Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis

Stella Gagliardi,Adam R Smith,Francesca Gerardi,Fabio Coppedè,Andrea Stoccoro,Katie Lunnon,Alessandro Marocchi,Christian Lunetta,Cristina Cereda,Lucia Migliore,Lorena Mosca

doi:10.1186/s13148-020-00933-2

Abstract

BackgroundMitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms.ResultsIn the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females.ConclusionsPresent data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms.

Highlights

Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA could contribute to the neurodegenerative process
More than 20 genes have been linked to familial ALS, including Chromosome 9 open reading frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP), and fused in sarcoma (FUS) that account for the majority of the cases, and representing 40%, 20%, 1–5%, and 1–5% of cases, respectively
We observed that D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females, while the mitochondrial DNA (mtDNA) copy number was higher in females than in males; after stratification of the sample into ALS patients and controls, age and sex effects were evident in control subjects, but not in ALS patients

Summary

Introduction

Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. Pyrosequencing analysis of Dloop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf that represent the major familial ALS forms. Several studies revealed global and gene-specific epigenetic changes in ALS animal models and in tissues derived from both familial and sporadic ALS patients [10,11,12,13,14,15] Most of these studies were focused on nuclear genes and on the epigenetic landscape of the nuclear genome, but recent evidence suggests that epigenetic modifications to the mitochondrial genome could contribute to neurodegeneration [16]

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