Mechanism of microglia proliferation - 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.
