A novel role of CDK2 inhibition in restraining microglial overactivation

Intrinsic immune checkpoints in microglia are vital for maintaining homeostatic immune response and preventing overactivation. Neuroinflammation, frequently driven by microglial overactivation, significantly influences a wide range of neurological disorders. While MEF2C has been associated with a syndromic form of autism spectrum disorder (ASD) and various other neurological disorders in humans, its specific role as an immune checkpoint remains poorly defined. By harnessing MEF2C-knockout (KO) induced microglia-like cells (iMGLs) through differentiation from human pluripotent stem cells (hPSCs), here we observed that these cells, following LPS stimulation, exhibited overactivation similar to patterns seen in various neurological disorders. High-throughput screening identified BMS265246, a CDK2 inhibitor, as effective in specifically suppressing the overactivated phenotypes of MEF2C-KO iMGLs, and in restoring inflammatory response closer to normal levels. Mechanistically, we uncovered that MEF2C regulates p21 transcription, a critical step in preventing CDK2 activation in microglia. Loss of MEF2C results in CDK2-induced RB phosphorylation and degradation, leading to enhanced NF-?B p65 subunit nuclear translocation and exacerbated inflammatory responses. Remarkably, BMS265246 treatment rectified microglial overactivation and ASD-like behaviors in both global and microglia-specific Mef2C heterozygous knockout mice. Overall, our findings elucidated a previously unknown immune checkpoint mechanism governed by MEF2C, and highlighted CDK2 as a potential key factor driving neuroinflammation through microglial overactivation. These insights positions CDK2 as a promising therapeutic target for treating various neurological diseases influenced by overactivated microglia. Overall design: mRNA profiles of WT and MEF2C-/- (KO) iMGLs, treated with or without 5 µM BMS, in the presence or absence of 100 ng/mL LPS for 12 hours, were generated through the Illumina Novaseq 6000 platform with 150 bp paired-ends.

小胶质细胞（microglia）的固有免疫检查点对于维持稳态免疫应答、防止过度激活至关重要。神经炎症通常由小胶质细胞过度激活驱动，对多种神经系统疾病均有显著影响。尽管MEF2C已被证实与人类的综合征性自闭症谱系障碍（Autism Spectrum Disorder, ASD）及其他多种神经系统疾病存在关联，但其作为免疫检查点的具体作用仍未明确。本研究通过人类多能干细胞（human pluripotent stem cells, hPSCs）定向分化获得MEF2C敲除（knockout, KO）诱导小胶质样细胞（induced microglia-like cells, iMGLs），并观察到经脂多糖（lipopolysaccharide, LPS）刺激后，此类细胞呈现出与多种神经系统疾病中相似的过度激活表型。通过高通量筛选（high-throughput screening），本研究发现细胞周期蛋白依赖性激酶2（cyclin-dependent kinase 2, CDK2）抑制剂BMS265246可特异性抑制MEF2C-KO iMGLs的过度激活表型，并使炎症应答恢复至接近正常水平。从机制层面而言，本研究揭示MEF2C可调控p21的转录，这是抑制小胶质细胞中CDK2激活的关键环节。MEF2C缺失会导致CDK2介导的RB蛋白磷酸化与降解，进而增强核因子κB（nuclear factor-κB, NF-κB）p65亚基的核转位，加剧炎症应答。值得注意的是，在全身性及小胶质细胞特异性Mef2C杂合敲除小鼠模型中，BMS265246给药均可纠正小胶质细胞过度激活，并改善类ASD行为表型。综上，本研究阐明了一种此前未被报道的、由MEF2C调控的免疫检查点机制，并证实CDK2是通过小胶质细胞过度激活驱动神经炎症的潜在关键靶点。上述研究结果将CDK2确立为治疗因小胶质细胞过度激活导致的多种神经系统疾病的潜在候选治疗靶点。实验整体设计：通过Illumina NovaSeq 6000平台进行150 bp双端测序，获取经或不经5 μM BMS处理、伴或不伴100 ng/mL LPS刺激12小时的野生型及MEF2C-KO iMGLs的mRNA转录组谱。