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

Abstract

Background 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 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. Results In 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. Conclusions Present 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.

Mitochondrial D-Loop Region Methylation and Copy Number in Peripheral Blood DNA of Parkinson’s Disease Patients

“…Genomic DNA was extracted from peripheral blood collected in EDTA tubes from each subject using the QIAmp DNA blood Mini Kit (Qiagen, Milan, Italy, Catalog N° 51106) following the manufacturer’s instructions, and quantified using a Nano Drop ND 2000c spectrophotometer (NanoDrop Thermo scientific, Wilmington, DE, USA). Methylation of the D-loop region was assessed by means of methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing techniques, as reported elsewhere [ 13 , 16 , 22 ].…”

“…Recent evidence suggests that also epigenetic modifications of the mitochondrial genome could contribute to neurodegeneration [ 10 ]. In this regard, dysregulation of mitochondrial DNA (mtDNA) methylation in the central nervous system and in peripheral blood samples of patients with AD, PD, and amyotrophic lateral sclerosis (ALS), as well as in transgenic mice modelling, has been reported [ 11 , 12 , 13 , 14 , 15 , 16 ]. The majority of these studies investigated the methylation levels of the mtDNA regulatory region (D-loop), which regulates mtDNA replication and transcription, revealing that D-loop methylation levels were dynamically dysregulated with disease progression in animal models of AD and were significantly different in human postmortem AD brains and in peripheral blood cells of living AD patients compared to healthy controls [ 12 , 13 , 15 ].…”

“…The majority of these studies investigated the methylation levels of the mtDNA regulatory region (D-loop), which regulates mtDNA replication and transcription, revealing that D-loop methylation levels were dynamically dysregulated with disease progression in animal models of AD and were significantly different in human postmortem AD brains and in peripheral blood cells of living AD patients compared to healthy controls [ 12 , 13 , 15 ]. Altered D-loop methylation levels were also detected in spinal cord and skeletal muscle cells of human- SOD1 transgenic ALS mice [ 11 ] as well as in peripheral blood of familial and sporadic ALS patients [ 14 , 16 ]. Moreover, an inverse correlation between D-loop methylation levels and mtDNA copy number was frequently observed [ 14 , 16 , 17 , 18 , 19 ], suggesting a role for methylation levels in this region in regulating mtDNA replication.…”

“…Altered D-loop methylation levels were also detected in spinal cord and skeletal muscle cells of human- SOD1 transgenic ALS mice [ 11 ] as well as in peripheral blood of familial and sporadic ALS patients [ 14 , 16 ]. Moreover, an inverse correlation between D-loop methylation levels and mtDNA copy number was frequently observed [ 14 , 16 , 17 , 18 , 19 ], suggesting a role for methylation levels in this region in regulating mtDNA replication. Regarding PD, a significant reduction of D-loop methylation levels was observed post-mortem in the substantia nigra of individuals with PD compared to healthy controls [ 12 ].…”

Mitochondrial DNA Methylation and Human Diseases

“…Subsequently, another study from the same research group revealed that mtDNA methylation patterns and mitochondrial DNMT3A levels were abnormal in the skeletal muscles and spinal cord of pre-symptomatic ALS mice carrying mutations in the human superoxide dismutase 1 gene (SOD1), which included DNMT3A up-regulation, increased MT-RNR2 gene methylation and decreased D-loop region methylation [49]. Altered D-loop methylation levels have also been observed in the peripheral blood of sporadic and SOD1 ALS patients when compared to both ALS patients with mutations in FUS, TARDBP and C9orf72 and to control subjects who are noncarriers of ALS-linked gene mutations [96,100]. Regarding Alzheimer's disease, a non-significant increase in 5-hmC levels was observed in post-mortem mtDNA brain samples of seven late-onset AD patients with respect to five control subjects [93].…”