Certain MAR sera label retinal rod and ON cone bipolar cells
We tested sera obtained from three well-characterized MAR patients who had a normal ERG a-wave but reduced b-wave. To identify the immunoreactive retinal cells, sera were first applied to monkey retinas. Both MAR1 and MAR2 sera, but not MAR3 serum, yielded distinct staining of bipolar cells somas and dendrites (). Applying control serum gave no staining (). To determine if only rod bipolar cells or all ON cone bipolar cells were stained, we co-stained for the G-protein subunit Gαo
(a marker for ON bipolar cells: Vardi, 1998
; Dhingra et al., 2002
), and found that practically all cell bodies labeled by Gαo
were also stained for MAR2 serum (149/151; 2 experiments) (). Eight cells that stained for MAR2 were unlabeled for Gαo
, likely because somatic staining for Gαo
is often weak (Vardi, 1998
). Thus, both rod bipolar and ON cone bipolar cells are targeted by MAR2. Although bipolar cell staining was robust, the relative intensity of the stain varied, e.g., the staining of bipolar dendrites in the mouse appeared more intense with MAR1 serum compared to MAR2, while MAR2 gave stronger staining in monkey. The intensity of additional stained structures that do not belong to bipolar cells also varied, e.g., in mouse, MAR1, but not MAR2, showed some staining in ganglion cells and axon fibers, and in monkey (but not in mouse) MAR2 stained cone outer segments and both sera stained cone terminals.
MAR sera target retinal rod and ON cone bipolar cells (monkey)
A similar pattern of bipolar cell staining was also obtained in baboon and rat. The autoantibodies in MAR1 serum are of the IgG type, because purified IgG gave similar labeling (not shown). To confirm purity of the fraction, we applied anti-IgM secondary antibodies, and this did not reveal any staining.
Autoantibodies in MAR1 and MAR2 sera target TRPM1
To determine whether MAR sera recognize TRPM1, we transfected HEK cells with human TRPM1 fused to myc. Immunostaining these cells with the sera showed that both MAR1 and MAR2 sera stained the TRPM1 transfected cells and, as with anti-TRPM1, the staining was not restricted to the membrane, but was present also in the cytosol (). We verified that each stained cell expressed TRPM1 by co-staining for myc: colocalization was approximately 100% (). MAR sera did not stain untransfected HEK cells, nor did the control serum stain TRPM-1-transfected cells (), To ensure that the MAR sera did not recognize the myc tag, we transfected HEK cells with an unrelated protein (neuropilin1) fused to myc; the anti-myc antibody stained transfected cells but the MAR sera did not ().
Autoantibodies in MAR1 and MAR2 sera recognize TRPM1 in cell culture
Next we tested whether MAR sera recognize TRPM1 by Western blotting. Applying the MAR sera to TRPM1-transfected HEK cell protein gave a band at the predicted size (180 kDa) of TRPM1 (Xu et al., 2001
; Koike et al., 2010
), and this was lacking from untransfected HEK cells (, middle and left lanes, arrow). The control serum did not show this band in TRPM1-transfected HEK cells (, right lane). When mouse and monkey retinas were probed with the MAR sera, several bands appeared, but none was at 180 kDa. We confirmed that the mobility of TRPM1 in monkey retina is 180 kDa by probing the Western blots of transfected HEK cells and monkey retina with anti-TRPM1 (). We conclude that the MAR sera do not easily recognize the denatured endogenous TRPM1.
The experiments presented so far establish that the sera of MAR1 and MAR2 recognize TRPM1, but do not prove that the staining of bipolar cells by the MAR serum represents recognition of TRPM1. Theoretically, the TRPM1 in these cells may go unrecognized, and the observed staining in bipolar cells may be due to an additional autoantibody in the serum. We addressed this issue in the mouse. First, we verified that MAR sera but not a control serum stain bipolar cells (not shown). Then we identified the type of bipolar cells by applying the MAR sera to retinal sections obtained from Grm6-GFP transgenic mice, which express GFP in all and only ON bipolar cells (). We noted almost complete overlap of MAR sera and GFP staining (184 cells out of 195 GFP positive cells showed MAR1 staining and 7 cells stained only for MAR1, 3 experiments; 52 out of 65 GFP-positive cells stained for MAR2, 1 cell stained only for MAR2, 2 experiments). Similar results were obtained by double staining for Gαo (not shown). This indicates that MAR staining in the INL is mostly restricted to ON bipolar cells.
In mouse ON bipolar cells, autoantibodies in MAR sera target only TRPM1
Next, we applied the MAR sera to retinal sections obtained from Trpm1−/− mice (3 different animals). In the Trpm1−/− retina, neither MAR1 nor MAR2 serum stained ON bipolar cells (). This suggests that, in bipolar cells, only TRPM1 is recognized.
The TRPM1 epitopes for the autoantibodies are likely to be in the intracellular domain
To determine whether the antibody recognizes an intracellular or extracellular domain, we incubated TRPM1-transfected HEK cells in the MAR sera with or without Triton X-100. In the absence of Triton X-100, only a weak background staining was seen (). This lack of staining may indicate that the TRPM1 epitope is intracellular, but it may also indicate a failure of the expressed TRPM1 to integrate into the transfected HEK cell surface. We have thus examined this question in retina, where TRPM1 must be localized to the cell surface to mediate the light response. Staining for MAR1 in the presence of Triton X-100 was strong in both somas and fine dendrites; however the antibody penetrated only down to a depth of about 9 μm. Omitting Triton X-100 retained somatic staining down to a depth of 5–6 μm (because these were cut open by sectioning), but it totally abolished dendritic staining (). Staining for MAR2 in the absence of Triton X-100 gave high background, but no specific staining. In contrast, staining for GABAA
receptor (raised against an extracellular epitope: Vardi and Sterling, 1994
), remains strong in dendritic tips of cone bipolar cells (and in the inner plexiform layer) even in the absence of Triton X-100 (). These experiments indicate that MAR antibodies recognize intracellular epitopes on the TRPM1 channel, but do not exclude the possibility that a small fraction of the antibody clones are against extracellular epitopes, which are too few to give above-background staining.
The epitope recognized by the MAR sera is intracellular and MAR serum is internalized
We have further tested whether the antibodies can be internalized into retinal cells. Incubation of live retinal slices in MAR2 revealed that many horizontal and amacrine cells internalized the antibodies within 1 hour while photoreceptors and bipolar cells required a longer period (). The obtained staining is not due to stickiness of the secondary antibody because incubation in normal horse or goat sera or just in Ames medium followed by anti-human secondary did not result in any staining. However, these sera, as well as normal human serum, were also internalized, as revealed by using the appropriate secondary antibodies. Injecting MAR2 intravitreally and observing the retina 24 hours later also showed internalization, albeit in a smaller fraction of cells.