Fig. 8.

CLDN19 mutations interfered with the intracellular transport of endogenous claudin-19 and down-regulated some retinal pigment epithelium (RPE) signature genes. a A mixture of AAV-GFP and AAV-CLDN19 viral vectors were injected into the subretinal space on postnatal day 0 (PN0) to co-express green fluorescent protein (GFP) and either CLDN19^WT, CLDN19^G20D, or CLDN19^R81W. The RPE monolayer was analyzed on PN30. The XZ plane displays the apical half of the cell imaged along the dotted line. Arrowheads at the right indicate the position of apical junctions. GFP indicated that most cells of the monolayer were transduced. Wild-type (WT) claudin-19 localized primarily to the tight junction, but G20D was mostly found in intracellular compartments. R81W displayed an intermediate phenotype. b Effect of claudin-19 on gene expression in human fetal RPE. Plasmid versions of the viral vectors were used to transfect human fetal RPE. Expression of WT claudin-19 had minimal effects, but G20D and to a lesser extent R81W down-regulated the expression of a subset of RPE signature genes; n = 3 cultures. c Effect of mutant CLDN19 on the intracellular transport of claudin-19. A red fluorescent protein (RFP) coding sequence was added to the 5’ end of claudin-19. In a second set of plasmids, a FLAG-tag (wild type) or HA-tag (mutants) was added to the 5’ end. The RFP and one of the antigen-tagged plasmids were co-transfected into ARPE-19 cells. Cells that were co-transfected are shown. Most of the wild-type claudin-19 reached the plasma membrane and the RFP and FLAG-tagged versions co-localized in plasma membrane and internal compartments. In the R81W cells, R81W and wild type also co-localized in the plasma membrane with an increase in the relative amount found in internal compartments. In the G20D cells, mutated claudin-19 did not reach the cell surface and most wild-type claudin-19 co-localized with the mutated claudin-19 in internal compartments. d Effect of claudin-19 on gene expression in ARPE-19. Expression of messenger RNAs (mRNAs) in transfected ARPE-19 was determined by quantitative-real-time RT-PCR (qRT²-PCR), normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and compared to expression in untransfected n = 4 culture ARPE-19. Dashed lines indicate a 2× difference. Arrowheads indicate regions enlarged 4× in the insets of the merged image. Scale bar, 20 µm. Error bars ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001