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Aim: The aim of this study was to detect misfolded Cu/Zn SOD1 as a potential biomarker for amyotrophic lateral sclerosis (ALS). Materials & methods: Two ultrasensitive immunodetection assays were developed for the quantification of total and misfolded SOD1. Results: The detection of total and misfolded SOD1 was possible in human serum and cerebrospinal fluid. Total SOD1 was increased in cerebrospinal fluid from ALS patients. Misfolded SOD1 had low and variable expression in both control and ALS patient samples. Conclusion: These assays hold promise for improving our understanding of ALS and its detection, and could lead to more effective treatment options in the future. Further studies in larger cohorts are now required.

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., (© 2023 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background : Data from published studies about the effect of HFE polymorphisms on ALS risk, phenotype, and survival are still inconclusive. We aimed at evaluating whether the p.H63D polymorphism is a modifier of phenotype and survival in SOD1 -mutated patients. Methods : We included 183 SOD1 -mutated ALS patients. Mutations were classified as severe or mild according to the median survival of the study population. Patients were screened for the HFE p.H63D polymorphism. Survival was calculated using the Kaplan-Meier modeling, and differences were measured by the log-rank test. Multivariable analysis was performed with the Cox proportional hazards model (stepwise backward). Results : SOD1 severe mutation carriers show more frequent familial history for ALS and shorter survival compared to mild mutation carriers. Carriers and non-carriers of the p.H63D polymorphism did not differ in terms of sex ratio, frequency of positive familial history, age at onset, and bulbar/spinal ratio. In univariate and in Cox multivariable analysis using sex, age at onset, site of onset, family history, country of origin, and mutation severity as covariates, p.H63D carriers had a longer survival ( p = 0.034 and p = 0.004). Conclusions : We found that SOD1 -mutated ALS patients carrying the p.H63D HFE polymorphism have a longer survival compared to non-carriers, independently of sex, age and site of onset, family history, nation of origin, and severity of mutations, suggesting a possible role as disease progression modifier for the p.H63D HFE polymorphism in SOD1 -ALS., Competing Interests: Antonio Canosa serves on the Editorial Board of Biomedicines and serves as Guest Editor for the Special Issues ‘Recent Advances in Amyotrophic Lateral Sclerosis Genetics and Pathophysiology’ and ‘Recent Advances in Amyotrophic Lateral Sclerosis Genetics and Pathophysiology 2.0′ of Biomedicines. Andrea Calvo received a research grant from Cytokinetics. Adriano Chiò serves on scientific advisory boards for Mitsubishi Tanabe, Roche, Biogen, Cytokinetics, Denali, and AveXis, and received a research grant from Italfarmaco. Jessica Mandrioli received research support from Pfizer. The other authors have no conflict of interest relevant for the manuscript.

The identity and role of environmental factors in the etiology of sporadic amyotrophic lateral sclerosis (sALS) is poorly understood outside of three former high-incidence foci of Western Pacific ALS and a hotspot of sALS in the French Alps. In both instances, there is a strong association with exposure to DNA-damaging (genotoxic) chemicals years or decades prior to clinical onset of motor neuron disease. In light of this recent understanding, we discuss published geographic clusters of ALS, conjugal cases, single-affected twins, and young-onset cases in relation to their demographic, geographic and environmental associations but also whether, in theory, there was the possibility of exposure to genotoxic chemicals of natural or synthetic origin. Special opportunities to test for such exposures in sALS exist in southeast France, northwest Italy, Finland, the U.S. East North Central States, and in the U.S. Air Force and Space Force. Given the degree and timing of exposure to an environmental trigger of ALS may be related to the age at which the disease is expressed, research should focus on the lifetime exposome (from conception to clinical onset) of young sALS cases. Multidisciplinary research of this type may lead to the identification of ALS causation, mechanism, and primary prevention, as well as to early detection of impending ALS and pre-clinical treatment to slow development of this fatal neurological disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spencer, Palmer, Kisby, Lagrange, Horowitz, Valdes Angues, Reis, Vernoux, Raoul and Camu.)

Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability., (© 2022. The Author(s).)

A 56-year-old man presented with rapidly evolving/sub-acute upper and lower motor neuron syndrome in 2015 with significant weakness in the four limbs and the bulbar region. Amyotrophic lateral sclerosis functional rating scale-revised (ALSFRS-r) was rated 34/48. On electromyography, there was a diffuse and active denervation in the four limbs and the tongue. A diagnosis of definite ALS according to international criteria was made. Six months later the patient stopped worsening. In the following years he progressively recovered. ALSFRS-r score improved to reach 48/48 in 2021. His neurological examination is normal and electromyography shows no denervation. Inquiry revealed that he presented a few months and, again a few days before onset, a mushroom poisoning. He was used to eating false morels either crude or undercooked and developed muscles cramps, nausea and vertigo. The relationships between this reversible sub-acute motor neuron syndrome and mushroom intoxication are discussed in the light of the recently described cluster in the Alps with a high incidence of ALS cases. Epidemiological investigations showed that all patients, but not their spouses, used to eat crude or undercooked false morels. Such a mushroom contains hydrazines, a known neurotoxic agent. We are not aware of another case of ALS reversal in that cluster area. We propose that a potential mushroom poisoning be thoroughly searched for when facing with a patient with sub-acute or rapidly worsening ALS syndrome.

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., (© 2022. Springer Nature B.V.)

Background: Pediatric investigation plans (PIPs) describe how adult drugs can be studied in children. In 2015, PIPs for Amyotrophic Lateral Sclerosis (ALS) became mandatory for European marketing-authorization of adult treatments, unless a waiver is granted by the European Medicines Agency (EMA). Objective: To assess the feasibility of clinical studies on the effect of therapy in children (<18 years) with ALS in Europe. Methods: The EMA database was searched for submitted PIPs in ALS. A questionnaire was sent to 58 European ALS centers to collect the prevalence of pediatric ALS during the past ten years, the recruitment potential for future pediatric trials, and opinions of ALS experts concerning a waiver for ALS. Results: Four PIPs were identified; two were waived and two are planned for the future. In total, 49 (84.5%) centers responded to the questionnaire. The diagnosis of 44,858 patients with ALS was reported by 46 sites; 39 of the patients had an onset < 18 years (prevalence of 0.008 cases per 100,000 or 0.087% of all diagnosed patients). The estimated recruitment potential (47 sites) was 26 pediatric patients within five years. A majority of ALS experts (75.5%) recommend a waiver should apply for ALS due to the low prevalence of pediatric ALS. Conclusions: ALS with an onset before 18 years is extremely rare and may be a distinct entity from adult ALS. Conducting studies on the effect of disease-modifying therapy in pediatric ALS may involve lengthy recruitment periods, high costs, ethical/legal implications, challenges in trial design and limited information.

Background: Little is known about the influence of Riluzole on serum neurofilament light chain (sNfL) levels, a biomarker of prognosis in amyotrophic lateral sclerosis (ALS), and variations with time of sNfL concentrations are controversial., Methods: Sera from ALS patients (n = 141) and controls (n = 33) were collected at inclusion (sNfL1) and second visit (sNfL2, mean delay 10.4 ± 8.7 months). sNfL levels, determined by single-molecule array, were compared between ALS and controls at both time points. sNfL concentration changes were compared between patients with Riluzole (w/Ril) at inclusion in the study and those who were treated by Riluzole following inclusion (w/o Ril). The factors influencing sNfL concentrations and changes were studied using linear regression and multivariate analysis., Results: sNfL levels were higher in ALS patients than in controls at the two time points (p < 0.00001). In ALS patients, sNfL concentrations were higher in females for both sNfL1 (p = 0.014) and sNfL2 (p < 0.001). In the whole ALS group, sNfL levels were higher at sNfL2 than at sNfL1 (p < 0.001). sNfL1 and sNfL2 concentrations were similar between the two ALS subgroups (w/ and w/o Ril). ALS functional rating scale-revised rate of decline and gender were the two main factors significantly influencing both sNfL1 and sNfL2 levels (p < 0.01). However, only gender was shown to significantly influence sNfL changes with time (p = 0.003)., Conclusions: In this study, sNfL levels increased with time in ALS patients and there was no difference between subjects already treated by Riluzole and those treated after sNfL1. Further studies with larger population samples and different sampling intervals are warranted to better determine the real potential of sNfL measurement as a tool to monitor treatment response in ALS., (© 2022 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Objectives : To describe a family with heterozygous P67S and D91A SOD1 mutations. Methods : The ALS profile of the proband was described. SOD1 gene sequencing was performed in the proband and his children. Results : The affected individual presented with progressive left peripheral facial palsy and slow progression with late limb involvement. Unequivocal upper and lower motor neuron signs were present, together with diffuse denervation at myography. The absence of trigeminal involvement excluded a FOSMN syndrome. Pedigree analysis did not show any other ALS case in the family. Genetic analysis of this patient showed P67S and D91A SOD1 mutations. The genetic analysis of the children showed that the mutations were each one carried by a different chromosome. Conclusions : P67S SOD1 mutation has been described in several ALS cases, either with familial or apparently sporadic ALS. The mutation is located in a mutational hotspot and was predicted pathogenic by in silico prediction software. The study of phylogenetic data show that at this codon, the proline is highly conserved throughout species reinforcing causality. Conversely, the D91A variant is known to have a recessive influence. Unilateral motor facial involvement, even after several years, in an ALS patient is unusual. The present case with compound heterozygosity and unusual onset in a patient with apparently sporadic ALS, widens the clinical spectrum of the disease and adds further arguments to support the systematic genetic screening of all ALS cases in referral ALS clinics.

Amyotrophic lateral sclerosis (ALS) is the third most frequent neurodegenerative disease after Alzheimer's and Parkinson's disease. ALS is characterized by the selective and progressive loss of motoneurons in the spinal cord, brainstem and cerebral cortex. Clinical manifestations typically occur in midlife and start with focal muscle weakness, followed by the rapid and progressive wasting of muscles and subsequent paralysis. As with other neurodegenerative diseases, the condition typically begins at an initial point and then spreads along neuroanatomical tracts. This feature of disease progression suggests the spreading of prion-like proteins called prionoids in the affected tissues, which is similar to the spread of prion observed in Creutzfeldt-Jakob disease. Intensive research over the last decade has proposed the ALS-causing gene products Cu/Zn superoxide dismutase 1, TAR DNA-binding protein of 43 kDa, and fused in sarcoma as very plausible prionoids contributing to the spread of the pathology. In this review, we will discuss the molecular and cellular mechanisms leading to the propagation of these prionoids in ALS., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

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., (© 2021. The Author(s), under exclusive licence to the American College of Medical Genetics and Genomics.)

Covid-19-related lockdown (LD) in France precluded in-person follow-up in referral ALS centers. ALS patients' evolution and worsening before and during LD were studied to analyze its impact. A total of 84 patients were identified. The monthly rate of ALSFRS-R decline during LD was 1.06 ± 1.42 and was significantly increased compared to the pre-LD period, 0.58 ± 0.73, corresponding to an 83% increase (p = 0.007). Weight loss was unchanged between pre-LD and LD, gender and site of onset did not influence the rates of change of ALSFRS-R score. Several factors may be implicated in this increased severity of ALS during LD, such as psychological consequences of LD, interruptions of physiotherapy and speech therapy, or in-patient visits both to the tertiary center and the GP. Physicians and health authorities should be aware of that, in order to prevent the consequences of future sanitary restrictions.

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., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation., Objective: To identify the genetic variants associated with juvenile ALS., Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism., Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members., Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway., Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Between 1990 and 2018, 14 cases of amyotrophic lateral sclerosis (ALS) were diagnosed in residents of, and in visitors with second homes to, a mountainous hamlet in the French Alps. Systematic investigation revealed a socio-professional network that connected ALS cases. Genetic risk factors for ALS were excluded. Several known environmental factors were scrutinized and eliminated, notably lead and other chemical contaminants in soil, water or home-grown vegetation used for food, radon and electromagnetic fields. Some lifestyle-related behavioral risk factors were identified: Prior to clinical onset of motor neuron disease, some patients had a high degree of athleticism and smoked tobacco. Recent investigations on site, based on a new hypothesis, showed that all patients had ingested wild mushrooms, notably poisonous False Morels. Half of the ALS cohort reported acute illness following Gyromitra gigas mushroom consumption. This finding supports the hypothesis that genotoxins of fungal origin may induce motor neuron degeneration., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hereditary spastic paraplegias (HSP) are heterogeneous disorders, with more than 70 causative genes. Variants in SPAST are the most frequent genetic etiology and are responsible for spastic paraplegia type 4 (SPG4). Age at onset can vary, even between patients from the same family, and incomplete penetrance is described. Somatic mosaicism is extremely rare with only three patients reported in the literature. We report here SPAST mosaic variants in four unrelated patients. We confirm that mosaicism in SPAST is a very rare event with only four identified cases on more than 300 patients with a SPAST variant previously described by our clinical diagnostic laboratory., (© 2021. The Author(s), under exclusive licence to European Society of Human Genetics.)

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

ANXA11 mutations have previously been discovered in amyotrophic lateral sclerosis (ALS) motor neuron disease. To confirm the contribution of ANXA11 mutations to ALS, a large exome data set obtained from 330 French patients, including 150 familial ALS index cases and 180 sporadic ALS cases, was analyzed, leading to the identification of 3 rare ANXA11 variants in 5 patients. The novel p.L254V variant was associated with early onset sporadic ALS. The novel p.D40Y mutation and the p.G38R variant concerned patients with predominant pyramidal tract involvement and cognitive decline. Neuropathologic findings in a p.G38R carrier associated the presence of ALS typical inclusions within the spinal cord, massive degeneration of the lateral tracts, and type A frontotemporal lobar degeneration. This mutant form of annexin A11 accumulated in various brain regions and in spinal cord motor neurons, although its stability was decreased in patients' lymphoblasts. Because most ANXA11 inclusions were not colocalized with transactive response DNA-binding protein 43 or p62 deposits, ANXA11 aggregation does not seem mandatory to trigger neurodegeneration with additional participants/partner proteins that could intervene., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Low-dose interleukin-2 (ld-IL-2) enhances regulatory T-cell (Treg) function in auto-inflammatory conditions. Neuroinflammation being a pathogenic feature of amyotrophic lateral sclerosis (ALS), we evaluated the pharmacodynamics and safety of ld-IL-2 in ALS subjects., Methods: We performed a single centre, parallel three-arm, randomised, double-blind, placebo-controlled study. Eligibility criteria included age < 75 years, disease duration < 5 years, riluzole treatment > 3 months, and a slow vital capacity ≥ 70% of normal. Patients were randomised (1:1:1) to aldesleukin 2 MIU, 1 MIU, or placebo once daily for 5 days every 4 weeks for 3 cycles. Primary outcome was change from baseline in Treg percentage of CD4 + T cells (%Tregs) following a first cycle. Secondary laboratory outcomes included: %Treg and Treg number following repeated cycles, and plasma CCL2 and neurofilament light chain protein (NFL) concentrations as surrogate markers of efficacy. Safety outcomes included motor-function (ALSFRS-R), slow vital capacity (SVC), and adverse event reports. This trial is registered with ClinicalTrials.gov, NCT02059759., Findings: All randomised patients (12 per group), recruited from October 2015 to December 2015, were alive at the end of follow-up and included in the intent-to-treat (ITT) analysis. No drug-related serious adverse event was observed. Non-serious adverse events occurred more frequently with the 1 and 2 MIU IL-2 doses compared to placebo, including injection site reactions and flu-like symptoms. Primary outcome analysis showed a significant increase (p < 0·0001) in %Tregs in the 2 MIU and 1 MIU arms (mean [SD]: 2 MIU: +6·2% [2·2]; 1 MIU: +3·9% [1·2]) as compared to placebo (mean [SD]: -0·49% [1·3]). Effect sizes (ES) were large in treated groups: 2 MIU ES=3·7 (IC95%: 2·3-4·9) and 1 MIU ES=3·5 (IC95%: 2·1-4·6). Secondary outcomes showed a significant increase in %Tregs following repeated cycles (p < 0·0001) as compared to placebo, and a dose-dependent decrease in plasma CCL2 (p = 0·0049). There were no significant differences amongst the three groups on plasma NFL levels., Interpretation: Ld-IL-2 is well tolerated and immunologically effective in subjects with ALS. These results warrant further investigation into their eventual therapeutic impact on slowing ALS disease progression., Funding: The French Health Ministry (PHRC-I-14-056), EU H2020 (grant #633413), and the Association pour la Recherche sur la SLA (ARSLA)., Competing Interests: Declaration of Competing Interest Drs. Camu, Mickunas, Payan, Juntas Morales, Pageot, Masseguin, Suehs, De Vos, Saker, Andreasson and Veyrune have nothing to disclose. Dr. Bensimon reports grants from French Health Ministry (PHRC-I), ARSLA and EU HORIZON 2020, during the conduct of the study; in addition, Dr. Bensimon has a patent (WO 2012123381 A1) with royalties paid to Assistance Publique Hopitaux de Paris (APHP), Institut National de la Sante et de la Recherche Medicale INSERM, and Sorbonne Universite. Drs. Bensimon, Tree, Leigh, Locati, Garlanda, Shaw, Kirby, Malaspina have a patent (B75649EPD40021) pending. Dr. Malaspina reports grants from EU HORIZON 2020, grants from MND Association UK, grants and other from Barts and the London Charity, and from UCB Pharma SPRL, during the conduct of the study; and from F. Hoffmann-La Roche outside the submitted work. Dr. Zetterberg reports personal fees from Samumed, Roche Diagnostics, Denali, CogRx and Wave, outside the submitted work. Dr. Kirby reports grants from The Nimes University Hospital Center (CHU Nimes) and grants from EU HORIZON 2020, during the conduct of the study. Dr. Shaw reports grants from EU HORIZON 2020, Sheffield component and MIROCALS (633413), outside the submitted work. Dr. Al-Chalabi reports involvement as Chief Investigator for LEVALS clinical trial and European CI for REFALS clinical trial for OrionPharma, as well as consultancy from Mitsubishi Tanabe Pharma, consultancy and involvement in debating panel for Cytokinetics Inc, consultancy from Chronos Therapeutics, GSK, Lilly, and from Biogen Idec, outside the submitted work., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

"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.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive degeneration of upper and lower motor neurons. Prognosis is highly variable, ranging from few months to more than 30 years. 25OH vitamin D (25OH VD) blood levels have been associated with worse prognosis of ALS, but these results remain in dispute. We addressed this controversy with a prospective study and multivariate analysis to study the influence of known clinical prognostic factors of the disease and 25OH VD levels on Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) severity score (ALS-SS), as defined by the monthly rate of decline of ALSFRS-R score, to identify the factors most closely linked to the risk of worsening of the disease. Results: This prospective cohort of ALS patients recruited 127 individuals, and 105 of them met inclusion criteria. Mean age of onset was 62.2 ± 12.1 years, 32% of subjects had bulbar onset, and gender ratio was 1.44 (male/female). Mean 25OH VD level was 26.8 ± 10.8 ng/ml and was similar between males and females. Patients with 25OH VD levels <15 ng/ml had significantly higher ALS-SS at inclusion (ALS-SSi) than those with normal levels (>30 ng/ml), p = 0.011. The study of ALS-SS as calculated at the end of follow-up (ALS-SSe) was not found correlated to initial 25OH VD levels ( r = -0.19; p = 0.084). Univariate analysis showed that ALS-SSe correlated with 25OH VD levels, ALS duration at inclusion, slow vital capacity (SVC) at inclusion, and SVC loss. Multivariate model showed that 25OH VD levels were independently associated with ALS-SSe: r = -0.0125, p = 0.033. Log rank test with Kaplan-Meier curves did not show significant differences of survival between the groups defined by 25OH VD levels: <15, >15 and <30, and > 30 ng/ml, p = 0.88. Conclusions: This prospective study in ALS patients confirmed previous retrospective results: ALS-SSi is significantly higher in patients with severe VD deficiency. For the first time, multivariate analysis showed that 25OH VD level was an independent prognostic factor correlated to ALS-SSe, suggesting that discrepancies between previous works could be due to confounders. It would be important that the present work be replicated in larger samples to confirm the present findings., (Copyright © 2020 Juntas-Morales, Pageot, Marin, Dupuy, Alphandery, Labar, Esselin, Picot and Camu.)

Background: In familial amyotrophic lateral sclerosis (ALS) cases, the presence of an abnormal C9ORF72 repeat expansion (C9RE) is the most frequent genetic cause identified. Various clinical phenotypes have been described in relation to the presence of C9RE, including psychiatric disorders or Huntington-like symptoms. In a subset of sporadic ALS, C9RE has also been described. In the present study, all index cases with ALS and C9RE identified in our center and their clinical profile, as well as neurological and psychiatric characteristics of identified family members, were described. Clinical characteristics of ALS patients were compared to 999 patients with sporadic ALS (SALS) from our database., Results: From the 70 index cases with ALS identified, a total of 200 individuals were studied, 118 with ALS, 32 with fronto-temporal lobe degeneration (FTD), 37 with ALS/FTD, and 13 with psychiatric disorders. A familial history was present in 57 of the index cases (81%). In ALS and ALS/FTD cases with C9RE, the age of onset (AoO) was earlier than that in SALS cases, p < 0.0001 and p = 0.008, respectively. Sporadic cases with C9REALS ( n = 13) had an earlier AoO compared to familial C9REALS ones, p < 0.0001. Within families, there was an earlier AoO in index cases and their siblings compared to their parental generation ( p < 0.01). There was also a significant intrafamilial correlation for bulbar onset of ALS. The parental generation had significant female predominance compared to index cases and their siblings (sex ratio 0.47 vs. 1.4, p = 0.004), and this predominance was also present when considering parent-child pairs. In the group with psychiatric disorders, suicide was prominent ( n = 9) and mean age was 54 years., Conclusion: Although our sample size is rather limited, the earlier AoO in index cases and their siblings compared to the parental generation may suggest an anticipation. Reasons for predominance of female transmission are unclear, but the hypothesis that gender influences transmission of the genetic trait or C9RE size variation may be taken into account. Intrafamilial correlation suggests that genetic aspects underlie the occurrence of bulbar onset in ALS patients. Studies on larger samples are warranted to confirm those results., (Copyright © 2020 Esselin, Mouzat, Polge, Juntas-Morales, Pageot, De la Cruz, Bernard, Lagrange, Danel, Alphandery, Labar, Nogué, Picot, Lumbroso and Camu.)

Pathophysiology of amyotrophic lateral sclerosis (ALS) remains partially understood even though it is accepted worldwide that motor neuron death results from a pluri-factorial process with a variable role of genetic factors. Although not distinguishable from a clinical point of view, familial forms of ALS (fALS, 10% of cases) and sporadic forms (sALS, 90% of cases) can be described. Since the identification of superoxide dismutase 1 gene (SOD1) mutations, more than 30 genes have been linked to fALS. Among these genes, five (C9ORF72, SOD1, TARDBP, FUS, TBK1) seem predominant with mutation frequencies of 40%, 20%, 5%, <5%, <5% in fALS and 6%, 3%, and <1% for the last three in sALS, respectively. The situation that classically leads to request genetic screening is the presence of a familial history of motor neuron disorders (MND) or fronto-temporal lobar dementia (FTLD). However, this dichotomy between fALS and sALS based on familial history can lead to mistakes since illegitimacy, ignorance of MND, FTD or psychiatric disorders within the family due to a familial censorship or lack of familial relationship, or a recessive autosomal inheritance could wrongly lead to failing to recognize a familial form. The significant development of genetic research and easier access to genetic tests in fALS increase the number of situations for which gene mutations are identified. The consequence is an increase in genetic requests from relatives of ALS patients who are eager to know their own genetic status and their own individual risk to develop ALS. Pre-symptomatic testing is thus becoming a daily issue in ALS Centers. This led us to propose a framework for such pre-symptomatic genetic testing for people at risk for developing ALS., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background and Purpose: The prognostic value of serum neurofilament light chain (sNfL), a biomarker of neurodegeneration, compared to other prognostic factors of amyotrophic lateral sclerosis (ALS) at the time of diagnosis, remains unclear., Methods: Sera from ALS patients were prospectively collected at the first diagnostic visit in our centre. sNfL levels were determined by single molecule array in 207 ALS patients and in 21 healthy controls. The prognostic value of sNfL was compared with that of other known clinical prognostic factors using a Cox regression model and multivariate analysis., Results: Serum neurofilament light chain levels were higher in ALS patients than in controls (P < 0.0001). Seven parameters were predictive of death in ALS: older age, bulbar onset, higher ALS Functional Rating Scale revised (ALSFRS-R) score, greater weight loss, lower maximal inspiratory pressure, forced vital capacity and higher sNfL levels. A Cox regression model showed that sNfL (P < 0.0001), weight loss (P = 0.040) and site at onset (P = 0.048) were independent predictive factors of death. In a sub-cohort restricted to 139 patients with complete spirometry data, sNfL level (P < 0.005) and forced vital capacity (P = 0.022) were independent factors predictive of death. In a subgroup of 142 patients in whom ALSFRS-R score was available at several time points, sNfL levels positively correlated with ALSFRS-R rate of decline (r = 0.571, P < 10 -12 )., Conclusions: Higher sNfL concentration is a strong and independent prognostic factor of death in ALS as early as the time of diagnosis., (© 2019 European Academy of Neurology.)

Background: Oligodendrocytes (OGs) provide metabolic support to motor neurons (MNs) and are implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). MD1003, or high-dose Pharmaceutical grade Biotin (hdPB), may improve disability in progressive multiple sclerosis patients via augmentation of OG or MN energy levels. Here, we assessed the safety and efficacy of MD1003 in ALS patients., Methods: This single centre, randomised, double-blind, placebo-controlled trial included patients aged 25-80 years with probable or definite ALS. Patients were assigned (2:1), using a computer-generated randomisation list, to receive oral MD1003 (300 mg/day) or placebo treatment for 24 weeks. The primary outcome, safety, was analysed in all patients who received at least one dose of study drug. This study, registered with ClinicalTrials.gov, NCT03114215, has been completed., Findings: Between June and December 2016, 30 patients were enrolled (MD1003, n  = 20; placebo, n  = 10). Baseline characteristics were representative of the ALS population. MD1003 and placebo groups were not well balanced at screening, with the MD1003-treated group having a higher rate of ALSFRS-R decline prior to screening versus placebo (-6·0 IQR [-8·5, -5·0] vs. -5·0 IQR [-5·0, -3·0]) and a predominance of ALS with upper limb onset compared to placebo (35% vs. 10%). MD1003 had a favourable safety profile and was well tolerated. The occurrence of adverse events was similar in both groups (60%). Two deaths occurred in the MD1003 group versus 1 in the placebo group. ALSFRS-R median change from baseline to month 6 was not significantly different between the two groups ( p  = 0·49); the mean difference between groups was -1·6 (SEM=3·3)., Interpretation: MD1003 treatment was safe and well tolerated. It was not possible to establish MD1003 efficacy in this relatively small study. Given the favourable safety profile of MD1003 and an imbalance between treatment groups favouring placebo, additional, larger studies in ALS are warranted., Funding: MedDay Pharmaceuticals., Competing Interests: WC received personal fees from Actelion, Effik, Merck, MedDay Pharmaceuticals, Novartis Pharma, Roche, and Sanofi. AB and FS are employees of MedDay Pharmaceuticals. RJM, NP and SA have nothing to disclose. SS was funded by MedDay Pharmaceuticals., (© 2019 Published by Elsevier Ltd.)

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., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Vitamin D and its main active metabolite 1,25-dihydroxyvitamin D serve a crucial role in maintenance of a healthy calcium metabolism, yet have additional roles in immune and central nervous system cell homeostasis. Serum levels of 25-hydroxyvitamin D are a biomarker of future disease activity in patients with early relapsing-remitting multiple sclerosis (RRMS), and vitamin D supplementation in patients with low circulating 25-dihydroxyvitamin D levels has been anticipated as a potential efficacious treatment strategy. The results of the first large randomized clinical trials (RCTs), the SOLAR and CHOLINE studies, have now been published. The SOLAR study compared 14,000 IU of vitamin D 3 (cholecalciferol) per day with placebo for 48 weeks in 232 randomized patients, whereas CHOLINE compared vitamin D 3 100,000 IU every other week with placebo for 96 weeks in 129 randomized patients. All patients in both studies also used interferon-β-1a. None of the studies met their primary endpoints, which were no evidence of disease activity (NEDA-3) at 48 weeks in SOLAR and annualized relapse rate at 96 weeks in CHOLINE. Both studies did, however, suggest modest effects on secondary endpoints. Thus, vitamin D reduced the number of new or enlarging lesions and new T2 lesions in SOLAR, and the annualized relapse rate and number of new T1 lesions, volume of hypointense T1 lesions, and disability progression in the 90 patients who completed 96 weeks' follow-up in CHOLINE. We conclude that none of the RCTs on vitamin supplementation in MS have met their primary clinical endpoint in the intention to treat cohorts. This contrasts the observation studies, where each 25 nmol/l increase in 25-hydroxyvitamin D levels were associated with 14-34% reduced relapse risk and 15-50% reduced risk of new lesions on magnetic resonnance imaging. This discrepancy may have several explanations, including confounding and reverse causality in the observational studies. The power calculations of the RCTs have been based on the observational studies, and the RCTs may have been underpowered to detect less prominent yet important effects of vitamin D supplementation. Although the effect of vitamin D supplementation is uncertain and less pronounced than suggested by observational studies, current evidence still support that people with MS should avoid vitamin D insufficiency, and preferentially aim for vitamin D levels around 100 nmol/L or somewhat higher.

A role for environmental factors in the etiology of amyotrophic lateral sclerocis (ALS) has been suspected for many years. A large body of work has implicated common exposures, conjugal cases, at-risk activities, heavy metals, organic solvents, and electric shocks, among others. One of the most demonstrative relationships between ALS and the environment is spatial clustering. We reviewed the most important and recent spatial clusters in a given area, whatever the geographical size, with either substantial epidemiological approaches or with highly significant associations, and with precise hypotheses. We present a broad, albeit incomplete overview of investigations in different areas, including examples of the difficulties and contradictions of some approaches. Most of the time, the implication of neurotoxins is suspected and, although not always strictly identified, some candidates are emerging: cycasin, MAM, L-BMAA, hydrazine, for example. One other important point is the possibility of interaction among risk/causal factors that increase the complexity of investigation. Additionally, with the exception of Western Pacific ALS, studies of spatial clustering are lacking a major methodological approach, namely a large cohort analysis extended over a long period of time, and probably for decades. Nevertheless, any spatial cluster needs to be identified, described and studied as deeply as possible to illuminate knowledge of the origin of this devastating disorder and to promote primary or secondary disease prevention., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: In 90% of Amyotrophic Lateral Sclerosis (ALS) cases, the disease is sporadic, the remaining 10% being familial. Many genes have been associated with the disease. The use of next generation sequencing has allowed increasing the number of genes analysed in routine diagnostics. However, this increase raises the issue of genetic variants interpretation within a growing number of ALS-associated-genes. Variant classification is based on a combinatory analysis of multiple factors. Among them, functional analyses provide strong arguments on pathogenicity interpretation. Objectives: We developed a simple animal model, the Zebrafish, for the functional analysis of candidate variants pathogenicity identified by routine genetic testing. Methods: Transient overexpression of different ALS associated genetic variants has been performed by mRNA injection in 1-cell stage zebrafish eggs. Validation of protein overexpression has been done by western blot. Embryos mortality, developmental delay and morphological abnormalities have been assessed within the first two days of development. Cellular phenotype has been investigated by the analysis of axonal length of 2-days old larvae with confocal microscopy. Motor phenotype of 5-days old larvae has been explored by touched-evoked response assay. Results: The model has been validated by the analysis of well-described ALS mutations, SOD1-Gly93Ala and OPTN Glu478Gly. Overexpression of this mutated protein was shown to provoke a shortening of axons and a premature axonal branching, as well as an impairment of motor performances as expected. We did not observe these aberrations in SOD1-WT injected fishes. Two candidate variants observed in ALS-patients have been explored with our model: SOD1 NM&#95;000454.4:c.400&#95;402del, p.Glu134del and OPTN NM&#95;021980.4:c.1475T > G, p. Leu492Arg. Overexpression of both variants induced morphological abnormalities and motor impairment, suggesting a pathogenic involvement of these variants in ALS-patients. Discussion and conclusions: We developed for the first time a simple animal model, the Zebrafish, useful for the functional analysis of variant pathogenicity in order to assist ALS molecular diagnosis. Our model has been used to assess the pathogenicity of SOD1 and OPTN candidate variants, allowing to improve genetic testing interpretation.

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.

Objective: To evaluate the safety and efficacy of cholecalciferol in patients with relapsing-remitting MS (RRMS)., Methods: In this double-blind, placebo-controlled parallel-group, 2-year study, 181 patients with RRMS were randomized 1:1. Key inclusion criteria were a low serum 25-hydroxy vitamin D (25OHD) concentration (<75 nmol/L), a treatment with interferon beta-1a 44 μg (SC 3 times per week) 4 months ± 2 months before randomization, and at least one documented relapse during the previous 2 years. Patients received high-dose oral cholecalciferol 100,000 IU or placebo every other week for 96 weeks. Primary outcome measure was the change in the annualized relapse rate (ARR) at 96 weeks. Secondary objectives included safety and tolerability of cholecalciferol and efficacy assessments (ARR, MRI parameters, and Expanded Disability Status Scale [EDSS])., Results: The primary end point was not met. In patients who completed the 2-year follow-up (45 with cholecalciferol and 45 with placebo), all efficacy parameters favored cholecalciferol with an ARR reduction ( p = 0.012), less new hypointense T1-weighted lesions ( p = 0.025), a lower volume of hypointense T1-weighted lesions ( p = 0.031), and a lower progression of EDSS ( p = 0.026). The overall rate of adverse events was well balanced between groups., Conclusions: Although the primary end point was not met, these data suggest a potential treatment effect of cholecalciferol in patients with RRMS already treated with interferon beta-1a and low serum 25OHD concentration. Together with the good safety profile, these data support the exploration of cholecalciferol treatment in such patients with RRMS., Clinicaltrialsgov Identifier: NCT01198132., Classification of Evidence: This study provides Class II evidence that for patients with RRMS and low serum 25OHD, cholecalciferol did not significantly affect ARRs., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Objectives: Disease-modifying therapies for amyotrophic lateral sclerosis (ALS) are still not satisfactory. The Rho kinase (ROCK) inhibitor fasudil has demonstrated beneficial effects in cell culture and animal models of ALS. For many years, fasudil has been approved in Japan for the treatment of vasospasm in patients with subarachnoid hemorrhage with a favorable safety profile. Here we describe a clinical trial protocol to repurpose fasudil as a disease-modifying therapy for ALS patients. Methods: ROCK-ALS is a multicenter, double-blind, randomized, placebo-controlled phase IIa trial of fasudil in ALS patients (EudraCT: 2017-003676-31, NCT: 03792490). Safety and tolerability are the primary endpoints. Efficacy is a secondary endpoint and will be assessed by the change in ALSFRS-R, ALSAQ-5, slow vital capacity (SVC), ECAS, and the motor unit number index (MUNIX), as well as survival. Efficacy measures will be assessed before (baseline) and immediately after the infusion therapy as well as on days 90 and 180. Patients will receive a daily dose of either 30 or 60 mg fasudil, or placebo in two intravenous applications for a total of 20 days. Regular assessments of safety will be performed throughout the treatment period, and in the follow-up period until day 180. Additionally, we will collect biological fluids to assess target engagement and evaluate potential biomarkers for disease progression. A total of 120 patients with probable or definite ALS (revised El Escorial criteria) and within 6-18 months of the onset of weakness shall be included in 16 centers in Germany, Switzerland and France. Results and conclusions: The ROCK-ALS trial is a phase IIa trial to evaluate the ROCK-inhibitor fasudil in early-stage ALS-patients that started patient recruitment in 2019.

Background: Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most frequent motor neuron disorders in adulthood and infancy, respectively. There is a growing literature supporting common pathophysiological patterns between those disorders. One important clinical issue for that is the co-occurrence of both diseases within a family., Objectives: To collect families in which ALS and SMA patients co-exist and describe the phenotype and the genotype of ALS patients., Patients and Methods: Nine families with co-occurrence of SMA and ALS have been gathered over the last 15 years. Epidemiological, phenotype and genetic status were collected., Results: Out of the nine families, six corresponded to the criteria of familial ALS (FALS). Clinical data were available for 11 patients out of the 15 ALS cases. Mean age of onset was 58.5 years, site of onset was lower limbs in nine cases (81.8%), median duration was 22 months. Four ALS patients carried a mutation: three mutations in SOD1 gene (G147N in two cases and one with E121G) and one repeat expansion in the C9ORF72 gene. Three patients had abnormal SMN1 copy numbers., Conclusions: While the high proportion of familial history of ALS cases in these ALS-SMA pedigrees could have suggested that these familial clusters of the two most frequent MND rely on a genetic background, we failed to exclude that this occurred by chance.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is one of the most severe motor neuron (MN) disorders in adults. Phenotype of ALS patients is highly variable and may be influenced by modulators of energy metabolism. Recent works have implicated the liver X receptors α and β (LXRs), either in the propagation process of ALS or in the maintenance of MN survival. LXRs are nuclear receptors activated by oxysterols, modulating cholesterol levels, a suspected modulator of ALS severity. In a cohort of 438 ALS patients and 330 healthy controls, the influence of LXR genes on ALS risk and phenotype was studied using single nucleotide polymorphisms (SNPs). The two LXRα SNPs rs2279238 and rs7120118 were shown to be associated with age at onset in ALS patients. Consistently, homozygotes were twice more correlated than were heterozygotes to delayed onset. The onset was thus delayed by 3.9 years for rs2279238 C/T carriers and 7.8 years for T/T carriers. Similar results were obtained for rs7120118 (+2.1 years and +6.7 years for T/C and C/C genotypes, respectively). The LXRβ SNP rs2695121 was also shown to be associated with a 30% increase of ALS duration (p = 0.0055, FDR = 0.044). The tested genotypes were not associated with ALS risk. These findings add further evidence to the suspected implication of LXR genes in the disease process of ALS and might open new perspectives in ALS therapeutics.

Objectives were: i) to describe the phenotypic heterogeneity of incident amyotrophic lateral sclerosis (ALS) patients diagnosed in 2012 in French ALS centres; ii) to look at the associations between ALSFRS-R score and ALSFRS-R slope (ΔFS) at time of diagnosis with diagnosis delay, ALS phenotypes and Airlie House diagnosis criteria (AHDC); iii) to describe the rate of progression on ΔFS, according to diagnosis delay., Methods: Incident ALS cases diagnosed in French ALS centres were included. The rate of progression was evaluated as follows: ΔFS = (48 - ALSFRS-R at time of diagnosis)/duration from onset to diagnosis (months). Fast and slow progressors were defined by ΔFS >1 and <0.5, respectively., Results: At time of diagnosis, 476 patients were classified into eight phenotypes: bulbar (33.0%), spinal lumbar (28.2%), spinal cervical (23.1%), flail leg (4.4%), ALS/FTD (4.2%), possible flail arm (4.0%), respiratory (2.1%), dropped-head (1.0%). Median ΔFS (n = 358/476) was 1.0 [0.5-2.0]. ΔFS was associated with AHDC (p = 0.009), but not with clinical phenotype (p = 0.902). Stratification on diagnosis delay (<12 months or ≥18 months) allowed to differentiate fast progressors from slow progressors., Conclusion: At time of inclusion in therapeutic trial closed to diagnosis, ΔFS or diagnosis delay may discriminate the rate of progression.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We report the third case of amyotrophic lateral sclerosis related to p.E121G Superoxide dismutase-1 (SOD1) mutation. Besides a sporadic presentation and a slow progressive course, as described in the 2 previously cases, our patient presented with prominent sensory and cerebellar signs. This case report strengthens that p.E121G should be considered as a causal mutation. Slowly upper and lower motor neuron degeneration, even with non-motor clinical features, should prompt a sequencing of SOD1.

Spinal muscular atrophy (SMA) refers to a group of disorders affecting lower motor neurons. The age of onset of these disorders is variable, ranging from the neonatal period to adulthood. Over the last few years, there has been enormous progress in the description of new genes and phenotypes that throw new light on the molecular pathways involved in motor neuron degeneration. Advances in our understanding of the pathophysiology of the most frequent forms, SMA linked to SMN1 gene mutations and Kennedy disease, has led to the development of therapeutic strategies currently being tested in clinical trials. This report provides a general overview of the clinical features and pathophysiological mechanisms in adult-onset genetic SMA disorders in which the causative gene has been identified (SMN1-related SMA, Kennedy disease, CHCHD10, TRPV4, DYNC1H1 and BICD2). Sporadic lower motor neuron disease, also known as progressive muscular atrophy (PMA), is also discussed. The finding of TDP-43 aggregates in immunohistochemical studies of PMA strongly supports the idea that it is a phenotypic variant of amyotrophic lateral sclerosis (ALS)., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

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., (© 2016 S. Karger AG, Basel.)

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.

Clinical evidence in patients with relapsing-remitting multiple sclerosis suggests an association between MRI outcome measures and disability progression (DP). Post hoc analysis to investigate the association and potential predictive value of brain volume loss (BVL) with long-term DP in FREEDOMS. Patients were categorized into quartiles by SIENA-calculated percent brain volume change from baseline to month (M) 24. Patient characteristics at baseline were determined for each quartile, as were the proportions of patients at M24 and M48 reaching Expanded Disability Status Scale (EDSS) scores of ≥4.0 or ≥6.0 or DP confirmed at 3 months (CDP3) or 6 months (CDP6), and change in EDSS and Multiple Sclerosis Functional Composite. MS disease activity and severity as well as brain volume at baseline were predictive of subsequent BVL over 24 months. The quartiles of patients with greater BVL at 24 months were at highest risk (odds ratio, p value) for reaching EDSS ≥4 (2.8, p = 0.001) or ≥6 (5.73, p = 0.0005) and experienced more DP at M24 (CDP3 2.13, p = 0.002; CDP6 2.17, p = 0.003) and M48 (CDP3 1.98, p = 0.006; CDP6 1.87, p = 0.018) compared to the quartile of patients with the least amount of BVL. These findings confirm the clinical relevance of early brain volume changes for long-term DP.

The Région Languedoc Roussillon is the umbrella organisation for an interconnected and integrated project on active and healthy ageing (AHA). It covers the 3 pillars of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA): (A) Prevention and health promotion, (B) Care and cure, (C) and (D) Active and independent living of elderly people. All sub-activities (poly-pharmacy, falls prevention initiative, prevention of frailty, chronic respiratory diseases, chronic diseases with multimorbidities, chronic infectious diseases, active and independent living and disability) have been included in MACVIA-LR which has a strong political commitment and involves all stakeholders (public, private, patients, policy makers) including CARSAT-LR and the Eurobiomed cluster. It is a Reference Site of the EIP on AHA. The framework of MACVIA-LR has the vision that the prevention and management of chronic diseases is essential for the promotion of AHA and for the reduction of handicap. The main objectives of MACVIA-LR are: (i) to develop innovative solutions for a network of Living labs in order to reduce avoidable hospitalisations and loss of autonomy while improving quality of life, (ii) to disseminate the innovation. The three years of MACVIA-LR activities are reported in this paper.