Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV–neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.

最近的证据表明，突触功能障碍是受神经炎性疾病（如多发性硬化症）影响的早期机制，其特征是慢性小胶质细胞活化。然而，尚未完全了解反应性小胶质细胞在引起突触缺陷中的作用方式。在这项研究中，我们表明炎性小胶质细胞产生细胞外囊泡（EVs），这些囊泡富含一组调控关键突触蛋白表达的miRNA。其中，miR-146a-5p是海马神经元中不存在的小胶质细胞特异性miRNA，它控制突触前突触前素1（Syt1）和突触后神经素1（Nlg1）的表达，黏附蛋白在树突棘的形成和突触中起关键作用稳定。使用雷尼利亚传感器，我们提供了正式的证据，证明炎症性电动车会将其miR-146a-5p货物转移至神经元。通过蛋白质印迹和免疫荧光分析，我们表明水泡miR-146a-5p抑制了接收神经元中Syt1和Nlg1的表达。当掩盖囊泡的磷脂酰丝氨酸残基抑制EV-神经元接触，并且神经元暴露于由miR-146a-5p耗尽的EV时，小胶质细胞至神经元miR-146a-5p的转移以及Syt1和Nlg1的下调均不会发生。 -再生小胶质细胞，或储存无活性的miR-146a-5p，由抗miR-146a-5p转染的细胞产生。形态学分析显示，长时间暴露于炎症性EV会导致体内和原代培养的海马神经元的树突棘密度显着降低，可通过转染miR不敏感的Nlg1形式在体外进行拯救。树突棘丧失伴随着兴奋性突触的密度和强度降低，如mEPSC频率和振幅降低所表明的。这些发现将炎症性小胶质细胞和增强的EV产生与兴奋性突触的丧失联系起来，揭示了先前未认识到的，与EV结合释放的富含小胶质的miRNA在关键突触基因沉默中的作用。