MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1^G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 ^G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 ^G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

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

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1^G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1^G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1^G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.     

Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A) mouse model of amyotrophic lateral sclerosis include sex‐dependent phenotypes

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper‐zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 ^G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 ^G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 ^G93A males displayed reduced locomotion, exploration and increased anxiety‐like behaviours compared with control males. Intermediate‐term spatial memory was impaired in SOD1 ^G93A females, whereas long‐term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 ^G93A mice of both sexes compared with respective controls. Interestingly, SOD1 ^G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 ^G93A when assessed in social paradigms. In conclusion, SOD1 ^G93A mice exhibit a variety of sex‐specific behavioural deficits beyond motor impairments supporting the notion of an ALS‐frontotemporal spectrum disorder. Thus, SOD1 ^G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Human Glial-Restricted Progenitor Transplantation into Cervical Spinal Cord of the SOD1G93A 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.     

Region-specific changes in the immunoreactivity of TRPV4 expression in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Transient receptor potential vanilloid 4 (TRPV4) is a broadly expressed Ca²⁺-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. It is activated by warm temperature, lipids downstream of arachidonic acid metabolism, hypoosmolarity, or mechanical stimulation. In the present study, we used SOD1^G93A mutant transgenic mice as the animal model of amyotrophic lateral sclerosis (ALS) and investigated the changes of TRPV4 immunoreactivity in the central nervous system of these mice by immunohistochemical studies. An increased expression of TRPV4 was pronounced in the cerebral cortex, hippocampal formation, thalamus, cerebellum and spinal cord of symptomatic SOD1^G93A transgenic mice. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of SOD1^G93A transgenic mice. In the hippocampal formation, pyramidal cells of the CA1-3 areas and in the granule cells of the dentate gyrus demonstrated increased TRPV4 immunoreactivity. In addition, TRPV4 immunoreactivity was increased in the spinal cord, thalamus and cerebellum of the symptomatic SOD1^G93A transgenic mice. This study, which showed increased TRPV4 in different brain and spinal cord regions of SOD1^G93A transgenic mice, may provide clues to the understanding of many basic neuronal functions in ALS. These findings suggest a role for TRPV4 in the neuronal functions in ALS but the mechanisms and functional implications of increased TRPV4 require elucidation.     

Spy1, a unique cell cycle regulator,alters viability in ALS motor neurons and cell lines in response to mutant SOD1-induced DNA damage

Increasing evidence indicates that DNA damage and p53 activation play major roles in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). Human SpeedyA1 (Spy1), a member of the Speedy/Ringo family, enhances cell proliferation and promotes tumorigenesis. Further studies have demonstrated that Spy1 promotes cell survival and inhibits DNA damage-induced apoptosis. We showed that the Spy1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro by qRT-PCR, western blotting, and Immunoassay tests. In addition, we established that over-expression of human SOD1 mutant G93A led to a decreased expression of Spy1. Furthermore, DNA damage response was activated in SOD1^G93A-transfected cells (mSOD1 cells). Moreover, decreased Spy1 expression reduced cell viability and further activated the DNA damage response in mSOD1 cells. In contrast, increased Spy1 expression improved cell viability and inhibited the DNA damage response in mSOD1 cells. These results suggest that Spy1 plays a protective role in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS as well as for trial designs, such as investigating the role of oncogenic proteins in ALS.     

Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1^G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1^G93A astrocytes. Treatment of SOD1^G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1⁺ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.     

A novel small molecule HSP90 inhibitor,NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1^G93A similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca²⁺ accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1^G93A transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.     

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation

Background

Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1^G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1^G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1^G93A animals.

Methods/Principal Findings

Presymptomatic SOD1^G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1^G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1^G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1^G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.

Conclusions/Significance

These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.     

Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease and it is the most common adult onset neurodegenerative disorder affecting motor neurons. There is currently no effective treatment for ALS and our understanding of the pathological mechanism is still far away from prevention and/or treatment of this devastating disease. Amyloid precursor protein (APP) is a transmembrane protein that undergoes processing either by β-secretase or α-secretase, followed by γ-secretase. In the present study, we show that APP levels, and aberrant phosphorylation, which is associated with enhanced β-secretase cleavage, are increased in SOD1^G93A ALS mouse model. Fluorescence resonance energy transfer (FRET) analysis suggests a close interaction between SOD1 and APP at hippocampal synapses. Notably, SOD1^G93A mutation induces APP-SOD1 conformational changes, indicating a crosstalk between these two signaling proteins. Inhibition of APP processing via monoclonal antibody called BBS that blocks APP β-secretase cleavage site, resulted in reduction of mutant SOD1^G93A levels in animal and cellular models of ALS, significantly prolonged life span of SOD1^G93A mice and diminished inflammation. Beyond its effect on toxic mutant SOD1^G93A, BBS treatment resulted in a reduction in the levels of APP, its processing product soluble APPβ and pro-apoptotic p53. This study demonstrates that APP and its processing products contribute to ALS pathology through several different pathways; thus BBS antibody could be a promising neuroprotective strategy for treatment of this disease.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

Enhanced Cortical Metabolic Activity in Females and Males of a Slow Progressing Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with selective degeneration of motor neurons in the central nervous system. The pathophysiology of ALS is not well understood. We have used ¹H-[¹³C]-NMR spectroscopy together with an administration of [1,6-¹³C₂]glucose and [2-¹³C]acetate in female and male SOD1^G37R mice to assess neuronal and astroglial metabolic activity, respectively, in the central nervous system in ALS condition. The female (p?=?0.0008) and male (p?<?0.0001) SOD1^G37R mice exhibited decreased forelimb strength when compared with wild-type mice. There was a reduction in N-acetylaspartylglutamate level, and elevation in myo-inositol in the spinal cord of female and male SOD1^G37R mice. The transgenic male mice exhibited increased acetate oxidation in the spinal cord (p?=?0.05) and cerebral cortex (p?=?0.03), while females showed an increase in the spinal cord (p?=?0.02) only. As acetate is transported and preferentially metabolized in the astrocytes, the finding of increased rate of acetate oxidation in the transgenic mice is suggestive of astrocytic involvement in the pathogenesis of ALS. The rates of glucose oxidation in glutamatergic (p?=?0.0004) and GABAergic neurons (p?=?0.0052) were increased in the cerebral cortex of male SOD1^G37R mice when compared with the controls. The female mice showed an increase in glutamatergic (p?=?0.039) neurometabolic activity only. The neurometabolic activity was unperturbed in the spinal cord of either sex. These data suggest differential changes in neurometabolic activity across the central nervous system in SOD1^G37R mice.

     

Defective Mitochondrial Dynamics Is an Early Event in Skeletal Muscle of an Amyotrophic Lateral Sclerosis Mouse Model

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

Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg(SOD1*G93A)dl1 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1 ^G93A. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1 ^G93A mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1 ^G93A mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2–3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + AGF can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.     

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.