Abstract
Purpose: :
Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of anti-bipolar cell autoantibodies. The electroretinogram (ERG) associated with MAR has a reduced b-wave, indicating a block in visual processing at the bipolar neurons in the retina. The goal of this study was to identify the antigen that is recognized by anti-bipolar cell autoantibodies in MAR.
Methods: :
The TRPM1 cation channel, which is expressed by both melanocytes and retinal bipolar cells, was selected as a candidate MAR antigen. Patient sera were tested by immunofluorescence using retinas from wild-type mice, TRPM1 knockout mice, and macaque. To confirm the specificity of binding, all sera were tested on CHO cells transfected with GFP-tagged human TRPM1. Immunoreactivity was visualized with anti-human IgG conjugated to Alexa 594, and images collected with an Olympus FV1000 confocal microscope.
Results: :
Of the MAR sera tested, 4/35 serum samples labeled CHO cells transfected with GFP-TRPM1. Furthermore, these sera also labeled bipolar cells in retina sections from macaque and mouse, and in primary cultures of dissociated mouse retinal cells. The labeling was very similar to that obtained with a commercial TRPM1 antibody. Double labeling for PKCα identified the majority of the labeled cells in mouse and macaque as rod bipolar cells. Presumed cone ON-bipolar cells were also labeled with the human sera. The identity of the antigen as TRPM1 was confirmed by the absence of labeling in sections from TRPM1 knockout mice. By comparing staining in fixed, permeabilized and non-permeabilized cells, the TRPM1 antigenic epitope was determined to be intracellular. Overnight incubation of live, dissociated retinal neurons at 37°C with TRPM1-positive MAR serum resulted in selective uptake and retention of the antibodies by ON-bipolar cells.
Conclusions: :
TRPM1 is the ON-BPC antigen in at least some MAR patients.
Keywords: bipolar cells • melanoma • ion channels