Mechanism of microglia proliferation

摘要 小胶质细胞增殖发生在许多神经系统疾病中，包括外周和视神经损伤， 神经变性疾病、中风、创伤性脑和脊髓损伤、多发性硬化症、癫痫发作、感染， 辐射和精神障碍。联体研究，沿着单细胞成像和细胞生物学研究 荧光命运绘图系统，已经证明，自我更新的居民小胶质细胞是负责 用于在神经系统疾病的小鼠模型中扩大小胶质细胞群体。事实上，新生儿 由小胶质细胞自我更新产生的小胶质细胞具有重要的神经功能， profile.此外，小胶质细胞增殖的失调显著影响了许多神经系统的发育。 神经系统疾病，表明小胶质细胞增殖在神经系统疾病的发展中起着关键作用， 疾病然而，小胶质细胞增殖的机制在很大程度上是未知的。我们做了一组初步的 实验探索小胶质细胞增殖的机制，并确定了一组胚胎基因， 在小胶质细胞发育过程中具有与增殖标记物Mki67相似的表达模式。与单细胞 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 在腰髓小胶质细胞增殖时上调。我们从分选的小胶质细胞的qRT-PCR 和小胶质细胞RiboTag证实了转录因子Myc的瞬时上调，Myc是胚胎干细胞中的一个转录因子。 小胶质细胞增殖相关基因，在腰髓小胶质细胞坐骨神经损伤后不久。我们还发现 成年小胶质细胞中Myc基因的缺失显著阻止了神经损伤诱导的早期阶段， 小胶质细胞增殖，小胶质细胞增殖的晚期阶段完整，表明成年小胶质细胞 增殖具有Myc依赖性起始阶段和Myc非依赖性维持阶段。探讨 在小胶质细胞Myc上调的上游信号通路中，我们分析了我们的scRNA-Seq，发现 编码小胶质细胞所需的DAP 12及其相关膜蛋白TREM2的基因， 在阿尔茨海默病中的增殖，在坐骨神经损伤后的腰髓小胶质细胞中上调。此外，本发明还提供了一种方法， 我们发现，Syk是一种位于TREM2和DAP 12下游、Myc上游的蛋白酪氨酸激酶， 坐骨神经损伤后不久，腰髓小胶质细胞磷酸化。在寻找Myc独立 在研究小胶质细胞增殖维持期的机制时，我们还发现Mafb，一种转录因子， 在成人中的表达水平比在胚胎小胶质细胞中高得多，抑制成人小胶质细胞增殖， 在新生儿中不存在，并且在神经损伤诱导的Myc上调后在腰髓小胶质细胞中下调。我们 现在提出一组实验来进一步阐明小胶质细胞增殖的机制。目标1：测试 Myc是小胶质细胞增殖所必需的假设。目的2：检验Mafb 有助于小胶质细胞增殖。目的3：验证CSF1R-TREM2-DAP 12-Syk通路与CSF1R-TREM2-DAP12-Syk通路相关的假设。 有助于小胶质细胞Myc和/或Mafb的表达调节以及小胶质细胞增殖。

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.