Role of MiR-204 in controlling metabolic functions of the subretinal microglia

<b>Rationale:</b> MicroRNA-204 (miR-204) is one of the most abundant miRNA species in the retinal pigment epithelium (RPE) and RPE-derived extracellular vesicles (EVs). Knockout (KO) of miR-204 leads to dysfunction and degeneration of both the RPE and the retina. In addition to previously reported retinal pathologies, we observed the accumulation of lipid-laden subretinal microglia in miR-204 KO mice. This study aimed to identify potential molecular targets of miR-204 involved in microglia lipid processing and to determine whether RPE-derived EVs can improve the function of miR-204-deficient retinal microglia. <b>Methods:</b> Lipid accumulation in microglia was detected by staining with LipidTox, a fluorescent dye specific for neutral lipids, followed by either flow cytometry analysis or direct visualization on RPE/choroid flat mounts. MiRNA database and target prediction tools, such as miRWalk and TargetScan, were used to search for potential target genes of miR-204 in microglia. The identified target mRNA was validated with a miRNA reporter assay. RPE EVs were prepared from <i>ex vivo</i> cultured mice eye cups and administered via retro-orbital injection in miR-204 knockout (KO) mice. RPE integrity was assessed by ERG c-wave measurement. <b>Results:</b> KO of miR-204 resulted in the accumulation of neutral lipids in subretinal microglia. MiR-204 targeted the TGF-β receptor 2 gene in microglia. TGF-β markedly suppressed the expression of genes related to microglia lipid clearance. Eyes injected with RPE-derived EVs showed improved ERG c-wave responses compared to the fellow eyes injected with saline. <b>Conclusions:</b> This study supports that TGF-β/TGF-β receptor 2 regulates microglia lipid metabolism primarily by suppressing lipid clearance. By modulating TGF-β signaling, miR-204 in RPE-derived EVs likely enhances the lipid metabolic activities of subretinal microglia, which are crucial for the structural integrity and proper function of the outer retina and RPE. RPE-derived EVs and their delivery of miRNAs represent a potential therapeutic approach for treating retinal diseases, such as age-related macular degeneration, which involve dysregulated lipid metabolism in subretinal microglia.