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

FXTAS 神经变性早期和晚期的人类 iPSC 神经元模型

• 批准号: 7938017
• 负责人: PAUL J HAGERMAN
• 金额: $ 39.38万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938017
• 关键词: Address; Age; Alleles; Architecture; Area; Astrocytes; Autopsy; CGG repeat; CGG repeat expansion; Case-Control Studies; Cell Aging; Cell Line; Cell Maturation; Cell model; Cells; Cellular Stress; Central Nervous System Diseases; Clinical; Coculture Techniques; Complex; Dementia; Development; Disease; Exhibits; FMR1; FMR1 Gene; FXTAS; Female; Fibroblasts; Functional RNA; Functional disorder; Funding; Gait Ataxia; General Population; Genes; Genetic; Grant; Growth; Harvest; Human; Impaired cognition; Impairment; Individual; Investigation; Knock-in Mouse; Knowledge; Lentivirus Vector; Longevity; Measures; Messenger RNA; Modeling; Motor; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Nuclear Pore Complex; Octamer Transcription Factor-3; Parkinsonian Disorders; Pathogenesis; Patients; Pattern; Phase; Phenotype; Population Control; Production; Public Health; Relative (related person); Research; Resources; Sampling; Skin; Staging; Stem cells; Subfamily lentivirinae; Synapses; Tissues; Toxic effect; Tremor; Woman; Work; X Chromosome; X Inactivation; active control; base; case control; cell age; cohort; density; induced pluripotent stem cell; innovation; male; men; molecular pathology; mouse model; nerve stem cell; neurodegenerative phenotype; neuron development; operation; population based; public health relevance; stem; stem cell technology; success; valproate; Address; Age; Alleles; Architecture; Area; Astrocytes; Autopsy; CGG repeat; CGG repeat expansion; Case-Control Studies; Cell Aging; Cell Line; Cell Maturation; Cell model; Cells; Cellular Stress; Central Nervous System Diseases; Clinical; Coculture Techniques; Complex; Dementia; Development; Disease; Exhibits; FMR1; FMR1 Gene; FXTAS; Female; Fibroblasts; Functional RNA; Functional disorder; Funding; Gait Ataxia; General Population; Genes; Genetic; Grant; Growth; Harvest; Human; Impaired cognition; Impairment; Individual; Investigation; Knock-in Mouse; Knowledge; Lentivirus Vector; Longevity; Measures; Messenger RNA; Modeling; Motor; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Nuclear Pore Complex; Octamer Transcription Factor-3; Parkinsonian Disorders; Pathogenesis; Patients; Pattern; Phase; Phenotype; Population Control; Production; Public Health; Relative (related person); Research; Resources; Sampling; Skin; Staging; Stem cells; Subfamily lentivirinae; Synapses; Tissues; Toxic effect; Tremor; Woman; Work; X Chromosome; X Inactivation; active control; base; case control; cell age; cohort; density; induced pluripotent stem cell; innovation; male; men; molecular pathology; mouse model; nerve stem cell; neurodegenerative phenotype; neuron development; operation; population based; public health relevance; stem; stem cell technology; success; valproate

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.

描述（由申请人提供）：此申请涉及广泛的挑战区域（14）：干细胞和特定挑战主题，14-AG-101，诱导多能茎（IPS）细胞用于衰老和神经变性研究。拟议研究的主要目的是使用人类神经元细胞通过诱导的多能干细胞（IPSC）技术从患者的成纤维细胞（IPSC）技术中对患者成纤维细胞重编程。 FXTA涉及进行性运动功能障碍（震颤，步态共济失调，帕金森氏症）和认知障碍/痴呆症。该疾病是由脆弱的X智力低下1（FMR1）基因中的非编码CGG重复扩展（预先摄入范围； 55-200重复序列）引起的，特别是由于扩展的CGG-重复FMR1 mRNA的毒性。我们最近在小鼠中使用CGG重复FMR1敲击的纯神经元培养物的工作表明，（小鼠）CGG重复诱导的发病机理涉及树突状生长改变，突触前和后突触后的改变，并减少神经元细胞的寿命。这些变化的功能出现在一日老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FXTA中神经元功能障碍的基础，我们建议使用IPSC技术重新编程来自FXTA的个体以及对照组的成纤维细胞，以产生培养的人类神经元和星形胶质细胞。这项工作将在Ki小鼠神经元中进行的平行研究。我们提出的研究的一个高度创新的特征是使用单个个体同时生产正常和预先享有的FMR1表达表型，要么通过将成纤维细胞从马赛克个体中缩减为各种等位基因大小，要么通过亚置IPSC衍生的神经祖细胞和基于哪些FMR1等位基因的子挂号来进行iPSC衍生的神经祖细胞和选择。在前一种情况下，将正常等位基因与单个（马赛克）个体的扩展等位基因进行比较，从而提供了同源性背景。在后一种情况下，等位基因在异体背景上进行比较。这种方法利用了我们的临床资源的功能（迄今为止超过100种成纤维细胞线），FMR1基因的独特特征在为异基因比较提供成纤维细胞底物方面，已知神经变性的触发因素是已知的，并且是CGG重复大小的函数。据我们所知，在神经退行性疾病的研究中，提出的个体内（等生，异源体）比较是独一无二的。基于UC Davis干细胞核心的当前运行，IPSC的生产和表征以及与神经干细胞的分化预计将花费大约三到六个月，最多长达18个月，用于表征神经干细胞成熟和变性的轨迹。因此，在拟议项目的两年窗口内，应很好地实现拟议研究的既定目标。与挑战赠款计划的目标一致，大部分要求的资金是为了保留和雇用研究人员。 公共卫生相关性：FXTA是一种领先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，具有帕金森主义和痴呆症的特征。因此，由于基因（FMR1）和致病性触发因素（mRNA）是已知的，并且可以操纵，因此该疾病代表了研究晚神经变性的范式。一般人口中约有1/130名女性和1/300名男性是预先享有的等位基因的载体，其中很大一部分将发展出FXTA的某些特征，从而强调了这种疾病的社会影响。