Impaired removal of dying brain cells by microglia in GPR34 deficient mice

GPR34, a G protein coupled receptor present selectively in microglia and other myeloid cells, is highly expressed in homeostatic microglia but is downregulated in disease associated microglia such as are found in Alzheimer's disease brain. However, little is known about GPR34's role in microglia function or brain development. Here, we studied Gpr34 knockout (KO) mice at postnatal 18-day (18 d) and 3 months (3 mo) age. In the brains of 18 d GPR34 KO mice, there were elevated numbers of neurons, oligodendrocytes and microglia, many of which were IHC-positive for cell death markers cleaved-caspase 3, phospho-RIP3, or annexin V. There was no increase in cell death markers or in steady state numbers of neurons, oligos and microglia at 3 months, indicating that GPR34-independent mechanisms are able to compensate during brain maturation. Based on RNA sequencing analysis and phagocytosis functional assays, as well as computational modeling, we provide evidence that Gpr34 KO microglia have deficiencies in chemotaxis, but not phagocytic efficiency, which leads to a slower clearance of dead cells from the developing brain. Collectively, these results indicate that Gpr34 is required for microglia to clear dead or dying cells. Overall design: GPR34 KO and WT mice were generated, and for each numerous brain regions were sequenced with RNA-seq. For more details see our publication.

GPR34是一种选择性表达于小胶质细胞及其他髓系细胞的G蛋白偶联受体（G protein coupled receptor），在稳态小胶质细胞中高表达，但在阿尔茨海默病患者大脑中发现的疾病相关小胶质细胞中表达下调。然而，目前对GPR34在小胶质细胞功能或大脑发育中的作用所知甚少。本研究针对出生后18日龄（18 d）及3月龄（3 mo）的Gpr34基因敲除（KO）小鼠展开实验。在18日龄GPR34 KO小鼠的脑组织中，神经元、少突胶质细胞与小胶质细胞的数量均出现升高，其中大量细胞的细胞死亡标志物——裂解型半胱氨酸天冬氨酸蛋白酶3（cleaved-caspase 3）、磷酸化RIP3（phospho-RIP3）或膜联蛋白V（annexin V）免疫组化（IHC）检测呈阳性。而在3月龄小鼠的脑组织中，细胞死亡标志物水平及神经元、少突胶质细胞与小胶质细胞的稳态数量均未出现升高，这提示大脑成熟过程中存在不依赖GPR34的代偿机制。通过RNA测序分析、吞噬功能实验及计算建模，本研究证实Gpr34 KO小胶质细胞存在趋化功能缺陷，但吞噬效率未受影响，进而导致发育中的大脑内死细胞清除速度减慢。综上，上述结果表明Gpr34是小胶质细胞清除死亡或濒死细胞所必需的基因。整体实验设计：构建了GPR34 KO及野生型（WT）小鼠，对每类小鼠的多个脑区开展RNA测序。更多细节可参阅本研究已发表的论文。