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Amyotrophic lateral sclerosis (ALS) is associated with impaired energy metabolism, including weight loss and decreased appetite which are negatively correlated with survival. Neural mechanisms underlying metabolic impairment in ALS remain unknown. ALS patients and presymptomatic gene carriers have early hypothalamic atrophy. The lateral hypothalamic area (LHA) controls metabolic homeostasis through the secretion of neuropeptides such as orexin/hypocretin and melanin-concentrating hormone (MCH). Here, we show loss of MCH-positive neurons in three mouse models of ALS based on SOD1 or FUS mutations. Supplementation with MCH (1.2 µg/d) through continuous intracerebroventricular delivery led to weight gain in male mutant Sod1G86R mice. MCH supplementation increased food intake, rescued expression of the key appetite-related neuropeptide AgRP (agouti-related protein) and modified respiratory exchange ratio, suggesting increased carbohydrate usage during the inactive phase. Importantly, we document pTDP-43 pathology and neurodegeneration in the LHA of sporadic ALS patients. Neuronal cell loss was associated with pTDP-43-positive inclusions and signs of neurodegeneration in MCH-positive neurons. These results suggest that hypothalamic MCH is lost in ALS and contributes to the metabolic changes, including weight loss and decreased appetite.

Background Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. Methods The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. Results Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. Conclusion This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype.

Large polyglutamine expansions in Ataxin-2 (ATXN2) cause multi-system nervous atrophy in Spinocerebellar Ataxia type 2 (SCA2). Intermediate size expansions carry a risk for selective motor neuron degeneration, known as Amyotrophic Lateral Sclerosis (ALS). Conversely, the depletion of ATXN2 prevents disease progression in ALS. Although ATXN2 interacts directly with RNA, and in ALS pathogenesis there is a crucial role of RNA toxicity, the affected functional pathways remain ill defined. Here, we examined an authentic SCA2 mouse model withAtxn2-CAG100-KnockIn for a first definition of molecular mechanisms in spinal cord pathology. Neurophysiology of lower limbs detected sensory neuropathy rather than motor denervation. Triple immunofluorescence demonstrated cytosolic ATXN2 aggregates sequestrating TDP43 and TIA1 from the nucleus. In immunoblots, this was accompanied by elevated CASP3, RIPK1 and PQBP1 abundance. RT-qPCR showed increase ofGrn,Tlr7andRnaset2mRNA versusEif5a2,Dcp2, Uhmk1andKif5adecrease. These SCA2 findings overlap well with known ALS features. Similar to other ataxias and dystonias, decreased mRNA levels forUnc80,Tacr1,Gnal,Ano3,Kcna2,Elovl5andCdr1contrasted withGpnmbincrease. Preterminal stage tissue showed strongly activated microglia containing ATXN2 aggregates, with parallel astrogliosis. Global transcriptome profiles from stages of incipient motor deficit versus preterminal age identified molecules with progressive downregulation, where a cluster of cholesterol biosynthesis enzymes includingDhcr24,Msmo1,Idi1andHmgcs1was prominent. Gas chromatography demonstrated a massive loss of crucial cholesterol precursor metabolites. Overall, the ATXN2 protein aggregation process affects diverse subcellular compartments, in particular stress granules, endoplasmic reticulum and receptor tyrosine kinase signaling. These findings identify new targets and potential biomarkers for neuroprotective therapies.

Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration., Mutations in the RNA-binding protein FUS contribute to ALS. Here the authors use CLIP-seq on synaptoneurosomes to identify proteins associated with synapse organization and plasticity that are differentially regulated in a knock-in ALS mouse model.

Degeneration of brainstem serotonin neurons has been demonstrated in ALS patients and mouse models and was found responsible for the development of spasticity. Consistent with involvement of central serotonin pathways, 5-HT2B receptor (5-HT2BR) was upregulated in microglia of ALS mice. Its deletion worsened disease outcome in the Sod1G86R mouse model and led to microglial degeneration. In ALS patients, a polymorphism in HTR2B gene leading to higher receptor expression in CNS, was associated with increased survival in patients as well as prevention of microglial degeneration. Thus, the aim of our study was to determine the effect of a 5-HT2BR agonist : BW723C86 (BW), in the Sod1G86R mouse model. Despite good pharmacokinetic and pharmacological profiles, BW did not ameliorate disease outcome or motor neuron degeneration in a fast progressing mouse model of ALS despite evidence of modulation of microglial gene expression.

Chemogenetic motoneuron excitation and astrocyte GPCR-Gi signaling restore blood–spinal cord barrier, disrupted in four ALS mouse models, revealing its role in disease progression but not initiation., Blood–spinal cord barrier (BSCB) disruption is thought to contribute to motoneuron (MN) loss in amyotrophic lateral sclerosis (ALS). It is currently unclear whether impairment of the BSCB is the cause or consequence of MN dysfunction and whether its restoration may be directly beneficial. We revealed that SOD1G93A, FUSΔNLS, TDP43G298S, and Tbk1+/− ALS mouse models commonly shared alterations in the BSCB, unrelated to motoneuron loss. We exploit PSAM/PSEM chemogenetics in SOD1G93A mice to demonstrate that the BSCB is rescued by increased MN firing, whereas inactivation worsens it. Moreover, we use DREADD chemogenetics, alone or in multiplexed form, to show that activation of Gi signaling in astrocytes restores BSCB integrity, independently of MN firing, with no effect on MN disease markers and dissociating them from BSCB disruption. We show that astrocytic levels of the BSCB stabilizers Wnt7a and Wnt5a are decreased in SOD1G93A mice and strongly enhanced by Gi signaling, although further decreased by MN inactivation. Thus, we demonstrate that BSCB impairment follows MN dysfunction in ALS pathogenesis but can be reversed by Gi-induced expression of astrocytic Wnt5a/7a.

International audience; Traumatic brain injury (TBI) has been proposed as a risk factor for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To determine whether TBI might trigger or exacerbate ALS-relevant pathology, we delivered a mild stab-wound injury to the motor cortex of three different ALS mouse models expressing mutations in SOD1, TDP-43 or FUS and scrutinized the effects on the formation of phospho-TDP-43 (pTDP-43) cytoplasmic granules. Stab-injury induced the formation of cytoplasmic TDP-43 granules in wt animals, peaking at 3dpi; a much larger response was seen in mutant TDP-43 mice, whose response peaked at 7dpi. The pTDP-43 granules did not colocalize with the stress markers TIAR-1 and FUS but colocalized with FMRP (35%) and with p62 (65%), suggesting their involvement in transport granules and their clearance by autophagy. A similar, albeit smaller effect, was seen in mutant FUS mice. In the SOD1G93A mouse model, neither increase in pTDP-43 granules nor in SOD1 aggregates were detected. In all cases, pTDP-43 granules were cleared and the number of pTDP-43-positive neurons returned to baseline by 40dpi. Neither injury-related neuronal loss nor motor performance or survival was significantly different in transgenic mice receiving injury vs sham mice. Thus, trauma can trigger ALS-related TDP-43 pathology, the extent of which is modulated by ALS-related mutations. However, the pathological findings prove reversible and do not affect disease progression and neuronal vulnerability.

Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with 'dying-back' neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.

International audience; Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

The primarily nuclear RNA-binding protein FUS (fused in sarcoma) forms pathological cytoplasmic inclusions in a subset of early-onset amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. In response to cellular stress, FUS is recruited to cytoplasmic stress granules, which are hypothesized to act as precursors of pathological inclusions. We monitored the stress-induced nucleocytoplasmic shuttling of endogenous FUS in an ex vivo mouse CNS model and human neural networks. We found that hyperosmolar, but not oxidative, stress induced robust cytoplasmic translocation of neuronal FUS, with transient nuclear clearance and loss of function. Surprisingly, this reaction is independent of stress granule formation and the molecular pathways activated by hyperosmolarity. Instead, it represents a mechanism mediated by cytoplasmic redistribution of Transportin 1/2 and is potentiated by transcriptional inhibition. Importantly, astrocytes, which remain unaffected in ALS/FTD-FUS, are spared from this stress reaction that may signify the initial event in the development of FUS pathology., Cell Reports, 24 (4), ISSN:2666-3864, ISSN:2211-1247

Mutations in components of the molecular motor dynein/dynactin lead to neurodegenerative diseases of the motor system or atypical parkinsonism. These mutations are associated with prominent accumulation of vesicles involved in autophagy and lysosomal pathways, and with protein inclusions. Whether alleviating these defects would affect motor symptoms remain unknown. Here, we show that a mouse model expressing low levels of disease linked-G59S mutant dynactin p150(Glued) develops motor dysfunction >8 months before loss of motor neurons or dopaminergic degeneration is observed. Abnormal accumulation of autophagosomes and protein inclusions were efficiently corrected by lowering dietary protein content, and this was associated with transcriptional upregulations of key players in autophagy. Most importantly this dietary modification partially rescued overall neurological symptoms in these mice after onset. Similar observations were made in another mouse strain carrying a point mutation in the dynein heavy chain gene. Collectively, our data suggest that stimulating the autophagy/lysosomal system through appropriate nutritional intervention has significant beneficial effects on motor symptoms of dynein/dynactin diseases even after symptom onset.

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.

International audience; FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.

International audience; Microglia are the resident mononuclear phagocytes of the central nervous system and have been implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). During neurodegeneration, microglial activation is accompanied by infiltration of circulating monocytes, leading to production of multiple inflammatory mediators in the spinal cord. Degenerative alterations in mononuclear phagocytes are commonly observed during neurodegenerative diseases, yet little is known concerning the mechanisms leading to their degeneration, or the consequences on disease progression. Here we observed that the serotonin 2B receptor (5-HT2B), a serotonin receptor expressed in microglia, is upregulated in the spinal cord of three different transgenic mouse models of ALS. In mutant SOD1 mice, this upregulation was restricted to cells positive for CD11b, a marker of mononuclear phagocytes. Ablation of 5-HT2B receptor in transgenic ALS mice expressing mutant SOD1 resulted in increased degeneration of mononuclear phagocytes, as evidenced by fragmentation of Iba1-positive cellular processes. This was accompanied by decreased expression of key neuroinflammatory genes but also loss of expression of homeostatic microglial genes. Importantly, the dramatic effect of 5-HT2B receptor ablation on mononuclear phagocytes was associated with acceleration of disease progression. To determine the translational relevance of these results, we studied polymorphisms in the human HTR2B gene, which encodes the 5-HT2B receptor, in a large cohort of ALS patients. In this cohort, the C allele of SNP rs10199752 in HTR2B was associated with longer survival. Moreover, patients carrying one copy of the C allele of SNP rs10199752 showed increased 5-HT2B mRNA in spinal cord and displayed less pronounced degeneration of Iba1 positive cells than patients carrying two copies of the more common A allele. Thus, the 5-HT2B receptor limits degeneration of spinal cord mononuclear phagocytes, most likely microglia, and slows disease progression in ALS. Targeting this receptor might be therapeutically useful.

Summary Studying ciliopathies, like the Bardet-Biedl syndrome (BBS), allow the identification of signaling pathways potentially involved in common diseases, sharing phenotypic features like obesity or type 2 diabetes. Given the close association between obesity and insulin resistance, obese BBS patients would be expected to be insulin resistant. Surprisingly, we found that a majority of obese BBS patients retained normal glucose tolerance and insulin sensitivity. Patient's adipose tissue biopsies revealed upregulation of adipogenic genes and decrease of inflammatory mediators. In vitro studies on human primary mesenchymal stem cells (MSCs) showed that BBS12 inactivation facilitated adipogenesis, increased insulin sensitivity, and glucose utilization. We generated a Bbs12 −/− mouse model to assess the impact of Bbs12 inactivation on adipocyte biology. Despite increased obesity, glucose tolerance was increased with specific enhanced insulin sensitivity in the fat. This correlated with an active recruitment of MSCs resulting in adipose tissue hyperplasia and decreased in inflammation.

International audience; The molecular motor dynein is regulated by the huntingtin protein, and Huntington's disease (HD) mutations of huntingtin disrupt dynein motor activity. Besides abnormalities in the central nervous system, HD animal models develop prominent peripheral pathology, with defective brown tissue thermogenesis and dysfunctional white adipocytes, but whether this peripheral phenotype is recapitulated by dynein dysfunction is unknown. Here, we observed prominently increased adiposity in mice harboring the legs at odd angles (Loa/+) or the Cramping mutations (Cra/+) in the dynein heavy chain gene. In Cra/+ mice, hyperadiposity occurred in the absence of energy imbalance and was the result of impaired norepinephrine-stimulated lipolysis. A similar phenotype was observed in 3T3L1 adipocytes upon chemical inhibition of dynein showing that loss of functional dynein leads to impairment of lipolysis. Ex vivo, dynein mutant adipose tissue displayed increased reactive oxygen species production that was, at least partially, responsible for the decreased cellular responses to norepinephrine and subsequent defect in stimulated lipolysis. Dynein mutation also affected norepinephrine efficacy to elicit a thermogenic response and led to morphological abnormalities in brown adipose tissue and cold intolerance in dynein mutant mice. Interestingly, protein levels of huntingtin were decreased in dynein mutant adipose tissue. Collectively, our results provide genetic evidence that dynein plays a key role in lipid metabolism and thermogenesis through a modulation of oxidative stress elicited by norepinephrine. This peripheral phenotype of dynein mutant mice is similar to that observed in various animal models of HD, lending further support for a functional link between huntingtin and dynein.

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntington's disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150(Glued). However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD.

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.

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.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar beta-amyloid (A beta), which is derived from the alternative processing of beta-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun N-terminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.

Nogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface.

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

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.

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.

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 devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 3-5 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1α, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1α leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1α-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1α as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1α in this neurodegenerative disorder.

Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that C. elegans and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the C. elegans VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, Vapb knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction., Author Summary ALS patients often present with systemic alterations in energy metabolism, such as dyslipidemia and hypermetabolism of unknown origin. Reduction of Vapb function is thought to cause motor neuron disease in ALS8 patients and may predispose individuals to ALS, in general. We have shown that neurons secrete the N-terminal VAPB vMSP into the extracellular environment. The secreted vMSPs signal through Roundabout and Lar-like receptors on striated muscles. This neuron to muscle signaling pathway localizes mitochondria to myofilament I-bands and promotes mitochondrial function. Here we show that loss of VAPB in C. elegans neurons causes metabolic changes in muscles, including altered fat metabolism and elevated DAF-16 FoxO transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels, and prolongs survival in Vapb mutants. However, it does not influence muscle mitochondrial localization nor does it affect oxygen consumption. We also show that Vapb knockout mice exhibit disrupted muscular triacylglycerol and FoxO target gene transcriptional responses to fasting and refeeding. These data indicate that impaired vMSP signaling to muscle triggers an energy deficiency, which induces a protective metabolic response involving FoxO. Hence, some energy metabolism alterations observed in ALS patients might be a consequence of striated muscle mitochondrial dysfunction.

Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.

BACKGROUND: Pioglitazone, an oral anti-diabetic that stimulates the PPAR-gamma transcription factor, increased survival of mice with amyotrophic lateral sclerosis (ALS). METHODS/PRINCIPAL FINDINGS: We performed a phase II, double blind, multicentre, placebo controlled trial of pioglitazone in ALS patients under riluzole. 219 patients were randomly assigned to receive 45 mg/day of pioglitazone or placebo (one: one allocation ratio). The primary endpoint was survival. Secondary endpoints included incidence of non-invasive ventilation and tracheotomy, and slopes of ALS-FRS, slow vital capacity, and quality of life as assessed using EUROQoL EQ-5D. The study was conducted under a two-stage group sequential test, allowing to stop for futility or superiority after interim analysis. Shortly after interim analysis, 30 patients under pioglitazone and 24 patients under placebo had died. The trial was stopped for futility; the hazard ratio for primary endpoint was 1.21 (95% CI: 0.71-2.07, p = 0.48). Secondary endpoints were not modified by pioglitazone treatment. Pioglitazone was well tolerated. CONCLUSION/SIGNIFICANCE: Pioglitazone has no beneficial effects on the survival of ALS patients as add-on therapy to riluzole. TRIAL REGISTRATION: Clinicaltrials.gov NCT00690118.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment.

Background Granulocyte colony stimulating factor (G-CSF) is a growth factor essential for generation of neutrophilic granulocytes. Apart from this hematopoietic function, we have recently uncovered potent neuroprotective and regenerative properties of G-CSF in the central nervous system (CNS). The G-CSF receptor and G-CSF itself are expressed in α motoneurons, G-CSF protects motoneurons, and improves outcome in the SOD1(G93A) transgenic mouse model for amyotrophic lateral sclerosis (ALS). In vitro, G-CSF acts anti-apoptotically on motoneuronal cells. Due to the pleiotrophic effects of G-CSF and the complexity of the SOD1 transgenic ALS models it was however not possible to clearly distinguish between directly mediated anti-apoptotic and indirectly protective effects on motoneurons. Here we studied whether G-CSF is able to protect motoneurons from purely apoptotic cell death induced by a monocausal paradigm, neonatal sciatic nerve axotomy. Results We performed sciatic nerve axotomy in neonatal mice overexpressing G-CSF in the CNS and found that G-CSF transgenic mice displayed significantly higher numbers of surviving lumbar motoneurons 4 days following axotomy than their littermate controls. Also, surviving motoneurons in G-CSF overexpressing animals were larger, suggesting additional trophic effects of this growth factor. Conclusions In this model of pure apoptotic cell death the protective effects of G-CSF indicate direct actions of G-CSF on motoneurons in vivo. This shows that G-CSF exerts potent anti-apoptotic activities towards motoneurons in vivo and suggests that the protection offered by G-CSF in ALS mouse models is due to its direct neuroprotective activity.

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; 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.

The Nogo-66 receptor (NgR) plays a critical role in restricting axon regeneration in the central nervous system. This inhibitory action is in part mediated by a neuronal receptor complex containing p75NTR, a multifunctional receptor also well known to trigger cell death upon binding to neurotrophins such as NGF. In the present study, we show that Pep4 and NEP1–40, which are two peptides derived from the Nogo-66 sequence that modulate NgR-mediated neurite outgrowth inhibition, prevent NGF-stimulated p75NTR-dependent death of cultured embryonic motor neurons. They also confer protection on spinal cord motor neurons after neonatal sciatic nerve axotomy. These findings demonstrate an as-yet-unknown function of NgR in maintaining neuronal survival that may be relevant for motor neuron development and degeneration.

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 (

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease, causing motor neuron degeneration, muscle atrophy, paralysis, and death. Despite this degenerative process, a stable hypermetabolic state has been observed in a large subset of patients. Mice expressing a mutant form of Cu/Zn-superoxide dismutase (mSOD1 mice) constitute an animal model of ALS that, like patients, exhibits unexpectedly increased energy expenditure. Counterbalancing for this increase with a high-fat diet extends lifespan and prevents motor neuron loss. Here, we investigated whether lipid metabolism is defective in this animal model. Hepatic lipid metabolism was roughly normal, whereas gastrointestinal absorption of lipids as well as peripheral clearance of triglyceride-rich lipoproteins were markedly increased, leading to decreased postprandial lipidemia. This defect was corrected by the high-fat regimen that typically induces neuroprotection in these animals. Together, our findings show that energy metabolism in mSOD1 mice shifts toward an increase in the peripheral use of lipids. This metabolic shift probably accounts for the protective effect of dietary lipids in this model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss and muscle wasting. In muscles of ALS patients, Nogo-A—a protein known to inhibit axon regeneration—is ectopically expressed at levels that correlate with the severity of the clinical symptoms. We now show that the genetic ablation of Nogo-A extends survival and reduces muscle denervation in a mouse model of ALS. In turn, overexpression of Nogo-A in wild-type muscle fibres leads to shrinkage of the postsynapse and retraction of the presynaptic motor ending. This suggests that the expression of Nogo-A occurring early in ALS skeletal muscle could cause repulsion and destabilization of the motor nerve terminals, and subsequent dying back of the axons and motor neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons and progressive muscle atrophy. A subset of patients harbors point mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1), which allowed the generation of transgenic mice that express different SOD1 mutations and develop an ALS-like pathology. Recently, we reported in these mice the occurrence of a characteristic defect in energy homeostasis and the beneficial effect on the course of the disease of a high-energy fat-enriched diet. In this review, we discuss the implication of these findings in the light of classical clinical observations concerning metabolic alterations in human ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by selective loss of motor neurons and progressive muscle wasting. Growing evidence indicates that mitochondrial dysfunction, not only occurring in motor neurons but also in skeletal muscle, may play a crucial role in the pathogenesis. In this regard, the life expectancy of the ALS G93A mouse line is extended by creatine, an intracellular energy shuttle that ameliorates muscle function. Moreover, a population of patients with sporadic ALS exhibits a generalized hypermetabolic state of as yet unknown origin. Altogether, these findings led us to explore whether alterations in energy homeostasis may contribute to the disease process. Here, we show important variations in a number of metabolic indicators in transgenic ALS mice, which in all shows a metabolic deficit. These alterations were accompanied early in the asymptomatic phase of the disease by reduced adipose tissue accumulation, increased energy expenditure, and concomitant skeletal muscle hypermetabolism. Compensating this energetic imbalance with a highly energetic diet extended mean survival by 20%. In conclusion, we suggest that hypermetabolism, mainly of muscular origin, may represent by itself an additional driven force involved in increasing motor neuron vulnerability.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily motor neurons. Growing evidence suggests a mitochondrial defect in ALS. The precise molecular mechanisms underlying those defects are unknown. We studied the expression of mitochondrial uncoupling proteins (UCPs), key regulators of mitochondrial functions, in tissues from a mouse model of ALS (SOD1 G86R transgenic mice) and from muscular biopsies of human sporadic ALS. Surprisingly, in SOD1 G86R mice, UCPs, and particularly UCP3, were upregulated in skeletal muscle but not in spinal cord. Consistent with this pattern of expression, ATP levels were selectively depleted in muscle but not in neural tissues 1 month before disease onset and the respiratory control ratio of isolated mitochondria is decreased. UCP3 up-regulation was not observed in experimentally denervated muscles, suggesting that changes in muscular UCP3 expression are associated with the physiopathological processes of ALS. This is further supported by our observation of increased UCP3 levels in human ALS muscular biopsies. We propose that UCP3 up-regulation in skeletal muscle contributes to the characteristic mitochondrial damage of ALS and to the onset of the disease. Moreover, since skeletal muscle is a key metabolic tissue, our findings suggest that ALS may not solely arise from neuronal events but also from more generalized metabolic defects.

Fumaric acid esters (FAE) are oral analogs of fumarate that have recently been shown to decrease relapse rate and disease progression in multiple sclerosis (MS), prompting to investigate their protective potential in other neurological diseases such as amyotrophic lateral sclerosis (ALS). Despite efficacy in MS, mechanisms of action of FAEs are still largely unknown. FAEs are known to activate the transcription factor Nrf2 and downstream anti-oxidant responses through the succination of Nrf2 inhibitor KEAP1. However, fumarate is also a known inhibitor of prolyl-hydroxylases domain enzymes (PhD), and PhD inhibition might lead to stabilization of the HIF-1α transcription factor under normoxic conditions and subsequent activation of a pseudo hypoxic response. Whether Nrf2 activation is associated with HIF-1α stabilization in response to FAEs in cell types relevant to MS or ALS remains unknown. Here, we show that FAEs elicit HIF-1α accumulation, and VEGF release as its expected consequence, in astrocytes but not in other cell types of the central nervous system. Reporter assays demonstrated that increased astrocytic VEGF release in response to FAEs was dependent upon both HIF-1α and Nrf2 activation. Last, astrocytes of transgenic mice expressing SOD1(G93A), an animal model of ALS, displayed reduced VEGF release in response to FAEs. These studies show that FAEs elicit different signaling pathways in cell types from the central nervous system, in particular a pseudo-hypoxic response in astrocytes. Disease relevant mutations might affect this response.

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles.