Signaling in the retina and retinal pigment epithelium

National Eye Institute

Investigators

Linked publications & trials

Abstract

We are studying RPE gene regulation and structural organization in the following ways: A) Analysis of RPE gene expression with special emphasis on differentiation/dedifferentiation pathways and protection against oxidative or inflammatory stress. Divergence from or convergence to the phenotype of native RPE is a common theme of RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In particular, given the likely importance of noncoding RNAs as regulators of gene expression in the response of RPE cells to various signals, we are studying how lncRNA expression affects RPE differentiation and dedifferentiation, and how their expression may be altered with manipulation of RPE cells; B) Analysis of RPE organellar sub-proteomes. The exact composition and stoichiometry of the visual cycle retinoid isomerization machinery (mostly located in the RPE smooth endoplasmic reticulum (ER)) is important to understanding this key RPE function. To answer this question, we plan to develop a complete catalog of the RPE smooth ER sub-proteome. Other complete RPE organellar sub-proteomes (lysosomes, melanosomes, mitochondria, etc.) will follow. These proteomes are most probably RPE-specific in particular ways, because of the specific functions of RPE, but are not completely known. This will be important to understanding the normal functions of RPE, as well as in RPE pathology such as AMD. A complete proteomics/mass spectrometry toolkit has been assembled and is being tested. In the past year we have made progress in the following areas: 1) We continued a project to study the role of the lncRNA LINC00276 in RPE. RNASeq analysis provided a comprehensive view of differentially expressed lncRNAs in differentiated 4-month (4-mo) old ARPE-19 cells relative to 4-day (4-da) old cells. From these data, we observed a number of lncRNAs that were differentially regulated with fold change of 2.5 in differentiated ARPE-19 cells and which show a differential expression pattern between 4-da and 4-mo cultured cells. The expression of one of these in particular, LINC00276, was increased >200-fold in 4-mo cells compared to 4-da cells. Knockdown of LINC00276 negatively affected expression of various RPE-preferential transcripts, while its over-expression enhanced expression. By silencing LINC00276, we observed a decrease in the expression of genes associated with RPE differentiation such as MITF, TRPM1, TRPM3 and miR-204/211, while LINC00276 over-expression increased their expression. Silencing LINC00276 also decreased RPE-characteristic genes such as RPE65, TYR and MERTK, while altering the expression of genes involved in Wnt signaling pathway. We used RNA-pulldown and mass spectrometry analysis to identify proteins potentially interacting with this lncRNA. Currently, we are validating hits by western blotting and RNA immunoprecipitation assay. We have determined that LINC00276 is preferentially expressed in native human RPE. These studies are ongoing. Additionally, we continue to investigate the possible role of lncRNAs in RPE inflammatory response induced by proinflammatory cytokine (PIC) mixture consisting of IFN-, IL-1 and TNF-. A more than 90% decrease in LINC00276 expression was observed with a very low concentration of PIC. The decrease in LINC00276 expression accompanied lower expression of RPE-specific genes (RPE65, MITF, TRPM1 and TRPM3), epithelial marker CDH1 and RPE-characteristic miRNAs (miR-204 and miR-211). Regulators of the NF-B pathway effectively modulated the PICs induced expression of LINC00276. Bay 11-7082, an irreversible inhibitor of IkB- phosphorylation, inhibited TNF- induced decrease in LINC00276 expression indicating the potential involvement of the NF-kB signaling pathway. These studies are ongoing. 2) We have initiated a new study to better understand the structural biology of the visual cycle. To delineate the potential interacting partners of RPE65 which may play a role in assisting the function or stability of RPE65 in the visual cycle, we are using pulldown, immunoprecipitation, and protein cross-linking techniques. These proteins will be analyzed by electrophoresis and immunoblot, and identified by LC-MS/MS, both before and after tryptic digestion. The overall stoichiometry of the complex is also important to elucidate. We will disrupt bovine RPE cells using nitrogen cavitation and use a sucrose gradient ultracentrifugation to separate smooth ER from crude RPE microsomal membranes, followed by Percoll gradient ultracentrifugation to isolate RPE65 and RPE65-associated protein complexes under native conditions. These will be analyzed by LC-MS/MS to develop a complete catalog of the RPE smooth ER sub-proteome. To facilitate this project, we have acquired a SCIEX 5600+ Triple-TOF mass spectrometer equipped with a nanospray source, and an Evosep nanoLC for peptide separation. These instruments will be utilized to develop the label-free quantitative sub-proteomes of RPE organelles, beginning with smooth ER. These studies are ongoing. 3) We continue to collaborate with sections in the LRCMB and with other laboratories and sections (e.g., Molecular Structure and Functional Genomics, Laboratory of Immunology), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of retinoid, lipids, and other compounds. Several separate collaborative studies are ongoing. A collaborative study (with a lab at University of Maryland Baltimore) was published during this reporting period.