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The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls.An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake.ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Background and purpose: Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles is currently incurable despite intense research and numerous unsuccessful clinical trials. Although considered as a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single target drug is not sufficient to improve the pathological phenotype. We therefore evaluated the therapeutic potential of the multi-target drug, trimetazidine, in SOD1G93A mice. Experimental approach: Trimetazidine is an anti-ischemic drug used for the treatment of coronary artery disease. As a metabolic modulator, Trimetazidine improves glucose metabolism. Furthermore, Trimetazidine enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. Here, we orally treated SOD1G93A mice with Trimetazidine, solubilized in drinking water at a dose of 20 mg/kg, from disease onset. We assessed the impact of Trimetazidine on disease progression by studying metabolic parameters, grip strength, and histological alterations in skeletal muscle, peripheral nerve and spinal cord. Key results: Trimetazidine administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1G93A mice (increased median survival of 16 days and 12.5 days for male and female respectively). Moreover, Trimetazidine prevents the dismantlement of neuromuscular junctions, attenuates motor neuron loss and reduces neuroinflammation in the spinal cord and in peripheral nerves. Conclusion and implications: In SOD1G93A mice, therapeutic effect of Trimetazidine is underpinned by its action on mitochondrial function in skeletal muscle and spinal cord. Keywords: Amyotrophic Lateral Sclerosis; Hypermetabolism; Mitochondria; Neurodegeneration; SOD1G93A mice; Trimetazidine.

CHMP2B is a protein that coordinates membrane scission events as a core component of the ESCRT machinery. Mutations in CHMP2B are an uncommon cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases with clinical, genetic, and pathological overlap. Different mutations have now been identified across the ALS-FTD spectrum. Disruption of the neuromuscular junction is an early pathogenic event in ALS. Currently, the links between neuromuscular junction functionality and ALS-associated genes, such as CHMP2B, remain poorly understood. We have previously shown that CHMP2B transgenic mice expressing the CHMP2B

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles and is currently incurable. Although considered to be a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single targeted drug is not sufficient to improve the pathological phenotype. We therefore evaluated the therapeutic potential of the multi-target drug used to treatment of coronary artery disease, trimetazidine, in SOD1As a metabolic modulator, trimetazidine improves glucose metabolism. Furthermore, trimetazidine enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. We orally treated SOD1Trimetazidine administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1In SOD1

International audience; Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons, amyotrophy and skeletal muscle paralysis usually leading to death due to respiratory failure. While generally considered an intrinsic motor neuron disease, data obtained in recent years, including our own, suggest that motor neuron protection is not sufficient to counter the disease. The dismantling of the neuromuscular junction is closely linked to chronic energy deficit found throughout the body. Metabolic (hypermetabolism and dyslipidemia) and mitochondrial alterations described in patients and murine models of ALS are associated with the development and progression of disease pathology and they appear long before motor neurons die. It is clear that these metabolic changes participate in the pathology of the disease. In this review, we summarize these changes seen throughout the course of the disease, and the subsequent impact of glucose–fatty acid oxidation imbalance on disease progression. We also highlight studies that show that correcting this loss of metabolic flexibility should now be considered a major goal for the treatment of ALS.

BackgroundThe cause of the motor neuron (MN) death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards motor neurons, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Since MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.MethodsSporadic ALS patients were confirmed to be ALS according to El Escorial criteria, were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n=27) or deltoids of aged-matched healthy subjects (n=30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RTqPCR and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC motor neurons and on healthy human myotubes, with untreated cells used as controls.ResultsAn accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared to vesicles from healthy control myotubes, when administered to healthy motor neurons the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient motor neurons was abolished by anti-CD63 immuno-blocking of vesicle uptake.ConclusionALS muscle vesicles are shown to be toxic to motor neurons, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.One Sentence SummaryMuscle cells of ALS patients secrete vesicles that are toxic to motor neurons

International audience; Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults. While it is primarily characterized by the death of upper and lower motor neurons, there is a significant metabolic component involved in the progression of the disease. Two-thirds of ALS patients have metabolic alterations that are associated with the severity of symptoms. In ALS, as in other neurodegenerative diseases, the metabolism of glycosphingolipids, a class of complex lipids, is strongly dysregulated. We therefore assume that this pathway constitutes an interesting avenue for therapeutic approaches. We have shown that the glucosylceramide degrading enzyme, glucocerebrosidase (GBA) 2 is abnormally increased in the spinal cord of the SOD1G86R mouse model of ALS. Ambroxol, a chaperone molecule that inhibits GBA2, has been shown to have beneficial effects by slowing the development of the disease in SOD1G86R mice. Currently used in clinical trials for Parkinson's and Gaucher disease, ambroxol could be considered as a promising therapeutic treatment for ALS.

Amyotrophic lateral sclerosis is characterized by the degeneration of upper and lower motor neurons, yet an increasing number of studies in both mouse models and patients with amyotrophic lateral sclerosis suggest that altered metabolic homeostasis is also a feature of disease. Pre-clinical and clinical studies have shown that modulation of energy balance can be beneficial in amyotrophic lateral sclerosis. However, the capacity to target specific metabolic pathways or mechanisms requires detailed understanding of metabolic dysregulation in amyotrophic lateral sclerosis. Here, using the superoxide dismutase 1, glycine to alanine substitution at amino acid 93 (SOD1G93A) mouse model of amyotrophic lateral sclerosis, we demonstrate that an increase in whole-body metabolism occurs at a time when glycolytic muscle exhibits an increased dependence on fatty acid oxidation. Using myotubes derived from muscle of amyotrophic lateral sclerosis patients, we also show that increased dependence on fatty acid oxidation is associated with increased whole-body energy expenditure. In the present study, increased fatty acid oxidation was associated with slower disease progression. However, within the patient cohort, there was considerable heterogeneity in whole-body metabolism and fuel oxidation profiles. Thus, future studies that decipher specific metabolic changes at an individual patient level are essential for the development of treatments that aim to target metabolic pathways in amyotrophic lateral sclerosis., In superoxide dismutase 1, glycine to alanine substitution at amino acid 93 (SOD1G93A) mice, increased whole-body metabolism occurs alongside fat depletion and increased fatty acid oxidation in glycolytic muscle. Myotubes from patients with amyotrophic lateral sclerosis have increased fatty acid oxidation, and this is associated with increased whole-body energy expenditure. Increased fatty acid oxidation may sustain energy supply to slow disease., Graphical Abstract Graphical Abstract

International audience; Sphingolipids are complex lipids. They play a structural role in neurons, but are also involved in regulating cellular communication, and neuronal differentiation and maturation. There is increasing evidence to suggest that dysregulated metabolism of sphingolipids is linked to neurodegenerative processes in amyotrophic lateral sclerosis (ALS), Parkinson's disease and Gaucher's disease. In this review, we provide an overview of the role of sphingolipids in the development and maintenance of the nervous system. We describe the implications of altered metabolism of sphingolipids in the pathophysiology of certain neurodegenerative diseases, with a primary focus on ALS. Finally, we provide an update of potential treatments that could be used to target the metabolism of sphingolipids in neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of upper and lower motor neurons, yet an increasing number of studies in both mouse models and patients with ALS suggest that altered metabolic homeostasis is a feature of disease. Pre-clinical and clinical studies have shown that modulation of energy balance can be beneficial in ALS. However, our capacity to target specific metabolic pathways or mechanisms requires detailed understanding of metabolic dysregulation in ALS. Here, using the SOD1G93Amouse model of ALS, we demonstrate that an increase in whole-body metabolism occurs at a time when glycolytic muscle exhibits an increased dependence on fatty acid oxidation. Using myotubes derived from muscle of ALS patients, we also show that increased dependence on fatty acid oxidation is associated with increased whole-body energy expenditure. In the present study, increased fatty acid oxidation was associated with slower disease progression. However, we observed considerable heterogeneity in whole-body metabolism and fuel oxidation profiles across our patient cohort. Thus, future studies that decipher specific metabolic changes at an individual patient level are essential for the development of treatments that aim to target metabolic pathways in ALS.

International audience; Amyotrophic lateral sclerosis (ALS) is a multifactorial and fatal neurodegenerative disease. Growing evidence connects sphingolipid metabolism to the pathophysiology of ALS. In particular, levels of ceramides, glucosylceramides, and gangliosides are dysregulated in the central nervous system and at the neuromuscular junctions of both animal models and patients. Glucosylceramide is the main precursor of complex glycosphingolipids that is degraded by lysosomal (GBA1) or non-lysosomal (GBA2) glucocerebrosidase. Here, we report that GBA2, but not GBA1, activity is markedly increased in the spinal cord, of SOD1G86R mice, an animal model of familial ALS, even before disease onset. We therefore investigated the effects of ambroxol hydrochloride, a known GBA2 inhibitor, in SOD1G86R mice. A presymptomatic administration of ambroxol hydrochloride, in the drinking water, delayed disease onset, protecting neuromuscular junctions, and the number of functional spinal motor neurons. When administered at disease onset, ambroxol hydrochloride delayed motor function decline, protected neuromuscular junctions, and extended overall survival of the SOD1G86R mice. In addition, ambroxol hydrochloride improved motor recovery and muscle re-innervation after transient sciatic nerve injury in non-transgenic mice and promoted axonal elongation in an in vitro model of motor unit. Our study suggests that ambroxol hydrochloride promotes and protects motor units and improves axonal plasticity, and that this generic compound is a promising drug candidate for ALS.

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with currently no cure. These two diseases share a clinical continuum with overlapping genetic causes. Mutations in the CHMP2B gene are found in patients with ALS, FTD and ALS-FTD. To highlight deregulated mechanisms occurring in ALS-FTD linked to the CHMP2B gene, we performed a whole transcriptomic study on lumbar spinal cord from CHMP2B

International audience; Mutations in the charged multivesicular body protein 2B (CHMP2B) are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and with a mixed ALS-FTD syndrome. To model this syndrome, we generated a transgenic mouse line expressing the human CHMP2Bintron5 mutant in a neuron-specific manner. These mice developed a dose-dependent disease phenotype. A longitudinal study revealed progressive gait abnormalities, reduced muscle strength and decreased motor coordination. CHMP2Bintron5 mice died due to generalized paralysis. When paralyzed, signs of denervation were present as attested by altered electromyographic profiles, by decreased number of fully innervated neuromuscular junctions, by reduction in size of motor endplates and by a decrease of sciatic nerve axons area. However, spinal motor neurons cell bodies were preserved until death. In addition to the motor dysfunctions, CHMP2Bintron5 mice progressively developed FTD-relevant behavioural modifications such as disinhibition, stereotypies, decrease in social interactions, compulsivity and change in dietary preferences. Furthermore, neurons in the affected spinal cord and brain regions showed accumulation of p62-positive cytoplasmic inclusions associated or not with ubiquitin and CHMP2Bintron5 As observed in FTD3 patients, these inclusions were negative for TDP-43 and FUS. Moreover, astrogliosis and microgliosis developed with age. Altogether, these data indicate that the neuronal expression of human CHMP2Bintron5 in areas involved in motor and cognitive functions induces progressive motor alterations associated with dementia symptoms and with histopathological hallmarks reminiscent of both ALS and FTD.

Besides loss of motor neurons in spinal cord, brainstem and cerebral cortex, patients with ALS show an abnormal depletion of energy stores and a parallel hypermetabolism in spite of simultaneous progression of skeletal muscle atrophy. Our results highlighted bioenergetic defects in skeletal muscle of ALS mice long before the disease onset that lead to deep modifications of muscle physiology. Muscle remodelling occurs in SOD1G93A mice before the activation of muscle denervation markers and this is followed by an increase of energy expenditure unrelated to physical activity. To this regard our attention has been focused on the identification of precocious biomarkers closely related to hypermetabolism, a phenomenon that possesses prognostic and diagnostic relevance. Finally, chronic Ranolazine treatment, a FDA-approved inhibitor of fatty acid b-oxidation, decreases energy expenditure in symptomatic SOD1G93A mice and this correlates with a robust, albeit temporary, recovery of pathological phenotype.

International audience; Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with currently no cure. These two diseases share a clinical continuum with overlapping genetic causes. Mutations in the CHMP2B gene are found in patients with ALS, FTD and ALS-FTD. To highlight deregulated mechanisms occurring in ALS-FTD linked to the CHMP2B gene, we performed a whole transcriptomic study on lumbar spinal cord from CHMP2Bintron5 mice, a model that develops progressive motor alterations associated with dementia symptoms reminiscent of both ALS and FTD. To gain insight into the transcriptomic changes taking place during disease progression this study was performed at three stages: asymptomatic, symptomatic and end stage. We showed that before appearance of motor symptoms, the major disrupted mechanisms were linked with the immune system/inflammatory response and lipid metabolism. These processes were progressively replaced by alterations of neuronal electric activity as motor symptoms appeared, alterations that could lead to motor neuron dysfunction. To investigate overlapping alterations in gene expression between two ALS-causing genes, we then compared the transcriptome of symptomatic CHMP2Bintron5 mice with the one of symptomatic SOD1G86R mice and found the same families deregulated providing further insights into common underlying dysfunction of biological pathways, disrupted or disturbed in ALS. Altogether, this study provides a database to explore potential new candidate genes involved in the CHMP2Bintron5-based pathogenesis of ALS, and provides molecular clues to further understand the functional consequences that diseased neurons expressing CHMP2B mutant may have on their neighbor cells.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets.

Spasticity is a common and disabling symptom observed in patients with central nervous system diseases, including amyotrophic lateral sclerosis, a disease affecting both upper and lower motor neurons. In amyotrophic lateral sclerosis, spasticity is traditionally thought to be the result of degeneration of the upper motor neurons in the cerebral cortex, although degeneration of other neuronal types, in particular serotonergic neurons, might also represent a cause of spasticity. We performed a pathology study in seven patients with amyotrophic lateral sclerosis and six control subjects and observed that central serotonergic neurons suffer from a degenerative process with prominent neuritic degeneration, and sometimes loss of cell bodies in patients with amyotrophic lateral sclerosis. Moreover, distal serotonergic projections to spinal cord motor neurons and hippocampus systematically degenerated in patients with amyotrophic lateral sclerosis. In SOD1 (G86R) mice, a transgenic model of amyotrophic lateral sclerosis, serotonin levels were decreased in brainstem and spinal cord before onset of motor symptoms. Furthermore, there was noticeable atrophy of serotonin neuronal cell bodies along with neuritic degeneration at disease onset. We hypothesized that degeneration of serotonergic neurons could underlie spasticity in amyotrophic lateral sclerosis and investigated this hypothesis in vivo using tail muscle spastic-like contractions in response to mechanical stimulation as a measure of spasticity. In SOD1 (G86R) mice, tail muscle spastic-like contractions were observed at end-stage. Importantly, they were abolished by 5-hydroxytryptamine-2b/c receptors inverse agonists. In line with this, 5-hydroxytryptamine-2b receptor expression was strongly increased at disease onset. In all, we show that serotonergic neurons degenerate during amyotrophic lateral sclerosis, and that this might underlie spasticity in mice. Further research is needed to determine whether inverse agonists of 5-hydroxytryptamine-2b/c receptors could be of interest in treating spasticity in patients with amyotrophic lateral sclerosis.

The mutations P56S and T46I in the gene encoding vesicle-associated membrane protein-associated protein B/C (VAPB) cause ALS8, a familial form of amyotrophic lateral sclerosis (ALS). Overexpression of mutant forms of VAPB leads to cytosolic aggregates, suggesting a gain of function of the mutant protein. However, recent work suggested that the loss of VAPB function could be the major mechanism leading to ALS8. Here, we used multiple genetic and experimental approaches to study whether VAPB loss of function might be sufficient to trigger motor neuron degeneration. In order to identify additional ALS-associated VAPB mutations, we screened the entire VAPB gene in a cohort of ALS patients and detected two mutations (A145V and S160Δ). To directly address the contribution of VAPB loss of function in ALS, we generated zebrafish and mouse models with either a decreased or a complete loss of Vapb expression. Vapb knockdown in zebrafish led to swimming deficits. Mice knocked-out for Vapb showed mild motor deficits after 18 months of age yet had innervated neuromuscular junctions (NMJs). Importantly, overexpression of VAPB mutations were unable to rescue the motor deficit caused by Vapb knockdown in zebrafish and failed to cause a toxic gain-of-function defect on their own. Thus, Vapb loss of function weakens the motor system of vertebrate animal models but is on its own unable to lead to a complete ALS phenotype. Our findings are consistent with the notion that VAPB mutations constitute a risk factor for motor neuron disease through a loss of VAPB function.

Amyotrophic lateral sclerosis (ALS) is characterised by the progressive degeneration of upper and lower motor neurons. Besides motor neuron degeneration, ALS is associated with several defects in energy metabolism, including weight loss, hypermetabolism, and hyperlipidaemia. Most of these abnormalities correlate with duration of survival, and available clinical evidence supports a negative contribution of defective energy metabolism to the overall pathogenic process. Findings from animal models of ALS support this view and provide insights into the underlying mechanisms. Altogether, these results have clinical consequences for the management of defective energy metabolism in patients with ALS and pave the way for future therapeutic interventions.

Motor neuron degeneration and progressive muscle atrophy characterize amyotrophic lateral sclerosis (ALS) in humans and related mutant superoxide dismutase-1 (SOD1) transgenic mice. Our previous microarray studies on ALS muscle revealed strong up-regulation of Ras-related associated with diabetes (Rad), an inhibitor of voltage-gated calcium channels. The mechanisms controlling Rad expression in disease are unknown. We analyzed Rad expression in skeletal muscle from ALS patients and animal models and investigated whether it is regulated by oxidative stress. In mutant SOD1 mice, Rad up-regulation preceded motor symptoms and markedly increased as disease progressed. Increased Rad expression was also obtained in surgically denervated muscle. No clinical signs of denervation were seen in asymptomatic mice, however. We therefore suspected that muscular mutant SOD1 toxicity causes precocious Rad up-regulation. We confirmed the accumulation of reactive oxygen species (ROS) at asymptomatic stages, coincident with the rise in Rad expression. By subjecting muscle to ischemia-reperfusion, we observed ROS accumulation and Rad overexpression. The cell-permeative antioxidant Tempol inhibited the stimulatory effect of ischemia-reperfusion. Tempol also reduced Rad up-regulation after experimental denervation. Our study provides strong evidence for the implication of oxidative stress in modulating Rad expression, in association with the initiation and progression of ALS muscle atrophy.

The efflux pumps located at the blood-brain barrier (BBB) prevent drugs entering the brain. As such, efflux pumps are a major obstacle to drug brain distribution. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with little therapeutics available: riluzole is the only drug approved in its treatment. The lack of response to treatment in ALS may be, at least in part, due to increased activities of efflux pumps in relation to disease, leading to subtherapeutic brain concentrations of drugs. In the present study, we used a transgenic mouse model of ALS (G86R mSOD1 mice) to test this hypothesis. Expression and functionality of P-glycoprotein (ABCB1, P-gp) and Breast Cancer Resistance Protein (ABCG2, BCRP), two major efflux pumps, were studied. We observed an increased P-gp expression (1.5-fold) in presymptomatic mSOD1 mice compared to wild-type controls. Consistent with this, P-gp function was also increased by 1.5-fold and riluzole brain disposition was decreased by 1.7-fold in mSOD1 mice. Contrasting with this, BCRP expression and function were unaltered by the pathology. These results demonstrate that BBB transport proteins are modified in G86R mSOD1 mice ALS model. Such findings underline potential problems in extrapolating the results of animal studies to humans and developing clinical trials, especially for drugs transported by P-gp.

Our objectives were to analyse carbohydrate metabolism in a series of ALS patients and to examine potential association with parameters of lipid metabolism and clinical features. Glucose tolerance was assessed by the oral glucose tolerance test in 21 non-diabetic ALS patients and compared with 21 age- and sex-matched normal subjects. Lipids and lactate/pyruvate ratio, levels of pro-inflammatory cytokines (tumour necrosis factor-alpha and interleukin-6) and adipocytokines (leptin and adiponectin) were also measured in ALS patients. Mann-Whitney U-tests analysed continuous data and Fisher's exact tests assessed categorical data. Blood glucose determined 120 min after the glucose bolus was significantly higher in patients with ALS (7.41 mmol/l+/-1.68) compared to controls (6.05+/-1.44, p=0.006). ALS patients with impaired glucose tolerance (IGT) according to WHO criteria (n=7, 33%) were more likely to have elevated free fatty acids (FFA) levels compared to patients with normal glucose tolerance (0.77 nmol/l+/-0.30 vs. 0.57+/-0.19, p=0.04). IGT was not associated with disease duration or severity. In conclusion, patients with ALS show abnormal glucose tolerance that could be associated with increased FFA levels, a key determinant of insulin resistance. The origin of glucose homeostasis abnormalities in ALS may be multifactorial and deserves further investigation.

The development of therapeutics for ALS/MND is largely based on work in experimental animals carrying human SOD mutations. However, translation of apparent therapeutic successes from in vivo to the human disease has proven difficult and a considerable amount of financial resources has been apparently wasted. Standard operating procedures (SOPs) for preclinical animal research in ALS/MND are urgently required. Such SOPs will help to establish SOPs for translational research for other neurological diseases within the next few years. To identify the challenges and to improve the research methodology, the European ALS/MND group held a meeting in 2006 and published guidelines in 2007 (1). A second international conference to improve the guidelines was held in 2009. These second and improved guidelines are dedicated to the memory of Sean F. Scott.

In neurons, cytoplasmic dynein functions as a molecular motor responsible for retrograde axonal transport. An impairment of axonal transport is thought to play a key role in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis, the most frequent motor neuron disease in the elderly. In this regard, previous studies described two heterozygous mouse strains bearing missense point mutations in the dynein heavy chain 1 gene that were reported to display late-onset progressive motor neuron degeneration. Here we show, however, that one of these mutant strains, the so-called Cra mice does not suffer from motor neuron loss, even in aged animals. Consistently, we did not observe electrophysiological or biochemical signs of muscle denervation, indicative of motor neuron disease. The "hindlimb clasping" phenotype of Cra mice could rather be due to the prominent degeneration of sensory neurons associated with a loss of muscle spindles. Altogether, these findings show that dynein heavy chain mutation triggers sensory neuropathy rather than motor neuron disease.

Objective A proportion of patients with pure lower motor neuron syndrome (LMNS) progress to amyotrophic lateral sclerosis (ALS). Early detection of this progression is impossible, which delays the patient's access to treatment. Muscle expression of Nogo-A is a new candidate marker of ALS. We tested whether detection of Nogo-A in a muscle biopsy from patients with LMNS predicts progression to ALS. Methods Thirty-three patients who had undergone a muscle biopsy during the diagnostic workup of spinal LMNS were observed for 12 months. Nogo-A expression was measured by Western blot in muscle biopsy samples and compared with the final diagnosis. Results Nogo-A expression was detected in 17 patients and was absent in 16 patients. The detection of Nogo-A in muscle biopsy samples from LMNS patients correctly identified patients who further progressed to ALS with 91% accuracy, 94% sensitivity, and 88% specificity. In patients who later developed typical ALS, Nogo-A may be detected as early as 3 months after the onset of symptoms. Interpretation Nogo-A test is able to identify ALS early in the course of the disease when diagnosis is difficult, requiring further progression. Use of the test in clinical practice may shorten the delay before introduction of neuroprotective drugs or inclusion in clinical trials. Ann Neurol 2007

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease characterized by degeneration of upper and lower motor neurons, generalized weakness and muscle atrophy. Most cases of ALS appear sporadically but some forms of the disease result from mutations in the gene encoding the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1). Several other mutated genes have also been found to predispose to ALS including, among others, one that encodes the regulator of axonal retrograde transport dynactin. As all roads lead to the proverbial Rome, we discuss here how distinct molecular pathways may converge to the same final result that is motor neuron death. We critically review the basic research on SOD1-linked ALS to propose a pioneering model of a 'systemic' form of the disease, causally involving multiple cell types, either neuronal or non-neuronal. Contrasting this, we also postulate that other neuron-specific defects, as those triggered by dynactin dysfunction, may account for a primary motor neuron disease that would represent 'pure' neuronal forms of ALS. Identifying different disease subtypes is an unavoidable step toward the understanding of the physiopathology of ALS and will hopefully help to design specific treatments for each subset of patients.

International audience; Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease in adults. Numerous studies indicate that ALS is a systemic disease that affects whole body physiology and metabolic homeostasis. Using a mouse model of the disease (SOD1 G86R), we investigated muscle physiology and motor behavior with respect to muscle metabolic capacity. We found that at 65 days of age, an age described as asymptomatic, SOD1 G86R mice presented with improved endurance capacity associated with an early inhibition in the capacity for glycolytic muscle to use glucose as a source of energy and a switch in fuel preference toward lipids. Indeed, in glycolytic muscles we showed progressive induction of pyruvate dehydrogenase kinase 4 expression. Phosphofructokinase 1 was inhibited, and the expression of lipid handling molecules was increased. This mechanism represents a chronic pathologic alteration in muscle metabolism that is exacerbated with disease progression. Further, inhibition of pyruvate dehydrogenase kinase 4 activity with dichloroacetate delayed symptom onset while improving mitochondrial dysfunction and ameliorating muscle denervation. In this study, we provide the first molecular basis for the particular sensitivity of glycolytic muscles to ALS pathology.

We performed repeated analysis of mitochondrial respiratory function in skeletal muscle (SM) of patients with early-stage sporadic amyotrophic lateral sclerosis (SALS) to determine whether mitochondrial function was altered as the disease advanced. SM biopsies were obtained from 7 patients with newly diagnosed SALS, the same 7 patients 3 months later, and 7 sedentary controls. Muscle fibers were permeabilized with saponin, then skinned and placed in an oxygraphic chamber to measure basal and maximal adenosine diphosphate (ADP)-stimulated respiration rates and to assess mitochondrial regulation by ADP. We found that the maximal oxidative phosphorylation capacity of muscular mitochondria significantly increased, and muscular mitochondrial respiratory complex IV activity significantly decreased as the disease advanced. This temporal study demonstrates for the first time that mitochondrial function in SM in human SALS is progressively altered as the disease develops.

Nogo, a protein inhibiting axonal regeneration, exhibits a characteristic isoform-specific pattern of expression in skeletal muscle of transgenic mice and patients with amyotrophic lateral sclerosis. Here, the increased levels of Nogo-A or Nogo-B in muscle biopsies of 15 amyotrophic lateral sclerosis patients significantly correlated with the severity of clinical disability and with the degree of muscle fiber atrophy. Nogo-A immunoreactivity was observed selectively in atrophic slow-twitch type I fibers. These results suggest that Nogo expression in muscle is a marker of amyotrophic lateral sclerosis severity.

A subset of familial ALS cases is associated with missense mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1), a free radical scavenging enzyme that protects cells against oxidative stress. Overexpression of these ALS-linked mutations confers an unidentified gain of function to the enzyme that triggers a series of neurological disorders characteristic of human ALS. To understand how skeletal muscle may counteract the progression of the disease, we explored the expression of different molecular effectors involved in antioxidant pathways. Our results are strongly indicative of the early and long-lasting activation of a series of molecular effectors thought to act coordinately in preventing the increased oxidative stress characteristic of ALS.

The nervous system plays a key role in the regulation of neuroendocrine axes and, in turn, the released neurohormones modulate the activity of different brain regions. Neurodegenerative diseases, which are known to affect specific neuronal populations, may provoke neuroendocrine dysfunctions that alter the intimate relationship between both systems. In addition, these modifications may influence the progression of the neurodegenerative process. In the present review, we summarise some of the endocrine changes characterising three major neurodegenerative diseases: Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Special attention is focused on the contribution of disease transgenic models to elucidate such alterations.

Amyotrophic lateral sclerosis is sporadic (SALS) in 90% of cases and has complex environmental and genetic influences. Nogo protein inhibits neurite outgrowth and is overexpressed in muscle in ALS. Our aims were to study the reticulon 4 receptor gene RTN4R which encodes Nogo 1 receptor (NgR1) in SALS, to test if the variants were associated with variable expression of the gene and whether NgR1 protein expression was modified in a transgenic mouse model of ALS. We genotyped three single nucleotide polymorphisms (SNPs; rs701421, rs701427, and rs1567871) of the RTN4R gene in 364 SALS French patients and 430 controls. We examined expression of RTN4R mRNA by quantitative PCR in control post mortem human brain tissue. We determined the expression of NgR1 protein in spinal motor neurons from a SOD1 G86R ALS mouse model. We observed significant associations between SALS and RTN4R alleles. Messenger RNA expression from RTN4R in human cortical brain tissue correlated significantly with the genotypes of rs701427. NgR1 protein expression was reduced in Nogo A positive motor neurons from diseased transgenic animals. In conclusion, these observations suggest that a functional RTN4R gene variant is associated with SALS. This variant may act in concert with other genetic variants or environmental influences.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by the selective degeneration of upper and lower motor neurons. The lack of a molecular diagnostic marker is of increasing concern in view of the therapeutic strategies in development. Using an unbiased subtractive suppressive hybridization screen we have identified a clone encoding the neurite outgrowth inhibitor Nogo and shown that its isoforms display a characteristic altered expression in ALS. This was first confirmed by analyzing Nogo isoform expression in a transgenic ALS model at early asymptomatic stages where we found increased levels of Nogo-A and decreased Nogo-C and importantly, not following experimentally induced denervation. Furthermore, we confirmed these changes in both post-mortem and biopsy samples from diagnosed ALS patients but not control patients. Thus, the alteration in Nogo expression pattern, common to sporadic and familial ALS, represents a potential diagnosis tool and points strongly to Nogo having a central role in disease.

International audience; Molecular mechanisms promoting neuronal death in amyotrophic lateral sclerosis (ALS) were investigated using transgenic mice that overexpressed the G86R mutated form of the Cu/Zn superoxide dismutase (SOD1) gene. We observed: (i) alteration of the Bcl-x/Bax ratio and (ii) activation of the transcription factor p53, as deduced from its location within neuron nuclei. We further demonstrated that ectopic expression of the G86R mutant SOD1 in PC12 cells enhanced both p53 expression and phosphorylation, leading to transcriptional stimulation of p53-responsive genes. These findings provide evidence that the p53 signaling pathway is activated in SOD1-linked familial ALS and may play a causative role in spinal cord neuron apoptosis by modulating the Bcl-x/Bax ratio.

In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

Amyotrophic lateral sclerosis (ALS) is a fatal, paralytic disorder that primarily affects motoneurons. By combining physiological and morphological approaches, we examined the effect of a murine superoxide dismutase 1 (SOD1) mutation (G86R), which induces neurological disorders resembling human familial ALS (FALS), on the arginine vasopressin (AVP) hypothalamo-neurohypophysial axis, an unmyelinated tract poor in neurofilaments. First, we observed that G86R mice progressively consumed more water than wild-type littermates. Furthermore, levels of plasma AVP and neurohypophysial AVP content were decreased in the SOD1 mutant mice, whereas the amount of hypothalamic AVP increased in an age-dependent manner. However, hypothalamic AVP mRNA levels were not significantly modified in these animals. At the ultrastructural level, we found that the neurohypophysis of G86R mice had a decreased number of neurosecretory axons. Conversely, the presence of large axon swellings was more pronounced in the SOD1 mutant mice. In addition, the size of neurosecretory granules was higher in G86R than in wild-type animals. All these findings strongly suggest that the FALS-associated SOD1 mutation injures the hypothalamo-neurohypophysial axis by provoking early, progressive disturbances in the axonal transport of neurosecretory products from neuronal perikarya to nerve terminals. This blockade could ultimately result in degeneration of the tract, as proposed for the myelinated, neurofilament-enriched motor axons affected by ALS.

Motor neuron diseases (MNDs) are characterized by selective death of motor neurons and include mainly adult-onset amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Neurodegeneration is not the single pathogenic event occurring during disease progression. There are multiple lines of evidence for the existence of defects in lipid metabolism at peripheral level. For instance, hypermetabolism is well characterized in ALS, and dyslipidemia correlates with better prognosis in patients. Lipid metabolism plays also a role in other MNDs. In SMA, misuse of lipids as energetic nutrients is described in patients and in related animal models. The composition of structural lipids in the central nervous system is modified, with repercussion on membrane fluidity and on cell signaling mediated by bioactive lipids. Here, we review the main epidemiologic and mechanistic findings that link alterations of lipid metabolism and motor neuron degeneration, and we discuss the rationale of targeting these modifications for therapeutic management of MNDs.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset degenerative disease characterized by the loss of upper and lower motor neurons leading to progressive muscle atrophy and paralysis. The lack of molecular markers of the progression of disease is detrimental to clinical practice and therapeutic trials. Objective: This study was designed to identify gene expression changes in skeletal muscle that could reliably define the degree of disease severity. Methods: Gene expression profiles were obtained from the deltoid muscles of ALS patients and healthy subjects. Changes in differentially expressed genes were compared to the status of deltoid muscle disability, as determined by manual muscle testing, electrophysiology and the degree of myofiber atrophy. Functionally related genes were grouped by annotation analysis, and deltoid muscle injury was predicted using binary tree classifiers. Results: Two sets of 25 and 70 transcripts appeared differentially regulated exclusively in early and advanced states of deltoid muscle impairment, respectively. The expression of another set of 198 transcripts correlated with a composite score of muscle injury combining manual muscle testing and histological examination. From the totality of these expression changes, 155 transcripts distinguished advanced from early deltoid muscle impairment with 80% sensitivity and 100% specificity. Nine of these transcripts, known also to be regulated in ALS mouse and surgically denervated muscle, predicted the advanced disease status with 100% sensitivity and specificity. Conclusion: We provide robust gene expression changes that can be of practical use when monitoring ALS status and the effects of disease-modifying drugs.

BackgroundAmyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease involving upper and lower motor neurons loss. Clinical features are highly variable among patients and there are currently few known disease-modifying factors underlying this heterogeneity. Serotonin is involved in a range of functions altered in ALS, including motor neuron excitability and energy metabolism. However, whether serotoninergic activity represents a disease modifier of ALS natural history remains unknown.MethodologyPlatelet and plasma unconjugated concentrations of serotonin and plasma 5-HIAA, the major serotonin metabolite, levels were measured using HPLC with coulometric detection in a cohort of 85 patients with ALS all followed-up until death and compared to a control group of 29 subjects.Principal findingsPlatelet serotonin levels were significantly decreased in ALS patients. Platelet serotonin levels did not correlate with disease duration but were positively correlated with survival of the patients. Univariate Cox model analysis showed a 57% decreased risk of death for patients with platelet serotonin levels in the normal range relative to patients with abnormally low platelet serotonin (p = 0.0195). This protective effect remained significant after adjustment with age, gender or site of onset in multivariate analysis. Plasma unconjugated serotonin and 5-HIAA levels were unchanged in ALS patients compared to controls and did not correlate with clinical parameters.Conclusions/significanceThe positive correlation between platelet serotonin levels and survival strongly suggests that serotonin influences the course of ALS disease.

Amyotrophic lateral sclerosis (ALS) represents the major adult-onset motor neuron disease. Analyses of ALS animal models have shown that motor neuron death starts with neuromuscular junction (NMJ) destruction and distal axonal degeneration. Most importantly, motor neuron death results from pathological events occurring outside the motor neuron, especially in glial cells and skeletal muscle and, surprisingly, is associated with pathological defects outside the motor system. In particular, ALS pathogenesis includes systemic defects such as muscle hypermetabolism, energy deficit, and widespread alterations of lipid metabolism that were shown to participate in motor neuron degeneration. Current research should now focus on understanding the relationships between these pathological hallmarks and how such global defects lead to the ALS-linked selective loss of motor neurons.

International audience; BACKGROUND: Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS. METHODOLOGY/PRINCIPAL FINDINGS: We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model. CONCLUSIONS/SIGNIFICANCE: These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases.

International audience; Amyotrophic lateral sclerosis (ALS) is the most frequent adult onset motor neuron disorder. A subset of ALS cases is linked to mutations in the copper/zinc superoxide dismutase (sod1) gene and detailed phenotypic analysis of transgenic mice overexpressing mutant forms of SOD1 (mSOD1) allowed a better understanding of the pathophysiological mechanisms leading to motor neuron death. The promising results obtained in these animal models however poorly translated into conclusive clinical trials. In this review, we summarize the main pathological mechanisms at work in mSOD1 mice. In particular, recent results showed that the key pathological event was the destruction of the neuromuscular junction rather than motor neuron death. Neuromuscular junction dismantlement is likely the result of a chronic energy deficiency at the level of the whole organism. These results, along with a comparative analysis between the phenotype of mSOD1 mice and ALS patients, suggest new therapeutic strategies and show the interests but also the limits of the animal models. double dagger.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of unknown origin affecting primarily motor neurons. The observation that point mutations in the copper/zinc superoxide dismutase

International audience; Muscle atrophy is a major hallmark of amyotrophic lateral sclerosis (ALS), the most frequent adult-onset motor neuron disease. To define the full set of alterations in gene expression in skeletal muscle during the course of the disease, we used the G86R superoxide dismutase-1 transgenic mouse model of ALS and performed high-density oligonucleotide microarrays. We compared these data to those obtained by axotomy-induced denervation. A major set of gene regulations in G86R muscles resembled those of surgically denervated muscles, but many others appeared specific to the ALS condition. The first significant transcriptional changes appeared in a subpopulation of mice before the onset of overt clinical symptoms and motor neuron death. These early changes affected genes involved in detoxification (e.g., ALDH3, metallothionein-2, and thioredoxin-1) and regeneration (e.g., BTG1, RB1, and RUNX1) but also tissue degradation (e.g., C/EBPdelta and DDIT4) and cell death (e.g., ankyrin repeat domain-1, CDKN1A, GADD45alpha, and PEG3). Of particular interest, metallothionein-1 and -2, ATF3, cathepsin-Z, and galectin-3 genes appeared, among others, commonly regulated in both skeletal muscle (our present data) and spinal motor neurons (as previously reported) of paralyzed ALS mice. The importance of these findings is twofold. First, they designate the distal part of the motor unit as a primary site of disease. Second, they identify specific gene regulations to be explored in the search for therapeutic strategies that could alleviate disease before motor neuron death manifests clinically.

Amyotrophic lateral sclerosis (ALS) is characterized by motoneuron (MN) degeneration, generalized weakness, and muscle atrophy. The premature death of MNs is thought to be a determinant in the onset of this disease. In a transgenic mouse model of ALS expressing the G86R mutant superoxide dismutase 1 (mSOD1), we demonstrated previously that CREB (cAMP response element-binding protein)-binding protein (CBP) and histone acetylation levels were specifically decreased in nuclei of degenerating MNs. We show here that oxidative stress and mSOD1 overexpression can both impinge on CBP levels by transcriptional repression, in an MN-derived cell line. Histone deacetylase inhibitor (HDACi) treatment was able to reset proper acetylation levels and displayed an efficient neuroprotective capacity against oxidative stressin vitro. Interestingly, HDACi also upregulated CBP transcriptional expression in MNs. Moreover, when injected to G86R micein vivo, the HDACi sodium valproate (VPA) maintained normal acetylation levels in the spinal cord, efficiently restored CBP levels in MNs, and significantly prevented MN death in these animals. However, despite neuroprotection, mean survival of treated animals was not significantly improved (