Exploring Disease-Toxicant Interactions in a Human Huntington's Disease Model

描述（由申请人提供）：环境因素被认为与个体的基因组成相互作用，从而影响或导致神经退行性疾病。亨廷顿氏病（HD）是一种衰弱的神经系统疾病，主要是单基因遗传模式。尽管如此，环境因素仍被认为对疾病的发病和发展有很大影响。我们假设环境导致细胞应激，HD大脑中的神经元容易受到氧化应激、线粒体功能障碍和钙调节失调等影响。我们假设突变的亨廷顿蛋白会影响细胞应激反应，从而产生这种疾病特异性的易感性。我们将通过将细胞暴露于已知引发相关细胞应激途径的毒物和测量毒理学表型来验证这一假设。在这里，我们提出用诱导多能干细胞分化成纹状体神经祖细胞作为我们的模型系统。这些细胞系含有突变的亨廷顿基因，具有完整的启动子区域以及神经细胞类型的患者特异性遗传背景。在Specific Aim 1中，我们将优化生成表达dlx2的神经祖细胞的技术。然后，我们将来自两名青少年HD受试者和两名对照受试者的hiPSCs中表达dlx2的培养物暴露于细胞应激模型毒物中，并评估其差异活力。在特异性靶2中，这些相同的细胞

DESCRIPTION (provided by applicant): Environmental agents are thought to interact with an individual's genetic makeup to influence or cause neurodegenerative disease. Huntington's disease (HD) is a debilitating neurological condition with a dominant monogenic inheritance pattern. Still, environmental factors are thought to strongly affect disease age at onset and progression. We postulate that the environment causes cellular stress that neurons in the HD brain are susceptible to such as oxidative stress, mitochondrial dysfunction, and calcium dysregulation. We hypothesize that mutant Huntingtin impinges upon the cellular stress response to produce this disease-specific vulnerability. We will test this hypothesis by exposing cells to toxicants known to elicit implicated cellular stress pathways and measure toxicological phenotypes. Here we propose using induced pluripotent stem cells differentiated into striatal neural progenitors from patients with Huntington's disease and controls as our model system. These cell lines contain the mutated Huntingtin gene with the complete promoter region as well as patient- specific genetic background in a neural cell type. In Specific Aim 1, we will optimize the technique for generating DLX2-expressing neural progenitors. We will then expose DLX2-expressing cultures from hiPSCs from two juvenile HD subjects and two control subjects to cell stress model toxicants and evaluate for differential viability. In Specific Aim 2, these same cells will be used to assay the effects toxicant exposure on reactive oxygen species production, mitochondrial membrane potential, ATP content, and cytosolic calcium concentration. The data generated in Aim 1 and Aim 2 will be analyzed by multivariate ANCOVA statistical modeling for disease-toxicant and/or patient-toxicant interactions. For toxicants eliciting a statistically significant disease-toxicant interaction, we will then perform protein and mRNA arrays for important cellular response factors. In Aim 3, any disease-toxicant interactions identified in Aim 1 and Aim 2 will be further tested in hiPSC-derived cells from HD patients with a range of different pathological repeat lengths. Results will also be analyzed by multivariate ANCOVA to determine if any of the phenotypes are repeat length dependent. This proposal will identify types of cellular stress and potential toxicants that could alter the progression of Huntington's disease and investigate the manner by which mutant Huntingtin causes this susceptibility. The proposed research will seek to identify patient-specific risk for these toxicants potentially providing individualized environmental health advice for patients (i.e. personalized environmental medicine). This funding will also contribute to my individual development as an independent researcher by allowing me to pursue these research aims and present the resulting data to the broader scientific community. As the principal investigator, I will have increased ownership of this research project and the freedom to take full advantage of my graduate training by reducing potential financial constraints.