Abstract
Purpose :
Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome in which impaired night vision is caused by an immune response against retinal ON-bipolar cells (ON-BCs). Pathogenic antibodies bind the cytoplasmic domain of TRPM1, a cation channel that mediates the light response of ON-BCs. This study explores the endocytic mechanisms used by ON-BCs to internalize the TRPM1 autoantibodies that cause MAR.
Methods :
Live mouse retinas (slices or split wholemount) were incubated with antibodies against TRPM1 or PKCα for 2 hours to allow for internalization with or without chemical inhibitors of endocytosis (pitstop2, dynasore). TRPM1 Fab fragments were used to assess dependency on membrane Fc receptors. Following internalization, retinas were fixed and internalized antibodies were visualized with anti-mouse IgG conjugated to AF488. Signal intensity from internalized antibodies was quantified with FIJI using images from a Leica TCS SP8 X confocal microscope. A viability dye (Zombie NIR; Biolegend) was used to exclude dead cells from analysis.
Results :
By comparing levels of antibody internalization in retinas treated with antibody Fabs and endocytosis inhibitors, this study evaluates the mechanism(s) responsible for antibody entry into the ON-BCs, and thus, MAR pathogenesis. Published single-cell RNA seq data and our immunolabeling of mouse retinas reveal no expression of membrane Fc receptors FcγII and FcγIII, which have been implicated in antibody uptake in related paraneoplastic syndromes. Evidence of antibody internalization in ON-BCs is visible within 15 minutes of incubation and is not limited to antibodies against TRPM1. Other retinal neurons, such as horizontal cells, do not appear capable of internalizing antibodies. The TRPM1 antibodies used in this study were custom made against epitopes targeted in MAR. We developed the split retina preparation as a model for studying antibody internalization in ON-BCs.
Conclusions :
Antibody internalization by ON-BCs in MAR is not dependent on FcγRII/III. Amongst retinal neurons, the ability to internalize antibodies may be unique to ON-BCs. Internalization can occur within 15 minutes and is not limited to antibodies that target TRPM1.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.