Genetic Biomarkers for ALS Disease in Transgenic SOD1G93A Mice

PubMed Central

Calvo, Ana C.; Manzano, Raquel; Atencia-Cibreiro, Gabriela; Oliván, Sara; Muñoz, María J.; Zaragoza, Pilar; Cordero-Vázquez, Pilar; Esteban-Pérez, Jesús; García-Redondo, Alberto; Osta, Rosario

2012-01-01

The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies. PMID:22412900

Preferential motor unit loss in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

PubMed Central

Hegedus, J; Putman, C T; Tyreman, N; Gordon, T

2008-01-01

The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast-twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1G93A and SOD1WT mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1G93A compared to SOD1WT mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1G93A mice (38 ± 7) compared with SOD1WT controls (95 ± 12); this was attributed to a lower proportion of the most forceful fast-fatigable (FF) MU in SOD1G93A mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross-sectional area (CSA) of serial sections of TA muscles stained with anti-neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1G93A, which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity-dependent fast-to-slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our

Muscle Expression of SOD1G93A Triggers the Dismantlement of Neuromuscular Junction via PKC-Theta.

PubMed

Dobrowolny, Gabriella; Martini, Martina; Scicchitano, Bianca Maria; Romanello, Vanina; Boncompagni, Simona; Nicoletti, Carmine; Pietrangelo, Laura; De Panfilis, Simone; Catizone, Angela; Bouchè, Marina; Sandri, Marco; Rudolf, Rüdiger; Protasi, Feliciano; Musarò, Antonio

2018-04-20

Neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve. Several chronic pathologies such as aging and neurodegenerative diseases, including muscular dystrophy and amyotrophic lateral sclerosis, display altered NMJ and functional denervation. However, the triggers and the molecular mechanisms underlying the dismantlement of NMJ remain unclear. Here we provide evidence that perturbation in redox signaling cascades, induced by muscle-specific accumulation of mutant SOD1 G93A in transgenic MLC/SOD1 G93A mice, is causally linked to morphological alterations of the neuromuscular presynaptic terminals, high turnover rate of acetylcholine receptor, and NMJ dismantlement. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 G93A revealed a causal link between protein kinase Cθ (PKCθ) activation and NMJ disintegration. The study discloses the molecular mechanism that triggers functional denervation associated with the toxic activity of muscle SOD1 G93A expression and suggests the possibility of developing a new strategy to counteract age- and pathology-associated denervation based on pharmacological inhibition of PKCθ activity. Collectively, these data indicate that muscle-specific accumulation of oxidative damage can affect neuromuscular communication and induce NMJ dismantlement through a PKCθ-dependent mechanism. Antioxid. Redox Signal. 28, 1105-1119.

Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats

PubMed Central

Satriotomo, Irawan; Grebe, Ashley M.

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model (SOD1G93A), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: Gq-protein-coupled 5-HT2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: Gs-protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male SOD1G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1G93A rats (∼30% survival; p < 0.001), BDNF and phosphorylated ERK expression were increased in spared phrenic motor neurons (p < 0.05), consistent with increased Q-pathway contributions to pLTF. Our results increase understanding of respiratory plasticity and its potential to preserve/restore breathing capacity in ALS. SIGNIFICANCE STATEMENT Since neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), end life via respiratory failure, the ability to harness respiratory motor plasticity to improve breathing

In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

PubMed

Bonifacino, Tiziana; Cattaneo, Luca; Gallia, Elena; Puliti, Aldamaria; Melone, Marcello; Provenzano, Francesca; Bossi, Simone; Musante, Ilaria; Usai, Cesare; Conti, Fiorenzo; Bonanno, Giambattista; Milanese, Marco

2017-09-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1 G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1 G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1 G93A mice with reduced expression of mGluR5 (SOD1 G93A Grm5 -/+ ) by crossing the SOD1 G93A mutant mouse with the mGluR5 heterozigous Grm5 -/+ mouse. SOD1 G93A Grm5 -/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca 2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1 G93A Grm5 -/+ mice. Unexpectedly, only male SOD1 G93A Grm5 -/+ mice showed improved motor skills during disease progression vs. SOD1 G93A mice, while SOD1 G93A Grm5 -/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

PubMed Central

Rudnick, Noam D.; Griffey, Christopher J.; Guarnieri, Paolo; Gerbino, Valeria; Wang, Xueyong; Piersaint, Jason A.; Tapia, Juan Carlos; Rich, Mark M.; Maniatis, Tom

2017-01-01

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression. PMID:28904095

Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1G93A mice

PubMed Central

2013-01-01

Background Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disorder characterized by selective motor neuron death in the spinal cord, brainstem, and motor cortex. Neuroinflammation is one of several pathological causes of degenerating motor neurons and is induced by activated microglial cells and astrocytes in ALS. Scolopendra subspinipes mutilans (SSM) is utilized in traditional Chinese and Korean medicine for the treatment of a variety of diseases, such as cancer, apoplexy, and epilepsy. However, the mechanisms underlying the effects of SSM are currently unclear, even though SSM increases immune and antibiotic activity. Methods To determine the effects of SSM on symptomatic hSOD1G93A transgenic mice, SSM (2.5 μℓ/g) was injected bilaterally at the Zusanli (ST36) acupoint three times per week for two weeks. The effects of SSM treatment on anti-neuroinflammation in the brainstem and spinal cord of hSOD1G93A mice were assessed via Nissl and Fluoro-Jade B (FJB) staining, and immunohistochemistry using Iba-1, CD14, HO1, and NQO1 proteins was evaluated by Western blotting. Results In this study, we investigated whether SSM affects neuroinflammation in the spinal cord of symptomatic hSOD1G93A transgenic mice. We found that SSM treatment attenuated the loss of motor neurons and reduced the activation of microglial cells and astrocytes. Furthermore, we demonstrated that SSM administration in this animal model of ALS suppressed oxidative stress in the brainstem and spinal cord by 1.6- and 1.8-fold, respectively. Conclusions Our findings suggest that SSM, which has previously been used in complementary and alternative medicine (CAM), might also be considered as an anti-neuroinflammatory therapy for neurodegenerative diseases. PMID:24168240

Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1(G93A) mice.

PubMed

Cai, MuDan; Choi, Sun-Mi; Song, Bong Keun; Son, Ilhong; Kim, Sungchul; Yang, Eun Jin

2013-10-29

Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disorder characterized by selective motor neuron death in the spinal cord, brainstem, and motor cortex. Neuroinflammation is one of several pathological causes of degenerating motor neurons and is induced by activated microglial cells and astrocytes in ALS.Scolopendra subspinipes mutilans (SSM) is utilized in traditional Chinese and Korean medicine for the treatment of a variety of diseases, such as cancer, apoplexy, and epilepsy. However, the mechanisms underlying the effects of SSM are currently unclear, even though SSM increases immune and antibiotic activity. To determine the effects of SSM on symptomatic hSOD1G93A transgenic mice, SSM (2.5 μℓ/g) was injected bilaterally at the Zusanli (ST36) acupoint three times per week for two weeks. The effects of SSM treatment on anti-neuroinflammation in the brainstem and spinal cord of hSOD1G93A mice were assessed via Nissl and Fluoro-Jade B (FJB) staining, and immunohistochemistry using Iba-1, CD14, HO1, and NQO1 proteins was evaluated by Western blotting. In this study, we investigated whether SSM affects neuroinflammation in the spinal cord of symptomatic hSOD1G93A transgenic mice. We found that SSM treatment attenuated the loss of motor neurons and reduced the activation of microglial cells and astrocytes. Furthermore, we demonstrated that SSM administration in this animal model of ALS suppressed oxidative stress in the brainstem and spinal cord by 1.6- and 1.8-fold, respectively. Our findings suggest that SSM, which has previously been used in complementary and alternative medicine (CAM), might also be considered as an anti-neuroinflammatory therapy for neurodegenerative diseases.

Human Glial-Restricted Progenitor Transplantation into Cervical Spinal Cord of the SOD1G93A Mouse Model of ALS

PubMed Central

Lepore, Angelo C.; O'Donnell, John; Kim, Andrew S.; Williams, Timothy; Tuteja, Alicia; Rao, Mahendra S.; Kelley, Linda L.; Campanelli, James T.; Maragakis, Nicholas J.

2011-01-01

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1G93A rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1G93A rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1G93A mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1G93A mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation. PMID:21998733

ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics.

PubMed

Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R; Zhu, Brian Z; Shissler, Susannah C; Korotkov, Konstantin V; Hayward, Lawrence J; Kasarskis, Edward J; Zhu, Haining

2016-10-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.

Imaging of glial cell morphology, SOD1 distribution and elemental composition in the brainstem and hippocampus of the ALS hSOD1G93A rat.

PubMed

Stamenković, Stefan; Dučić, Tanja; Stamenković, Vera; Kranz, Alexander; Andjus, Pavle R

2017-08-15

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor and cognitive domains of the CNS. Mutations in the Cu,Zn-superoxide dismutase (SOD1) cause 20% of familial ALS and provoke formation of intracellular aggregates and copper and zinc unbinding, leading to glial activation and neurodegeneration. Therefore, we investigated glial cell morphology, intracellular SOD1 distribution, and elemental composition in the brainstem and hippocampus of the hSOD1 G93A transgenic rat model of ALS. Immunostaining for astrocytes, microglia and SOD1 revealed glial proliferation and progressive tissue accumulation of SOD1 in both brain regions of ALS rats starting already at the presymptomatic stage. Glial cell morphology analysis in the brainstem of ALS rats revealed astrocyte activation occurring before disease symptoms onset, followed by activation of microglia. Hippocampal ALS astrocytes exhibited an identical reactive profile, while microglial morphology was unchanged. Additionally, ALS brainstem astrocytes demonstrated progressive SOD1 accumulation in the cell body and processes, while microglial SOD1 levels were reduced and its distribution limited to distal cell processes. In the hippocampus both glial cell types exhibited SOD1 accumulation in the cell body. X-ray fluorescence imaging revealed decreased P and increased Ca, Cl, K, Ni, Cu and Zn in the brainstem, and higher levels of Cl, Ni and Cu, but lower levels of Zn in the hippocampus of symptomatic ALS rats. These results bring new insights into the glial response during disease development and progression in motor as well as in non-motor CNS structures, and indicate disturbed tissue elemental homeostasis as a prominent hallmark of disease pathology. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Soma size and Cav1.3 channel expression in vulnerable and resistant motoneuron populations of the SOD1G93A mouse model of ALS

PubMed Central

Shoenfeld, Liza; Westenbroek, Ruth E.; Fisher, Erika; Quinlan, Katharina A.; Tysseling, Vicki M.; Powers, Randall K.; Heckman, Charles J.; Binder, Marc D.

2014-01-01

Abstract Although the loss of motoneurons is an undisputed feature of amyotrophic lateral sclerosis (ALS) in man and in its animal models (SOD1 mutant mice), how the disease affects the size and excitability of motoneurons prior to their degeneration is not well understood. This study was designed to test the hypothesis that motoneurons in mutant SOD1G93A mice exhibit an enlargement of soma size (i.e., cross‐sectional area) and an increase in Cav1.3 channel expression at postnatal day 30, well before the manifestation of physiological symptoms that typically occur at p90 (Chiu et al. 1995). We made measurements of spinal and hypoglossal motoneurons vulnerable to degeneration, as well as motoneurons in the oculomotor nucleus that are resistant to degeneration. Overall, we found that the somata of motoneurons in male SOD1G93A mutants were larger than those in wild‐type transgenic males. When females were included in the two groups, significance was lost. Expression levels of the Cav1.3 channels were not differentiated by genotype, sex, or any interaction of the two. These results raise the intriguing possibility of an interaction between male sex steroid hormones and the SOD1 mutation in the etiopathogenesis of ALS. PMID:25107988

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity

PubMed Central

Weisberg, Sarah J.; Lyakhovetsky, Roman; Werdiger, Ayelet-chen; Gitler, Aaron D.; Soen, Yoav; Kaganovich, Daniel

2012-01-01

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability. PMID:22967507

Effects of tongue force training on bulbar motor function in the female SOD1-G93A rat model of Amyotrophic Lateral Sclerosis

PubMed Central

Ma, Delin; Shuler, Jeffrey M.; Kumar, Aishwarya; Stanford, Quincy R.; Tungtur, Sudheer; Nishimune, Hiroshi; Stanford, John A.

2016-01-01

Background The use of exercise in Amyotrophic Lateral Sclerosis (ALS) is controversial. Although moderate exercise appears to be beneficial for limb muscles in ALS, the effects of exercise on bulbar muscles such as the tongue have not been studied. Objective The aim of this study was to determine the effects of tongue force training on bulbar motor function in the SOD1-G93A rat model of ALS. Methods We compared the effects of tongue force training on bulbar motor function and neuromuscular junction (NMJ) innervation in female SOD1-G93A rats and age-matched female wild-type controls. Half of each group underwent afternoon tongue force training sessions, while all rats were tested under minimal force conditions in the mornings. Results Tongue force did not differ between the SOD1-G93A rats and healthy controls during the morning testing sessions, nor was it affected by training. Surprisingly, decreases in tongue motility, the number of licks per session, and body weight were greater in the tongue force-trained SOD1-G93A rats. Forelimb grip force, survival, and denervation of the genioglossus muscle did not differ between the trained and untrained SOD1-G93A rats. Genioglossus innervation was correlated with changes in tongue force but not tongue motility in SOD1-G93A rats at end stage. Conclusions The results indicate a potential deleterious effect of tongue force training on tongue motility in female SOD1-G93A rats. The lack of relationship between genioglossus innervation and tongue motility suggest that factors other than lower motor neuron integrity likely accounted for this effect. PMID:27573800

Presymptomatic electrophysiological tests predict clinical onset and survival in SOD1(G93A) ALS mice.

PubMed

Mancuso, Renzo; Osta, Rosario; Navarro, Xavier

2014-12-01

We assessed the predictive value of electrophysiological tests as a marker of clinical disease onset and survival in superoxide-dismutase 1 (SOD1)(G93A) mice. We evaluated the accuracy of electrophysiological tests in differentiating transgenic versus wild-type mice. We made a correlation analysis of electrophysiological parameters and the onset of symptoms, survival, and number of spinal motoneurons. Presymptomatic electrophysiological tests show great accuracy in differentiating transgenic versus wild-type mice, with the most sensitive parameter being the tibialis anterior compound muscle action potential (CMAP) amplitude. The CMAP amplitude at age 10 weeks correlated significantly with clinical disease onset and survival. Electrophysiological tests increased their survival prediction accuracy when evaluated at later stages of the disease and also predicted the amount of lumbar spinal motoneuron preservation. Electrophysiological tests predict clinical disease onset, survival, and spinal motoneuron preservation in SOD1(G93A) mice. This is a methodological improvement for preclinical studies. © 2014 Wiley Periodicals, Inc.

Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1G93A mouse model of amyotrophic lateral sclerosis

PubMed Central

Lerman, Bruce J; Hoffman, Eric P; Sutherland, Margaret L; Bouri, Khaled; Hsu, Daniel K; Liu, Fu-Tong; Rothstein, Jeffrey D; Knoblach, Susan M

2012-01-01

Galectins are pleiotropic carbohydrate-binding lectins involved in inflammation, growth/differentiation, and tissue remodeling. The functional role of galectins in amyotrophic lateral sclerosis (ALS) is unknown. Expression studies revealed increases in galectin-1 mRNA and protein in spinal cords from SOD1G93A mice, and in galectin-3 and -9 mRNAs and proteins in spinal cords of both SOD1G93A mice and sporadic ALS patients. As the increase in galectin-3 appeared in early presymptomatic stages and increased progressively through to end stage of disease in the mouse, it was selected for additional study, where it was found to be mainly expressed by microglia. Galectin-3 antagonists are not selective and do not readily cross the blood–brain barrier; therefore, we generated SOD1G93A/Gal-3−/− transgenic mice to evaluate galectin-3 deletion in a widely used mouse model of ALS. Disease progression, neurological symptoms, survival, and inflammation were assessed to determine the effect of galectin-3 deletion on the SOD1G93A disease phenotype. Galectin-3 deletion did not change disease onset, but resulted in more rapid progression through functionally defined disease stages, more severely impaired neurological symptoms at all stages of disease, and expiration, on average, 25 days earlier than SOD1G93A/Gal-3+/+ cohorts. In addition, microglial staining, as well as TNF-α, and oxidative injury were increased in SOD1G93A/Gal-3−/− mice compared with SOD1G93A/Gal-3+/+ cohorts. These data support an important functional role for microglial galectin-3 in neuroinflammation during chronic neurodegenerative disease. We suggest that elevations in galectin-3 by microglia as disease progresses may represent a protective, anti-inflammatory innate immune response to chronic motor neuron degeneration. PMID:23139902

Palmitoylation of superoxide dismutase 1 (SOD1) is increased for familial amyotrophic lateral sclerosis-linked SOD1 mutants.

PubMed

Antinone, Sarah E; Ghadge, Ghanashyam D; Lam, Tukiet T; Wang, Lijun; Roos, Raymond P; Green, William N

2013-07-26

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.

Early Detection of Motor Dysfunction in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis (ALS) Using Home Cage Running Wheels

PubMed Central

Bennett, Ellen J.; Mead, Richard J.; Azzouz, Mimoun; Shaw, Pamela J.; Grierson, Andrew J.

2014-01-01

The SOD1G93A mouse has been used since 1994 for preclinical testing in amyotrophic lateral sclerosis (ALS). Despite recent genetic advances in our understanding of ALS, transgenic mice expressing mutant SOD1 remain the best available, and most widely used, vertebrate model of the disease. We previously described an optimised and rapid approach for preclinical studies in the SOD1G93A mouse. Here we describe improvements to this approach using home cage running wheels to obtain daily measurements of motor function, with minimal intervention. We show that home cage running wheels detect reductions in motor function at a similar time to the rotarod test, and that the data obtained are less variable allowing the use of smaller groups of animals to obtain satisfactory results. This approach refines use of the SOD1G93A model, and reduces the number of animals undergoing procedures of substantial severity, two central principles of the 3Rs (replacement, reduction and refinement of animal use in research). The small group sizes and rapid timescales enable affordable large-scale therapeutic pre-screening in the SOD1G93A mouse, as well as rapid validation of published positive effects in a second laboratory, one of the major stumbling blocks in ALS preclinical therapy development. PMID:25268710

A Cystine-Rich Whey Supplement (Immunocal®) Delays Disease Onset and Prevents Spinal Cord Glutathione Depletion in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

PubMed Central

Ross, Erika K.; Winter, Aimee N.; Wilkins, Heather M.; Sumner, Whitney A.; Duval, Nathan; Patterson, David; Linseman, Daniel A.

2014-01-01

Depletion of the endogenous antioxidant, glutathione (GSH), underlies progression of the devastating neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Thus, strategies aimed at elevating GSH may yield new therapeutics for ALS. Here, we investigated the effects of a unique non-denatured whey protein supplement, Immunocal®, in the transgenic Gly position 93 to Ala (G93A) mutant hSOD1 (hSOD1G93A) mouse model of ALS. Immunocal® is rich in the GSH precursor, cystine, and is therefore capable of bolstering GSH content. Transgenic hSOD1G93A mice receiving Immunocal® displayed a significant delay in disease onset compared to untreated hSOD1G93A controls. Additionally, Immunocal® treatment significantly decreased the rate of decline in grip strength and prevented disease-associated reductions in whole blood and spinal cord tissue GSH levels in end-stage hSOD1G93A mice. However, Immunocal® did not extend survival, likely due to its inability to preserve the mitochondrial GSH pool in spinal cord. Combination treatment with Immunocal® and the anti-glutamatergic compound, riluzole, delayed disease onset and extended survival in hSOD1G93A mice. These findings demonstrate that sustaining tissue GSH with Immunocal® only modestly delays disease onset and slows the loss of skeletal muscle strength in hSOD1G93A mice. Moreover, the inability of Immunocal® to rescue mitochondrial GSH in spinal cord provides a possible mechanism for its lack of effect on survival and is a limiting factor in the potential utility of this supplement as a therapeutic for ALS. PMID:26785244

The Effects of Bee Venom Acupuncture on the Central Nervous System and Muscle in an Animal hSOD1G93A Mutant

PubMed Central

Cai, MuDan; Choi, Sun-Mi; Yang, Eun Jin

2015-01-01

Amyotrophic lateral sclerosis (ALS) is caused by the degeneration of lower and upper motor neurons, leading to muscle paralysis and respiratory failure. However, there is no effective drug or therapy to treat ALS. Complementary and alternative medicine (CAM), including acupuncture, pharmacopuncture, herbal medicine, and massage is popular due to the significant limitations of conventional therapy. Bee venom acupuncture (BVA), also known as one of pharmacopunctures, has been used in Oriental medicine to treat inflammatory diseases. The purpose of this study is to investigate the effect of BVA on the central nervous system (CNS) and muscle in symptomatic hSOD1G93A transgenic mice, an animal model of ALS. Our findings show that BVA at ST36 enhanced motor function and decreased motor neuron death in the spinal cord compared to that observed in hSOD1G93A transgenic mice injected intraperitoneally (i.p.) with BV. Furthermore, BV treatment at ST36 eliminated signaling downstream of inflammatory proteins such as TLR4 in the spinal cords of symptomatic hSOD1G93A transgenic mice. However, i.p. treatment with BV reduced the levels of TNF-α and Bcl-2 expression in the muscle hSOD1G93A transgenic mice. Taken together, our findings suggest that BV pharmacopuncture into certain acupoints may act as a chemical stimulant to activate those acupoints and subsequently engage the endogenous immune modulatory system in the CNS in an animal model of ALS. PMID:25781653

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2011-05-15

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

Expression and Distribution of Arylsulfatase B are Closely Associated with Neuron Death in SOD1 G93A Transgenic Mice.

PubMed

Zhang, Jie; Liang, Huiting; Zhu, Lei; Gan, Weiming; Tang, Chunyan; Li, Jiao; Xu, Renshi

2018-02-01

The known proteins only explained the partial pathogenesis of amyotrophic lateral sclerosis (ALS). Therefore, this study aimed to search the novel proteins possibly involved in ALS. In this study, we analyzed the expression and distribution of the candidate protein arylsulfatase B (ARSB) in the different segments, anatomic regions, and neural cells of spinal cord at the different stages of the wild-type and [Cu/Zn] superoxide dismutase 1 (SOD1) G93A transgenic mice using the fluorescent immunohistochemistry and the western blot. The results revealed that the ARSB was extensively expressed and distributed in the entire spinal cord; the expression and distribution of ARSB was significantly different in the different regions of spinal cord, the anterior horn of gray matter (AHGM) was significantly more than that in the posterior horn of gray matter (PHGM) and significantly more than that in the central canal, and ARSB was mainly distributed in the microglia and neuron cells in the wild-type mice. The expression of ARSB significantly increased in other anatomic regions besides the thoracic PHGM, significantly decreased at the progression stage, occurred in the redistribution from the AHGM and the PHGM to the central canal at the onset and progression stages, and no any alteration of ARSB expression and distribution occurred between the different neural cells in the SOD1 G93A mice compared with the wild-type mice. The increase of ARSB expression and distribution followed with the increased of neuron death. Our data suggested that the abnormal expression and distribution of ARSB were closely associated with the neuron death in the SOD1 G93A transgenic mice.

State of the field: An informatics-based systematic review of the SOD1-G93A amyotrophic lateral sclerosis transgenic mouse model

PubMed Central

Kim, Renaid B.; Irvin, Cameron W.; Tilva, Keval R.; Mitchell, Cassie S.

2016-01-01

Numerous sub-cellular through system-level disturbances have been identified in over 1300 articles examining the superoxide dismutase-1 guanine 93 to alanine (SOD1-G93A) transgenic mouse amyotrophic lateral sclerosis (ALS) pathophysiology. Manual assessment of such a broad literature base is daunting. We performed a comprehensive informatics-based systematic review or ‘field analysis’ to agnostically compute and map the current state of the field. Text mining of recaptured articles was used to quantify published data topic breadth and frequency. We constructed a nine-category pathophysiological function-based ontology to systematically organize and quantify the field's primary data. Results demonstrated that the distribution of primary research belonging to each category is: systemic measures an motor function, 59%; inflammation, 46%; cellular energetics, 37%; proteomics, 31%; neural excitability, 22%; apoptosis, 20%; oxidative stress, 18%; aberrant cellular chemistry, 14%; axonal transport, 10%. We constructed a SOD1-G93A field map that visually illustrates and categorizes the 85% most frequently assessed sub-topics. Finally, we present the literature-cited significance of frequently published terms and uncover thinly investigated areas. In conclusion, most articles individually examine at least two categories, which is indicative of the numerous underlying pathophysiological interrelationships. An essential future path is examination of cross-category pathophysiological interrelationships and their co-correspondence to homeostatic regulation and disease progression. PMID:25998063

Redox susceptibility of SOD1 mutants is associated with the differential response to CCS over-expression in vivo.

PubMed

Son, Marjatta; Fu, Qiao; Puttaparthi, Krishna; Matthews, Christina M; Elliott, Jeffrey L

2009-04-01

Over-expression of CCS in G93A SOD1 mice accelerates neurological disease and enhances mitochondrial pathology. We studied the effect of CCS over-expression in transgenic mice expressing G37R, G86R or L126Z SOD1 mutations in order to understand factors which influence mitochondrial dysfunction. Over-expression of CCS markedly decreased survival and produced mitochondrial vacuolation in G37R SOD1 mice but not in G86R or L126Z SOD1 mice. Moreover, CCS/G37R SOD1 spinal cord showed specific reductions in mitochondrial complex IV subunits consistent with an isolated COX deficiency, while no such reductions were detected in CCS/G86R or CCS/L126Z SOD1 mice. CCS over-expression increased the ratio of reduced to oxidized SOD1 monomers in the spinal cords of G37R SOD1 as well as G93A SOD1 mice, but did not influence the redox state of G86R or L126Z SOD1 monomers. The effects of CCS on disease are SOD1 mutation dependent and correlate with SOD1 redox susceptibility.

Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

PubMed

Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

2014-02-01

Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

Identification of compounds protective against G93A-SOD1 toxicity for the treatment of amyotrophic lateral sclerosis.

PubMed

Benmohamed, Radhia; Arvanites, Anthony C; Kim, Jinho; Ferrante, Robert J; Silverman, Richard B; Morimoto, Richard I; Kirsch, Donald R

2011-03-01

The underlying cause of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, remains unknown. However, there is strong evidence that one pathophysiological mechanism, toxic protein misfolding and/or aggregation, may trigger motor neuron dysfunction and loss. Since the clinical and pathological features of sporadic and familial ALS are indistinguishable, all forms of the disease may be better understood and ultimately treated by studying pathogenesis and therapy in models expressing mutant forms of SOD1. We developed a cellular model in which cell death depended on the expression of G93A-SOD1, a mutant form of superoxide dismutase found in familial ALS patients that produces toxic protein aggregates. This cellular model was optimized for high throughput screening to identify protective compounds from a >50,000 member chemical library. Three novel chemical scaffolds were selected for further study following screen implementation, counter-screening and secondary testing, including studies with purchased analogs. All three scaffolds blocked SOD1 aggregation in high content screening assays and data on the optimization and further characterization of these compounds will be reported separately. These data suggest that optimization of these chemicals scaffolds may produce therapeutic candidates for ALS patients.

Structures of the G85R Variant of SOD1 in Familial Amyotrophic Lateral Sclerosis

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

Cao, Xiaohang; Antonyuk, Svetlana V.; Seetharaman, Sai V.

2008-07-21

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ionmore » deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.« less

SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system.

PubMed

Watanabe, Seiji; Ageta-Ishihara, Natsumi; Nagatsu, Shinji; Takao, Keizo; Komine, Okiru; Endo, Fumito; Miyakawa, Tsuyoshi; Misawa, Hidemi; Takahashi, Ryosuke; Kinoshita, Makoto; Yamanaka, Koji

2014-08-29

Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system. We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1G93A-H) or a low expression line with a milder phenotype (SOD1G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1G93A-H transgenic mice partly because their HSP70i level had peaked out. The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis.

PubMed

Zhu, Cheng; Beck, Matthew V; Griffith, Jack D; Deshmukh, Mohanish; Dokholyan, Nikolay V

2018-05-01

Aberrant accumulation of misfolded Cu, Zn superoxide dismutase (SOD1) is a hallmark of SOD1-associated amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disease. While recent discovery of nonnative trimeric SOD1-associated neurotoxicity has suggested a potential pathway for motor neuron impairment, it is yet unknown whether large, insoluble aggregates are cytotoxic. Here we designed SOD1 mutations that specifically stabilize either the fibrillar form or the trimeric state of SOD1. The designed mutants display elevated populations of fibrils or trimers correspondingly, as demonstrated by gel filtration chromatography and electron microscopy. The trimer-stabilizing mutant, G147P, promoted cell death, even more potently in comparison with the aggressive ALS-associated mutants A4V and G93A. In contrast, the fibril-stabilizing mutants, N53I and D101I, positively impacted the survival of motor neuron-like cells. Hence, we conclude the SOD1 oligomer and not the mature form of aggregated fibril is critical for the neurotoxic effects in the model of ALS. The formation of large aggregates is in competition with trimer formation, suggesting that aggregation may be a protective mechanism against formation of toxic oligomeric intermediates.

Multiple intracerebroventricular injections of human umbilical cord mesenchymal stem cells delay motor neurons loss but not disease progression of SOD1G93A mice.

PubMed

Sironi, Francesca; Vallarola, Antonio; Violatto, Martina Bruna; Talamini, Laura; Freschi, Mattia; De Gioia, Roberta; Capelli, Chiara; Agostini, Azzurra; Moscatelli, Davide; Tortarolo, Massimo; Bigini, Paolo; Introna, Martino; Bendotti, Caterina

2017-12-01

Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1β) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression. Copyright © 2017. Published by Elsevier B.V.

Axonal Mitochondrial Clusters Containing Mutant SOD1 in Transgenic Models of ALS

PubMed Central

Lepanto, Paola; Elizondo, Victoria; Horjales, Sofia; Palacios, Florencia; Martinez-Palma, Laura; Marin, Monica; Beckman, Joseph S.

2009-01-01

Abstract We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS. Antioxid. Redox Signal. 11, 1535–1545. PMID:19344250

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

PubMed

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

2005-09-15

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

Tempol Moderately Extends Survival in a hSOD1G93A ALS Rat Model by Inhibiting Neuronal Cell Loss, Oxidative Damage and Levels of Non-Native hSOD1G93A Forms

PubMed Central

Linares, Edlaine; Seixas, Luciana V.; dos Prazeres, Janaina N.; Ladd, Fernando V. L.; Ladd, Aliny A. B. L.; Coppi, Antonio A.; Augusto, Ohara

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS. PMID:23405225

Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype.

PubMed

Pinto, Sara; Cunha, Carolina; Barbosa, Marta; Vaz, Ana R; Brites, Dora

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs). Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase 1 (mSOD1) from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs) that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A) and extended the study into their derived exosomes (mSOD1 exosomes). Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells [either transfected with either wild-type (wt) human SOD1 or mutant SOD1(G93A)] showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP-2, and MMP-9 activation, as well as upregulation of IL-1β, TNF-α, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Muscle Expression of SOD1(G93A) Modulates microRNA and mRNA Transcription Pattern Associated with the Myelination Process in the Spinal Cord of Transgenic Mice.

PubMed

Dobrowolny, Gabriella; Bernardini, Camilla; Martini, Martina; Baranzini, Mirko; Barba, Marta; Musarò, Antonio

2015-01-01

A crucial system severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. One of the best examples of impaired interplay between muscle and nerve is Amyotrophic Lateral Sclerosis, a neurodegenerative disease characterized by degeneration of motor neurons and muscle atrophy. Increasing evidences suggest that damage to motor neurons is enhanced by alterations in the neighboring non-neuronal cells and indicate that altered skeletal muscle might be the source of signals that impinge motor neuron activity and survival. Here we investigated whether muscle selective expression of SOD1(G93A) mutant gene modulates mRNAs and miRNAs expression at the level of spinal cord of MLC/SOD1(G93A) mice. Using a Taqman array, the Affymetrix Mouse Gene 2.0 ST approach and the MiRwalk 2.0 database, which provides information on miRNA and their predicted target genes, we revealed that muscle specific expression of SOD1(G93A) modulates relevant molecules of the genetic and epigenetic circuitry of myelin homeostasis in spinal cord of transgenic mice. Our study provides insights into the pathophysiological interplay between muscle and nerve and supports the hypothesis that muscle is a source of signals that can either positively or negatively affect the nervous system.

Extra virgin olive oil intake delays the development of amyotrophic lateral sclerosis associated with reduced reticulum stress and autophagy in muscle of SOD1G93A mice.

PubMed

Oliván, Sara; Martínez-Beamonte, Roberto; Calvo, Ana C; Surra, Joaquín C; Manzano, Raquel; Arnal, Carmen; Osta, Rosario; Osada, Jesús

2014-08-01

Amyotrophic lateral sclerosis is a neurodegenerative disease associated with mutations in antioxidant enzyme Cu/Zn-superoxide dismutase 1. Albeit there is no treatment for this disease, new insights related to an exacerbated lipid metabolism have been reported. In connection with the hypermetabolic lipid status, the hypothesis whether nature of dietary fat might delay the progression of the disease was tested by using a transgenic mouse that overexpresses the human SOD1G93A variant. For this purpose, SOD1G93A mice were assigned randomly to one of the following three experimental groups: (1) a standard chow diet (control, n=21), (2) a chow diet enriched with 20% (w/w) extra virgin olive oil (EVOO, n=22) and (3) a chow diet containing 20% palm oil (palm, n=20). They received the diets for 8 weeks and the progression of the disease was assessed. On the standard chow diet, average plasma cholesterol levels were lower than those mice receiving the high-fat diets. Mice fed an EVOO diet showed a significant higher survival and better motor performance than control mice. EVOO group mice survived longer and showed better motor performance and larger muscle fiber area than animals receiving palm. Moreover, the EVOO-enriched diet improved the muscle status as shown by expression of myogenic factors (Myod1 and Myog) and autophagy markers (LC3 and Beclin1), as well as diminished endoplasmic reticulum (ER) stress through decreasing Atf6 and Grp78. Our results demonstrate that EVOO may be effective in increasing survival rate, improving motor coordination together with a potential amelioration of ER stress, autophagy and muscle damage. Copyright © 2014 Elsevier Inc. All rights reserved.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

PubMed

Abdolvahabi, Alireza; Shi, Yunhua; Rasouli, Sanaz; Croom, Corbin M; Aliyan, Amir; Martí, Angel A; Shaw, Bryan F

2017-06-21

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼10 3 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R 2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R 2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R 2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

PubMed

Şahin, Aslı; Held, Aaron; Bredvik, Kirsten; Major, Paxton; Achilli, Toni-Marie; Kerson, Abigail G; Wharton, Kristi; Stilwell, Geoff; Reenan, Robert

2017-02-01

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies. Copyright © 2017 by the Genetics Society of America.

Acquisition of pro-oxidant activity of fALS-linked SOD1 mutants as revealed using circular dichroism and UV-resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Fujimaki, Nobuhiro; Nishiya, Ken; Miura, Takashi; Nakabayashi, Takakazu

2016-11-01

The acquisition of pro-oxidant activity of the mutated form of human Cu, Zn-superoxide dismutase (SOD1) has been investigated to clarify the relationship between mutations in SOD1 and the pathogenesis of amyotrophic lateral sclerosis (ALS). Ala4 → Val (A4V) and Gly93 → Ala (G93A) mutants, which are representative ALS-linked SOD1 mutants, have been found to exhibit both the denaturation and the gain of pro-oxidant activity after incubation in the apo-form at a physiological condition of 37 °C and pH 7.4 and the rebinding of Cu2+. These characteristics are similar to those previously reported for the His43 → Arg (H43R) mutant. UV-resonance Raman spectra indicated that the coordination structure of the Cu-binding site catalyzing the oxidation reaction is the same among the denatured A4V, G93A, and H43R. Since wild-type SOD1 does not exhibit the denaturation in its apo-form at 37 °C and pH 7.4, the instability of the protein structure due to mutation can be considered as a significant factor that induces the denaturation and the subsequent pro-oxidant activity.

Galactooligosaccharide improves the animal survival and alleviates motor neuron death in SOD1G93A mouse model of amyotrophic lateral sclerosis.

PubMed

Song, L; Gao, Y; Zhang, X; Le, W

2013-08-29

Amyotrophic lateral sclerosis (ALS) is a progressive and devastating neurodegenerative disease caused by selective degeneration and death of motor neurons. So far very limited therapeutic options have emerged to treat this fatal disease. Homocysteine (Hcy) lowering drugs have been suggested to be a palliative therapy of this disease. Folate, Vitamin B12 (VitB12) and Vitamin B6 (VitB6) are important elements involved in the Hcy metabolism and we proposed that medications which could promote the absorption of folate, VitB12 and VitB6 might have benefit for ALS. Galactooligosaccharides (GOS) is a prebiotic which could significantly improve the absorption and syntheses of B Vitamins. To investigate whether GOS could provide neuroprotective effect in ALS, we applied GOS and GOS-rich prebiotic yogurt in SOD1(G93A) mice and assessed their effects on the disease progression of ALS. Our results showed that GOS and prebiotics yogurt administration significantly delayed the disease onset and prolonged the lifespan in SOD1(G93A) mice. Also, these products increased the concentration of folate, VitB12 and reduced the level of Hcy. Moreover, we found that both GOS and prebiotics yogurt attenuated motor neurons loss, improved the atrophy and mitochondrial activity in myocyte. Furthermore, we demonstrated that GOS and GOS-rich prebiotic treatment suppressed the activation of astrocytes and microglia and regulated several inflammatory- and apoptosis-related factors. Our findings suggested that GOS might have therapeutic potential for ALS, and GOS-rich prebiotic yogurt might be considered as a nutritional therapy for this disease. Copyright © 2013 IBRO. All rights reserved.

Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability

PubMed Central

Fritz, Elsa; Izaurieta, Pamela; Weiss, Alexandra; Mir, Franco R.; Rojas, Patricio; Gonzalez, David; Rojas, Fabiola; Brown, Robert H.; Madrid, Rodolfo

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1G93A) increases persistent sodium inward currents (PCNa), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitability by other specific (mexiletine) and nonspecific (spermidine and riluzole) blockers of voltage-sensitive sodium (Nav) channels restored basal calcium transients and prevented motoneuron death induced by ACM-hSOD1G93A. These findings suggest that riluzole, the only FDA-approved drug with known benefits for ALS patients, acts by inhibiting hyperexcitability. Together, our data document that a critical element mediating the non-cell-autonomous toxicity of ACM-hSOD1G93A on motoneurons is increased excitability, an observation with direct implications for therapy of ALS. PMID:23486205

Observation of c.260A > G mutation in superoxide dismutase 1 that causes p.Asn86Ser in Iranian amyotrophic lateral sclerosis patient and absence of genotype/phenotype correlation.

PubMed

Khani, Marzieh; Alavi, Afagh; Nafissi, Shahriar; Elahi, Elahe

2015-07-06

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder in European populations. ALS can be sporadic ALS (SALS) or familial ALS (FALS). Among 20 known ALS genes, mutations in C9orf72 and superoxide dismutase 1 (SOD1) are the most common genetic causes of the disease. Whereas C9orf72 mutations are more common in Western populations, the contribution of SOD1 to ALS in Iran is more than C9orf72. At present, a clear genotype/phenotype correlation for ALS has not been identified. We aimed to perform mutation screening of SOD1 in a newly identified Iranian FALS patient and to assess whether a genotype/phenotype correlation for the identified mutation exists. The five exons of SOD1 and flanking intronic sequences of a FALS proband were screened for mutations by direct sequencing. The clinical features of the proband were assessed by a neuromuscular specialist (SN). The phenotypic presentations were compared to previously reported patients with the same mutation. Heterozygous c.260A > G mutation in SOD1 that causes Asn86Ser was identified in the proband. Age at onset was 34 years and site of the first presentation was in the lower extremities. Comparisons of clinical features of different ALS patients with the same mutation evidenced variable presentations. The c.260A > G mutation in SOD1 that causes Asn86Ser appears to cause ALS with variable clinical presentations.

FUS and TARDBP but Not SOD1 Interact in Genetic Models of Amyotrophic Lateral Sclerosis

PubMed Central

Kabashi, Edor; Bercier, Valérie; Lissouba, Alexandra; Liao, Meijiang; Brustein, Edna; Rouleau, Guy A.; Drapeau, Pierre

2011-01-01

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS–related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS–related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1. PMID:21829392

FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Bercier, Valérie; Lissouba, Alexandra; Liao, Meijiang; Brustein, Edna; Rouleau, Guy A; Drapeau, Pierre

2011-08-01

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.

Astrocytes expressing mutant SOD1 and TDP43 trigger motoneuron death that is mediated via sodium channels and nitroxidative stress

PubMed Central

Rojas, Fabiola; Cortes, Nicole; Abarzua, Sebastian; Dyrda, Agnieszka; van Zundert, Brigitte

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1G93A contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Nav) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1G93A and ACM-SOD1G86R) or TDP43 (ACM-TDP43A315T) mutants; we show that such exposure rapidly (within 30–60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Nav channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Nav channel activity. PMID:24570655

Combined fulminant frontotemporal dementia and amyotrophic lateral sclerosis associated with an I113T SOD1 mutation.

PubMed

Katz, Jonathan S; Katzberg, Hans D; Woolley, Susan C; Marklund, Stefan L; Andersen, Peter M

2012-10-01

Mutations in the gene for superoxide dismutase type 1 cause amyotrophic lateral sclerosis (ALS), but are not thought to be associated with frontotemporal dementia (FTD). A lack of detailed case reports is one reason, among others, for this skepticism. This case report comments on a patient with familial ALS caused by I113T mutation in the SOD1 gene presenting with progressive cognitive and behavioral decline two years before developing progressive motor degeneration. In conclusion, this case provides evidence that SOD1 mutations can be associated with FTD.

Bee venom effects on ubiquitin proteasome system in hSOD1(G85R)-expressing NSC34 motor neuron cells.

PubMed

Kim, Seon Hwy; Jung, So Young; Lee, Kang-Woo; Lee, Sun Hwa; Cai, MuDan; Choi, Sun-Mi; Yang, Eun Jin

2013-07-18

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from a progressive loss of motor neurons. Familial ALS (fALS) is caused by missense mutations in Cu, Zn-superoxide dismutase 1 (SOD1) that frequently result in the accumulation of mutant protein aggregates that are associated with impairments in the ubiquitin-proteasome system (UPS). UPS impairment has been implicated in many neurological disorders. Bee venom (BV) extracted from honey bees has been used as a traditional medicine for treating inflammatory diseases and has been shown to attenuate the neuroinflammatory events that occur in a symptomatic ALS animal model. NSC34 cells were transiently transfected with a WT or G85R hSOD1-GFP construct for 24 hrs and then stimulated with 2.5 μg/ml BV for 24 hrs. To determine whether a SOD1 mutation affects UPS function in NSC34 cells, we examined proteasome activity and performed western blotting and immunofluorescence using specific antibodies, such as anti-misfolded SOD1, anti-ubiquitin, anti-GRP78, anti-LC3, and anti-ISG15 antibodies. We found that GFP-hSOD1G85R overexpression induced SOD1 inclusions and reduced proteasome activity compared with the overexpression of GFP alone in NSC34 motor neuronal cells. In addition, we also observed that BV treatment restored proteasome activity and reduced the accumulation of ubiquitinated and misfolded SOD1 in GFP-hSOD1G85R-overexpressing NSC34 motor neuronal cells. However, BV treatment did not activate the autophagic pathway in these cells. Our findings suggest that BV may rescue the impairment of the UPS in ALS models.

Copper Homeostasis as a Therapeutic Target in Amyotrophic Lateral Sclerosis with SOD1 Mutations

PubMed Central

Tokuda, Eiichi; Furukawa, Yoshiaki

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease affecting both upper and lower motor neurons, and currently, there is no cure or effective treatment. Mutations in a gene encoding a ubiquitous antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1), have been first identified as a cause of familial forms of ALS. It is widely accepted that mutant SOD1 proteins cause the disease through a gain in toxicity but not through a loss of its physiological function. SOD1 is a major copper-binding protein and regulates copper homeostasis in the cell; therefore, a toxicity of mutant SOD1 could arise from the disruption of copper homeostasis. In this review, we will briefly review recent studies implying roles of copper homeostasis in the pathogenesis of SOD1-ALS and highlight the therapeutic interventions focusing on pharmacological as well as genetic regulations of copper homeostasis to modify the pathological process in SOD1-ALS. PMID:27136532

Copper Homeostasis as a Therapeutic Target in Amyotrophic Lateral Sclerosis with SOD1 Mutations.

PubMed

Tokuda, Eiichi; Furukawa, Yoshiaki

2016-04-28

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease affecting both upper and lower motor neurons, and currently, there is no cure or effective treatment. Mutations in a gene encoding a ubiquitous antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1), have been first identified as a cause of familial forms of ALS. It is widely accepted that mutant SOD1 proteins cause the disease through a gain in toxicity but not through a loss of its physiological function. SOD1 is a major copper-binding protein and regulates copper homeostasis in the cell; therefore, a toxicity of mutant SOD1 could arise from the disruption of copper homeostasis. In this review, we will briefly review recent studies implying roles of copper homeostasis in the pathogenesis of SOD1-ALS and highlight the therapeutic interventions focusing on pharmacological as well as genetic regulations of copper homeostasis to modify the pathological process in SOD1-ALS.

In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1G93A ALS rat model.

PubMed

Stamenković, Stefan; Pavićević, Aleksandra; Mojović, Miloš; Popović-Bijelić, Ana; Selaković, Vesna; Andjus, Pavle; Bačić, Goran

2017-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1 G93A ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t 1/2 =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1 G93A rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of

Degenerative myelopathy in the Collie breed: a retrospective immunohistochemical analysis of superoxide dismutase 1 in an affected Rough Collie, and a molecular epidemiological survey of the SOD1: c.118G>A mutation in Japan

PubMed Central

KOHYAMA, Moeko; KITAGAWA, Masato; KAMISHINA, Hiroaki; KOBATAKE, Yui; YABUKI, Akira; SAWA, Mariko; KAKITA, Shusaku; YAMATO, Osamu

2016-01-01

Canine degenerative myelopathy (DM) is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds. A DM-associated mutation of the canine superoxide dismutase 1 (SOD1) gene, designated as c.118G>A (p.E40K), has been implicated as one of pathogenetic determinants of the disease in many breeds, but it remains to be determined whether the c.118G>A mutation is responsible for development or progression of DM in Collies. Previously, a Rough Collie was diagnosed clinically and histopathologically as having DM in Japan, suggesting the possibility that the Collie breed may be predisposed to DM due to the high frequency of c.118G>A in Japan. In this study, accumulation and aggregate formation of SOD1 protein were retrospectively demonstrated in the spinal cord of the DM-affected dog by immunohistochemical analysis. Furthermore, a molecular epidemiological survey revealed a high carrier rate (27.6%) and mutant allele frequency (0.138) of c.118G>A in a population of Collies in Japan, suggesting that the Collie breed may be predisposed to DM associated with c.118G>A, and the prevention of DM in Collies in Japan should be addressed through epidemiological and genetic testing strategies. PMID:27941298

Intramuscular transplantation of bone marrow cells prolongs the lifespan of SOD1G93A mice and modulates expression of prognosis biomarkers of the disease.

PubMed

Rando, Amaya; Pastor, Diego; Viso-León, Mari Carmen; Martínez, Anna; Manzano, Raquel; Navarro, Xavier; Osta, Rosario; Martínez, Salvador

2018-04-06

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive muscle weakness, paralysis and death. There is no effective treatment for ALS and stem cell therapy has arisen as a potential therapeutic approach. SOD1 mutant mice were used to study the potential neurotrophic effect of bone marrow cells grafted into quadriceps femoris muscle. Bone marrow intramuscular transplants resulted in increased longevity with improved motor function and decreased motoneuron degeneration in the spinal cord. Moreover, the increment of the glial-derived neurotrophic factor and neurotrophin 4 observed in the grafted muscles suggests that this partial neuroprotective effect is mediated by neurotrophic factor release at the neuromuscular junction level. Finally, certain neurodegeneration and muscle disease-specific markers, which are altered in the SOD1 G93A mutant mouse and may serve as molecular biomarkers for the early detection of ALS in patients, have been studied with encouraging results. This work demonstrates that stem cell transplantation in the muscle prolonged the lifespan, increased motoneuron survival and slowed disease progression, which was also assessed by genetic expression analysis.

The Overexpression of TDP-43 Protein in the Neuron and Oligodendrocyte Cells Causes the Progressive Motor Neuron Degeneration in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.

PubMed

Lu, Yi; Tang, Chunyan; Zhu, Lei; Li, Jiao; Liang, Huiting; Zhang, Jie; Xu, Renshi

2016-01-01

The recent investigation suggested that the TDP-43 protein was closely related to the motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the pathogenesis contributed to motor neuron degeneration largely remained unknown. Therefore, we detected the alteration of TDP-43 expression and distribution in the adult spinal cord of the SOD1 G93A transgenic mouse model for searching the possible pathogenesis of ALS. We examined the TDP-43 expression and distribution in the different anatomic regions, segments and neural cells in the adult spinal cord at the different stages of the SOD1 wild-type and G93A transgenic model by the fluorescent immunohistochemical technology. We revealed that the amount of TDP-43 positive cell was cervical>lumbar>thoracic segment, that in the ventral horn was more than that in the dorsal horn, a few of TDP-43 protein sparsely expressed and distributed in the other regions, the TDP-43 protein weren't detected in the white matter and the central canal. The TDP-43 protein was mostly expressed and distributed in the nuclear of neuron cells and the cytoplasm of oligodendrocyte cells of the gray matter surrounding the central canal of spinal cord by the granular shape in the SOD1 wild-type and G93A transgenic mice. The amount of TDP-43 positive cell significantly increased at the onset and progression stages of ALS following with the increase of neuron death in spinal cord, particularly in the ventral horn of cervical segment at the progression stage. Our results suggested that the overexpression of TDP-43 protein in the neuron and oligodendrocyte cell causes the progressive motor neuron degeneration in the ALS-like mouse model.

A Phase I, Randomised, First-in-Human Study of an Antisense Oligonucleotide Directed Against SOD1 Delivered Intrathecally in SOD1-Familial ALS Patients

PubMed Central

Miller, Timothy; Pestronk, Alan; David, William; Rothstein, Jeffrey; Simpson, Ericka; Appel, Stanley H.; Andres, Patricia L.; Mahoney, Katy; Allred, Peggy; Alexander, Katie; Ostrow, Lyle W.; Schoenfeld, David; Macklin, Eric A.; Norris, Daniel A.; Manousakis, Georgios; Crisp, Matthew; Smith, Richard; Bennett, C.F.; Bishop, Kathie; Cudkowicz, Merit E

2013-01-01

Objective To evaluate the safety, tolerability, and pharmacokinetics of an antisense oligonucleotide designed to inhibit SOD1 expression (ISIS 333611) following intrathecal administration in patients with SOD1-related familial amyotrophic lateral sclerosis (ALS). Background Mutations in SOD1 cause 13% of familial ALS. In animal studies, ISIS 333611 delivered to the cerebrospinal fluid (CSF) distributed to the brain and spinal cord, decreased SOD1 mRNA and protein levels in spinal cord tissue, and prolonged survival in the SOD1G93A rat ALS model. Methods In a randomized, placebo controlled Phase 1 trial, ISIS 333611 was delivered by intrathecal infusion using an external pump over 11.5 hours at increasing doses to four cohorts of eight SOD1 positive ALS subjects (randomized 6 drug: 2 placebo/cohort). Subjects were allowed to re-enroll in subsequent cohorts. Safety and tolerability assessments were made during the infusion and periodically over 28 days following the infusion. CSF and plasma drug levels were measured. Findings No dose-limiting toxicities were identified at doses up to 3.0 mg. No safety or tolerability concerns related to ISIS 333611 were identified. There were no serious adverse events (AEs) in ISIS 333611-treated subjects. Re-enrollment and re-dosing of subjects with ISIS 333611 was also well tolerated. Dose-dependent CSF and plasma concentrations were observed. Interpretation In this first clinical study to report intrathecal delivery of an antisense oligonucleotide, ISIS 333611 was well tolerated when administered as an intrathecal infusion in subjects with SOD1 familial ALS. CSF and plasma drug levels were consistent with levels predicted from preclinical studies. These results suggest that antisense oligonucleotide delivery to the central nervous system may be a feasible therapeutic strategy for neurological disorders. Source of funding ALS Association, Muscular Dystrophy Association, Isis Pharmaceuticals PMID:23541756

Fisetin Exerts Antioxidant and Neuroprotective Effects in Multiple Mutant hSOD1 Models of Amyotrophic Lateral Sclerosis by Activating ERK.

PubMed

Wang, T H; Wang, S Y; Wang, X D; Jiang, H Q; Yang, Y Q; Wang, Y; Cheng, J L; Zhang, C T; Liang, W W; Feng, H L

2018-05-21

Oxidative stress exhibits a central role in the course of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease commonly found to include a copper/zinc superoxide dismutase (SOD1) gene mutation. Fisetin, a natural antioxidant, has shown benefits in varied neurodegenerative diseases. The possible effect of fisetin in ALS has not been clarified as of yet. We investigated whether fisetin affected mutant hSOD1 ALS models. Three different hSOD1-related mutant models were used: Drosophila expressing mutant hSOD1 G85R , hSOD1 G93A NSC34 cells, and transgenic mice. Fisetin treatment provided neuroprotection as demonstrated by an improved survival rate, attenuated motor impairment, reduced ROS damage and regulated redox homeostasis compared with those in controls. Furthermore, fisetin increased the expression of phosphorylated ERK and upregulated antioxidant factors, which were reversed by MEK/ERK inhibition. Finally, fisetin reduced the levels of both mutant and wild-type hSOD1 in vivo and in vitro, as well as the levels of detergent-insoluble hSOD1 proteins. The results indicate that fisetin protects cells from ROS damage and improves the pathological behaviors caused by oxidative stress in disease models related to SOD1 gene mutations probably by activating ERK, thereby providing a potential treatment for ALS. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Resveratrol Ameliorates Motor Neuron Degeneration and Improves Survival in SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

PubMed Central

Song, Lin; Zhang, Xiaojie; Li, Jia; Le, Weidong

2014-01-01

Resveratrol has recently been used as a supplemental treatment for several neurological and nonneurological diseases. It is not known whether resveratrol has neuroprotective effect on amyotrophic lateral sclerosis (ALS). To assess the effect of resveratrol on the disease, we tested this agent on an ALS model of SOD1G93A transgenic mouse. Rotarod measurement was performed to measure the motor function of the ALS mice. Nissl staining and SMI-32 immunofluorescent staining were used to determine motor neurons survival in the spinal cord of the ALS mice. Hematoxylin-eosin (H&E), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) staining were applied to pathologically analyze the skeletal muscles of the ALS mice. We found that resveratrol treatment significantly delayed the disease onset and prolonged the lifespan of the ALS mice. Furthermore, resveratrol treatment attenuated motor neuron loss, relieved muscle atrophy, and improved mitochondrial function of muscle fibers in the ALS mice. In addition, we demonstrated that resveratrol exerted these neuroprotective effects mainly through increasing the expression of Sirt1, consequently suppressing oxidative stress and downregulating p53 and its related apoptotic pathway. Collectively, our findings suggest that resveratrol might provide a promising therapeutic intervention for ALS. PMID:25057490

Role of disulfide cross-linking of mutant SOD1 in the formation of inclusion-body-like structures.

PubMed

Roberts, Brittany L T; Patel, Kinaree; Brown, Hilda H; Borchelt, David R

2012-01-01

Pathologic aggregates of superoxide dismutase 1 (SOD1) harboring mutations linked to familial amyotrophic lateral sclerosis (fALS) have been shown to contain aberrant intermolecular disulfide cross-links. In prior studies, we observed that intermolecular bonding was not necessary in the formation of detergent- insoluble SOD1 complexes by mutant SOD1, but we were unable to assess whether this type of bonding may be important for pathologic inclusion formation. In the present study, we visually assess the formation of large inclusions by fusing mutant SOD1 to yellow fluorescent protein (YFP). Experimental constructs possessing mutations at all cysteine residues in SOD1 (sites 6, 57, 111, and 146 to F,S,Y,R or G,S,Y,R, respectively) were shown to maintain a high propensity of inclusion formation despite the inability to form disulfide cross-links. Interestingly, although aggregates form when all cysteines were mutated, double mutants of the ALS mutation C6G with an experimental mutation C111S exhibited low aggregation propensity. Overall, this study is an extension of previous work demonstrating that cysteine residues in mutant SOD1 play a role in modulating aggregation and that intermolecular disulfide bonds are not required to produce large intracellular inclusion-like structures.

Biological effects of CCS in the absence of SOD1 enzyme activation: implications for disease in a mouse model for ALS.

PubMed

Proescher, Jody B; Son, Marjatta; Elliott, Jeffrey L; Culotta, Valeria C

2008-06-15

The CCS copper chaperone is critical for maturation of Cu, Zn-superoxide dismutase (SOD1) through insertion of the copper co-factor and oxidization of an intra-subunit disulfide. The disulfide helps stabilize the SOD1 polypeptide, which can be particularly important in cases of amyotrophic lateral sclerosis (ALS) linked to misfolding of mutant SOD1. Surprisingly, however, over-expressed CCS was recently shown to greatly accelerate disease in a G93A SOD1 mouse model for ALS. Herein we show that disease in these G93A/CCS mice correlates with incomplete oxidation of the SOD1 disulfide. In the brain and spinal cord, CCS over-expression failed to enhance oxidation of the G93A SOD1 disulfide and if anything, effected some accumulation of disulfide-reduced SOD1. This effect was mirrored in culture with a C244,246S mutant of CCS that has the capacity to interact with SOD1 but can neither insert copper nor oxidize the disulfide. In spite of disulfide effects, there was no evidence for increased SOD1 aggregation. If anything, CCS over-expression prevented SOD1 misfolding in culture as monitored by detergent insolubility. This protection against SOD1 misfolding does not require SOD1 enzyme activation as the same effect was obtained with the C244,246S allele of CCS. In the G93A SOD1 mouse, CCS over-expression was likewise associated with a lack of obvious SOD1 misfolding marked by detergent insolubility. CCS over-expression accelerates SOD1-linked disease without the hallmarks of misfolding and aggregation seen in other mutant SOD1 models. These studies are the first to indicate biological effects of CCS in the absence of SOD1 enzymatic activation.

Acute Traumatic Brain Injury Does Not Exacerbate Amyotrophic Lateral Sclerosis in the SOD1G93A Rat Model1,2,3

PubMed Central

Thomsen, Gretchen M.

2015-01-01

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease in which upper and lower motor neurons degenerate, leading to muscle atrophy, paralysis, and death within 3 to 5 years of onset. While a small percentage of ALS cases are genetically linked, the majority are sporadic with unknown origin. Currently, etiological links are associated with disease onset without mechanistic understanding. Of all the putative risk factors, however, head trauma has emerged as a consistent candidate for initiating the molecular cascades of ALS. Here, we test the hypothesis that traumatic brain injury (TBI) in the SOD1 G93A transgenic rat model of ALS leads to early disease onset and shortened lifespan. We demonstrate, however, that a one-time acute focal injury caused by controlled cortical impact does not affect disease onset or survival. Establishing the negligible involvement of a single acute focal brain injury in an ALS rat model increases the current understanding of the disease. Critically, untangling a single focal TBI from multiple mild injuries provides a rationale for scientists and physicians to increase focus on repeat injuries to hopefully pinpoint a contributing cause of ALS. PMID:26464984

Neuronal glucose metabolism is impaired while astrocytic TCA cycling is unaffected at symptomatic stages in the hSOD1G93A mouse model of amyotrophic lateral sclerosis.

PubMed

Tefera, Tesfaye W; Borges, Karin

2018-01-01

Although alterations in energy metabolism are known in ALS, the specific mechanisms leading to energy deficit are not understood. We measured metabolite levels derived from injected [1- 13 C]glucose and [1,2- 13 C]acetate (i.p.) in cerebral cortex and spinal cord extracts of wild type and hSOD1 G93A mice at onset and mid disease stages using high-pressure liquid chromatography, 1 H and 13 C nuclear magnetic resonance spectroscopy. Levels of spinal and cortical CNS total lactate, [3- 13 C]lactate, total alanine and [3- 13 C]alanine, but not cortical glucose and [1- 13 C]glucose, were reduced mostly at mid stage indicating impaired glycolysis. The [1- 13 C]glucose-derived [4- 13 C]glutamate, [4- 13 C]glutamine and [2- 13 C]GABA amounts were diminished at mid stage in cortex and both time points in spinal cord, suggesting decreased [3- 13 C]pyruvate entry into the TCA cycle. Lack of changes in [1,2- 13 C]acetate-derived [4,5- 13 C]glutamate, [4,5- 13 C]glutamine and [1,2- 13 C]GABA levels indicate unchanged astrocytic 13 C-acetate metabolism. Reduced levels of leucine, isoleucine and valine in CNS suggest compensatory breakdown to refill TCA cycle intermediate levels. Unlabelled, [2- 13 C] and [4- 13 C]GABA concentrations were decreased in spinal cord indicating that impaired glucose metabolism contributes to hyperexcitability and supporting the use of treatments which increase GABA amounts. In conclusion, CNS glucose metabolism is compromised, while astrocytic TCA cycling appears to be normal in the hSOD1 G93A mouse model at symptomatic disease stages.

Reduction in hSOD1 copy number significantly impacts ALS phenotype presentation in G37R (line 29) mice: implications for the assessment of putative therapeutic agents.

PubMed

Zwiegers, Pierre; Lee, Grace; Shaw, Christopher A

2014-08-08

In vivo animal models of familial amyotrophic lateral sclerosis (fALS) are widely used to delineate the potential role that genetic mutations play in the neurodegenerative process. While these models are extensively used for establishing the safety and efficacy of putative therapeutics during pre-clinical development, effective clinical translation of pharmacological interventions has been largely unsuccessful. In this report we compare a recent cohort of G37R (line 29) mice generated from mating wild-type females with transgenic males obtained commercially to a previous set of offspring produced with transgenic male breeders from a colony established at a local collaborator's facility. Commercially derived progeny presented with a tightly clustered genomic signature for the mutant human superoxide dismutase1 transgene (hSOD1) locus, and exhibited a greater than two-fold reduction in the number of transgene copies present in the genome compared to offspring derived locally. Decrease in transgene levels corresponded with delayed ALS progression and a significant increase in overall lifespan (146%). These results highlight some key challenges inherent to the use of G37R (line 29) animals in pre-clinical studies for the development of ALS therapeutics. Without stringent assessment of mutant SOD1 copy number/protein levels, heterogeneity of transgene levels within cohorts may influence the behavioural and pathological presentation of disease and thus calls to question the validity of any detected therapeutic effects. Nuanced changes in mutant SOD1 copy number that currently remain unreported may undermine research endeavours, delay efforts for clinical translation, and compromise the rigor of animal studies by limiting reproducibility amongst research groups.

Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

PubMed

Lange, Dale J; Shahbazi, Mona; Silani, Vincenzo; Ludolph, Albert C; Weishaupt, Jochen H; Ajroud-Driss, Senda; Fields, Kara G; Remanan, Rahul; Appel, Stanley H; Morelli, Claudia; Doretti, Alberto; Maderna, Luca; Messina, Stefano; Weiland, Ulrike; Marklund, Stefan L; Andersen, Peter M

2017-06-01

Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848. © 2017 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

The VPAC2 agonist peptide histidine isoleucine (PHI) up-regulates glutamate transport in the corpus callosum of a rat model of amyotrophic lateral sclerosis (hSOD1G93A) by inhibiting caspase-3 mediated inactivation of GLT-1a.

PubMed

Goursaud, Stéphanie; Focant, Marylène C; Berger, Julie V; Nizet, Yannick; Maloteaux, Jean-Marie; Hermans, Emmanuel

2011-10-01

Degeneration of corpus callosum appears in patients with amyotrophic lateral sclerosis (ALS) before clinical signs of upper motor neuron death. Considering the ALS-associated impairment of astrocytic glutamate uptake, we have characterized the expression and activity of the glutamate transporter isoforms GLT-1a and GLT-1b in the corpus callosum of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A)). We have also studied the effect of peptide histidine isoleucine (PHI), a vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 2 (VPAC(2)) agonist on glutamate transporters both in vivo and in callosal astrocytes. Before the onset of motor symptoms, the expression of both transporter isoforms was correlated with a constitutive activity of caspase-3. This enzyme participates in the down-regulation of GLT-1 in ALS, and here we demonstrated its involvement in the selective degradation of GLT-1a in the white matter. A single stereotactic injection of PHI into the corpus callosum of symptomatic rats decreased caspase-3 activity and promoted GLT-1a expression and uptake activity. Together, with evidence for a reduced expression of prepro-VIP/PHI mRNA in the corpus callosum of transgenic animals, these data shed light on the modulatory role of the VIP/PHI system on the glutamatergic transmission in ALS.

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

PubMed Central

Qualls, David A.; Crosby, Keith; Brown, Hilda; Borchelt, David R.

2013-01-01

Background By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures. Methodology/Principal Findings In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures. Conclusions/Significance Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells. PMID:24391857

Screening of SOD1, FUS and TARDBP genes in patients with amyotrophic lateral sclerosis in central-southern China.

PubMed

Hou, Lihua; Jiao, Bin; Xiao, Tingting; Zhou, Lu; Zhou, Zhifan; Du, Juan; Yan, Xinxiang; Wang, Junling; Tang, Beisha; Shen, Lu

2016-09-08

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons of the brain, brainstem and spinal cord. To date, mutations in more than 30 genes have been linked to the pathogenesis of ALS. Among them, SOD1, FUS and TARDBP are ranked as the three most common genes associated with ALS. However, no mutation analysis has been reported in central-southern China. In this study, we sequenced SOD1, FUS and TARDBP in a central-southern Chinese cohort of 173 patients with ALS (15 familial ALS and 158 sporadic ALS) to detect mutations. As a result, five missense mutations in SOD1, namely, p.D101N, p.D101G, p.C111Y, p.N86S and p.V87A, were identified in three unrelated familial probands and three sporadic cases; two mutations in FUS were found in two unrelated familial probands, including an insertion mutation (p.P525_Y526insY) and a missense mutation (p.R521H); no variants of TARDBP were observed in patients. Therefore, SOD1 mutations were present in 20.0% of familial ALS patients and 1.9% of sporadic ALS patients, while FUS mutations were responsible for 13.3% of familial ALS cases, and TARDBP mutations were rare in either familial or sporadic ALS cases. This study broadens the known mutational spectrum in patients with ALS and further demonstrates the necessity for genetic screening in ALS patients from central-southern China.

Accumulation of misfolded SOD1 in dorsal root ganglion degenerating proprioceptive sensory neurons of transgenic mice with amyotrophic lateral sclerosis.

PubMed

Sábado, Javier; Casanovas, Anna; Tarabal, Olga; Hereu, Marta; Piedrafita, Lídia; Calderó, Jordi; Esquerda, Josep E

2014-01-01

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs). Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1) gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1(G93A) mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG) proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons.

Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration

PubMed Central

Banks, Courtney J.; Rodriguez, Nathan W.; Gashler, Kyle R.; Pandya, Rushika R.; Mortenson, Jeffrey B.; Whited, Matthew D.; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur; Reddi, Amit R.; Tessem, Jeffery S.; Torres, Matthew P.; Bikman, Benjamin T.

2017-01-01

ABSTRACT In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival. PMID:28739857

Metal-deficient SOD1 in amyotrophic lateral sclerosis.

PubMed

Hilton, James B; White, Anthony R; Crouch, Peter J

2015-05-01

Mutations to the ubiquitous antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) were the first established genetic cause of the fatal, adult-onset neurodegenerative disease amyotrophic lateral sclerosis (ALS). It is widely accepted that these mutations do not cause ALS via a loss of antioxidant function, but elucidating the alternate toxic gain of function has proven to be elusive. Under physiological conditions, SOD1 binds one copper ion and one zinc ion per monomer to form a highly stable and functional homodimer, but there is now ample evidence to indicate aberrant persistence of SOD1 in an intermediate metal-deficient state may contribute to the protein's involvement in ALS. This review briefly discusses some of the data to support a role for metal-deficient SOD1 in the development of ALS and some of the outcomes from drug development studies that have aimed to modify the symptoms of ALS by targeting the metal state of SOD1. The implications for the metal state of SOD1 in cases of sporadic ALS that do not involve mutant SOD1 are also discussed.

Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.

PubMed

Madji Hounoum, Blandine; Mavel, Sylvie; Coque, Emmanuelle; Patin, Franck; Vourc'h, Patrick; Marouillat, Sylviane; Nadal-Desbarats, Lydie; Emond, Patrick; Corcia, Philippe; Andres, Christian R; Raoul, Cédric; Blasco, Hélène

2017-04-01

The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605. © 2017 Wiley Periodicals, Inc.

Defining SOD1 ALS natural history to guide therapeutic clinical trial design.

PubMed

Bali, Taha; Self, Wade; Liu, Jingxia; Siddique, Teepu; Wang, Leo H; Bird, Thomas D; Ratti, Elena; Atassi, Nazem; Boylan, Kevin B; Glass, Jonathan D; Maragakis, Nicholas J; Caress, James B; McCluskey, Leo F; Appel, Stanley H; Wymer, James P; Gibson, Summer; Zinman, Lorne; Mozaffar, Tahseen; Callaghan, Brian; McVey, April L; Jockel-Balsarotti, Jennifer; Allred, Peggy; Fisher, Elena R; Lopate, Glenn; Pestronk, Alan; Cudkowicz, Merit E; Miller, Timothy M

2017-02-01

Understanding the natural history of familial amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations (ALS SOD1 ) will provide key information for optimising clinical trials in this patient population. To establish an updated natural history of ALS SOD1 . Retrospective cohort study from 15 medical centres in North America evaluated records from 175 patients with ALS with genetically confirmed SOD1 mutations, cared for after the year 2000. Age of onset, survival, ALS Functional Rating Scale (ALS-FRS) scores and respiratory function were analysed. Patients with the A4V (Ala-Val) SOD1 mutation (SOD1 A4V ), the largest mutation population in North America with an aggressive disease progression, were distinguished from other SOD1 mutation patients (SOD1 non-A4V ) for analysis. Mean age of disease onset was 49.7±12.3 years (mean±SD) for all SOD1 patients, with no statistical significance between SOD1 A4V and SOD1 non-A4V (p=0.72, Kruskal-Wallis). Total SOD1 patient median survival was 2.7 years. Mean disease duration for all SOD1 was 4.6±6.0 and 1.4±0.7 years for SOD1 A4V . SOD1 A4V survival probability (median survival 1.2 years) was significantly decreased compared with SOD1 non-A4V (median survival 6.8 years; p<0.0001, log-rank). A statistically significant increase in ALS-FRS decline in SOD1 A4V compared with SOD1 non-A4V participants (p=0.02) was observed, as well as a statistically significant increase in ALS-forced vital capacity decline in SOD1 A4V compared with SOD1 non-A4V (p=0.02). SOD1 A4V is an aggressive, but relatively homogeneous form of ALS. These SOD1-specific ALS natural history data will be important for the design and implementation of clinical trials in the ALS SOD1 patient population. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Defining SOD1 ALS natural history to guide therapeutic clinical trial design

PubMed Central

Bali, Taha; Self, Wade; Liu, Jingxia; Siddique, Teepu; Wang, Leo H; Bird, Thomas D; Ratti, Elena; Atassi, Nazem; Boylan, Kevin B; Glass, Jonathan D; Maragakis, Nicholas J; Caress, James B; McCluskey, Leo F; Appel, Stanley H; Wymer, James P; Gibson, Summer; Zinman, Lorne; Mozaffar, Tahseen; Callaghan, Brian; McVey, April L; Jockel-Balsarotti, Jennifer; Allred, Peggy; Fisher, Elena R; Lopate, Glenn; Pestronk, Alan; Cudkowicz, Merit E; Miller, Timothy M

2016-01-01

Importance Understanding the natural history of familial amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations (ALSSOD1) will provide key information for optimising clinical trials in this patient population. Objective To establish an updated natural history of ALSSOD1. Design, setting and participants Retrospective cohort study from 15 medical centres in North America evaluated records from 175 patients with ALS with genetically confirmed SOD1 mutations, cared for after the year 2000. Main outcomes and measures Age of onset, survival, ALS Functional Rating Scale (ALS-FRS) scores and respiratory function were analysed. Patients with the A4V (Ala-Val) SOD1 mutation (SOD1A4V), the largest mutation population in North America with an aggressive disease progression, were distinguished from other SOD1 mutation patients (SOD1non-A4V) for analysis. Results Mean age of disease onset was 49.7 ±12.3 years (mean±SD) for all SOD1 patients, with no statistical significance between SOD1A4V and SOD1non-A4V (p=0.72, Kruskal-Wallis). Total SOD1 patient median survival was 2.7 years. Mean disease duration for all SOD1 was 4.6±6.0 and 1.4±0.7 years for SOD1A4V. SOD1A4V survival probability (median survival 1.2 years) was significantly decreased compared with SOD1non-A4V (median survival 6.8 years; p<0.0001, log-rank). A statistically significant increase in ALS-FRS decline in SOD1A4V compared with SOD1non-A4V participants (p=0.02) was observed, as well as a statistically significant increase in ALS-forced vital capacity decline in SOD1A4V compared with SOD1non-A4V (p=0.02). Conclusions and relevance SOD1A4V is an aggressive, but relatively homogeneous form of ALS. These SOD1-specific ALS natural history data will be important for the design and implementation of clinical trials in the ALSSOD1 patient population. PMID:27261500

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

PubMed

Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

2014-04-01

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Overexpression of metallothionein-I, a copper-regulating protein, attenuates intracellular copper dyshomeostasis and extends lifespan in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase-1.

PubMed

Tokuda, Eiichi; Okawa, Eriko; Watanabe, Shunsuke; Ono, Shin-Ichi

2014-03-01

Over 170 mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS), a lethal motor neuron disease. Although the molecular properties of SOD1 mutants differ considerably, we have recently shown that intracellular copper dyshomeostasis is a common pathogenic feature of different SOD1 mutants. Thus, the potentiation of endogenous copper regulation could be a therapeutic strategy. In this study, we investigated the effects of the overexpression of metallothionein-I (MT-I), a major copper-regulating protein, on the disease course of a mouse model of ALS (SOD1(G93A)). Using double transgenic techniques, we found that the overexpression of MT-I in SOD1(G93A) mice significantly extended the lifespan and slowed disease progression, but the effects on disease onset were modest. Genetically induced MT-I normalized copper dyshomeostasis in the spinal cord without influencing SOD1 enzymatic activity. The overexpression of MT-I in SOD1(G93A) mice markedly attenuated the pathological features of the mice, including the death of motor neurons, the degeneration of ventral root axons, the atrophy of skeletal muscles, and the activation of glial cells. Double transgenic mice also showed a decreased level of SOD1 aggregates within the glial cells of the spinal cord. Furthermore, the overexpression of MT-I in SOD1(G93A) mice reduced the number of spheroid-shaped astrocytes cleaved by active caspase-3. We concluded that therapeutic strategies aimed at the potentiation of copper regulation by MT-I could be of benefit in cases of ALS caused by SOD1 mutations.

In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress.

PubMed

Klöppel, Christine; Michels, Christine; Zimmer, Julia; Herrmann, Johannes M; Riemer, Jan

2010-12-03

The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS. Copyright © 2010 Elsevier Inc. All rights reserved.

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS

PubMed Central

Nichols, Nicole L.; Satriotomo, Irawan; Harrigan, Daniel J.; Mitchell, Gordon S.

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated superoxide dismutase-1 (SOD1G93A), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1G93A (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600µM; 12µL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms preserving

HSPB1 mutations causing hereditary neuropathy in humans disrupt non-cell autonomous protection of motor neurons.

PubMed

Heilman, Patrick L; Song, SungWon; Miranda, Carlos J; Meyer, Kathrin; Srivastava, Amit K; Knapp, Amy; Wier, Christopher G; Kaspar, Brian K; Kolb, Stephen J

2017-11-01

Heat shock protein beta-1 (HSPB1), is a ubiquitously expressed, multifunctional protein chaperone. Mutations in HSPB1 result in the development of a late-onset, distal hereditary motor neuropathy type II (dHMN) and axonal Charcot-Marie Tooth disease with sensory involvement (CMT2F). The functional consequences of HSPB1 mutations associated with hereditary neuropathy are unknown. HSPB1 also displays neuroprotective properties in many neuronal disease models, including the motor neuron disease amyotrophic lateral sclerosis (ALS). HSPB1 is upregulated in SOD1-ALS animal models during disease progression, predominately in glial cells. Glial cells are known to contribute to motor neuron loss in ALS through a non-cell autonomous mechanism. In this study, we examined the non-cell autonomous role of wild type and mutant HSPB1 in an astrocyte-motor neuron co-culture model system of ALS. Astrocyte-specific overexpression of wild type HSPB1 was sufficient to attenuate SOD1(G93A) astrocyte-mediated toxicity in motor neurons, whereas, overexpression of mutHSPB1 failed to ameliorate motor neuron toxicity. Expression of a phosphomimetic HSPB1 mutant in SOD1(G93A) astrocytes also reduced toxicity to motor neurons, suggesting that phosphorylation may contribute to HSPB1 mediated-neuroprotection. These data provide evidence that astrocytic HSPB1 expression may play a central role in motor neuron health and maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of ligand binding at a site relevant to SOD1 oxidation and aggregation.

PubMed

Manjula, Ramu; Wright, Gareth S A; Strange, Richard W; Padmanabhan, Balasundaram

2018-05-01

Cu/Zn superoxide dismutase-1 (SOD1) mutations are causative for a subset of amyotrophic lateral sclerosis (ALS) cases. These mutations lead to structural instability, aggregation and ultimately motor neuron death. We have determined crystal structures of SOD1 in complex with a naphthalene-catechol-linked compound which binds with low micro-molar affinity to a site important for oxidative damage-induced aggregation. SOD1 Trp32 oxidation is indeed significantly inhibited by ligand binding. Our work shows how compound linking can be applied successfully to ligand interactions on the SOD1 surface to generate relatively good binding strength. The ligand, positioned in a region important for SOD1 fibrillation, offers the possibility that it, or a similar compound, could prevent the abnormal self-association that drives SOD1 toxicity in ALS. © 2018 Federation of European Biochemical Societies.

An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

PubMed

Zhang, Yalan; Ni, Weiming; Horwich, Arthur L; Kaczmarek, Leonard K

2017-02-22

Mutations that alter levels of Slack (KCNT1) Na + -activated K + current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica , a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na + from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na + -activated K + channels in neurons. SIGNIFICANCE STATEMENT Slack Na + -activated K + channels (KCNT1, K Na 1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Taylor, David M; Minotti, Sandra; Durham, Heather D

2004-06-01

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.

S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

PubMed

Antinone, Sarah E; Ghadge, Ghanashyam D; Ostrow, Lyle W; Roos, Raymond P; Green, William N

2017-01-25

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects

PubMed Central

Antinone, Sarah E.; Ghadge, Ghanashyam D.; Ostrow, Lyle W.; Roos, Raymond P.; Green, William N.

2017-01-01

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord. PMID:28120938

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

PubMed Central

Bosco, Daryl A.; Morfini, Gerardo; Karabacak, N. Murat; Song, Yuyu; Gros-Louis, Francois; Pasinelli, Piera; Goolsby, Holly; Fontaine, Benjamin A.; Lemay, Nathan; McKenna-Yasek, Diane; Frosch, Matthew P.; Agar, Jeffery N.; Julien, Jean-Pierre; Brady, Scott T.; Brown, Robert H.

2010-01-01

Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS. PMID:20953194

A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants.

PubMed

Luchinat, Enrico; Barbieri, Letizia; Banci, Lucia

2017-12-12

Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

Unraveling ALS due to SOD1 mutation through the combination of brain and cervical cord MRI.

PubMed

Agosta, Federica; Spinelli, Edoardo Gioele; Marjanovic, Ivan V; Stevic, Zorica; Pagani, Elisabetta; Valsasina, Paola; Salak-Djokic, Biljana; Jankovic, Milena; Lavrnic, Dragana; Kostic, Vladimir S; Filippi, Massimo

2018-02-20

To explore structural and functional changes of the brain and cervical cord in patients with amyotrophic lateral sclerosis (ALS) due to mutation in the superoxide dismutase ( SOD1 ) gene compared with sporadic ALS. Twenty patients with SOD1 ALS, 11 with sporadic ALS, and 33 healthy controls underwent clinical evaluation and brain MRI. Cortical thickness analysis, diffusion tensor MRI of the corticospinal tracts (CST) and corpus callosum, and resting-state functional connectivity were performed. Patients with ALS also underwent cervical cord MRI to evaluate cord cross-sectional area and magnetization transfer ratio (MTR). Patients with SOD1 ALS showed longer disease duration and slower rate of functional decline relative to those with sporadic ALS. No cortical thickness abnormalities were found in patients with ALS compared with controls. Fractional anisotropy showed that sporadic ALS patients had significant CST damage relative to both healthy controls ( p = 0.001-0.02) and SOD1-related ALS ( p = 0.05), although the latter showed alterations that were intermediate between controls and sporadic ALS. Functional hyperconnectivity of the motor cortex in the sensorimotor network was observed in patients with sporadic ALS relative to controls. Conversely, patients with SOD1 ALS showed lower cord cross-sectional area along the whole cervical cord relative to those with sporadic ALS ( p < 0.001). No cord MTR differences were found between patient groups. Patients with SOD1 ALS showed cervical cord atrophy relative to those with sporadic ALS and a relative preservation of brain motor structural and functional networks. Neurodegeneration in SOD1 ALS is likely to occur primarily in the spinal cord. An objective and accurate estimate of spinal cord damage has potential in the future assessment of preventive SOD1 ALS therapies. © 2018 American Academy of Neurology.

A Drosophila Model for Amyotrophic Lateral Sclerosis Reveals Motor Neuron Damage by Human SOD1*♦

PubMed Central

Watson, Melanie R.; Lagow, Robert D.; Xu, Kexiang; Zhang, Bing; Bonini, Nancy M.

2008-01-01

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, we have generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These studies uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS. PMID:18596033

Is SOD1 loss of function involved in amyotrophic lateral sclerosis?

PubMed Central

Saccon, Rachele A.; Bunton-Stasyshyn, Rosie K. A.; Fisher, Elizabeth M.C.; Fratta, Pietro

2013-01-01

Mutations in the gene superoxide dismutase 1 (SOD1) are causative for familial forms of the neurodegenerative disease amyotrophic lateral sclerosis. When the first SOD1 mutations were identified they were postulated to give rise to amyotrophic lateral sclerosis through a loss of function mechanism, but experimental data soon showed that the disease arises from a—still unknown—toxic gain of function, and the possibility that loss of function plays a role in amyotrophic lateral sclerosis pathogenesis was abandoned. Although loss of function is not causative for amyotrophic lateral sclerosis, here we re-examine two decades of evidence regarding whether loss of function may play a modifying role in SOD1–amyotrophic lateral sclerosis. From analysing published data from patients with SOD1–amyotrophic lateral sclerosis, we find a marked loss of SOD1 enzyme activity arising from almost all mutations. We continue to examine functional data from all Sod1 knockout mice and we find obvious detrimental effects within the nervous system with, interestingly, some specificity for the motor system. Here, we bring together historical and recent experimental findings to conclude that there is a possibility that SOD1 loss of function may play a modifying role in amyotrophic lateral sclerosis. This likelihood has implications for some current therapies aimed at knocking down the level of mutant protein in patients with SOD1–amyotrophic lateral sclerosis. Finally, the wide-ranging phenotypes that result from loss of function indicate that SOD1 gene sequences should be screened in diseases other than amyotrophic lateral sclerosis. PMID:23687121

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

PubMed Central

Perera, Nirma D.; Sheean, Rebecca K.; Lau, Chew L.; Shin, Yea Seul; Beart, Philip M.; Horne, Malcolm K.; Turner, Bradley J.

2018-01-01

ABSTRACT Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS. PMID:28980850

Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia.

PubMed

Kinsella, Sinéad; König, Hans-Georg; Prehn, Jochen H M

2016-01-01

Mutations in the superoxide dismutase 1 (SOD1) gene contribute to motoneuron degeneration and are evident in 20% of familial amyotrophic lateral sclerosis cases. Mutant SOD1 induces microglial activation through a stimulation of Toll-like receptors 2 and 4 (TLR2 and TLR4). In the present study, we identified the proapoptotic Bcl-2 family protein Bid as a positive regulator of mutant SOD1-induced TLR-nuclear factor-κB (NF-κB) signaling in microglia. bid-deficient primary mouse microglia showed reduced NF-κB signaling in response to TLR4 activation or exposure to conditioned medium derived from SOD1 (G93A) expressing NSC-34 cells. Attenuation of NF-κB signaling in bid-deficient microglia was associated with lower levels of phosphorylated IKKα/β and p65, with a delayed degradation of IκBα and enhanced degradation of Peli1. Upstream of IKK, we found that Bid interacted with, and promoted, the K63-linked polyubiquitination of the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) in microglia. Our study suggests a key role for Bid in the regulation of TLR4-NF-κB proinflammatory signaling during mutant SOD1-induced disease pathology. Bid promotes TLR4-NF-κB signaling by interacting with TRAF6 and promoting TRAF6 K63-linked polyubiquitination in microglia.

Solid-state NMR studies of metal-free SOD1 fibrillar structures.

PubMed

Banci, Lucia; Blaževitš, Olga; Cantini, Francesca; Danielsson, Jens; Lang, Lisa; Luchinat, Claudio; Mao, Jiafei; Oliveberg, Mikael; Ravera, Enrico

2014-06-01

Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90%) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central β-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia123

PubMed Central

Kinsella, Sinéad

2016-01-01

Mutations in the superoxide dismutase 1 (SOD1) gene contribute to motoneuron degeneration and are evident in 20% of familial amyotrophic lateral sclerosis cases. Mutant SOD1 induces microglial activation through a stimulation of Toll-like receptors 2 and 4 (TLR2 and TLR4). In the present study, we identified the proapoptotic Bcl-2 family protein Bid as a positive regulator of mutant SOD1-induced TLR-nuclear factor-κB (NF-κB) signaling in microglia. bid-deficient primary mouse microglia showed reduced NF-κB signaling in response to TLR4 activation or exposure to conditioned medium derived from SOD1 G93A expressing NSC-34 cells. Attenuation of NF-κB signaling in bid-deficient microglia was associated with lower levels of phosphorylated IKKα/β and p65, with a delayed degradation of IκBα and enhanced degradation of Peli1. Upstream of IKK, we found that Bid interacted with, and promoted, the K63-linked polyubiquitination of the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) in microglia. Our study suggests a key role for Bid in the regulation of TLR4-NF-κB proinflammatory signaling during mutant SOD1-induced disease pathology. Bid promotes TLR4-NF-κB signaling by interacting with TRAF6 and promoting TRAF6 K63-linked polyubiquitination in microglia. PMID:27257617

Riluzole But Not Melatonin Ameliorates Acute Motor Neuron Degeneration and Moderately Inhibits SOD1-Mediated Excitotoxicity Induced Disrupted Mitochondrial Ca2+ Signaling in Amyotrophic Lateral Sclerosis

PubMed Central

Jaiswal, Manoj Kumar

2017-01-01

Selective motoneurons (MNs) degeneration in the brain stem, hypoglossal motoneurons (HMNs), and the spinal cord resulting in patients paralysis and eventual death are prominent features of amyotrophic lateral sclerosis (ALS). Previous studies have suggested that mitochondrial respiratory impairment, low Ca2+ buffering and homeostasis and excitotoxicity are the pathological phenotypes found in mice, and cell culture models of familial ALS (fALS) linked with Cu/Zn-superoxide dismutase 1 (SOD1) mutation. In our study, we aimed to understand the impact of riluzole and melatonin on excitotoxicity, neuronal protection and Ca2+ signaling in individual HMNs ex vivo in symptomatic adult ALS mouse brain stem slice preparations and in WT and SOD1-G93A transfected SH-SY5Y neuroblastoma cell line using fluorescence microscopy, calcium imaging with high speed charged coupled device camera, together with immunohistochemistry, cell survival assay and histology. In our experiments, riluzole but not melatonin ameliorates MNs degeneration and moderately inhibit excitotoxicity and cell death in SH-SY5YWT or SH-SY5YG93A cell lines induced by complex IV blocker sodium azide. In brain stem slice preparations, riluzole significantly inhibit HMNs cell death induced by inhibiting the mitochondrial electron transport chain by Na-azide. In the HMNs of brainstem slice prepared from adult (14–15 weeks) WT, and corresponding symptomatic SOD1G93A mice, we measured the effect of riluzole and melatonin on [Ca2+]i using fura-2 AM ratiometric calcium imaging in individual MNs. Riluzole caused a significant decrease in [Ca2+]i transients and reversibly inhibited [Ca2+]i transients in Fura-2 AM loaded HMNs exposed to Na-azide in adult symptomatic SOD1G93A mice. On the contrary, melatonin failed to show similar effects in the HMNs of WT and SOD1G93A mice. Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence, an indicator of mitochondrial metabolism and health in MNs, showed enhanced

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

PubMed

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

2014-06-04

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

ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

PubMed

Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

2017-11-04

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

Androgens affect muscle, motor neuron, and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis.

PubMed

Aggarwal, Tanya; Polanco, Maria J; Scaramuzzino, Chiara; Rocchi, Anna; Milioto, Carmelo; Emionite, Laura; Ognio, Emanuela; Sambataro, Fabio; Galbiati, Mariarita; Poletti, Angelo; Pennuto, Maria

2014-08-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of MLH1 -93G>A on gene expression in patients with colorectal cancer.

PubMed

Funck, Alexandre; Santos, Juliana C; Silva-Fernandes, Isabelle J L; Rabenhorst, Silvia H B; Martinez, Carlos A R; Ribeiro, Marcelo L

2014-09-01

The DNA repair machinery plays a key role in maintaining genomic stability by preventing the emergence of mutations. Furthermore, the -93G>A polymorphism in the MLH1 gene has been associated with an increased risk of developing colorectal cancer. Therefore, the aim of this study was to examine the expression pattern and effect of this polymorphism in normal and tumour samples from patients with colorectal cancer. The MLH1 -93G>A (rs1800734) polymorphism was detected by PCR-RFLP in 49 cases of colorectal cancer. MLH1 expression was investigated using real-time quantitative PCR. The results indicate a significant decrease in MLH1 expression in tumour samples compared to their normal counterparts. The MLH1 gene was also significantly repressed in samples from patients who had some degree of tumour invasion into other organs. Similarly, those patients who were in a more advanced tumour stage (TNM III and IV) exhibited a significant reduction in MLH1 gene expression. Finally, the mutant genotype AA of MLH1 was associated with a significant decrease in the expression of this gene. This finding suggests that this polymorphism could increase the risk of developing colorectal cancer by a defective mismatch repair system, particularly through the loss of MLH1 expression in an allele-specific manner.

Computational investigation of the human SOD1 mutant, Cys146Arg, that directs familial amyotrophic lateral sclerosis.

PubMed

Srinivasan, E; Rajasekaran, R

2017-07-25

The genetic substitution mutation of Cys146Arg in the SOD1 protein is predominantly found in the Japanese population suffering from familial amyotrophic lateral sclerosis (FALS). A complete study of the biophysical aspects of this particular missense mutation through conformational analysis and producing free energy landscapes could provide an insight into the pathogenic mechanism of ALS disease. In this study, we utilized general molecular dynamics simulations along with computational predictions to assess the structural characterization of the protein as well as the conformational preferences of monomeric wild type and mutant SOD1. Our static analysis, accomplished through multiple programs, predicted the deleterious and destabilizing effect of mutant SOD1. Subsequently, comparative molecular dynamic studies performed on the wild type and mutant SOD1 indicated a loss in the protein conformational stability and flexibility. We observed the mutational consequences not only in local but also in long-range variations in the structural properties of the SOD1 protein. Long-range intramolecular protein interactions decrease upon mutation, resulting in less compact structures in the mutant protein rather than in the wild type, suggesting that the mutant structures are less stable than the wild type SOD1. We also presented the free energy landscape to study the collective motion of protein conformations through principal component analysis for the wild type and mutant SOD1. Overall, the study assisted in revealing the cause of the structural destabilization and protein misfolding via structural characterization, secondary structure composition and free energy landscapes. Hence, the computational framework in our study provides a valuable direction for the search for the cure against fatal FALS.

Carnitine-acylcarnitine translocase deficiency with c.199-10 T>G and novel c.1A>G mutation

PubMed Central

Yan, Hui-ming; Hu, Hao; Ahmed, Aisha; Feng, Bing-bing; Liu, Jing; Jia, Zheng-jun; Wang, Hua

2017-01-01

Abstract Rationale: Carnitine-acylcarnitine translocate deficiency (CACTD) is a rare and life-threatening, autosomal recessive disorder of fatty acid β-oxidation characterized by hypoketotic hypoglycemia, hyperammonemia, cardiomyopathy, liver dysfunction, and muscle weakness; culminating in early death. To date, CACTD cases screened from the Chinese mainland population, especially patient with compound heterozygote with c.199-10T>G and a novel c.1A>G mutation in the SLC25A20 gene has never been described. Patient concerns: Herein, we report 2 neonatal cases of CACTD identified from the mainland China. These 2 patients were presented with severe metabolic crisis and their clinical conditions deteriorate rapidly and both died of cardiorespiratory collapse in the first week of life. We present the clinical and biochemical features of 2 probands and a brief literature review of previously reported CACTD cases with the c.199-10T>G mutation. Diagnoses: The acylcarnitine profiles by tandem-mass-spectrometry and the mutation analysis of SLC25A20 gene confirmed the diagnosis of CACTD in both patients. Mutation analysis demonstrated that patient No. 1 was homozygous for c.199-10T>G mutation, while patient No. 2 was a compound heterozygote for 2 mutations, a maternally-inherited c.199-10T>G and a paternally-inherited, novel c.1A>G mutation. Interventions: Both patients were treated with an aggressive treatment regimen include high glucose and arginine infusion, respiratory, and circulatory support. Outcomes: The first proband died 3 days after delivery due to sudden cardiac arrest. The second patient's clinical condition, at one time, was improved by high glucose infusion, intravenous arginine, and circulatory support. However, the patient failed to wean from mechanical ventilation. Unfortunately, her parents refused further treatment due to fear of financial burdens. The patient died of congestive heart failure in the 6th day of life. Lessons: We report the first 2 cases of

Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

PubMed

Dupuis, L; de Tapia, M; René, F; Lutz-Bucher, B; Gordon, J W; Mercken, L; Pradier, L; Loeffler, J P

2000-08-01

In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology. Copyright 2000 Academic Press.

Marked synergism between mutant SOD1 and glutamate transport inhibition in the induction of motor neuronal degeneration in spinal cord slice cultures.

PubMed

Yin, Hong Z; Weiss, John H

2012-04-11

Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1 week old pups, and after an additional week in vitro, some were exposed to either a low level (30 μM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3 weeks, or a higher level (50 μM) for 48 h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury. Copyright © 2012 Elsevier B.V. All rights reserved.

Induction of parkinsonism-related proteins in the spinal motor neurons of transgenic mouse carrying a mutant SOD1 gene.

PubMed

Morimoto, Nobutoshi; Nagai, Makiko; Miyazaki, Kazunori; Ohta, Yasuyuki; Kurata, Tomoko; Takehisa, Yasushi; Ikeda, Yoshio; Matsuura, Tohru; Asanuma, Masato; Abe, Koji

2010-06-01

Amyotrophic lateral sclerosis is a progressive and fatal disease caused by selective death of motor neurons, and a number of these patients carry mutations in the superoxide dismutase 1 (SOD1) gene involved in ameliorating oxidative stress. Recent studies indicate that oxidative stress and disruption of mitochondrial homeostasis is a common mechanism for motor neuron degeneration in amyotrophic lateral sclerosis and the loss of midbrain dopamine neurons in Parkinson's disease. Therefore, the present study investigated the presence and alterations of familial Parkinson's disease-related proteins, PINK1 and DJ-1, in spinal motor neurons of G93ASOD1 transgenic mouse model of amyotrophic lateral sclerosis. Following onset of disease, PINK1 and DJ-1 protein expression increased in the spinal motor neurons. The activated form of p53 also increased and translocated to the nuclei of spinal motor neurons, followed by increased expression of p53-activated gene 608 (PAG608). This is the first report demonstrating that increased expression of PAG608 correlates with activation of phosphorylated p53 in spinal motor neurons of an amyotrophic lateral sclerosis model. These results provide further evidence of the profound correlations between spinal motor neurons of amyotrophic lateral sclerosis and parkinsonism-related proteins.

Parkin Protects Against Misfolded SOD1 Toxicity by Promoting Its Aggresome Formation and Autophagic Clearance.

PubMed

Yung, Cheryl; Sha, Di; Li, Lian; Chin, Lih-Shen

2016-11-01

Mutations in Cu/Zn superoxide dismutase (SOD1) cause autosomal dominant amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease with no effective treatment. Despite ample evidence indicating involvement of mutation-induced SOD1 protein misfolding and aggregation in ALS pathogenesis, the molecular mechanisms that control cellular management of misfolded, aggregation-prone SOD1 mutant proteins remain unclear. Here, we report that parkin, an E3 ubiquitin-protein ligase which is linked to Parkinson's disease, is a novel regulator of cellular defense against toxicity induced by ALS-associated SOD1 mutant proteins. We find that parkin mediates K63-linked polyubiquitination of SOD1 mutants in cooperation with the UbcH13/Uev1a E2 enzyme and promotes degradation of these misfolded SOD1 proteins by the autophagy-lysosome system. In response to strong proteotoxic stress associated with proteasome impairment, parkin promotes sequestration of misfolded and aggregated SOD1 proteins to form perinuclear aggresomes, regulates positioning of lysosomes around misfolded SOD1 aggresomes, and facilitates aggresome clearance by autophagy. Our findings reveal parkin-mediated cytoprotective mechanisms against misfolded SOD1 toxicity and suggest that enhancing parkin-mediated cytoprotection may provide a novel therapeutic strategy for treating ALS.

Tryptophan 32 potentiates aggregation and cytotoxicity of a copper/zinc superoxide dismutase mutant associated with familial amyotrophic lateral sclerosis.

PubMed

Taylor, David M; Gibbs, Bernard F; Kabashi, Edor; Minotti, Sandra; Durham, Heather D; Agar, Jeffrey N

2007-06-01

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp-32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures.

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

NASA Astrophysics Data System (ADS)

Luchinat, Enrico; Barbieri, Letizia; Rubino, Jeffrey T.; Kozyreva, Tatiana; Cantini, Francesca; Banci, Lucia

2014-11-01

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration.

PubMed

Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Usarek, Ewa; Barańczyk-Kuźma, Anna

2011-01-01

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

Absence of Nrf2 or Its Selective Overexpression in Neurons and Muscle Does Not Affect Survival in ALS-Linked Mutant hSOD1 Mouse Models

PubMed Central

Vargas, Marcelo R.; Burton, Neal C.; Gan, Li; Johnson, Delinda A.; Schäfer, Matthias; Werner, Sabine; Johnson, Jeffrey A.

2013-01-01

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS. PMID:23418589

Absence of Nrf2 or its selective overexpression in neurons and muscle does not affect survival in ALS-linked mutant hSOD1 mouse models.

PubMed

Vargas, Marcelo R; Burton, Neal C; Kutzke, Jennifer; Gan, Li; Johnson, Delinda A; Schäfer, Matthias; Werner, Sabine; Johnson, Jeffrey A

2013-01-01

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A) mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.

Defective mitochondrial dynamics is an early event in skeletal muscle of an amyotrophic lateral sclerosis mouse model.

PubMed

Luo, Guo; Yi, Jianxun; Ma, Changling; Xiao, Yajuan; Yi, Frank; Yu, Tian; Zhou, Jingsong

2013-01-01

Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1(G93A)). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1(G93A) in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1(G93A) forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1(G93A) model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1(G93A) action on mitochondrial dynamics, indicating SOD1(G93A) likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1(G93A) inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may

Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS.

PubMed

Watanabe, Seiji; Ilieva, Hristelina; Tamada, Hiromi; Nomura, Hanae; Komine, Okiru; Endo, Fumito; Jin, Shijie; Mancias, Pedro; Kiyama, Hiroshi; Yamanaka, Koji

2016-12-01

A homozygous mutation in the gene for sigma 1 receptor (Sig1R) is a cause of inherited juvenile amyotrophic lateral sclerosis (ALS16). Sig1R localizes to the mitochondria-associated membrane (MAM), which is an interface of mitochondria and endoplasmic reticulum. However, the role of the MAM in ALS is not fully elucidated. Here, we identified a homozygous p.L95fs mutation of Sig1R as a novel cause of ALS16. ALS-linked Sig1R variants were unstable and incapable of binding to inositol 1,4,5-triphosphate receptor type 3 (IP 3 R3). The onset of mutant Cu/Zn superoxide dismutase (SOD1)-mediated ALS disease in mice was accelerated when Sig1R was deficient. Moreover, either deficiency of Sig1R or accumulation of mutant SOD1 induced MAM disruption, resulting in mislocalization of IP 3 R3 from the MAM, calpain activation, and mitochondrial dysfunction. Our findings indicate that a loss of Sig1R function is causative for ALS16, and collapse of the MAM is a common pathomechanism in both Sig1R- and SOD1-linked ALS Furthermore, our discovery of the selective enrichment of IP 3 R3 in motor neurons suggests that integrity of the MAM is crucial for the selective vulnerability in ALS. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Data set for phylogenetic tree and RAMPAGE Ramachandran plot analysis of SODs in Gossypium raimondii and G. arboreum.

PubMed

Wang, Wei; Xia, Minxuan; Chen, Jie; Deng, Fenni; Yuan, Rui; Zhang, Xiaopei; Shen, Fafu

2016-12-01

The data presented in this paper is supporting the research article "Genome-Wide Analysis of Superoxide Dismutase Gene Family in Gossypium raimondii and G. arboreum" [1]. In this data article, we present phylogenetic tree showing dichotomy with two different clusters of SODs inferred by the Bayesian method of MrBayes (version 3.2.4), "Bayesian phylogenetic inference under mixed models" [2], Ramachandran plots of G. raimondii and G. arboreum SODs, the protein sequence used to generate 3D sructure of proteins and the template accession via SWISS-MODEL server, "SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information." [3] and motif sequences of SODs identified by InterProScan (version 4.8) with the Pfam database, "Pfam: the protein families database" [4].

Glycogen storage disease type 1a in three siblings with the G270V mutation.

PubMed

Parvari, R; Isam, J; Moses, S W

1999-04-01

Glycogen storage disease type 1a (von Gierke disease, GSD1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity. The cloning of G6Pase cDNA and characterization of the human G6Pase gene enabled the identification of the mutations causing GSD1a. Here we report on the clinical and biochemical features of three GSD1a siblings of a Muslin Arab family with a G270V mutation. Two older patients presented with an unusually mild clinical and biochemical course.

Haplotype defined by the MLH1-93G/A polymorphism is associated with MLH1 promoter hypermethylation in sporadic colorectal cancers.

PubMed

Miyakura, Yasuyuki; Tahara, Makiko; Lefor, Alan T; Yasuda, Yoshikazu; Sugano, Kokichi

2014-11-24

Methylation of the MLH1 promoter region has been suggested to be a major mechanism of gene inactivation in sporadic microsatellite instability-positive (MSI-H) colorectal cancers (CRCs). Recently, single-nucleotide polymorphism (SNP) in the MLH1 promoter region (MLH1-93G/A; rs1800734) has been proposed to be associated with MLH1 promoter methylation, loss of MLH1 protein expression and MSI-H tumors. We examined the association of MLH1-93G/A and six other SNPs surrounding MLH1-93G/A with the methylation status in 210 consecutive sporadic CRCs in Japanese patients. Methylation of the MLH1 promoter region was evaluated by Na-bisulfite polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis. The genotype frequencies of SNPs located in the 54-kb region surrounding the MLH1-93G/A SNP were examined by SSCP analysis. Methylation of the MLH1 promoter region was observed in 28.6% (60/210) of sporadic CRCs. The proportions of MLH1-93G/A genotypes A/A, A/G and G/G were 26% (n=54), 51% (n=108) and 23% (n=48), respectively, and they were significantly associated with the methylation status (p=0.01). There were no significant associations between genotype frequency of the six other SNPs and methylation status. The A-allele of MLH1-93G/A was more common in cases with methylation than the G-allele (p=0.0094), especially in females (p=0.0067). In logistic regression, the A/A genotype of the MLH1-93G/A SNP was shown to be the most significant risk factor for methylation of the MLH1 promoter region (odds ratio 2.82, p=0.003). Furthermore, a haplotype of the A-allele of rs2276807 located -47 kb upstream from the MLH1-93G/A SNP and the A-allele of MLH1-93G/A SNP was significantly associated with MLH1 promoter methylation. These results indicate that individuals, and particularly females, carrying the A-allele at the MLH1-93G/A SNP, especially in association with the A-allele of rs2276807, may harbor an increased risk of methylation of the MLH1 promoter

Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression.

PubMed

Tortarolo, Massimo; Vallarola, Antonio; Lidonnici, Dario; Battaglia, Elisa; Gensano, Francesco; Spaltro, Gabriella; Fiordaliso, Fabio; Corbelli, Alessandro; Garetto, Stefano; Martini, Elisa; Pasetto, Laura; Kallikourdis, Marinos; Bonetto, Valentina; Bendotti, Caterina

2015-10-01

Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies. © 2015 International Society for Neurochemistry.

Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

PubMed

Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

2013-08-01

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Novel PSEN1 G209A mutation in early-onset Alzheimer dementia supported by structural prediction.

PubMed

An, Seong Soo A; Bagyinszky, Eva; Kim, Hye Ryoun; Seok, Ju-Won; Shin, Hae-Won; Bae, SeunOh; Kim, SangYun; Youn, Young Chul

2016-05-20

Three main genes are described as causative genes for early-onset Alzheimer dementia (EOAD): APP, PSEN1 and PSEN2. We describe a woman with EOAD had a novel PSEN1 mutation. A 54-year-old right-handed woman presented 12-year history of progressive memory decline. She was clinically diagnosed as familial Alzheimer's disease due to a PSEN1 mutation. One of two daughters also has the same mutation, G209A in the TM-IV of PS1 protein. Her mother had unspecified dementia that began at the age of 40s. PolyPhen2 and SIFT prediction suggested that G209A might be a damaging variant with high scores. 3D modeling revealed that G209A exchange could result significant changes in the PS1 protein. We report a case of EOAD having probable novel PSEN1 (G209A) mutation verified with structural prediction.

Regulation of Intracellular Copper by Induction of Endogenous Metallothioneins Improves the Disease Course in a Mouse Model of Amyotrophic Lateral Sclerosis.

PubMed

Tokuda, Eiichi; Watanabe, Shunsuke; Okawa, Eriko; Ono, Shin-ichi

2015-04-01

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease. The pathogenesis of the disease is poorly understood, but intracellular copper dyshomeostasis has been implicated as a key process in the disease. We recently observed that metallothioneins (MTs) are an excellent target for the modification of copper dyshomeostasis in a mouse model of ALS (SOD1(G93A)). Here, we offer a therapeutic strategy designed to increase the level of endogenous MTs. The upregulation of endogenous MTs by dexamethasone, a synthetic glucocorticoid, significantly improved the disease course and rescued motor neurons in SOD1(G93A) mice, even if the induction was initiated when peak body weight had decreased by 10%. Neuroprotection was associated with the normalization of copper dyshomeostasis, as well as with decreased levels of SOD1(G93A) aggregates. Importantly, these benefits were clearly mediated in a MT-dependent manner, as dexamethasone did not provide any protection when endogenous MTs were abolished from SOD1(G93A) mice. In conclusion, the upregulation of endogenous MTs represents a promising strategy for the treatment of ALS linked to mutant SOD1.

cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

PubMed

Choi, Jin Sun; Kim, Kidae; Lee, Do Hee; Cho, Sayeon; Ha, Jae Du; Park, Byoung Chul; Kim, Sunhong; Park, Sung Goo; Kim, Jeong-Hoon

2016-11-18

Although the ubiquitin-proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H 2 O 2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

SOD1 Function and Its Implications for Amyotrophic Lateral Sclerosis Pathology: New and Renascent Themes.

PubMed

Bunton-Stasyshyn, Rosie K A; Saccon, Rachele A; Fratta, Pietro; Fisher, Elizabeth M C

2015-10-01

The canonical role of superoxide dismutase 1 (SOD1) is as an antioxidant enzyme protecting the cell from reactive oxygen species toxicity. SOD1 was also the first gene in which mutations were found to be causative for the neurodegenerative disease amyotrophic lateral sclerosis (ALS), more than 20 years ago. ALS is a relentless and incurable mid-life onset disease, which starts with a progressive paralysis and usually leads to death within 3 to 5 years of diagnosis; in the majority of cases, the intellect appears to remain intact while the motor system degenerates. It rapidly became clear that when mutated SOD1 takes on a toxic gain of function in ALS. However, this novel function remains unknown and many cellular systems have been implicated in disease. Now it seems that SOD1 may play a rather larger role in the cell than originally realized, including as a key modulator of glucose signaling (at least so far in yeast) and in RNA binding. Here, we consider some of the new findings for SOD1 in health and disease, which may shed light on how single amino acid changes at sites throughout this protein can cause devastating neurodegeneration in the mammalian motor system. © The Author(s) 2014.

Novel Mutations in HESX1 and PROP1 Genes in Combined Pituitary Hormone Deficiency.

PubMed

Avbelj Stefanija, Magdalena; Kotnik, Primož; Bratanič, Nina; Žerjav Tanšek, Mojca; Bertok, Sara; Bratina, Nataša; Battelino, Tadej; Trebušak Podkrajšek, Katarina

2015-01-01

The HESX1 gene is essential in forebrain development and pituitary organogenesis, and its mutations are the most commonly identified genetic cause of septo-optic dysplasia (SOD). The PROP1 gene is involved in anterior pituitary cell lineage specification and is commonly implicated in non-syndromic combined pituitary hormone deficiency (CPHD). We aimed to assess the involvement of HESX1 and PROP1 mutations in a cohort of patients with SOD and CPHD. Six patients with sporadic SOD and 16 patients with CPHD from 14 pedigrees were screened for mutations in HESX1 and PROP1 genes by exon sequencing. Half of the CPHD patients had variable associated clinical characteristics, such as hearing loss, orofacial cleft, kidney disorder or developmental delay. Novel variants were evaluated in silico and verified in SNP databases. A novel heterozygous p.Glu102Gly mutation in the HESX1 gene and a novel homozygous p.Arg121Thr mutation in the PROP1 gene were detected in 2 pedigrees with CPHD. A small previously reported deletion in PROP1 c.301_302delAG was detected in a separate patient with CPHD, in heterozygous state. No mutations were identified in patients with SOD. Our results expand the spectrum of mutations implicated in CPHD. The frequency of 15% of the PROP1 mutations in CPHD was low, likely due to the clinical heterogeneity of the cohort. © 2015 S. Karger AG, Basel.

Antioxidant enzymes GSR, SOD1, SOD2, and CAT gene variants and bone mineral density values in postmenopausal women: a genetic association analysis.

PubMed

Mlakar, Simona Jurkovic; Osredkar, Josko; Prezelj, Janez; Marc, Janja

2012-03-01

Oxidative stress participates in decreasing bone formation and stimulating bone resorption. Furthermore, antioxidant enzymes have been observed to have low protective activity in women with osteoporosis.The aim of the present study was to examine any association of selected gene polymorphisms of the glutathione S-reductase (GSR), superoxide dismutase (SOD1 and SOD2), and catalase (CAT) genes, alone or in combination, with the bone mineral density (BMD) values of femoral neck (fn), lumbar spine (ls), and total hip (th) in Slovenian postmenopausal women. The gene polymorphisms of CAT, GSR, SOD1, and SOD2 genes in 468 postmenopausal women were analyzed using restriction fragment length polymorphism and a fluorescent 5'-exonuclease genotyping method. BMD_fn, BMD_ls, and BMD_th were measured using dual-energy x-ray absorptiometry. Moreover, univariate statistic analysis and two-way analysis of variance for interaction testing were performed. A significant association of BMD_th values (P = 0.027) was found in genotype subgroups of 423-287G>A GSR polymorphism located in the third intron among postmenopausal women. Furthermore, women with at least one G allele showed significantly higher levels of BMD_fn (P = 0.044), BMD_th (P = 0.009), and BMD_ls (P = 0.043) than those that are AA homozygotes. Interestingly, the 423-287G>A_GSR*1154-393T>A_GSR combination was significantly associated with BMD_fn (P = 0.013) and BMD_th (P = 0.002) in postmenopausal women. The results of our study demonstrate for the first time that antioxidant enzyme GSR gene polymorphisms are significantly associated with BMD, suggesting that the A allele of 423-287G>A GSR polymorphism could contribute to decreased BMD values in postmenopausal women.

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

PubMed Central

Crisp, Matthew J.; Mawuenyega, Kwasi G.; Patterson, Bruce W.; Reddy, Naveen C.; Chott, Robert; Self, Wade K.; Weihl, Conrad C.; Jockel-Balsarotti, Jennifer; Varadhachary, Arun S.; Bucelli, Robert C.; Yarasheski, Kevin E.; Bateman, Randall J.; Miller, Timothy M.

2015-01-01

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins. PMID:26075819

In vivo kinetic approach reveals slow SOD1 turnover in the CNS.

PubMed

Crisp, Matthew J; Mawuenyega, Kwasi G; Patterson, Bruce W; Reddy, Naveen C; Chott, Robert; Self, Wade K; Weihl, Conrad C; Jockel-Balsarotti, Jennifer; Varadhachary, Arun S; Bucelli, Robert C; Yarasheski, Kevin E; Bateman, Randall J; Miller, Timothy M

2015-07-01

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.

FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis

PubMed Central

Deng, Han-Xiang; Zhai, Hong; Bigio, Eileen H.; Yan, Jianhua; Fecto, Faisal; Ajroud, Kaouther; Mishra, Manjari; Ajroud-Driss, Senda; Heller, Scott; Sufit, Robert; Siddique, Nailah; Mugnaini, Enrico; Siddique, Teepu

2014-01-01

Objective Amyotrophic lateral sclerosis (ALS) is a fatal disorder of motor neuron degeneration. Most cases of ALS are sporadic (SALS), but about 5-10% of ALS cases are familial (FALS). Recent studies have shown that mutations in FUS are causal in approximately 4-5% of FALS and some apparent SALS cases. The pathogenic mechanism of the mutant FUS-mediated ALS and potential roles of FUS in non-FUS ALS remain to be investigated. Methods Immunostaining was performed on postmortem spinal cords from 78 ALS cases, including SALS (n=52), ALS with dementia (ALS/dementia, n=10) and FALS (n=16). In addition, postmortem brains or spinal cords from 22 cases with or without frontotemporal lobar degeneration (FTLD) were also studied. In total, 100 cases were studied. Results FUS-immunoreactive inclusions were observed in spinal anterior horn neurons in all sporadic and familial ALS cases, except for those with SOD1 mutations. The FUS-containing inclusions were also immunoreactive with antibodies to TDP43, p62 and ubiquitin. A fraction of tested FUS antibodies recognized FUS inclusions and an unusual antigen retrieval appeared to be important for detection of the skein-like FUS inclusions. Interpretation Although mutations in FUS account for only a small fraction of FALS and SALS, our data suggest that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS and some other neurodegenerative conditions, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/dementia and related disorders. Our data also indicate that SOD1-linked ALS may have a distinct pathogenic pathway from SALS and other types of FALS. PMID:20517935

MLH1-93 G/a polymorphism is associated with MLH1 promoter methylation and protein loss in dysplastic sessile serrated adenomas with BRAFV600E mutation.

PubMed

Fennell, Lochlan J; Jamieson, Saara; McKeone, Diane; Corish, Tracie; Rohdmann, Megan; Furner, Tori; Bettington, Mark; Liu, Cheng; Kawamata, Futoshi; Bond, Catherine; Van De Pols, Jolieke; Leggett, Barbara; Whitehall, Vicki

2018-01-05

Sessile serrated adenomas with BRAF mutation progress rapidly to cancer following the development of dysplasia (SSAD). Approximately 75% of SSADs methylate the mismatch repair gene MLH1, develop mismatch repair deficiency and the resultant cancers have a good prognosis. The remaining SSADs and BRAF mutant traditional serrated adenomas (TSA) develop into microsatellite stable cancers with a poor prognosis. The reason for this dichotomy is unknown. In this study, we assessed the genotypic frequency of the MLH1-93 polymorphism rs1800734 in SSADs and TSAs to determine if the uncommon variant A allele predisposes to MLH1 promoter hypermethylation. We performed genotyping for the MLH1-93 polymorphism, quantitative methylation specific PCR, and MLH1 immunohistochemistry on 124 SSAD, 128 TSA, 203 BRAF mutant CRCs and 147 control subjects with normal colonoscopy. The minor A allele was significantly associated with a dose dependent increase in methylation at the MLH1 promoter in SSADs (p = 0.022). The AA genotype was only observed in SSADs with MLH1 loss. The A allele was also overrepresented in BRAF mutant cancers with MLH1 loss. Only one of the TSAs showed loss of MLH1 and the overall genotype distribution in TSAs did not differ from controls. The MLH1-93 AA genotype is significantly associated with promoter hypermethylation and MLH1 loss in the context of SSADs. BRAF mutant microsatellite stable colorectal cancers with the AA genotype most likely arise in TSAs since the A allele does not predispose to methylation in this context.

Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis.

PubMed

Álvarez-Zaldiernas, Cristina; Lu, Jun; Zheng, Yujuan; Yang, Hongqian; Blasi, Juan; Solsona, Carles; Holmgren, Arne

2016-08-12

Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The identification of HESX1 mutations in Kallmann syndrome

PubMed Central

Newbern, Kayce; Natrajan, Nithya; Kim, Hyung-Goo; Chorich, Lynn .P.; Halvorson, Lisa; Cameron, Richard S.; Layman, Lawrence C.

2013-01-01

Objective To determine if HESX1 mutations are present in patients with idiopathic hypogonadotropic hypogonadism (IHH)/Kallmann syndrome (KS). HESX1 mutations have previously been characterized in patients with septo-optic dysplasia (SOD), isolated growth hormone deficiency (IGHD), and combined pituitary hormone deficiency (CPHD). We hypothesized that IHH/KS represents a milder phenotypic variant of SOD. Design PCR-based DNA sequencing was performed on 217 well-characterized IHH/KS patients. Putative missense mutations were analyzed by sorting intolerant from tolerant (SIFT) and Clustal Ω. Setting An academic medical center Patients 217 IHH/KS and 192 controls Interventions DNA was extracted from patients and controls; genotype/phenotype comparisons were made Main Outcome Measures DNA sequence of HESX1, SIFT analysis, and ortholog alignment Results Two novel heterozygous missense mutations (p.H42Y and p.V75L) and previously reported heterozygous missense mutation p.Q6H in HESX1 were identified in 3/217 (1.4%) patients. All were males with KS. Both p.Q6H and p.H42Y were predicted to be deleterious by SIFT, while p.V75L was conserved in 8/9 species. No other IHH/KS gene mutations were present. Conclusions HESX1 mutations may cause KS in addition to more severe phenotypes. Our findings expand the phenotypic spectrum of HESX1 mutations in humans, thereby broadening its role in development. PMID:23465708

Genetic Polymorphisms in SOD (rs2070424, rs7880) and CAT (rs7943316, rs1001179) Enzymes Are Associated with Increased Body Fat Percentage and Visceral Fat in an Obese Population from Central Mexico.

PubMed

Hernández-Guerrero, César; Hernández-Chávez, Paulina; Romo-Palafox, Inés; Blanco-Melo, Grecia; Parra-Carriedo, Alicia; Pérez-Lizaur, Ana

2016-07-01

Oxidative disturbance is an important factor involved in the etiology of comorbidities associated with obesity. Genetic polymorphisms such as SOD1 -251A>G, SOD2 47 C>T, CAT -21A>T and CAT -262 C>T have been described to alter the activity of antioxidant enzymes. The aim of the present work was to analyze the association of the mentioned SNPs with obesity and their relationship with anthropometric and clinical variables in this group. The study included 416 Mexican women (208 normal weight, NW and 208 subjects with obesity, OB). Dietary intake, anthropometric, biochemical and clinical features were evaluated and then analyzed in function of the genotypes. The mutated carriers (GA+GG) of SOD -251 were significantly higher in the OB group (0.24) compared to the NW group (0.08). The other SNPs showed no differences compared with control group. When comparing carrier mutated subjects with obesity vs. wild-type obese participants with the SNPs SOD1 -251, SOD2 47 and CAT -262, the carriers showed a significantly (p <0.05) higher value in body fat percentage. Also, carriers of SOD2 47 and CAT-262 showed significantly higher values (p = 0.002) and (p = 0.01), respectively, when visceral fat was compared between groups. Systolic blood pressure was significantly higher (p = 0.02) in carriers of mutated CAT-21. SOD1 -251A>G is associated with obesity independent of the presence of diabetes or dyslipidemia. Mutated obese carries of SOD1 -251, SOD2 47 and CAT -262 are associated with a higher distribution of fat in comparison with obese wild-type carriers. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

Carnitine-acylcarnitine translocase deficiency with c.199-10 T>G and novel c.1A>G mutation: Two case reports and brief literature review.

PubMed

Yan, Hui-Ming; Hu, Hao; Ahmed, Aisha; Feng, Bing-Bing; Liu, Jing; Jia, Zheng-Jun; Wang, Hua

2017-11-01

Carnitine-acylcarnitine translocate deficiency (CACTD) is a rare and life-threatening, autosomal recessive disorder of fatty acid β-oxidation characterized by hypoketotic hypoglycemia, hyperammonemia, cardiomyopathy, liver dysfunction, and muscle weakness; culminating in early death. To date, CACTD cases screened from the Chinese mainland population, especially patient with compound heterozygote with c.199-10T>G and a novel c.1A>G mutation in the SLC25A20 gene has never been described. Herein, we report 2 neonatal cases of CACTD identified from the mainland China. These 2 patients were presented with severe metabolic crisis and their clinical conditions deteriorate rapidly and both died of cardiorespiratory collapse in the first week of life. We present the clinical and biochemical features of 2 probands and a brief literature review of previously reported CACTD cases with the c.199-10T>G mutation. The acylcarnitine profiles by tandem-mass-spectrometry and the mutation analysis of SLC25A20 gene confirmed the diagnosis of CACTD in both patients. Mutation analysis demonstrated that patient No. 1 was homozygous for c.199-10T>G mutation, while patient No. 2 was a compound heterozygote for 2 mutations, a maternally-inherited c.199-10T>G and a paternally-inherited, novel c.1A>G mutation. Both patients were treated with an aggressive treatment regimen include high glucose and arginine infusion, respiratory, and circulatory support. The first proband died 3 days after delivery due to sudden cardiac arrest. The second patient's clinical condition, at one time, was improved by high glucose infusion, intravenous arginine, and circulatory support. However, the patient failed to wean from mechanical ventilation. Unfortunately, her parents refused further treatment due to fear of financial burdens. The patient died of congestive heart failure in the 6th day of life. We report the first 2 cases of CACTD identified from the mainland China. Apart from a founder mutation c.199-10T>G

A novel mutation in HESX1 causes combined pituitary hormone deficiency without septo optic dysplasia phenotypes.

PubMed

Takagi, Masaki; Takahashi, Mai; Ohtsu, Yoshiaki; Sato, Takeshi; Narumi, Satoshi; Arakawa, Hirokazu; Hasegawa, Tomonobu

2016-04-25

Heterozygous and/or homozygous HESX1 mutations have been reported to cause isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD), in association with septo optic dysplasia (SOD). We report a novel heterozygous HESX1 mutation in a CPHD patient without SOD phenotypes. The propositus was a one-year-old Japanese girl. Shortly after birth, she was found to be hypoglycemic. She was diagnosed with central adrenal insufficiency based on low cortisol and ACTH at a time of severe hypoglycemia. Further endocrine studies indicated that the patient also had central hypothyroidism and growth hormone deficiency. Using a next-generation sequencing strategy, we identified a novel heterozygous HESX1 mutation, c.326G>A (p.Arg109Gln). Western blotting and subcellular localization revealed no significant difference between wild type and mutant HESX1. Electrophoretic mobility shift assays showed that the mutant HESX1 abrogated DNA-binding ability. Mutant HESX1 was unable to repress PROP1-mediated activation. In conclusion, this study identified Arg109 as a critical residue in the HESX1 protein and extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in HESX1. When multiple genes need to be analyzed for mutations simultaneously, targeted sequence analysis of interesting genomic regions is an attractive approach.

Interference-free determination of sub ng kg-1 levels of long-lived 93Zr in the presence of high concentrations (μg kg-1) of 93Mo and 93Nb using ICP-MS/MS.

PubMed

Petrov, Panayot; Russell, Ben; Douglas, David N; Goenaga-Infante, Heidi

2018-01-01

Long-lived high abundance radionuclides are of increasing interest with regard to decommissioning of nuclear sites and longer term nuclear waste storage and disposal. In many cases, no routine technique is available for their measurement in nuclear waste and low-level (ng kg -1 ) environmental samples. Recent advances in ICP-MS technology offer attractive features for the selective and sensitive determination of a wide range of long-lived radionuclides. In this work, inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS)-based methodology, suitable for accurate routine determinations of 93 Zr at very low (ng kg -1 ) levels in the presence of high levels (μg kg -1 ) of the isobaric interferents 93 Nb and 93 Mo (often present in nuclear waste samples), is reported for the first time. Additionally, a novel and systematic strategy for method development based on the use of non-radioactive isotopes is proposed. It relies on gas-phase chemical reactions for different molecular ion formation to achieve isobaric interference removal. Using cell gas mixtures of NH 3 /He/H 2 or H 2 /O 2 , and suitable mass shifts, the signal from the 93 Nb and 93 Mo isobaric interferences on 93 Zr were suppressed by up to 5 orders of magnitude. The achieved limit of detection for 93 Zr was 1.3 × 10 -5  Bq g -1 (equivalent to 0.14 ng kg -1 ). The sample analysis time is 2 min, which represents a significant improvement in terms of sample throughput, compared to liquid scintillation counting methods. The method described here can be used for routine measurements of 93 Zr at environmentally relevant levels. It can also be combined with radiometric techniques for use towards the standardisation of 93 Zr measurements. Graphical abstract Interference-free determination of 93 Zr in the presence of high concentrations of isobaric 93 Mo and 93 Nb by ICP-MS/MS.

Base substitution mutations in uridinediphosphate-dependent glycosyltransferase 76G1 gene of Stevia rebaudiana causes the low levels of rebaudioside A: mutations in UGT76G1, a key gene of steviol glycosides synthesis.

PubMed

Yang, Yong-Heng; Huang, Su-Zhen; Han, Yu-Lin; Yuan, Hai-Yan; Gu, Chun-Sun; Zhao, Yan-Hai

2014-07-01

Steviol glycosides, extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, are calorie-free sugar substitute of natural origin with intensely sweet (Boileau et al., 2012). Stevioside and rebaudioside A are the two main kinds of the diterpenic glycosides. We analyzed the concentration of stevioside and rebaudioside A in Stevia leaves of about 500 samples (hybrid progenies) and discovered a mutation plant "Z05" with very low levels of rebaudioside A. Because UGT76G1, a uridinediphosphate-dependent glycosyltransferases, is responsible for the conversion from stevioside to rebaudioside A (Richman et al., 2005), so mutation identification was done by sequencing the candidate gene, UGT76G1. In this study molecular analysis of two strains revealed a heterozygotic nonsense mutation of c.389T > G (p.L121X) in UGT76G1. Meanwhile, we found some amino acid substitutions significant change the protein structure. And the difference of enzyme activity between two strains proved the lack of functionality of UGT76G1 of the mutation "Z05". So the nonsense mutation and amino acid substitution mutation resulted in the low levels of rebaudioside A. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Import, maturation, and function of SOD1 and its copper chaperone CCS in the mitochondrial intermembrane space.

PubMed

Kawamata, Hibiki; Manfredi, Giovanni

2010-11-01

Cu, Zn, superoxide dismutase (SOD1) is a ubiquitous enzyme localized in multiple cellular compartments, including mitochondria, where it concentrates in the intermembrane space (IMS). Similar to other small IMS proteins, the import and retention of SOD1 in the IMS is linked to its folding and maturation, involving the formation of critical intra- and intermolecular disulfide bonds. Therefore, the cysteine residues of SOD1 play a fundamental role in its IMS localization. IMS import of SOD1 involves its copper chaperone, CCS, whose mitochondrial distribution is regulated by the Mia40/Erv1 disulfide relay system in a redox-dependent manner: CCS promotes SOD1 maturation and retention in the IMS. The function of SOD1 in the IMS is still unknown, but it is plausible that it serves to remove superoxide released from the mitochondrial respiratory chain. Mutations in SOD1 cause familial amyotrophic lateral sclerosis (ALS), whose pathologic features include mitochondrial bioenergetic dysfunction. Mutant SOD1 localization in the IMS is not dictated by oxygen concentration and the Mia40/Erv1 system, but is primarily dependent on aberrant protein folding and aggregation. Mutant SOD1 localization and aggregation in the IMS might cause the mitochondrial abnormalities observed in familial ALS and could play a significant role in disease pathogenesis.

Association of breast-fed neonatal hyperbilirubinemia with UGT1A1 polymorphisms: 211G>A (G71R) mutation becomes a risk factor under inadequate feeding.

PubMed

Sato, Hiroko; Uchida, Toshihiko; Toyota, Kentaro; Kanno, Miyako; Hashimoto, Taeko; Watanabe, Masashi; Nakamura, Tomohiro; Tamiya, Gen; Aoki, Kuraaki; Hayasaka, Kiyoshi

2013-01-01

Breastfeeding jaundice is a well-known phenomenon, but its pathogenesis is still unclear. Increased production of bilirubin, impaired hepatic uptake and metabolism of bilirubin, and increased enterohepatic circulation of bilirubin account for most cases of pathological neonatal hyperbilirubinemia. We previously reported that 211G>A (G71R) mutation of the UGT1A1 gene is prevalent in East Asians and is associated with the development of neonatal hyperbilirubinemia. Recently, significant association of G71R mutation with hyperbilirubinemia in breast-fed neonates was reported. We enrolled 401 full-term Japanese infants, who were exclusively breast-fed without supplementation of formula before developing hyperbilirubinemia, and classified them into two groups based on the degree of maximal body weight loss during the neonatal period. We analyzed the sex, gestational age, delivery mode, body weight at birth, maximal body weight loss and genotypes of G71R and (TA)(7) polymorphic mutations of UGT1A1. Statistical analysis revealed that maximal body weight loss during the neonatal period is the only independent risk factor for the development of neonatal hyperbilirubinemia. The effect of G71R mutation on neonatal hyperbilirubinemia is significant in neonates with 5% or greater maximal body weight loss and its influence increases in parallel with the degree of maximal body weight loss. Our study indicates that G71R mutation is a risk factor for neonatal hyperbilirubinemia only in infants with inadequate breastfeeding and suggests that adequate breastfeeding may overcome the genetic predisposing factor, G71R mutation, for the development of neonatal hyperbilirubinemia.

CHEK2 1100delC, IVS2+1G>A and I157T mutations are not present in colorectal cancer cases from Turkish population.

PubMed

Bayram, Süleyman; Topaktaş, Mehmet; Akkız, Hikmet; Bekar, Aynur; Akgöllü, Ersin

2012-10-01

The cell cycle checkpoint kinase 2 (CHEK2) protein participates in the DNA damage response in many cell types. Germline mutations in CHEK2 (1100delC, IVS2+1G>A and I157T) have been impaired serine/threonine kinase activity and associated with a range of cancer types. This hospital-based case-control study aimed to investigate whether CHEK2 1100delC, IVS2+1G>A and I157T mutations play an important role in the development of colorectal cancer (CRC) in Turkish population. A total of 210 CRC cases and 446 cancer-free controls were genotyped for CHEK2 mutations by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific-polymerase chain reaction (AS-PCR) methods. We did not find the CHEK2 1100delC, IVS2+1G>A and I157T mutations in any of the Turkish subjects. Our result demonstrate for the first time that CHEK2 1100delC, IVS2+1G>A and I157T mutations have not been agenetic susceptibility factor for CRC in the Turkish population. Overall, our data suggest that genotyping of CHEK2 mutations in clinical settings in the Turkish population should not be recommended. However, independent studies are need to validate our findings in a larger series, as well as in patients of different ethnic origins. Copyright © 2012 Elsevier Ltd. All rights reserved.

[Diabetes mellitus associated with the mitochondrial mutation A3243G: frequency and clinical presentation].

PubMed

Salles, João Eduardo N; Kalinin, Larissa Bresgunov; Ferreira, Sandra Roberta G; Kasamatsu, Teresa; Moisés, Regina S

2007-06-01

Maternal inherited diabetes and deafness (MIDD) has been related to an A to G transition in the mitochondrial RNA Leu (UUR) at base pair 3243. The prevalence of MIDD in the diabetes population ranges between 0.5-3.0% depending on the ethnic background. To examine the frequency and clinical features of diabetes associated with this mutation in Brazilian patients with glucose intolerance. The study population comprised: 78 type 1 diabetic subjects (group I), 148 patients with type 2 diabetes (group II), 15 patients with either type 1 or type 2 diabetes and hearing loss (group III) and 492 Japanese Brazilians with varying degrees of glucose intolerance. DNA was extracted from peripheral blood leucocytes and the A3243G mutation was determined by PCR amplification and Apa 1 digestion. In some individuals DNA was also extracted from buccal mucosa and hair follicles. The 3243 bp mutation was found in three individuals, all from group III, resulting in a prevalence of 0.4%. These subjects had an early age of diagnosis of diabetes, low or normal body mass index and requirement of insulin therapy. In conclusion MIDD is rare in our population and should be investigate in patients with diabetes and deafness.

R248G cystic fibrosis transmembrane conductance regulator mutation in three siblings presenting with recurrent acute pancreatitis and reproductive issues: a case series.

PubMed

Villalona, Seiichi; Glover-López, Guillermo; Ortega-García, Juan Antonio; Moya-Quiles, Rosa; Mondejar-López, Pedro; Martínez-Romero, Maria C; Rigabert-Montiel, Mariano; Pastor-Vivero, María D; Sánchez-Solís, Manuel

2017-02-15

Mutational combinations of the cystic fibrosis transmembrane conductance regulator, CFTR, gene have different phenotypic manifestations at the molecular level with varying clinical consequences for individuals possessing such mutations. Reporting cystic fibrosis transmembrane conductance regulator mutations is important in understanding the genotype-phenotype correlations and associated clinical presentations in patients with cystic fibrosis. Understanding the effects of mutations is critical in developing appropriate treatments for individuals affected with cystic fibrosis, non-classic cystic fibrosis, or cystic fibrosis transmembrane conductance regulator-related disorders. This is the first report of related individuals possessing the R248G missense cystic fibrosis transmembrane conductance regulator mutation and we present their associated clinical histories. All three patients are of Spanish descent. Deoxyribonucleic acid analysis revealed that all three siblings possessed a novel c.742A>G mutation, resulting in a p.Arg248Gly (R248G) amino acid change in exon 6 in trans with the known N1303K mutant allele. Case 1 patient is a 39-year-old infertile man presenting with congenital unilateral absence of the vas deferens and recurrent episodes of epigastric pain. Case 2 patient is a 32-year-old woman presenting with periods of infertility, two previous spontaneous abortions, recurrent epigastric pain, and recurrent pancreatitis. Case 3 patient is a 29-year-old woman presenting with recurrent pancreatitis and epigastric pain. We report the genotype-phenotype correlations and clinical manifestations of a novel R248G cystic fibrosis transmembrane conductance regulator mutation: congenital unilateral absence of the vas deferens in males, reduced female fertility, and recurrent acute pancreatitis. In addition, we discuss the possible functional consequences of the mutations at the molecular level.

SOD1 oxidation and formation of soluble aggregates in yeast: Relevance to sporadic ALS development

PubMed Central

Martins, Dorival; English, Ann M.

2014-01-01

Misfolding and aggregation of copper–zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS. PMID:24936435

Contribution of the MLH1 -93G>a promoter polymorphism in modulating susceptibility risk in Malaysian colorectal cancer patients.

PubMed

Nizam, Zahary Mohd; Abdul Aziz, Ahmad Aizat; Kaur, Gurjeet; Abu Hassan, Muhammad Radzi; Mohd Sidek, Ahmad Shanwani; Yeh, Lee Yeong; Mazuwin, Maya; Ankathil, Ravindran

2013-01-01

Colorectal cancer (CRC) exists in a more common sporadic form and less common hereditary forms, associated with the Lynch syndrome, familial adenomatous polyposis (FAP) and other rare syndromes. Sporadic CRC is believed to arise as a result of close interaction between environmental factors, including dietary and lifestyle habits, and genetic predisposition factors. In contrast, hereditary forms such as those related to the Lynch syndrome result from inheritance of germline mutations of mismatch repair (MMR) genes. However, in certain cases, the influence of low penetrance alleles in familial colorectal cancer susceptibility is also undeniable. To investigate the genotype frequencies of MLH1 promoter polymorphism -93G>A and to determine whether it could play any role in modulating familial and sporadic CRC susceptibility risk. A case-control study comprising of 104 histopathologically confirmed CRC patients as cases (52 sporadic CRC and 52 Lynch syndrome patients) and 104 normal healthy individuals as controls was undertaken. DNA was extracted from peripheral blood and the polymorphism was genotyped employing PCR-RFLP methods. The genotypes were categorized into homozygous wild type, heterozygous and homozygous variants. The risk association between these polymorphisms and CRC susceptibility risk was calculated using binary logistic regression analysis and deriving odds ratios (ORs). When risk association was investigated for all CRC patients as a single group, the heterozygous (G/A) genotype showed a significantly higher risk for CRC susceptibility with an OR of 2.273, (95%CI: 1.133-4.558 and p-value=0.021). When analyzed specifically for the 2 types of CRC, the heterozygous (G/A) genotype showed significantly higher risk for sporadic CRC susceptibility with and OR of 3.714, (95%CI: 1.416-9.740 and p-value=0.008). Despite high OR value was observed for Lynch syndrome (OR: 1.600, 95%CI: 0.715-3.581), the risk was not statistically significant (P=0.253). Our results

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

PubMed Central

Tang, Xiaohu; Seyb, Kathleen I.; Huang, Mickey; Schuman, Eli R.; Shi, Ping; Zhu, Haining; Glicksman, Marcie A.

2013-01-01

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells. PMID:22140121

Superoxide Dismutases, SOD1 and SOD2, Play a Distinct Role in the Fat Body during Pupation in Silkworm Bombyx mori

PubMed Central

Nojima, Yosui; Ito, Katsuhiko; Ono, Hiromasa; Nakazato, Takeru; Bono, Hidemasa; Yokoyama, Takeshi; Sato, Ryoichi; Suetsugu, Yoshitaka; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Tabunoki, Hiroko; Fugo, Hajime

2015-01-01

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation. PMID:25714339

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation*

PubMed Central

Girotto, Stefania; Cendron, Laura; Bisaglia, Marco; Tessari, Isabella; Mammi, Stefano; Zanotti, Giuseppe; Bubacco, Luigi

2014-01-01

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis. PMID:24567322

Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.

PubMed

Bhinge, Akshay; Namboori, Seema C; Zhang, Xiaoyu; VanDongen, Antonius M J; Stanton, Lawrence W

2017-04-11

Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

PubMed

Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The mitochondrial DNA 10197 G > A mutation causes MELAS/Leigh overlap syndrome presenting with acute auditory agnosia.

PubMed

Leng, Yinglin; Liu, Yuhe; Fang, Xiaojing; Li, Yao; Yu, Lei; Yuan, Yun; Wang, Zhaoxia

2015-04-01

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes/Leigh (MELAS/LS) overlap syndrome is a mitochondrial disorder subtype with clinical and magnetic resonance imaging (MRI) features that are characteristic of both MELAS and Leigh syndrome (LS). Here, we report an MELAS/LS case presenting with cortical deafness and seizures. Cranial MRI revealed multiple lesions involving bilateral temporal lobes, the basal ganglia and the brainstem, which conformed to neuroimaging features of both MELAS and LS. Whole mitochondrial DNA (mtDNA) sequencing and PCR-RFLP revealed a de novo heteroplasmic m.10197 G > A mutation in the NADH dehydrogenase subunit 3 gene (ND3), which was predicted to cause an alanine to threonine substitution at amino acid 47. Although the mtDNA m.10197 G > A mutation has been reported in association with LS, Leber hereditary optic neuropathy and dystonia, it has never been linked with MELAS/LS overlap syndrome. Our patient therefore expands the phenotypic spectrum of the mtDNA m.10197 G > A mutation.

STS-93 Post Flight Presentation

NASA Technical Reports Server (NTRS)

1999-01-01

An overview of Flight STS-93 is presented. The primary objective of the STS-93 mission was to deploy the Advanced X-Ray Astrophysics Facility (AXAF), also known as the Chandra X-ray Observatory. The mission flew on the Columbia Shuttle, on July 22, 1999. This facility is the most sophisticated X-ray observatory ever built. Other payloads on STS-93 were: (1) the Midcourse Space Experiment (MSX), (2) Shuttle Ionospheric Modification with Pulsed Local Exhaust (SIMPLEX), (3) Southwest Ultraviolet Imaging System (SWUIS), (4) Gelation of Sols: Applied Microgravity Research (GOSAMR), Space Tissue Loss-B (STL-B), (5) Light Weight Flexible Solar Array Hinge (LFSAH), (6) Cell Culture Module (CCM), and (7) the Shuttle Amateur Radio Experiment-II (SAREX-II), (8) EarthKam, (9) Plant Growth Investigations in Microgravity (PGIM), (10) Commercial Generic Bioprocessing Apparatus (CGBA), (11) Micro-Electrical Mechanical System (MEMS), and (12) the Biological Research in Canisters (BRIC). The crew was: Eileen M. Collins, Mission Commander, the first female shuttle commander; Jeffrey S. Ashby, Pilot; Steven A. Hawley , Mission Specialist; Catherine G. Coleman, Mission Specialist; Michel Tognini (CNES), Mission Specialist. The video contains views of life aboard the space shuttle. This mission featured both a night launching and a night landing at the Kennedy Space Center.

Identification of a novel COL1A1 frameshift mutation, c.700delG, in a Chinese osteogenesis imperfecta family

PubMed Central

Wang, Xiran; Pei, Yu; Dou, Jingtao; Lu, Juming; Li, Jian; Lv, Zhaohui

2015-01-01

Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI. PMID:25983617

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle.

PubMed

Sakellariou, Giorgos K; McDonagh, Brian; Porter, Helen; Giakoumaki, Ifigeneia I; Earl, Kate E; Nye, Gareth A; Vasilaki, Aphrodite; Brooks, Susan V; Richardson, Arlan; Van Remmen, Holly; McArdle, Anne; Jackson, Malcolm J

2018-02-01

Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1 -/- ) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1 -/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1 -/- mice, we characterized neuromuscular changes in the Sod1 -/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. In contrast to mSod1KO mice, myofiber atrophy in Sod1 -/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1 -/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1 -/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1 -/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1 -/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1 -/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS)

PubMed Central

Sangwan, Smriti; Zhao, Anni; Adams, Katrina L.; Jayson, Christina K.; Sawaya, Michael R.; Guenther, Elizabeth L.; Pan, Albert C.; Ngo, Jennifer; Moore, Destaye M.; Soriaga, Angela B.; Do, Thanh D.; Goldschmidt, Lukasz; Nelson, Rebecca; Bowers, Michael T.; Koehler, Carla M.; Shaw, David E.; Novitch, Bennett G.; Eisenberg, David S.

2017-01-01

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS. PMID:28760994

Novel mutations of MYO7A and USH1G in Israeli Arab families with Usher syndrome type 1.

PubMed

Rizel, Leah; Safieh, Christine; Shalev, Stavit A; Mezer, Eedy; Jabaly-Habib, Haneen; Ben-Neriah, Ziva; Chervinsky, Elena; Briscoe, Daniel; Ben-Yosef, Tamar

2011-01-01

This study investigated the genetic basis for Usher syndrome type 1 (USH1) in four consanguineous Israeli Arab families. Haplotype analysis for all known USH1 loci was performed in each family. In families for which haplotype analysis was inconclusive, we performed genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array. For mutation analysis, specific primers were used to PCR amplify the coding exons of the MYO7A, USH1C, and USH1G genes including intron-exon boundaries. Mutation screening was performed with direct sequencing. A combination of haplotype analysis and genome-wide homozygosity mapping indicated linkage to the USH1B locus in two families, USH1C in one family and USH1G in another family. Sequence analysis of the relevant genes (MYO7A, USH1C, and USH1G) led to the identification of pathogenic mutations in all families. Two of the identified mutations are novel (c.1135-1147dup in MYO7A and c.206-207insC in USH1G). USH1 is a genetically heterogenous condition. Of the five USH1 genes identified to date, USH1C and USH1G are the rarest contributors to USH1 etiology worldwide. It is therefore interesting that two of the four Israeli Arab families reported here have mutations in these two genes. This finding further demonstrates the unique genetic structure of the Israeli population in general, and the Israeli Arab population in particular, which due to high rates of consanguinity segregates many rare autosomal recessive genetic conditions.

The Omega-3 Fatty Acid Eicosapentaenoic Acid Accelerates Disease Progression in a Model of Amyotrophic Lateral Sclerosis

PubMed Central

Gladman, Stacy; Biggio, Maria Luigia; Marino, Marianna; Jayasinghe, Maduka; Ullah, Farhan; Dyall, Simon C.; Malaspina, Andrea; Bendotti, Caterina; Michael-Titus, Adina

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease characterised by loss of motor neurons that currently has no cure. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have many health benefits including neuroprotective and myoprotective potential. We tested the hypothesis that a high level of dietary EPA could exert beneficial effects in ALS. The dietary exposure to EPA (300 mg/kg/day) in a well-established mouse model of ALS expressing the G93A superoxide dismutase 1 (SOD1) mutation was initiated at a pre-symptomatic or symptomatic stage, and the disease progression was monitored until the end stage. Daily dietary EPA exposure initiated at the disease onset did not significantly alter disease presentation and progression. In contrast, EPA treatment initiated at the pre-symptomatic stage induced a significantly shorter lifespan. In a separate group of animals sacrificed before the end stage, the tissue analysis showed that the vacuolisation detected in G93A-SOD1 mice was significantly increased by exposure to EPA. Although EPA did not alter motor neurone loss, EPA reversed the significant increase in activated microglia and the astrocytic activation seen in G93A-SOD1 mice. The microglia in the spinal cord of G93A-SOD1 mice treated with EPA showed a significant increase in 4-hydroxy-2-hexenal, a highly toxic aldehydic oxidation product of omega-3 fatty acids. These data show that dietary EPA supplementation in ALS has the potential to worsen the condition and accelerate the disease progression. This suggests that great caution should be exerted when considering dietary omega-3 fatty acid supplements in ALS patients. PMID:23620776

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid

PubMed Central

Leal, Sónia S.; Cardoso, Isabel; Valentine, Joan S.; Gomes, Cláudio M.

2013-01-01

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca2+ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca2+. We show that at physiological pH, Ca2+ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca2+ boosts the onset of SOD1 aggregation. In agreement, Ca2+ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca2+ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca2+-induced aggregates, thus indicating that Ca2+ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca2+ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca2+ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions. PMID:23861388

Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons.

PubMed

Petrosyan, Agavni; Hsieh, I-Hui; Phillips, John P; Saberi, Kourosh

2015-03-01

Mutation of the human gene superoxide dismutase (hSOD1) is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig's disease). Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophila's motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50 days). Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking) behavior in late life when flies had lost the ability to fly (age ≥ 60 d). hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life.

Mitochondrial dynamics and bioenergetic dysfunction is associated with synaptic alterations in mutant SOD1 motor neurons

PubMed Central

Magrané, Jordi; Sahawneh, Mary Anne; Przedborski, Serge; Estévez, Álvaro G.; Manfredi, Giovanni

2012-01-01

Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Taken together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS. PMID:22219285

Transcriptomic indices of fast and slow disease progression in two mouse models of amyotrophic lateral sclerosis.

PubMed

Nardo, Giovanni; Iennaco, Raffaele; Fusi, Nicolò; Heath, Paul R; Marino, Marianna; Trolese, Maria C; Ferraiuolo, Laura; Lawrence, Neil; Shaw, Pamela J; Bendotti, Caterina

2013-11-01

Amyotrophic lateral sclerosis is heterogeneous with high variability in the speed of progression even in cases with a defined genetic cause such as superoxide dismutase 1 (SOD1) mutations. We reported that SOD1(G93A) mice on distinct genetic backgrounds (C57 and 129Sv) show consistent phenotypic differences in speed of disease progression and life-span that are not explained by differences in human SOD1 transgene copy number or the burden of mutant SOD1 protein within the nervous system. We aimed to compare the gene expression profiles of motor neurons from these two SOD1(G93A) mouse strains to discover the molecular mechanisms contributing to the distinct phenotypes and to identify factors underlying fast and slow disease progression. Lumbar spinal motor neurons from the two SOD1(G93A) mouse strains were isolated by laser capture microdissection and transcriptome analysis was conducted at four stages of disease. We identified marked differences in the motor neuron transcriptome between the two mice strains at disease onset, with a dramatic reduction of gene expression in the rapidly progressive (129Sv-SOD1(G93A)) compared with the slowly progressing mutant SOD1 mice (C57-SOD1(G93A)) (1276 versus 346; Q-value ≤ 0.01). Gene ontology pathway analysis of the transcriptional profile from 129Sv-SOD1(G93A) mice showed marked downregulation of specific pathways involved in mitochondrial function, as well as predicted deficiencies in protein degradation and axonal transport mechanisms. In contrast, the transcriptional profile from C57-SOD1(G93A) mice with the more benign disease course, revealed strong gene enrichment relating to immune system processes compared with 129Sv-SOD1(G93A) mice. Motor neurons from the more benign mutant strain demonstrated striking complement activation, over-expressing genes normally involved in immune cell function. We validated through immunohistochemistry increased expression of the C3 complement subunit and major histocompatibility

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

NASA Astrophysics Data System (ADS)

Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

2009-11-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

A novel WFS1 mutation in a family with dominant low frequency sensorineural hearing loss with normal VEMP and EcochG findings

PubMed Central

Bramhall, Naomi F; Kallman, Jeremy C; Verrall, Aimee M; Street, Valerie A

2008-01-01

Background Low frequency sensorineural hearing loss (LFSNHL) is an uncommon clinical finding. Mutations within three different identified genes (DIAPH1, MYO7A, and WFS1) are known to cause LFSNHL. The majority of hereditary LFSNHL is associated with heterozygous mutations in the WFS1 gene (wolframin protein). The goal of this study was to use genetic analysis to determine if a small American family's hereditary LFSNHL is linked to a mutation in the WFS1 gene and to use VEMP and EcochG testing to further characterize the family's audiovestibular phenotype. Methods The clinical phenotype of the American family was characterized by audiologic testing, vestibular evoked myogenic potentials (VEMP), and electrocochleography (EcochG) evaluation. Genetic characterization was performed by microsatellite analysis and direct sequencing of WFS1 for mutation detection. Results Sequence analysis of the WFS1 gene revealed a novel heterozygous mutation at c.2054G>C predicting a p.R685P amino acid substitution in wolframin. The c.2054G>C mutation segregates faithfully with hearing loss in the family and is absent in 230 control chromosomes. The p.R685 residue is located within the hydrophilic C-terminus of wolframin and is conserved across species. The VEMP and EcochG findings were normal in individuals segregating the WFS1 c.2054G>C mutation. Conclusion We discovered a novel heterozygous missense mutation in exon 8 of WFS1 predicting a p.R685P amino acid substitution that is likely to underlie the LFSNHL phenotype in the American family. For the first time, we describe VEMP and EcochG findings for individuals segregating a heterozygous WFS1 mutation. PMID:18518985

A novel WFS1 mutation in a family with dominant low frequency sensorineural hearing loss with normal VEMP and EcochG findings.

PubMed

Bramhall, Naomi F; Kallman, Jeremy C; Verrall, Aimee M; Street, Valerie A

2008-06-02

Low frequency sensorineural hearing loss (LFSNHL) is an uncommon clinical finding. Mutations within three different identified genes (DIAPH1, MYO7A, and WFS1) are known to cause LFSNHL. The majority of hereditary LFSNHL is associated with heterozygous mutations in the WFS1 gene (wolframin protein). The goal of this study was to use genetic analysis to determine if a small American family's hereditary LFSNHL is linked to a mutation in the WFS1 gene and to use VEMP and EcochG testing to further characterize the family's audiovestibular phenotype. The clinical phenotype of the American family was characterized by audiologic testing, vestibular evoked myogenic potentials (VEMP), and electrocochleography (EcochG) evaluation. Genetic characterization was performed by microsatellite analysis and direct sequencing of WFS1 for mutation detection. Sequence analysis of the WFS1 gene revealed a novel heterozygous mutation at c.2054G>C predicting a p.R685P amino acid substitution in wolframin. The c.2054G>C mutation segregates faithfully with hearing loss in the family and is absent in 230 control chromosomes. The p.R685 residue is located within the hydrophilic C-terminus of wolframin and is conserved across species. The VEMP and EcochG findings were normal in individuals segregating the WFS1 c.2054G>C mutation. We discovered a novel heterozygous missense mutation in exon 8 of WFS1 predicting a p.R685P amino acid substitution that is likely to underlie the LFSNHL phenotype in the American family. For the first time, we describe VEMP and EcochG findings for individuals segregating a heterozygous WFS1 mutation.

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu, Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

PubMed Central

Ibrahim, Osama M. A.; Dogru, Murat; Matsumoto, Yukihiro; Igarashi, Ayako; Kojima, Takashi; Wakamatsu, Tais Hitomi; Inaba, Takaaki; Shimizu, Takahiko; Shimazaki, Jun; Tsubota, Kazuo

2014-01-01

Purpose The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 −/−) mouse. Methods Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 −/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed. Results Corneal vital staining scores in the Sod1 −/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 −/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 −/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 −/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 −/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 −/− mice. Conclusions Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 −/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations. PMID:25036096

Identification of Bangladeshi domestic cats with GM1 gangliosidosis caused by the c.1448G>C mutation of the feline GLB1 gene: case study.

PubMed

Uddin, Mohammad Mejbah; Hossain, Mohammad Alamgir; Rahman, Mohammad Mahbubur; Chowdhury, Morshedul Alam; Tanimoto, Takeshi; Yabuki, Akira; Mizukami, Keijiro; Chang, Hye-Sook; Yamato, Osamu

2013-01-01

GM1 gangliosidosis is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the β-galactosidase (GLB1) gene. In feline GM1 gangliosidosis, a pathogenic mutation (c.1448G>C) in the feline GLB1 gene was identified in Siamese cats in the United States and Japan and in Korat cats in Western countries. The present study found the homozygous c.1448G>C mutation in 2 apparent littermate native kittens in Bangladesh that were exhibiting neurological signs. This is the first identification of GM1 gangliosidosis in native domestic cats in Southeast Asia. This pathogenic mutation seems to have been present in the domestic cat population in the Siamese region and may have been transferred to pure breeds such as Siamese and Korat cats originating in this region.

Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors

PubMed

Mohamed Yusoff, Abdul Aziz; Zulfakhar, Fatin Najwa; Sul’ain, Mohd Dasuki; Idris, Zamzuri; Abdullah, Jafri Malin

2016-12-01

Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors. Creative Commons Attribution License

Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors

PubMed Central

Yusoff, Abdul Aziz Mohamed; Zulfakhar, Fatin Najwa; Sul’ain, Mohd Dasuki; Idris, Zamzuri; Abdullah, Jafri Malin

2016-01-01

Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors. PMID:28125199

Adult onset Leigh syndrome with mitochondrial DNA 8344 A>G mutation.

PubMed

Han, Jee-Young; Sung, Jung-Joon; Park, Hong-Kyun; Yoon, Byung-Nam; Lee, Kwang-Woo

2014-11-01

We report a pedigree of adult-onset Leigh syndrome (LS) with mitochondrial mutation 8344 A>G. A 38-year-old woman presented with optic neuropathy, weakness and cognitive impairment. Family history of optic neuropathy and systemic involvement was suggestive of mitochondrial encephalopathy. Genetic and radiologic studies showed m.8344 A>G mutation with characteristics of LS. To our knowledge this is the first case of adult-onset LS demonstrating the m.8344 A>G mutation. Copyright © 2014 Elsevier Ltd. All rights reserved.

MicroRNA-206: A Potential Circulating Biomarker Candidate for Amyotrophic Lateral Sclerosis

PubMed Central

Toivonen, Janne M.; Manzano, Raquel; Oliván, Sara; Zaragoza, Pilar; García-Redondo, Alberto; Osta, Rosario

2014-01-01

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. Biomarkers are urgently needed to facilitate ALS diagnosis and prognosis, and as indicators of therapeutic response in clinical trials. microRNAs (miRNAs), small posttranscriptional modifiers of gene expression, are frequently altered in disease conditions. Besides their important regulatory role in variety of biological processes, miRNAs can also be released into the circulation by pathologically affected tissues and display remarkable stability in body fluids. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. To find biomarkers for ALS, we studied miRNA alterations from skeletal muscle and plasma of SOD1-G93A mice, and subsequently tested the levels of the affected miRNAs in the serum from human ALS patients. Fast-twitch and slow-twitch muscles from symptomatic SOD1-G93A mice (age 90 days) and their control littermates were first studied using miRNA microarrays and then evaluated with quantitative PCR from five age groups from neonatal to the terminal disease stage (10–120 days). Among those miRNA changed in various age/gender/muscle groups (miR-206, -1, -133a, -133b, -145, -21, -24), miR-206 was the only one consistently altered during the course of the disease pathology. In both sexes, mature miR-206 was increased in fast-twitch muscles preferably affected in the SOD1-G93A model, with highest expression towards the most severely affected animals. Importantly, miR-206 was also increased in the circulation of symptomatic animals and in a group of 12 definite ALS patients tested. We conclude that miR-206 is elevated in the circulation of symptomatic SOD1-G93A mice and possibly in human ALS patients. Although larger scale studies on ALS patients are warranted, miR-206 is a promising candidate

The dynamic DNA methylation landscape of the mutL homolog 1 shore is altered by MLH1-93G>A polymorphism in normal tissues and colorectal cancer.

PubMed

Savio, Andrea J; Mrkonjic, Miralem; Lemire, Mathieu; Gallinger, Steven; Knight, Julia A; Bapat, Bharat

2017-01-01

Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 ( MLH1 ), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 ( MLH1 -93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites . To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs ( P  < 0.01). When shore methylation levels were stratified by SNP genotype, normal colorectal DNA and PBMC DNA were significantly hypomethylated in association with variant SNP genotype ( P  < 0.05). However, this association was lost in tumour DNA. Among distinct stages of CRC, metastatic stage IV CRC tumours incurred significant hypomethylation compared to stage I-III cases, irrespective of genotype status. Shore methylation of MLH1 was not associated with MSI status or promoter CpG island hypermethylation, regardless of genotype. To confirm these results, bisulfite sequencing was performed in matched tumour and normal colorectal specimens from six CRC cases, including two cases per genotype (wildtype, heterozygous, and homozygous variant). Bisulfite sequencing

The anabolic/androgenic steroid nandrolone exacerbates gene expression modifications induced by mutant SOD1 in muscles of mice models of amyotrophic lateral sclerosis

PubMed Central

Galbiati, Mariarita; Onesto, Elisa; Zito, Arianna; Crippa, Valeria; Rusmini, Paola; Mariotti, Raffaella; Bentivoglio, Marina; Bendotti, Caterina; Poletti, Angelo

2012-01-01

Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS. PMID:22178654

The anabolic/androgenic steroid nandrolone exacerbates gene expression modifications induced by mutant SOD1 in muscles of mice models of amyotrophic lateral sclerosis.

PubMed

Galbiati, Mariarita; Onesto, Elisa; Zito, Arianna; Crippa, Valeria; Rusmini, Paola; Mariotti, Raffaella; Bentivoglio, Marina; Bendotti, Caterina; Poletti, Angelo

2012-02-01

Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS. Copyright © 2011 Elsevier Ltd. All rights reserved.

PROP1 gene mutations in a 36-year-old female presenting with psychosis

PubMed Central

Rijal, Tshristi; Jha, Kunal Kishor; Saluja, Harpreet

2017-01-01

Summary Combined pituitary hormonal deficiency (CPHD) is a rare disease that results from mutations in genes coding for transcription factors that regulate the differentiation of pituitary cells. PROP1 gene mutations are one of the etiological diagnoses of congenital panhypopituitarism, however symptoms vary depending on phenotypic expression. We present a case of psychosis in a 36-year-old female with congenital panhypopituitarism who presented with paranoia, flat affect and ideas of reference without a delirious mental state, which resolved with hormone replacement and antipsychotics. Further evaluation revealed that she had a homozygous mutation of PROP1 gene. In summary, compliance with hormonal therapy for patients with hypopituitarism appears to be effective for the prevention and treatment of acute psychosis symptoms. Learning points: Patients with PROP1 gene mutation may present with psychosis with no impairment in orientation and memory. There is currently inadequate literature on this topic, and further study on the possible mechanisms of psychosis as a result of endocrine disturbance is required. Compliance with hormonal therapy for patients with hypopituitarism appears to be effective for prevention and treatment of acute psychosis symptoms. PMID:28458894

Decreased mutation frequencies among immunoglobulin G variable region genes during viremic HIV-1 infection.

PubMed

Bowers, Elisabeth; Scamurra, Ronald W; Asrani, Anil; Beniguel, Lydie; MaWhinney, Samantha; Keays, Kathryne M; Thurn, Joseph R; Janoff, Edward N

2014-01-01

HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10±5% vs. 13.5±6%, p = 0.03) and amino acids (CDR: 20%±10 vs. 25%±12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

Gaucher disease: A G[sup +1][yields]A[sup +1] IVS2 splice donor site mutation causing exon 2 skipping in the acid [beta]-glucosidase mRNA

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

He, Guo-Shun; Grabowski, G.A.

1992-10-01

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid [beta]-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-years-old, enzyme-deficient, 1226G (Asn[sup 370][yields]Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 ([Delta] EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 ([Delta] EX2-3), or a completely normal sequence. Aboutmore » 50% of the cDNAs were the [Delta] EX2, the [Delta] EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5[prime] and 3[prime] intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G[sup +1][yields]A[sup +1] transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed [open quotes]IVS2 G[sup +1],[close quotes] is the first in the Ashkenazi Jewish population. The occurrence of this [open quotes]pseudogene[close quotes]-type mutation in the structural gene indicates the role of acid [beta]-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease. 33 refs., 8 figs., 1 tab.« less

Polymorphism of MSH2 Gly322Asp and MLH1 –93G>A in non-familial colon cancer – a case-controlled study

PubMed Central

Dziki, Lukasz; Malinowska, Katarzyna; Trzcinski, Radzislaw; Majsterek, Ireneusz; Dziki, Adam

2017-01-01

Introduction Our aim was to determine the effect of the single nucleotide polymorphisms (SNP) –93G>A of the MLH1 gene (rs1800734) and Gly322Asp of the MSH2 gene (rs4987188) on the risk of colon cancer (CC) and identify any relationship with clinical factors. Material and methods The study included 144 unrelated patients with sporadic CC (71 males; mean age: 61.7 ±11 years) and 151 control patients (74 males; mean age: 63 ±11 years). DNA was extracted from peripheral blood lymphocytes, and genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Results In our population, the homozygous G/G genotype of the –93G>AMLH1 gene increased the risk of sporadic CC (OR = 2.07; 95% CI: 1.11–3.83; p < 0.02). For A/G and A/A genotypes, the MLH1-93G>A polymorphism was significantly more common in women (p = 0.034). The SNP demonstrated differences in allele distribution according to the location of the tumor, i.e. right vs. left side (p = 0.014), and disease recurrence (p = 0.022). Significant differences were found in the occurrence of Gly322Asp of MSH2 with regard to primary and recurrent disease (p = 0.001). Conclusions The –93G>AMLH1 polymorphism plays an important role in evaluating the risk of sporadic CC. It can also be used as an indicator in some patients with left-sided and recurrent tumors. MSH2 Gly322Asp is a potential marker in patients with risk of recurrence. PMID:29181059

Compound Heterozygosity for Hb Alperton (HBB: c.407C>T) and IVS-I-5 (G>C) (HBB: c.92+5G>C) Mutations Presenting as a Moderate Anemia in an Indian Family.

PubMed

Godbole, Koumudi G; Ramachandran, Angelina; Karkamkar, Ashwini S; Dalal, Ashwin B

2018-04-13

While knowledge of HBB gene mutations is necessary for offering prenatal diagnosis (PND) of β-thalassemia (β-thal), a genotype-phenotype correlation may not always be available for rare variants. We present for the first time, genotype-phenotype correlation for a compound heterozygous status with IVS-I-5 (G>C) (HBB: c.92+5G>C) and HBB: c.407C>T (Hb Alperton) mutations on the HBB gene in an Indian family. Hb Alperton is a very rare hemoglobin (Hb) variant with scant published information about its clinical presentation, especially when accompanied with another HBB gene mutation. Here we provide biochemical as well as clinical details of this variant.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways

PubMed Central

Sekhar, Ashok; Rumfeldt, Jessica AO; Broom, Helen R; Doyle, Colleen M; Bouvignies, Guillaume; Meiering, Elizabeth M; Kay, Lewis E

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD12SH). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD12SH has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization. DOI: http://dx.doi.org/10.7554/eLife.07296.001 PMID:26099300

Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

PubMed Central

Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Goldstein, Lawrence S. B.

2008-01-01

Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment containing variable numbers of non-mutant, non-motor neurons. Despite high-level mutant expression within 100% of motor neurons and oligodendrocytes, in most of these chimeras, the presence of WT non-motor neurons substantially delayed onset of motor neuron degeneration, increasing disease-free life by 50%. Disease onset is therefore non-cell autonomous, and mutant SOD1 damage within cell types other than motor neurons and oligodendrocytes is a central contributor to initiation of motor neuron degeneration. PMID:18492803