mazo, Tamara; Muñoz, Marina C.; Dominici, Fernando P.; Carreras, María C.; Poderoso, Juan José; Sadoshima, Junichi; Gelpi, Ricardo J.
Thioredoxin-1 (Trx1) protects the heart from ischemia/reperfusion (I/R) injury. Given that the age at which the first episode of coronary disease takes place has considerably decreased, life at middle-aged (MA) emerges as a new field of study. The aim was determine whether infarct size, Trx1 expression and activity, Akt and GSK-3β were altered in young (Y) and MA mice overexpressing cardiac Trx1, and in a dominant negative (DN-Trx1) mutant of Trx1. Langendorff-perfused hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (R). We used 3 and 12 month-old male of wild type (WT), Trx1, and DN-Trx1. Trx1 overexpression reduced infarct size in young mice (WT-Y: 46.8±4.1% vs. Trx1-Y: 27.6±3.5%, p < 0.05). Trx1 activity was reduced by 52.3±3.2% (p < 0.05) in Trx1-MA, accompanied by an increase in nitration by 17.5±0.9%, although Trx1 expression in transgenic mice was similar between young and middle-aged. The expression of p-Akt and p-GSK-3β increased during reperfusion in Trx1-Y. DN-Trx1 mice showed neither reduction in infarct size nor Akt and GSK-3β phosphorylation. Our data suggest that the lack of protection in Trx1 middle-aged mice even with normal Trx1 expression may be associated to decreased Trx1 activity, increased nitration and inhibition of p-Akt and p-GSK-3β. PMID:26933812
Lopatina, Olga; Yoshihara, Toru; Nishimura, Tomoko; Zhong, Jing; Akther, Shirin; Fakhrul, Azam A. K. M.; Liang, Mingkun; Higashida, Chiharu; Sumi, Kohei; Furuhara, Kazumi; Inahata, Yuki; Huang, Jian-Jung; Koizumi, Keita; Yokoyama, Shigeru; Tsuji, Takahiro; Petugina, Yulia; Sumarokov, Andrei; Salmina, Alla B.; Hashida, Koji; Kitao, Yasuko; Hori, Osamu; Asano, Masahide; Kitamura, Yoji; Kozaka, Takashi; Shiba, Kazuhiro; Zhong, Fangfang; Xie, Min-Jue; Sato, Makoto; Ishihara, Katsuhiko; Higashida, Haruhiro
CD157, known as bone marrow stromal cell antigen-1, is a glycosylphosphatidylinositol-anchored ADP-ribosyl cyclase that supports the survival and function of B-lymphocytes and hematopoietic or intestinal stem cells. Although CD157/Bst1 is a risk locus in Parkinson's disease (PD), little is known about the function of CD157 in the nervous system and contribution to PD progression. Here, we show that no apparent motor dysfunction was observed in young knockout (CD157−/−) male mice under less aging-related effects on behaviors. CD157−/− mice exhibited anxiety-related and depression-like behaviors compared with wild-type mice. These behaviors were rescued through treatment with anti-psychiatric drugs and oxytocin. CD157 was weakly expressed in the amygdala and c-Fos immunoreactivity in the amygdala was less evident in CD157−/− mice than in wild-type mice. These results demonstrate for the first time that CD157 plays a role as a neuro-regulator and suggest a potential role in pre-motor symptoms in PD. PMID:24795584
Lopatina, Olga; Yoshihara, Toru; Nishimura, Tomoko; Zhong, Jing; Akther, Shirin; Fakhrul, Azam A K M; Liang, Mingkun; Higashida, Chiharu; Sumi, Kohei; Furuhara, Kazumi; Inahata, Yuki; Huang, Jian-Jung; Koizumi, Keita; Yokoyama, Shigeru; Tsuji, Takahiro; Petugina, Yulia; Sumarokov, Andrei; Salmina, Alla B; Hashida, Koji; Kitao, Yasuko; Hori, Osamu; Asano, Masahide; Kitamura, Yoji; Kozaka, Takashi; Shiba, Kazuhiro; Zhong, Fangfang; Xie, Min-Jue; Sato, Makoto; Ishihara, Katsuhiko; Higashida, Haruhiro
CD157, known as bone marrow stromal cell antigen-1, is a glycosylphosphatidylinositol-anchored ADP-ribosyl cyclase that supports the survival and function of B-lymphocytes and hematopoietic or intestinal stem cells. Although CD157/Bst1 is a risk locus in Parkinson's disease (PD), little is known about the function of CD157 in the nervous system and contribution to PD progression. Here, we show that no apparent motor dysfunction was observed in young knockout (CD157 (-/-)) male mice under less aging-related effects on behaviors. CD157 (-/-) mice exhibited anxiety-related and depression-like behaviors compared with wild-type mice. These behaviors were rescued through treatment with anti-psychiatric drugs and oxytocin. CD157 was weakly expressed in the amygdala and c-Fos immunoreactivity in the amygdala was less evident in CD157 (-/-) mice than in wild-type mice. These results demonstrate for the first time that CD157 plays a role as a neuro-regulator and suggest a potential role in pre-motor symptoms in PD.
Inouye, M.; Matsumoto, N.; Galvin, M.J.; McRee, D.I.
The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm2 and 19 mW/cm2 on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 degrees C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.
Savonenko, A.; Munoz, P.; Melnikova, T.; Wang, Q.; Liang, X.; Breyer, R. M.; Montine, T. J.; Kirkwood, A.; Andreasson, K.
Cyclooxygenase-2 (COX-2) is a neuronal immediate early gene that is regulated by N-methyl D aspartate (NMDA) receptor activity. COX-2 enzymatic activity catalyzes the first committed step in prostaglandin synthesis. Recent studies demonstrate an emerging role for the downstream PGE2 EP2 receptor in diverse models of activity-dependent synaptic plasticity and a significant function in models of neurological disease including cerebral ischemia, Familial Alzheimer’s disease, and Familial amyotrophic lateral sclerosis. Little is known, however, about the normal function of the EP2 receptor in behavior and cognition. Here we report that deletion of the EP2 receptor leads to significant cognitive deficits in standard tests of fear and social memory. EP2 −/− mice also demonstrated impaired prepulse inhibition (PPI) and heightened anxiety, but normal startle reactivity, exploratory behavior, and spatial reference memory. This complex behavioral phenotype of EP2−/− mice was associated with a deficit in long-term depression (LTD) in hippocampus. Our findings suggest that PGE2 signaling via the EP2 receptors plays an important role in cognitive and emotional behaviors that recapitulate some aspects of human psychopathology related to schizophrenia. PMID:19416671
Garten, Antje; Popkova, Yulia; Penke, Melanie; Franke, Christin; Ricken, Albert; Schulz, Angela; Kiess, Wieland; Huster, Daniel; Schöneberg, Torsten; Schiller, Jürgen
Obesity is often associated with dyslipidemia and hepatosteatosis. A number of animal models of non-alcoholic fatty liver disease (NAFLD) are established but they significantly differ in the molecular and biochemical changes depending on the genetic modification and diet used. Mice deficient for melanocortin type 4 receptor (Mc4rmut) develop hyperphagia, obesity, and subsequently NAFLD already under regular chow and resemble more closely the energy supply-driven obesity found in humans. This animal model was used to assess the molecular and biochemical consequences of hyperphagia-induced obesity on hepatic lipid metabolism. We analyzed transcriptome changes in Mc4rmut mice by RNA sequencing and used high resolution 1H magic angle spinning NMR spectroscopy and MALDI-TOF mass spectrometry to assess changes in the lipid composition. On the transcriptomic level we found significant changes in components of the triacylglycerol metabolism, unsaturated fatty acids biosynthesis, peroxisome proliferator-activated receptor signaling pathways, and lipid transport and storage compared to the wild-type. These findings were supported by increases in triacylglycerol, monounsaturated fatty acid, and arachidonic acid levels. The transcriptome signatures significantly differ from those of other NAFLD mouse models supporting the concept of hepatic subphenotypes depending on the genetic background and diet. Comparative analyses of our data with previous studies allowed for the identification of common changes and genotype-specific components and pathways involved in obesity-associated NAFLD. PMID:28207798
Schnell, Anna; Sandrelli, Federica; Ranc, Vaclav; Ripperger, Jürgen A; Brai, Emanuele; Alberi, Lavinia; Rainer, Gregor; Albrecht, Urs
Genomic studies suggest an association of circadian clock genes with bipolar disorder (BD) and lithium response in humans. Therefore, we tested mice mutant in various clock genes before and after lithium treatment in the forced swim test (FST), a rodent behavioral test used for evaluation of depressive-like states. We find that expression of circadian clock components, including Per2, Cry1 and Rev-erbα, is affected by lithium treatment, and thus, these clock components may contribute to the beneficial effects of lithium therapy. In particular, we observed that Cry1 is important at specific times of the day to transmit lithium-mediated effects. Interestingly, the pathways involving Per2 and Cry1, which regulate the behavior in the FST and the response to lithium, are distinct as evidenced by the phosphorylation of GSK3β after lithium treatment and the modulation of dopamine levels in the striatum. Furthermore, we observed the co-existence of depressive and mania-like symptoms in Cry1 knock-out mice, which resembles the so-called mixed state seen in BD patients. Taken together our results strengthen the concept that a defective circadian timing system may impact directly or indirectly on mood-related behaviors.
Goodfellow, Nathalie M; Sargin, Derya; Ansorge, Mark S; Gingrich, Jay A; Lambe, Evelyn K
The activity of the prefrontal cortex is essential for normal emotional processing and is strongly modulated by serotonin (5-HT). Yet, little is known about the regulatory mechanisms that control the activity of the prefrontal 5-HT receptors. Here, we found and characterized a deregulation of prefrontal 5-HT receptor electrophysiological signaling in mouse models of disrupted serotonin transporter (5-HTT) function, a risk factor for emotional and cognitive disturbances. We identified a novel tyrosine kinase-dependent mechanism that regulates 5-HT-mediated inhibition of prefrontal pyramidal neurons. We report that mice with compromised 5-HTT, resulting from either genetic deletion or brief treatment with selective serotonin reuptake inhibitors during development, have amplified 5-HT1A receptor-mediated currents in adulthood. These greater inhibitory effects of 5-HT are accompanied by enhanced downstream coupling to Kir3 channels. Notably, in normal wild-type mice, we found that these larger 5-HT1A responses can be mimicked through inhibition of Src family tyrosine kinases. By comparison, in our 5-HTT mouse models, the larger 5-HT1A responses were rapidly reduced through inhibition of tyrosine phosphatases. Our findings implicate tyrosine phosphorylation in regulating the electrophysiological effects of prefrontal 5-HT1A receptors with implications for neuropsychiatric diseases associated with emotional dysfunction, such as anxiety and depressive disorders.
Utting, Oliver; Sedgmen, Bradley J.; Watts, Tania H.; Shi, Xiaoshu; Rottapel, Robert; Iulianella, Angelo; Lohnes, David; Veillette, André
The SLP-76 family of immune cell-specific adaptors is composed of three distinct members named SLP-76, Blnk, and Clnk. They have been implicated in the signaling pathways coupled to immunoreceptors such as the antigen receptors and Fc receptors. Previous studies using gene-targeted mice and deficient cell lines showed that SLP-76 plays a central role in T-cell development and activation. Moreover, it is essential for normal mast cell and platelet activation. In contrast, Blnk is necessary for B-cell development and activation. While the precise function of Clnk is not known, it was reported that Clnk is selectively expressed in mast cells, natural killer (NK) cells, and previously activated T-cells. Moreover, ectopic expression of Clnk was shown to rescue T-cell receptor-mediated signal transduction in an SLP-76-deficient T-cell line, suggesting that, like its relatives, Clnk is involved in the positive regulation of immunoreceptor signaling. Stimulatory effects of Clnk on immunoreceptor signaling were also reported to occur in transfected B-cell and basophil leukemia cell lines. Herein, we attempted to address the physiological role of Clnk in immune cells by the generation of Clnk-deficient mice. The results of our studies demonstrated that Clnk is dispensable for normal differentiation and function of T cells, mast cells, and NK cells. Hence, unlike its relatives, Clnk is not essential for normal immune functions. PMID:15199160
Logan, Angela; Shabalina, Irina G; Prime, Tracy A; Rogatti, Sebastian; Kalinovich, Anastasia V; Hartley, Richard C; Budd, Ralph C; Cannon, Barbara; Murphy, Michael P
In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria-targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro-apoptotic and pro-inflammatory redox signaling pathways.
Woolbright, Benjamin L.; Antoine, Daniel J.; Jenkins, Rosalind E.; Bajt, Mary Lynn; Park, B. Kevin; Jaeschke, Hartmut
Cholestasis is a pathological common component of numerous liver diseases that results in hepatotoxicity, inflammation, and cirrhosis when untreated. While the predominant hypothesis in cholestatic liver injury remains hepatocyte apoptosis due to direct toxicity of hydrophobic bile acid exposure, recent work suggests that the injury occurs through inflammatory necrosis. In order to resolve this controversy, we used novel plasma biomarkers to assess the mechanisms of cell death during early cholestatic liver injury. C57Bl/6 mice underwent bile duct ligation (BDL) for 6–72 h, or sham operation. Another group of mice were given D-galactosamine and endotoxin as a positive control for apoptosis and inflammatory necrosis. Plasma levels of full length cytokeratin-18 (FL-K18), microRNA-122 (miR-122) and high mobility group box-1 protein (HMGB1) increased progressively after BDL with peak levels observed after 48 h. These results indicate extensive cell necrosis after BDL, which is supported by the time course of plasma alanine aminotransferase activities and histology. In contrast, plasma caspase-3 activity, cleaved caspase-3 protein and caspase-cleaved cytokeratin-18 fragments (cK18) were not elevated at any time during BDL suggesting the absence of apoptosis. In contrast, all plasma biomarkers of necrosis and apoptosis were elevated 6 h after Gal/End treatment. In addition, acetylated HMGB1, a marker for macrophage and monocyte activation, was increased as early as 12 h but mainly at 48–72 h. However, progressive neutrophil accumulation in the area of necrosis started at 6 h after BDL. In conclusion, these data indicate that early cholestatic liver injury in mice is an inflammatory event, and occurs through necrosis with little evidence for apoptosis. - Highlights: • The mechanism of cell death during cholestasis remains a controversial topic. • Plasma biomarkers offer new insight into cell death after bile duct ligation. • Cytokeratin-18, microRNA-122 and HMGB
Wang, Qing M.; Meng, Zhaoxiang; Yin, Zhenglu
Objective To investigate whether gait dysfunction is a predictor of severe spatial learning and memory impairment in aged mice. Methods A total of 100 12-month-old male mice that had no obvious abnormal motor ability and whose Morris water maze performances were not significantly different from those of two-month-old male mice were selected for the study. The selected aged mice were then divided into abnormal or normal gait groups according to the results from the quantitative gait assessment. Gaits of aged mice were defined as abnormal when the values of quantitative gait parameters were two standard deviations (SD) lower or higher than those of 2-month-old male mice. Gait parameters included stride length, variability of stride length, base of support, cadence, and average speed. After nine months, mice exhibiting severe spatial learning and memory impairment were separated from mice with mild or no cognitive dysfunction. The rate of severe spatial learning and memory impairment in the abnormal and normal gait groups was tested by a chi-square test and the correlation between gait dysfunction and decline in cognitive function was tested using a diagnostic test. Results The 12-month-old aged mice were divided into a normal gait group (n = 75) and an abnormal gait group (n = 25). Nine months later, three mice in the normal gait group and two mice in the abnormal gait group had died. The remaining mice were subjected to the Morris water maze again, and 17 out of 23 mice in the abnormal gait group had developed severe spatial learning and memory impairment, including six with stride length deficits, 15 with coefficient of variation (CV) in stride length, two with base of support (BOS) deficits, five with cadence dysfunction, and six with average speed deficits. In contrast, only 15 out of 72 mice in the normal gait group developed severe spatial learning and memory impairment. The rate of severe spatial learning and memory impairment was significantly higher in
Selesniemi, Kaisa; Lee, Ho-Joon; Muhlhauser, Ailene; Tilly, Jonathan L
Increased meiotic spindle abnormalities and aneuploidy in oocytes of women of advanced maternal ages lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Despite the significance of the problem, strategies to sustain oocyte quality with age have remained elusive. Here we report that adult female mice maintained under 40% caloric restriction (CR) did not exhibit aging-related increases in oocyte aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, or mitochondrial dysfunction (aggregation, impaired ATP production), all of which occurred in oocytes of age-matched ad libitum-fed controls. The effects of CR on oocyte quality in aging females were reproduced by deletion of the metabolic regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Thus, CR during adulthood or loss of PGC-1α function maintains female germline chromosomal stability and its proper segregation during meiosis, such that ovulated oocytes of aged female mice previously maintained on CR or lacking PGC-1α are comparable to those of young females during prime reproductive life.
Srivastava, Amit K.; Renusch, Samantha R.; Naiman, Nicole E.; Gu, Shuping; Sneh, Amita; Arnold, W. David; Sahenk, Zarife; Kolb, Stephen J.
The small heat shock protein HSPB1 is a multifunctional, α-crystallin-based protein that has been shown to be neuroprotective in animal models of motor neuron disease and peripheral nerve injury. Missense mutations in HSPB1 result in axonal Charcot-Marie-Tooth disease with minimal sensory involvement (CMT2F) and distal hereditary motor neuropathy type 2 (dHMN-II). These disorders are characterized by a selective loss of motor axons in peripheral nerve resulting in distal muscle weakness and often severe disability. To investigate the pathogenic mechanisms of HSPB1 mutations in motor neurons in vivo, we have developed and characterized transgenic PrP-HSPB1 and PrP-HSPB1(R136W) mice. These mice express the human HSPB1 protein throughout the nervous system including in axons of peripheral nerve. Although both mouse strains lacked obvious motor deficits, the PrP-HSPB1(R136W) mice developed an age-dependent motor axonopathy. Mutant mice showed axonal pathology in spinal cord and peripheral nerve with evidence of impaired neurofilament cytoskeleton, associated with organelle accumulation. Accompanying these findings, increases in the number of Schmidt-Lanterman incisures, as evidence of impaired axon-Schwann cell interactions, were present. These observations suggest that overexpression of HSPB1(R136W) in neurons is sufficient to cause pathological and electrophysiological changes in mice that are seen in patients with hereditary motor neuropathy. PMID:22521462
Jeong, J-J; Kim, K A; Hwang, Y-J; Han, M J; Kim, D-H
In the present study, lactic acid bacteria (LAB) strains were collected from kimchi and were screened to isolate strains that inhibit lipopolysaccharide (LPS) production by Escherichia coli and p16 expression and nuclear factor-kappa B (NF-κB) activation in LPS-stimulated macrophages. Oral administration of Lactobacillus brevis OW38 (1×10(9) cfu/mouse) to aged mice (male, 18 months old) for 8 weeks reduced the LPS level in colon fluid and blood. In addition, OW38 treatment also reduced the ratio of Firmicutes or Proteobacteria to Bacteroidetes, which was significantly higher in aged mice than in young mice. Treatment with OW38 in aged mice inhibited the expression of inflammatory markers, such as myeloperoxidase, tumour necrosis factor (TNF), and interleukin (IL)-1β, and inhibited NF-κB activation. Furthermore, it induced the expression of colonic tight junction proteins zonula occludens-1, occludin, and claudin-1. OW38 treatment also suppressed the expression of senescence markers p16, p53, and SAMHD1 in the colon and the hippocampus of aged mice. In addition, it significantly restored spontaneous alternation as well as the expression of brain-derived neurotrophic factor and doublecortin in aged mice compared to that in young mice (P<0.05). Based on these findings, we conclude that OW38 treatment may ameliorate aging-associated colitis and memory impairment by inhibiting gut microbiota LPS production, NF-κB activation, and p16 expression.
Koh, Ming Teng; Spiegel, Amy M; Gallagher, Michela
Episodic memory impairment due to aging has been linked to hippocampal dysfunction. Evidence exists for alterations in specific circuits within the hippocampal system that are closely coupled to individual differences in the presence and severity of such memory loss. Here, we used the newly developed Diversity Outbred (DO) mouse that was designed to model the genetic diversity in human populations. Young and aged DO mice were tested in a hippocampal-dependent water maze task. Young mice showed higher proficiency and more robust memory compared to the overall performance of aged mice. A substantial number of the older mice, however, performed on par with the normative performance of the younger mice. Stereological quantification of somatostatin-immunoreactive neurons in the dentate hilus showed that high-performing young and unimpaired aged mice had similar numbers of somatostatin-positive interneurons, while aged mice that were impaired in the spatial task had significantly fewer such neurons. These data in the DO model tie loss of hilar inhibitory network integrity to age-related memory impairment, paralleling data in other rodent models.
Ang, Hooi Hoon; Lee, Kheng Leng; Kiyoshi, Matsumoto
Eurycoma longifolia Jack was investigated for sexual motivation activity in adult, middle-aged male mice and in retired breeders, using the modified open field and the modified runway choice methods. Each mouse received 500 mg/kg of one of 4 fractions of E. longifolia Jack, viz. chloroform, methanol, butanol, and water, whereas the mice in the control and yohimbine groups received 3 ml/kg of normal saline and 30 mg/kg of yohimbine daily respectively for 10 d. The results show a transient increase in the percentage of male mice responding to the right choice after chronic consumption of the fractions with 50 percent of the adult middle-aged male mice treated with E. longifolia Jack and yohimbine scoring the right choice after 8 and 5 days post-treatment respectively. In conclusion, this study has shown that E. longifolia Jack continues to enhance sexual motivation in adult, middle-aged male mice and in retired breeders.
Rho, Jong M; Sarnat, Harvey B; Sullivan, Patrick G; Robbins, Carol A; Kim, Dong W
Although there is increasing awareness of adverse effects associated with use of the high-fat ketogenic diet, very little is known regarding its long-term clinical consequences, especially in relation to cardiovascular health. Recent reports have highlighted rare but significant cardiac problems in patients treated with the ketogenic diet. Given the inherent limitations in conducting detailed pathologic assessments in patients, we asked whether histologic changes might develop in the brain and skeletal muscle of mice fed a high-fat diet for 2 to 3 months. We found no evidence of gross morphologic or histochemical alterations in muscle or brain after administration of the ketogenic diet. Further, there was no abnormal lipid storage or mitochondrial enzymatic staining. Our data suggest that patients chronically treated with a ketogenic diet are not likely to develop a lipid myopathy or neuronal inclusions.
Rauner, Martina; Franke, Kristin; Murray, Marta; Singh, Rashim Pal; Hiram-Bab, Sahar; Platzbecker, Uwe; Gassmann, Max; Socolovsky, Merav; Neumann, Drorit; Gabet, Yankel; Chavakis, Triantafyllos; Hofbauer, Lorenz C; Wielockx, Ben
The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2(f/f) ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2(f/f) ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2(f/f) ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.
Littlefield, Alyssa; Kohman, Rachel A
Normal aging is associated with low-grade neuroinflammation that results from age-related priming of microglial cells. Further, aging alters the response to several anti-inflammatory factors, including interleukin (IL)-4 and IL-13. One intervention that has been shown to modulate microglia activation in the aged brain, both basally and following an immune challenge, is exercise. However, whether engaging in exercise can improve responsiveness to anti-inflammatory cytokines is presently unknown. The current study evaluated whether prior exercise training increases sensitivity to anti-inflammatory cytokines that promote the M2 (alternative) microglia phenotype in adult (5-month-old) and aged (23-month-old) C57BL/6J mice. After 8weeks of exercise or control housing, mice received bilateral hippocampal injections of an IL-4/IL-13 cocktail or vehicle. Twenty-four hours later hippocampal samples were collected and analyzed for expression of genes associated with the M1 (inflammatory) and M2 microglia phenotypes. Results show that IL-4/IL-13 administration increased expression of the M2-associated genes found in inflammatory zone 1 (Fizz1), chitinase-like 3 (Ym1), Arginase-1 (Arg1), SOCS1, IL-1ra, and CD206. In response to IL-4/IL-13 administration, aged mice showed increased hippocampal expression of the M2-related genes Arg1, SOCS1, Ym1, and CD206 relative to adult mice. Aged mice also showed increased expression of IL-1β relative to adults, which was unaffected by wheel running or IL-4/IL-13. Wheel running was found to have modest effects on expression of Ym1 and Fizz1 in aged and adult mice. Collectively, our findings indicate that aged mice show a differential response to anti-inflammatory cytokines relative to adult mice and that exercise has limited effects on modulating this response.
King-Himmelreich, Tanya S.; Möser, Christine V.; Wolters, Miriam C.; Olbrich, Katrin; Geisslinger, Gerd; Niederberger, Ellen
The processing of pain undergoes several changes in aging that affect sensory nociceptive fibers and the endogenous neuronal inhibitory systems. So far, it is not completely clear whether age-induced modifications are associated with an increase or decrease in pain perception. In this study, we assessed the impact of age on inflammatory nociception in mice and the role of the hormonal inhibitory systems in this context. We investigated the nociceptive behavior of 12-month-old versus 6–8-week-old mice in two behavioral models of inflammatory nociception. Levels of TRP channels, and cortisol as well as cortisol targets, were measured by qPCR, ELISA, and Western blot in the differently aged mice. We observed an age-related reduction in nociceptive behavior during inflammation as well as a higher level of cortisol in the spinal cord of aged mice compared to young mice, while TRP channels were not reduced. Among potential cortisol targets, the NF-κB inhibitor protein alpha (IκBα) was increased, which might contribute to inhibition of NF-κB and a decreased expression and activity of the inducible nitric oxide synthase (iNOS). In conclusion, our results reveal a reduced nociceptive response in aged mice, which might be at least partially mediated by an augmented inflammation-induced increase in the hormonal inhibitory system involving cortisol. PMID:26593904
Wu, Jing; Li, Huihui; Sun, Xiaoru; Zhang, Hui; Hao, Shuangying; Ji, Muhuo; Yang, Jianjun; Li, Kuanyu
Isoflurane possesses neurotoxicity and can induce cognitive deficits, particularly in aging mammals. Mitochondrial reactive oxygen species (mtROS) have been linked to the early pathogenesis of this disorder. However, the role of mtROS remains to be evaluated due to a lack of targeted method to treat mtROS. Here, we determined in aging mice the effects of the mitochondrion-targeted antioxidant SS-31, on cognitive deficits induced by isoflurane, a general inhalation anesthetic. We further investigated the possible mechanisms underlying the effects of SS-31 on hippocampal neuro-inflammation and apoptosis. The results showed that isoflurane induced hippocampus-dependent memory deficit, which was associated with mitochondrial dysfunction including reduced ATP contents, increased ROS levels, and mitochondrial swelling. Treatment with SS-31 significantly ameliorated isoflurane-induced cognitive deficits through the improvement of mitochondrial integrity and function. Mechanistically, SS-31 treatment suppressed pro-inflammatory responses by decreasing the levels of NF-κB, NLRP3, caspase 1, IL-1β, and TNF-α; and inhibited the apoptotic pathway by decreasing the Bax/Bcl-2 ratio, reducing the release of cytochrome C, and blocking the cleavage of caspase 3. Our results indicate that isoflurane-induced cognitive deficits may be attenuated by mitochondrion-targeted antioxidants, such as SS-31. Therefore, SS-31 may have therapeutic potentials in preventing injuries from oxidative stresses that contribute to anesthetic-induced neurotoxicity.
Streicher, Carmen; Zeitz, Ute; Andrukhova, Olena; Rupprecht, Anne; Pohl, Elena; Larsson, Tobias E.; Windisch, Wilhelm; Lanske, Beate
It is still controversial whether the bone-derived hormone fibroblast growth factor-23 (FGF23) has additional physiological functions apart from its well-known suppressive actions on renal phosphate reabsorption and vitamin D hormone synthesis. Here we analyzed premature aging, mineral homeostasis, carbohydrate metabolism, and fat metabolism in 9-month-old male wild-type (WT) mice, vitamin D receptor mutant mice (VDRΔ/Δ) with a nonfunctioning vitamin D receptor, and Fgf23−/−/VDRΔ/Δ compound mutant mice on both a standard rodent chow and a rescue diet enriched with calcium, phosphorus, and lactose. Organ atrophy, lung emphysema, and ectopic tissue or vascular calcifications were absent in compound mutants. In addition, body weight, glucose tolerance, insulin tolerance, insulin secretory capacity, pancreatic beta cell volume, and retroperitoneal and epididymal fat mass as well as serum cholesterol and triglycerides were indistinguishable between vitamin D receptor and compound mutants. In contrast to VDRΔ/Δ and Fgf23−/−/VDRΔ/Δ mice, which stayed lean, WT mice showed obesity-induced insulin resistance. To rule out alopecia and concomitantly elevated energy expenditure present in 9-month-old VDRΔ/Δ and Fgf23−/−/VDRΔ/Δ mice as a confounding factor for the lacking effect of Fgf23 deficiency on fat mass, we analyzed whole-body composition in WT, Fgf23−/−, VDRΔ/Δ, and Fgf23−/−/VDRΔ/Δ mice at the age of 4 wk, when the coat in VDRΔ/Δ mice is still normal. Whole-body fat mass was reduced in Fgf23−/− mice but almost identical in WT, VDRΔ/Δ, and Fgf23−/−/VDRΔ/Δ mice. In conclusion, our data indicate that Fgf23 has no molecular vitamin D-independent role in aging, insulin signaling, or fat metabolism in mice. PMID:22294750
Ohgami, Nobutaka; Yajima, Ichiro; Iida, Machiko; Li, Xiang; Oshino, Reina; Kumasaka, Mayuko Y.; Kato, Masashi
Despite the fact that manganese (Mn) is known to be a neurotoxic element relevant to age-related disorders, the risk of oral exposure to Mn for age-related hearing loss remains unclear. In this study, we orally exposed wild-type young adult mice to Mn (Mn-exposed WT-mice) at 1.65 and 16.50 mg/L for 4 weeks. Mn-exposed WT-mice showed acceleration of age-related hearing loss. Mn-exposed WT-mice had neurodegeneration of spiral ganglion neurons (SGNs) with increased number of lipofuscin granules. Mn-exposed WT-mice also had increased hypoxia-inducible factor-1 alpha (Hif-1α) protein with less hydroxylation at proline 564 and decreased c-Ret protein in SGNs. Mn-mediated acceleration of age-related hearing loss involving neurodegeneration of SGNs was rescued in RET-transgenic mice carrying constitutively activated RET. Thus, oral exposure to Mn accelerates age-related hearing loss in mice with Ret-mediated neurodegeneration of SGNs. PMID:27824154
Guo, Wen; Miller, Andrew D; Pencina, Karol; Wong, Siu; Lee, Amanda; Yee, Michael; Toraldo, Gianluca; Jasuja, Ravi; Bhasin, Shalender
Since its discovery as a potent inhibitor for muscle development, myostatin has been actively pursued as a drug target for age- and disease-related muscle loss. However, potential adverse effects of long-term myostatin deficiency have not been thoroughly investigated. We report herein that male myostatin null mice (mstn(-/-)), in spite of their greater muscle mass compared to wild-type (wt) mice, displayed more significant functional decline from young (3-6months) to middle age (12-15months) than age-matched wt mice, measured as gripping strength and treadmill endurance. Mstn(-/-) mice displayed markedly restricted ankle mobility and degenerative changes of the ankle joints, including disorganization of bone, tendon and peri-articular connective tissue, as well as synovial thickening with inflammatory cell infiltration. Messenger RNA expression of several pro-osteogenic genes was higher in the Achilles tendon-bone insertion in mstn(-/-) mice than wt mice, even at the neonatal age. At middle age, higher plasma concentrations of growth factors characteristic of excessive bone remodeling were found in mstn(-/-) mice than wt controls. These data collectively indicate that myostatin may play an important role in maintaining ankle and wrist joint health, possibly through negative regulation of the pro-osteogenic WNT/BMP pathway.
Kerjan, Géraldine; Koizumi, Hiroyuki; Han, Edward B; Dubé, Celine M; Djakovic, Stevan N; Patrick, Gentry N; Baram, Tallie Z; Heinemann, Stephen F; Gleeson, Joseph G
Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.
Arnold, Thomas D; Niaudet, Colin; Pang, Mei-Fong; Siegenthaler, Julie; Gaengel, Konstantin; Jung, Bongnam; Ferrero, Gina M; Mukouyama, Yoh-suke; Fuxe, Jonas; Akhurst, Rosemary; Betsholtz, Christer; Sheppard, Dean; Reichardt, Louis F
Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVβ8 activates angiosuppressive TGFβ gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVβ8 in the brain or downstream TGFβ1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfb(ret/ret) mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage.
Chen, Yei-Tsung; Collins, Loretta L.; Uno, Hideo; Chang, Chawnshang
Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4−/−) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4−/− cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4−/− cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4−/− cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4−/− cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development. PMID:15767677
Pitari, G; Malergue, F; Martin, F; Philippe, J M; Massucci, M T; Chabret, C; Maras, B; Duprè, S; Naquet, P; Galland, F
Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.
Wang, Ying; Park, Sangeun; Bajpayee, Neil S; Nagaoka, Yoshiko; Boulay, Guylain; Birnbaumer, Lutz; Jiang, Meisheng
Insulin secretion by pancreatic β cells is a complex and highly regulated process. Disruption of this process can lead to diabetes mellitus. One of the various pathways involved in the regulation of insulin secretion is the activation of heterotrimeric G proteins. Bordetella pertussis toxin (PTX) promotes insulin secretion, suggesting the involvement of one or more of three G(i) and/or two G(o) proteins as suppressors of insulin secretion from β cells. However, neither the mechanism of this inhibitory modulation of insulin secretion nor the identity of the G(i/o) proteins involved has been elucidated. Here we show that one of the two splice variants of G(o), G(o2), is a key player in the control of glucose-induced insulin secretion by β cells. Mice lacking G(o2)α, but not those lacking α subunits of either G(o1) or any G(i) proteins, handle glucose loads more efficiently than wild-type (WT) mice, and do so by increased glucose-induced insulin secretion. We thus provide unique genetic evidence that the G(o2) protein is a transducer in an inhibitory pathway that prevents damaging oversecretion of insulin.
Wallace, M. E.; MacSwiney, F. M.
In a warfarin-resistant population of wild mice reared in the laboratory, a dominant gene for adiposity, Ad, was found to segregate. The onset of obesity is at 4--6 months, and adipose mice suffer from hyperinsulinaemia; the sexes differ in penetrance, males having greater penetrance then females. Linkage backcrosses show the gene to be situated on chromosome 7 with about 25% recombination with the closely linked warfarin-resistance genes War, and frizzy, fr. The finding of adipose in two other wild populations also carrying War is discussed as an ecological and physiological problem. PMID:429792
Michelsen, Kathrin S; Wong, Michelle H; Shah, Prediman K; Zhang, Wenxuan; Yano, Juliana; Doherty, Terence M; Akira, Shizuo; Rajavashisth, Tripathi B; Arditi, Moshe
Toll-like receptors (TLRs) and the downstream adaptor molecule myeloid differentiation factor 88 (MyD88) play an essential role in the innate immune responses. Here, we demonstrate that genetic deficiency of TLR4 or MyD88 is associated with a significant reduction of aortic plaque areas in atherosclerosis-prone apolipoprotein E-deficient mice, despite persistent hypercholesterolemia, implying an important role for the innate immune system in atherogenesis. Apolipoprotein E-deficient mice that also lacked TLR4 or MyD88 demonstrated reduced aortic atherosclerosis that was associated with reductions in circulating levels of proinflammatory cytokines IL-12 or monocyte chemoattractant protein 1, plaque lipid content, numbers of macrophage, and cyclooxygenase 2 immunoreactivity in their plaques. Endothelial-leukocyte adhesion in response to minimally modified low-density lipoprotein was reduced in aortic endothelial cells derived from MyD88-deficient mice. Taken together, our results suggest an important role for TLR4 and MyD88 signaling in atherosclerosis in a hypercholesterolemic mouse model, providing a pathophysiologic link between innate immunity, inflammation, and atherogenesis.
Naik, Amish A; Xie, Chao; Zuscik, Michael J; Kingsley, Paul; Schwarz, Edward M; Awad, Hani; Guldberg, Robert; Drissi, Hicham; Puzas, J Edward; Boyce, Brendan; Zhang, Xinping; O'Keefe, Regis J
The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX-2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX-2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX-2 is expressed primarily in early cartilage precursors that co-express col-2. COX-2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX-2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX-2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging. PMID:18847332
Wang, Hongkai; Li, Chengren; Wang, Hanzhi; Mei, Feng; Liu, Zhi; Shen, Hai-Ying; Xiao, Lan
Schizophrenia is a mental disease that mainly affects young individuals (15 to 35 years old) but its etiology remains largely undefined. Recently, accumulating evidence indicated that demyelination and/or dysfunction of oligodendrocytes is an important feature of its pathogenesis. We hypothesized that the vulnerability of young individuals to demyelination may contribute to the onset of schizophrenia. In the present study, three different age cohorts of mice, i.e. juvenile (3 weeks), young-adult (6 weeks) and middle-aged (8 months), were subjected to a 6-week diet containing 0.2% cuprizone (CPZ) to create an animal model of acute demyelination. Then, age-related vulnerability to CPZ-induced demyelination, behavioral outcomes, and myelination-related molecular biological changes were assessed. We demonstrated: (1) CPZ treatment led to more severe demyelination in juvenile and young-adult mice than in middle-aged mice in the corpus callosum, a region closely associated with the pathophysiology of schizophrenia; (2) the higher levels of demyelination in juvenile and young-adult mice were correlated with a greater reduction of myelin basic protein, more loss of CC-1-positive mature oligodendrocytes, and higher levels of astrocyte activation; and (3) CPZ treatment resulted in a more prominent exploratory behavior deficit in juvenile and young-adult mice than in middle-aged mice. Together, our data demonstrate an age-related vulnerability to demyelination with a concurrent behavioral deficit, providing supporting evidence for better understanding the susceptibility of the young to the onset of schizophrenia.
Noppe, Gauthier; Dufeys, Cécile; Buchlin, Patricia; Marquet, Nicolas; Castanares-Zapatero, Diego; Balteau, Magali; Hermida, Nerea; Bouzin, Caroline; Esfahani, Hrag; Viollet, Benoit; Bertrand, Luc; Balligand, Jean-Luc; Vanoverschelde, Jean-Louis; Beauloye, Christophe; Horman, Sandrine
Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-β1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.
Nassar, Mohammed A; Levato, Alessandra; Stirling, L Caroline; Wood, John N
Two voltage gated sodium channel α-subunits, Nav1.7 and Nav1.8, are expressed at high levels in nociceptor terminals and have been implicated in the development of inflammatory pain. Mis-expression of voltage-gated sodium channels by damaged sensory neurons has also been implicated in the development of neuropathic pain, but the role of Nav1.7 and Nav1.8 is uncertain. Here we show that deleting Nav1.7 has no effect on the development of neuropathic pain. Double knockouts of both Nav1.7 and Nav1.8 also develop normal levels of neuropathic pain, despite a lack of inflammatory pain symptoms and altered mechanical and thermal acute pain thresholds. These studies demonstrate that, in contrast to the highly significant role for Nav1.7 in determining inflammatory pain thresholds, the development of neuropathic pain does not require the presence of either Nav1.7 or Nav1.8 alone or in combination. PMID:16111501
Aso, Kazuyoshi; Tsuruhara, Akitoshi; Takagaki, Kentaro; Oki, Katsuyuki; Ota, Megumi; Nose, Yasuhiro; Tanemura, Hideki; Urushihata, Naoki; Sasanuma, Jinichi; Sano, Masayuki; Hirano, Atsuyuki; Aso, Rio; McGhee, Jerry R.; Fujihashi, Kohtaro
It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice. PMID:26840058
Lund, Troy C; Grange, Robert W; Lowe, Dawn A
The progression of Duchenne muscular dystrophy (DMD) is, in part, due to satellite cell senescence driven by high replicative pressure as these muscle stem cells repeatedly divide and fuse to damaged muscle fibers. We hypothesize that telomere shortening in satellite cells underlies their senescence. To test this hypothesis, we evaluated the diaphragm and a leg muscle from dystrophic mice of various ages for telomere dynamics. We found 30% telomere shortening in tibialis anterior muscles from 600-day-old mdx mice relative to age-matched wildtype mice. We also found a more severe shortening of telomere length in diaphragm muscles of old mdx mice. In those muscles, telomeres were shortened by approximately 15% and 40% in 100- and 600-day-old mdx mice, respectively. These findings indicate that satellite cells undergo telomere erosion, which may contribute to the inability of these cells to perpetually repair DMD muscle.
Golan, H; Stilman, M; Lev, V; Huleihel, M
Intrauterine inflammation is a major risk for offspring neurodevelopmental brain damage and may result in cognitive limitations and poor cognitive and perceptual outcomes. In the present study we tested the possibility that prenatal exposure to a high level of inflammatory factors may increase the risk for neurodegeneration in aging. The effect of systemic maternal inflammation (MI), induced by lipopolysaccharide (LPS) on offspring brain aging, was examined in 8 month old (adult) and 20 month old (aged) offspring mice. A significant effect of age was found in the distance and velocity of exploration in the open field in both groups. In addition, MI aged offspring covered longer distances and enter frequently to the center of the field compared with the aged control group. Although only little difference was found in the aged MI offspring compared with the control offspring, the overall profile of behavior of these mice differs from that of the control group, as detected by clustering analysis. The expression of the death-associated protein FAS-ligand and the amount of apoptotic cell death were examined in the brains of aged offspring. Similar levels of FAS-ligand expression and parallel density of apoptotic cells were detected in the brains of aged mice of control and MI groups. Altogether, moderate systemic MI was not found to increase the risk for cell death in the aged offspring; limited effect was found in mice profile of behavior.
Popovich, Irina G.; Zabezhinski, Mark A.; Panchenko, Andrei V.; Piskunova, Tatiana S.; Semenchenko, Anna V.; Tyndyk, Maragriata L.; Yurova, Maria N.; Anisimov, Vladimir N.
The effect of the constant illumination on the development of spontaneous tumors in female 129/Sv mice was investigated. Forty-six female 129/Sv mice starting from the age of 2 mo were kept under standard light/dark regimen [12 h light (70 lx):12hr dark; LD, control group], and 46 of 129/Sv mice were kept under constant illumination (24 h a day, 2,500 lx, LL) from the age of 5 mo until to natural death. The exposure to the LL regimen significantly accelerated body weight gain, increased body temperature as well as acceleration of age-related disturbances in estrous function, followed by significant acceleration of the development of the spontaneous uterine tumors in female 129/Sv mice. Total tumor incidence as well as a total number of total or malignant tumors was similar in LL and LD group (p > 0.05). The mice from the LL groups survived less than those from the LD group (χ2 = 8.5; p = 0.00351, log-rank test). According to the estimated parameters of the Cox’s regression model, constant light regimen increased the relative risk of death in female mice compared with the control (LD) group (p = 0.0041). The data demonstrate in the first time that the exposure to constant illumination was followed by the acceleration of aging and spontaneous uterine tumorigenesis in female 129/Sv mice. PMID:23656779
Grieb, Brian C.; Boyd, Kelli; Mitra, Ramkrishna; Eischen, Christine M.
Alterations of specific genes can modulate aging. Myc, a transcription factor that regulates the expression of many genes involved in critical cellular functions was shown to have a role in controlling longevity. Decreased expression of Myc inhibited many of the deleterious effects of aging and increased lifespan in mice. Without altering Myc expression, reduced levels of Mtbp, a recently identified regulator of Myc, limit Myc transcriptional activity and proliferation, while increased levels promote Myc-mediated effects. To determine the contribution of Mtbp to the effects of Myc on aging, we studied a large cohort of Mtbp heterozygous mice and littermate matched wild-type controls. Mtbp haploinsufficiency significantly increased longevity and maximal survival in mice. Reduced levels of Mtbp did not alter locomotor activity, litter size, or body size, but Mtbp heterozygous mice did exhibit elevated markers of metabolism, particularly in the liver. Mtbp+/− mice also had a significant delay in spontaneous cancer development, which was most prominent in the hematopoietic system, and an altered tumor spectrum compared to Mtbp+/+ mice. Therefore, the data suggest Mtbp is a regulator of longevity in mice that mimics some, but not all, of the properties of Myc in aging. PMID:27803394
Huitrón-Reséndiz, Salvador; Sánchez-Alavez, Manuel; Gallegos, Roger; Berg, Greta; Crawford, Elena; Giacchino, Jeannie L; Games, Dora; Henriksen, Steven J; Criado, José R
Recent studies demonstrated that mice overexpressing the human mutant beta-amyloid precursor protein (hbetaAPP; PDAPP mice) show age-independent and age-related deficits in spatial learning. We used behavioral and electrophysiological techniques to determine in young and aged PDAPP mice whether deficits in spatial learning also involve alterations in sleep-wake states, thermoregulation and motor activity. Consistent with earlier studies, young PDAPP mice exhibited selective age-independent deficits using spatial, but not random and serial strategies in the circular maze. Aged PDAPP mice exhibited deficits using all search strategies. The core body temperature (Tb) in young and aged PDAPP mice was significantly lower than in age-matched non-transgenic (non-Tg) littermates. During the dark period, the motor activity (LMA) was significantly increased in young PDAPP mice, but not in aged PDAPP mice. During the light period, young PDAPP mice showed a reduction in the generation of rapid-eye-movement (REM) sleep. In contrast, aged PDAPP mice exhibited a reduction in the amount of time spent in W and an increase in SWS during the light period. Aged PDAPP mice also showed an increase in the amount of time spent in W and a reduction in REM sleep during the dark period. Our findings support previous reports indicating deficits in spatial learning in young and aged PDAPP mice. These data also suggest that PDAPP mice exhibit age-independent and age-related deficits in neural mechanisms regulating visuospatial learning, the total amount and the circadian distribution of sleep-wake states, thermoregulation and motor activity.
Gurkar, Aditi U.; Niedernhofer, Laura J.
Aging is the primary risk factor for numerous chronic, debilitating diseases. These diseases impact quality of life of the elderly and consume a large portion of health care costs. The cost of age-related diseases will only increase as the world's population continues to live longer. Thus it would be advantageous to consider aging itself as a therapeutic target, potentially stemming multiple age-related diseases simultaneously. While logical, this is extremely challenging as the molecular mechanisms that drive aging are still unknown. Furthermore, clinical trials to treat aging are impractical. Even in preclinical models, testing interventions to extend healthspan in old age is lengthy and therefore costly. One approach to expedite aging studies is to take advantage of mouse strains that are engineered to age rapidly. These strains are genetically and phenotypically quite diverse. This review aims to offer a comparison of several of these strains to highlight their relative strengths and weaknesses as models of mammalian and more specifically human aging. Additionally, careful identification of commonalities amongst the strains may lead to the identification of fundamental pathways of aging. PMID:25617508
Garfinkel, Benjamin P; Arad, Shiri; Le, Phuong T; Bustin, Michael; Rosen, Clifford J; Gabet, Yankel; Orly, Joseph
Heterochromatin protein 1 binding protein 3 (HP1BP3) is a recently described histone H1-related protein with roles in chromatin structure and transcriptional regulation. To explore the potential physiological role of HP1BP3, we have previously described an Hp1bp3(-/-) mouse model with reduced postnatal viability and growth. We now find that these mice are proportionate dwarfs, with reduction in body weight, body length, and organ weight. In addition to their small size, microcomputed tomography analysis showed that Hp1bp3(-/-) mice present a dramatic impairment of their bone development and structure. By 3 weeks of age, mice of both sexes have severely impaired cortical and trabecular bone, and these defects persist into adulthood and beyond. Primary cultures of both osteoblasts and osteoclasts from Hp1bp3(-/-) bone marrow and splenocytes, respectively, showed normal differentiation and function, strongly suggesting that the impaired bone accrual is due to noncell autonomous systemic cues in vivo. One major endocrine pathway regulating both body growth and bone acquisition is the IGF regulatory system, composed of IGF-1, the IGF receptors, and the IGF-binding proteins (IGFBPs). At 3 weeks of age, Hp1bp3(-/-) mice exhibited a 60% reduction in circulating IGF-1 and a 4-fold increase in the levels of IGFBP-1 and IGFBP-2. These alterations were reflected in similar changes in the hepatic transcripts of the Igf1, Igfbp1, and Igfbp2 genes. Collectively, these results suggest that HP1BP3 plays a key role in normal growth and bone development by regulating transcription of endocrine IGF-1 components.
Ali, Sameh S; Young, Jared W; Wallace, Chelsea K; Gresack, Jodi; Jeste, Dilip V; Geyer, Mark A; Dugan, Laura L; Risbrough, Victoria B
Unregulated production of reactive oxygen species (ROS) is a marker of cellular and organismal aging linked to cognitive decline in humans and rodents. The sources of elevated ROS contributing to cognitive decline are unknown. Because NADPH oxidase (Nox) inhibition may prevent memory decline with age, we hypothesized that Nox and not mitochondrial sources of synaptic ROS production are linked to individual variance in cognitive performance in aged mice. Young (8 months) and aged (26 months) mice were tested in the novel object recognition task (NORT). Mitochondrial and Nox ROS production was assayed in isolated synaptosomes using spin trapping electron paramagnetic resonance (EPR) spectroscopy. Aged mice exhibited variance in NORT performance, with some performing similar to young mice while others exhibited poorer short-term memory. EPR studies indicated that Nox rather than mitochondria was the major ROS source at the synapse, and Nox-induced but not mitochondrial-induced ROS levels correlated with NORT performance in aged mice. Our findings support the hypothesis that variance in Nox-specific synaptic ROS production may predict short-term memory deficits with age.
Turner, Michael J; Guderian, Sophie; Wikstrom, Erik A; Huot, Joshua R; Peck, Bailey D; Arthur, Susan T; Marino, Joseph S; Hubbard-Turner, Tricia
We assessed the impact of differing physical activity levels throughout the lifespan, using a musculoskeletal injury model, on the age-related changes in left ventricular (LV) parameters in active mice. Forty male mice (CBA/J) were randomly placed into one of three running wheel groups (transected CFL group, transected ATFL/CFL group, SHAM group) or a SHAM Sedentary group (SHAMSED). Before surgery and every 6 weeks after surgery, LV parameters were measured under 2.5 % isoflurane inhalation. Group effects for daily distance run was significantly greater for the SHAM and lesser for the ATLF/CFL mice (p = 0.013) with distance run decreasing with age for all mice (p < 0.0001). Beginning at 6 months of age, interaction (group × age) was noted with LV posterior wall thickness-to-radius ratios (h/r) where h/r increased with age in the ATFL/CFL and SHAMSED mice while the SHAM and CFL mice exhibited decreased h/r with age (p = 0.0002). Passive filling velocity (E wave) was significantly greater in the SHAM mice and lowest for the ATFL/CFL and SHAMSED mice (p < 0.0001) beginning at 9 months of age. Active filling velocity (A wave) was not different between groups (p = 0.10). Passive-to-active filling velocity ratio (E/A ratio) was different between groups (p < 0.0001), with higher ratios for the SHAM mice and lower ratios for the ATFL/CFL and SHAMSED mice in response to physical activity beginning at 9 months of age. Passive-to-active filling velocity ratio decreased with age (p < 0.0001). Regular physical activity throughout the lifespan improved LV structure, passive filling velocity, and E/A ratio by 6 to 9 months of age and attenuated any negative alterations throughout the second half of life. The diastolic filling differences were found to be significantly related to the amount of activity performed by 9 months and at the end of the lifespan.
Hong, Wei; Xu, Xiao-ya; Qiu, Zhao-hui; Gao, Jian-jun; Wei, Zhan-ying; Zhen, Li; Zhang, Xiao-li; Ye, Zhi-bing
Aim: Apolipoprotein E (ApoE) plays an important role in the transport and metabolism of lipids. Recent studies show that bone mass is increased in young apoE−/− mice. In this study we investigated the bone phenotype and metabolism in aged apoE−/− mice. Methods: Femurs and tibias were collected from 18- and 72-week-old apoE−/− mice and their age-matched wild-type (WT) littermates, and examined using micro-CT and histological analysis. Serum levels of total cholesterol, oxidized low-density lipoprotein (ox-LDL) and bone turnover markers were measured. Cultured bone mesenchymal stem cells (BMSCs) from tibias and femurs of 18-week-old apoE−/− mice were used in experiments in vitro. The expression levels of Sirt1 and Runx2 in bone tissue and BMSCs were measured using RT-PCR and Western blot analysis. Results: Compared with age-matched WT littermates, young apoE−/− mice exhibited high bone mass with increased bone formation, accompanied by higher serum levels of bone turnover markers OCN and TRAP5b, and higher expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. In contrast, aged apoE−/− mice showed reduced bone formation and lower bone mass relative to age-matched WT mice, accompanied by lower serum OCN levels, and markedly reduced expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. After BMSCs were exposed to ox-LDL (20 μg/mL), the expression of Sirt1 and Runx2 proteins was significantly increased at 12 h, and then decreased at 72 h. Treatment with the Sirt1 inhibitor EX527 (10 μmol/L) suppressed the expression of Runx2, ALP and OCN in BMSCs. Conclusion: In contrast to young apoE−/− mice, aged apoE−/− mice showe lower bone mass than age-matched WT mice. Long-lasting exposure to ox-LDL decreases the expression of Sirt1 and Runx2 in BMSCs, which may explain the decreased bone formation in aged apoE−/− mice. PMID:26592520
Jongbloed, Franny; de Bruin, Ron W. F.; Pennings, Jeroen L. A.; Payán-Gómez, César; van den Engel, Sandra; van Oostrom, Conny T.; de Bruin, Alain; Hoeijmakers, Jan H. J.; van Steeg, Harry; IJzermans, Jan N. M.; Dollé, Martijn E. T.
Ischemia-reperfusion injury (IRI) is inevitable during kidney transplantation leading to oxidative stress and inflammation. We previously reported that preoperative fasting in young-lean male mice protects against IRI. Since patients are generally of older age with morbidities possibly leading to a different response to fasting, we investigated the effects of preoperative fasting on renal IRI in aged-overweight male and female mice. Male and female F1-FVB/C57BL6-hybrid mice, average age 73 weeks weighing 47.2 grams, were randomized to preoperative ad libitum feeding or 3 days fasting, followed by renal IRI. Body weight, kidney function and survival of the animals were monitored until day 28 postoperatively. Kidney histopathology was scored for all animals and gene expression profiles after fasting were analyzed in kidneys of young and aged male mice. Preoperative fasting significantly improved survival after renal IRI in both sexes compared with normal fed mice. Fasted groups had a better kidney function shown by lower serum urea levels after renal IRI. Histopathology showed less acute tubular necrosis and more regeneration in kidneys from fasted mice. A mRNA analysis indicated the involvement of metabolic processes including fatty acid oxidation and retinol metabolism, and the NRF2-mediated stress response. Similar to young-lean, healthy male mice, preoperative fasting protects against renal IRI in aged-overweight mice of both genders. These findings suggest a general protective response of fasting against renal IRI regardless of age, gender, body weight and genetic background. Therefore, fasting could be a non-invasive intervention inducing increased oxidative stress resistance in older and overweight patients as well. PMID:24959849
Lim, Jinhwan; Nakamura, Brooke N.; Mohar, Isaac; Kavanagh, Terrance J.
Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hy