Hypothesis-free evaluation of circulating metabolome provides cell-specific insights regarding the role of energy substrate availability in amyotrophic lateral sclerosis

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited therapeutic options. The metabolome comprises small-molecules (typically < 1 kDa) present in biological tissues and fluids, which are the intermediates and end-products of cellular metabolism. The plasma metabolome has been linked to ALS pathogenesis, particularly with respect to energy substrates. Methods We conducted a hypothesis-free two-sample Mendelian randomisation (MR) analysis of the concentration of 575 circulating plasma/serum metabolites, to determine which are causally linked to risk of ALS. Genetic instruments used to infer metabolite concentrations were measured in ~ 20,000 individuals; and genetic association with ALS was sourced from a GWAS including 29,612 ALS cases and 122,656 controls. Significant metabolites were validated in an independent GWAS of plasma/serum metabolite concentrations and evaluated for sex-specific effects. Correlations between directly measured plasma and cerebrospinal fluid (CSF) metabolite concentrations and ALS risk and severity were examined in 94 ALS patients and 40 controls. Finally, we experimentally assessed metabolic function in a patient-derived neuronal model under stress conditions. Results MR identified five metabolites causally associated with ALS risk after Bonferroni multiple-testing correction. Higher serum concentration of glycoprotein acetyls (P = 9.7e-9, β = 0.21) and the peptide DSGEGDFXAEGGGVR (P = 8.0e-6, β = 0.22) were associated with increased ALS risk, whereas higher serum concentration of phenylalanylserine, isobutyrylcarnitine, and acetylcarnitine were protective (P < 5e-5, β − 0.29 to − 0.72). Independent validation via MR in a different cohort confirmed the protective effects of carnitines and the harmful effect of DSGEGDFXAEGGGVR. Fine mapping implicated SLC2A3 (GLUT3), a neuronal glucose transporter, as a genetic mediator of ALS risk linked to DSGEGDFXAEGGGVR. Direct measurement of metabolite concentrations in patient biofluids revealed elevated acetylcarnitine levels in patients with ALS, which were associated with delayed symptom onset (HR = 0.41, P = 0.019). Similarly serum lactate is elevated in ALS patients with longer survival time (HR = 0.33, P = 0.03) and serum fructose is elevated in ALS patients with shorter survival time (Cox regression, P = 0.015, HR = 1.1). In vitro , neurons carrying an ALS-associated G4C2-repeat expansion within C9orf72 demonstrated reduced metabolic flexibility, and an impaired ability to use alternative energy substrates when relatively deprived of glucose (P < 0.05). Conclusions Our multilayered analysis provides convergent evidence that impaired energy substrate availability contributes to ALS risk and severity. CNS cell-types differ in their use of energy substrates and therefore we can infer the relative importance of different cell-types for different stages of disease: Astrocytes, which preferentially utilise β-oxidation of fatty acids (requiring carnitines), are a key determinant of disease onset. Energy supply to CNS neurons, particularly under conditions of energetic stress when they utilise lactate, is a key determinant of disease progression. Importantly, diseased neurons are selectively impaired in their ability to utilise diverse energy substrates and altered neuronal glucose uptake via GLUT3 may increase vulnerability to disease. Finally microglia, which utilise fructose, contribute primarily to disease progression. Our findings support further investigation of metabolic interventions, such as dietary supplementation, to treat or prevent ALS.