Epigenetic Mechanisms Contributing to the Pathogenesis of ALS/FTD with GGGGCC Repeat Expansion Mutation at the C9orf72 Locus

C9orf72基因座的GGGGCC(G4C2)六核苷酸重复序列的扩展是最常见的遗传 肌萎缩侧索硬化症(ALS)的病因和相关的神经退行性疾病，额颞部 痴呆症(FTD)。已记录的与C9orf72突变相关的细胞缺陷包括功能丧失 通过单倍体不足，由包含G4C2重复的C9orf72转录本变体触发的RNA焦点，以及 有毒的二肽重复蛋白(Dprs)的积累。目前还没有有效的治疗方法，因为 复杂的病因。然而，并不是所有C9orf72突变携带者都会患上ALS或FTD，这表明还有其他因素 修改疾病的发病和进展。双胞胎研究估计表观遗传因素在其中起作用 发生ALS/FTD的风险约为40%。DNA甲基化是一种基本的表观遗传机制，它使 我们的基因组整合外在信号，而DNA甲基化失调在 疾病的发生和发展。有趣的是，G4C2重复序列和CpG岛的高甲基化 在C9orf72 ALS/FTD患者中观察到C9orf72启动子G4C2两侧的CGI，提示 DNA甲基化的病理作用。我们假设G4C2的甲基化重复和两个CGI 是决定ALS/FTD的体征和症状发生和发展的关键因素 C9ORF72突变携带者。为了验证这一假设，我们将应用DNA甲基化编辑工具 (CRISPR/dCas9-TET1/DNMT3)来操纵来自 C9ORF72 ALS IPSCs和来源于C9orf72 FTD iPSCs的皮质神经元以及转基因小鼠 携带人类C9orf72突变细菌人工染色体，然后表现为 甲基化编辑的神经元和小鼠。精确编辑CGI和G4C2甲基化将评估 这些表观遗传学事件在C9orf72 ALS/FTD发病机制中的作用 展示编辑甲基化对ALS/FTD最常见原因的治疗潜力。 为了确定改变C9orf72突变携带者疾病发病的表观遗传因素，我们将比较 单细胞DNA甲基化和马达核糖核酸测序研究脑区病变与非病变 以及来自同一C9orf72突变携带者的前额叶皮质样本，这些携带者被诊断为ALS或FTD。我们还将 比较从皮肤成纤维细胞直接转化的运动神经元的甲基组和转录组 相同的C9orf72 ALS患者在症状前期和症状阶段。这些比较在两种情况下 受疾病影响和未受影响的脑区以及症状前期和症状神经元将 确定CGI和/或G4C2的DNA甲基化是否是改变 C9ORF72 ALS或FTD。此外，这些比较将确定其他新的DNA甲基化和 在C9ofr72突变携带者中导致疾病发生的转录事件，从而指导未来的研究 在C9orf72突变携带者中，这些因素可以预防ALS或FTD的发生。

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.