The Hexokinase 1 5'-UTR Mutation in Charcot-Marie-Tooth 4G Disease Alters Hexokinase 1 Binding to Voltage-Dependent Anion Channel-1 and Leads to Dysfunctional Mitochondrial Calcium Buffering.

Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance.

Serum neurofilament light chain cut-off definition for clinical diagnosis and prognosis of amyotrophic lateral sclerosis.

BACKGROUND: The neurofilament light chain (NfL) assay is gradually becoming an essential diagnostic tool for the diagnosis of many neurological diseases including amyotrophic lateral sclerosis (ALS). Different methods for the determination of this biomarker in serum have been developed in recent years. METHODS: We measured blood NfL in 429 patients referred to the tertiary ALS center of Montpellier, France using two different ultrasensitive methods (Ella™ and Simoa™) and we compared the clinical performances of these two approaches. We also converted NfL values into age and body mass index-adjusted Z-scores to assess cut-off values of this biomarker in this clinical context. RESULTS: We show comparable diagnostic and prognostic performance of Ella™ and Simoa™ technologies in ALS, with specificities and sensitivities exceeding 80% for both. We propose cut-off values for serum NfL in this clinical context, thus enabling the routine clinical use of this biomarker. CONCLUSION: The use of NfL in routine clinical practice will help predict survival and improve diagnostic accuracy by distinguishing ALS from other neurological diseases and motor neuron disease mimics.

SLITRK2, an X-linked modifier of the age at onset in C9orf72 frontotemporal lobar degeneration.

The G4C2-repeat expansion in C9orf72 is the most common cause of frontotemporal dementia and of amyotrophic lateral sclerosis. The variability of age at onset and phenotypic presentations is a hallmark of C9orf72 disease. In this study, we aimed to identify modifying factors of disease onset in C9orf72 carriers using a family-based approach, in pairs of C9orf72 carrier relatives with concordant or discordant age at onset. Linkage and association analyses provided converging evidence for a locus on chromosome Xq27.3. The minor allele A of rs1009776 was associated with an earlier onset (P = 1 × 10-5). The association with onset of dementia was replicated in an independent cohort of unrelated C9orf72 patients (P = 0.009). The protective major allele delayed the onset of dementia from 5 to 13 years on average depending on the cohort considered. The same trend was observed in an independent cohort of C9orf72 patients with extreme deviation of the age at onset (P = 0.055). No association of rs1009776 was detected in GRN patients, suggesting that the effect of rs1009776 was restricted to the onset of dementia due to C9orf72. The minor allele A is associated with a higher SLITRK2 expression based on both expression quantitative trait loci (eQTL) databases and in-house expression studies performed on C9orf72 brain tissues. SLITRK2 encodes for a post-synaptic adhesion protein. We further show that synaptic vesicle glycoprotein 2 and synaptophysin, two synaptic vesicle proteins, were decreased in frontal cortex of C9orf72 patients carrying the minor allele. Upregulation of SLITRK2 might be associated with synaptic dysfunctions and drives adverse effects in C9orf72 patients that could be modulated in those carrying the protective allele. How the modulation of SLITRK2 expression affects synaptic functions and influences the disease onset of dementia in C9orf72 carriers will require further investigations. In summary, this study describes an original approach to detect modifier genes in rare diseases and reinforces rising links between C9orf72 and synaptic dysfunctions that might directly influence the occurrence of first symptoms.

Spatio-temporal clustering of amyotrophic lateral sclerosis in France: A population-based study.

OBJECTIVE: To assess spatial aggregates of amyotrophic lateral sclerosis (ALS) incident cases, using a solid geo-epidemiological statistical method, in France. METHODS: This population-based study (2003-2011) investigated 47.1 million person-years of follow-up (PYFU). Case ascertainment of incident ALS cases was based on multiple sources (ALS referral centers, hospital centres and health insurance data). Neurologists confirmed all ALS diagnoses. Exhaustiveness was estimated through capture-recapture. Aggregates were investigated in four steps: (a) geographical modelling (standardized incidence ratio (SIR) calculation), (b) analysis of the spatial distribution of incidence (Phothoff-Winttinghill's test, Global Moran's Index, Kulldorf's spatial scan statistic, Local Moran's Index), (c) classification of the level of certainty of spatial aggregates (i.e. definite cluster; probable over-incidence area; possible over-incidence area) and (d) evaluation of the robustness of the results. RESULTS: The standardized incidence of ALS was 2.46/100,000 PYFU (95% CI 2.31-2.63, European population as reference) based on 1199 incident cases. We identified 13 areas of spatial aggregates: one cluster (stable in robustness analysis), five probable over-incidence areas (2 stable in robustness analysis) and seven possible over-incidence areas (including 4 stable areas in robustness analysis). A cluster was identified in the Rhône-Alpes region: 100 observed vs 54.07 expected cases for 2,411,514 PYFU, SIR: 1.85 (95% CI 1.50-2.25). CONCLUSION: We report here one of the largest investigations of incidence and spatial aggregation of ALS ever performed in a western country. Using a solid methodology framework for case ascertainment and cluster analysis, we identified 13 areas that warrant further investigation.

High rate of hypomorphic variants as the cause of inherited ataxia and related diseases: study of a cohort of 366 families.

PURPOSE: Diagnosis of inherited ataxia and related diseases represents a real challenge given the tremendous heterogeneity and clinical overlap of the various causes. We evaluated the efficacy of molecular diagnosis of these diseases by sequencing a large cohort of undiagnosed families. METHODS: We analyzed 366 unrelated consecutive patients with undiagnosed ataxia or related disorders by clinical exome-capture sequencing. In silico analysis was performed with an in-house pipeline that combines variant ranking and copy-number variant (CNV) searches. Variants were interpreted according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. RESULTS: We established the molecular diagnosis in 46% of the cases. We identified 35 mildly affected patients with causative variants in genes that are classically associated with severe presentations. These cases were explained by the occurrence of hypomorphic variants, but also rarely suspected mechanisms such as C-terminal truncations and translation reinitiation. CONCLUSION: A significant fraction of the clinical heterogeneity and phenotypic overlap is explained by hypomorphic variants that are difficult to identify and not readily predicted. The hypomorphic C-terminal truncation and translation reinitiation mechanisms that we identified may only apply to few genes, as it relies on specific domain organization and alterations. We identified PEX10 and FASTKD2 as candidates for translation reinitiation accounting for mild disease presentation.

Effect of familial clustering in the genetic screening of 235 French ALS families.

OBJECTIVES: To determine whether the familial clustering of amyotrophic lateral sclerosis (ALS) cases and the phenotype of the disease may help identify the pathogenic genes involved. METHODS: We conducted a targeted next-generation sequencing analysis on 235 French familial ALS (FALS), unrelated probands to identify mutations in 30 genes linked to the disease. The genealogy, that is, number of cases and generations with ALS, gender, age, site of onset and the duration of the disease were analysed. RESULTS: Regarding the number of generations, 49 pedigrees had only one affected generation, 152 had two affected generations and 34 had at least three affected generations. Among the 149 pedigrees (63.4%) for which a deleterious variant was found, an abnormal G4C2 expansion in C9orf72 was found in 98 cases as well as SOD1, TARBP or FUS mutations in 30, 9 and 7 cases, respectively. Considering pedigrees from the number of generations, abnormal G4C2 expansion in C9orf72 was more frequent in pedigrees with pairs of affected ALS cases, which represented 65.2% of our cohort. SOD1 mutation involved all types of pedigrees. No TARDBP nor FUS mutation was present in monogenerational pedigrees. TARDBP mutation predominated in bigenerational pedigrees with at least three cases and FUS mutation in multigenerational pedigrees with more than seven cases, on average, and with an age of onset younger than 45 years. CONCLUSION: Our results suggest that familial clustering, phenotypes and genotypes are interconnected in FALS, and thus it might be possible to target the genetic screening from the familial architecture and the phenotype of ALS cases.

Impact of a frequent nearsplice SOD1 variant in amyotrophic lateral sclerosis: optimising SOD1 genetic screening for gene therapy opportunities.

OBJECTIVE: Mutations in superoxide dismutase 1 gene (SOD1), encoding copper/zinc superoxide dismutase protein, are the second most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) motor neuron disease in populations of European descent. More than 200 missense variants are reported along the SOD1 protein. To limit the production of these aberrant and deleterious SOD1 species, antisense oligonucleotide approaches have recently emerged and showed promising effects in clinical trials. To offer the possibility to any patient with SOD1-ALS to benefit of such a gene therapy, it is necessary to ascertain whether any variant of unknown significance (VUS), detected for example in SOD1 non-coding sequences, is pathogenic. METHODS: We analysed SOD1 mutation distribution after SOD1 sequencing in a large cohort of 470 French familial ALS (fALS) index cases. RESULTS: We identified a total of 27 SOD1 variants in 38 families including two SOD1 variants located in nearsplice or intronic regions of the gene. The pathogenicity of the c.358-10T>G nearsplice SOD1 variant was corroborated based on its high frequency (as the second most frequent SOD1 variant) in French fALS, the segregation analysis confirmed in eight affected members of a large pedigree, the typical SOD1-related phenotype observed (with lower limb onset and prominent lower motor neuron involvement), and findings on postmortem tissues showing SOD1 misaccumulation. CONCLUSIONS: Our results highlighted nearsplice/intronic mutations in SOD1 are responsible for a significant portion of French fALS and suggested the systematic analysis of the SOD1 mRNA sequence could become the method of choice for SOD1 screening, not to miss these specific cases.

Oligogenicity, C9orf72 expansion, and variant severity in ALS.

"Oligogenic inheritance" is used to describe cases where more than one rare pathogenic variant is observed in the same individual. While multiple variants can alter disease presentation, the necessity of multiple variants to instigate pathogenesis has not been addressed in amyotrophic lateral sclerosis (ALS). We sequenced ALS-associated genes in C9orf72-expansion-positive and negative ALS patients, alongside unaffected controls, to test the importance of oligogenicity and variant deleteriousness in ALS. We found that all groups had similar numbers of rare variants, but that variant severity was significantly higher in C9orf72-negative ALS cases, suggesting sufficiency of C9orf72 expansion to cause ALS alone.

Mechanism of action of s1p receptor modulators in multiple sclerosis: The double requirement.

The ideal treatment for multiple sclerosis (MS) would target both the neuroinflammatory component of the disease (peripheral and central) and its neurodegenerative component, via modulation of a ubiquitous and pleiotropic common target. Sphingosine-1-phosphate (S1P), a product of sphingosine metabolism, regulates many biological functions (including cell proliferation and survival, cell migration, the immune response and cardiovascular function) via five subtypes of receptor. These receptors are expressed in all types of brain cells where they modulate a number of processes involved in neuronal plasticity, including myelination, neurogenesis and neuroprotection. This profile has aroused interest in modulation of S1P function as a therapeutic target in many brain diseases, particularly those in which the immune system plays a role in the development of brain lesions. Fingolimod, a S1P receptor modulator, exerts its beneficial effects in MS through its anti-inflammatory and anti-neurodegenerative effects. This review discusses recent evidence indicating that fingolimod may target both the inflammatory and neurodegenerative components of the disease process in MS.

Regulation of Brain Cholesterol: What Role Do Liver X Receptors Play in Neurodegenerative Diseases?

Liver X Receptors (LXR) alpha and beta are two members of nuclear receptor superfamily documented as endogenous cholesterol sensors. Following conversion of cholesterol in oxysterol, both LXR isoforms detect intracellular concentrations and act as transcription factors to promote expression of target genes. Among their numerous physiological roles, they act as central cholesterol-lowering factors. In the central nervous system (CNS), cholesterol has been shown to be an essential determinant of brain function, particularly as a major constituent of myelin and membranes. In the brain, LXRs act as cholesterol central regulators, and, beyond this metabolic function, LXRs have additional roles such as providing neuroprotective effects and lowering neuroinflammation. In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis (MS), dysregulations of cholesterol and oxysterol have been reported. In this paper, we propose to focus on recent advances in the knowledge of the LXRs roles on brain cholesterol and oxysterol homeostasis, neuroinflammation, neuroprotection, and their putative involvement in neurodegenerative disorders. We will discuss their potential use as candidates for both molecular diagnosis and as promising pharmacological targets in the treatment of ALS, AD, or MS patients.

KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

Deleterious mutations in the essential mRNA metabolism factor, hGle1, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Liver X receptors: from cholesterol regulation to neuroprotection-a new barrier against neurodegeneration in amyotrophic lateral sclerosis?

Cholesterol plays a central role in numerous nervous system functions. Cholesterol is the major constituent of myelin sheaths, is essential for synapse and dendrite formation, axon guidance as well as neurotransmission. Among regulators of cholesterol homeostasis, liver X receptors (LXRs), two members of the nuclear receptor superfamily, play a determinant role. LXRs act as cholesterol sensors and respond to high intracellular cholesterol concentration by decreasing plasmatic and intracellular cholesterol content. Beyond their cholesterol-lowering role, LXRs have been proposed as regulators of immunity and anti-inflammatory factors. Dysregulation of cholesterol metabolism combined to neuroinflammatory context have been described in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). ALS is characterized by the progressive loss of motoneurons in the brain and spinal cord, leading to severe paralytic condition and death of patients in a median time of 3 years. Motoneuron degeneration is accompanied by chronic neuroinflammatory response, involving microglial and astrocytic activation, infiltration of blood-derived immune cells and release of pro-inflammatory factors. We propose to discuss here the role of LXRs as a molecular link between the central nervous system cholesterol metabolism, neuroinflammation, motoneuron survival and their potential as promising therapeutic candidates for ALS therapy.

The Use of Peripherally Inserted Central Catheter in Amyotrophic Lateral Sclerosis Patients at a Later Stage.

BACKGROUND/AIMS: To describe the use of peripherally inserted central catheter (PICC), in amyotrophic lateral sclerosis (ALS) at a later stage. METHODS: Twenty-five ALS patients in the later stages of the disease underwent PICC insertion followed by parenteral nutrition (PN). For all of them, gastrostomy was non-feasible. Patients were followed until death and monitored for complications. RESULTS: PICC insertion was successful in all patients. Three months after insertion, the mean body weight increased by 4.5% (p = 0.0057). PICC could be maintained until death in all but 1 patient. The mean delay between insertion and death was 4.5 months, but PN was administered for more than 1 year in 2 patients. Complications were noted in 6 patients: sepsis (n = 4), venous thrombosis (n = 1), and upper limb oedema (n = 1), none of them resulting in death. CONCLUSION: PICC insertion for PN at a later stage of ALS, in patients for whom gastrostomy is non-feasible, appears to be a useful option compared to the central venous catheter.

Investigating the contribution of VAPB/ALS8 loss of function in 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.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories.

BACKGROUND: The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. METHODS: The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. RESULTS: Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9-100%), and the mean specificity was 98.0% (87.5-100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. CONCLUSIONS: Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

Corrected speciation and gyromitrin content of false morels linked to ALS patients with mostly slow-acetylator phenotypes.

A case-control study of sporadic amyotrophic lateral sclerosis (ALS) in a mountainous village in the French Alps discovered an association of cases with a history of eating wild fungi (false morels) collected locally and initially identified and erroneously reported as Gyromitra gigas. Specialist re-examination of dried specimens of the ALS-associated fungi demonstrated they were members of the G. esculenta group, namely G. venenata and G. esculenta, species that have been reported to contain substantially higher concentrations of gyromitrin than present in G. gigas. Gyromitrin is metabolized to monomethylhydrazine, which is responsible not only for the acute oral toxic and neurotoxic properties of false morels but also has genotoxic potential with proposed mechanistic relevance to the etiology of neurodegenerative disease. Most ALS patients had a slow- or intermediate-acetylator phenotype predicted by N-acetyltransferase-2 (NAT2) genotyping, which would increase the risk for neurotoxic and genotoxic effects of gyromitrin metabolites.

Pharmacometabolomics applied to low-dose interleukin-2 treatment in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The immunosuppressive functions of regulatory T lymphocytes (Tregs) are impaired in ALS, and correlate to disease progression. The phase 2a IMODALS trial reported an increase in Treg number in ALS patients following the administration of low-dose (ld) interleukin-2 (IL-2). We propose a pharmacometabolomics approach to decipher metabolic modifications occurring in patients treated with ld-IL-2 and its relationship with Treg response. Blood metabolomic profiles were determined on days D1, D64, and D85 from patients receiving 2 MIU of IL-2 (n = 12) and patients receiving a placebo (n = 12). We discriminated the three time points for the treatment group (average error rate of 42%). Among the important metabolites, kynurenine increased between D1 and D64, followed by a reduction at D85. The percentage increase of Treg number from D1 to D64, as predicted by the metabolome at D1, was highly correlated with the observed value. This study provided a proof of concept for metabolic characterization of the effect of ld-IL-2 in ALS. These data could present advances toward a personalized medicine approach and present pharmacometabolomics as a key tool to complement genomic and transcriptional data for drug characterization, leading to systems pharmacology.

Safety and efficacy of arimoclomol in patients with early amyotrophic lateral sclerosis (ORARIALS-01): a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial.

BACKGROUND: Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder leading to muscle weakness and respiratory failure. Arimoclomol, a heat-shock protein-70 (HSP70) co-inducer, is neuroprotective in animal models of amyotrophic lateral sclerosis, with multiple mechanisms of action, including clearance of protein aggregates, a pathological hallmark of sporadic and familial amyotrophic lateral sclerosis. We aimed to evaluate the safety and efficacy of arimoclomol in patients with amyotrophic lateral sclerosis. METHODS: ORARIALS-01 was a multinational, randomised, double-blind, placebo-controlled, parallel-group trial done at 29 centres in 12 countries in Europe and North America. Patients were eligible if they were aged 18 years or older and met El Escorial criteria for clinically possible, probable, probable laboratory-supported, definite, or familial amyotrophic lateral sclerosis; had an ALS Functional Rating Scale-Revised score of 35 or more; and had slow vital capacity at 70% or more of the value predicted on the basis of the participant's age, height, and sex. Patients were randomly assigned (2:1) in blocks of 6, stratified by use of a stable dose of riluzole or no riluzole use, to receive oral arimoclomol citrate 1200 mg/day (400 mg three times per day) or placebo. The Randomisation sequence was computer generated centrally. Investigators, study personnel, and study participants were masked to treatment allocation. The primary outcome was the Combined Assessment of Function and Survival (CAFS) rank score over 76 weeks of treatment. The primary outcome and safety were analysed in the modified intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT03491462, and is completed. FINDINGS: Between July 31, 2018, and July 17, 2019, 287 patients were screened, 245 of whom were enrolled in the trial and randomly assigned. The modified intention-to-treat population comprised 239 patients (160 in the arimoclomol group and 79 in the placebo group): 151 (63%) were male and 88 (37%) were female; mean age was 57·6 years (SD 10·9). CAFS score over 76 weeks did not differ between groups (mean 0·51 [SD 0·29] in the arimoclomol group vs 0·49 [0·28] in the placebo group; p=0·62). Cliff's delta comparing the two groups was 0·039 (95% CI -0·116 to 0·194). Proportions of participants who died were similar between the treatment groups: 29 (18%) of 160 patients in the arimoclomol group and 18 (23%) of 79 patients in the placebo group. Most deaths were due to disease progression. The most common adverse events were gastrointestinal. Adverse events were more often deemed treatment-related in the arimoclomol group (104 [65%]) than in the placebo group (41 [52%]) and more often led to treatment discontinuation in the arimoclomol group (26 [16%]) than in the placebo group (four [5%]). INTERPRETATION: Arimoclomol did not improve efficacy outcomes compared with placebo. Although available biomarker data are insufficient to preclude future strategies that target the HSP response, safety data suggest that a higher dose of arimoclomol would not have been tolerated. FUNDING: Orphazyme.

A rare motor neuron deleterious missense mutation in the DPYSL3 (CRMP4) gene is associated with ALS.

The dihydropyrimidinase-like 3 (DPYSL3) or Collapsin Response Mediator Protein 4a (CRMP4a) expression is modified in neurodegeneration and is involved in several ALS-associated pathways including axonal transport, glutamate excitotoxicity, and oxidative stress. The objective of the study was to analyze CRMP4 as a risk factor for ALS. We analyzed the DPYSL3/CRMP4 gene in French ALS patients (n = 468) and matched-controls (n = 394). We subsequently examined a variant in a Swedish population (184 SALS, 186 controls), and evaluated its functional effects on axonal growth and survival in motor neuron cell culture. The rs147541241:A>G missense mutation occurred in higher frequency among French ALS patients (odds ratio = 2.99) but the association was not confirmed in the Swedish population. In vitro expression of mutated DPYSL3 in motor neurons reduced axonal growth and accelerated cell death compared with wild type protein. Thus, the association between the rs147541241 variant and ALS was limited to the French population, highlighting the geographic particularities of genetic influences (risks, contributors). The identified variant appears to shorten motor neuron survival through a detrimental effect on axonal growth and CRMP4 could act as a key unifier in transduction pathways leading to neurodegeneration through effects on early axon development.