Gupta Neelima, Jadhav Shweta, Tan Kai-Leng, Saw Genevieve, Mallilankaraman Karthik Babu, Dheen S Thameem
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Front Cell Neurosci. 2020 May 21;14:132. doi: 10.3389/fncel.2020.00132. eCollection 2020.
Microglia, the innate immune effector cells of the mammalian central nervous system (CNS), are involved in the development, homeostasis, and pathology of CNS. Microglia become activated in response to various insults and injuries and protect the CNS by phagocytosing the invading pathogens, dead neurons, and other cellular debris. Recent studies have demonstrated that the epigenetic mechanisms ensure the coordinated regulation of genes involved in microglial activation. In this study, we performed a microRNA (miRNA) microarray in activated primary microglia derived from rat pup's brain and identified differentially expressed miRNAs targeting key genes involved in cell survival, apoptosis, and inflammatory responses. Interestingly, miR-142-3p, one of the highly up-regulated miRNAs in microglia upon lipopolysaccharide (LPS)-mediated activation, compared to untreated primary microglia cells was predicted to target Ca/calmodulin dependent kinase 2a (CAMK2A). Further, luciferase reporter assay confirmed that miR-142-3p targets the 3'UTR of . CAMK2A has been implicated in regulating the expression of brain-derived neurotrophic factor (BDNF) and long-term potentiation (LTP), a cellular mechanism underlying memory and learning. Given this, this study further focused on understanding the miR-142-3p mediated regulation of the CAMK2A-BDNF pathway Cyclic AMP-responsive element-binding protein (CREB) in activated microglia. The results revealed that CAMK2A was downregulated in activated microglia, suggesting an inverse relationship between miR-142-3p and in activated microglia. Overexpression of miR-142-3p in microglia was found to decrease the expression of CAMK2A and subsequently BDNF through regulation of CREB phosphorylation. Functional analysis through shRNA-mediated stable knockdown of CAMK2A in microglia confirmed that the regulation of BDNF by miR-142-3p is CAMK2A. Overall, this study provides a database of differentially expressed miRNAs in activated primary microglia and reveals that microglial miR-142-3p regulates the CAMK2A-CREB-BDNF pathway which is involved in synaptic plasticity.
小胶质细胞是哺乳动物中枢神经系统（CNS）的固有免疫效应细胞，参与中枢神经系统的发育、稳态和病理过程。小胶质细胞会对各种损伤和伤害做出反应而被激活，并通过吞噬入侵的病原体、死亡的神经元和其他细胞碎片来保护中枢神经系统。最近的研究表明，表观遗传机制确保了参与小胶质细胞激活的基因的协调调控。在本研究中，我们对源自大鼠幼崽大脑的活化原代小胶质细胞进行了微小RNA（miRNA）微阵列分析，并鉴定了靶向参与细胞存活、凋亡和炎症反应的关键基因的差异表达miRNA。有趣的是，与未处理的原代小胶质细胞相比，脂多糖（LPS）介导的激活后小胶质细胞中高度上调的miRNA之一miR-142-3p被预测靶向钙/钙调蛋白依赖性激酶2a（CAMK2A）。此外，荧光素酶报告基因检测证实miR-142-3p靶向CAMK2A的3'非翻译区。CAMK2A已被证明参与调节脑源性神经营养因子（BDNF）的表达和长时程增强（LTP），这是一种记忆和学习的细胞机制。鉴于此，本研究进一步聚焦于了解miR-142-3p介导的激活小胶质细胞中CAMK2A-BDNF途径的环磷酸腺苷反应元件结合蛋白（CREB）的调控。结果显示，激活的小胶质细胞中CAMK2A表达下调，表明激活的小胶质细胞中miR-142-3p与CAMK2A之间存在负相关关系。在小胶质细胞中过表达miR-142-3p被发现通过调节CREB磷酸化来降低CAMK2A的表达，进而降低BDNF的表达。通过小干扰RNA（shRNA）介导的小胶质细胞中CAMK2A的稳定敲低进行的功能分析证实，miR-142-3p对BDNF的调控是通过CAMK2A实现的。总体而言，本研究提供了活化原代小胶质细胞中差异表达miRNA的数据库，并揭示小胶质细胞miR-142-3p调节参与突触可塑性的CAMK2A-CREB-BDNF途径。
