Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1 mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1^G93A mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1^G93A mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.     

Lead exposure stimulates VEGF expression in the spinal cord and extends survival in a mouse model of ALS

Exposure to environmental lead (Pb) is a mild risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive degeneration of motor neurons. However, recent evidence has paradoxically linked higher Pb levels in ALS patients with longer survival. We investigated the effects of low-level Pb exposure on survival of mice expressing the ALS-linked superoxide dismutase-1 G93A mutation (SOD1^G93A). SOD1^G93A mice exposed to Pb showed longer survival and increased expression of VEGF in the ventral horn associated with reduced astrocytosis. Pretreatment of cultured SOD1^G93A astrocytes with low, non toxic Pb concentrations upregulated VEGF expression and significantly abrogated motor neuron loss in coculture, an effect prevented by neutralizing antibodies to VEGF. The actions of Pb on astrocytes might explain its paradoxical slowing of disease progression in SOD1^G93A mice and the improved survival of ALS patients. Understanding how Pb stimulates astrocytic VEGF production and reduces neuroinflammation may yield a new therapeutic approach for treating ALS.     

Early-Stage Treatment with Withaferin A Reduces Levels of Misfolded Superoxide Dismutase 1 and Extends Lifespan in a Mouse Model of Amyotrophic Lateral Sclerosis

Approximately 20 % of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1^G93A and SOD1^G37R. Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1^G93A mice, and from 9 months until end stage in SOD1^G37R mice. The beneficial effects of WA in the SOD1^G93A mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1^G93A mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1^G93A mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0311-0) contains supplementary material, which is available to authorized users.Key Words: ALS, Neuroinflammation, Withaferin A, SOD1^G93A, SOD1^G37R     

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

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.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-015-0346-x) contains supplementary material, which is available to authorized users.     

Effects of chronic caffeine intake in a mouse model of amyotrophic lateral sclerosis

Caffeine is a nonselective adenosine receptor antagonist; chronic consumption has proved protective toward neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The present study was designed to determine whether caffeine intake affected survival and/or motor performance in a transgenic model of amyotrophic lateral sclerosis (ALS). SOD1^G93A mice received caffeine through drinking water from 70 days of age until death. Body weight, motor performance and survival were evaluated. Furthermore, the expression of adenosine A_2A receptors (A_2ARs), glial glutamate transporter (GLT1), and glial fibrillar acidic protein (GFAP) were evaluated by Western blotting. The results showed that caffeine intake significantly shortened the survival of SOD1^G93A mice (log rank test, P = 0.01) and induced a nonsignificant advancing of disease onset. The expression of A_2AR, GLT1, and GFAP was altered in the spinal cords of ALS mice, but caffeine did not influence their expression in either wild‐type or SOD1^G93 mice. These data indicate that adenosine receptors may play an important role in ALS. © 2013 Wiley Periodicals, Inc.     

Glycoprotein nonmetastatic melanoma protein B ameliorates skeletal muscle lesions in a SOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and subsequent muscular atrophy. The quality of life of patients with ALS is significantly improved by ameliorating muscular symptoms. We previously reported that glycoprotein nonmetastatic melanoma protein B (GPNMB; osteoactivin) might serve as a target for ALS therapy. In the present study, superoxide dismutase 1/glycine residue 93 changed to alanine (SOD1^G93A) transgenic mice were used as a model of ALS. Expression of the C‐terminal fragment of GPNMB was increased in the skeletal muscles of SOD1^G93A mice and patients with sporadic ALS. SOD1^G93A/GPNMB transgenic mice were generated to determine whether GPNMB expression ameliorates muscular symptoms. The weight and cross‐sectional area of the gastrocnemius muscle, number and cross‐sectional area of myofibers, and denervation of neuromuscular junctions were ameliorated in SOD1^G93A/GPNMB vs. SOD1^G93A mice. Furthermore, direct injection of a GPNMB expression plasmid into the gastrocnemius muscle of SOD1^G93A mice increased the numbers of myofibers and prevented myofiber atrophy. These findings suggest that GPNMB directly affects skeletal muscle and prevents muscular pathology in SOD1^G93A mice and may therefore serve as a target for therapy of ALS. © 2015 Wiley Periodicals, Inc.     

Isoform‐selective as opposed to complete depletion of fibroblast growth factor 2 (FGF‐2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice

We have previously shown that total knockout of fibroblast growth factor‐2 (FGF‐2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1^G93A) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1^G93A mice, with distinct regulation patterns of FGF‐2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF‐2‐isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1^G93AFGF‐2 high molecular weight‐ and SOD1^G93AFGF‐2 low molecular weight‐knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform‐dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1^G93AFGF‐2 low molecular weight knockout mice compared to single mutant SOD1^G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1^G93AFGF‐2 low molecular weight knockout mice implies that FGF‐2 low molecular weight knockout (or the presence of the FGF‐2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.     

SOD1G93A transgenic mouse CD4+ T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving motoneuron (MN) axonal withdrawal and cell death. Previously, we established that facial MN (FMN) survival levels in the SOD1^G93A transgenic mouse model of ALS are reduced and nerve regeneration is delayed, similar to immunodeficient RAG2^−/− mice, after facial nerve axotomy. The objective of this study was to examine the functionality of SOD1^G93A splenic microenvironment, focusing on CD4⁺ T cells, with regard to defects in immune-mediated neuroprotection of injured MN. We utilized the RAG2^−/− and SOD1^G93A mouse models, along with the facial nerve axotomy paradigm and a variety of cellular adoptive transfers, to assess immune-mediated neuroprotection of FMN survival levels. We determined that adoptively transferred SOD1^G93A unfractionated splenocytes into RAG2^−/− mice were unable to support FMN survival after axotomy, but that adoptive transfer of isolated SOD1^G93A CD4⁺ T cells could. Although WT unfractionated splenocytes adoptively transferred into SOD1^G93A mice were able to maintain FMN survival levels, WT CD4⁺ T cells alone could not. Importantly, these results suggest that SOD1^G93A CD4⁺ T cells retain neuroprotective functionality when removed from a dysfunctional SOD1^G93A peripheral splenic microenvironment. These results also indicate that the SOD1^G93A central nervous system microenvironment is able to re-activate CD4⁺ T cells for immune-mediated neuroprotection when a permissive peripheral microenvironment exists. We hypothesize that a suppressive SOD1^G93A peripheral splenic microenvironment may compromise neuroprotective CD4⁺ T cell activation and/or differentiation, which, in turn, results in impaired immune-mediated neuroprotection for MN survival after peripheral axotomy in SOD1^G93A mice.     

A Novel Iron Chelator-Radical Scavenger Ameliorates Motor Dysfunction and Improves Life Span and Mitochondrial Biogenesis in SOD1<Superscript>G93A</Superscript> ALS Mice

The aim of the present study was to evaluate the therapeutic effect of the novel neuroprotective multitarget brain permeable monoamine oxidase inhibitor/iron chelating-radical scavenging drug, VAR10303 (VAR), co-administered with high-calorie/energy-supplemented diet (ced) in SOD1^G93A transgenic amyotrophic lateral sclerosis (ALS) mice. Administration of VAR-ced was initiated after the appearance of disease symptoms (at day 88), as this regimen is comparable with the earliest time at which drug therapy could start in ALS patients. Using this rescue protocol, we demonstrated in the current study that VAR-ced treatment provided several beneficial effects in SOD1^G93A mice, including improvement in motor performance, elevation of survival time, and attenuation of iron accumulation and motoneuron loss in the spinal cord. Moreover, VAR-ced treatment attenuated neuromuscular junction denervation and exerted a significant preservation of myofibril regular morphology, associated with a reduction in the expression levels of genes related to denervation and atrophy in the gastrocnemius (GNS) muscle in SOD1^G93A mice. These effects were accompanied by upregulation of mitochondrial DNA and elevated activities of complexes I and II in the GNS muscle. We have also demonstrated that VAR-ced treatment upregulated the mitochondrial biogenesis master regulator, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and increased PGC-1α-targeted metabolic genes and proteins, such as, PPARγ, UCP1/3, NRF1/2, Tfam, and ERRα in GNS muscle. These results provide evidence of therapeutic potential of VAR-ced in SOD1^G93A mice with underlying molecular mechanisms, further supporting the importance role of multitarget iron chelators in ALS treatment.     

Epothilone D accelerates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Aims

Degeneration of the distal neuromuscular circuitry is a hallmark pathology of Amyotrophic Lateral Sclerosis (ALS). The potential for microtubule dysfunction to be a critical pathophysiological mechanism in the destruction of this circuitry is increasingly being appreciated. Stabilization of microtubules to improve neuronal integrity and pathology has been shown to be a particularly favourable approach in other neurodegenerative diseases. We present evidence here that treatment with the microtubule‐targeting compound Epothilone D (EpoD) both positively and negatively affects the spinal neuromuscular circuitry in the SOD1^G93A mouse model of ALS.

Methods

SOD1^G93A mice were treated every 5 days with 2 mg/kg EpoD. Evaluation of motor behaviour, neurological phenotype and survival was completed, with age‐dependent histological characterization also conducted, using the thy1‐YFP mouse. Motor neuron degeneration, axonal integrity, neuromuscular junction (NMJ) health and gliosis were also assessed.

Results

EpoD treatment prevented loss of the spinal motor neuron soma, and distal axon degeneration, early in the disease course. This, however, was not associated with protection of the NMJ synapse and did not improve motor phenotype or clinical progression. EpoD administration was also found to be neurotoxic at later disease stages. This was evidenced by accelerated motor neuron cell body loss, increasing gliosis, and was associated with detrimental outcomes to motor behaviour, clinical assessment and survival.

Conclusions

The results suggest that EpoD accelerates disease progression in the SOD1^G93A mouse model of ALS, and highlights that the pathophysiological involvement of microtubules in ALS is an evolving and underappreciated phenomenon.     

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte‐mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1^G93A mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1^G93A mice as well as human induced pluripotent stem cell (iPSC)‐derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co‐culture. Increased Cx43 expression led to important functional consequences when tested in SOD1^G93A astrocytes when compared to control astrocytes over‐expressing wild‐type SOD1 (SOD1^WT). We observed SOD1^G93A astrocytes exhibited enhanced gap junction coupling, increased hemichannel‐mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1^G93A astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS‐related motor neuron loss. GLIA 2016;64:1154–1169     

Specific Expression of Glial-Derived Neurotrophic Factor in Muscles as Gene Therapy Strategy for Amyotrophic Lateral Sclerosis

Glial cell line–derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1^G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1^G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1^G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1^G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01025-6.Key Words: GDNF, Amyotrophic lateral sclerosis, Motoneuron, Gene therapy, AAV, Neuromuscular junction     

ROCK inhibition improves axonal regeneration in a preclinical model of amyotrophic lateral sclerosis

Alteration of the RhoA/ROCK (Rho kinase) pathway has been shown to be neuroprotective in SOD1^G93A mice, the most commonly used animal model of ALS. Since previous studies indicate that, apart from neuroprotection, ROCK inhibitor Y-27632 can also accelerate regeneration of motor axons, we here assessed the regenerative capability of axons in SOD1^G93A mice with and without treatment with Y-27632. Regeneration of axons was examined after sciatic nerve crush in pre- and symptomatic SOD1^G93A mice. Proregenerative effects of Y-27632 were studied during the disease course in the SOD1^G93A mouse model. In symptomatic SOD1^G93A mice, axonal regeneration was markedly reduced compared to presymptomatic SOD1^G93A mice and wild types. Treatment with Y-27632 improved functional and morphological measures of motor axons after sciatic crush in all tested conditions. Y-27632 treatment did not increase the lifespan of symptomatic SOD1^G93A mice, but did improve axonal (re)innervation of neuromuscular junctions. Our study provides proof of concept that axonal regeneration of motor neurons harboring SOD1^G93A is impaired, but amenable for pharmacological interventions aiming to accelerate axonal regeneration. Given the lack of treatments for ALS, approaches to improve axonal regeneration, including by inhibiting ROCK, should be further explored.     

Expression of the ALS-causing variant hSOD1G93A leads to an impaired integrity and altered regulation of claudin-5 expression in an in vitro blood–spinal cord barrier model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood–spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood–spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood–spinal cord barrier endothelial cells of presymptomatic hSOD1^G93A mice and bEnd.3 cells stably expressing hSOD1^G93A. In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1^G93A cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood–spinal cord barrier.     

Chronic Administration of Pimozide Fails to Attenuate Motor and Pathological Deficits in Two Mouse Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which presently does not have any efficient therapeutic approach. Pimozide, a Food and Drug Administration (FDA)-approved neuroepileptic drug, has been recently proposed as a promising treatment for ALS patients based on apparent stabilization of right hand muscles after a short-time administration. A new clinical trial started at the end of 2017 to recruit patients with a prolonged drug delivery schedule. Here, our aim was to investigate the effects of chronic administration of pimozide on disease progression and pathological events in two mouse models of ALS. Pimozide was administered every 2 days to transgenic mice bearing the ALS-linked A315T mutation on the human TAR DNA-binding protein 43 (TDP-43) gene and to mice carrying the human superoxide dismutase 1 (SOD1) gene with the ALS-linked G93A mutation. Chronic administration of pimozide exacerbated motor performances in both animal models and reduced survival in SOD1^G93A mice. In TDP-43^A315T, it decreased the percentage of innervated neuromuscular junctions (NMJs) and increased the accumulation of insoluble TDP-43. In SOD1^G93A mice, pimozide had no effects on NMJ innervation or motoneuron loss, but it increased the levels of misfolded SOD1. We conclude that a chronic administration of pimozide did not confer beneficial effects on disease progression in two mouse models of ALS. In light of a new clinical trial on ALS patients with a chronic regime of pimozide, these results with mouse models suggest prudence and careful monitoring of ALS patients subjected to pimozide treatment.     

Monitoring systemic oxidative stress in an animal model of amyotrophic lateral sclerosis

A mutant form of the ubiquitous copper/zinc superoxide dismutase (SOD1) protein has been found in some patients with amyotrophic lateral sclerosis (ALS). We monitored oxidative stress in an animal model of ALS, the SOD^G93A mouse, which develops a disease similar to ALS with an accelerated course. The aim of this work was to show that ALS damages several organs and tissues, from an oxidative stress point of view. We measured lipid and protein oxidative damage in different tissue homogenates of SOD^G93A mice. The biomarkers that we analyzed were malondialdehyde + 4-hydroxyalkenal (MDA + 4-HDA) and carbonyls, respectively. The spinal cord and brain of SOD^G93A mice showed increased lipid peroxidation after 100 or 130 days compared to age-matched littermate controls. The CNS was most affected, but lipid peroxidation was also detected in the skeletal muscle and liver on day 130. No changes were observed in protein carbonylation in the homogenates. Our results are consistent with a multisystem etiology of ALS and suggest that oxidative stress may play a primary role in ALS pathogenesis. Thus, oxidative stress represents a potential biomarker that might be useful in developing new therapeutic strategies for ALS.     

Acute glial activation by stab injuries does not lead to overt damage or motor neuron degeneration in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Recently, a correlation between head trauma and the incidence of ALS has been reported. Furthermore, new invasive neurosurgical studies are being planned which involve inserting needles directly to the spinal cord. We therefore tested whether acute trauma to the spinal cord via a knife wound injury would lead to accelerated disease progression in rodent models of ALS (SOD1^G93A rats). A longitudinal stab injury using a small knife was performed within the lumbar spinal cord region of presymptomatic SOD1^G93A rats. Host glial activation was detected in the lumbar area surrounding a micro-knife lesion at 2 weeks after surgery in both wild type and SOD1^G93A animals. However, there was no sign of motor neuron loss in the injured spinal cord of any animal and normal motor function was maintained in the ipsilateral limb. These results indicate that motor neurons in presymptomatic G93A animals are not affected by an invasive puncture wound injury involving reactive astrocytes. Furthermore, acute trauma alone does not accelerate disease onset or progression in this ALS model which is important for future strategies of gene and cell therapies directly targeting the spinal cord of ALS patients.     

Ammonium tetrathiomolybdate delays onset, prolongs survival, and slows progression of disease in a mouse model for amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) cause a form of familial amyotrophic lateral sclerosis (ALS). The pathogenesis of familial ALS may be associated with aberrant copper chemistry through a cysteine residue in mutant SOD1. Ammonium tetrathiomolybdate (TTM) is a copper-chelating drug that is capable of removing a copper ion from copper-thiolate clusters, such as SOD1. We found that TTM exerted therapeutic benefits in a mouse model of familial ALS (SOD1^G93A). TTM treatment significantly delayed disease onset, slowed disease progression and prolonged survival by approximately 20%, 42% and 25%, respectively. TTM also effectively depressed the spinal copper ion level and inhibited lipid peroxidation, with a significant suppression of SOD1 enzymatic activity in SOD1^G93A. These results support the hypothesis that aberrant copper chemistry through a cysteine residue plays a critical role in mutant SOD1 toxicity and that TTM may be a promising therapy for familial ALS with SOD1 mutants.     

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

DJ-1 Changes in G93A-SOD1 Transgenic Mice: Implications for Oxidative Stress in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder. The causes of ALS are still obscure. Accumulating evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. DJ-1 plays an important role in the oxidative stress response. The aim of this study was to discover whether there are changes in DJ-1 expression or in DJ-1-oxidized isoforms in an animal model of ALS. We used mutant SOD1^G93A transgenic mice, a commonly used animal model for ALS. Upregulation of DJ-1 mRNA and protein levels were identified in the brains and spinal cords of SOD1^G93A transgenic mice as compared to wild-type controls, evident from an early disease stage. Furthermore, an increase in DJ-1 acidic isoforms was detected, implying that there are more oxidized forms of DJ-1 in the CNS of SOD1^G93A mice. This is the first report of possible involvement of DJ-1 in ALS. Since DJ-1 has a protective role against oxidative stress, it may suggest a possible therapeutic target in ALS.