Lipid Accumulation Induced by APOE4 Impairs Microglial Surveillance of Neuronal-Network Activity

Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for developing late-onset Alzheimer’s disease and its expression in microglia is associated with pro-inflammatory states. How the interaction of APOE4 microglia with neurons differs from microglia expressing the disease-neutral allele APOE3 is currently unknown. Here, we employ CRISPR-edited induced pluripotent stem cells (iPSCs) to dissect the impact of APOE4 in neuron-microglia communication. Our results reveal that APOE4 induces a distinct metabolic program in microglia that is marked by the accumulation of intracellular neutral lipid stores through impaired lipid catabolism. Importantly, this altered lipid-accumulated state shifts microglia away from homeostatic surveillance and renders APOE4 microglia weakly responsive to neuronal activity. By examining the transcriptional signatures of APOE3 versus APOE4 microglia before and after exposure to neuronal conditioned media, we further established that neuronal soluble cues differentially induce a lipogenic program in APOE4 microglia that exacerbates pro-inflammatory signals. Pharmacological blockade of lipogenesis in APOE4 microglia is sufficient to diminish intracellular lipid accumulation and restore microglial homeostasis. Remarkably, unlike APOE3 microglia that support neuronal network activity, co-culture of APOE4 microglia with neurons disrupts the coordinated activity of neuronal ensembles. We identified that through decreased uptake of extracellular fatty acids and lipoproteins, APOE4 microglia disrupts the net flux of lipids which results in decreased neuronal activity via the potentiation of the lipid-gated K+ channel, GIRK3. These findings suggest that neurological diseases that exhibit abnormal neuronal network-level disturbances may in part be triggered by impairment in lipid homeostasis in non-neuronal cells, underscoring a novel therapeutic route to restore circuit function in the diseased brain. After 4 weeks in culture, APOE3 or APOE4 iMGLs pre-conditioned in unspent neuronal media for 24hrs were further incubated with spheroid conditioned neuronal media for 2 hours. Cells were lysed and harvested for RNA extraction, library preparation and bulk sequencing. Biological triplicates were analyzed for 4 groups: APOE3 vs APOE4 iMGLs with or without exposure to APOE3 spheroid conditioned media (+CM)

载脂蛋白E4（Apolipoprotein E4, APOE4）是目前已知的晚发型阿尔茨海默病最强遗传风险因子，其在小胶质细胞（microglia）中的表达与促炎状态密切相关。目前学界尚不清楚，表达APOE4的小胶质细胞与携带疾病中性等位基因APOE3的小胶质细胞，在与神经元的相互作用模式上存在何种差异。本研究借助经CRISPR编辑的诱导多能干细胞（induced pluripotent stem cells, iPSCs），解析APOE4对神经元-小胶质细胞通讯的调控效应。研究结果显示，APOE4可在小胶质细胞中诱导出独特的代谢程序，其核心特征为脂质分解代谢受损，进而导致胞内中性脂质贮积。值得注意的是，这种脂质蓄积异常的状态会使小胶质细胞脱离稳态监视功能，并使APOE4阳性小胶质细胞对神经元活动的应答能力显著减弱。通过比对APOE3与APOE4阳性小胶质细胞在暴露于神经元条件培养基前后的转录组特征，本研究进一步证实，神经元分泌的可溶性信号可差异性诱导APOE4阳性小胶质细胞激活脂生成程序，进而加剧促炎信号通路的活化。对APOE4阳性小胶质细胞的脂生成过程进行药理学阻断，足以减少胞内脂质蓄积并恢复小胶质细胞的稳态功能。值得注意的是，与能够支持神经元网络活动的APOE3阳性小胶质细胞不同，APOE4阳性小胶质细胞与神经元共培养时，会破坏神经元集群的协同活动。本研究发现，APOE4阳性小胶质细胞通过减少细胞外脂肪酸与脂蛋白的摄取，打破脂质净流动平衡，进而通过激活脂质门控钾离子通道GIRK3，导致神经元活动降低。上述研究结果表明，表现出神经元网络水平异常扰动的神经系统疾病，可能部分由非神经元细胞的脂质稳态受损所触发，这为恢复患病大脑的神经环路功能提供了全新的治疗途径。在体外培养4周后，将在未使用过的神经元培养基中预培养24小时的APOE3或APOE4阳性诱导小胶质样细胞（induced microglia-like cells, iMGLs），与神经球条件神经元培养基共孵育2小时。随后收集细胞并裂解，用于RNA提取、文库构建及批量测序。本研究对四组样本开展了三次生物学重复分析：分别为暴露或未暴露于APOE3神经球条件培养基（+CM）的APOE3与APOE4阳性诱导小胶质样细胞。