Dissect regulation of glial nets surrounding amyloid plaques in Alzheimer's disease

项目总结 阿尔茨海默病(AD)的病理生理学机制尚不清楚。淀粉样斑块周围有反应性 星形胶质细胞和小胶质细胞，形成影响淀粉样蛋白扩散和炎症环境的神经胶质网。然而， 神经胶质网络中的神经胶质间通讯机制还知之甚少。我们最近的综合网络 来自晚发性AD患者的组学数据分析确定轴突引导受体Plexin-B1为中枢 星形胶质细胞特异子网络中的基因。我们已经证实，丛状蛋白-B1主要在 AD患者星形胶质细胞表达上调。值得注意的是，我们的试点研究 淀粉样变性AD小鼠模型显示Plexin-B1缺失显著改变斑块周围结构 神经胶质网。Plexin-B1缺失导致星形胶质细胞反应性减弱，胶质细胞网细胞间距减小， 小胶质细胞对斑块的覆盖率较高，导致斑块向致密核心型转变。这些变化 神经胶质网的减少与斑块负担和神经性营养不良的总体减少有关。在这里，我们将 扩展我们的初步研究，以进一步检验胶质细胞激活和细胞相互作用这一中心假设 在神经胶质网络中，受丛状蛋白-B1调节，影响淀粉样蛋白聚集和AD的神经毒性。阻断丛状蛋白-B1 从而提供了一个新的机会来减弱星形胶质细胞在神经胶质网络中的反应性，减少淀粉样蛋白的负担和 神经炎症，从而减缓AD的进展。在目标1中，我们的目标是建立特定细胞类型的胶质细胞 AD中的信号网络，并确定神经胶质网络中由Plexin-B1介导的基因模块。我们将分析单曲 AD患者和AD小鼠(带有和不带有Plexin-B1缺失)的细胞转录数据来确定 反应性星形胶质细胞和激活的小胶质细胞与丛蛋白-B1相关的共调控基因网络 发信号。在目标2中，我们将在神经胶质细胞培养中进行一系列功能分析，以研究丛状蛋白-B1的作用。 在调节星形胶质细胞在淀粉样蛋白攻击时的激活。含Plexin-B1的人iPSC来源星形胶质细胞 CRISPR-CAS的缺失将与Plexin-B1突变小鼠的原代星形胶质细胞进行比较。然后我们会做模特 在星形胶质细胞/小胶质细胞共培养中，星形胶质细胞与淀粉样蛋白的相互作用依赖于神经丛蛋白。 B1.在目标3中，我们将使用小鼠AD模型进行体内研究，以详细研究Plexin- 神经胶质网络B1缺失、斑块沉积、神经元功能和认知功能。两者都是淀粉样变性 并将在早期和晚期对AD的变态反应模型进行评估。我们希望证明这一点 Plexin-B1缺失导致星形胶质细胞在胶质网络中的反应性减弱，细胞间距减小，增加 小胶质细胞覆盖淀粉样斑块，并转向致密核心斑块，因此神经毒性较低，如 飞行员数据表明。总之，我们的研究将为神经胶质网络和神经丛蛋白的贡献提供新的见解。 B1对阿尔茨海默病的神经退行性变过程的影响，从而提供了新的治疗角度。

PROJECT SUMMARY The pathophysiology of Alzheimer’s disease (AD) remains unclear. Amyloid plaques are surrounded by reactive astrocytes and microglia, forming glial nets that affect amyloid spreading and inflammatory milieu. However, the mechanisms of inter-glial communication in glial nets are poorly understood. Our recent integrated network analysis of -omics data from late-onset AD patients identified the axon guidance receptor Plexin-B1 as a hub gene in an astrocyte-specific subnetwork. We have confirmed that Plexin-B1 is predominantly expressed in astrocytes and that it is upregulated in glial nets of AD patients. Remarkably, our pilot study with an amyloidogenic mouse model of AD showed that Plexin-B1 deletion markedly altered the structure of peri-plaque glial nets. Plexin-B1 deletion resulted in attenuated astrocyte reactivity, reduced cellular spacing of glial nets, and a higher coverage of plaques by microglia, leading to a shift of plaques to a dense core type. These changes of glial nets were associated with an overall reduction in plaque burden and neuritic dystrophy. Here we will expand our preliminary studies to further test the central hypothesis that glial activation and cellular interactions in glial nets, as regulated by Plexin-B1, affect amyloid aggregates and neurotoxicity in AD. Blocking Plexin-B1 may thus present a new opportunity to attenuate astrocyte reactivity in glial nets, reducing amyloid burden and neuroinflammation, thereby slowing down AD progression. In Aim 1, we aim to build cell type-specific glial signaling networks in AD, and to identify Plexin-B1-mediated gene modules in glial nets. We will analyze single cell transcriptomic data from both AD patients and AD mice (with and without Plexin-B1 deletion) to define coregulated gene networks in reactive astrocytes and activated microglia that are associated with Plexin-B1 signaling. In Aim 2, we will carry out a series of functional assays in glial cultures to study the role of Plexin-B1 in mediating astrocyte activation upon amyloid challenge. Human iPSC-derived astrocytes with Plexin-B1 deletion by CRISPR-Cas will be compared to primary astrocytes from Plexin-B1 mutant mice. We will then model in astrocyte/microglia co-cultures glial interactions in response to amyloid challenge in dependence of Plexin- B1. In Aim 3, we will conduct in vivo studies using mouse AD models to investigate in detail the impact of Plexin- B1 deletion on glial nets, plaque deposition, neuronal function and cognitive performance. Both an amyloidogenic and a tauopathy model of AD will be evaluated at early and advanced stages. We expect to demonstrate that Plexin-B1 deletion leads to attenuated astrocyte reactivity in glial nets, reduced cellular spacing, increased microglial coverage of amyloid plaques, and a shift to dense-core plaques and thus less neurotoxicity, as indicated by pilot data. Altogether, our study will provide new insights into the contribution of glial nets and Plexin- B1 to the neurodegenerative processes in AD, thus providing new therapeutic angles.