A Comparative Analysis of Reactive Müller Glia Gene Expression After Light Damage and microRNA-Depleted Müller Glia—Focus on microRNAs

Abstract

Müller glia (MG) are the predominant glia in the neural retina and become reactive after injury or in disease. microRNAs (miRNAs) are translational repressors that regulate a variety of processes during development and are required for MG function. However, no data is available about the MG miRNAs in reactive gliosis. Therefore, in this study, we aimed to profile miRNAs and mRNAs in reactive MG 7 days after light damage. Light damage was performed for 8 h at 10,000 lux; this leads to rapid neuronal loss and strong MG reactivity. miRNAs were profiled using the Nanostring platform, gene expression analysis was conducted via microarray. We compared the light damage dataset with the dataset of Dicer deleted MG in order to find similarities and differences. We found: (1) The vast majority of MG miRNAs declined in reactive MG 7 days after light damage. (2) Only four miRNAs increased after light damage, which included miR-124. (3) The top 10 genes found upregulated in reactive MG after light damage include Gfap, Serpina3n, Ednrb and Cxcl10. (4) The miRNA decrease in reactive MG 7 days after injury resembles the profile of Dicer-depleted MG after one month. (5) The comparison of both mRNA expression datasets (light damage and Dicer-cKO) showed 1,502 genes were expressed under both conditions, with Maff , Egr2, Gadd45b, and Atf3 as top upregulated candidates. (6) The DIANA-TarBase v.8 miRNA:RNA interaction tool showed that three miRNAs were found to be present in all networks, i.e., after light damage, and in the combined data set; these were miR-125b-5p, let-7b and let-7c. Taken together, results show there is an overlap of gene regulatory events that occur in reactive MG after light damage (direct damage of neurons) and miRNA-depleted MG (Dicer-cKO), two very different paradigms. This suggests that MG miRNAs play an important role in a ubiquitous MG stress response and manipulating these miRNAs could be a first step to attenuate gliosis.

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury

“…We confirmed that vascular permeability in IR retinas was also significantly decreased by 3 days of treatment, to a similar extent of that obtained after 7 days of treatment (data not shown). The mRNA examined included Tnfa, Il1b, Ptgs2, and Nos2, which are all considered markers of M1 microglial activation [ 59 , 60 ]; Cd68, which is considered a marker of microglia phagocyte phenotype [ 54 , 56 ]; Cyba and Cybb, corresponding to the NADPH oxidase complex members P22-pox and Gp91-phox, respectively; Lcn2 and Serpina3n, which are markers of reactive astrogliosis [ 61 ] and are greatly increased in the rat retinal IR injury model [ 34 ]; Arg1 (Arginase-1), Cd200r1 (CD200 receptor), Tgm2 (transglutaminase-2), and Mrc1 (CD206), which are all considered M2 markers [ 62 – 64 ]. Tnfa, Il1b, and Ptgs2 (Cox2) mRNAs were all significantly upregulated in IR injured retinas relative to Sham retinas, and all these inducements were significantly diminished by minocycline treatment.…”

“…However, this may not have been due to a direct effect of minocycline on microglia. Lcn2 and Serpina3n were among the most highly upregulated mRNAs in reactive astrocytes following experimental stroke in mice [ 90 ], were both upregulated in Müller cells undergoing astrogliosis [ 62 , 91 , 92 ], and were recently identified as being highly upregulated in endothelial cells in a mouse experimental autoimmune uveitis model [ 93 ].…”

Regulations of Retinal Inflammation: Focusing on Müller Glia

“…But they are not the most sensitive ones in regarding inflammation. MiR-124, normally localizing to both INL and OLM of the retina, redistributes from neurons to MG with increased expression under photo-oxidative damage, targeting Ccl2 for retinal inflammation regulation ( Chu-Tan et al, 2018 ; Kang et al, 2020 ), suggesting its role behind the retinal inflammatory responses. The inflammation-related genes Fgf2, Mt2 and Ccl2 are regulated by MG miRNAs and predominantly by members of the let-7 family.…”

Disruption ofmiR-18aalters proliferation, photoreceptor replacement kinetics, inflammatory signaling and microglia/macrophage numbers during retinal regeneration in zebrafish

“…In addition to regulating inflammation, miRNA profiling studies combined with broad blocking of miRNA function (e.g. Dicer knockout) are revealing the potential importance of many miRNAs in regulating neuronal regeneration in the zebrafish retina [11] and in reactive gliosis and understanding the mechanisms that prevent Müller glia reprogramming and neuronal regeneration in the mammalian retina [78]. Studies like these are bringing miRNA research to the forefront and showing the complexities of understanding pathways that govern neuronal regeneration.…”