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.     

Immunohistochemical study on the distribution of glycogen synthase kinase 3α in the central nervous system of SOD1G93A transgenic mice

Abstract

Objective: To identify glycogen synthase kinase (GSK) 3α expression in a mouse model of familial amyotrophic lateral sclerosis (ALS), we investigated the changes of GSK3α in the central nervous system of SOD1^G93A transgenic mice by immunohistochemistry.

Methods: We used 12 SOD1^G93A transgenic and ten wild-type (wt) SOD1 transgenic mice bred by 'The Jackson Laboratory' under the strain designations B6SJL-TgN (SOD1^G93A) 1 Gur/J and B6SJL-TgN (SOD1) 2 Gur/J, respectively. Immunohistochemistry was performed in accordance with the free-floating method described earlier.

Results: In symptomatic transgenic mice, GSK3α-immunoreactive astrocytes were detected in the spinal cord, brainstem and cerebellum of symptomatic SOD1^G93A transgenic mice. In contrast to symptomatic mice, no GSK3α-immunoreactive astrocytes were observed in any brain region of wtSOD1 and pre-symptomatic mice, and the number and intensity of stained cells were not different at the age of 8 and 13 weeks.

Discussion: These results provide the first evidence that GSK3α-immunoreactive astrocytes were found in the CNS of SOD1^G93A transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for GSK3α and the functional implications require elucidation.     

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.     

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.     

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.     

The influence of metallothionein treatment and treadmill running exercise on disease onset and survival in SOD1G93A amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate‐intensity treadmill exercise and/or treatment with metallothionein‐2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1^G93A familial ALS mice. Six‐week‐old female SOD1^G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2‐treated mice survived around 3% longer than vehicle‐treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2‐treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline‐treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1^G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.     

Expression of the axon‐guidance protein receptor Neuropilin 1 is increased in the spinal cord and decreased in muscle of a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co‐receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de‐/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS‐patients and controls as well as transgenic SOD1^G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real‐time PCR (qRT‐PCR), western blot and immunohistochemistry. Groups were compared using either Student t‐test or Mann–Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1^G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.     

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.     

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     

Degeneration of proprioceptive sensory nerve endings in mice harboring amyotrophic lateral sclerosis–causing mutations

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily targets the motor system. Although much is known about the effects of ALS on motor neurons and glial cells, little is known about its effect on proprioceptive sensory neurons. This study examines proprioceptive sensory neurons in mice harboring mutations associated with ALS, in SOD1^G93A and TDP43^A315T transgenic mice. In both transgenic lines, we found fewer proprioceptive sensory neurons containing fluorescently tagged cholera toxin in their soma five days after injecting this retrograde tracer into the tibialis anterior muscle. We asked whether this is due to neuronal loss or selective degeneration of peripheral nerve endings. We found no difference in the total number and size of proprioceptive sensory neuron soma between symptomatic SOD1^G93A and control mice. However, analysis of proprioceptive nerve endings in muscles revealed early and significant alterations at Ia/II proprioceptive nerve endings in muscle spindles before the symptomatic phase of the disease. Although these changes occur alongside those at α‐motor axons in SOD1^G93A mice, Ia/II sensory nerve endings degenerate in the absence of obvious alterations in α‐motor axons in TDP43^A315T transgenic mice. We next asked whether proprioceptive nerve endings are similarly affected in the spinal cord and found that nerve endings terminating on α‐motor neurons are affected during the symptomatic phase and after peripheral nerve endings begin to degenerate. Overall, we show that Ia/II proprioceptive sensory neurons are affected by ALS‐causing mutations, with pathological changes starting at their peripheral nerve endings. J. Comp. Neurol. 523:2477–2494, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

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     

Presymptomatic electrophysiological tests predict clinical onset and survival in SOD1G93A ALS mice

Introduction: We assessed the predictive value of electrophysiological tests as a marker of clinical disease onset and survival in superoxide‐dismutase 1 (SOD1)^G93A mice. Methods: 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. Results: 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. Conclusions: Electrophysiological tests predict clinical disease onset, survival, and spinal motoneuron preservation in SOD1^G93A mice. This is a methodological improvement for preclinical studies. Muscle Nerve 50: 943–949, 2014     

Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.     

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.     

Excitability properties of mouse motor axons in the mutant SOD1G93A model of amyotrophic lateral sclerosis

Non‐invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1^G93A mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild‐type mice were compared before onset of signs and during disease progression (4–19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1^G93A axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model. Muscle Nerve, 2010     

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 Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1^G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1^G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1^G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00953-z.Key Words: ALS, NF-κB-p65, microglia, BANA.     

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.