Mechanism of microglia proliferation

摘要 小胶质细胞的增殖出现在许多神经疾病中，包括外周和视神经损伤， 神经退行性疾病，中风，创伤性脑和脊髓损伤，多发性硬化症，癫痫，感染， 辐射和精神障碍。副生物研究，以及单细胞成像和多色研究 荧光命运图谱系统，已经证明了驻留的小胶质细胞的自我更新是负责的 用于在神经系统疾病的小鼠模型中扩大小胶质细胞数量。事实上，新生儿 小胶质细胞自我更新产生的小胶质细胞具有显著的神经功能，具有独特的转录 侧写。此外，小胶质细胞增殖的失调显著影响许多疾病的发展。 神经性疾病，表明小胶质细胞增殖在神经性疾病的发展中起关键作用 疾病。然而，小胶质细胞增殖的机制在很大程度上是未知的。我们做了一组预赛 探索小胶质细胞增殖机制的实验，并确定了一组胚胎基因 在小胶质细胞发育过程中与增殖标记物Mki67有相似的表达模式。使用单电池 RNA测序(scRNA-Seq)，我们发现这些胚胎小胶质细胞增殖相关基因是 腰髓小胶质细胞在增殖时表达上调。我们从分离的小胶质细胞中提取的qRT-PCR 小胶质细胞RiboTag证实了胚胎之一转录因子Myc的瞬时上调 坐骨神经损伤后不久腰髓小胶质细胞中的小胶质细胞增殖相关基因。我们还发现 成年小胶质细胞Myc基因的缺失显著阻止了神经损伤的早期阶段 小胶质细胞增殖，晚期小胶质细胞增殖完好，提示成年小胶质细胞 增殖有一个Myc依赖的起始阶段和一个Myc非依赖的维持阶段。要探索 小胶质细胞Myc上调的上游信号通路，我们分析了我们的scRNA-Seq，发现 编码小胶质细胞所需的DAP12及其相关膜蛋白TREM2的基因 在阿尔茨海默病中，在坐骨神经损伤后，腰髓小胶质细胞中的增殖被上调。此外, 我们发现，TREM2和DAP12下游、Myc上游的蛋白酪氨酸激酶Syk是 坐骨神经损伤后不久，腰髓小胶质细胞中的磷酸化。在搜索Myc非依赖时 对于小胶质细胞增殖维持阶段的机制，我们还发现了转录因子Mafb 在成人中的表达水平远远高于胚胎小胶质细胞，但在成人中抑制小胶质细胞的增殖 在新生儿中不表达，在神经损伤诱导的Myc上调后，在腰髓小胶质细胞中下调。我们 现在提出一系列实验，以进一步阐明小胶质细胞增殖的机制。目标1：测试 一般认为小胶质细胞增殖需要Myc的假设。目标2：检验Mafb的假设 有助于小胶质细胞的增殖。目的3：验证CSF1R-TREM2-DAP12-Syk通路的假说 参与小胶质细胞Myc和/或Mafb的表达调控和小胶质细胞的增殖。

ABSTRACT Microglia proliferation occurs in numerous neurological disorders, including peripheral and optic nerve injuries, neurodegenerative diseases, stroke, traumatic brain and spinal cord injuries, multiple sclerosis, seizure, infection, irradiation, and mental disorders. Parabiotic studies, along with studies with single-cell imaging and multicolor fluorescence fate mapping system, have demonstrated that the self-renewal of resident microglia is responsible for the expansion of microglia population in mouse models of neurological diseases. In fact, the newborn microglia generated from microglial self-renewal have significant neurological function with unique transcriptional profile. Moreover, dysregulation of microglia proliferation significantly impacts the development of many neurological diseases, indicating that microglia proliferation plays critical roles in the development of neurological diseases. However, the mechanism of microglia proliferation is largely unknown. We did a group of preliminary experiments to explore the mechanisms of microglia proliferation, and identified a group of embryonic genes that have similar expression pattern as proliferation marker Mki67 during microglia development. With single-cell RNA sequencing (scRNA-Seq), we found that these embryonic microglial proliferation-associated genes were upregulated in lumbar cord microglia around the time they proliferate. Our qRT-PCR from the sorted microglia and microglial RiboTag confirmed the transient upregulation of transcription factor Myc, one of the embryonic microglial proliferation-associated genes, in lumbar cord microglia shortly after sciatic nerve injury. We also found that the deletion of Myc from adult microglia significantly prevented the early phase of nerve injury-induced microglia proliferation, with the late phase of microglia proliferation intact, suggesting that adult microglia proliferation has a Myc-dependent initiation phase and a Myc-independent maintenance phase. To explore the signaling pathway upstream of microglial Myc upregulation, we analyzed our scRNA-Seq and found that the genes encoding DAP12 and its associated membrane protein TREM2, which is required for microglia proliferation in Alzheimer's disease, are upregulated in lumbar cord microglia after sciatic nerve injury. In addition, we found that Syk, a protein tyrosine kinase downstream of TREM2 and DAP12, and upstream of Myc, is phosphorylated in lumbar cord microglia shortly after sciatic nerve injury. In searching for Myc independent mechanism for the maintenance phase of microglia proliferation, we also found that Mafb, a transcription factor expressed at much higher level in adult than in embryonic microglia, inhibits microglia proliferation in adult but not in neonate, and is downregulated in lumbar cord microglia after nerve injury-induced Myc upregulation. We now propose a set of experiments to further delineate the mechanism of microglia proliferation. Aim 1: To test the hypothesis that Myc is required for microglia proliferation in general. Aim 2: To test the hypothesis that Mafb contributes to microglia proliferation. Aim 3: To test the hypothesis that CSF1R-TREM2-DAP12-Syk pathway contributes to the expression regulation of microglial Myc and/or Mafb and to microglia proliferation.