SOD1G93A Mouse Model Of Amyotrophic Lateral Sclerosis Research Articles

Sex-Specific Markers of Neuroinflammation and Neurodegeneration in the Spinal Cord Proteome of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that has no cure. The underlying mechanistic details of sex differences in the ALS spinal cord, the site of disease onset, are not understood to an extent that could guide novel drug development. To address this, the spinal cords of 120-day-old wild-type (WT) and SOD1G93A (familial mouse model of ALS with mutant superoxide dismutase 1) mice were subjected to untargeted, quantitative proteomics using tandem mass tag acquisition on high-resolution mass spectrometric instrumentation. Compared to WT, both male and female SOD1G93A spinal cords exhibited an upregulation of neuroinflammatory cascades of both peripheral and central origins, as well as a downregulation of proteins reflective of death and dysfunction of cells within the spinal cord. However, female and male SOD1G93A mouse spinal cords exhibited sex-specific differences in proteins compared to respective WT that related to immune response, as well as cellular structure, function, and homeostasis. The proteomic datasets presented provide entire cohort and sex-specific spinal cord drug targets and disease biomarkers in the SOD1G93A mouse model of ALS that may guide future drug development and sex selection in preclinical study designs utilizing the SOD1G93A model.

Amyotrophic Lateral Sclerosis, the Endocannabinoid System, and Exogenous Cannabinoids: Current State and Clinical Implications.

A unifying mechanistic cause for amyotrophic lateral sclerosis (ALS) remains uncertain. Multiple pathophysiological processes appear to occur simultaneously. Cannabinoids, including delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), and others found in cannabis, and cannabis extracts (CEs), appear to have activity in these pathogenic pathways, which have led to increasing interest in cannabinoids as therapeutic agents for ALS. The use of cannabinoids as a treatment strategy is substantiated by preclinical evidence suggesting a role for the endocannabinoid system (ECS) in ALS and other neurodegenerative disorders. Preclinical data indicate that cannabis and CEs have powerful antioxidative, anti-inflammatory, and neuroprotective effects in the SOD1G93A mouse model of ALS. The use of CEs in SOD1G93A murine models has been shown to prolong neuronal cell survival, which leads to delayed onset of the disease state, and slows progression of the disease. Although research in humans remains limited, a few studies suggest that cannabis and CBD, in humans, provide benefits for both motor symptoms, including rigidity, cramps, and fasciculations, and non-motor symptoms including sleep quality, pain, emotional state, quality of life, and depression. There remains a need for further, well-designed clinical trials to validate further the use of an individual cannabinoid, or a combination of cannabinoids, as a disease-modifying therapy for ALS.

Brown Adipose Tissue undergoes pathological perturbations and shapes C2C12 myoblast homeostasis in the SOD1-G93A mouse model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons. The contribution of peripheral organs remains incompletely understood. We focused our attention on brown adipose tissue (BAT) and its secreted extracellular vesicles (EVs) given their role in regulating systemic energy balance. In this study, we employed a multi-omics approach, including RNA sequencing (GEO identifier GSE273052) and proteomics (ProteomeXchange identifier PXD054147), to investigate the alterations in BAT and its EVs in the SOD1-G93A mouse model of ALS. Our results revealed consistent changes in the proteomic and transcriptomic profiles of BAT from SOD1-G93A mice, highlighting alterations such as mitochondrial dysfunction and impaired differentiation capacity. Specifically, primary brown adipocytes (PBAs) from SOD1-G93A mice exhibited differentiation impairment, respiratory defects, and alterations in mitochondrial dynamics. Furthermore, the BAT-derived EVs from SOD1-G93A mice displayed distinct changes in size distribution and cargo content. In parallel, such EVs negatively impacted the differentiation and homeostasis of C2C12 murine myoblasts, as well as induced atrophy in C2C12-derived myotubes. These findings suggest that BAT undergoes pathological perturbations in ALS mouse model and could impact on skeletal muscle homeostasis through the secretion of dysfunctional EVs.

Polyamine metabolism dysregulation contributes to muscle fiber vulnerability in ALS.

Polyamine metabolism dysregulation contributes to muscle fiber vulnerability in ALS.

Overexpression of autophagy enhancer PACER/RUBCNL in neurons accelerates disease in the SOD1G93A ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal paralytic disorder associated with motor neuron death. Mutant superoxide dismutase 1 (SOD1) misfolding and aggregation have been linked to familial ALS, with the accumulation of abnormal wild-type SOD1 species being also observed in postmortem tissue of sporadic ALS cases. Both wild-type and mutated SOD1 are reported to contribute to motoneuron cell death. The autophagic pathway has been shown to be dysregulated in ALS. Recent evidence suggests a dual time-dependent role of autophagy in the progression of the disease. PACER, also called RUBCNL (Rubicon-like), is an enhancer of autophagy and has been found diminished in its levels during ALS pathology in mice and humans. Pacer loss of function disturbs the autophagy process and leads to the accumulation of SOD1 aggregates, as well as sensitizes neurons to death. Therefore, here we investigated if constitutive overexpression of PACER in neurons since early development is beneficial in an in vivo model of ALS. We generated a transgenic mouse model overexpressing human PACER in neurons, which then was crossbred with the mutant SOD1G93A ALS mouse model. Unexpectedly, PACER/SOD1G93A double transgenic mice exhibited an earlier disease onset and shorter lifespan than did littermate SOD1G93A mice. The overexpression of PACER in neurons in vivo and in vitro increased the accumulation of SOD1 aggregates, possibly due to impaired autophagy. These results suggest that similar to Pacer loss-of function, Pacer gain-of function is detrimental to autophagy, increases SOD1 aggregation and worsens ALS pathogenesis. In a wider context, our results indicate the requirement to maintain a fine balance of PACER protein levels to sustain proteostasis.

Exercise, disease state and sex influence the beneficial effects of Fn14-depletion on survival and muscle pathology in the SOD1G93A amyotrophic lateral sclerosis (ALS) mouse model

BackgroundAmyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1G93A ALS mice could lead to differential and potentially improved benefits.MethodsWe thus investigated the contribution of Fn14 to disease phenotypes in the SOD1G93A ALS mice. To do so, Fn14 knockout mice (Fn14−/−) were crossed onto the SOD1G93A background to generate SOD1G93A;Fn14−/− mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14−/−, SOD1G93A and SOD1G93A;Fn14−/−).ResultsHere, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1G93A mice. We then show that Fn14-depleted SOD1G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size.ConclusionsOur study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies.

Acute stress differently modulates interneurons excitability and synaptic plasticity in the primary motor cortex of wild-type and SOD1G93A mouse model of ALS.

Primary motor cortex (M1) network stability depends on activity of inhibitory interneurons, for which susceptibility to stress was previously demonstrated in limbic regions. Hyperexcitability in M1 following changes in the excitatory/inhibitory balance is a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Using electrophysiological approaches, we assessed the impact of acute restraint stress on inhibitory interneurons excitability and global synaptic plasticity in M1 of the SOD1G93A ALS mouse model at a late pre-symptomatic stage (10-12.5weeks). Based on their firing type (continuous, discontinuous, with accommodation or not) and electrophysiological characteristics (resting potential, rheobase, firing frequency), interneurons from M1slices were separated into four clusters, labelled from 1 to 4. Among them, only interneurons from the first cluster, presenting continuous firing with few accommodations, tended to show increased excitability in wild-type (WT) and decreased excitability in SOD1G93A animals following stress. In vivo analyses of evoked field potentials showed that stress suppressed the theta burst-induced plasticity of an excitatory component (N1) recorded in the superficial layers of M1 in WT, with no impact on an inhibitory complex (N2-P1) from the deeper layers. In SOD1G93A mice, stress did not affect N1 but suppressed the N2-P1 plasticity. These data suggest that stress can alter M1 network functioning in a different manner in WT and SOD1G93A mice, possibly through changes of inhibitory interneurons excitability and synaptic plasticity. This suggests that stress-induced activity changes in M1may therefore influence ALS outcomes. KEY POINTS: Disruption of the excitatory/inhibitory balance in the primary motor cortex (M1) has been linked to cortical hyperexcitability development, a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Psychological stress was reported to influence excitatory/inhibitory balance in limbic regions, but very little is known about its influence on the M1 functioning under physiological or pathological conditions. Our study revealed that acute stress influences the excitatory/inhibitory balance within the M1, through changes in interneurons excitability along with network plasticity. Such changes were different in pathological (SOD1G93A ALS mouse model) vs. physiological (wild-type) conditions. The results of our study help us to better understand how stress modulates the M1 and highlight the need to further characterize stress-induced motor cortex changes because it may be of importance when evaluating ALS outcomes.

Montelukast improves disease outcome in SOD1G93A female mice by counteracting oligodendrocyte dysfunction and aberrant glial reactivity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss and consequent muscle atrophy, for which no effective therapies are available. Recent findings reveal that disease progression is fuelled by early aberrant neuroinflammation and the loss of oligodendrocytes with neuroprotective and remyelinating properties. On this basis, pharmacological interventions capable of restoring a pro-regenerative local milieu and re-establish proper oligodendrocyte functions may be beneficial. Here, we evaluated the in vivo therapeutic effects of montelukast (MTK), an antagonist of the oligodendroglial G protein-coupled receptor 17 (GPR17) and of cysteinyl-leukotriene receptor 1 (CysLT1R) receptors on microglia and astrocytes, in the SOD1G93A ALS mouse model. We chronically treated SOD1G93A mice with MTK, starting from the early symptomatic disease stage. Disease progression was assessed by behavioural and immunohistochemical approaches. Oral MTK treatment significantly extended survival probability, delayed body weight loss and ameliorated motor functionalityonly in female SOD1G93A mice. Noteworthy, MTK significantly restored oligodendrocyte maturation and induced significant changes in the reactive phenotype and morphological features of microglia/macrophages and astrocytes in the spinal cord of female SOD1G93A mice, suggesting enhanced pro-regenerative functions. Importantly, concomitant MN preservation has been detected after MTK administration. No beneficial effects were observed in male mice, highlighting a sex-based difference in the protective activity of MTK. Our results provide the first preclinical evidence indicating that repurposing of MTK, a safe and marketed anti-asthmatic drug, may be a promising sex-specific strategy for personalized ALS treatment.

Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.

IRAK-M Plays A Role in the Pathology of Amyotrophic Lateral Sclerosis Through Suppressing the Activation of Microglia.

Microglial activation plays a crucial role in the disease progression in amyotrophic lateral sclerosis (ALS). Interleukin receptor-associated kinases-M (IRAK-M) is an important negative regulatory factor in the Toll-like receptor 4 (TLR4) pathway during microglia activation, and its mechanism in this process is still unclear. In the present study, we aimed to investigate the dynamic changes of IRAK-M and its protective effects for motor neurons in SOD1-G93A mouse model of ALS. qPCR (Real-time Quantitative PCR Detecting System) were used to examine the mRNA levels of IRAK-M in the spinal cord in both SOD1-G93A mice and their age-matched wild type (WT) littermates at 60, 100 and 140days of age. We established an adeno-associated virus 9 (AAV9)-based platform by which IRAK-M was targeted mostly to microglial cells to investigate whether this approach could provide a protection in the SOD1-G93A mouse. Compared with age-matched WT mice, IRAK-M mRNA level was elevated at 100 and 140days in the anterior horn region of spinal cords in the SOD1-G93A mouse. AAV9-IRAK-M treated SOD1-G93A mice showed reduction of IL-1β mRNA levels and significant improvements in the numbers of spinal motor neurons in spinal cord. Mice also showed previously reduction of muscle atrophy. Our data revealed the dynamic changes of IRAK-M during ALS pathological progression and demonstrated that an AAV9-IRAK-M delivery was an effective and translatable therapeutic approach for ALS. These findings may help identify potential molecular targets for ALS therapy.

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

Excitatory action of low frequency depolarizing GABA/glycine synaptic inputs is prevalent in prenatal spinal SOD1G93A motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onsetneurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. Recent evidence suggests the dysfunction of inhibitory signalling in ALS motor neurons. We have shown that embryonic day (E)17.5spinal motoneurons (MNs) of the SOD1G93A mouse model of ALS exhibit an altered chloride homeostasis. At this prenatal stage, inhibition of spinal motoneurons (MNs) is mediated by depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs). Here, using an ex vivo preparation and patch clamp recording from MNs with a chloride equilibrium set below spike threshold, we report that low input resistance (Rin ) E17.5 MNs from the SOD1G93A ALS mouse model do not correctly integrate dGPSPs evoked by electrical stimulations of GABA/glycine inputs at different frequencies. Indeed, firing activity of most wild-type (WT) MNs with low Rin was inhibited by incoming dGPSPs, whereas low Rin SOD1G93A MNs were excited or exhibited a dual response (excited by low frequency dGPSPs and inhibited by high frequency dGPSPs). Simulation highlighted the importance of the GABA/glycine input density and showed that pure excitation could be obtained in SOD-like MNs by moving GABA/glycine input away from the cell body to dendrites. This was in agreement with confocal imaging showing a lack of peri-somatic inhibitory terminals in SOD1G93A MNs compared to WT littermates. Putative fast ALS-vulnerable MNs with low Rin are therefore lacking functional inhibition at the near-term prenatal stage. KEY POINTS: We analysed the integration of GABAergic/glycinergic synaptic events by embryonic spinal motoneurons (MNs) in a mouse model of the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. We found that GABAergic/glycinergic synaptic events do not properly inhibit ALS MNs with low input resistance, most probably corresponding to future vulnerable MNs. We used a neuron model to highlight the importance of the GABA/glycine terminal location and density in the integration of the GABAergic/glycinergic synaptic events. Confocal imaging showed a lack of GABA/glycine terminals on the cell body of ALS MNs. The present study suggests that putative ALS vulnerable MNs with low Rin lack functional inhibition at the near-term stage.

TwinF interface inhibitor FP802 stops loss of motor neurons and mitigates disease progression in a mouse model of ALS

Toxic signaling by extrasynaptic NMDA receptors (eNMDARs) is considered an important promoter of amyotrophic lateral sclerosis (ALS) disease progression. To exploit this therapeutically, we take advantage of TwinF interface (TI) inhibition, a pharmacological principle that, contrary to classical NMDAR pharmacology, allows selective elimination of eNMDAR-mediated toxicity via disruption of the NMDAR/TRPM4 death signaling complex while sparing the vital physiological functions of synaptic NMDARs. Post-disease onset treatment of the SOD1G93A ALS mouse model with FP802, a modified TI inhibitor with a safe pharmacology profile, stops the progressive loss of motor neurons in the spinal cord, resulting in a reduction in the serum biomarker neurofilament light chain, improved motor performance, and an extension of life expectancy. FP802 also effectively blocks NMDA-induced death of neurons in ALS patient-derived forebrain organoids. These results establish eNMDAR toxicity as a key player in ALS pathogenesis. TI inhibitors may provide an effective treatment option for ALS patients.

Dietary NMN supplementation enhances motor and NMJ function in ALS

Dietary NMN supplementation enhances motor and NMJ function in ALS

An optogenetic cell therapy to restore control of target muscles in an aggressive mouse model of amyotrophic lateral sclerosis

Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, no therapies exist that can prevent progressive motor neuron degeneration, muscle denervation, or paralysis in ALS. Here, we report important advances in the development of an optogenetic, neural replacement strategy that can effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1G93A mouse model of ALS, thus providing an interface to selectively control the function of targeted muscles using optical stimulation. We also identify a specific approach to confer complete survival of allogeneic replacement motor neurons. Furthermore, we demonstrate that an optical stimulation training paradigm can prevent atrophy of reinnervated muscle fibers and results in a tenfold increase in optically evoked contractile force. Together, these advances pave the way for an assistive therapy that could benefit all ALS patients.

An optogenetic cell therapy to restore control of target muscles in an aggressive mouse model of amyotrophic lateral sclerosis.

Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, no therapies exist that can prevent progressive motor neuron degeneration, muscle denervation, or paralysis in ALS. Here, we report important advances in the development of an optogenetic, neural replacement strategy that can effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1G93A mouse model of ALS, thus providing an interface to selectively control the function of targeted muscles using optical stimulation. We also identify a specific approach to confer complete survival of allogeneic replacement motor neurons. Furthermore, we demonstrate that an optical stimulation training paradigm can prevent atrophy of reinnervated muscle fibers and results in a tenfold increase in optically evoked contractile force. Together, these advances pave the way for an assistive therapy that could benefit all ALS patients.

The Secretome of Human Dental Pulp Stem Cells and Its Components GDF15 and HB-EGF Protect Amyotrophic Lateral Sclerosis Motoneurons against Death.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.

Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

Glycogen accumulation modulates life span in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord. Glial cells, including astrocytes and microglia, have been shown to contribute to neurodegeneration in ALS, and metabolic dysfunction plays an important role in the progression of the disease. Glycogen is a soluble polymer of glucose found at low levels in the central nervous system that plays an important role in memory formation, synaptic plasticity, and the prevention of seizures. However, its accumulation in astrocytes and/or neurons is associated with pathological conditions and aging. Importantly, glycogen accumulation has been reported in the spinal cord of human ALS patients and mouse models. In the present work, using the SOD1G93A mouse model of ALS, we show that glycogen accumulates in the spinal cord and brainstem during symptomatic and end stages of the disease and that the accumulated glycogen is associated with reactive astrocytes. To study the contribution of glycogen to ALS progression, we generated SOD1G93A mice with reduced glycogen synthesis (SOD1G93A GShet mice). SOD1G93A GShet mice had a significantly longer life span than SOD1G93A mice and showed lower levels of the astrocytic pro-inflammatory cytokine Cxcl10, suggesting that the accumulation of glycogen is associated with an inflammatory response. Supporting this, inducing an increase in glycogen synthesis reduced life span in SOD1G93A mice. Altogether, these results suggest that glycogen in reactive astrocytes contributes to neurotoxicity and disease progression in ALS.

Development of abnormalities at the neuromuscular junction in the SOD1-G93A mouse model of ALS: dysfunction then disruption of postsynaptic structure precede overt motor symptoms.

The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.