Sun Hao, Wang Heng, Wu Chaoran, Liu Gang, He Meijun, Zhang Hao, Hou Fengsheng, Liao Hong
New Drug Screening Center, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China (H.S., H.W., C.W., G.L., M.H., H.Z., F.H., H.L.).
Chongqing Innovation Institute of China Pharmaceutical University, Chongqing, China (H.S., H.L.).
Stroke. 2025 Feb;56(2):505-516. doi: 10.1161/STROKEAHA.124.049265. Epub 2025 Jan 8.
Activating glutamatergic neurons in the ipsilesional motor cortex can promote functional recovery after stroke. However, the underlying molecular mechanisms remain unclear. Clarifying key molecular mechanisms involved in recovery could help understand the development of neuromodulation strategies after stroke.
Adeno-associated virus 2/9-CamKIIa-hM3Dq-mCherry was injected into ipsilesional motor cortex by stereotaxic in the photothrombotic stroke model. Starting from the third day after the stroke, male mice were injected intraperitoneally with clozapine-N-oxide every day to activate excitatory neurons. C1q-blocking antibody and annexin V were used to inhibit C1q and exposed phosphatidylserine (EPS), respectively. The cylinder test and grid-walking test were performed to evaluate functional recovery. The potential molecular mechanisms of excitatory neuronal activation on microglia-mediated synaptic pruning after stroke by immunofluorescence, real-time polymerase chain reaction, Western blotting, and RNA sequencing.
Activating excitatory neurons significantly promoted functional recovery and inhibited microglia-mediated synaptic pruning after stroke. Furthermore, it decreased EPS and C1q levels in synapses. On the contrary, inhibiting excitatory neurons aggravated functional defects, promoted microglia-mediated synaptic pruning, and increased EPS and C1q levels in synapses. Selective blocking of EPS repressed C1q tagging of synapses and microglia-mediated synaptic pruning and improved functional recovery. Meanwhile, blocking EPS markedly rescued synaptic density, and motor function deteriorated by chemogenetic inhibition. In addition, C1q-blocking antibody prevented phosphatidylserine engulfment by microglia.
Together, these data provide mechanistic insight into microglia-mediated synapse pruning after neuronal activation after stroke and identify the role of C1q binding to EPS in stroke treatment during the repair phase.
激活患侧运动皮层中的谷氨酸能神经元可促进中风后的功能恢复。然而，其潜在的分子机制仍不清楚。阐明恢复过程中涉及的关键分子机制有助于理解中风后神经调节策略的发展。
在光血栓性中风模型中，通过立体定位将腺相关病毒2/9-CamKIIa-hM3Dq-mCherry注射到患侧运动皮层。从中风后的第三天开始，每天给雄性小鼠腹腔注射氯氮平氮氧化物以激活兴奋性神经元。分别使用C1q阻断抗体和膜联蛋白V抑制C1q和暴露的磷脂酰丝氨酸（EPS）。进行圆筒试验和网格行走试验以评估功能恢复情况。通过免疫荧光、实时聚合酶链反应、蛋白质印迹和RNA测序研究中风后兴奋性神经元激活对小胶质细胞介导的突触修剪的潜在分子机制。
激活兴奋性神经元可显著促进中风后的功能恢复并抑制小胶质细胞介导的突触修剪。此外，它降低了突触中EPS和C1q的水平。相反，抑制兴奋性神经元会加重功能缺陷，促进小胶质细胞介导的突触修剪，并增加突触中EPS和C1q的水平。选择性阻断EPS可抑制突触的C1q标记和小胶质细胞介导的突触修剪，并改善功能恢复。同时，阻断EPS可显著挽救突触密度，并改善化学遗传抑制导致的运动功能恶化。此外，C1q阻断抗体可防止小胶质细胞吞噬磷脂酰丝氨酸。
总之，这些数据为中风后神经元激活后小胶质细胞介导的突触修剪提供了机制性见解，并确定了C1q与EPS结合在修复阶段中风治疗中的作用。
