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.

淀粉样蛋白级联假说认为，细胞外Aβ斑块的沉积是AD的主要事件 促进过度磷酸化的tau（pTau）的积累，神经元缠结（NFT）的形成， 会导致随后的神经退化小胶质细胞活化是病理性脑损伤的最早反应。 大脑中的变化，并且已知会塑造神经元活动和邻近神经元的免疫激活。 神经胶质细胞因此，Aβ驱动的小胶质细胞活化如何塑造AD中的神经退行性信号传导是一个重要的研究课题。 在目前的治疗方法中是未知的。小胶质细胞和神经元之间的通讯 小胶质细胞CX 3CR 1及其神经元配体是一个中央信号检查点，它塑造了免疫系统。 AD中的niche。RNA测序研究推测，小胶质细胞CX 3CR 1的下调是一个重要的机制。 神经保护反应相比之下，我们的数据表明，CX 3CR 1的缺失增加了 毒性、可溶性Aβ物质，与突触功能障碍、pTau病理学和 神经变性事实上，最近的报道已经将CX 3CR 1-V249 I，一种损害 在AD患者中，CX 3CR 1功能增加，NFT病理学增加，神经退行性变恶化。这 该提案旨在测试CX 3CR 1信号转导的下调/缺失改变小胶质细胞的独特假设。 活化并导致神经毒性可溶性Aβ寡聚体的清除受损和/或蓄积增加。 微环境中毒性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 提出了一个公正的遗传筛选，以评估CX 3CR 1如何改变神经退行性，吞噬和 AD的早期与晚期阶段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APPPS 1转基因AD小鼠对疾病进行深层病理表型分析来验证 CX 3CR 1信号传导缺陷（Aim 1）。为了研究在不存在CX 3CR 1的情况下毒性Aβ环境如何能够 驱动神经变性，我们将评估病理性tau蛋白种类的传播和扩散， 将来自死后AD组织的毒性tau立体定位注射到5xFAD小鼠的脑中， CX 3CR 1（目标2）。最后，为了了解人类V249 I变体如何影响神经退行性反应， 我们将使用CRISPER-Cas9技术来产生具有CX 3CR 1的人类iPSC衍生的小胶质细胞，所述CX 3CR 1具有 V429 I（功能丧失）突变和表达M146 L和L286 V的同基因iPS衍生的AD神经元 PSEN 1基因突变。人源性小胶质细胞与同基因AD神经元的共培养将用于 评估a）V249 I突变如何塑造Aβ驱动的神经毒性小胶质细胞活化和B）Aβ激活的神经毒性小胶质细胞活化如何影响V249 I突变。 V249 I+小胶质细胞改变神经元活性并影响神经退行性信号传导/病理学（目的3）。结合 使用转基因AD小鼠模型和基于人iPSC的体外试验，该提议将阐明 CX 3CR 1对AD发病机制级联中Aβ驱动的神经变性的总体作用。