microRNAs are required for maintenance of Müller glia homeostasis and retinal architecture: The role of Brevican.

Abstract

Purpose : microRNAs (miRNAs) are negative regulators of gene expression and known to play a role in retinal development and regeneration. However, there is not much known about the role of miRNAs in Müller glia function and whether or not they are required for glial state per se.

Methods : We created a conditional knock out (CKO) of Dicer1 (Dicer-CKO_MG) specific for MG (Rlbp-CreER: Dicer^fl/fl : Stop^fl/fl tdTomato; wild type: Rlbp-CreER: Stop^fl/fl tdTomato). To activate the CreER in the Dicer-CKO_MG mice, we injected tamoxifen intraperitoneally at postnatal days (P) 11-14. Retinal cross sections of eyes were used to analyze the Dicer-CKO_MG phenotype one or six months after Dicer deletion by means of immunofluorescent labeling and confocal microscopy. For RNA analysis, Müller glia from wild type and Dicer-CKO_MG mice were purified by FACS and analyzed using the NanoStrings nCounter® System and RNA-Seq. In addition, we used in vitro (Müller glia cell culture) and ex vivo (retinal explants) cultures to confirm observations made in vivo and to identity a possible underlying mechanism.

Results : In the Dicer-CKO_MG, we found a reduction in miRNA levels in Müller glia by an average of 70% and a temporary increase in the number of Müller glia due to increased proliferation. However, after six months, we observed a significant decline in Müller glia number. Moreover, we saw Müller glial migration from their normal position in the inner nuclear layer, ultimately disrupting normal retinal architecture within six months. RNA-Seq analysis and in vitro experiments suggest that an increase in the expression of Brevican in the Dicer-CKO_MG partly explains the observed changes in their migratory behavior and that miR-9 is a potential regulator of Brevican. Brevican is a chondroitin sulfate proteoglycan, usually absent in MG, and over-expression of Brevican causes Müller glia to migrate and form aggregations in vitro and ex vivo. These aggregations can be prevented/resolved in the glia from Dicer-CKO_MG mice by miR-9 mimics or Brevican inhibitory antibodies.

Conclusions : Our results show that miRNAs and downstream targets are important for the maintenance of the stable Müller glia state and these results could have implications for the retinal remodeling that occurs in long-term degenerative diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.