Project description:Purpose: The present work investigated changes in the gene expression, molecular mechanisms, and pathogenesis of inherited retinal degeneration (RD) in three different disease models to identify predictive biomarkers for their varied phenotypes and to provide a better scientific basis for their diagnosis, treatment, and prevention. Methods: Differentially expressed genes (DEGs) between retinal tissue from RD mouse models obtained during the photoreceptor cell death peak period (Pde6brd1 at post-natal (PN) day 13, Pde6brd10 at PN23, Prphrd2 at PN29) and retinal tissue from C3H wild-type mice were identified using Illumina high-throughput RNA-sequencing. Co-expression gene modules were identified using a combination of GO and KEGG enrichment analyses and gene co-expression network analysis. CircRNA-miRNA-mRNA network interactions were studied by genome-wide circRNA screening. Results: Pde6brd1, Pde6brd10, and Prphrd2 mice had 1,926, 3,096, and 375 DEGs, respectively. Genes related to ion channels, stress, inflammatory processes, tumor necrosis factor (TNF) production, and microglial cell activation were up-regulated, while genes related to endoplasmic reticulum regulation, metabolism, and homeostasis were down-regulated. Differential expression of transcription factors and non-coding RNAs generally implicated in other human diseases was detected (e.g Gloucoma, diabetes retinopathy and inherited retinal degeneration). CircRNA-miRNA-mRNA network analysis indicated that these factors may be involved in photoreceptor cell death. Moreover, excessive cGMP accumulation causes photoreceptor cell death, and cGMP-related genes were generally affected by different pathogenic gene mutations. Conclusions: We screened genes and pathways related to photoreceptor cell death. Additionally, up-stream regulatory factors, such as transcription factors and non-coding RNA and their interaction networks were analyzed. Furthermore, RNAs involved in RDs were functionally annotated. Overall, this study lays a foundation for future studies on photoreceptor cell death mechanisms.

Project description:The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNAseq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd). were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67≥FC≥1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, andtight junctions were down regulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNAseq results. Thus, the RPE quickly down-regulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space and the opsonization of orphaned outer segments following RD.

Project description:The vitreous humor can provide important information about physiological and pathophysiological processes within the eye, particularly concerning the retina. Despite the widespread prevalence of various vitreoretinal diseases, that can ultimately lead to blindness, there is not a clear understanding of many molecular processes underlying these conditions. Diabetic retinopathy (DR) is the leading cause of blindness among working-age adults in U.S. and developed world. It can result in macular edema and tractional retinal detachment (TRD). Hyperglycemia can induce damage to retinal cells and capillary closure, leading to retinal ischemia and elevated levels of nitric oxide (NO). This, in turn, increases the activity of protein kinase C and the levels of different growth factors, including vascular endothelial growth factor (VEGF). These events can stimulate the development of new blood vessels from existing matured vessels, which are subsequently encompassed by glial cells and fibrous proliferation, potentially leading to TRD. Rhegmatogenous retinal detachment (RD) is the separation of neuroretinal layers from the retinal pigment epithelium (RPE), induced by a break in the neuroretina that allows the influx of liquefied vitreous. In the detached retina outer layers become ischemic and photoreceptors undergo cell death mainly by apoptosis. Moreover, 3–4 days after retinal detachment, a peak of proliferation of non-neuronal cells, such as astrocytes, endothelial cells, pericytes, and microglia, can be observed. A possible complication of rhegmatogenous retinal detachment is proliferative vitreoretinopathy (PVR), a condition defined by the growth and contraction of cellular membranes within the vitreous cavity. Fibroblast or fibrocyte cells have been detected in epiretinal membranes in PVR, and the origin of these cells is debated. Different authors have suggested that they could be transformed RPE cells or metaplastic vascular endothelial cells, glial cells, macrophages, or hyalocytes. Macular pucker is a condition defined by an epiretinal membrane described as a semi-translucent avascular membrane over the internal limiting membrane (ILM) on the surface of the retina. It can be idiopathic or secondary to various conditions, such as trauma, intraocular inflammation, etc. The initial event that can induce an idiopathic pucker is posterior vitreous detachment (PVD), which can cause dehiscence in the internal limiting membrane. Through this opening, microglial cells move to the preretinal surface, where they can interact with hyalocytes and laminocytes. Subsequent transdifferentiation to fibroblast-like cells leads to the development of an epiretinal membrane.

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