ALS/FTD mutant C9orf72-induced genetic and nuclear pathology in iPS cell models

描述（由申请人提供）：本提案的总体目标是利用人类iPS分化的神经元和胶质细胞研究C9orf72基因中扩增的六核苷酸重复序列的病理学。在染色体9p21上的C9orf72基因的非编码区扩增的GGGGCC六核苷酸重复已被发现是大约30-50%的家族性和高达10%的散发性ALS病例以及12%的家族性FTD病例的原因，使其成为迄今为止已知的ALS/FTD最常见的遗传原因。扩增的重复序列是高度不稳定的，可能产生有毒的rna，这些rna积聚在细胞核中，并被假设通过异常的DNA和蛋白质结合导致细胞功能障碍。我们实验室的初步数据证实（GGGGCC）在C9orf72患者来源的成纤维细胞和iPS分化的神经元中存在核RNA聚焦、异常基因表达和RNA结合蛋白（rbp）的核保留。因此，我们建议通过使用微阵列生成ALS/FTD人类iPS神经元和胶质细胞的广泛遗传图谱，并通过与人类尸检C9orf72脑组织进行比较来验证iPS变化是否相关，从而详细阐述这些早期发现。此外，我们将利用C9orf72 iPS细胞株，通过鉴定RNA结合蛋白的异常积累和结合来研究RNA毒性/病理。最后，我们将确定是否可以用我们实验室已经设计和验证的反义寡核苷酸来消除C9orf72的基因组毒性和病理学。通过协作工作关系、利用iPS细胞和人体组织的可用性和经验，成功的可能性将大大提高。iPS细胞广泛应用于疾病建模，与人体组织交叉关联，并用于验证改良反义治疗，为理解疾病病理生理和治疗发展提供了重要的和可能的新方向。！

DESCRIPTION (provided by applicant): The overall aim of this proposal is to study the pathology of the expanded hexanucleotide repeats in the C9orf72 gene using human iPS differentiated neurons and glia cells. The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene on chromosome 9p21 has been discovered as the cause of approximately 30-50% of familial and up to 10% of sporadic ALS cases as well as 12% of familial FTD cases, making this the most common known genetic cause of ALS/FTD to date. Expanded repeats are highly unstable and potentially yield toxic RNAs that accumulate in the nucleus and are hypothesized to cause cellular dysfunction via aberrant DNA and protein binding. Preliminary data from our laboratory confirm (GGGGCC)n nuclear RNA foci, aberrant gene expression and nuclear retention of RNA binding proteins (RBPs) in C9orf72 patient derived fibroblasts and iPS differentiated neurons. We therefore propose to elaborate on these early findings by generating an extensive genetic profile of ALS/FTD human iPS neurons and glia cells through the use of microarray and validate whether the iPS changes are relevant by comparing to human autopsy C9orf72 brain tissues. Furthermore, we will use C9orf72 iPS cell lines to investigate the RNA toxicity/pathology thru the identification of aberrant accumulation and binding of RNA binding proteins. Finally we will determine if we can abrogate C9orf72 genomic toxicity and pathology with antisense oligonucleotides already designed and validated in our laboratory. The likelihood of success will be greatly enhanced through a collaborative working relationship, the availability and experience of using iPS cells and human tissues. The extensive use of iPS cells to model disease, to cross correlate with human tissues and their use to validate ameliorative antisense therapy provides an important and possibly new direction for understanding disease pathophysiology and therapeutics development. !