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

描述（由申请人提供）：本提案的总体目的是使用人iPS分化的神经元和神经胶质细胞研究C9 orf 72基因中扩增的六核苷酸重复序列的病理学。已发现染色体9 p21上C9 orf 72基因非编码区中扩展的GGGGCC六核苷酸重复序列是约30-50%的家族性ALS病例和高达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ALS/FTD的最常见已知遗传原因。扩增的重复序列是高度不稳定的，并可能产生毒性RNA，这些RNA在细胞核中积累，并被假设通过异常的DNA和蛋白质结合引起细胞功能障碍。来自我们实验室的初步数据证实了C9 orf 72患者来源的成纤维细胞和iPS分化的神经元中的（GGGGCC）n核RNA灶、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iPS神经元和神经胶质细胞的广泛遗传图谱来详细说明这些早期发现，并通过与人尸检C9 orf 72脑组织进行比较来验证iPS变化是否相关。此外，我们将使用C9 orf 72 iPS细胞系通过鉴定RNA结合蛋白的异常积累和结合来研究RNA毒性/病理学。最后，我们将确定我们是否可以废除C9 orf 72基因组毒性和病理与反义寡核苷酸已经设计和验证在我们的实验室。通过合作的工作关系，使用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细胞广泛用于疾病建模、与人体组织交叉相关以及它们用于验证改善性反义疗法的用途为理解疾病病理生理学和疗法开发提供了重要且可能是新的方向。!

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. !