BackgroundOcular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction.MethodologyBy using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis.ConclusionOur results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.

Extracellular vesicles (EVs) released by virtually every cell of all organisms are involved in processes of intercellular communication through the delivery of their functional mRNAs, proteins and bioactive lipids. We previously demonstrated that mouse embryonic stem cell-released EVs (mESEVs) are able to transfer their content to different target retinal cells, inducing morphological and biochemical changes in them. The main objective of this paper is to characterize EVs derived from human embryonic stem cells (hESEVs) and investigate the effects that they have on cultured retinal glial, progenitor Müller cells, which are known to give rise to retinal neurons under specific conditions. This would allow us to establish if hESEVs have a pro-regenerative potential not yet described that could be used in the future for treatment of human retinal degenerative diseases. Initially, we showed that hESEVs are heterogeneous in size, contain mRNAs and proteins involved in the induction and maintenance of stem cell pluripotency and can be internalized by cultured Müller cells. After a single exposure to hESEVs these cells display changes in their gene expression profile, and with multiple exposures they de-differentiate and trans-differentiate into retinal neuronal precursors. hESEVs were then fractionated into microvesicles (MVs) and exosomes (EXOs), which were characterized by size, specific surface proteins and biochemical/molecular components. We demonstrate that despite the similar internalization of non-fractionated hESEVs, MVs and EXOs by Müller progenitor cells, in vitro, only the release of MVs’ cargo into the cells’ cytoplasm induces specific changes in their levels of pluripotency mRNAs and early retinal proteins. EXOs do not produce any detectable effect. Thus, we conclude that MVs and MVs-containing hESEVs are promising agents that possibly could promote the regeneration of diseased or damaged retinas in vivo through inducing glial Müller cells to become replacement neurons.

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.

Background: RHBDD2 is distantly related to rhomboids, membrane-bound proteases. Results: In retina, RHBDD2 exists as a monomer in all cells throughout life and a homotrimer only in cone outer segments; a mutation in RHBDD2 possibly leads to retinitis pigmentosa. Conclusion: RHBDD2 plays important roles in development and normal retinal function. Significance: This is the first characterization of RHBDD2 and its association with retinal disease.