Abstract
Purpose :
Melanoma-associated retinopathy (MAR) results from an autoimmune response against the retina in which autoantibodies penetrate ON-bipolar cells (ON-BCs) and bind to the cation channel, TRPM1, blocking the ON-BC light response. Patients experience night blindness, photopsia, and reduced contrast sensitivity. How these autoantibodies enter the cell remains unknown and represents an avenue for therapeutic intervention. Using an ex vivo mouse retina preparation, we investigated the contribution of various endocytic pathways to antibody internalization by ON-BCs.
Methods :
Two retina preparations were used: a wholemount with photoreceptors removed (split retina) and dissociated neuronal cultures. Retina preps were incubated for 1 hour with antibodies against TRPM1 (found in all ON-BCs) or PKCα (a marker of rod-BCs, the most abundant type of ON-BC in mouse) to allow for internalization with or without a chemical inhibitor of dynamin-dependent endocytosis (Dyngo-4a). Incubation with PKCα Fab fragments was performed to determine whether internalization depends on binding of IgG by Fc receptors. After incubation, retinas were fixed and internalized antibodies were visualized with AF488-conjugated secondary antibodies. Signal intensity from internalized antibodies was quantified with FIJI using images from a Leica TCS SP8 X confocal microscope. A viability dye (Zombie Violet; Biolegend) was used to exclude dead cells from analysis.
Results :
After 1 hour, internalized antibodies in the split wholemount retina were localized to ON-BC dendrites and somas, with the strongest signal present in the dendrites. Preliminary experiments reveal that antibody internalization is Dyngo-4a insensitive, as treatment with 30µM Dyngo-4a did not significantly reduce levels of IgG in ON-BCs. In contrast, 30µM Dyngo-4a significantly inhibits clathrin-mediated endocytosis of transferrin in HEK293T cells. Incubation with PKCα Fab fragments yielded comparable levels of internalized antibody as PKCα IgG, indicating antibody internalization is not mediated by Fc receptors.
Conclusions :
Retinal ON-BCs are capable of internalizing antibodies in their dendrites and cell bodies. Preliminary evidence suggests this mechanism is dynamin-independent and does not depend on antibody interaction with Fc receptors. Therefore, antibody uptake in MAR may be mediated by macropinocytosis or caveolae-dependent endocytosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.