DNA damage accumulates and responses are engaged in human ALS brain and spinal motor neurons and DNA repair is activatable in iPSC-derived motor neurons with SOD1 mutations

Abstract

DNA damage is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, relationships between DNA damage accumulation, DNA damage response (DDR), and upper and lower motor neuron vulnerability in human ALS are unclear; furthermore, it is unknown whether epigenetic silencing of DNA repair pathways contributes to ALS pathogenesis. We tested the hypotheses that DNA damage accumulates in ALS motor neurons along with diminished DDR, and that DNA repair genes undergo hypermethylation. Human postmortem CNS tissue was obtained from ALS cases (N = 34) and age-matched controls without neurologic disease (N = 15). Compared to age-matched controls, abasic sites accumulated in genomic DNA of ALS motor cortex and laser capture microdissection-acquired spinal motor neurons but not in motor neuron mitochondrial DNA. By immunohistochemistry, DNA damage accumulated significantly in upper and lower motor neurons in ALS cases as single-stranded DNA and 8-hydroxy-deoxyguanosine (OHdG) compared to age-matched controls. Significant DDR was engaged in ALS motor neurons as evidenced by accumulation of c-Abl, nuclear BRCA1, and ATM activation. DNA damage and DDR were present in motor neurons at pre-attritional stages and throughout the somatodendritic attritional stages of neurodegeneration. Motor neurons with DNA damage were also positive for activated p53 and cleaved caspase-3. Gene-specific promoter DNA methylation pyrosequencing identified the DNA repair genes Ogg1, Apex1, Pnkp and Aptx as hypomethylated in ALS. In human induced-pluripotent stem cell (iPSC)-derived motor neurons with familial ALS SOD1 mutations, DNA repair capacity was similar to isogenic control motor neurons. Our results show that vulnerable neurons in human ALS accumulate DNA damage, and contrary to our hypothesis, strongly activate and mobilize response effectors and DNA repair genes. This DDR in ALS motor neurons involves recruitment of c-Abl and BRCA1 to the nucleus in vivo, and repair of DNA double-strand breaks in human ALS motor neurons with SOD1 mutations in cell culture.

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

“…Indeed, we previously suggested that the reduced D-loop methylation levels leading to an increased mtDNA copy number in carriers of ALS-linked SOD1 mutations could represent a compensatory mechanism to counteract the increased oxidative stress, impaired mitochondrial respiration, and increased mtDNA damage in those subjects [23]. In this regard, it has also been recently demonstrated that DNA repair genes are hypomethylated and overexpressed to counteract oxidative DNA damage in human ALS motor neurons with SOD1 mutations [47]. Defective mitochondrial respiration and ATP production are also well documented in the spinal cord and lymphocytes of sporadic ALS patients, so that the reduced D-loop methylation levels that we observed in the present study could represent a compensatory mechanism to counteract the mitochondrial impairment in sporadic ALS too.…”

The role of TDP-43 mislocalization in amyotrophic lateral sclerosis

“…Loss of function due to mislocalization results in a loss of VPS4B repression leading to an increased interaction with the ALS-linked protein Charged Multivesicular Body Protein 2B (CHMP2B) thereby disrupting dendritic recycling-endosome trafficking and reducing ALS-linked ERB-B2 Receptor Tyrosine Kinase 4 (ERBB4) surface expression [191][192][193]. Another nuclear role for TDP-43 is in its response to genomic double stranded breaks (DSBs) which accumulate in ALS patients [163,[194][195][196][197][198][199][200]. Mislocalization of TDP-43 through an ALS-causing mutation impair the nuclear localization of DSB-repair proteins and result in the accumulation of DNA damage promoting cell death [163,194,201,202].…”

Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration

“…Studies have shown that DNA damage, particularly DSBs, is elevated in the brains of AD, PD, and ALS patients (Kim et al, 2020;Mitra et al, 2019;Schaser et al, 2019;Shanbhag et al, 2019). While genomic instability is considered a contributor in the pathogenesis of neurodegenerative disorders, the causes of neuronal DSBs and the mechanism by which DSBs drive the degenerative brain pathology remain to be defined.…”