Peroxisome proliferator activator receptor gamma coactivator-1alpha (PGC-1α) improves motor performance and survival in a mouse model of amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects spinal cord and cortical motor neurons. An increasing amount of evidence suggests that mitochondrial dysfunction contributes to motor neuron death in ALS. Peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) is a principal regulator of mitochondrial biogenesis and oxidative metabolism.

Results

In this study, we examined whether PGC-1α plays a protective role in ALS by using a double transgenic mouse model where PGC-1α is over-expressed in an SOD1 transgenic mouse (TgSOD1-G93A/PGC-1α). Our results indicate that PGC-1α significantly improves motor function and survival of SOD1-G93A mice. The behavioral improvements were accompanied by reduced blood glucose level and by protection of motor neuron loss, restoration of mitochondrial electron transport chain activities and inhibition of stress signaling in the spinal cord.

Conclusion

Our results demonstrate that PGC-1α plays a beneficial role in a mouse model of ALS, suggesting that PGC-1α may be a potential therapeutic target for ALS therapy.     

Metabolic Therapy with Deanna Protocol Supplementation Delays Disease Progression and Extends Survival in Amyotrophic Lateral Sclerosis (ALS) Mouse Model

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and eventual death from respiratory failure. There is currently no cure or effective treatment for ALS. Besides motor neuron degeneration, ALS is associated with impaired energy metabolism, which is pathophysiologically linked to mitochondrial dysfunction and glutamate excitotoxicity. The Deanna Protocol (DP) is a metabolic therapy that has been reported to alleviate symptoms in patients with ALS. In this study we hypothesized that alternative fuels in the form of TCA cycle intermediates, specifically arginine-alpha-ketoglutarate (AAKG), the main ingredient of the DP, and the ketogenic diet (KD), would increase motor function and survival in a mouse model of ALS (SOD1-G93A). ALS mice were fed standard rodent diet (SD), KD, or either diets containing a metabolic therapy of the primary ingredients of the DP consisting of AAKG, gamma-aminobutyric acid, Coenzyme Q10, and medium chain triglyceride high in caprylic triglyceride. Assessment of ALS-like pathology was performed using a pre-defined criteria for neurological score, accelerated rotarod test, paw grip endurance test, and grip strength test. Blood glucose, blood beta-hydroxybutyrate, and body weight were also monitored. SD+DP-fed mice exhibited improved neurological score from age 116 to 136 days compared to control mice. KD-fed mice exhibited better motor performance on all motor function tests at 15 and 16 weeks of age compared to controls. SD+DP and KD+DP therapies significantly extended survival time of SOD1-G93A mice by 7.5% (p = 0.001) and 4.2% (p = 0.006), respectively. Sixty-three percent of mice in the KD+DP and 72.7% of the SD+DP group lived past 125 days, while only 9% of the control animals survived past that point. Targeting energy metabolism with metabolic therapy produces a therapeutic effect in ALS mice which may prolong survival and quality of life in ALS patients.     

Oxidative Stress and Autophagic Alteration in Brainstem of SOD1-G93A Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease involving both upper and lower motor neurons. The mechanism of motor neuron degeneration is still unknown. Although many studies have been performed on spinal motor neurons, few have been reported on brainstem and its motor nuclei. The aim of this study was to investigate oxidative stress and autophagic changes in the brainstem and representative motor nuclei of superoxide dismutase 1 (SOD1)-G93A mouse model of ALS. The expression levels of cluster of differentiation molecule 11b (CD11b), glial fibrillary acidic protein, glutamate–cysteine ligase catalytic subunit, heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, voltage-dependent anion-selective channel protein 1, Sequestosome 1/p62 (p62), microtubule-associated protein 1 light chain 3B (LC3), and SOD1 proteins in brainstem were examined by Western blot analysis. Immunohistochemistry and immunofluorescence were performed to identify the cellular localization of SOD1, p62, and LC3B, respectively. The results showed that there were progressive asctrocytic proliferation and microglial activation, induction of antioxidant proteins, and increased p62 and LC3II expression in brainstem of SOD1-G93A mice. Additionally, SOD1 and p62 accumulated in hypoglossal, facial, and red nuclei, but not in oculomotor nucleus. Furthermore, electron microscope showed increased autophagic vacuoles in affected brainstem motor nuclei. Our results indicate that brainstem share similar gliosis, oxidative stress, and autophagic changes as the spinal cord in SOD1-G93A mice. Thus, SOD1 accumulation in astrocytes and neurons, oxidative stress, and altered autophagy are involved in motor neuron degeneration in the brainstem, similar to the motor neurons in spinal cord. Therefore, therapeutic trials in the SOD1G93A mice need to target the brainstem in addition to the spinal cord.     

Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice

The SOD1-G93A transgenic mouse is a widely used ALS model, but the death of lower motor neurons is the hallmark. Here, we show that the SOD1-G93A transgene and HO-1 are preferentially over-expressed in the lumbar spinal cord, particularly in the activated astrocytes of the transgenic mice. We also show down-regulation of GLT-1 in spite of the proliferating astrocytes. However, GLT-1, SOD1-G93A transgene and HO-1 expression were not obviously changed in the motor cortex. Our data link spinal cord vulnerability to relatively decreased expression of GLT-1, and high expression of the transgene and HO-1 in astrocytes in SOD1-G93A transgenic mice.     

The Deanna protocol supplement complex supports mitochondrial energy metabolism and prolongs lifespan in preclinical models of amyotrophic lateral sclerosis (ALS)

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and eventual death from respiratory failure. There is currently no cure or effective treatment for ALS. The Deanna protocol (DP) is a comprehensive treatment approach that includes a metabolic therapy in the form of a supplement complex that improved neurological function, increased motor function and survival in SOD1-G93A mice and has been reported to alleviate symptoms in patients with ALS; therefore, it has been proposed as a treatment for the disease.

Objectives

We hypothesized that the major components of the DP, including arginine alpha-ketoglutarate, gamma amino butyric acid (GABA), medium chain triglycerides (MCT), and soluble coenzyme Q10 (ubiquinol) supports energy metabolism by increasing energy intermediates of the tricarboxylic acid cycle in a mouse model of ALS (SOD1-G93A).

Methods

We explored the potential therapeutic use of DP by testing the effects of DP supplementation on the metabolomics profile of SOD1-G93A mice. In addition, we assessed time to paralysis in a Caenorhabditis elegans model of ALS (TDP-43) given DP supplementation. SOD1-G93A mice were fed a standard rodent diet (SD) or SD with low dose (LOW) or high dose of DP (HIGH). Global metabolomics analysis was performed on blood plasma from treated and untreated animals. Additionally, the time to paralysis of TDP-43 ALS C. elegans treated with and without the individual and combination DP supplements was measured.

Results

30 and 49 biochemicals were significantly altered in the plasma of LOW and HIGH groups, respectively. Metabolites associated with mitochondrial energy metabolism, arginine metabolism, as well as long- and medium-chain fatty acids, GABA and related intermediates were elevated in response to DP. Elements of DP, arginine and alpha-ketoglutarate, GABA, and MCTs prolonged the rate of final paralysis of C. elegans TDP-43 disease models.

Conclusion

Targeting energy metabolism with the DP supplement as a metabolic therapy produces a change in the global metabolic profile of ALS mice that support the role of the DP for enhanced mitochondrial energy metabolism and prolongs time to paralysis of ALS C. elegans.

     

Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the in vivo level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium methane sulfonate (MitoQ) can modify disease progression in the SOD1^G93A mouse model of ALS. To do this, we administered MitoQ (500 µM) in the drinking water of SOD1^G93A mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, in both the spinal cord and the quadriceps muscle, as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated with a significant increase in hindlimb strength. Finally, MitoQ treatment significantly prolonged the life span of SOD1^G93A mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.     

Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60-day-old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild-type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.     

Modulation of astrocytic mitochondrial function by dichloroacetate improves survival and motor performance in inherited amyotrophic lateral sclerosis

Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1(G93A) mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1(G93A) mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1(G93A) astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1(G93A) mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1(G93A) mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1(G93A) mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS.     

Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.     

Male-Specific Differences in Proliferation,Neurogenesis, and Sensitivity to Oxidative Stress in Neural Progenitor Cells Derived from a Rat Model of ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1^G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1^G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1^G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1^G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1^G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1^G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.     

Diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)) protects against peroxynitrite-induced nitrosative damage and prolongs survival in amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.     

Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1-5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1(G93A)). Arimoclomol-treated SOD1(G93A) mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.     

Human glial-restricted progenitor transplantation into cervical spinal cord of the SOD1 mouse model of ALS

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1(G93A) rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1(G93A) mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1(G93A) mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.     

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

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

Dihydrotestosterone ameliorates degeneration in muscle, axons and motoneurons and improves motor function in amyotrophic lateral sclerosis model mice

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by a progressive loss of motoneurons. The clinical symptoms include skeletal muscle weakness and atrophy, which impairs motor performance and eventually leads to respiratory failure. We tested whether dihydrotestosterone (DHT), which has both anabolic effects on muscle and neuroprotective effects on axons and motoneurons, can ameliorate clinical symptoms in ALS. A silastic tube containing DHT crystals was implanted subcutaneously in SOD1-G93A mice at early symptomatic age when decreases in body weight and grip-strength were observed as compared to wild-type mice. DHT-treated SOD1-G93A mice demonstrated ameliorated muscle atrophy and increased body weight, which was associated with stronger grip-strength. DHT treatment increased the expression of insulin-like growth factor-1 in muscle, which can exert myotrophic as well as neurotrophic effects through retrograde transport. DHT treatment attenuated neuromuscular junction denervation, and axonal and motoneuron loss. DHT-treated SOD1-G93A mice demonstrated improvement in motor behavior as assessed by rota-rod and gait analyses, and an increased lifespan. Application of DHT is a relatively simple and non-invasive procedure, which may be translated into therapy to improve the quality of life for ALS patients.     

RNAi knockdown of Par-4 inhibits neurosynaptic degeneration in ALS-linked mice

Evidence from human amyotrophic lateral sclerosis (ALS) patients and ALS-linked Cu/Zn superoxide dismutase (Cu/Zn-SOD) transgenic mice bearing the mutation of glycine to alanine at position 93 (G93A) suggests that the pro-apoptotic protein prostate apoptosis response-4 (Par-4) might be a critical link in the chain of events leading to motor neuron degeneration. We now report that Par-4 is enriched in synaptosomes and post-synaptic density from the ventral horn of the spinal cord. Levels of Par-4 in synaptic compartments increased significantly during rapid and slow declining stages of muscle strength in hSOD1 G93A mutant mice. In the pre-muscle weakness stage, hSOD1 G93A mutation sensitized synaptosomes from the ventral horn of the spinal cord to increased levels of Par-4 expression following excitotoxic and apoptotic insults. In ventral spinal synaptosomes, Par-4-mediated production of pro-apoptotic cytosolic factor(s) was significantly enhanced by the hSOD1 G93A mutation. RNA interference (RNAi) knockdown of Par-4 inhibited mitochondrial dysfunction and caspase-3 activation induced by G93A mutation in synaptosomes from the ventral horn of the spinal cord, and protected spinal motor neurons from apoptosis. These results identify the synapse as a crucial cellular site for the cell death promoting actions of Par-4 in motor neurons, and suggest that targeted inhibition of Par-4 by RNAi may prove to be a neuroprotective strategy for motor neuron degeneration.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.     

Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective degeneration of motor neurons, atrophy, and paralysis of skeletal muscle. Although a significant proportion of familial ALS results from a toxic gain of function associated with dominant SOD1 mutations, the etiology of the disease and its specific cellular origins have remained difficult to define. Here, we show that muscle-restricted expression of a localized insulin-like growth factor (Igf) -1 isoform maintained muscle integrity and enhanced satellite cell activity in SOD1(G93A) transgenic mice, inducing calcineurin-mediated regenerative pathways. Muscle-specific expression of local Igf-1 (mIgf-1) isoform also stabilized neuromuscular junctions, reduced inflammation in the spinal cord, and enhanced motor neuronal survival in SOD1(G93A) mice, delaying the onset and progression of the disease. These studies establish skeletal muscle as a primary target for the dominant action of inherited SOD1 mutation and suggest that muscle fibers provide appropriate factors, such as mIgf-1, for neuron survival.     

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.