Supplement for: NanoGenomics to study molecular mechanisms of neurodegeneration in Human cells

描述（由申请人提供）： 阿尔茨海默氏症、帕金森氏症、亨廷顿氏症（HD）和ALS等神经退行性疾病是美国的主要健康风险，影响着数百万人。所有这些疾病都是fata，没有治疗方法可以阻止任何神经退行性疾病的进展。没有有效疗法的原因之一是我们缺乏对导致神经退行性变和疾病进展的特定分子机制的理解。我们的目标是采用新技术来开始更好地理解神经退行性变的分子基础。为此，我们将与Gladstone神经退行性疾病研究所的Steven Finkbeiner博士合作，利用HD和ALS患者的诱导多能干细胞（iPSC）进行研究。Finkbeiner博士，作为HD iPSC联盟的一部分，最近描述了HD患者来源的iPSC的发展，这些iPSC分化为纹状体神经元（i-神经元），这是疾病中受影响最严重的细胞。i-神经元表达疾病表型，包括与来自健康个体的i-神经元相比更短的存活时间。使用一种新的技术，纳米基因组学，我们建议开始，以确定在HD神经元，可能与神经退行性疾病被破坏的细胞通路。 为此，我们将在个体HD患者和对照i神经元中进行全转录组分析（WTA）。 这种单细胞分析将使我们能够直接识别在i-神经元的神经变性的不同阶段的遗传ad分子通路变化的进展。我们的nanoGenomics技术的基础是我们发明的纳米移液管平台，该平台允许单个i神经元内容的电压控制抽吸。 我们将联合收割机这种单细胞分析与单个细胞RNA测序的独特策略相结合，该策略需要少于500 pg的总RNA，我们已经验证了该策略可以测量单个乳腺癌细胞中的基因表达谱。WTA将揭示基因簇的差异表达， 控制和HD i-神经元。使用信息学，我们将把这种聚类分析与HD i-神经元中失调的细胞通路联系起来。将通过qPCR验证WTA结果。使用一种新的基于纳米移液管的纳米注射器技术，我们将随着时间的推移提取细胞内容物的小样本（15毫微微摩尔）。该过程不影响细胞活力，并将使我们能够识别个体HD i-神经元中这些细胞通路的时间变化，以将分子变化与神经变性联系起来。我们将把nanoGenomics的应用扩展到其他神经退行性疾病，如ALS。Finkbeiner博士及其同事最近报道了从具有TDP 43突变的ALS患者中开发iPSC衍生的运动神经元（i-MN），该患者表现出疾病表型。ALS与HD的nanoGenomics的比较分析将提供对导致神经变性的常见分子破坏以及每种疾病独特机制的见解。

DESCRIPTION (provided by applicant): Neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's disease (HD) and ALS are a major health risk in the US affecting millions of people. All of these diseases are fata and no therapeutic is available that blocks the progression of any neurodegenerative disease. One of the reasons that no effective therapies exist is our lack of understanding of the specific molecular mechanisms that cause neurodegeneration and disease progression. Our goal is to employ novel technologies to begin to better understand the molecular basis of neurodegeneration. To do this, we will collaborate with Dr. Steven Finkbeiner of the Gladstone Institute of Neurodegenerative Diseases in studies employing induced Pluripotent Stem Cells (iPSC) from patients with HD and ALS. Dr. Finkbeiner, as part of the HD iPSC Consortium, recently described the development of HD patient derived iPSCs that were differentiated to striatal neurons (i-neurons) the cells most affected in the disease. The i-neurons expressed disease phenotypes including shorter survival times compared to i-neurons from healthy individuals. Using a novel technology, nanoGenomics, we propose begin to identify cellular pathways which are disrupted in HD neurons that may be linked to neurodegeneration. To do this, we will conduct whole transcriptome analysis (WTA) in individual HD patient and control i-neurons. This single cell analysis will allow us to directly identify the progression of genetic ad molecular pathway changes at different stages of neurodegeneration of i-neurons. The basis of our nanoGenomics technology is a nanopipette platform that we invented that allows for voltage controlled aspiration of single i-neuron content. We combine this single cell analysis with a unique strategy for RNA sequencing of individual cells that requires less than 500 pg of total RNA which we have already validated to measure gene expression profiles in single breast cancer cells. The WTA will reveal clusters of genes that are differentially expressed in the control and HD i-neurons. Using informatics, we will relate this cluster analysis to cellular pathways dysregulated in HD i- neurons. The WTA results will be verified by qPCR. Using a novel nanopipette-based nanosyringe technology, we will extract small samples (15 femtomoles) of cellular content over time. This procedure does not affect cell viability and will allow us to identify temporal changes in these cellular pathways in individuals HD i-neurons to link molecular changes to neurodegeneration. We will expand the utility of nanoGenomics to other neurodegenerative diseases such as ALS. Dr. Finkbeiner and associates recently reported the development of iPSC-derived motor neurons (i-MN) from an ALS patient with a mutation in TDP43 which exhibited disease phenotypes. Comparative analysis of the nanoGenomics of ALS with HD will provide insights into common molecular disruptions that cause neurodegeneration as well as mechanisms unique to each disease.