Matrix-metalloproteinase expression and gelatinase activity in the avian retina and their influence on Müller glia proliferation.

Gelatinases are a class of matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) to regulate intercellular signaling and cell migration. Gelatinase activity is tightly regulated via proteolytic activation and through the expression of tissue inhibitors of matrix metalloproteinases (TIMPs). Gelatinase activity has been implicated in retinal pathophysiology in different animal models and human disease. However, the role of gelatinases in retinal regeneration remains uncertain. In this study we investigated the dynamic changes in gelatinase activity in response to excitotoxic damage and how this enzymatic activity influenced the formation of Müller glia progenitor cells (MGPCs) in the avian retina. This study used hydrogels containing a gelatinase-degradable fluorescent peptide to measure gelatinase activity in vitro and dye quenched gelatin to localize enzymatic activity in situ. These data were corroborated by using single cell RNA sequencing (scRNA-seq). Gelatinase mRNA, specifically MMP2, was detected in oligodendrocytes and Non-Astrocytic Inner Retinal Glia (NIRG). Total retinal gelatinase activity was reduced following NMDA-treatment, and sustained inhibition of MMP2 prior to damage or growth factor treatment increased the formation of proliferating MGPCs and c-fos signaling. We observed that microglia, Müller glia (MG), and NIRG cells were involved in regulating changes in gelatinase activity through TIMP2 and TIMP3. Collectively, these findings implicate MMP2 in reprogramming of Muller glia into MGPCs.

BMP- and TGFß-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina.

Müller glia-derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell-signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8- and TGFß/Smad2/3-signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up-regulated in the nuclei of Müller glia following treatment with BMP4, FGF2, or NMDA-induced damage, and this up-regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA- or FGF2-treatment. These findings suggest a decrease in TGFß- and increase in BMP-signaling when MGPCs are known to form. In both NMDA-damaged and FGF2-treated retinas, inhibition of BMP-signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFß-signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFß2 suppressed the formation of MGPCs in NMDA-damaged retinas. Our findings indicate that BMP/TGFß/Smad-signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFß/Smad2/3 suppresses the formation of MGPCs.