The Endocrine Program, Department of Animal Sciences, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901-1573.
Endocrinology and Animal Biosciences Graduate Program, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901-1573.
J Neurosci. 2020 Oct 7;40(41):7965-7979. doi: 10.1523/JNEUROSCI.0284-20.2020. Epub 2020 Sep 4.
Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the β-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the β-endorphin neuronal killing activity of microglial exosomes both and Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in β-endorphin neuronal cells in both and systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of β-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced β-endorphin neuronal death. Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.
小胶质细胞是中枢神经系统免疫细胞的一种，已经被证明在胎儿酒精谱系障碍的新生大鼠模型中，有助于乙醇激活下丘脑应激调节 proopiomelanocortin (POMC) 神经元产生 β-内啡肽肽的神经元死亡。我们确定了小胶质细胞细胞外囊泡外泌体是否参与了乙醇诱导的 β-内啡肽神经元的神经元死亡。从小鼠（雄性和雌性）的下丘脑组织中提取细胞外囊泡，这些小鼠每天接受 2.5mg/kg 乙醇或对照牛奶配方喂养 5 天，或从小鼠的下丘脑小胶质细胞中提取，在培养物中培养数天，然后用乙醇或载体处理 24 小时。纳米颗粒跟踪分析和透射电子显微镜表明，这些囊泡具有外泌体的大小范围和形状。乙醇处理增加了小胶质细胞外泌体的数量和 β-内啡肽神经元杀伤活性，并且 蛋白质组学分析培养的小胶质细胞的外泌体鉴定出大量蛋白质，包括各种补体，这些蛋白质在乙醇处理后升高。使用定量蛋白质测定法重新确认了涉及补体的蛋白质组学数据。乙醇处理还增加了 β-内啡肽神经元细胞中补体蛋白 C1q 的沉积，这两种情况都是在 和 系统中。重组 C1q 蛋白增加，而 C1q 阻断剂减少乙醇诱导的 C3a/b、C4 和膜攻击复合物/C5b9 形成；ROS 产生；最终β-内啡肽神经元的细胞死亡。这些数据表明，涉及 C1q-C3-C4-膜攻击复合物和 ROS 的补体系统调节外泌体介导的、乙醇诱导的 β-内啡肽神经元死亡。发育期酒精的神经毒性作用因其参与胎儿酒精谱系障碍而被认识，但由于大脑中小胶质细胞免疫细胞的激活，对酒精作用机制的缺乏清晰理解阻碍了这种疾病的治疗干预进展。已知调节应激、能量平衡和免疫功能的 proopiomelanocortin 神经元因发育性酒精暴露而被杀死，因为大脑中的小胶质细胞免疫细胞被激活。虽然小胶质细胞被认为使用细胞外囊泡与神经元进行通讯以维持内稳态，但我们在这里表明，发育期的乙醇暴露劫持了这个系统，传播凋亡因子，包括补体蛋白 C1q，以诱导膜攻击复合物和活性超氧自由基来杀死 proopiomelanocortin 神经元。
