Uncovering Alzheimer's disease risk mechanisms through neuron-specific analysis of autophagy and endosomal-lysosomal function

摘要 这个PPG已经证实，溶酶体网络(LN)中多个阶段的功能障碍， 由自噬和内体-溶酶体(EL)途径组成，发生于阿尔茨海默病(AD)。 发病机制。贯穿整个PPG的一个假设是，增加β位点裂解羧基的水平- 淀粉样蛋白β前体蛋白(βCTF)末端片段与遗传和环境AD风险的关系 包括载脂蛋白E4等位基因、胆固醇和最近GWAS发现的危险基因在内的因素会破坏LN 在早发性阿尔茨海默病和更常见的晚发性阿尔茨海默病中起作用。在这个项目4(P4)中，我们重点关注 由晚发性AD危险因素驱动的自噬改变，特别是调查β细胞毒性T细胞因子及其 通过激活Rab5对LN的影响。这些目标将通过全面的 转录学方法评估同种易损神经元自噬的所有阶段 使用RNA测序(RNA-SEQ)和生物信息学的种群应用于定制设计的管道 自噬和溶酶体基因。这些信息性和功能预测性的基因分析管道具有 已经得到证实，并得到了自噬的免疫学和生化功能分析的补充， 包括使用一种新的神经元特异性自噬报告构建的小鼠。我们的实验设计 克服了与脑组织显著的细胞异质性相关的问题，在这些组织中，脆弱的神经元 与备用神经元和神经胶质细胞混合在一起，我们在初步数据中显示 不同的自噬途径程序。这种方法的强有力的验证来自于我们长期的 自噬和溶酶体途径在AD中的研究及其在体内的创新应用 涉及特定脆弱神经元群体的研究。与P1-P3一起，我们将雇用一个队列 模型，包括一种过度表达小鼠的野生型应用程序，一种新型的Rab5转基因小鼠 内体形态和行为异常，以及人源化的ApoE4小鼠，以及其他显示 与AD相关的LN缺陷，使我们能够检验与βCTF致病作用相关的多个假说 自噬和EL途径。在目标1中，我们将评估体内自噬的所有阶段的功能 通过一种新的细胞特异性转录切割、荧光报告结构的跟踪和 易损胆碱能神经元、海马神经元和皮质神经元的平行免疫化学功能分析。 增加自噬周转和诱导溶酶体生物发生是一种有吸引力的治疗方法 提高层粘连蛋白在脆弱细胞中的效率，已知的诱导自噬的方式包括 卡路里限制(CR)。在目标2中，我们将检验这样一种假设，即限制卡路里(CR)和 已建立的CR模拟物，表没食子儿茶素没食子酸酯(EGCG)，涉及增加自噬周转和 体内溶酶体的生物发生，促进包括βctf在内的溶酶体对底物的清除，从而避免 致病后果。

ABSTRACT This PPG has established that dysfunction at multiple stages throughout the lysosomal network (LN), comprised of autophagy and the endosomal-lysosomal (EL) pathway, occurs in Alzheimer’s disease (AD) pathogenesis. A hypothesis throughout the PPG is that increasing the levels of β-site cleaved carboxyl- terminal fragment of the amyloid-β precursor protein (βCTF) through genetic and environmental AD risk factors, including the ApoE4 allele, cholesterol, and recently identified risk genes found by GWAS, disrupts LN function in early-onset AD and in the more common late-onset AD form. In this project 4 (P4), we focus on autophagy alterations driven by late-onset AD risk factors, investigating particularly the role of the βCTF and its effects on the LN mediated through rab5 activation. The goals will be attained through a comprehensive transcriptomic approach that evaluates all stages of autophagy in homogeneous vulnerable neuronal populations using RNA-sequencing (RNA-seq) and bioinformatics applied to custom-designed pipelines of autophagy and lysosomal genes. These informative and functionally predictive gene analysis pipelines have been confirmed, and are complemented by immunological and biochemical functional analyses of autophagy, including the use of a novel neuron-specific autophagy reporter-construct mouse. Our experimental design overcomes issues associated with the marked cellular heterogeneity of brain tissue, where vulnerable neurons are intermixed with spared neuronal populations and glia, which we show in our preliminary data to have different autophagy pathway programs. The strong validation of this approach derives from our longstanding investigations of autophagy and lysosomal pathways in AD and supports its innovative application to in vivo studies involving specific vulnerable neuronal populations. In conjunction with P1-P3, we will employ a cohort of models, including a wild-type APP overexpressing mouse, a novel rab5 transgenic mouse that displays endosome morphologic and behavioral abnormalities, and humanized ApoE4 mice, among others that display LN deficits relevant to AD, enabling us to test multiple hypotheses related to the pathogenic effects of βCTF on the autophagy and EL pathways. In Aim 1 we will evaluate the function of all stages of autophagy in vivo through a novel combination of cell-specific transcriptomics, tracking of fluorescence reporter constructs, and parallel immunochemical functional analyses in vulnerable cholinergic, hippocampal, and cortical neurons. Increasing autophagy turnover and inducing lysosomal biogenesis is an appealing therapeutic approach for improving the efficiency of the LN in vulnerable cells, and modalities known to induce autophagy include calorie restriction (CR). In Aim 2 we will test the hypothesis that the benefits of calorie restriction (CR) and an established CR mimetic, epigallocatechin-3-gallate (EGCG), involve increased autophagy turnover and lysosomal biogenesis in vivo, enhancing clearance of substrates by lysosomes including βCTF, thus averting pathogenic consequences.