C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

Abstract

Hexanucleotide repeat expansions of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. The mutation is associated with reduced C9orf72 expression and the accumulation of potentially toxic RNA and protein aggregates. CpG methylation is known to protect the genome against unstable DNA elements and to stably silence inappropriate gene expression. Using bisulfite cloning and restriction enzyme-based methylation assays on DNA from human brain and peripheral blood, we observed CpG hyper-methylation involving the C9orf72 promoter in cis to the repeat expansion mutation in approximately one-third of C9orf72 repeat expansion mutation carriers. Promoter hypermethylation of mutant C9orf72 was associated with transcriptional silencing of C9orf72 in patient-derived lymphoblast cell lines, resulting in reduced accumulation of intronic C9orf72 RNA and reduced numbers of RNA foci. Furthermore, demethylation of mutant C9orf72 with 5-aza-deoxycytidine resulted in increased vulnerability of mutant cells to oxidative and autophagic stress. Promoter hypermethylation of repeat expansion carriers was also associated with reduced accumulation of RNA foci and dipeptide repeat protein aggregates in human brains. These results indicate that C9orf72 promoter hypermethylation prevents downstream molecular aberrations associated with the hexanucleotide repeat expansion, suggesting that epigenetic silencing of the mutant C9orf72 allele may represent a protective counter-regulatory response to hexanucleotide repeat expansion.

Isoform‐specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis

“…39 Alternatively, or in combination, epigenetic modifications, such as hypermethylation of CpG islands juxtaposing the repeats or binding of trimethylated histones, may underlie decreased abundance of C9orf72 transcripts. 24,31,[38][39][40] At the immunohistochemical level, C9-L antibody showed a diffuse labeling in the cytoplasm of cerebellar Purkinje cells, with a striking labeling of numerous speckles that were observed both in the neuronal perikarya and dendritic processes. The identity of these speckles remains unknown, and it should be noted that they were observed in both c9-ALS and non-c9-ALS cases.…”

“…24 Haploinsufficiency was initially suggested as a disease mechanism owing to the decreased abundance of V2 and V3 transcripts in c9-ALS cases, and consistent with this hypothesis, reduced expression of selected or total C9orf72 transcripts in C9orf72 repeat expansion carrier-derived cells or tissues have been widely reported. 1,[24][25][26][27][28][29][30][31] Although there is little known about the effects of C9orf72 haploinsufficiency, the development of motor phenotypes in zebrafish and Caenorhabditis elegans owing to downregulation of C9orf72 orthologues supports its role as a disease pathomechanism. 30,32 It has been predicted that C9orf72 proteins may belong to the DENN-like protein family and play roles in Rabmediated cellular trafficking; however, the precise functions and properties of C9orf72 protein are largely unknown owing to a lack of specific antibodies.…”

Evaluation of methylation status of the 5’-promoter region of C9orf72 gene in Russian patients with neurodegenerative diseases

“…Возросший интерес к этой области науки при БАС и ЛВД связан с возможностью иссле-дования новых теорий развития заболевания, что мо-жет обеспечить понимание механизмов этих 2 леталь-ных патологий. Существуют противоречивые данные о том, что эпигенетическая модификация гена C9orf72 посредством гиперметилирования значимо коррели-рует с более быстрым течением заболевания, однако в других исследованиях показан нейропротективный эффект для этой категории пациентов [14][15][16][17]. Гипер-метилирование CpG-островков 5' -промоторной области гена C9orf72 продемонстрировано различными группами исследователей и встречается примерно в 10-30 % случаев среди пациентов с экспансией в ге-не C9orf72 [14,17], вероятно, приводя к снижению уровня экспрессии C9orf72.…”

“…Изменение клинического фенотипа у нашего пациента может быть результатом снижения уровня экспрессии патологического аллеля вследствие метилирования 5' -промоторной области гена C9orf72, что может способствовать уменьшению токсического эффекта экспансии. По данным других исследований, четкой корреляции статуса метилирова-ния с фенотипическим вариантом нейродегенератив-ного процесса либо продолжительностью заболевания не выявлено [14][15][16][17]. Для поиска и анализа более де-тальных клинико-эпигенетических корреляций необ-ходимы дальнейшие исследования на большей когорте пациентов с БАС и другой C9orf72-ассоциированной нейродегенеративной патологией.…”

Emerging role of RNA•DNA hybrids inC9orf72-linked neurodegeneration

“…62,68,[80][81][82] There are 2 cytosine-phosphate-guanine (CpG) islands flanking the C9orf72 repeat locus. The CpG island upstream of the repeat has been found to have increased methylation in a subset of expanded repeat carriers, 62,68,81 while the CpG island downstream of the repeat is unmethylated. The expanded repeat itself is not apparently hypermethylated.…”