Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells.

The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology.

Neurofilament accumulations in amyotrophic lateral sclerosis patients' motor neurons impair axonal initial segment integrity.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient' fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.

Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

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

Phenoconversion from Spastic Paraplegia to ALS/FTD Associated with CYP7B1 Compound Heterozygous Mutations.

Biallelic mutations in the CYP7B1 gene lead to spastic paraplegia-5 (SPG5). We report herein the case of a patient whose clinical symptoms began with progressive lower limb spasticity during childhood, and who secondly developed amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) at the age of 67 years. Hereditary spastic paraplegia (HSP) gene analysis identified the compound heterozygous mutations c.825T>A (pTyr275*) and c.1193C>T (pPro398Leu) in CYP7B1 gene. No other pathogenic variant in frequent ALS/FTD causative genes was found. The CYP7B1 gene seems, therefore, to be the third gene associated with the phenoconversion from HSP to ALS, after the recently described UBQLN2 and ERLIN2 genes. We therefore expand the phenotype associated with CYP7B1 biallelic mutations and make an assumption about a link between cholesterol dyshomeostasis and ALS/FTD.

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.

Deletion of the inflammatory S100-A9/MRP14 protein does not influence survival in hSOD1G93A ALS mice.

Neuroinflammation is a hallmark of Amyotrophic Lateral Sclerosis (ALS) in hSOD1G93A mouse models where microglial cells contribute to the progressive motor neuron degenerative process. S100-A8 and S100-A9 (also known as MRP8 and MRP14, respectively) are cytoplasmic proteins expressed by inflammatory myeloid cells, including microglia and macrophages. Mainly acting as a heterodimer, S100-A8/A9, when secreted, can activate Toll-like Receptor 4 on immune cells, leading to deleterious proinflammatory cytokine production. Deletion of S100a9 in Alzheimer's disease mouse models showed a positive outcome, reducing pathology. We now assessed its role in ALS. Unexpectedly, our results show that deleting S100a9 in hSOD1G93A ALS mice had no impact on mouse survival, but rather accelerated symptoms with no impact on microglial activation and motor neuron survival, suggesting that blocking S100-A9 would not be a valuable strategy for ALS.

Genetic screening of ANXA11 revealed novel mutations linked to amyotrophic lateral sclerosis.

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.

Modifying macrophages at the periphery has the capacity to change microglial reactivity and to extend ALS survival.

Microglia and peripheral macrophages have both been implicated in amyotrophic lateral sclerosis (ALS), although their respective roles have yet to be determined. We now show that macrophages along peripheral motor neuron axons in mouse models and patients with ALS react to neurodegeneration. In ALS mice, peripheral myeloid cell infiltration into the spinal cord was limited and depended on disease duration. Targeted gene modulation of the reactive oxygen species pathway in peripheral myeloid cells of ALS mice, using cell replacement, reduced both peripheral macrophage and microglial activation, delayed symptoms and increased survival. Transcriptomics revealed that sciatic nerve macrophages and microglia reacted differently to neurodegeneration, with abrupt temporal changes in macrophages and progressive, unidirectional activation in microglia. Modifying peripheral macrophages suppressed proinflammatory microglial responses, with a shift toward neuronal support. Thus, modifying macrophages at the periphery has the capacity to influence disease progression and may be of therapeutic value for ALS.

Clinical and Molecular Landscape of ALS Patients with SOD1 Mutations: Novel Pathogenic Variants and Novel Phenotypes. A Single ALS Center Study.

Mutations in the copper zinc superoxide dismutase 1 (SOD1) gene are the second most frequent cause of familial amyotrophic lateral sclerosis (ALS). Nearly 200 mutations of this gene have been described so far. We report all SOD1 pathogenic variants identified in patients followed in the single ALS center of Lyon, France, between 2010 and 2020. Twelve patients from 11 unrelated families are described, including two families with the not yet described H81Y and D126N mutations. Splice site mutations were detected in two families. We discuss implications concerning genetic screening of SOD1 gene in familial and sporadic ALS.

ATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization.

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10-7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.

Increased prevalence of granulovacuolar degeneration in C9orf72 mutation.

Granulovacuolar degeneration (GVD) is usually found in Alzheimer's disease (AD) cases or in elderly individuals. Its severity correlates positively with the density of neurofibrillary tangles (NFTs). Mechanisms underlying GVD formation are unknown. We assessed the prevalence and distribution of GVD in cases with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Consecutively autopsied cases with FTLD/ALS-TDP and C9orf72 mutations (FTLD/ALS-C9; N = 29), cases with FTLD/ALS-TDP without C9orf72 mutations (FTLD/ALS-nonC9; N = 46), and age-matched healthy controls (N = 40) were studied. The prevalence of GVD was significantly higher in the FTLD/ALS-C9 cases (26/29 cases) than in the FTLD/ALS-nonC9 cases (15/46 cases; Fisher exact test; p < 2×10-6) or in the control group (12/40 individuals; p < 1×10-6). Average Braak stages and ages of death were not significantly different among the groups. The CA2 sector was most frequently affected in the FTLD/ALS-C9 group, whereas the CA1/subiculum was the most vulnerable area in the other groups. Extension of GVD correlated with the clinical duration of the disease in the FTLD/ALS-C9 cases but not in the FTLD/ALS-nonC9 cases. The GVD-containing neurons frequently had dipeptide repeat (DPR) protein inclusions. GVD granules labeled with antibodies directed against charged multivesicular body protein 2B or casein kinase 1δ were attached to DPR inclusions within GVD. Our results suggest that development of GVD and DPR inclusions is related to common pathogenic mechanisms and that GVD is not only associated with NFTs seen in AD cases or aging individuals.

Spastic paraplegia due to recessive or dominant mutations in ERLIN2 can convert to ALS.

OBJECTIVE: The aim of this study was to evaluate whether mutations in ERLIN2, known to cause SPG18, a recessive hereditary spastic paraplegia (SP) responsible for the degeneration of the upper motor neurons leading to weakness and spasticity restricted to the lower limbs, could contribute to amyotrophic lateral sclerosis (ALS), a distinct and more severe motor neuron disease (MND), in which the lower motor neurons also profusely degenerates, leading to tetraplegia, bulbar palsy, respiratory insufficiency, and ultimately the death of the patients. METHODS: Whole-exome sequencing was performed in a large cohort of 200 familial ALS and 60 sporadic ALS after a systematic screening for C9orf72 hexanucleotide repeat expansion. ERLIN2 variants identified by exome analysis were validated using Sanger analysis. Segregation of the identified variant with the disease was checked for all family members with available DNA. RESULTS: Here, we report the identification of ERLIN2 mutations in patients with a primarily SP evolving to rapid progressive ALS, leading to the death of the patients. These mutations segregated with the disease in a dominant (V168M) or recessive (D300V) manner in these families or were found in apparently sporadic cases (N125S). CONCLUSIONS: Inheritance of ERLIN2 mutations appears to be, within the MND spectrum, more complex that previously reported. These results expand the clinical phenotype of ERLIN2 mutations to a severe outcome of MND and should be considered before delivering a genetic counseling to ERLIN2-linked families.

Electrophysiological Characterization of C9ORF72-Associated Amyotrophic Lateral Sclerosis: A Retrospective Study.

OBJECTIVE: C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS). The aim of the present study was to determine whether C9ORF72-associated ALS (C9-ALS) patients present distinctive electrophysiological characteristics that could differentiate them from non C9ORF72-associated ALS (nonC9-ALS) patients. METHODS: Clinical and electrodiagnostic data from C9-ALS patients and nonC9-ALS patients were collected retrospectively. For electroneuromyography, the mean values of motor conduction, myography, and the mean values of sensory conduction were considered. Furthermore, the proportion of ALS patients with electrophysiological sensory neuropathy was determined. RESULTS: No significant difference was observed between 31 C9-ALS patients and 22 nonC9-ALS patients for mean motor conduction and myography. For sensory conduction analyses, mean sensory conduction was not significantly different between both groups. In total, 38% of -C9-ALS patient and 21% of nonC9-ALS patients presented electrophysiological sensory neuropathy (p = 0.33). In -C9-ALS patients with electrophysiological sensory neuropathy, 80% (8/10) were male and 67% (6/9) presented spinal onset compare to 25% (4/16, p = 0.014) male and 25% (4/16, p = 0.087) with spinal onset in those without electrophysiological sensory neuropathy. CONCLUSION: Although not different from nonC9-ALS, these results suggest that sensory involvement is a frequent feature of C9-ALS patients, expanding the phenotype of the disease beyond the motor and cognitive domains.

Phenotypic and genotypic studies of ALS cases in ALS-SMA families.

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.

Adolescence/adult onset MTHFR deficiency may manifest as isolated and treatable distinct neuro-psychiatric syndromes.

5,10-Methylene-tetrahydrofolate reductase (MTHFR) deficiency is a genetic disorder that can occur at any age and can be easily detected by increased homocysteinemia. In adolescence/adult onset forms, the clinical picture is often complex with association of various neurological features and thrombosis.Here we report the cases of two adult siblings who experienced focal epilepsy at 18 years old as a first disease manifestation, without other symptom during several years. Upon diagnosis, both patients received metabolic treatment comprising B9, B12 and betaine which has stopped the occurrence of seizures, allowing discontinuation of anti-epileptic drugs.Among 24 reviewed adolescent/adult onset patients with MTHFR deficiency in the literature, clinical manifestations included gait disorder (96%, from motor central or peripheral origin), cognitive decline (74%), epileptic syndromes (50%), encephalopathy (30%), psychotic symptoms (17%), and thrombotic events (21%). A total of 41% presented a single neurological manifestation that could stay isolated during at least 3 years, delaying achievement of the diagnosis. Brain MRI showed a mostly periventricular white matter changes in 71% of cases. All patients stabilized or improved following metabolic treatment.Despite being rare, adolescence/adult onset MTHFR deficiency can nevertheless be successfully treated. Therefore, homocysteinemia should be tested in various unexplained neuro-psychiatric syndromes like epilepsy or spastic paraparesis, even if isolated, since waiting for completion of the clinical picture is likely to increase the risk of irreversible neurological damage.

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.