Abstract
Purpose :
With neurodegeneration, the glial scar that forms can be detrimental. microRNAs (miRNAs) are negative regulators of gene expression and known to change in injured or diseased tissue. At present, there is not much known about the role of miRNAs in the response of Müller glia (MG) to neuronal loss, and so we characterized miRNAs following light damage.
Methods :
We crossed the MG specific reporter mouse (Rlbp-CreER: Stopfl/fl tdTomato) onto albino mice with a RPE-65 mutation and light-damaged the mice for 8h. For the conditional knock out (CKO) of Dicer1 we crossed the tdTomato reporter mouse to a Dicerfl/fl mouse (Rlbp-CreER: Dicerfl/fl: Stopfl/fl tdTomato)). To activate the CreER in the mice, we injected tamoxifen intraperitoneally. Retinal cross sections at 1, 6, 12, and 18 weeks after light damage and 1, 3, 6, and 12 months after Dicer deletion were analyzed. For miRNA analysis, MG from wild type, light damaged, and Dicer-CKOMG mice were purified by FACS and analyzed using the NanoStrings nCounter® System. Microarrays to identify the genes changing after light damage and compared that to RNA-Seq data performed after Dicer-CKO.
Results :
One week after light damage, we found a reduction of photoreceptors and a clear thinning in the ONL accompanied by MG activation (GFAP upregulation). This phenotype was similar to the phenotype we observed 6 months after Dicer-CKOMG in the retinal periphery, although migrating MG in the Dicer-CKO did not express GFAP.
By analyzing the microRNA profile, already one week after light damage, there was a rapid decline of the miRNAs highly expressed in the MG by an average of 60%. One month after Dicer deletion, we found a reduction in levels of the same miRNA by an average of 70%. Interestingly, after light damage, there was also an increase in a set of miRNAs not present in the Dicer-CKO. By comparing genes affected in both, light-damaged and Dicer-deleted MG, we found that similar genes were upregulated such as the transcription factors Maff and Egr2. However, a strongly upregulated gene in the Dicer-CKOMG did not increase after the injury.
Conclusions :
Our results show neuronal death due to light damage leads to a rapid decline of the MG specific miRNAs and to upregulation of disease-associated genes, similar to what occurs in the DIcer-CKO. Therefore, MG miRNAs might be important for the maintenance of the physiological function might also be relevant for late-staged degenerative diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.