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

C9orf72 基因座 GGGGCC 重复扩增突变导致 ALS/FTD 发病机制的表观遗传机制

• 批准号: 10792462
• 负责人: X. Shawn Liu
• 金额: $ 40.61万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10792462
• 关键词: 5' Untranslated Regions; ALS patients; Acceleration; Address; Affect; Amyotrophic Lateral Sclerosis; Bacterial Artificial Chromosomes; Biological Assay; Brain region; C9ORF72; CGG repeat; CGG repeat expansion; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; CpG Islands; DNA Methylation; DNA methylation profiling; DNMT3a; Defect; Development; Diagnosis; Dipeptides; Disease; Disease Progression; Electrophysiology (science); Enzymes; Epigenetic Process; Etiology; Event; FMR1; Fibroblasts; Fragile X Syndrome; Frontotemporal Dementia; Future; Genetic; Genetic Transcription; Genome; Human; Hypermethylation; Methylation; Molecular; Motor; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Onset of illness; Pathogenesis; Pathologic; Patients; Phenotype; Play; Prefrontal Cortex; Production; Property; Protein Isoforms; Proteins; Public Health; RNA; RNA-Binding Proteins; Research; Resolution; Risk; Role; Safety; Sampling; Signal Transduction; Signs and Symptoms; Skin; Testing; Therapeutic; Transcript; Transgenic Mice; Twin Studies; Variant; Viral Vector; Work; candidate validation; delivery vehicle; demethylation; disease phenotype; effective therapy; efficacy evaluation; epigenetic silencing; experimental study; frontotemporal lobar dementia amyotrophic lateral sclerosis; gain of function; in vivo; induced pluripotent stem cell; loss of function; methylome; mouse model; multi-electrode arrays; mutant; novel; novel therapeutic intervention; promoter; protective factors; tool; transcriptome; transcriptome sequencing

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.

C9orf72 基因座上的 GGGGCC (G4C2) 六核苷酸重复扩增是最常见的遗传 肌萎缩侧索硬化症 (ALS) 和相关神经退行性疾病（额颞叶）的病因 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，由含有 G4C2 重复序列的 C9orf72 转录变体触发的 RNA 焦点，以及 有毒二肽重复蛋白（DPR）的积累。目前，尚无有效的治疗方法，因为 病因复杂。然而，并非所有 C9orf72 突变携带者都会出现 ALS 或 FTD，这表明其他因素 改变疾病的发作和进展。双胞胎研究估计表观遗传因素有助于 大约 40% 的风险发生 ALS/FTD。 DNA 甲基化是一种重要的表观遗传机制，使 我们的基因组整合外在信号，DNA甲基化的失调在 疾病的发生和进展。有趣的是，G4C2 重复序列和 CpG 岛的超甲基化 在 C9orf72 ALS/FTD 患者中观察到 C9orf72 启动子中 G4C2 侧翼（CGI），这表明 DNA甲基化的病理作用。我们假设 G4C2 重复甲基化和两个 CGI 是确定 ALS/FTD 体征和症状发生和进展的关键因素 C9orf72 突变体携带者。为了检验这个假设，我们将应用我们的 DNA 甲基化编辑工具 (CRISPR/dCas9-TET1/DNMT3) 操纵运动神经元中的这些甲基化事件 C9orf72 ALS iPSC 和源自 C9orf72 FTD iPSC 以及转基因小鼠的皮质神经元 携带人类 C9orf72 突变细菌人工染色体，然后对其进行表型表征 甲基化编辑的神经元和小鼠。 CGI 和 G4C2 甲基化的精确编辑将评估 这些表观遗传事件在 C9orf72 ALS/FTD 发病机制中的功能意义和 证明了编辑甲基化对于最常见的 ALS/FTD 病因的治疗潜力。 为了确定改变 C9orf72 突变携带者疾病发作的表观遗传因素，我们将比较 通过单细胞 DNA 甲基化和运动 RNA 测序分析受疾病影响和未受影响的大脑区域 以及来自诊断患有 ALS 或 FTD 的相同 C9orf72 突变携带者的前额皮质样本。我们也会 比较直接从皮肤成纤维细胞转化的运动神经元的甲基化组和转录组 相同的 C9orf72 ALS 患者在症状前和症状阶段。这些比较 受疾病影响和未受影响的大脑区域以及症状前和有症状的神经元将 确定 CGI 和/或 G4C2 的 DNA 甲基化是否是修饰起始的表观遗传因素 C9orf72 ALS 或 FTD。此外，这些比较将鉴定其他新的 DNA 甲基化和 导致 C9ofr72 突变携带者疾病发作的转录事件，从而指导未来的研究 这些因素可防止 C9orf72 突变携带者发生 ALS 或 FTD。