The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the-protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance

Combining Gene-Disease Associations with Single-Cell Gene Expression Data Provides Anatomy-Specific Subnetworks in Age-Related Macular Degeneration

Background: Age-related macular degeneration (AMD) is the most common cause of visual impairment in the developed world. Despite some treatment options for late AMD, there is no intervention that blocks early AMD proceeding to the late and blinding forms. This is partly due to the lack of precise drug targets, despite great advances in genetics, epidemiology, and protein-protein interaction (PPI) networks proposed to be driving the disease pathology. A systems approach to narrow down PPI networks to specific protein drug targets would provide new therapeutic options. Materials and Methods: In this study we analyzed single cell RNAseq (RNA sequencing) datasets of 17 cell types present in choroidal, retinal pigment epithelium (RPE), and neural retina (NR) tissues to explore if a more granular analysis incorporating different cell types exposes more specific pathways and relationships. Furthermore, we developed a novel and systematic gene ontology database (SysGO) to explore if a subcellular classification of processes will further enhance the understanding of the pathogenesis of this complex disorder and its comorbidities with other age-related diseases. Results: We found that 57% of the AMD (risk) genes are among the top 25% expressed genes in ∼1 of the 17 choroidal/RPE/NR cell types, and 9% were among the top 1% of expressed genes. Using SysGO, we identified an enrichment of AMD genes in cell membrane and extracellular anatomical locations, and we found both functional enrichments (e.g., cell adhesion) and cell types (e.g., fibroblasts, microglia) not previously associated with AMD pathogenesis. We reconstructed PPI networks among the top expressed AMD genes for all 17 choroidal/RPE/NR cell types, which provides molecular and anatomical definitions of AMD phenotypes that can guide therapeutic approaches to target this complex disease. Conclusion: We provide mechanism-based AMD endophenotypes that can be exploited in vitro, using computational models and for drug discovery/repurposing

P2X7R modulation of visually evoked synaptic responses in the retina.

P2X7Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X7R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X7R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg(2+)-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X7R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X7Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment

Form, shape and function: segmented blood flow in the choriocapillaris

The development of fluid transport systems was a key event in the evolution of animals and plants. While within vertebrates branched geometries predominate, the choriocapillaris, which is the microvascular bed that is responsible for the maintenance of the outer retina, has evolved a planar topology. Here we examine the flow and mass transfer properties associated with this unusual geometry. We show that as a result of the form of the choriocapillaris, the blood flow is decomposed into a tessellation of functional vascular segments of various shapes delineated by separation surfaces across which there is no flow, and in the vicinity of which the transport of passive substances is diffusion-limited. The shape of each functional segment is determined by the distribution of arterioles and venules and their respective relative flow rates. We also show that, remarkably, the mass exchange with the outer retina is a function of the shape of each functional segment. In addition to introducing a novel framework in which the structure and function of the metabolite delivery system to the outer retina may be investigated in health and disease, the present work provides a general characterisation of the flow and transfers in multipole Hele-Shaw configurations

Retinal Neuronal Ectopia: a new entity in the differential diagnosis of retinoblastoma

BACKGROUND: To present a rare retinal disorder that should be considered in the differential diagnosis of retinoblastoma. METHODS: A 2-year-old boy presented with left ocular discomfort, leukocoria, and a left divergent squint. Examination of the left eye revealed abnormalities in the anterior segment, and fundoscopy showed an irregular white calcified mass with fibrosis and traction toward the lens. As the ocular discomfort worsened, enucleation of the left eye was performed. RESULTS: Histopathological and immunohistochemical assessment of the enucleated eye established the diagnosis of retinal neuronal ectopia. CONCLUSION: We believe that this case is unique in the human retina and highlights the need for specialist differential diagnosis. Although rare, retinal neuronal ectopia should be considered in the differential diagnosis of retinoblastoma

Mathematical and computational models of the retina in health, development and disease

The retina confers upon us the gift of vision, enabling us to perceive the world in a manner unparalleled by any other tissue. Experimental and clinical studies have provided great insight into the physiology and biochemistry of the retina; however, there are questions which cannot be answered using these methods alone. Mathematical and computational techniques can provide complementary insight into this inherently complex and nonlinear system. They allow us to characterise and predict the behaviour of the retina, as well as to test hypotheses which are experimentally intractable. In this review, we survey some of the key theoretical models of the retina in the healthy, developmental and diseased states. The main insights derived from each of these modelling studies are highlighted, as are model predictions which have yet to be tested, and data which need to be gathered to inform future modelling work. Possible directions for future research are also discussed. Whilst the present modelling studies have achieved great success in unravelling the workings of the retina, they have yet to achieve their full potential. For this to happen, greater involvement with the modelling community is required, and stronger collaborations forged between experimentalists, clinicians and theoreticians. It is hoped that, in addition to bringing the fruits of current modelling studies to the attention of the ophthalmological community, this review will encourage many such future collaborations

Increased Epithelial Expression of CTGF and S100A7 with Elevated Subepithelial Expression of IL-1 beta in Trachomatous Trichiasis

Purpose: To characterize the histological appearance and expression of pro-inflammatory mediators, growth factors, matrix metalloproteinases and biomarkers of epithelial-mesenchymal transition (EMT) in healthy control and trachomatous trichiasis (TT) conjunctival tissue. Methods: Conjunctival biopsies were taken from 20 individuals with TT and from 16 individuals with healthy conjunctiva, which served as controls. Study participants were of varying ethnicity and were living in a trachoma-endemic region of northern Tanzania. Formalin-fixed paraffin-embedded tissue sections were stained using hematoxylin and eosin or by immunohistochemistry using antibodies against: IL-1β, IL-6, IL-17A, IL-22, CXCL5, S100A7, cleaved caspase 1 (CC1), PDGF, CTGF, TGFβ2, MMP7, MMP9, E-cadherin, vimentin, and αSMA. Results: Tissue from TT cases had a greater inflammatory cell infiltrate relative to controls and greater disruption of collagen structure. CTGF and S100A7 were more highly expressed in the epithelium and IL-1β was more highly expressed in the substantia propria of TT cases relative to controls. Latent TGFβ2 was slightly more abundant in the substantia propria of control tissue. No differences were detected between TT cases and controls in the degree of epithelial atrophy, the number of myofibroblasts or expression of EMT biomarkers. Conclusions: These data indicate that the innate immune system is active in the immunopathology of trachoma, even in the absence of clinical inflammation. CTGF might provide a direct link between inflammation and fibrosis and could be a suitable target for therapeutic treatment to halt the progression of trachomatous scarring

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents

Detection of mutations in SF3B1, EIF1AX and GNAQ in primary orbital melanoma by candidate gene analysis

BACKGROUND: Ocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM. METHODS: DNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression. RESULTS: MLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease. CONCLUSIONS: EIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing 'omic technologies will provide novel insights into tumour pathogenesis