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

Using induced pluripotent stem cells to understand retinal ciliopathy disease mechanisms and develop therapies

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies

The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration

Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor

Modelling the Health and Economic Impacts of Population-Wide Testing, Contact Tracing and Isolation (PTTI) Strategies for COVID-19 in the UK

Background: The COVID-19 epidemic in the UK has resulted in over 280,000 reported cases and over 40,000 deaths as of 5th June 2020. In the context of a slower increase in reported cases and deaths associated with COVID-19 over the last few weeks compared to earlier in the epidemic, the UK is starting to relax the physical restrictions (‘lockdown’) that have been imposed since 23 March 2020. This has been accompanied by the announcement of a strategy to test people for infection, trace contacts of those tested positive, and isolate positive diagnoses. While such policies are expected to be impactful, there is no conclusive evidence of which approach to this is likely to achieve the most appropriate balance between benefits and costs. This study combines mathematical and economic modelling to estimate the impact, costs, feasibility, and health and economic effects of different strategies. / Methods: We provide detailed description, impact, costing, and feasibility assessment of population-scale testing, tracing, and isolation strategies (PTTI). We estimate the impact of different PTTI strategies with a deterministic mathematical model for SARS-CoV-2 transmission that accurately captures tracing and isolation of contacts of individuals exposed, infectious, and diagnosed with the virus. We combine this with an economic model to project the mortality, intensive care, hospital, and non-hospital case outcomes, costs to the UK National Health Service, reduction in GDP, and intervention costs of each strategy. Model parameters are derived from publicly available data, and the model is calibrated to reported deaths associated with COVID-19. We modelled 31 scenarios in total (Panel 2). The first 18 comprised nine with ‘triggers’ (labelled with the -Trig suffix) for subsequent lockdown periods (>40,000 new infections per day) and lockdown releases (<10,000 new infections per day), and nine corresponding scenarios without triggers, namely: no large-scale PTTI (scenario 1); scale-up of PTTI to testing the whole population every week, with May–July 2020 lockdown release (scenario 2b), or delayed lockdown release until scale-up complete on 31 August 2020 (scenario 2a); these two scenarios with mandatory use of face coverings (scenarios 3a and 3b); and scenarios 2a, 2b, 3a, 3b replacing untargeted PTTI with testing of symptomatic people only (scenarios 4a, 4b, 4c, 4d). The final 13 scenarios looked at: whole population weekly testing to suppress the epidemic with lower tracing success (scenarios 3b-Trig00, 3b-Trig10, 3b-Trig20, 3b-Trig30) and switched to targeted testing after two months when it may suppress the epidemic (scenarios 3b-Trig00-2mo and 3b-Trig30-2mo), and targeted testing with lower tracing success (scenarios 4d-Trig10, 4dTrig20, 4d-Trig30, 4d-Trig40, 4d-Trig50, 4d-Trig60, 4d-Trig70). / Findings: Given that physical distancing measures have already been relaxed in the UK, scenario 4d-Trig (targeted testing of symptomatic people only, with a mandatory face coverings policy and subsequent lockdown triggered to enable PTTI to suppress the epidemic), is a strategy that will result in the fewest deaths (~52,000) and has the lowest intervention costs (~£8bn). The additional lockdown results in total reduction in GDP of ~£503bn, less than half the cost to the economy of subsequent lockdowns triggered in a scenario without PTTI (scenario 1-Trig, ~£1180bn reduction in GDP, ~105,000 deaths). In summer months, with lower cold and flu prevalence, approximately 75,000 symptomatic people per day need to be tested for this strategy to work, assuming 64% of their contacts are effectively traced (~80% traced with 80% success) within the infectious period (most within the first two days and nearly all by seven days) and all are isolated – including those without any symptoms – for 14 days. Untargeted testing of everyone every week, if it were feasible, may work without tracing, but at a higher cost (scenario 3b-Trig00). This cost could be reduced by switching to targeted testing after the epidemic is suppressed (scenario 3b-Trig30-2mo), though we note the epidemic could be suppressed with targeted testing itself providing tracing and isolation has at least a 32% success rate (scenario 4dTrig40). / Interpretation: PTTI strategies to suppress the COVID-19 epidemic within the context of a relaxation of lockdown will necessitate subsequent lockdowns to keep the epidemic suppressed during PTTI scale-up. Targeted testing of symptomatic people only can suppress the epidemic if accompanied by mandated use of face coverings. The feasibility of PTTI depends on sufficient capacity, capabilities, infrastructure and integrated systems to deliver it. The political and public acceptability of alternative scenarios for subsequent lockdowns needs to take account of crucial implications for employment, personal and national debt, education, population mental health and non-COVID-19 disease. Our model is able to incorporate additional scenarios as the situation evolves

Treatments for irritable bowel syndrome: patients' attitudes and acceptability

<p>Abstract</p> <p>Background</p> <p>Irritable Bowel Syndrome, a highly prevalent chronic disorder, places significant burden on the health service and the individual. Symptomatic distress and reduced quality of life are compounded by few efficacious treatments available. As researchers continue to demonstrate the clinical efficacy of alternative therapies, it would be useful to gain a patient-perspective of treatment acceptability and identify patient's attitudes towards those modalities considered not acceptable.</p> <p>Methods</p> <p>Six hundred and forty-five participants identified from an earlier IBS-prevalence study received a postal questionnaire to evaluate preferences and acceptability of nine forms of treatment. Proportions accepting each form of treatment were calculated and thematic analysis of qualitative data undertaken.</p> <p>Results</p> <p>A total of 256 (39.7%) of 645 potential respondents completed the questionnaire (mean age 55.9 years, 73% female). Tablets were most acceptable (84%), followed by lifestyle changes (diet (82%), yoga (77%)). Acupuncture (59%) and suppositories (57%) were less acceptable.</p> <p>When explaining lack of acceptability, patient views fell into four broad categories: dislike treatment modality, do not perceive benefit, general barriers and insufficient knowledge. Scepticism, lack of scientific rationale and fear of CAM were mentioned, although others expressed a dislike of conventional medical treatments. Past experiences, age and health concerns, and need for proof of efficacy were reported.</p> <p>Conclusion</p> <p>Most patients were willing to accept various forms of treatment. However, the reservations expressed by this patient-population must be recognised with particular focus directed towards allaying fears and misconceptions, seeking further evidence base for certain therapies and incorporating physician support and advice.</p

Functional Electrical Stimulation of Intrinsic Laryngeal Muscles under Varying Loads in Exercising Horses

Bilateral vocal fold paralysis (BVCP) is a life threatening condition and appears to be a good candidate for therapy using functional electrical stimulation (FES). Developing a working FES system has been technically difficult due to the inaccessible location and small size of the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle. A naturally-occurring disease in horses shares many functional and etiological features with BVCP. In this study, the feasibility of FES for equine vocal fold paralysis was explored by testing arytenoid abduction evoked by electrical stimulation of the PCA muscle. Rheobase and chronaxie were determined for innervated PCA muscle. We then tested the hypothesis that direct muscle stimulation can maintain airway patency during strenuous exercise in horses with induced transient conduction block of the laryngeal motor nerve. Six adult horses were instrumented with a single bipolar intra-muscular electrode in the left PCA muscle. Rheobase and chronaxie were within the normal range for innervated muscle at 0.55±0.38 v and 0.38±0.19 ms respectively. Intramuscular stimulation of the PCA muscle significantly improved arytenoid abduction at all levels of exercise intensity and there was no significant difference between the level of abduction achieved with stimulation and control values under moderate loads. The equine larynx may provide a useful model for the study of bilateral fold paralysis

Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells.

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gβ1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients