Deciphering the role of CX3CR1 in Modulating Mechanisms of Amyloid driven Neurodegeneration in Alzheimer's Disease (Diversity Supplement)

破译 CX3CR1 在阿尔茨海默氏病淀粉样蛋白驱动的神经变性调节机制中的作用（多样性补充）

• 批准号: 10524900
• 负责人: Bruce T Lamb
• 金额: $ 13.61万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524900
• 关键词: AD transgenic mice; Affect; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Autopsy; Award; Binding; Brain; CRISPR/Cas technology; CX3CL1 gene; CX3CR1 gene; Cell Line; Cell Nucleus; Chronic; Coculture Techniques; Communication; Data; Deposition; Disease; Down-Regulation; Event; Foundations; Frameshift Mutation; Functional disorder; Generations; Genes; Genetic Screening; Homeostasis; Human; Immunologics; Impaired cognition; Impairment; Inflammatory; Injections; Knock-out; Ligands; Light; Microglia; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neuroglia; Neurons; Parents; Pathogenesis; Pathologic; Pathology; Patients; Phagocytes; Phenotype; Population; Proteins; Reporting; Risk Factors; Role; Senile Plaques; Shapes; Signal Transduction; Synapses; Technology; Testing; Therapeutic; Tissues; Variant; abeta deposition; abeta oligomer; base; design; disease phenotype; extracellular; genetic analysis; genetic variant; homologous recombination; hyperphosphorylated tau; immune activation; in vitro Assay; induced pluripotent stem cell; insertion/deletion mutation; intercellular communication; loss of function mutation; mouse model; neurofibrillary tangle formation; neuroinflammation; neuron loss; neurotoxic; presenilin-1; prodromal Alzheimer' s disease; receptor; receptor function; response; tau Proteins; transcriptome sequencing

The amyloid-cascade hypothesis states that deposition of extracellular Aβ plaques is the primary event in AD that facilitates accumulation of hyperphosphorylated tau (pTau), formation of neurofibrillary tangles (NFTs) and drives subsequent neurodegeneration. Microglial activation is the earliest response to pathological changes in the brain and is known to shape neuronal activity and immune activation of neighboring glial cells. Thus, how Aβ-driven microglial activation shapes neurodegenerative signaling in AD is a critical unknown in current therapeutic approaches. Communication between microglia and neurons via microglial CX3CR1 and its neuronal ligand is a central signaling checkpoint that shapes the immunological niche in AD. RNA sequencing studies have postulated that downregulation of microglial CX3CR1 is a neuroprotective response. By contrast, our data suggests that the loss of CX3CR1 increases accumulation of toxic, soluble Aβ species which correlate with increased synaptic dysfunction, pTau pathology and neurodegeneration. Indeed, recent reports have associated CX3CR1-V249I, a mutation which impairs CX3CR1 function, with increased NFT pathology and worsened neurodegeneration in AD patients. This proposal aims to test the unique hypothesis that downregulation/loss of CX3CR1 signaling alters microglial activation and results in impaired clearance and/or increased accumulation of neurotoxic soluble Aβ oligomers. Enrichment of toxic Aβ in the micro-environment triggers a cascade of neurodegenerative signaling including the generation and spread of neurotoxic species of pTau. Using the single-nuclei sequencing approach we propose an unbiased genetic screen to assess how CX3CR1 alters neurodegenerative, phagocytic and neuroinflammatory signaling in early vs. late stages of AD. Results of this broad genetic analysis will be validated by a deep pathological phenotyping of the disease using 5xFAD and APPPS1 transgenic AD mice deficient in CX3CR1 signaling (Aim 1). To investigate how a toxic Aβ milieu in the absence of CX3CR1 can drive neurodegeneration, we will assess the propagation and spread of pathological tau species following stereotaxic injection of toxic tau from post-mortem AD tissue into the brains of 5xFAD mice with and without CX3CR1 (Aim 2). Lastly, to understand how the human V249I variant affects neurodegenerative responses, we will use CRISPER-Cas9 technology to generate human iPSC derived microglia with CX3CR1 harboring the V429I (loss-of-function) mutation and isogenic iPS-derived AD neurons expressing M146L and L286V mutations in the PSEN1 gene. Co-culture of human-derived microglia with isogenic AD neurons will be used to assess a) how the V249I mutation shapes Aβ driven neurotoxic microglial activation and b) how Aβ-activated V249I+ microglia alter neuronal activity and affect neurodegenerative signalling/pathology (Aim 3). Combining the use of transgenic AD mouse models and human iPSC based in-vitro assays, this proposal will shed light on the overarching effects of CX3CR1 on Aβ-driven neurodegeneration in the cascade of AD pathogenesis.

淀粉样蛋白局体假设指出，细胞外Aβ斑块的沉积是AD的主要事件 这促进了高磷酸化tau（PTAU）的积累，神经原纤维缠结（NFTS）的形成和 驱动随后的神经变性。小胶质细胞激活是对病理的最早反应 大脑的变化，已知会塑造神经元活性和相邻的免疫激活 神经胶质细胞。那就是Aβ驱动的小胶质细胞激活在AD中塑造神经退行性信号是A 当前的治疗方法中的关键未知。通过小胶质细胞和神经元之间的交流 小胶质细胞CX3CR1及其神经元配体是一个中央信号检查点，可塑造免疫学 广告中的利基市场。 RNA测序研究已经发布了小胶质细胞CX3CR1的下调是 神经保护反应。相比之下，我们的数据表明CX3CR1的损失增加了 有毒，可溶性Aβ物种与突触功能障碍增加，PTAU病理和 神经变性。实际上，最近的报告与CX3CR1-V249I有关，这是一个损害的突变 CX3CR1功能，NFT病理增加，AD患者的神经退行性变化恶化。这 提案旨在检验独特的假设，即CX3CR1信号的下调/丢失会改变小胶质 激活并导致神经毒性固体Aβ低聚物的清除和/或增加的积累。 微环境中有毒Aβ的富集触发了一系列神经退行性信号传导 PTAU神经毒性物种的产生和传播。使用单核测序方法我们 提案一个无偏的遗传筛查，以评估CX3CR1如何改变神经退行性，吞噬细胞和 早期与AD晚期的神经炎症信号传导。这种广泛的遗传分析的结果将是 通过使用5XFAD和APPPS1转基因AD小鼠对疾病的深层病理表型验证 缺乏CX3CR1信号传导（AIM 1）。在没有CX3CR1的情况下，如何研究有毒的Aβ环境 驱动神经变性，我们将评估病理tau物种的传播和传播 立体定位注射有毒后的tau从验尸后组织到有或没有的5xfad小鼠的大脑 CX3CR1（AIM 2）。最后，了解人类V249i变体如何影响神经退行性反应， 我们将使用Crisper-Cas9技术来产生人类IPSC衍生的小胶质细胞，并带有CX3CR1 V429i（功能丧失）突变和表达M146L和L286V的同源IPS衍生的AD神经元 PSEN1基因中的突变。人类衍生的小胶质细胞共培养与等源性AD神经元将用于 评估a）V249i突变如何塑造Aβ驱动的神经毒性小胶质细胞激活和b）Aβ激活如何激活 V249i+小胶质细胞改变神经元活性并影响神经退行性信号传导/病理（AIM 3）。结合 使用转基因AD鼠标模型和基于人IPSC的体外测定法，该建议将揭示 CX3CR1对AD发病机理级联Aβ驱动的神经变性的总体作用。