Functional Dissection of Alzheimer's Disease Networks in Drosophila: from Association to Causal Modulators of Age-Dependent Neurodegeration

这个补充项目的目标是扩展生物信息学分析和实验验证， AMP-AD目标网络，这是父母赠款的重点。首先（AIM 1），我们将生成单细胞 表达人tau和分泌的淀粉样蛋白-β的果蝇AD模型的纵向转录组学谱。 这些数据将允许区分来自不同神经元群体的基因表达谱， 不同的神经胶质亚型。这些研究也将有力地补充正在进行的AMP-AD单细胞基因 人类死后组织的表达谱。我们将包括严格的实验控制tau / β淀粉样蛋白表达以及纵向取样，以分析年龄和AD的具体贡献。 病理物种对脑中细胞类型特异性基因表达变化的影响。最初，我们将包括2次 疾病进展的早期和晚期。在拟议补编的第一年（ 我们将产生适当基因型的果蝇，提取mRNA， scRNAseq，并开始分析结果。在补贴的第二年期间（母公司R 01的Y 5 我们将完成对数据的分析，结合我们在全脑RNAseq中的发现， 与AMP-AD scRNAseq进行跨物种比较。我们还将独立执行 实验证实了scRNAseq最有前途的结果，如免疫荧光共聚焦显微镜， 显微镜，利用可用的抗体和试剂。选定的候选因果驱动因素还将 可以使用细胞类型特异性驱动器来操纵，包括神经胶质和/或谷氨酸能、GABA能、胆碱能和 果蝇中枢神经系统内的多巴胺能神经元，以检查大脑维护和/或功能的要求。 第二（AIM 2），我们将扩展计算预测的AD因果基因的实验验证 从果蝇到哺乳动物细胞。在tau果蝇筛选中鉴定的所有因果驱动因素都将在 使用shRNA培养小鼠和人类神经祖细胞。具体而言，我们将评估 考虑到tau积累在AD发病机制中的关键作用，tau蛋白水平上的因果驱动因素。我们的数据显示 鉴定的tau修饰基因的子集（例如，Hippo通路组分，Nuak 1）低tau蛋白 果蝇的水平。因此，我们假设正在进行的AMP-AD中确定的因果驱动因素的子集 筛选调节tau积累，并且从治疗的观点来看，该亚组可能特别令人感兴趣。 在拟议补充的第一年（父R 01补助金的Y 4），我们将测试Hippo途径 Neuro 2A和人神经前体细胞中的基因加上所有44个tau修饰基因，用于它们调节 使用免疫印迹和均相时间分辨法测定果蝇脑中的tau蛋白水平 荧光接近。在补助金的第二年（父R 01补助金的Y 5），我们将测试 在Neuro 2A和人类神经前体细胞中，所有额外的基因都降低了果蝇大脑中的tau水平。 第三（AIM 3），所有项目数据将通过Synapse/AMP-AD知识门户网站提供。

The goal of this supplemental project is to extend the bioinformatic analyses and experimental validation of AMP-AD target networks, which are the focus of the parent grant. First (AIM1), we will generate single-cell longitudinal transcriptomic profiles of Drosophila AD models expressing human tau and secreted amyloid-β. These data will allow to distinguish gene expression profiles from distinct neuronal populations as well as different glia subtypes. These studies will also strongly complement ongoing AMP-AD single-cell gene expression profiles from human postmortem tissue. We will include rigorous experimental controls for tau / amyloid-β expression as well as longitudinal sampling to dissect out the specific contributions of age and AD pathologic species on cell-type specific gene expression changes in the brain. Initially we will include 2 time points (early and late) during disease progression. During the first year of the proposed supplement (Y4 of the parent R01 grant) we will generate Drosophila of the appropriate genotypes, extract mRNA, perform scRNAseq, and begin analyses of the results. During the second year of the supplement (Y5 of the parent R01 grant) we will complete analysis of the data, integrating with our findings from whole brain RNAseq and performing cross-species comparisons with AMP-AD scRNAseq. We will also perform independent experiments to confirm the most promising results from scRNAseq, such as immunofluorescence confocal microscopy, taking advantage of available antibodies, and reagents. Selected candidate causal drivers will also be manipulated using cell-type specific drivers, including glia and/or glutamatergic, GABAergic, cholinergic and dopaminergic neurons within Drosophila CNS to examine requirements for brain maintenance and/or function. Second (AIM2), we will extend experimental validation of computationally predicted AD causal genes from Drosophila to mammalian cells. All causal drivers identified in the tau Drosophila screen, will be tested in cultured mouse and human neural progenitor cells using shRNAs. Specifically, we will assess the impact of causal drivers on tau protein levels given the key role of tau accumulation AD pathogenesis. Our data shows that a subset of the identified tau modifier genes (e.g., Hippo pathway components, Nuak1) lower tau protein levels in Drosophila. Thus, we hypothesize that a subset of causal drivers identified in the ongoing AMP-AD screen modulate tau accumulation and this subset may be especially interesting from a therapeutic standpoint. During the first year of the proposed supplement (Y4 of the parent R01 grant) we will test the Hippo pathway genes in Neuro2A and human neural precursor cells plus all 44 tau modifier genes for their ability to modulate tau protein levels in the Drosophila brain using immunoblotting and Homogeneous Time Resolved Fluorescence approaches. During the second year of the supplement (Y5 of the parent R01 grant) we will test in Neuro2A and human neural precursor cells all additional genes lowering tau levels in Drosophila brains. Third (AIM 3), all project data will be made available via the Synapse/AMP-AD Knowledge Portal.