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.

Motor neuron-extrinsic mechanisms have been shown to participate in the pathogenesis of ALS-SOD1, one familial form of amyotrophic lateral sclerosis (ALS). It remains unclear whether such mechanisms contribute to other familial forms, such as TDP-43 and FUS-associated ALS. Here, we characterize a single-copy mouse model of ALS-FUS that conditionally expresses a disease-relevant truncating FUS mutant from the endogenous murine Fus gene. We show that these mice, but not mice heterozygous for a Fus null allele, develop similar pathology as ALS-FUS patients and a mild motor neuron phenotype. Most importantly, CRE-mediated rescue of the Fus mutation within motor neurons prevented degeneration of motor neuron cell bodies, but only delayed appearance of motor symptoms. Indeed, we observed downregulation of multiple myelin-related genes, and increased numbers of oligodendrocytes in the spinal cord supporting their contribution to behavioral deficits. In all, we show that mutant FUS triggers toxic events in both motor neurons and neighboring cells to elicit motor neuron disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1687-9) contains supplementary material, which is available to authorized users.

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, the most common adult-onset motor neuron disease, leads to death within 3 to 5 years after onset. Beyond progressive motor impairment, patients with amyotrophic lateral sclerosis suffer from major defects in energy metabolism, such as weight loss, which are well correlated with survival. Indeed, nutritional intervention targeting weight loss might improve survival of patients. However, the neural mechanisms underlying metabolic impairment in patients with amyotrophic lateral sclerosis remain elusive, in particular due to the lack of longitudinal studies. Here we took advantage of samples collected during the clinical trial of pioglitazone (GERP-ALS), and characterized longitudinally energy metabolism of patients with amyotrophic lateral sclerosis in response to pioglitazone, a drug with well-characterized metabolic effects. As expected, pioglitazone decreased glycaemia, decreased liver enzymes and increased circulating adiponectin in patients with amyotrophic lateral sclerosis, showing its efficacy in the periphery. However, pioglitazone did not increase body weight of patients with amyotrophic lateral sclerosis independently of bulbar involvement. As pioglitazone increases body weight through a direct inhibition of the hypothalamic melanocortin system, we studied hypothalamic neurons producing proopiomelanocortin (POMC) and the endogenous melanocortin inhibitor agouti-related peptide (AGRP), in mice expressing amyotrophic lateral sclerosis-linked mutant SOD1(G86R). We observed lower Pomc but higher Agrp mRNA levels in the hypothalamus of presymptomatic SOD1(G86R) mice. Consistently, numbers of POMC-positive neurons were decreased, whereas AGRP fibre density was elevated in the hypothalamic arcuate nucleus of SOD1(G86R) mice. Consistent with a defect in the hypothalamic melanocortin system, food intake after short term fasting was increased in SOD1(G86R) mice. Importantly, these findings were replicated in two other amyotrophic lateral sclerosis mouse models based on TDP-43 (Tardbp) and FUS mutations. Finally, we demonstrate that the melanocortin defect is primarily caused by serotonin loss in mutant SOD1(G86R) mice. Altogether, the current study combined clinical evidence and experimental studies in rodents to provide a mechanistic explanation for abnormalities in food intake and weight control observed in patients with amyotrophic lateral sclerosis. Importantly, these results also show that amyotrophic lateral sclerosis progression impairs responsiveness to classical drugs leading to weight gain. This has important implications for pharmacological management of weight loss in amyotrophic lateral sclerosis.

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.

Abstract:Microglia are the resident 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 overall increased phagocytic activity and production of multiple inflammatory mediators in the spinal cord. Degenerative alterations in monocytes and microglia 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-HT 2B ), 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-HT 2B 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-HT 2B 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-HT 2B 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-HT 2B mRNA in spinal cord and displayed less pronounced degeneration of mononuclear phagocytes than patients carrying two copies of the more common A allele. Thus functional 5-HT 2B receptors limit degeneration of spinal cord macrophages (most likely microglia), and slow disease progression in ALS. Targeting this receptor might be therapeutically useful.

Degeneration of serotonin neurons is necessary to trigger spasticity through the 5-HT receptor. The wide range of drugs targeting the serotonergic system could be useful to treat spasticity in neurological diseases. Ann Neurol 2017;82:444-456.

Classical and systems genetics have identified wide networks of genes associated with cognitive and neurodevelopmental diseases. In parallel to deciphering the role of each of these genes in neuronal or synaptic function, evaluating the response of neuronal and molecular networks to gene loss of function could reveal some pathophysiological mechanisms potentially accessible to nongenetic therapies. Loss of function of the Rho-GAP oligophrenin-1 is associated with cognitive impairments in both human and mouse. Upregulation of both PKA and ROCK has been reported in mice, but it remains unclear whether kinase hyperactivity contributes to the behavioral phenotypes. In this study, we thoroughly characterized a prominent perseveration phenotype displayed by-deficient mice using a Y-maze spatial working memory (SWM) test. We report that deficiency in the mouse generated severe cognitive impairments, characterized by both a high occurrence of perseverative behaviors and a lack of deliberation during the SWM test. and pharmacological experiments suggest that PKA dysregulation in the mPFC underlies cognitive dysfunction in-deficient mice, as assessed using a delayed spatial alternation task results. Functionally, mPFC neuronal networks appeared to be affected in a PKA-dependent manner, whereas hippocampal-PFC projections involved in SWM were not affected in mice. Thus, we propose that discrete gene mutations in intellectual disability might generate "secondary" pathophysiological mechanisms, which are prone to become pharmacological targets for curative strategies in adult patients. Here we report that deficiency generates severe impairments in performance at spatial working memory tests, characterized by a high occurrence of perseverative behaviors and a lack of decision making. This cognitive deficit is consecutive to PKA deregulation in the mPFC that prevents KO mice to exploit a correctly acquired rule. Functionally, mPFC neuronal networks appear to be affected in a PKA-dependent manner, whereas behaviorally important hippocampal projections were preserved by the mutation. Thus, we propose that discrete gene mutations in intellectual disability can generate "secondary" pathophysiological mechanisms prone to become pharmacological targets for curative strategies in adults.