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.     

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.     

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.     

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.     

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.     

Increased Axonal Ribosome Numbers Is an Early Event in the Pathogenesis of Amyotrophic Lateral Sclerosis

Myelinating glia cells support axon survival and functions through mechanisms independent of myelination, and their dysfunction leads to axonal degeneration in several diseases. In amyotrophic lateral sclerosis (ALS), spinal motor neurons undergo retrograde degeneration, and slowing of axonal transport is an early event that in ALS mutant mice occurs well before motor neuron degeneration. Interestingly, in familial forms of ALS, Schwann cells have been proposed to slow disease progression. We demonstrated previously that Schwann cells transfer polyribosomes to diseased and regenerating axons, a possible rescue mechanism for disease-induced reductions in axonal proteins. Here, we investigated whether elevated levels of axonal ribosomes are also found in ALS, by analysis of a superoxide dismutase 1 (SOD1)^G93A mouse model for human familial ALS and a patient suffering from sporadic ALS. In both cases, we found that the disorder was associated with an increase in the population of axonal ribosomes in myelinated axons. Importantly, in SOD1^G93A mice, the appearance of axonal ribosomes preceded the manifestation of behavioral symptoms, indicating that upregulation of axonal ribosomes occurs early in the pathogenesis of ALS. In line with our previous studies, electron microscopy analysis showed that Schwann cells might serve as a source of axonal ribosomes in the disease-compromised axons. The early appearance of axonal ribosomes indicates an involvement of Schwann cells early in ALS neuropathology, and may serve as an early marker for disease-affected axons, not only in ALS, but also for other central and peripheral neurodegenerative disorders.     

A CRMP4‐dependent retrograde axon‐to‐soma death signal in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal non‐cell‐autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1^G93A‐ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72‐mutant patients, and the SOD1^G93A‐ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS‐affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4‐dynein interaction reduces MN loss in human‐derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4‐dependent retrograde death signal that underlies MN loss in ALS.     

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.     

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.     

The Human G93A-Superoxide Dismutase-1 Mutation,Mitochondrial Glutathione and Apoptotic Cell Death

Mutations in Cu/Zn superoxide dismutase are a cause of motor neuron death in about 20% of cases of familial amyotrophic lateral sclerosis (ALS). Although the molecular mechanism of which these mutations induce motor neuron cell death is to a large extent unknown, there is significant evidence that effects on mitochondrial function and development of oxidative stress make a major contribution to the selective death of motor neurons in this disease. In this overview article we review the current understanding of mutant SOD1-mediated motor neuron degeneration in ALS with focus on oxidative damage and mitochondrial dysfunction. We also present novel information on the role of mitochondrial glutathione for the survival of NSC-34 cells stably transfected with the human SOD1^G93A mutation, putting forward the hypothesis that this antioxidant pool provides a potentially useful target for therapeutic intervention. Special issue article in Honor of Dr. Graham Johnston.     

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.     

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.     

Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background

Motor neuron degeneration in SOD1^G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers.

Methodology/Principal Findings

Hindlimb muscles were transplanted between wild-type and SOD1^G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1^G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1^G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1^G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles.

Conclusions/Significance

These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1^G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient.     

Plasma neurofilament heavy chain levels correlate to markers of late stage disease progression and treatment response in SOD1(G93A) mice that model ALS

Background

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder characterised by progressive degeneration of motor neurons leading to death, typically within 3–5 years of symptom onset. The diagnosis of ALS is largely reliant on clinical assessment and electrophysiological findings. Neither specific investigative tools nor reliable biomarkers are currently available to enable an early diagnosis or monitoring of disease progression, hindering the design of treatment trials.

Methodology/Principal Findings

In this study, using the well-established SOD1^G93A mouse model of ALS and a new in-house ELISA method, we have validated that plasma neurofilament heavy chain protein (NfH) levels correlate with both functional markers of late stage disease progression and treatment response. We detected a significant increase in plasma levels of phosphorylated NfH during disease progression in SOD1^G93A mice from 105 days onwards. Moreover, increased plasma NfH levels correlated with the decline in muscle force, motor unit survival and, more significantly, with the loss of spinal motor neurons in SOD1 mice during this critical period of decline. Importantly, mice treated with the disease modifying compound arimoclomol had lower plasma NfH levels, suggesting plasma NfH levels could be validated as an outcome measure for treatment trials.

Conclusions/Significance

These results show that plasma NfH levels closely reflect later stages of disease progression and therapeutic response in the SOD1^G93A mouse model of ALS and may potentially be a valuable biomarker of later disease progression in ALS.     

Vascular endothelial growth factor prevents G93A‐SOD1‐induced motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A‐SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase‐3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1‐induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A‐SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A‐SOD1 toxicity and neuroprotection involves phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

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.     

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.     

DJ-1 Knockout Augments Disease Severity and Shortens Survival in a Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder, characterized by the degeneration of motor neurons. Oxidative stress plays a central role in the disease progression, in concert with an enhanced glutamate excitotoxicity and neuroinflammation. DJ-1 mutations, leading to the loss of functional protein, cause familial Parkinson’s disease and motor neuron disease in several patients. DJ-1 responds to oxidative stress and plays an important role in the cellular defense mechanisms. We aimed to investigate whether loss of functional DJ-1 alters the disease course and severity in an ALS mouse model. To this end we used mice that express the human SOD1^G93A mutation, the commonly used model of ALS and knockout of DJ-1 mice to generate SOD1 DJ-1 KO mice. We found that knocking out DJ-1in the ALS model led to an accelerated disease course and shortened survival time. DJ-1 deficiency was found to increase neuronal loss in the spinal cord associated with increased gliosis in the spinal cord and reduced antioxidant response that was regulated by the Nrf2 mechanism.The importance of DJ-1 in ALS was also illustrated in a motor neuron cell line that was exposed to glutamate toxicity and oxidative stress. Addition of the DJ-1 derived peptide, ND-13, enhanced the resistance to glutamate and SIN-1 induced toxicity. Thus, our results maintain that DJ-1 plays a role in the disease process and promotes the necessity of further investigation of DJ-1 as a therapeutic target for ALS.     

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.     

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.