Epigenetic mechanisms contributing to the pathogenesis of ALS/FTD with GGGGCC repeat expansion mutation at the C9orf72 locus

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The GGGGCC (G4C2) hexanucleotide repeat expansion at the C9orf72 locus is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and the related neurodegenerative disorder, frontotemporal dementia (FTD). Documented cellular defects associated with the C9orf72 mutation include loss-of-function through haploinsufficiency, RNA foci triggered by the C9orf72 transcript variants containing G4C2 repeats, and the accumulation of toxic dipeptide repeat proteins (DPRs). Currently, there is no effective treatment due to the complex etiology. However, not all C9orf72 mutant carriers develop ALS or FTD, suggesting that other factors modify the disease onset and progression. Twin studies have estimated that epigenetic factors contribute about 40% of risk to develop ALS/FTD. DNA methylation is an essential epigenetic mechanism that enables our genome to integrate extrinsic signals, and dysregulation of DNA methylation plays important roles in disease onset and progression. Interestingly, hypermethylation of the G4C2 repeats and the CpG islands (CGIs) flanking G4C2 in the C9orf72 promoter were observed in C9orf72 ALS/FTD patients, suggesting a pathological role for DNA methylation. We hypothesize that the methylation of G4C2 repeats and two CGIs represents a critical factor in determining the onset and progression of signs and symptoms of ALS/FTD in C9orf72 mutant carriers. To test this hypothesis, we will apply our DNA methylation editing tool (CRISPR/dCas9-TET1/DNMT3) to manipulate these methylation events in motor neurons derived from C9orf72 ALS iPSCs and cortical neurons derived from C9orf72 FTD iPSCs as well as in transgenic mice carrying a human C9orf72 mutant bacterial artificial chromosome, and then phenotypically characterize the methylation-edited neurons and mice. Precise editing of CGIs and G4C2 methylations will evaluate the functional significance of these epigenetic events during the pathogenesis of C9orf72 ALS/FTD and demonstrate the therapeutic potential of editing methylation for the most commonly known cause of ALS/FTD. To identify epigenetic factors that modify the disease onset of C9orf72 mutant carriers, we will compare the disease-affected and non-affected brain regions by single-cell DNA methylation and RNA sequencing of motor and prefrontal cortex samples from the same C9orf72 mutant carriers diagnosed with ALS or FTD. We will also compare the methylome and transcriptome of motor neurons converted directly from the skin fibroblasts of the same C9orf72 ALS patients at the pre-symptomatic and symptomatic stages. These comparisons between the disease-affected and non-affected brain regions and the pre-symptomatic and symptomatic neurons will determine whether the DNA methylation of CGIs and/or G4C2 is an epigenetic factor modifying the onset of C9orf72 ALS or FTD. Additionally, these comparisons will identify other novel DNA methylation and transcriptional events contributing to the disease onset in C9ofr72 mutant carriers, thus guiding the future study of these factors protective against the development of ALS or FTD in C9orf72 mutant carriers.