Functional analysis of the Parkinson's disease associated genes, parkin and PINK1, in vitro

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Our understanding of the disease has been enhanced by the discovery of gene mutations in rare families with eariy-onset autosomal recessive juvenile parkinsonism (ARJP). The first gene to be identified was parkin and two further genes have been identified namely, DJ-1 and PINK1. The latter was discovered in our laboratory. The work of this thesis has aimed to increase our understanding of how these newly discovered genes play a role in neuronal survival and how disease-causing mutations result in neurodegenerarJon. Utilising gene transfer techniques and transient and stable cell culture expression systems, the function of parkin and PINK1 was investigated in human dopaminergic SH-SY5Y neuroblastoma cell lines. The work of this thesis has confirmed and extended previous reports that parkin over-expression confers neuroprotection against a variety of cellular stresses implicated in PD. Moreover, this work has showed for the first time that endogenous parkin protein can localize to aggregates known as "aggresomes' following stress and that the formation of these parkin positive aggresomes can be dissociated from parkin's effect on neuronal survival. Furthermore, this work describes the first functional characterization of PINK1. It demonstrates that PINK1 localizes to the mitochondria in neuronal cells where it may play a neuroprotective role against cellular stress. Moreover, this effect is abrogated by disease causing mutations in PINK1. This work also reports on the characterisation of novel PINK1 antibodies and shows for the first time that PINK1 can localize to aggresomes and the mechanism is linked to mitochondrial recruitment. The work of this thesis sheds light on the increasing importance of the ubiquitin proteasome system and mitochondria in the pathogenesis of PD. Improved understanding of these cellular processes should lead to more effective treatments for this devastating disease

Vitrectomy for endophthalmitis: 5-year study of outcomes and complications

Background/Aims: To analyse the complications and outcomes of vitrectomy surgery for endophthalmitis. Methods This was a retrospective case series. All cases that underwent 23-gauge vitrectomy surgery for endophthalmitis at a tertiary centre between 1 February 2013 and 1 February 2018 were included. Main outcome measures were as follows: visual acuity (VA) at final visit and post-vitrectomy complications. / Results: 33 patients were included in the study with 20 men and 13 women, average age 63 years. Main post-surgical causes for endophthalmitis included phacoemulsification (n=9), trabeculectomy (n=5), intravitreal injection (n=5), corneal graft (n=4), vitreoretinal surgery (n=3) and endogenous endophthalmitis (n=6). Average follow-up was 18 months (SD 14). 21/33 (64%) patients had baseline perception of light VA. Analysis of exogenous endophthalmitis cases only demonstrated: mean LogMAR VA improved significantly from 2.68 to 1.66 (p=0.001). At final follow-up, 12% had VA of 6/12 or better, and 28% had VA of 6/36 or better. Vitrectomy within 7 days resulted in improved final VA outcomes (1.49 vs 2.16 LogMAR, p=0.032). Complications included retinal detachment (24.2%), macular hole (3%), hypotony (6%), suprachoroidal haemorrhage (3%) and enucleation/ evisceration (6%). / Conclusion: Vitrectomy for endophthalmitis leads to VA gains in some cases. Surgical outcomes may be improved with early vitrectomy performed within 7 days of the initial event for exogenous endophthalmitis. Patients should be advised of the potential risk of severe complications with/ and without surger

Early vitrectomy for exogenous endophthalmitis following surgery

This is a protocol for a Cochrane Review (intervention). The objectives are as follows: To assess the potential role of combined pars plana vitrectomy and intravitreal antibiotics in the acute management of exogenous endophthalmitis, versus the standard of care, defined as vitreous tap and intravitreal antibiotics

Retinal detachment in retinitis pigmentosa

Objective: Retinitis pigmentosa-related retinal detachment (RPRD) is rare, and the full spectrum of retinal complications is not well defined. To describe the types of retinal detachment in patients with retinitis pigmentosa and the surgical outcomes of RPRD. Methods: This is a non-comparative, retrospective case series. An electronic database search was performed using Moorfields OpenEyes electronic health records. We identified 90 patients with RPRD between January 2000 and August 2017. Main outcome and measures are visual acuity (VA), surgical outcomes and classification of RPRD. Results: Of the 90 patients/detachments, 61 (67.8%) were rhegmatogenous retinal detachment (RRD), 19 (21.1%) were exudative, 3 (3.3%) were tractional retinal detachment (TRD) and 7 (7.8%) had combined. 37.5% (9/24) of patients with exudative retinal detachment were treated with either cryotherapy or laser, and one patient underwent vitrectomy for vitreous haemorrhage. 56/90 patients underwent surgical intervention. Nine patients presented late and were deemed inoperable (two exudative and seven RRD). Of the RRD patients with full operative record, the primary attachment rate was 76.2% (16/21) and final reattachment rate was 85.7% (18/21) over a mean 15.4-year follow-up period. Mean VA for RRD surgery improved from 6/190 (1.51 logMAR) to 6/120 (1.31 logMAR) (p=0.194). In the TRD group, the mean VA was 6/300 (1.66 logMAR) at baseline and improved after surgery to 6/48 (0.90 logMAR) (p=0.421). Conclusions: We demonstrated a final reattachment rate of 85.7% with a trend toward better vision following intervention for patients with RPRD. However, the final long-term vision may be poor due to the natural progression of retinitis pigmentosa-associated macular degeneration

Trends in Diabetic Retinopathy, Visual Acuity, and Treatment Outcomes for Patients Living With Diabetes in a Fundus Photograph-Based Diabetic Retinopathy Screening Program in Bangladesh

IMPORTANCE: Diabetic retinopathy (DR) is the leading cause of low vision among working-age adults. An estimated 6.9 million people in Bangladesh were living with diabetes in 2017, which is projected to increase to more than 10 million people in 2025. Currently, no standardized and/or large-scale DR screening program exists in Bangladesh. OBJECTIVE: To develop a novel fundus photograph–based eye screening model for early detection of DR to prevent vision loss in Bangladeshi individuals with diabetes. DESIGN, SETTING, AND PARTICIPANTS: In this cross-sectional study, 49 264 patients with diabetes underwent opportunistic eye screening at 2 eye hospitals and 1 diabetic hospital in Bangladesh between June 1, 2010, and September 30, 2017. The data set was analyzed from April 8 to December 30, 2018. Technicians were trained to obtain 2-field digital fundus photographs and to grade each according to a standardized DR severity scale. Each patient was counseled and triaged for treatment using defined DR referral criteria. MAIN OUTCOMES AND MEASURES: Primary DR grading outcomes, visual acuity, and treatment outcomes. RESULTS: A total of 49 264 patients (54.3% male; mean [SD] age, 50.8 [12.3] years) underwent DR screening during a 7-year period. The DR prevalence rate across all 3 sites was 33% (95% CI, 33%-33%). Prevalence rates varied by center (Chittagong, 64.6% [95% CI, 64.0%-65.0%]; Dhaka, 39.8% [95% CI, 39.0%-41.0%]; and Feni, 13.0% [95% CI, 13.0%-14.0%]). Across all age groups, male patients were at higher risk of prevalent DR than female patients (odds ratio, 1.99; 95% CI, 1.90-2.07). The prevalence was 3.9% for preproliferative DR, 7.8% for proliferative DR, and 19.2% for maculopathy. Individuals with DR had significantly worse visual acuity than those with no DR (bestcorrected visual acuity, 0.35 vs 0.21 logMAR; P < .001). The rate of moderate visual impairment was 12.2%, and the rate of blindness was 2.5%. Primary treatments included laser photocoagulation (n = 1637), intravitreal injection (n = 1440), and vitrectomy (n = 309). CONCLUSIONS AND RELEVANCE: Screening Bangladeshi individuals known to have diabetes using fundus photography identified large numbers of patients with sight-threatening proliferative DR, maculopathy, and visual impairment or blindness. Expansion of eye screening services in Bangladesh is warranted as part of a national government eye care and diabetes health polic

An update on retinal prostheses

Retinal prostheses are designed to restore a basic sense of sight to people with profound vision loss. They require a relatively intact posterior visual pathway (optic nerve, lateral geniculate nucleus and visual cortex). Retinal implants are options for people with severe stages of retinal degenerative disease such as retinitis pigmentosa and age-related macular degeneration. There have now been three regulatory-approved retinal prostheses. Over five hundred patients have been implanted globally over the past 15 years. Devices generally provide an improved ability to localize high-contrast objects, navigate, and perform basic orientation tasks. Adverse events have included conjunctival erosion, retinal detachment, loss of light perception, and the need for revision surgery, but are rare. There are also specific device risks, including overstimulation (which could cause damage to the retina) or delamination of implanted components, but these are very unlikely. Current challenges include how to improve visual acuity, enlarge the field-of-view, and reduce a complex visual scene to its most salient components through image processing. This review encompasses the work of over 40 individual research groups who have built devices, developed stimulation strategies, or investigated the basic physiology underpinning retinal prostheses. Current technologies are summarized, along with future challenges that face the field

PRIMA subretinal wireless photovoltaic microchip implantation in non-human primate and feline models

PURPOSE: To evaluate the surgical technique for subretinal implantation of two sizes of PRIMA photovoltaic wireless microchip in two animal models, and refine these surgical procedures for human trials. METHODS: Cats and Macaca fascicularis primates with healthy retina underwent vitrectomy surgery and were implanted with subretinal wireless photovoltaic microchip at the macula/central retina. The 1.5mm PRIMA chip was initially studied in feline eyes. PRIMA implant (2mm,1.5mm sizes) arrays were studied in primates. Feasibility of subretinal chip implantation was evaluated with a newly-developed surgical technique, with surgical complications and adverse events recorded. RESULTS: The 1.5mm implant was placed in the central retina of 11 feline eyes, with implantation duration 43-106 days. The 1.5mm implant was correctly positioned into central macula of 11 primate eyes, with follow-up periods of minimum 6 weeks (n = 11), 2 years (n = 2), and one eye for 3 years. One primate eye underwent multi-chip 1.5mm implantation using two 1.5mm chips. The 2mm implant was delivered to 4 primate eyes. Optical coherence tomography confirmed correct surgical placement of photovoltaic arrays in the subretinal space in all 26 eyes. Intraoperative complications in primate eyes included retinal tear, macular hole, retinal detachment, and vitreous hemorrhage that resolved spontaneously. Postoperatively, there was no case of significant ocular inflammation in the 1.5mm implant group. CONCLUSIONS: We report subretinal implantation of 1.5mm and 2mm photovoltaic arrays in the central retina of feline and central macula of primate eyes with a low rate of device-related complications. The in vivo PRIMA implantation technique has been developed and refined for use for a 2mm PRIMA implant in ongoing human trials

Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells.

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD

Single-Session vs Multiple-Session Pattern Scanning Laser Panretinal Photocoagulation in Proliferative Diabetic Retinopathy

Objective: To investigate the effects of pattern scanning laser (Pascal; OptiMedica, Santa Clara, California) multispot panretinal photocoagulation given in a single-session (SS-PRP) vs single-spot multiple-session PRP (MS-PRP) on proliferative diabetic retinopathy (PDR). Methods: Single-center, randomized clinical trial of 40 eyes. Proliferative diabetic retinopathy was treated with a 400-μm spot size in 1500 burns given either as Pascal in 20-millisecond SS-PRP or in 3 sessions (100-millisecond MS-PRP) during a 4-week period. Visual acuity, central subfield retinal thickness (CRT), and 24-2 Swedish interactive thresholding algorithm visual fields were recorded at baseline and 4 and 12 weeks. Main Outcome: Measures Central subfield retinal thickness, mean deviation, and PDR grade at 12 weeks. Results: There was a significant increase in mean CRT with MS-PRP (22 μm at 4 weeks, 95% CI, −32.25 to −10.75; 20 μm at 12 weeks, 95% CI, −28.75 to −10.82; P < .001) and no significant increase in the SS-PRP group. The mean deviation increased significantly in the SS-PRP group after 4 weeks (0.73 dB, P = .048), with no significant changes in either group at other points. A positive effect on PDR was observed in 74% of eyes in the SS-PRP group vs 53% in the MS-PRP group (P = .31). Mean treatment time for SS-PRP was 5.04 minutes (SD, 1.5 minutes) compared with 59.3 (SD, 12.7 minutes) in the MS-PRP group (P < .001). Conclusions: There were no adverse outcomes (CRT, visual acuity, or visual field) from using multispot SS-PRP vs single-spot MS-PRP at 12 weeks postlaser, and treatment times were significantly shorter for multispot SS-PRP. Pascal SS-PRP was as effective as MS-PRP in the treatment of PDR. Application to Clinical Practice: Twenty-millisecond Pascal SS-PRP may be safely and rapidly performed in 1500 burns with a similar efficacy to conventional MS-PRP. Trial Identifier: Research and Development Office PIN R00037, Central Manchester University Hospitals Foundation Trust