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，这是父母拨款的重点。首先(AIM1)，我们将生成单细胞 表达人tau和分泌型淀粉样蛋白-β的果蝇AD模型的纵向转录图谱。 这些数据将允许区分不同神经元群体的基因表达谱以及 不同的胶质细胞亚型。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。我们将包括对tau/的严格实验控制。 淀粉样蛋白-β的表达以及纵向取样以剖析AGE和AD的特定贡献 病理种对脑组织细胞类型特异性基因表达的影响。最初我们将包括2次 疾病进展期间的积分(早期和晚期)。在拟议补编的第一年( 父母R01赠款)我们将产生合适的基因类型的果蝇，提取mRNA，执行 ScRNAseq，并开始分析结果。在补充的第二年(母公司R01的Y5 Grant)我们将完成对数据的分析，结合我们来自全脑RNAseq和 使用AMP-AD scRNAseq进行跨物种比较。我们也将独立表演 证实scRNAseq最有希望的结果的实验，如免疫荧光共聚焦 显微镜，利用可用的抗体和试剂。选定的候选因果驱动程序也将 使用细胞类型的特定驱动因素，包括胶质细胞和/或谷氨酸、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/或功能的需求。 其次(AIM2)，我们将扩大对计算预测的AD病因基因的实验验证 从果蝇到哺乳动物细胞。在tau果蝇屏幕中确定的所有原因驱动程序将在 使用shRNAs培养小鼠和人类神经前体细胞。具体地说，我们将评估 因果驱动因素对tau蛋白水平的影响赋予tau蓄积在AD发病机制中的关键作用。我们的数据显示 已鉴定的tau修饰基因的一个子集(例如，河马途径组件NUAK1)降低了tau蛋白 果蝇体内的含量。因此，我们假设在正在进行的AMP-AD中确定的因果驱动因素的子集 屏幕调节tau的积累，从治疗的角度来看，这个子集可能特别有趣。 在拟议补充资金的第一年(父母R01拨款的Y4)，我们将测试河马路径 Neuro2A和人类神经前体细胞中的基因加上所有44个tau修饰物基因的调节能力 免疫印迹和均相时间分辨技术研究果蝇脑内tau蛋白水平 荧光接近了。在补贴的第二年(家长R01拨款的Y5)，我们将测试 在Neuro2A和人类神经前体细胞中，所有额外的基因都会降低果蝇大脑中tau的水平。 第三，所有项目数据将通过Synapse/AMP-AD知识门户提供。

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.