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

描述（由申请人提供）：本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，用于衰老和神经退行性变研究的诱导多能干细胞（iPS）。拟议研究的主要目的是阐明一种领先的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，使用通过诱导多能干细胞（iPSC）技术从患者成纤维细胞重新编程的人类神经元细胞。FXTAS涉及进行性运动功能障碍（震颤、步态共济失调、帕金森综合征）和认知障碍/痴呆。该疾病由脆性X智力低下1（FMR 1）基因中的非编码CGG重复扩增（前突变范围; 55- 200个重复）引起，特别是由扩增的CGG重复FMR 1 mRNA的毒性引起。我们最近的工作与纯神经元文化的前突变CGG-重复Fmr 1敲入小鼠揭示，（小鼠）CGG-重复诱导的发病机制涉及改变树突状细胞的生长，改变前和突触后的体积，减少神经元细胞寿命。这些改变的功能出现在从一天大的小鼠培养的神经元中的一到三周内，提高了人类神经退行性表型之前的发育成分的可能性。为了研究FXTAS中神经元功能障碍的基础，我们提出使用iPSC技术来重编程来自FXTAS个体的成纤维细胞，沿着对照，以产生培养的人神经元和星形胶质细胞。这项工作之后，将进行平行研究，以那些在KI小鼠神经元。我们提出的研究的一个高度创新的特征是使用单个个体来产生正常和前突变FMR 1表达表型，或者通过亚克隆来自嵌合个体的具有各种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基于FMR 1等位基因（正常或前突变）是活性的来选择亚克隆。在前一种情况下，将正常等位基因与来自单个（嵌合体）个体的扩增等位基因进行比较，从而提供等基因背景;在后一种情况下，等位基因在等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，FMR 1基因在提供用于同基因比较的成纤维细胞底物方面的独特特征，以及神经变性的触发因素是已知的并且是CGG重复大小的函数的事实。据我们所知，提出的个体内（同基因，同常染色体）比较是唯一的神经退行性疾病的研究。根据加州大学戴维斯分校干细胞核心的当前操作，iPSC的生产和表征以及向神经干细胞的分化预计需要大约三到六个月的时间，其中长达十八个月的时间用于表征神经干细胞成熟和变性的轨迹。因此，拟议研究的既定目标应在拟议项目的两年窗口期内实现。根据挑战赠款倡议的目标，所要求的资金大部分用于留住和雇用研究人员。 公共卫生相关性：FXTAS是一种主要的单基因神经退行性疾病，除了其核心运动功能障碍（震颤和步态共济失调）外，还具有帕金森综合征和痴呆的特征。因此，这种疾病代表了研究迟发性神经变性的范例，因为基因（FMR 1）和致病触发因子（mRNA）是已知的，可以被操纵。一般人群中约1/130的女性和1/300的男性是前突变等位基因的携带者，其中很大一部分将发展出FXTAS的一些特征，强调了这种疾病的社会影响。

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.