Metabolomics: A Tool to Understand the Impact of Genetic Mutations in Amyotrophic Lateral Sclerosis.

Metabolomics studies performed in patients with amyotrophic lateral sclerosis (ALS) reveal a set of distinct metabolites that can shed light on the pathological alterations taking place in each individual. Metabolites levels are influenced by disease status, and genetics play an important role both in familial and sporadic ALS cases. Metabolomics analysis helps to unravel the differential impact of the most common ALS-linked genetic mutations (as C9ORF72, SOD1, TARDBP, and FUS) in specific signaling pathways. Further, studies performed in genetic models of ALS reinforce the role of TDP-43 pathology in the vast majority of ALS cases. Studies performed in differentiated cells from ALS-iPSC (induced Pluripotent Stem Cells) reveal alterations in the cell metabolism that are also found in ALS models and ultimately in ALS patients. The development of metabolomics approaches in iPSC derived from ALS patients allow addressing and ultimately understanding the pathological mechanisms taking place in any patient. Lately, the creation of a "patient in a dish" will help to identify patients that may benefit from specific treatments and allow the implementation of personalized medicine.

Metabolomics signatures of a subset of RET variants according to their oncogenic risk level.

Thirty percent of medullary thyroid carcinomas (MTCs) are related to dominant germline pathogenic variants in the RET proto-oncogene. According to their aggressiveness, these pathogenic variants are classified in three risk levels: 'moderate', 'high' and 'highest'. The present study compares the metabolomics profiles of five pathogenic variants, whether already classified or not. We have generated six stable murine fibroblast cell lines (NIH3T3) expressing the WT allele or variants of the human RET gene, with different levels of pathogenicity, including the M918V variant that is yet to be accurately classified. We carried out a targeted metabolomics study of the cell extracts with a QTRAP mass spectrometer, using the Biocrates Absolute IDQ p180 kit, which allows the quantification of 188 endogenous molecules. The data were then subjected to multivariate statistical analysis. One hundred seventy three metabolites were accurately measured. The metabolic profiles of the cells expressing the RET variants were found to be correlated with their oncogenic risk. In addition, the statistical model we constructed for predicting the oncogenic risk attributed a moderate risk to the M918V variant. Our results indicate that metabolomics may be useful for characterizing the pathogenicity of the RET gene variants and their levels of aggressiveness.

The Metabolomic Bioenergetic Signature of Opa1-Disrupted Mouse Embryonic Fibroblasts Highlights Aspartate Deficiency.

OPA1 (Optic Atrophy 1) is a multi-isoform dynamin GTPase involved in the regulation of mitochondrial fusion and organization of the cristae structure of the mitochondrial inner membrane. Pathogenic OPA1 variants lead to a large spectrum of disorders associated with visual impairment due to optic nerve neuropathy. The aim of this study was to investigate the metabolomic consequences of complete OPA1 disruption in Opa1-/- mouse embryonic fibroblasts (MEFs) compared to their Opa1+/+ counterparts. Our non-targeted metabolomics approach revealed significant modifications of the concentration of several mitochondrial substrates, i.e. a decrease of aspartate, glutamate and α-ketoglutaric acid, and an increase of asparagine, glutamine and adenosine-5'-monophosphate, all related to aspartate metabolism. The signature further highlighted the altered metabolism of nucleotides and NAD together with deficient mitochondrial bioenergetics, reflected by the decrease of creatine/creatine phosphate and pantothenic acid, and the increase in pyruvate and glutathione. Interestingly, we recently reported significant variations of five of these molecules, including aspartate and glutamate, in the plasma of individuals carrying pathogenic OPA1 variants. Our findings show that the disruption of OPA1 leads to a remodelling of bioenergetic pathways with the central role being played by aspartate and related metabolites.

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions.

OBJECTIVE: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. METHODS: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. RESULTS: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. CONCLUSIONS: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.

A Plasma Metabolomic Signature Involving Purine Metabolism in Human Optic Atrophy 1 (OPA1)-Related Disorders.

Purpose: Dominant optic atrophy (DOA; MIM [Mendelian Inheritance in Man] 165500), resulting in retinal ganglion cell degeneration, is mainly caused by mutations in the optic atrophy 1 (OPA1) gene, which encodes a dynamin guanosine triphosphate (GTP)ase involved in mitochondrial membrane processing. This work aimed at determining whether plasma from OPA1 pathogenic variant carriers displays a specific metabolic signature. Methods: We applied a nontargeted clinical metabolomics pipeline based on ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) allowing the exploration of 500 polar metabolites in plasma. We compared the plasma metabolic profiles of 25 patients with various OPA1 pathogenic variants and phenotypes to those of 20 healthy controls. Statistical analyses were performed using univariate and multivariate (principal component analysis [PCA], orthogonal partial least-squares discriminant analysis [OPLS-DA]) methods and a machine learning approach, the Biosigner algorithm. Results: A robust and relevant predictive model characterizing OPA1 individuals was obtained, based on a complex panel of metabolites with altered concentrations. An impairment of the purine metabolism, including significant differences in xanthine, hypoxanthine, and inosine concentrations, was at the foreground of this signature. In addition, the signature was characterized by differences in urocanate, choline, phosphocholine, glycerate, 1-oleoyl-rac-glycerol, rac-glycerol-1-myristate, aspartate, glutamate, and cystine concentrations. Conclusions: This first metabolic signature reported in the plasma of patient carrying OPA1 pathogenic variants highlights the unexpected involvement of purine metabolism in the pathophysiology of DOA.

Could Conservative Iron Chelation Lead to Neuroprotection in Amyotrophic Lateral Sclerosis?

Iron accumulation has been observed in mouse models and in both sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Iron chelation could reduce iron accumulation and the related excess of oxidative stress in the motor pathways. However, classical iron chelation would induce systemic iron depletion. We assess the safety and efficacy of conservative iron chelation (i.e., chelation with low risk of iron depletion) in a murine preclinical model and pilot clinical trial. In Sod1G86R mice, deferiprone increased the mean life span compared with placebo. The safety was good, without anemia after 12 months of deferiprone in the 23 ALS patients enrolled in the clinical trial. The decreases in the ALS Functional Rating Scale and the body mass index were significantly smaller for the first 3 months of deferiprone treatment (30 mg/kg/day) than for the first treatment-free period. Iron levels in the cervical spinal cord, medulla oblongata, and motor cortex (according to magnetic resonance imaging), as well as cerebrospinal fluid levels of oxidative stress and neurofilament light chains were lower after deferiprone treatment. Our observation leads to the hypothesis that moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality of neuroprotection for ALS. Antioxid. Redox Signal. 29, 742-748.

Nationwide French Study of RET Variants Detected from 2003 to 2013 Suggests a Possible Influence of Polymorphisms as Modifiers.

BACKGROUND: The presence of single nucleotide polymorphisms (SNPs) in the REarranged during Transfection (RET) gene has been investigated with regard to their potential role in the development or progression of medullary thyroid cancer or pheochromocytomas (PHEO) in patients with the multiple endocrine neoplasia type 2 (MEN2) syndrome. The aim of this study was to evaluate the spectrum of RET variants in France between 2003 and 2013, and to evaluate the impact of SNPs on the MEN2 A phenotype. METHODS: In this retrospective cohort study, RET variants were screened in 5109 index cases, and RET pathogenic variants were screened in 2214 relatives. Exons 5, 8, 10, 11, 13, 14, 15, and 16 were characterized by Sanger sequencing. RET pathogenic variants, RET variants with unknown functional significance (VUS), and four RET SNP variants-G691S (rs1799939), L769L (rs1800861), S836S (rs1800862), and S904S (rs1800863)-were characterized and are reported in index cases. In silico analysis and classification following the recommendation of the American College of Medical Genetics and Genomics was performed for RET VUS. Each patient's age at the time of diagnosis, sex, and the endocrine neoplasias present at molecular diagnosis were recorded. RESULTS: Twenty-six single VUS in RET without any well-defined risk profiles were found in 33 patients. Nine of these were considered probably pathogenic, 11 of uncertain significance, and six as probably benign. Three double pathogenic variants found in three patients were classified as pathogenic. A study of the entire cohort showed that patients carrying pathogenic variants or VUS in RET together with PHEO were diagnosed earlier than the others. The presence of the G691S SNP, or a combination of SNPs, increased the risk of developing PHEO but did not modify the date of the diagnosis. No association was found between SNPs and medullary thyroid cancer or hyperparathyroidism. CONCLUSIONS: The findings propose a classification of 15 of the 26 VUS in RET without any well-defined risk profiles and suggest that the G691S SNP, or a combination of SNPs, may be associated with the development of PHEO.

Iron metabolism disturbance in a French cohort of ALS patients.

OBJECTIVE: The aim of this study was to assess iron status in a cohort of amyotrophic lateral sclerosis (ALS) patients compared to controls in order to evaluate these parameters as a risk factor or a modifying factor of ALS. METHODS: We collected serum iron, ferritin, transferrin, total iron-binding capacity, and transferrin saturation coefficient (TSC) from 104 ALS patients at the time of diagnosis and from 145 controls. We reported phenotypic characteristics and evolution parameters such as ALSFRS-R and forced vital capacity at diagnosis and after one year of follow-up. In a first step we compared iron status between ALS patients and controls, and then we evaluated the relation between iron status and disease evolution of ALS patients using univariate and multivariate analysis. RESULTS: We observed increased concentrations of serum iron (P = 0.002) and ferritin (P < 0.0001) and increased TSC (P = 0.017) in ALS patients. We also showed an association between markers of iron status and high body weight loss in ALS patients. The multivariate analysis of survival highlighted a significant relation between ferritin level and disease duration (P = 0.038). CONCLUSION: This is the first study showing a higher concentration of serum iron in ALS patients, strengthening the involvement of a deregulation of iron metabolism in ALS.

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.