Visual cycle proteins: Structure, function, and roles in human retinal disease

Here, we seek to summarize the current understanding of the biochemical and molecular events mediated by visual cycle molecules in the eye. The structures and functions of selected visual cycle proteins and their roles in human retinal diseases are also highlighted. Genetic mutations and malfunctions of these proteins provide etiological evidence that many ocular diseases arise from anomalies of retinoid (vitamin A) metabolism and related visual processes. Genetic retinal disorders such as retinitis pigmentosa, Leber\u27s congenital amaurosis, and Stargardt\u27s disease are linked to structural changes in visual cycle proteins. Moreover, recent reports suggest that visual cycle proteins may also play a role in the development of diabetic retinopathy. Basic science has laid the groundwork for finding a cure for many of these blindness-causing afflictions, but much work remains. Some translational research projects have advanced to the clinical trial stage, while many others are still in progress, and more are at the ideas stage and remain yet to be tested. Some examples of these studies are discussed. Recent and future progress in our understanding of the visual cycle will inform intervention strategies to preserve human vision and prevent blindness

Expression of Integrin and TGFBI in Human Retinal Pericytes

Purpose: The aim for this study is to investigate the expression of integrin α3, β1 and TGF-β induced protein (TGFBI) and the secretion of TGFBI by primary culture of human retinal pericytes (pHRP). Evidence suggests that chronic diabetes associate with HRP apoptosis leading to the development of diabetic retinopathy. Methods: pHRP (Cell Systems) were cultured in complete media (15mM glucose) in a humidified, 5% CO2, 37°C condition. Cells were seeded at passage 6 to 8 into a 24 well-plate with coverslips or P10 dishes. Cells (85% confluence) media were then replaced by DMEM media with euglycemic glucose (5.5mM) or hyperglycemic glucose (30mM) and cells were incubated for 48 or 72 hours. Gene and protein expressions of α3, β1 were detected by Real-Time PCR and flow cytometry. TGFBI gene expression was detected by Real-Time PCR and ELISA was used to measure protein level in cell media. Results: Real-Time PCR showed expression of α3, β1 and TGFBI in pHRP at 48 hrs of incubation in both glucose concentrations. Expression of a3 in pHRP in 30 mM glucose was 1.3 times higher than cells in 5.5mM glucose whereas expressions of b1 and TGFBI were comparable in two glucose concentrations. Flow cytometry results also showed expression of integrin subunits in pHRP at 72 hr of incubation. Expression of a3 in pHRP in 30mM glucose was similar to those in cells in 5.5m M (MFI of 251 vs 221 respectively). However, expression of b1 was higher in cells in the higher glucose concentration (MFI: 422 vs 343). ELISA data showed secretion TGFBI protein by HRP at 48 hr of incubation. Protein concentration in media of cell in 30mM glucose was significantly higher than those in 5.5mM (97 vs 57 pg/ml; p=0.0318). Conclusions: This is the first report on the expression of integrin subunits in HPR in euglycemic and hyperglycemic conditions. Both RT-PCR and flow cytometry results show α3, β1 subunits expressions, the level of which may be affected by glucose concentration in the cell media. Furthermore, our ELISA results confirm the secretion of TGFB1 by HRP and a significantly higher protein secretion in hyperglycemic condition. Overall, our data support the hypothesis of integrin and TGFBI expression in HRP. The increase in TGFBI secretion in hyperglycemia suggest a possible role of diabetes. Further studies will provide insight into the role of integrin and TGFBI interaction on the signaling pathway of HRP apoptosis and diabetic retinopathy

Recent advances on visual cycle protein research and progress on clinical translation

Since the publication of our previous paper, Visual cycle proteins: Structure, function, and roles in human retinal disease (Tsin, et.al, JBC 293:13016, 2018) there has been significant progress on multiple topics discussed in this paper. In the present communication, we further explore research advances on two visual cycle proteins: DES1 and IRBP. In addition, we emphasize the progress of clinical translation of other visual cycle protein research, including the breakthrough of FDA-approved gene therapy for Leber’s congenital amaurosis, and additional gene therapies at different stages of clinical trials for various retinal diseases such as retinitis pigmentosa, diabetic retinopathy, and Stargardt’s disease

The Role of Microglia in Diabetic Retinopathy

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patient’s susceptibility to diabetic retinopathy

The role of microglia in diabetic retinopathy

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patient's susceptibility to diabetic retinopathy

Cellular and Molecular Mechanisms of Neuronal Degeneration In Early Stage Diabetic Retinopathy

DR is a common complication of diabetes in which every cell type in the retina is affected or damaged, resulting in irreversible vision loss. Although DR was classically considered a vascular disease, evidence now supports DR as a neurovascular disease, in which neuronal damage is present much earlier than vascular pathology or clinically observable abnormalities. The mechanisms by which neurons are damaged in early DR are linked to the effects of reactive oxygen species, apoptotic signaling by capase-dependent, caspase-independent, and Fas/FasL pathways and glial cell reactivity resulting in excitotoxicity, neurotrophin imbalance, and breakdown of neurovascular coupling. Although some features of neurodegeneration in early DR have been uncovered, research must continue to elucidate gaps in understanding the neurovascular unit, effects of neurotrophins on the diabetic retina, and the exact mechanisms by which ROS encourage neuronal apoptosis. These mechanisms are crucial to prevent progression of DR and vision loss. Currently treatment for DR treats vascular symptoms but cannot stall the course of the disease. As discussed in this paper, strict glycemic control in early stages of DR is not sufficient to prevent progression of neuronal apoptosis. Thus understanding mechanisms of neurodegeneration in DR must guide future avenues of treatment development