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斑块沉积是AD的主要事件 这有助于过度磷酸化tau(Ptau)的积累，形成神经原纤维缠结(NFTs)和 会导致随后的神经退化。小胶质细胞激活是病理性反应的最早反应 大脑的变化，并已知塑造神经活动和邻近的免疫激活 神经胶质细胞。因此，β驱动的小胶质细胞激活如何在AD中塑造神经退行性信号是一个 在目前的治疗方法中，危急未知。小胶质细胞和神经元之间的通讯通过 小胶质细胞CX3CR1及其神经元配体是形成免疫学的中枢信号检查点 公元后的利基市场。RNA测序研究推测，小胶质细胞CX3CR1的下调是一种 神经保护性反应。相比之下，我们的数据表明，CX3CR1的丢失增加了 毒性，可溶性Aβ与突触功能障碍增加，PTAU病理和 神经退行性变。事实上，最近的报道与CX3CR1-V249I有关，这是一种损害 CX3CR1功能异常，伴有NFT病理改变，加重AD患者神经退行性变。这 该提案旨在测试CX3CR1信号下调/丢失改变小胶质细胞这一独特假设 激活并导致神经毒性的可溶性Aβ寡聚体的清除受损和/或积累增加。 微环境中有毒Aβ的浓缩触发了一系列神经退行性信号，包括 Ptau神经毒性物种的产生和传播。使用单核测序方法，我们 提出一种公正的基因筛查方法，以评估CX3CR1如何改变神经退行性变、吞噬功能和 阿尔茨海默病早期与晚期的神经炎性信号。这一广泛的遗传分析的结果将是 用5xFAD和APPPS1转基因AD小鼠对该病进行深层病理表型验证 缺乏CX3CR1信号(目标1)。要研究在没有CX3CR1的情况下有毒的Aβ环境如何 推动神经退化，我们将评估病理性tau物种的繁殖和扩散 立体定向将死后AD组织中的有毒tau注射到5xFAD小鼠的脑内 CX3CR1(目标2)。最后，为了了解人类V249I变异如何影响神经退行性反应， 我们将使用CRISper-Cas9技术来产生人IPSC来源的小胶质细胞，CX3CR1携带有 V429I(功能丧失)突变和表达M146L和L286V的等基因诱导型AD神经元 PSEN1基因的突变。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)V249I突变如何影响Aβ诱导的神经毒性小胶质细胞激活以及b)Aβ如何激活 V249I+小胶质细胞改变神经元活动并影响神经退行性信号/病理(目标3)。组合 使用转基因AD小鼠模型和基于人IPSC的体外检测，这一建议将有助于 CX3CR1在AD发病级联反应中对Aβ诱导的神经退行性变的主要作用