Abstract
Purpose :
Retinal metabolic homeostasis and RPE function relies on numerous autophagy-associated processes. Among these, LC3b associated phagocytosis (LAP) is critical to maintain retinal function; mice lacking LC3b (LC3b-/-) show loss of retinal lipid metabolic homeostasis and increased inflammation. We sought a non-biased approach to determine if loss of LAP mediated processes modulates the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation.
Methods :
Total RNA was extracted from RPE cells from C57BL6/J (WT) and the LC3b-/- mouse eyes. Libraries prepared using Illumina truSeq stranded mRNA kit were assessed for quality and sequenced on a NovaSeq 6000 system (NGSC, University of Pennsylvania). Raw data was imported into Salmon to count hits against the transcriptome defined in Gencode vM26 and further analyzed using R and DESeq2 (Penn Genomics Analysis Core). Genes showing > 1.5-fold change, and an adjusted p-value < 0.05 were considered significantly different. The up- and down-regulated differentially expressed genes (DEGs) were subjected to pathway enrichment analysis using Metascape. Additionally, an unfiltered list of genes ranked by their DESeq2 statistic were analyzed with GSEA against gene collections in the Molecular Signatures Database (MSigDB).
Results :
The comparison between the RPE transcriptome of WT and LC3b-/- revealed 1533 DEGs, with ~73% up-regulated and 27% down-regulated. Enriched GO terms included inflammatory response (up-regulated DEGs), fatty acid metabolic process and vascular transport (down-regulated DEGs). GSEA identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families/ groups identified significant difference for genes in the solute carrier family, RPE signature genes, and genes with potential role in AMD.
Conclusions :
Loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy and inflammation. Expression of genes for oxidative metabolic pathways decreases, expression of genes associated with cholesterol homeostasis increases. Given the AMD-like phenotype of the LC3b-/- mouse, these results suggest that lipid handling plays a major role in the observed pathophysiology.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.