Abstract
Purpose :
The ILM of the retina separates the retina and the vitreous body, serving as developmental substrate and barrier. Deletion of Lamb2-containing laminins causes severe defects in the ILM. Müller glia (MG) respond to these ILM changes. Here, we analyzed MG responses to ILM lacking Lamb2-containing laminins.
Methods :
Control Lamb2-loxP/loxP (CTL), conditional Lamb2-loxP/loxP, dkk3-cre (cKO) mice were used. MG were studied in vivo with IHC, OCT and single cell RNA sequencing. Microglia-depleted fractions were sorted, sequenced, aligned against the mm39 genome database, normalized and dimensionally reduced to UMAP. MG were identified by genetic markers and separated as the MG-enriched (MG+) and MG-depleted (MG-) fractions. Both fractions were analyzed with ANOVA to produce Differentially Expressed Genes (DEGs) and enriched KEGG/GO pathways.
Results :
In cKO retina, β2-laminins are absent from the ILM but present in vascular basement membranes. Glial and vasculature protrude into the vitreous. The overall retinal structure is basically intact at P20 but degenerates by 5 months (thickness CTL/cKO: 190+/-1.6 – 146.2+/-5.3. p<0.013; ONL thickness: 58.7+/-0.3 – 40.3+/-0.3, P<0.017). KEGG pathway analysis demonstrated that the Enrichment Scores (ES) and Rich Factors (RF) of MG(+) is higher than MG(-) cKO: endocytosis (ES=28, RF=0.4/ES=9.5, RF=0.2), regulation of cytoskeleton (ES=21, RF=0.4/ES=6.3, RF=0.2), focal adhesion (ES=18, RF=0.4/ES=9.3, RF=0.2) adherens junction (ES=10, RF=0.45/ES=7.5, RF=0.2) pathways (FDR<0.05). For MG(+), GO for endocytosis pathways was enriched in receptor- and clathrin- mediated endocytosis (ES 20 and 16 respectively, P<0.003) and for MG(-) GO was enriched for receptor-mediated endocytosis (ES 19, P<0). ECM (collagen, fibronectin, laminin chains) and receptor (integrins), focal adhesion (Talin, FAK), adhesion junction (cadherin and protocadherin), cytoskeletal (Actin family, β-catenin) and endocytosis-associated genes (clathrin) genes were upregulated in MG(+) and downregulated or not changed in MG(-).
Conclusions :
Disruption of ILM integrity produces a fibrosis consisting of MG, vasculature and matrix. MG morphologic changes become more apparent with age. Gene expression changes (cytoskeleton, endocytic processes, and junctional pathways) precede gross anatomical changes. These glial responses are likely relevant in the pathobiology of diabetic and proliferative retinopathies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.