Study of Ubiquitin Pathway Genes in a French Population with Amyotrophic Lateral Sclerosis: Focus on HECW1 Encoding the E3 Ligase NEDL1.

The ubiquitin pathway, one of the main actors regulating cell signaling processes and cellular protein homeostasis, is directly involved in the pathophysiology of amyotrophic lateral sclerosis (ALS). We first analyzed, by a next-generation sequencing (NGS) strategy, a series of genes of the ubiquitin pathway in two cohorts of familial and sporadic ALS patients comprising 176 ALS patients. We identified several pathogenic variants in different genes of this ubiquitin pathway already described in ALS, such as FUS, CCNF and UBQLN2. Other variants of interest were discovered in new genes studied in this disease, in particular in the HECW1 gene. We have shown that the HECT E3 ligase called NEDL1, encoded by the HECW1 gene, is expressed in neurons, mainly in their somas. Its overexpression is associated with increased cell death in vitro and, very interestingly, with the cytoplasmic mislocalization of TDP-43, a major protein involved in ALS. These results give new support for the role of the ubiquitin pathway in ALS, and suggest further studies of the HECW1 gene and its protein NEDL1 in the pathophysiology of ALS.

TAR DNA-binding protein of 43 kDa (TDP-43) and amyotrophic lateral sclerosis (ALS): a promising therapeutic target.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that lacks an effective treatment. Aggregates of the TAR DNA-binding protein-43 (TDP-43) are observed in 97% of all ALS cases, thus making this protein a major therapeutic target in ALS. . AREAS COVERED: The authors describe the major cellular functions of TDP-43 and the features and consequences of TDP-43 proteinopathy. Drawing from fundamental and preclinical studies on cellular and animal TDP-43 models of ALS and selected clinical trials, the major pathways that have been targeted for the mitigation of TDP-43 pathology in ALS are discussed. The authors provide insights on the approaches targeting the tendency of TDP-43 for aggregation, defective nucleocytoplasmic transport, dysfunctional proteostasis, abnormal stress granule dynamics, and pathological post-translational modifications of TDP-43. EXPERT OPINION: The complexity of ALS and TDP-43 proteinopathy generates challenges for the development of novel therapeutic approaches. However, the critical involvement of TDP-43 in the initiation and progression of ALS, makes it a promising therapeutic target. Further research should be centered on the development of precision strategies, consideration of patient subgroups, the prevention of the mislocalization of TDP-43 and restoration of the lost functions of TPD-43. .

Dysregulations of Expression of Genes of the Ubiquitin/SUMO Pathways in an In Vitro Model of Amyotrophic Lateral Sclerosis Combining Oxidative Stress and SOD1 Gene Mutation.

Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.

Genes containing hexanucleotide repeats resembling C9ORF72 and expressed in the central nervous system are frequent in the human genome.

More than 40 human diseases, mainly diseases affecting the central nervous system, are caused by the expansion of unstable nucleotide repeats. Repeats of sequences like (CAG)n present in different genes can be responsible for various diseases of the central nervous system. An expanded hexanucleotide repeat (GGGGCC)n in the C9ORF72 gene has been characterized as the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar dementia. In this study, we performed a genome-wide analysis in the human genome and identified 74 genes containing this precise hexanucleotide repeat, with a preference for a location in exon 1 or intron 1, similar to the C9ORF72 gene. A total of 36 of these 74 genes may be of interest as candidates in neurodevelopmental or neurodegenerative diseases, based on their function.

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.

Inhibition of Pathogenic Mutant SOD1 Aggregation in Cultured Motor Neuronal Cells by Prevention of Its SUMOylation on Lysine 75.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons. Mutations in the SOD1 gene encoding the superoxide dismutase 1 are present in 15% of familial ALS cases and in 2% of sporadic cases. These mutations are associated with the formation of SOD1-positive aggregates. The mechanisms of aggregation remain unknown, but posttranslational modifications of SOD1 may be involved. Here, we report that NSC-34 motor neuronal cells expressing mutant SOD1 contained aggregates positive for small ubiquitin modifier-1 (SUMO-1), and in parallel a reduced level of free SUMO-1. CLEM (correlative light and electron microscopy) analysis showed nonorganized cytosolic aggregates for all mutations tested (SOD1A4V, SOD1V31A, and SOD1G93C). We next show that preventing the SUMOylation of mutant SOD1 by the substitution of lysine 75, the SUMOylation site of SOD1, significantly reduces the number of motor neuronal cells with aggregates. These results support the need for further research on the SUMOylation pathways, which may be a potential therapeutic target in ALS.

Disruption of TCA Cycle and Glutamate Metabolism Identified by Metabolomics in an In Vitro Model of Amyotrophic Lateral Sclerosis.

This study aims to develop a cellular metabolomics model that reproduces the pathophysiological conditions found in amyotrophic lateral sclerosis in order to improve knowledge of disease physiology. We used a co-culture model combining the motor neuron-like cell line NSC-34 and the astrocyte clone C8-D1A, with each over-expressing wild-type or G93C mutant human SOD1, to examine amyotrophic lateral sclerosis (ALS) physiology. We focused on the effects of mutant human SOD1 as well as oxidative stress induced by menadione on intracellular metabolism using a metabolomics approach through gas chromatography coupled with mass spectrometry (GC-MS) analysis. Preliminary non-supervised analysis by Principal Component Analysis (PCA) revealed that cell type, genetic environment, and time of culture influenced the metabolomics profiles. Supervised analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) on data from intracellular metabolomics profiles of SOD1G93C co-cultures produced metabolites involved in glutamate metabolism and the tricarboxylic acid cycle (TCA) cycle. This study revealed the feasibility of using a metabolomics approach in a cellular model of ALS. We identified potential disruption of the TCA cycle and glutamate metabolism under oxidative stress, which is consistent with prior research in the disease. Analysis of metabolic alterations in an in vitro model is a novel approach to investigation of disease physiology.

A novel p.E121G SOD1 mutation in slowly progressive form of amyotrophic lateral sclerosis induces cytoplasmic aggregates in cultured motor neurons and reduces cell viability.

Mutations in the SOD1 gene encoding the Cu/Zn superoxide dismutase-1 protein are responsible for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. To date a large number of mutations have been reported in SOD1, but only few of them have been studied and validated by functional studies. We present a novel mutation in SOD1 in a female suffering from slowly progressive ALS. This dominant mutation (c.365A > G) in exon 5 resulted in a substitution of a highly conserved amino acid (p.E121G) of the protein. Functional studies in the motor neuronal cell line NSC34 and in primary culture of mouse motor neurons revealed that this mutation p.E121G induced aggregates positive for SOD1 and ubiquitin, as well as reduced cell viability. These findings identified a novel causal mutation in ALS in close proximity with one of the three histidine residues (H120) of SOD1 interacting with copper.

Novel SOD1 mutation p.V31A identified with a slowly progressive form of amyotrophic lateral sclerosis.

The SOD1 gene encoding the superoxide dismutase 1 (SOD1) protein is mutated in approximately 15% of familial amyotrophic lateral sclerosis (ALS) and 3% of sporadic ALS. We identified a novel mutation in SOD1 in a man who presented at age 49 with lower limb stiffness, and at age 53, a spastic paraparesia with distal muscular atrophy in the lower limbs and fasciculations in the quadriceps. A diagnosis of ALS was established. Eleven years after disease onset his condition continues gradually and slowly to deteriorate. The heterozygous mutation observed in exon 2 resulted in a valine to alanine substitution at position 31 in the ß-barrel domain of the SOD1 protein. Functional analysis in NSC34 cells showed that the overexpression of the mutant form of SOD1(V31A) induced aggregates and decreased cell viability. This mutation is located outside of the regions carrying most of the ALS-related mutations (i.e., the catalytic center, the region of dimerization, and the loops between the ß-strands of the ß-barrel). In conclusion, we identified a novel SOD1 mutation in a patient with slow disease progression and supported the idea that different SOD1 mutations can lead to distinct ALS phenotypes.

Association of a paraneoplastic motor neuron disease with anti-Ri antibodies and a novel SOD1 I18del mutation.

INTRODUCTION: Whether motor neuron diseases (MNDs) can be considered in some cases of paraneoplastic syndromes is controversial. We report a case of rapidly progressive motor neuronopathy following a diagnosis of breast carcinoma, with a presence of anti-Ri antibodies, and a novel SOD1 gene mutation. OBSERVATION: An 80-year-old woman with mucinous adenocarcinoma of the left breast for 4 years developed sub-acute quadriparesis. Myography revealed chronic denervation signs. The patient had serum anti-Ri onconeural antibodies. The diagnosis of paraneoplastic MND was established. Because of a familial history of ALS, a genetic analysis for familial ALS was performed. We identified a novel heterozygous mutation in SOD1 gene, SOD I18del. This mutation may reflect a genetic predisposition to develop a MND, inducing fragility of motor neurons. Neurological improvement was observed after three months of both intravenous gamma globulin and corticosteroids. CONCLUSION: The present observation supports the idea that MND can be considered as a paraneoplastic syndrome. A combination of anti-Ri onconeural antibodies and a particular SOD1 gene mutation, consisting in risk factor, might be in cause in the process of motor neuron death. When in doubt, paraneoplastic cause should be suspected in the differential diagnosis of MND. Immunotherapy treatment may lead to a favorable outcome.

Protein SUMOylation, an emerging pathway in amyotrophic lateral sclerosis.

The covalent attachment of SUMO proteins (small ubiquitin-like modifier) to specific proteins or SUMOylation regulates their functional properties in the nucleus and cytoplasm of neurons. Recent studies reported dysfunction of the SUMO pathway in molecular and cellular abnormalities associated with amyotrophic lateral sclerosis (ALS). Furthermore, several observations support a direct role for SUMOylation in diverse pathogenic mechanisms involved in ALS, such as response to hypoxia, oxidative stress, glutamate excitotoxicity and proteasome impairment. Recent results also suggest that SUMO modifications of superoxide dismutase 1, transactive response DNA-binding protein 43, CTE (COOH terminus of EAAT2) (proteolytic C-terminal fragment of the glutamate transporter excitatory amino acid transporter 2, EAAT2) and proteins regulating the turnover of ALS-related proteins can participate in the pathogenesis of ALS. Moreover, the fused in sarcoma (FUS) gene, mutated in ALS, encodes a protein with a SUMO E3 ligase activity. In this review, we summarize the functioning of the SUMO pathway in normal conditions and in response to stresses, its action on ALS-related proteins and discuss the need for further research on this pathway in ALS.