Assessing cellular aging in old and rejuvenated neurons from Alzheimer patients

项目概述：阿尔茨海默病（AD）影响着全球超过5000万人1。对于绝大多数 AD是散发性的，除了高龄之外没有已知的病因。神经科学领域 可以理解的是，神经元细胞的细胞内在特性是神经退行性疾病的基础。 疾病，如AD。然而，大脑中大约一半的细胞是神经胶质细胞，大约15%的细胞在大脑中。 由小胶质细胞占2.小胶质细胞是中枢神经系统的特化巨噬细胞， 清除碎片病原体和神经突触在稳态条件下，小胶质细胞为 神经元，而在病理条件下，小胶质细胞经常过度修剪突触，激活星形胶质细胞， 增加炎症，减少对神经元的支持3。目前，对小胶质细胞的机械理解， 阿尔茨海默病（AD）尚不清楚。为了推进aim 4的科学目标，本项目将研究代谢 三维多细胞培养中的神经元-小胶质细胞相互作用。这个项目将测试这个假设 异常的神经元PKM 2挑战神经元-胶质细胞偶联，并且小胶质细胞修剪和 对炎症刺激的反应受其载脂蛋白E（Apolipoprotein E）单体型的影响。原提案 表明在没有小胶质细胞的情况下星形胶质细胞活化可能缺乏以适当地活化它们。因为， Gage实验室在解决这一问题方面取得了重大进展， 将小胶质细胞转化为类器官（In press，Cell. 2023年5月），这将补充微载体数据，并提供 单独的证据APOE单倍型引起的神经胶质细胞代谢变化已得到充分证实4，5，但 对神经元的影响还不太清楚。目前尚不清楚APOE ε4等位基因如何影响AD的发病和进展， APOE ε2等位基因提供保护6。这个实验室最近获得了商业上可用的人类同基因 已被遗传改变以代表每个APOE单倍型的iPSC系。小神经胶质细胞会生成 如前所述7从这些细胞系中的每一个中分离，并在多细胞微载体和类器官中培养。 该实验室的初步数据表明，携带APOE 4/4的小胶质细胞具有AD相关的代谢缺陷。 与脂质代谢、溶酶体和线粒体功能相关。这个项目的目标是分析 具有不同APOE单倍型的小胶质细胞的转录组和功能。通过使用单细胞RNA 通过测序，该项目将确定这些群体之间基于细胞类型的基因表达差异。 这些数据将有助于整体的建议，并揭示涉及AD和年龄的特定基因。 相关的溶酶体功能和代谢被APOE下调。通过这种机制，APOE 4 小胶质细胞可能积极参与AD的发病机制，而不仅仅是对AD的发病机制作出反应。 该项目的假设是，APOE 4小胶质细胞将表现出减少的线粒体自噬和溶酶体吞噬。 损伤，从而增加细胞内的毒性，并最终影响APOE 4/4小胶质细胞的作用方式 为神经元提供支持，本项目将通过微载体和类器官培养进行研究。

Project Summary: Alzheimer’s disease (AD) affects over 50 million people worldwide1. For the vast majority of patients, AD is sporadic, with no known etiology other than advanced age. The field of neuroscience has understandably focused on cell intrinsic properties of neuronal cells as the basis for neurodegenerative diseases, such as AD. However, about half of the cells in the brain are glial cells with ~15% of cells in the brain accounted for by microglia2. Microglia are specialized macrophages of the central nervous system that help to clear debris, pathogens, and neuronal synapses. Under homeostatic conditions, microglia provide support to neurons, while under pathological conditions, microglia often prune synapses excessively, activate astrocytes, increase inflammation, and reduce support to neurons3. Currently, a mechanistic understanding of microglia in Alzheimer’s disease (AD) is unclear. To advance the scientific goals of aim 4, this project will study metabolic neuron-microglia interactions in three-dimensional multicellular cultures. This project will test the hypothesis that aberrant neuronal PKM2 challenges neuron-glia coupling, and that the microglial capacity to prune and respond to inflammatory stimuli is influenced by their APOE (Apolipoprotein E) haplotype. The original proposal indicated that astrocyte activation may be lacking in the absence of microglia to properly activate them. Since, significant advances have been made in the Gage lab to address this issue through the incorporation of microglia into organoids (In press, Cell. May 2023), which will complement the microcarrier data and provide a separate piece of evidence. Metabolic changes in glia by APOE haplotype is well documented4,5, but the impact on neurons is less clear. It is unknown how APOE ε4 allele influences AD onset and progression or how APOE ε2 allele provides protection6. This lab has recently acquired commercially available human isogenic iPSC lines that have been genetically altered to represent each APOE haplotype. Microglia will be generated from each of these lines as previously described7 and cultured in multi-cell microcarriers and organoids. Preliminary data from this lab suggests that microglia with APOE4/4 have AD related metabolic defects associated with lipid metabolism, lysosomal and mitochondrial function. The goal of this project is to analyze the transcriptome and functionality of microglia with different APOE haplotypes. By employing single cell RNA sequencing, this project will identify differences in gene expression on a cell-type basis, between these groups. These data will contribute to the overall proposal and reveal specific genes involved implicated in AD and age- related lysosomal function and metabolism are downregulated by APOE. Through this mechanism, APOE4 microglia may actively contribute to the pathogenesis of AD, rather than solely reacting to AD pathogenesis. The hypothesis of this project is that APOE4 microglia will exhibit decreased mitophagy and lysosomal impairment, therefore increasing toxicity within cells, and ultimately impacting the way APOE4/4 microglia can provide support to neurons, which this project will investigate through the microcarriers and organoid cultures.