Abstract
Purpose :
In dAMD, dysfunctional retinal pigmented epithelial (RPE) cell trafficking of lipids and photoreceptor outer segments is followed by RPE degeneration. The sigma-2 receptor (S2R, TMEM97) has been linked to dAMD in genome-wide association studies, and small molecule modulators of S2R rescue RPE functional deficits. S2R interacts with proteins involved in lipid trafficking, such as low-density lipoprotein receptor (LDLR) and Niemann-Pick Protein 1 (NPC1). Given that disruption in lipid trafficking is a key factor in dAMD, we hypothesize that S2R plays a functional role in this process and we interrogate this relationship in a model of human, mature RPE cells.
Methods :
Undifferentiated or differentiated, using nicotinamide, ARPE-19 cells were harvested for RNA and protein analysis after 7 or 14 days in vitro (DIV). Levels of RPE-specific markers, TMEM97, PGRMC1, and LDLR were assessed via RT-qPCR. To disrupt lipid trafficking, DIV14 cultures were treated with NPC1 inhibitor U18666A (U18; 1-30μM for 16 hr). Expression of TMEM97 was assessed by RT-qPCR and western blot. LDLR-mediated lipid trafficking was assessed by LDL-Dylight550 uptake assay. Ability of S2R modulators including CT2074 and a tool compound, Z4857158944, to regulate LDL uptake was also assessed (1, 3, 10μM for 16 hr).
Results :
Differentiated cells showed moderate mRNA expression of mature RPE markers at DIV7, with BEST1, RPE65, and MERTK increasing to 1500-, 900-, and 6.5-fold, respectively, compared to control cells by DIV14 (p<0.0001). At DIV7, mRNA levels of S2R, PGRMC1, and LDLR were higher than control, with 1.8-, 1.3-, and 3.5-fold increases in expression observed at DIV14 (p<0.0001). At DIV 14, disruption of lipid trafficking via U18 increased LDL-Dylight fluorescence, which corresponded with increased TMEM97 and LDLR mRNA expression. Under basal conditions, treatment with S2R modulators CT2074 and Z4857158944 led to concentration-dependent increases in LDL uptake. Taken together, these results support a model in which S2R plays a role in lipid trafficking in mature RPE.
Conclusions :
Differentiated ARPE-19s may be a useful model for exploring relationships between LDL trafficking, S2R-LDLR interactions, and RPE cell function. Elucidating these mechanisms may be beneficial for devising new therapeutic approaches and investigating S2R modulation as a means of restoring RPE function in dAMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.