A novel role for astrocyte-secreted synaptogenic factor Hevin/SPARCL1 in microglia-mediated synaptic pruning in response to visual experience

摘要 在高等动物中，突触回路是行为和认知的基础。适当地建立和维护 这些电路需要在生命早期形成和移除的连接数量之间取得平衡。渐增 有证据表明，突触的形成和消除受损与突触病理有关。 存在于许多神经系统疾病中。我们实验室和其他实验室进行了严格的体外和体内研究 证明了星形胶质细胞和小胶质细胞在调节突触形成和消除中的关键作用， 分别进行了分析。这一领域的进一步研究已经开始描述这两种神经胶质细胞如何进行通讯 平衡突触的形成和消除。然而，对信号的详细分子理解 调解这种交流以及这种交流在电路发展中的作用还没有完全得到 地址。在我的初步研究中，我已经确定了星形胶质细胞衍生的突触生成因子， Hevin/Sparcl1，作为对小胶质细胞的直接信号。Hevin在睁开眼睛后立即被蛋白质分解， 与丘脑皮质突触密度净减少的时期相吻合。此外，Hevin的C-末端 结合并激活TLR4，导致小胶质细胞TLR2表达增加。使用最近的 发展了TLR2敲入报告系，进一步表明TLR2的表达仅限于小胶质细胞。 TLR2-LOW和TLR2-HIGH可用于鉴定两类小胶质细胞。 有趣的是，我发现高TLR2的小胶质细胞有一个高溶酶体室，这表明它们代表一个 小胶质细胞吞噬群增多。基于这些发现，这项提议将检验这一假设 星形胶质细胞分泌的Hevin以感觉经验依赖的方式被切割，以产生C- 突触发育所需的末端片段。我进一步建议Hevin在当地发出信号 通过小胶质细胞表达TLR4来启动周围的小胶质细胞亚群 吞噬细胞标记TLR2的表达增加。这一假设将分两个阶段进行检验。 目标。目的1阐明Hevin蛋白降解裂解在突触发育和可塑性中的作用。 将这一事件与感官体验联系起来。目标2将定义一种分子机制，将 星形胶质细胞衍生的突触生成因子与高密度的小胶质细胞介导的突触消除。调查结果 这项研究将首次确定从星形胶质细胞到小胶质细胞的分子信号通路 协调星形胶质细胞和小胶质细胞的功能，以响应感官体验。

ABSTRACT Synaptic circuits underlie behavior and cognition in higher animals. Proper establishment and maintenance of these circuits requires a balance in the number of connections formed and removed early in life. Increasing evidence suggests that impaired synapse formation and elimination contribute to the synaptic pathologies found in many neurological disorders. Rigorous in vitro and in vivo work from our lab and others have demonstrated the critical role for astrocytes and microglia in mediating synapse formation and elimination, respectively. Further work in the field has begun to describe how these two glial cell types may communicate to balance synapse formation and elimination. However, a detailed molecular understanding of the signals that mediate this communication and the role of such communication in circuit development have not been fully addressed. In my preliminary studies, I have identified the astrocyte-derived synaptogenic factor, Hevin/Sparcl1, as a direct signal to microglia. Hevin is proteolytically cleaved just after eye opening which coincides with a period of net decrease in thalamocortical synapse density. Additionally, Hevin’s C-terminal binds and activates TLR4 leading to an increase in the expression of microglia TLR2. Using a recently developed TLR2 knock in reporter line, I further show that TLR2 expression is restricted to microglia in the developing cortex and can be used to identify two populations of microglia, TLR2-low vs. TLR2-high. Intriguingly, I found that TLR2-high microglia have a high lysosomal compartment suggesting they represent a more phagocytic population of microglia. Based on these findings, this proposal will test the hypothesis that astrocyte secreted Hevin is cleaved in a sensory experience dependent manner to produce a C- terminal fragment that is required for synapse development. I further propose that Hevin signals locally through microglia expressed TLR4 to prime a subpopulation of the surrounding microglia to be more phagocytic marking them with an increased expression of TLR2. This hypothesis will be tested in two aims. Aim 1 will elucidate the role of Hevin proteolytic cleavage in synapse development and plasticity and link this event to sensory experience. Aim 2 will define a molecular mechanism that links processing of an astrocyte-derived synaptogenic factor with heightened microglia mediated synapse elimination. The findings from this study will for the first time identify a molecular signaling pathway from astrocytes to microglia that coordinates astrocyte and microglia function in response to sensory experience.