Abstract
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 Ca2+/calmodulin dependent kinase 2a (CAMK2A). Further, luciferase reporter assay confirmed that miR-142-3p targets the 3′UTR of Camk2a. 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 via 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 Camk2a 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 via 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.
Highlights
Microglia, the resident macrophages of the central nervous system (CNS), serve as the first line of defense in the brain and contributes to the maintenance of brain homeostasis (Li and Barres, 2018)
A consistent downregulation of calmodulin dependent kinase 2a (CAMK2A), phosphorylation of CREB (pCREB), and brain-derived neurotrophic factor (BDNF) at the protein level was observed in microglia after knockdown of CAMK2A compared to a negative control. This observation was further confirmed by immunocytochemistry showing a decrease in the expression level of CAMK2A and BDNF in shRNA-mediated CAMK2A knockdown cells compared to the empty vector transduced cells (Figures 6D,E). These results demonstrate that miR-142-3p-mediated downregulation of CAMK2A in microglia leads to the repression of BDNF, which is involved in synaptic plasticity
Given that microglial activation is a hallmark of neuroinflammation, there is an urgent need for understanding the molecular and epigenetic mechanisms that underpin microglial functions in the healthy and pathological brain
Summary
The resident macrophages of the central nervous system (CNS), serve as the first line of defense in the brain and contributes to the maintenance of brain homeostasis (Li and Barres, 2018). The activated microglia appear morphologically hypertrophic, migrate to the site of injury and release several proinflammatory cytokines, chemokines, cytotoxic molecules such as nitric oxide and reactive oxygen species (Chao et al, 1992; Banati et al, 1993; Fetler and Amigorena, 2005; Thameem Dheen et al, 2007) In addition to their immune functions, microglial cells play an important role in sculpting the neuronal circuits during brain development (Wu et al, 2015). Because of the pivotal role of microglial neurotrophins (such as BDNF), an insight into the miRNA-mediated regulation of neurotrophic factors may provide promising biomarkers for various brain diseases. We focused on understanding the role of microRNA-mediated regulation of CAMK2A-CREB-BDNF signaling pathway in activated microglia
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