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Thomas Langmann, Christoph Moehle, Marcus Karlstetter, Bernhard Weber; Loss of MicroRNA-124 and MicroRNA-126 expression regulates inflammatory microglial activation in inherited retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4517.
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© ARVO (1962-2015); The Authors (2016-present)
Activation of microglia is a common hallmark of inherited and induced retinal degenerations. It is currently unknown whether MicroRNAs (miRNAs) play a role in gene regulation associated with this immune process. The purpose of this study was to identify key miRNAs and their target genes using a complementary genome-wide analysis of miRNAs and mRNAs in purified retinal microglia from retinoschisin-deficient mice, a prototypic model for inherited retinal degeneration.
Transgenic MacGreen reporter mice were crossed with retinoschisin-deficient animals to label alerted, amoeboid retinal microglia with GFP. Microglial cells from MacGreen mice served as ramified control cells. Small RNA fractions containing miRNAs as well as total RNA were isolated from both GFP-positive retinal microglia populations at postnatal day 21, an early phase of immune activation. Affymetrix miRNA 2.0 and Gene 1.0 microarrays were performed and selected transcripts were confirmed by qRT-PCR. Candidate miRNAs and their targets were deduced from microarry expression profiles and bioinformatic analyses using a compendium of 11 established miRNA target prediction programs. 3’ UTR regions containing miRNA seed matches were verified using the luciferase reporter system.
Alerted microglial cells purified from MacGreen/retinoschisin-deficient retinas showed a strongly reduced expression of both precursor and mature forms of miRNA-124 and miRNA-126. The loss of miRNA-124 and miRNA-126 expression correlated well with predicted effects on over-expressed inflammatory mRNA targets. miRNA-124 targets include PKR, the key mediator of the NLRP3 inflammasome, the NfkB-regulator IkBzeta, and Irf1. miRNA-126 seed sequences targets were identified in Vcam1 and Adam33, which control cell-cell and cell-matrix interactions of alerted microglia. A microglial cell culture system with low endogenous miRNA-124 and miRNA-126 expression was successfully used to verify 3’ UTR target sequences by transfection with miRNA mimetics.
Our results demonstrate a loss of miRNA-124 and miRNA-126 expression in alerted retinal microglia of retinoschisin-deficient mice. This correlated with an induction of direct target genes related to inflammation and cell adhesion. Manipulation of both microRNAs could be potentially used as a therapeutic tool to dampen retinal microglia activation in the future.
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