Mutations in rhodopsin, the light sensitive protein of rod cells that mediates night vision, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations such as P23H lead to misfolding of rod opsin and to its accumulation within the cell, with detrimental effects on photoreceptor function and viability. Therefore, it is important to address the role of molecular chaperones in rod opsin biogenesis and subsequently in the disease. This study shows that BiP, the hsp70 protein of the ER, is important for rod opsin biogenesis. Depletion of BiP with the subtilase cytotoxin (SubAB) or inhibition of BiP function results in aggregation of wild-type (WT) rod opsin in the ER. By contrast, BiP overxpression improves P23H rod opsin ER mobility suggesting that it can be used as a tool to reduce rod opsin aggregation and highlighting a role of BiP in maintaining rod opsin solubility in the ER. The reductase protein ERdj5 that acts as a co-chaperone for BiP can promote P23H rod opsin degradation and its overexpression can improve P23H mobility in the ER. Moreover, loss of the reductase activity of ERdj5 can impair WT rod opsin traffic to the plasma membrane and decrease its mobility in the ER. Misfolded P23H rod opsin is characterised by the formation of an incorrect disulphide bond between C185-C187 (as opposed to the correct C110-C187). Therefore, the role of the reductase activity of ERdj5 in dealing with misfolded rod opsin was further investigated by studying WT rod opsin or P23H mutation in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds. These mutants confirmed a role of the disulphide bond formation/reduction in rod opsin biogenesis and disease, but illustrated that the situation is complex. Finally, improvement of P23H traffic and P23H cell viability was achieved by treatment with compounds that activate AMP kinase, the master regulator of cellular energy homeostasis. The effect of these compounds and chaperones can now be tested in animal models of ADRP.

Ocular mucous membrane pemphigoid (ocular MMP) is a visually devastating disease where up to 30% of patients become blind due to the consequences of conjunctival inflammation and aggressive fibrosis. Standard systemic immunosuppression controls inflammation, but is limited by toxicity. How well immunosuppressive therapy prevents fibrosis is unknown. The cellular and molecular mechanisms that lead to excessive conjunctival fibrosis in ocular MMP are incompletely understood. Chronic inflammation and repair play an important role, but the conjunctival fibroblasts may also be autonomously activated. This thesis aimed to investigate potential mechanisms involved in conjunctival fibrosis in ocular MMP, for the purpose of proposing future anti-fibrotic therapies, to be used in conjunction with systemic immunosuppression. A retrospective review established that whilst conjunctival inflammation appeared to be controlled in 70% of patients, fibrosis progressed in 53%. A pilot randomised trial of adjunctive pulse intravenous methylprednisolone in patients with severe ocular MMP commencing both oral cyclophosphamide and oral corticosteroids did not reduce the time to control of inflammation, which had been hypothesised to prevent the progression of scarring. Both tumour necrosis factor-alpha (TNFα) and the T cell-derived fibrogenic cytokine interleukin-13 (IL-13) were expressed in active ocular MMP. Although this expression was reduced after treatment in clinically uninflamed ocular MMP, it was still significantly elevated compared to normal conjunctiva. Both TNFα and IL-13 stimulated migration and altered matrix metalloproteinase expression by normal conjunctival fibroblasts. IL-13 also stimulated collagen contraction. Both TNFα and IL-13 upregulated surface expression of costimulatory molecules by conjunctival fibroblasts, which suggests facilitation of a potential mechanism for fibroblast-T cell cross talk. Finally, pemphigoid conjunctival fibroblasts showed an altered phenotype compared to normal conjunctival fibroblasts, with increased cell division, migration, collagen contraction, type I collagen secretion, and secretion of eotaxin and matrix metalloproteinase-3. This thesis increases our understanding of the mechanisms involved in conjunctival fibrosis in ocular MMP, and provides new avenues for investigation of potential adjuvant therapies which could improve the prognosis in this poorly understood disease.

Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells. They form a paracellular diffusion barrier and interact with a network of intracellular signalling mechanisms that control junction function, gene expression and cell behaviour. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. In this thesis I have identified a novel fourpass transmembrane protein of the tight junction called MarvelD3 and begun to analyse its function in the regulation of intracellular signalling pathways from the junction. There are two isoforms of MarvelD3, both of which show a broad tissue distribution and are expressed in different types of epithelial and endothelial cells. MarvelD3 co-localises with occludin at the tight junction in epithelial cells. I have found that MarvelD3 is not necessary for junction formation, but may have a role in the regulation of ion conductance properties of the tight junction. Functional analyses combining loss- and gain-of-function approaches in epithelial cell lines have further identified a role for MarvelD3 in the regulation of cell proliferation, migration and the cellular response to hyperosmotic shock. MarvelD3 expression regulates levels of active c-Jun N-terminal kinase (JNK) and AP1 signalling, possibly via an interaction between its N-terminus and the MAP kinase kinase kinase MEKK1. I have also shown MarvelD3 to be implicated in regulation of the actin cytoskeleton, affecting leading edge formation in migrating cells and cytoskeletal rearrangements in response to hyperosmotic shock. I will also describe some initial studies conducted in Xenopus laevis embryos in which depletion of Xenopus MarvelD3 by morpholino injection results in curvature of the anterioposterior axis and reduced pigmentation, possibly resulting from a defect in neural crest cell migration.

MacTel type 2 is a slowly progressive neurodegenerative condition primarily affecting the tem- poral area of the central retina. Subtle generalised increases in foveal autofluorescence are early sign of the disease. During the course of examining autofluorescence imaging at the Reading Centre of Moorfields Eye Hospital, we identified a new hitherto undescribed pattern of autofluorescence in patients with MacTel type 2. This pattern consists of focal hyperauto- fluorescent deposits (HADs). Cross-sectionally, we studied HADs prevalence and patterns of distribution in MacTel type 2 patients, family members and controls in fundus autofluorescence and fundus colour images. We carried out a detailed phenomenological description of HADs using a multimodal approach consisting of point-to-point correlative analysis of fundus auto- fluorescence, colour fundus imaging, fluorescein angiography imaging and high-definition OCT. Longitudinally, we examined HADs progression over a 5-year-period and HADs in the clinical progression of MacTel type 2. In parallel, we carried out in vivo investigations to examine our hypothesis that hyperautofluorescent deposits have an autofluorescent signature distinct from that of Bruch’s membrane and the RPE and that this autofluorescent signature could mark specific pathological states. We used the microprobe synchrotron X-ray fluorescence and X-ray diffraction analysis to examine the effect of trace metals and elements on drusen autofluorescence. We demonstrated that HADs are a newly identified pattern of autofluores- cence change in patients with MacTel type 2 by showing that HADs prevalence, topographical distribution, progression; all strongly correlate with the disease. We explored the potential of autofluorescence imaging as technique to derive specific information on deposits/drusen and RPE layer in pathological states. We discovered the presence of calcium is associated with the precipitation of hydroxyapatite, a constituent of drusen that had not been described before. Future studies are required to confirm the potential for the use of HADs as a biomarker the in clinical settings.

Exudative age-related macular degeneration, characterised by choroidal neovascularisation (CNV), is the leading cause of severe visual impairment in developed societies. Until recently, established treatments were of limited efficacy, and associated with other disadvantages including being inherently destructive therapies. Although novel anti-VEGF pharmacotherapy has since revolutionised the management and prognosis of many patients, current treatment regimens have distinct limitations, particularly in terms of the probable requirement for life-long, frequent invasive dosing, and associated cumulative medical, financial and logistical consequences. Furthermore, many patients respond suboptimally despite frequent administration. Continued development of superior therapies, therefore remains essential. Targeted angiostatic gene delivery may achieve many of the characteristics required of an ideal treatment modality. Work is presented which further expands the possibility of safe and efficacious retinal gene therapy by viral methods, for the ultimate intention of controlling human CNV. Proof of principle is demonstrated for in vivo intraocular expression from equine infectious anaemia virus-based vectors and non-integrating HIV-1-based vectors, which both represent significant advances in biosafety. The angiostatic efficacies of sFlt-1, endostatin, angiostatin and Pedf are then evaluated in an established murine laser model of CNV. This model is further optimised to quantify CNV-associated hyperpermeability in addition to CNV area. Lentiviral transfer of sFlt-1, endostatin or angiostatin, and Pedf upregulation by bespoke zinc finger transcription factors delivered by adeno-associated viral vectors potently inhibited angiogenesis, with sFlt-1, endostatin and angiostatin additionally inhibiting CNV-associated hyperpermeability. Finally, a novel angiogenic role of sonic hedgehog signalling in experimental CNV is identified, and its pharmacological inhibition demonstrated to be angiostatic. These results complement the current body of experimental evidence, which coupled with the demonstration of efficacious molecular targeting of angiogenic pathways in humans, support the further development of this technology to provide novel treatments which may be used as adjuncts or as superior alternatives to existing therapies.

Retinal vascular disease and breakdown of the inner blood retinal barrier are an important cause of visual morbidity in a number of multisystem disorders. The pathological processes underlying these retinal changes include immune, metabolic and genetically mediated mechanisms, and this thesis examines an example of each of these aetiologies. Behçet’s disease is a multisystem inflammatory disorder. Intraocular inflammation occurs in the majority of patients. The associated retinal vasculitis can be occlusive and carries a high risk of severe and permanent visual loss. A longitudinal study of 107 patients with ocular Behçet’s disease presenting over the last decade was undertaken. Long term visual outcomes were found to be improved when compared to earlier published series which may in part reflect a treatment benefit of newer biological agents. Of the metabolic disorders, diabetic retinopathy is the most common retinal vascular disease encountered in ophthalmic practice. Reports in the literature suggest that in patients with coexisting diabetes mellitus and uveitis there may be disease interactions that could potentially augment their respective clinical manifestations and modify disease progression. A retrospective case note review of uveitic eyes that developed diabetes mellitus found stable visual acuity up to 4 years after the onset of coexisting disease and no change in the treatment required to control the intraocular inflammation. No increase in the complications of uveitis was demonstrated. In a comparable study there was no evidence found to suggest that the onset of uveitis in patients with diabetes mellitus resulted in an increase in the development or progression of diabetic retinopathy. von Hippel-Lindau disease is an autosomal dominant inherited cancer syndrome. Retinal capillary haemangioblastoma is the characteristic ocular finding and can cause visual loss as a consequence of leakage through the abnormal vascular endothelium of the tumour. Patients with and without cystic visceral lesions were compared and found not to differ in relation to their respective ocular phenotype. A phenotype comprising pancreatic cysts and central nervous system haemangioblastoma linked to VHL gene deletions was suggested.

Epidermolysis bullosa (EB) describes a group of inherited bullous disorders affecting the basement membrane zone of skin and mucous membranes. It is characterised by fragility and blistering of the skin and mucosa following friction or mechanical trauma. Ocular features are thought to result from a lack of adherence and disruption of the corneal and/or conjunctival epithelium. This thesis aimed to prospectively evaluate the ocular complications occurring in children with EB via comprehensive ocular examination including assessment of the anterior segment, posterior segment and ocular movements. Findings demonstrated the majority of EB patients exhibit signs of meibomian gland dysfunction (MGD) and decreased tear break up time (TBUT), abnormal tear films on Tearscope® evaluation and ocular surface anomalies. Two further cohorts were studied; a) Age-matched control patients with no anterior segment pathology to provide normative data on MGD and TBUT and b) Children diagnosed with MGD who did not suffer from EB to compare the ocular surface phenotype in these, with that in children with EB and MGD. Having identified MGD and an abnormal ocular surface in children with EB, and shown effective management of MGD in otherwise healthy children, translational research was performed to evaluate the effect of potential therapeutic options at a cellular level. Skills in tissue culture and flow cytometry were developed to work with two untransfected conjunctival cell lines (Immortalized Cell Line (IOBA-NHC) and the Wong-Kilbourne derivative of Chang cells) as an in vitro method to investigate the effect of the current therapeutic agents for MGD on the conjunctival epithelial inflammatory response. In conclusion this thesis furthers our understanding of the ocular phenotype in EB subtypes, the aetiology of ocular features and the appropriate management. Findings may allow future therapeutic developments with greater specificity for children with EB.

No description available.

Retinal degenerative disorders are the leading cause of blindness in the developed world, resulting in loss of the photoreceptor cells and vision. Few treatments are available and none can reverse the loss of sight. Photoreceptor transplantation offers the potential to restore vision by replacing cells lost in disease. Previous work has demonstrated that, following transplantation, rod-precursor cells can migrate into the retina, differentiate into mature phenotypes and confer increased sensitivity to light in the rod range. However, rigorous assessment of whether transplanted photoreceptors can actually restore vision is required. In order to do this, we first optimized rod photoreceptor transplantation. To test the functionality of the transplanted rod photoreceptors, we selected a model in which improvements could be assessed unambiguously. The Gnat1-/- mouse, a model of stationary night-blindness, lacks rod a-transducin and thus has no rod function. We demonstrate that transplanted cells robustly integrate forming synaptic connections with the recipient. Integrated cells form inner/outer segments that appropriately expressed phototransduction proteins. Single cell recordings demonstrate that integrated cells are light responsive and intrinsic imaging of the visual cortex shows that visual signals generated by transplanted rods project to higher visual areas. These cells are also capable of restoring optokinetic head-tracking and visually-guided behavior in response to scotopic visual stimuli. A major question remains as to how amenable the heterogeneous diseases encompassed within retinal degenerative disorders will be to photoreceptor replacement and if treatment of late-stage disease is feasible. We performed a comprehensive assessment of photoreceptor transplantation in 6 murine models of inherited retinal degeneration encompassing different types and stages of degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase, decrease or remain constant with disease progression, depending upon the gene defect, with no correlation with disease severity. Robust integration into late-stage disease is possible in some disease types. We assessed features of the recipient microenvironment known to change during degeneration, namely gliosis, outer nuclear layer cyto-architecture and outer limiting membrane (OLM) integrity. Disruption of glial scarring and OLM integrity significantly increased integration to levels sufficient to restore optokinetic head-tracking responses in a model with an otherwise poor transplantation outcome. Together, these findings demonstrate the feasibility of photoreceptor transplantation as a strategy for the restoration of vision in retinal disease.

Epithelial cells adhere to each other and are connected via a series of junctions. Tight junctions (TJs) are a specific type of junction consisting of heteromeric protein complexes that are linked to the actin cytoskeleton and are important in regulating paracellular permeability and cell polarity. RhoGTPases are small molecular switch proteins that are important regulators of the cytoskeleton and modulators of gene expression. RhoGTPases have thus been identified as being major signalling components associated with TJs. However little is known about how RhoGTPases are regulated to control junction formation and gene expression in corneal epithelial cells. I used a siRNA screening approach combined with functional assays to identify components of RhoGTPase signalling that affect the assembly of junctions and gene expression in Human corneal epithelial cells (HCE). I identified and validated several candidates that regulate junction assembly. One of these candidates was p114RhoGEF, a novel TJ localised guanine nucleotide exchange factor (GEF) important for the assembly of functional TJs. p114RhoGEF is a widely expressed and I discovered its depletion effects junction formation and morphogenesis in three dimensional culture systems in different epithelial cell types. p114RhoGEF is required for activation of RhoA at cell-cell junctions and junctional actinomyosin activity, p114RhoGEF is present in a complex containing Myosin II-A, the RhoA effector Rock II and the junctional adaptor protein Cingulin; indicating p114RhoGEF is a component of a junction associated RhoA-signalling module. p114RhoGEF, thus regulates spatial activation of RhoA at cell-cell junctions and organisation of the junctional cytoskeleton. p114RhoGEF may also have a role in cell migration, as depletion in HCE cells, caused cells to migrate at a slower rate during wound healing assays. I have also started to explore the function of a putative p114RhoGEF ortholog, cg10188 in Drosophila melanogaster. Preliminary experiments have identified cg10188 to be important in larval development.