Human iPSC neuronal models for early and late phases of FXTAS neurodegeneration

描述（由申请人提供）：该申请涉及广泛的挑战领域（14）：干细胞和特定的挑战主题，14- ag -101，用于衰老和神经变性研究的诱导多能干细胞（iPS）。该研究的主要目的是通过诱导多能干细胞（iPSC）技术，利用从患者成纤维细胞重编程的人类神经细胞，阐明单基因神经退行性疾病脆性x相关震颤/共济失调综合征（FXTAS）的致病机制。FXTAS包括进行性运动功能障碍（震颤、步态共济失调、帕金森病）和认知障碍/痴呆。这种疾病是由脆性X智力迟钝1 （FMR1）基因的非编码CGG-重复扩增（预突变范围；55- 200重复）引起的，特别是由扩增的CGG-重复FMR1 mRNA的毒性引起的。我们最近对突变前CGG-repeat Fmr1敲入小鼠的纯神经元培养物的研究表明，CGG-repeat诱导的小鼠发病机制涉及树突生长改变、突触前和突触后体积改变以及神经元细胞寿命缩短。这些改变的功能在一到三周内出现在从一岁大的小鼠培养的神经元中，这增加了在人类神经退行性表型之前存在发育成分的可能性。为了研究FXTAS神经元功能障碍的基础，我们建议使用iPSC技术对来自FXTAS个体的成纤维细胞进行重编程，以产生培养的人类神经元和星形胶质细胞。这项工作之后将进行类似于在KI小鼠神经元中进行的研究。我们提出的研究的一个高度创新的特点是使用单个个体来产生正常和预突变的FMR1表达表型，或者通过亚克隆来自拼接个体的具有不同等位基因大小的成纤维细胞，或者通过亚克隆ipsc衍生的神经祖细胞并选择基于FMR1等位基因（正常或预突变）活跃的亚克隆。在前一种情况下，将正常等位基因与来自单个（马赛克）个体的扩展等位基因进行比较，从而提供等基因背景；在后一种情况下，等位基因在同常染色体背景上进行比较。这种方法利用了我们的临床资源（迄今为止超过100个成纤维细胞系）的力量，FMR1基因在为等基因比较提供成纤维细胞底物方面的独特特征，以及已知的触发神经变性的事实，并且是CGG重复大小的功能。据我们所知，在神经退行性疾病的研究中，提出的个体内（等基因，同常染色体）比较是独特的。根据加州大学戴维斯分校干细胞中心目前的运作情况，多能干细胞的生产和表征，以及向神经干细胞的分化，预计需要大约3到6个月的时间，而神经干细胞成熟和退化的轨迹表征则需要18个月的时间。因此，拟议研究的既定目标应在拟议项目的两年窗口内很好地实现。与挑战资助计划的目标一致，大部分请求的资金用于保留和雇用研究人员。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14): Stem Cells and specific Challenge Topic, 14-AG-101, Induced Pluripotent Stem (iPS) Cells for Aging and Neurodegeneration Research. The main objective of the proposed research is the elucidation of the pathogenic mechanism of a leading single-gene neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), using human neuronal cells reprogrammed from patient fibroblasts through induced pluripotent stem cell (iPSC) technology. FXTAS involves both progressive motor dysfunction (tremor, gait ataxia, parkinsonism) and cognitive impairment/dementia. The disorder is caused by non-coding CGG-repeat expansions (premutation range; 55- 200 repeats) in the fragile X mental retardation 1 (FMR1) gene, specifically by toxicity of the expanded CGG- repeat FMR1 mRNA. Our recent work with pure neuronal cultures from premutation CGG-repeat Fmr1 knock- in mice has revealed that (mouse) CGG-repeat-induced pathogenesis involves altered dendritic growth, altered pre- and post-synaptic volumes, and decreased neuronal cell longevity. These altered functions appear within one to three weeks in neurons cultured from one-day old mice, raising the possibility of a developmental component that precedes the neurodegenerative phenotype in humans. To investigate the basis of neuronal dysfunction in FXTAS, we propose to use iPSC technology to reprogram fibroblasts from individuals with FXTAS, along with controls, to produce cultured human neurons and astrocytes. This effort will be followed by parallel studies to those performed in the KI mouse neurons. A highly innovative feature of our proposed studies is the use of single individuals to produce both normal and premutation FMR1 expression phenotypes, either by sub-cloning fibroblasts with various allele sizes from mosaic individuals, or by sub-cloning iPSC-derived neural progenitor cells and selecting sub-clones based on which FMR1 allele (normal or premutation) is active. In the former case, the normal alleles are compared to expanded alleles from single (mosaic) individuals, thus providing an isogenic background; in the latter case, alleles are compared on an isoautosomal background. This approach exploits both the power of our clinical resources (over 100 fibroblast lines to date), the unique features of the FMR1 gene in providing the fibroblast substrates for isogenic comparison, and the fact that the trigger for neurodegeneration is known and is a function of the CGG repeat size. To our knowledge the proposed intra-individual (isogenic, isoautosomal) comparisons are unique among studies of neurodegenerative disorders. Based on current operation of the UC Davis Stem Cell Core, production and characterization of the iPSCs, and differentiation to neural stem cells, is expected to take approximately three to six months, with up to eighteen months afforded for the characterization of the trajectory for neural stem cell maturation and degeneration. Thus, the stated aims of the proposed research should be realized well within the two-year window of the proposed project. Consistent with the objective of the Challenge Grant Initiative, much of the requested funding is for purpose of retention and hiring of research staff. PUBLIC HEALTH RELEVANCE: FXTAS is a leading single-gene neurodegenerative disorder, with features of parkinsonism and dementia in addition to its core motor dysfunction (tremor and gait ataxia). Thus, the disorder represents a paradigm for studying late-onset neurodegeneration, since the gene (FMR1) and the pathogenic trigger (mRNA) are known and can be manipulated. Approximately 1/130 women and 1/300 men in the general population are carriers of premutation alleles, and a significant fraction of these will develop some features of FXTAS, underscoring the societal impact of this disorder.