Investigating CD47-SIRPa as novel protective signals during CNS synaptic pruning

 描述(申请人提供)：小胶质细胞，大脑的常驻免疫细胞和吞噬细胞，正在成为健康大脑中发展突触电路的关键调节器。我们实验室和其他实验室最近的研究表明，小胶质细胞吞噬了发育中的大脑中的突触；然而，小胶质细胞如何知道以哪些突触为目标仍然是一个主要的悬而未决的问题。我们以前的工作表明，小胶质细胞介导的修剪是发育突触细化的基础，这是形成成熟回路所必需的过程，在成熟回路中，弱小或不适当的突触被消除，剩余的连接被维持和加强。我们发现，小胶质细胞吞噬突触前输入是活动依赖的，并受到补体分子C1q和C3以及小胶质细胞补体受体CR3的驱动。这些分子是已知的促进巨噬细胞吞噬凋亡细胞或碎片的先天免疫“吃我”信号，缺乏这些信号的小鼠表现出突触输入的小胶质细胞吞噬减少和精致受损。这表明，小胶质细胞介导的修剪可能类似于免疫系统中吞噬细胞去除非我物质。然而，我们还不知道小胶质细胞如何准确地决定吞噬和避免哪些输入，这是一个关于塑造精确、成熟的连接所需的特异性的重要决定。我们认为，保护性的“不要吃我”信号是必要的，以防止小胶质细胞在突触细化期间不适当地吞噬必要的连接，就像它们防止在免疫反应期间被吞噬细胞不适当地吞噬健康的自我细胞一样。我们的初步数据支持这一假说，因为发育中的大脑中存在“不要吃我”信号CD47和Sirp，需要它们来防止过度的小胶质细胞吞噬突触输入。我们将研究缺乏CD47和Sirp的小鼠的解剖、功能和行为异常，以更好地了解过度吞噬小胶质细胞的后果。我们还将调查这些“不要吃我”信号是否以及如何受到活动的调节，以确定它们是否指示小胶质细胞以依赖于活动的方式吞噬特定的突触。最后，众所周知，在神经退行性疾病患者的大脑中，“不要吃我”信号被下调，我们将检查这些分子在亨廷顿病(HD)小鼠模型中是否调节失调，从而可能是由异常的小胶质细胞吞噬导致的突触丢失的原因。这项研究将第一次证明突触保护是必要的，以防止小胶质细胞在发育过程中不适当地吞噬必要的连接。这项研究计划不仅将为调节小胶质细胞吞噬特定突触的机制提供洞察力，而且将为研究中枢神经系统退行性疾病突触丢失的可能机制提供依据。

 DESCRIPTION (provided by applicant): Microglia, the brain's resident immune cells and phagocytes, are emerging as critical regulators of developing synaptic circuits in the healthy brain. Recent studies from our lab and others indicate that microglia engulf synapses in the developing brain; however, how microglia know which synapses to target remains a major open question. Our previous work demonstrates that microglia-mediated pruning underlies developmental synaptic refinement, an essential process required for the formation of mature circuits in which weak or inappropriate synapses are eliminated and remaining connections are maintained and strengthened. We found that microglial engulfment of presynaptic inputs is activity-dependent and driven by complement molecules C1q and C3, and microglial complement receptor CR3. These molecules are innate immune "eat me" signals known for promoting macrophage phagocytosis of apoptotic cells or debris, and mice lacking these signals exhibit reduced microglial engulfment of synaptic inputs and impaired refinement. This suggests that microglia-mediated pruning may be analogous to the removal of non-self material by phagocytes in the immune system. However, we do not yet know how microglia precisely determine which inputs to engulf and which to avoid, an important decision regarding the specificity needed to sculpt precise, mature connections. We propose that protective "don't eat me" signals are required to prevent inappropriate microglial engulfment of necessary connections during synaptic refinement, just as they prevent inappropriate engulfment of healthy self-cells by phagocytes during an immune response. Our preliminary data support this hypothesis, as "don't eat me" signals CD47 and SIRP are present in the developing brain and required to prevent excess microglial engulfment of synaptic inputs. We will investigate the anatomical, functional, and behavioral abnormalities in mice lacking CD47 and SIRP to better understand the consequences of excess microglial engulfment. We will also investigate whether and how these "don't eat me" signals are regulated by activity to determine if they direct microglia to engulf specific synapses in an activity-dependent manner. Finally, as "don't eat me" signals are known to be downregulated in the brains of patients with neurodegenerative diseases, we will examine whether these molecules are dysregulated in mouse models of Huntington's disease (HD) and could thereby underlie synapse loss caused by aberrant microglial engulfment. This study would be the first to demonstrate that synaptic protection is required to prevent inappropriate microglial engulfment of necessary connections during development. This research program will provide insight not only into the mechanisms regulating microglial engulfment of specific synapses, but also into possible mechanisms underlying synapse loss in CNS neurodegenerative diseases.