Dr. Levin is a visual scientist and neuro-ophthalmologist at the University of Wisconsin School of Medicine and Public Health and the University of Montreal. He specializes in diseases of the optic nerve, and studies optic neuropathies and retinal ganglion cell death at the molecular, biochemical, and cellular level. He serves as a consultant to pharmaceutical companies in the areas of neuroprotection and its application to human disease, including glaucoma.
Modeling Reactive Oxygen Species-Induced Axonal Loss in Leber Hereditary Optic Neuropathy
Biomolecules. 2022 Oct 2;12(10):1411. doi: 10.3390/biom12101411.
Leber hereditary optic neuropathy (LHON) is a rare syndrome that results in vision loss. A necessary but not sufficient condition for its onset is the existence of known mitochondrial DNA mutations that affect complex I biomolecular structure. Cybrids with LHON mutations generate higher rates of reactive oxygen species (ROS). This study models how ROS, particularly H2O2, could signal and execute the axonal degeneration process that underlies LHON. We modeled and explored several hypotheses regarding the influence of H2O2 on the dynamics of propagation of axonal degeneration in LHON. Zonal oxidative stress, corresponding to H2O2 gradients, correlated with the morphology of injury exhibited in the LHON pathology. If the axonal membrane is highly permeable to H2O2 and oxidative stress induces larger production of H2O2, small injuries could trigger cascading failures of neighboring axons. The cellular interdependence created by H2O2 diffusion, and the gradients created by tissue variations in H2O2 production and scavenging, result in injury patterns and surviving axonal loss distributions similar to LHON tissue samples. Specifically, axonal degeneration starts in the temporal optic nerve, where larger groups of small diameter fibers are located and propagates from that region. These findings correlate well with clinical observations of central loss of visual field, visual acuity, and color vision in LHON, and may serve as an in silico platform for modeling the mechanism of action for new therapeutics.
PMID:36291620 | PMC:PMC9599876 | DOI:10.3390/biom12101411
Equity, diversity, and inclusion landscape in Canadian postgraduate medical education for ophthalmology
Can J Ophthalmol. 2022 Sep 24:S0008-4182(22)00272-1. doi: 10.1016/j.jcjo.2022.08.015. Online ahead of print.
OBJECTIVE: To collect information on the current equity, diversity, and inclusion (EDI) landscape at Canadian ophthalmology academic centres.
DESIGN: Cross-sectional survey.
PARTICIPANTS: Faculty representatives from 15 accredited Canadian ophthalmology postgraduate training programs and 57 ophthalmology resident survey respondents.
METHOD: A three-phase virtual EDI initiative was conducted by the Association of Canadian University Professors of Ophthalmology in 2021. A qualitative survey of Canadian academic ophthalmology programs and an anonymous survey of Canadian ophthalmology residents were completed.
RESULTS: All Canadian ophthalmology programs provided information on their current EDI strategies, yielding a response rate of 100%. The majority (73%) of Canadian academic ophthalmology centres identified as being in the beginning stages of building an EDI framework. Of the 57 responding resident physicians, 44% identified as a woman and 51% as a visible minority. There were no respondents who identified as Indigenous. Three respondents identified as having a disability per the Accessible Canada Act. Most respondents either agreed or strongly agreed that their programs are equal opportunity providers (80%), trustworthy (72%), respectful (74%), and of diverse opinions and ideas (71%). There were no statistically significant associations between responses to EDI questions and gender across population groups.
CONCLUSIONS: Most Canadian ophthalmology academic centres are in the beginning stages of implementing EDI initiatives. Multiple priorities have been identified as areas for improvement, such as increasing EDI education for patients, learners, and physicians, and implementing EDI policies in selection and recruitment.
PMID:36162441 | DOI:10.1016/j.jcjo.2022.08.015
Increasing equity, diversity, and inclusion in the ophthalmology CaRMS selection process: ACUPO recommendations
Can J Ophthalmol. 2023 Apr;58(2):e58-e60. doi: 10.1016/j.jcjo.2022.08.004. Epub 2022 Aug 31.
PMID:36057339 | DOI:10.1016/j.jcjo.2022.08.004
Neuroprotection in neurodegenerations of the brain and eye: Lessons from the past and directions for the future
Front Neurol. 2022 Aug 12;13:964197. doi: 10.3389/fneur.2022.964197. eCollection 2022.
BACKGROUND: Neurological and ophthalmological neurodegenerative diseases in large part share underlying biology and pathophysiology. Despite extensive preclinical research on neuroprotection that in many cases bridges and unifies both fields, only a handful of neuroprotective therapies have succeeded clinically in either.
MAIN BODY: Understanding the commonalities among brain and neuroretinal neurodegenerations can help develop innovative ways to improve translational success in neuroprotection research and emerging therapies. To do this, analysis of why translational research in neuroprotection fails necessitates addressing roadblocks at basic research and clinical trial levels. These include optimizing translational approaches with respect to biomarkers, therapeutic targets, treatments, animal models, and regulatory pathways.
CONCLUSION: The common features of neurological and ophthalmological neurodegenerations are useful for outlining a path forward that should increase the likelihood of translational success in neuroprotective therapies.
PMID:36034312 | PMC:PMC9412944 | DOI:10.3389/fneur.2022.964197
Author Correction: Regenerative and restorative medicine for eye disease
Nat Med. 2022 Oct;28(10):2218. doi: 10.1038/s41591-022-01996-9.
PMID:35945286 | DOI:10.1038/s41591-022-01996-9
Pivotal roles for membrane phospholipids in axonal degeneration
Int J Biochem Cell Biol. 2022 Sep;150:106264. doi: 10.1016/j.biocel.2022.106264. Epub 2022 Jul 19.
Membrane phospholipids are critical components of several signaling pathways. Maintained in a variety of asymmetric distributions, their trafficking across the membrane can be induced by intra-, extra-, and intercellular events. A familiar example is the externalization of phosphatidylserine from the inner leaflet to the outer leaflet in apoptosis, inducing phagocytosis of the soma. Recently, it has been recognized that phospholipids in the axonal membrane may be a signal for axonal degeneration, regeneration, or other processes. This review focuses on key recent developments and areas for ongoing investigations. KEY FACTS: Phosphatidylserine externalization propagates along an axon after axonal injury and is delayed in the Wallerian degeneration slow (WldS) mutant. The ATP8A2 flippase mutant has spontaneous axonal degeneration. Microdomains of axonal degeneration in spheroid bodies have differential externalization of phosphatidylserine and phosphatidylethanolamine. Phospholipid trafficking could represent a mechanism for coordinated axonal degeneration and elimination, i.e. axoptosis, analogous to apoptosis of the cell body.
PMID:35868612 | DOI:10.1016/j.biocel.2022.106264
Regenerative and restorative medicine for eye disease
Nat Med. 2022 Jun;28(6):1149-1156. doi: 10.1038/s41591-022-01862-8. Epub 2022 Jun 17.
Causes of blindness differ across the globe; in higher-income countries, most blindness results from the degeneration of specific classes of cells in the retina, including retinal pigment epithelium (RPE), photoreceptors, and retinal ganglion cells. Advances over the past decade in retinal regenerative medicine have allowed each of these cell types to be produced ex vivo from progenitor stem cells. Here, we review progress in applying these technologies to cell replacement - with the goal of vision restoration in degenerative disease. We discuss the landscape of human clinical trials for RPE transplantation and advanced preclinical studies for other cell types. We also review progress toward in situ repair of retinal degeneration using endogenous progenitor cells. Finally, we provide a high-level overview of progress toward prosthetic ocular vision restoration, including advanced photovoltaic devices, opsin-based gene therapy, and small-molecule photoswitches. Progress in each of these domains is at or near the human clinical-trial stage, bringing the audacious goal of vision restoration within sight.
PMID:35715505 | DOI:10.1038/s41591-022-01862-8
A quantitative approach to the spread of variance in translational research using Monte Carlo simulation
Sci Rep. 2022 Apr 15;12(1):6274. doi: 10.1038/s41598-022-09921-3.
The translation of promising preclinical research into successful trials often fails. One contributing factor is the "Princess and the Pea" problem, which refers to how an initially significant effect size dissipates as research transitions to more complex systems. This work aimed to quantify the effects of spreading variability on sample size requirements. Sample size estimates were performed by Monte Carlo simulation. To simulate the process of progressing from preclinical to clinical studies, nested sigmoidal dose-response transformations with modifiable input parameter variability were used. The results demonstrated that adding variabilty to the dose-response parameters substantially increases sample size requirements compared to standared calculations. Increasing the number of consecutive studies further increases the sample size. These results quantitatively demonstrate how the spread of variability in translational research, which is not typically accounted for, can result in drastic increases in the sample size required to maintain a desired study power.
PMID:35428790 | PMC:PMC9012853 | DOI:10.1038/s41598-022-09921-3
Report From the National Eye Institute Workshop on Neuro-Ophthalmic Disease Clinical Trial Endpoints: Optic Neuropathies
Invest Ophthalmol Vis Sci. 2021 Nov 1;62(14):30. doi: 10.1167/iovs.62.14.30.
PMID:34846515 | PMC:PMC8648055 | DOI:10.1167/iovs.62.14.30
Exploring Ophthalmologists' Adoption of Telemedicine during the COVID-19 Pandemic: A Mixed Methods Study
Ophthalmic Epidemiol. 2022 Dec;29(6):595-603. doi: 10.1080/09286586.2021.2008454. Epub 2021 Nov 25.
INTRODUCTION: The COVID-19 pandemic promoted hitherto unseen uptake of telemedicine by ophthalmologists. We performed a mixed methods study to explore patters of utilization during the pandemic and perceived future utility.
METHODS: Ophthalmologists practicing in Canada between March and July 2020 were invited to complete an online questionnaire assessing demographics, clinical practice characteristics and telemedicine utilization prior to and during the pandemic. Descriptive and bivariate statistics were used to analyze the data. Agglomerative hierarchical cluster analysis was used to identify groups who varied on the types of visits offered using telemedicine. Ten one-on-one interviews were conducted and analyzed using thematic content analysis to explain trends observed in the survey data.
RESULTS: Seventy-three ophthalmologists completed the survey. Six percent reported using telemedicine prior to the pandemic compared to 80% during the pandemic. A significant majority (81%) primarily used the telephone for telemedicine visits. Overall, visit volumes during the pandemic declined to 40% of pre-pandemic levels, with a smaller decline for ophthalmologists who used telemedicine than those who did not. Those who used telemedicine for all visit types were more likely to use telemedicine software and to anticipate a modest-to-large role for telemedicine in their future practice.
DISCUSSION: For many ophthalmologists, integrating telemedicine into clinical practice may have partially offset the disruption to normal clinical activities during the pandemic. While the majority saw telemedicine as a temporary solution, a sizeable minority appear to have made considerable use of the technology and see an ongoing role for it once regular clinical activities resume.
PMID:34821531 | DOI:10.1080/09286586.2021.2008454
Neuroprotection of retinal ganglion cells by the sigma-1 receptor agonist pridopidine in models of experimental glaucoma
Sci Rep. 2021 Nov 9;11(1):21975. doi: 10.1038/s41598-021-01077-w.
Optic neuropathies such as glaucoma are characterized by retinal ganglion cell (RGC) degeneration and death. The sigma-1 receptor (S1R) is an attractive target for treating optic neuropathies as it is highly expressed in RGCs, and its absence causes retinal degeneration. Activation of the S1R exerts neuroprotective effects in models of retinal degeneration. Pridopidine is a highly selective and potent S1R agonist in clinical development. We show that pridopidine exerts neuroprotection of retinal ganglion cells in two different rat models of glaucoma. Pridopidine strongly binds melanin, which is highly expressed in the retina. This feature of pridopidine has implications to its ocular distribution, bioavailability, and effective dose. Mitochondria dysfunction is a key contributor to retinal ganglion cell degeneration. Pridopidine rescues mitochondrial function via activation of the S1R, providing support for the potential mechanism driving its neuroprotective effect in retinal ganglion cells.
PMID:34753986 | PMC:PMC8578336 | DOI:10.1038/s41598-021-01077-w
Axonal degeneration induces distinct patterns of phosphatidylserine and phosphatidylethanolamine externalization
Cell Death Discov. 2021 Sep 17;7(1):247. doi: 10.1038/s41420-021-00641-7.
Axonal degeneration is a common feature of multiple neurodegenerative diseases, yet the mechanisms underlying its various manifestations are incompletely understood. We previously demonstrated that axonal degeneration is associated with externalization of phosphatidylserine (PS), which precedes morphological evidence of degeneration, is redox-sensitive, and is delayed in Wallerian degeneration slow (WldS) mutant animals. Phosphatidylethanolamine (PE) is the other major membrane phospholipid in the inner leaflet of the cell membrane, and given that PS signals apoptosis, phagocytosis, and degeneration, we hypothesized that PS and PE membrane dynamics play distinct roles in axonal degeneration. To test this hypothesis, axonal degeneration was induced with calcium ionophores in postnatal rat retinal ganglion cells, and PS- and PE-specific fluorescent probes used to measure their externalization over time. In untreated cells, cell-surface PS was prominent in the cell body alone. Elevation of intracellular calcium with calcium ionophores resulted in significantly increased levels of PS externalization in the cell body, axon, and axon growth cone. Unlike PS, cell-surface PE was diffusely distributed in untreated cells, with comparable levels across the soma, axons, and axon terminals. After exposure to calcium ionophores, PE externalization significantly increased in the cell body and axon. Elevated intracellular calcium also resulted in the formation of axonal blebs which exclusively contained externalized PS, but not PE. Together, these results indicated distinct patterns of externalized PS and PE in normal and degenerating neurons, suggesting a differential role for these phospholipids in transducing neuronal injury.
PMID:34535640 | PMC:PMC8448818 | DOI:10.1038/s41420-021-00641-7
Predicting Absorption-Distribution Properties of Neuroprotective Phosphine-Borane Compounds Using In Silico Modeling and Machine Learning
Molecules. 2021 Apr 25;26(9):2505. doi: 10.3390/molecules26092505.
Phosphine-borane complexes are novel chemical entities with preclinical efficacy in neuronal and ophthalmic disease models. In vitro and in vivo studies showed that the metabolites of these compounds are capable of cleaving disulfide bonds implicated in the downstream effects of axonal injury. A difficulty in using standard in silico methods for studying these drugs is that most computational tools are not designed for borane-containing compounds. Using in silico and machine learning methodologies, the absorption-distribution properties of these unique compounds were assessed. Features examined with in silico methods included cellular permeability, octanol-water partition coefficient, blood-brain barrier permeability, oral absorption and serum protein binding. The resultant neural networks demonstrated an appropriate level of accuracy and were comparable to existing in silico methodologies. Specifically, they were able to reliably predict pharmacokinetic features of known boron-containing compounds. These methods predicted that phosphine-borane compounds and their metabolites meet the necessary pharmacokinetic features for orally active drug candidates. This study showed that the combination of standard in silico predictive and machine learning models with neural networks is effective in predicting pharmacokinetic features of novel boron-containing compounds as neuroprotective drugs.
PMID:33923006 | PMC:PMC8123347 | DOI:10.3390/molecules26092505
Crohn-related Chronic Relapsing Inflammatory Optic Neuropathy
Can J Neurol Sci. 2021 Sep;48(5):740-741. doi: 10.1017/cjn.2020.270. Epub 2020 Dec 14.
PMID:33308333 | DOI:10.1017/cjn.2020.270
Author Correction: Propagation and Selectivity of Axonal Loss in Leber Hereditary Optic Neuropathy
Sci Rep. 2020 Mar 10;10(1):4646. doi: 10.1038/s41598-020-61398-0.
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
PMID:32157129 | PMC:PMC7064521 | DOI:10.1038/s41598-020-61398-0
Randomized Controlled Phase 2a Study of RPh201 in Previous Nonarteritic Anterior Ischemic Optic Neuropathy
J Neuroophthalmol. 2019 Sep;39(3):291-298. doi: 10.1097/WNO.0000000000000786.
BACKGROUND: No proven treatment exists for nonarteritic anterior ischemic optic neuropathy (NAION), either in the acute or late phase.
OBJECTIVE: To assess safety and changes in visual function and structure after RPh201/placebo treatment in participants with previous NAION.
DESIGN AND SETTING: Phase 2a, single-site, prospective, randomized, placebo-controlled, double-masked trial (registration NCT02045212).
MAIN OUTCOMES MEASURES: Early Treatment Diabetic Retinopathy Study best-corrected visual acuity (BCVA), visual fields, retinal nerve fiber layer, and visual evoked potential at weeks 13, 26, and after a 13-week wash-out ("off-drug") period; and safety.
STUDY POPULATION: Twenty-two participants aged 18 years or older with previous NAION.
INTERVENTION(S): RPh201 (20 mg) or placebo (cottonseed oil vehicle) administered subcutaneously twice weekly at the study site.
RESULTS: Thirteen men and 9 women were randomized, of which 20 completed all visits. The mean (±SD) age was 61.0 ± 7.6 years. In a post hoc analysis, after 26 weeks of treatment, BCVA improved by ≥15 letters in 4/11 (36.4%) eyes with RPh201, compared to 1/8 (12.5%) eyes with placebo (P = 0.24). Overall, 7/11 (63.6%) of participants on RPh201 showed some improvement in BCVA, compared with 3/8 (37.5%) on placebo (P = 0.26). Improvement in BCVA from a calculated baseline was 14.8 ± 15.8 letters for RPh201 and 6.6 ± 15.3 for placebo (P = 0.27). Of the 154 adverse effects (AEs), 52 were considered related to the study procedures/treatment. Across the study and 1,017 injections, the most frequently reported AE was injection site pain (23 events in 5 participants). There were no clinically significant changes in vital signs or laboratory values.
CONCLUSIONS: This Phase 2a was designed to assess safety, feasibility, and explore potential efficacy signals in treating previous NAION with RPh201. No safety concerns were raised. The results support a larger trial in patients with previous NAION.
PMID:31430268 | PMC:PMC6705418 | DOI:10.1097/WNO.0000000000000786
A First-in-Human Phase 1 Randomized Single and Multiple Ascending Dose Study of RPh201 in Healthy Volunteers
Clin Pharmacol Drug Dev. 2020 Apr;9(3):366-374. doi: 10.1002/cpdd.720. Epub 2019 Jun 28.
RPh201 is a drug extracted from gum mastic that has been studied for its anti-inflammatory and antibacterial properties. Preclinical studies of RPh201 demonstrated neuroprotective and neuroenhancing effects. Toxicology studies in animals did not reveal safety concerns or genotoxic effects. This single-center, phase 1, randomized, placebo-controlled, double-masked study in healthy volunteers assessed the safety and tolerability of RPh201, and determined the highest tolerated dose. There were 2 parts: a single ascending dose (SAD) stage, followed by a multiple ascending dose (MAD) stage. Three dosing arms were included in each stage (5 mg, 10 mg, and 20 mg). Safety data in the lower dosing arms were evaluated before higher doses were initiated. Eighteen participants were randomized in the SAD stage: 12 to RPh201 (4 at each dose) and 4 to placebo. Twenty-one participants were randomized in the MAD stage, of which 13 received RPh201. All 18 participants in the SAD stage completed treatment. Sixteen of the 21 participants in the MAD stage completed treatment. The most frequently reported adverse events were local injection site pain and erythema. No deaths or adverse events related to changes in vital signs or electrocardiograms were reported. No occurrences of suicidal behavior or ideation were reported.
PMID:31250992 | PMC:PMC7187404 | DOI:10.1002/cpdd.720
Neuroprotection: Pro-survival and Anti-neurotoxic Mechanisms as Therapeutic Strategies in Neurodegeneration
Front Cell Neurosci. 2019 Jun 6;13:231. doi: 10.3389/fncel.2019.00231. eCollection 2019.
Neurotrophins (NTs) are a subset of the neurotrophic factor family. These growth factors were originally named based on the nerve growth functional assays used to identify them. NTs act as paracrine or autocrine factors for cells expressing NT receptors. The receptors and their function have been studied primarily in cells of the nervous system, but are also present in the cardiovascular, endocrine, and immune systems, as well as in many neoplastic cells. The signals activated by NTs can be varied, depending on cellular stage and context, healthy or disease states, and depending on whether the specific NTs and their receptors are expressed in the relevant cells. In the healthy central and peripheral adult nervous systems, NTs drive neuronal survival, phenotype, synaptic maintenance, and function. Deficiencies of the NT/NT receptor axis are causally associated with disease onset or disease progression. Paradoxically, NTs can also drive synaptic loss and neuronal death. In the embryonic stage this activity is essential for proper developmental pruning of the nervous system, but in the adult it can be associated with neurodegenerative disease. Given their key role in neuronal survival and death, NTs and NT receptors have long been considered therapeutic targets to achieve neuroprotection. The first neuroprotective approaches consisted of enhancing neuronal survival signals using NTs. Later strategies selectively targeted receptors to induce survival signals specifically, while avoiding activation of death signals. Recently, the concept of selectively targeting receptors to reduce neuronal death signals has emerged. Here, we review the rationale of each neuroprotective strategy with respect to the complex cell biology and pharmacology of each target receptor.
PMID:31244606 | PMC:PMC6563757 | DOI:10.3389/fncel.2019.00231
Propagation and Selectivity of Axonal Loss in Leber Hereditary Optic Neuropathy
Sci Rep. 2019 Apr 30;9(1):6720. doi: 10.1038/s41598-019-43180-z.
Leber hereditary optic neuropathy (LHON) is a syndrome of subacute loss of central vision associated with mutations in mitochondrial DNA coding for components of complex I. LHON preferentially involves small axons in the temporal optic nerve, but the reason is unclear. We performed a Monte Carlo simulation of the spread of injury in LHON axons to better understand the predilection for small axons. Optic nerve slices were modeled as grids containing axons with sizes from reported regional distributions. The propagation of injury from a localized concentration of superoxide was simulated as the spread via passive diffusion from one axon to adjacent axons, with basal production and scavenging rate proportional to axonal area and volume, respectively. Axonal degeneration occurred when intra-axonal concentrations reached a toxic threshold. Simulations demonstrated that almost all small and medium axons degenerated by the time steady-state was reached, but about 50% of large axons were preserved. The location of initial injury affected time to steady state, with nasal injuries reaching steady state faster than temporal injuries. The pattern of axonal degeneration in the simulations mirrored both visual fields and optic nerve histology from patients with LHON. These results provide insight into the nature of axonal loss in LHON.
PMID:31040363 | PMC:PMC6491426 | DOI:10.1038/s41598-019-43180-z
Neuroprotection in Optic Neuropathy
Asia Pac J Ophthalmol (Phila). 2018 Jul-Aug;7(4):246-250. doi: 10.22608/APO.2018299. Epub 2018 Aug 1.
Almost all optic neuropathies are untreatable, motivating the search for new therapies that address the final common pathway of optic nerve disease, retinal ganglion cell loss. These neuroprotective strategies have been primarily studied in glaucoma, the most common optic neuropathy, but increasing also tested at the laboratory and animal model level in nonglaucomatous optic neuropathies. More recently, several clinical trials, most of which are randomized, have begun to examine whether neuroprotection is efficacious in human optic nerve disease. Many of these trials are reviewed, along with the critical issues in the major areas of optic neuropathy, particularly the site of injury, the mechanism of axonal damage, and disease-specific features relevant to neuroprotection studies.
PMID:30066502 | DOI:10.22608/APO.2018299
The Academic-Industrial Complexity: Failure to Launch
Trends Pharmacol Sci. 2017 Dec;38(12):1052-1060. doi: 10.1016/j.tips.2017.10.003. Epub 2017 Oct 27.
The pharmaceutical industry has long known that ∼80% of the results of academic laboratories cannot be reproduced when repeated in industry laboratories. Yet academic investigators are typically unaware of this problem, which severely impedes the drug development process. This academic-industrial complication is not one of deception, but rather a complex issue related to how scientific research is carried out and translated in strikingly different enterprises. This Opinion describes the reasons for inconsistencies between academic and industrial laboratories and what can be done to repair this failure of translation.
PMID:29111229 | PMC:PMC5696093 | DOI:10.1016/j.tips.2017.10.003
Cobalamin-Associated Superoxide Scavenging in Neuronal Cells Is a Potential Mechanism for Vitamin B<sub>12</sub>-Deprivation Optic Neuropathy
Am J Pathol. 2018 Jan;188(1):160-172. doi: 10.1016/j.ajpath.2017.08.032. Epub 2017 Oct 14.
Chronic deficiency of vitamin B12 is the only nutritional deficiency definitively proved to cause optic neuropathy and loss of vision. The mechanism by which this occurs is unknown. Optic neuropathies are associated with death of retinal ganglion cells (RGCs), neurons that project their axons along the optic nerve to the brain. Injury to RGC axons causes a burst of intracellular superoxide, which then signals RGC apoptosis. Vitamin B12 (cobalamin) was recently shown to be a superoxide scavenger, with a rate constant similar to superoxide dismutase. Given that vitamin B12 deficiency causes an optic neuropathy through unknown mechanisms and that it is a potent superoxide scavenger, we tested whether cobalamin, a vitamin B12 vitamer, would be neuroprotective in vitro and in vivo. We found that cobalamin scavenged superoxide in neuronal cells in vitro treated with the reduction-oxidation cycling agent menadione. In vivo confocal scanning laser ophthalmoscopy demonstrated that optic nerve transection in Long-Evans rats increased superoxide levels in RGCs. The RGC superoxide burst was significantly reduced by intravitreal cobalamin and resulted in increased RGC survival. These data demonstrate that cobalamin may function as an endogenous neuroprotectant for RGCs through a superoxide-associated mechanism.
PMID:29037851 | PMC:PMC5745528 | DOI:10.1016/j.ajpath.2017.08.032
Glaucoma and the brain: Trans-synaptic degeneration, structural change, and implications for neuroprotection
Surv Ophthalmol. 2018 May-Jun;63(3):296-306. doi: 10.1016/j.survophthal.2017.09.010. Epub 2017 Oct 3.
A recent hypothesis to enter the literature suggests that glaucoma is a neurodegenerative disease. The basis for this has been the finding of central nervous system changes in glaucoma patients on histology and neuroimaging. It is known that retinal ganglion cell pathology of any cause leads to anterograde and retrograde retinal ganglion cell degeneration, as well as trans-synaptic (transneuronal) anterograde degeneration. Trans-synaptic degeneration has been demonstrated in a range of optic neuropathies including optic nerve transection, optic neuritis, and hereditary optic neuropathies. More recently, similar changes have been confirmed in glaucoma patients using the neuroimaging techniques of voxel-based morphometry and diffusion tensor imaging. Some studies have reported brain changes in glaucoma outside the retino-geniculo-cortical pathway; however, these are preliminary and exploratory in nature. Further research is required to identify whether the degenerative brain changes in glaucoma are entirely secondary to the optic neuropathy or whether there is additional primary central nervous system pathology. This has critical implications for neuroprotective and regenerative treatment strategies and our basic understanding of glaucoma.
PMID:28986311 | DOI:10.1016/j.survophthal.2017.09.010
Corticosteroid Therapy in Nonarteritic Anterior Ischemic Optic Neuropathy: Response
J Neuroophthalmol. 2017 Sep;37(3):350. doi: 10.1097/WNO.0000000000000558.
PMID:28759500 | DOI:10.1097/WNO.0000000000000558
Neuroprotection in Glaucoma: Animal Models and Clinical Trials
Annu Rev Vis Sci. 2017 Sep 15;3:91-120. doi: 10.1146/annurev-vision-102016-061422. Epub 2017 Jul 21.
Glaucoma is a progressive neurodegenerative disease that frequently results in irreversible blindness. Glaucoma causes death of retinal ganglion cells (RGCs) and their axons in the optic nerve, resulting in visual field deficits and eventual loss of visual acuity. Glaucoma is a complex optic neuropathy, and a successful strategy for its treatment requires not only better management of known risk factors such as elevated intraocular pressure and the development of improved tools for detecting RGC injury but also treatments that address this injury (i.e., neuroprotection). Experimental models of glaucoma provide insight into the cellular and molecular mechanisms of glaucomatous optic neuropathy and aid the development of neuroprotective therapies.
PMID:28731838 | DOI:10.1146/annurev-vision-102016-061422
Polyester-based microdisc systems for sustained release of neuroprotective phosphine-borane complexes
Pharm Dev Technol. 2018 Nov;23(9):882-889. doi: 10.1080/10837450.2017.1333516. Epub 2017 Jun 9.
Phosphine-borane complexes are recently developed redox-active drugs that are neuroprotective in models of optic nerve injury and radioprotective in endothelial cells. However, a single dose of these compounds is short-lived, necessitating the development of sustained-release formulations of these novel molecules. We screened a library of biodegradable co- and non-block polyester polymer systems for release of incorporated phosphine-borane complexes to evaluate them as drug delivery systems for use in chronic disease. Bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) was combined with biodegradable polymers based on poly(D,L-lactide) (PDLLA), poly(L-lactide) (PLLA), poly(caprolactone) (PCL), poly(lactide-co-glycide) (PLGA), or poly(dioxanone-co-caprolactone) (PDOCL) to make polymer microdiscs, and release over time quantified. Of 22 polymer-PB1 formulations tested, 17 formed rigid polymers. Rates of release differed significantly based on the chemical structure of the polymer. PB1 released from PLGA microdiscs released most slowly, with the most linear release in polymers of 60:40 LA:GA, acid endcap, Mn 15 000-25 000 and 75:25 LA:GA, acid endcap, Mn 45 000-55 000. Biodegradable polymer systems can, therefore, be used to produce sustained-release formulations for redox-active phosphine-borane complexes, with PLGA-based systems most suitable for very slow release. The sustained release could enable translation to a clinical neuroprotective strategy for chronic diseases such as glaucoma.
PMID:28524719 | PMC:PMC6218243 | DOI:10.1080/10837450.2017.1333516
Special Commentary: Early Clinical Development of Cell Replacement Therapy: Considerations for the National Eye Institute Audacious Goals Initiative
Ophthalmology. 2017 Jul;124(7):926-934. doi: 10.1016/j.ophtha.2017.02.017. Epub 2017 Mar 29.
The National Eye Institute launched the Audacious Goals Initiative (AGI) in 2013 with the aim "to restore vision through the regeneration of neurons and neural connections in the eye and visual system." An AGI Town Hall held at the Association for Research in Vision and Ophthalmology Annual Meeting in 2016 brought together basic, translational, and clinical scientists to address the clinical implications of the AGI, with a particular emphasis on diseases amenable to regenerative medicine and strategies to deal with barriers to progess. An example of such a barrier is that replacement of lost neurons may be insufficient because damage to other neurons and non-neuronal cells is common in retinal and optic nerve disease. Reparative processes such as gliosis and fibrosis also can make it difficult to replenish and regenerate neurons. Other issues include choice of animal models, selecting appropriate endpoints, ethics of informed consent, and regulatory issues. Another area critical to next steps in the AGI is the choice of target diseases and the stage at which early development studies should be focused. For example, an advantage of doing clinical trials in patients with early disease is that supporting cellular and structural constituents are still likely to be present. However, regenerative studies in patients with late disease make it easier to detect the effects of replacement therapy against the background of severe visual loss, whereas it may be harder to detect incremental improvement in visual function in those with early disease and considerable remaining visual function. Achieving the goals of the AGI also requires preclinical advances, new imaging techniques, and optimizing translational issues. The work of the AGI is expected to take at least 10 years but should eventually result in therapies to restore some degree of vision to the blind.
PMID:28365209 | PMC:PMC5483186 | DOI:10.1016/j.ophtha.2017.02.017
Axonal Degeneration in Retinal Ganglion Cells Is Associated with a Membrane Polarity-Sensitive Redox Process
J Neurosci. 2017 Apr 5;37(14):3824-3839. doi: 10.1523/JNEUROSCI.3882-16.2017. Epub 2017 Mar 8.
Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (WldS) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and WldS rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized WldS RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the WldS phenotype, whereas extracellular redox modulation reversed the WldS phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.SIGNIFICANCE STATEMENT Axonal degeneration is a neuronal process independent of somal apoptosis, the propagation of which is unclear. We combined single-cell laser axotomy with time-lapse imaging to study the dynamics of phosphatidylserine externalization immediately after axonal injury in purified retinal ganglion cells. The extension of phosphatidylserine externalization was slowed and delayed in Wallerian degeneration slow (WldS) axons and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in WldS axons. These results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.
PMID:28275163 | PMC:PMC5394899 | DOI:10.1523/JNEUROSCI.3882-16.2017
New Treatments in Neuro-Ophthalmology: The Role for Evidence
J Neuroophthalmol. 2017 Mar;37(1):1-2. doi: 10.1097/WNO.0000000000000482.
PMID:28107253 | DOI:10.1097/WNO.0000000000000482
Neuroprotection for glaucoma: Requirements for clinical translation
Exp Eye Res. 2017 Apr;157:34-37. doi: 10.1016/j.exer.2016.12.005. Epub 2016 Dec 9.
Within the field of glaucoma research, neuroprotection is defined as slowing the functional loss in glaucoma by a mechanism independent of lowering of intraocular pressure. There is currently a great potential for research surrounding neuroprotection as it relates to glaucoma. Anatomical targets for neuroprotection should focus on upstream rather than downstream factors, and could include any part of the retinal ganglion cell, the glia, especially astrocytes or Muller cells, and vasculature. The great number of anatomical targets is exceeded only by the number of possible biochemical pathways and potential treatments. Successful treatment may be accomplished through the targeting of one or even a combination of multiple pathways. Once a treatment is shown effective in vitro, it should be evaluated in vivo with carefully chosen animal models and studied in sufficient numbers to detect statistically and clinically significant effects. Such a drug should have few systemic side effects and its delivery should be optimized so as to encourage compliance. There are still a multitude of possible screens available to test the efficacy of a neuroprotective drug and a single gold standard is ideal for the accurate assessment and comparison of new drugs. Future studies in neuroprotection should investigate the genetic component of the disease, novel pharmaceutical agents for new or known pathways, modulations of scleral biomechanics, and relation to research of other complex disorders of the central nervous system.
PMID:27955999 | PMC:PMC5413382 | DOI:10.1016/j.exer.2016.12.005
Detection and measurement of clinically meaningful visual field progression in clinical trials for glaucoma
Prog Retin Eye Res. 2017 Jan;56:107-147. doi: 10.1016/j.preteyeres.2016.10.001. Epub 2016 Oct 20.
Glaucomatous visual field progression has both personal and societal costs and therefore has a serious impact on quality of life. At the present time, intraocular pressure (IOP) is considered to be the most important modifiable risk factor for glaucoma onset and progression. Reduction of IOP has been repeatedly demonstrated to be an effective intervention across the spectrum of glaucoma, regardless of subtype or disease stage. In the setting of approval of IOP-lowering therapies, it is expected that effects on IOP will translate into benefits in long-term patient-reported outcomes. Nonetheless, the effect of these medications on IOP and their associated risks can be consistently and objectively measured. This helps to explain why regulatory approval of new therapies in glaucoma has historically used IOP as the outcome variable. Although all approved treatments for glaucoma involve IOP reduction, patients frequently continue to progress despite treatment. It would therefore be beneficial to develop treatments that preserve visual function through mechanisms other than lowering IOP. The United States Food and Drug Administration (FDA) has stated that they will accept a clinically meaningful definition of visual field progression using Glaucoma Change Probability criteria. Nonetheless, these criteria do not take into account the time (and hence, the speed) needed to reach significant change. In this paper we provide an analysis based on the existing literature to support the hypothesis that decreasing the rate of visual field progression by 30% in a trial lasting 12-18 months is clinically meaningful. We demonstrate that a 30% decrease in rate of visual field progression can be reliably projected to have a significant effect on health-related quality of life, as defined by validated instruments designed to measure that endpoint.
PMID:27773767 | PMC:PMC5313392 | DOI:10.1016/j.preteyeres.2016.10.001
Translational Pharmacology in Glaucoma Neuroprotection
Handb Exp Pharmacol. 2017;242:209-230. doi: 10.1007/164_2016_20.
Glaucoma is both the most common optic neuropathy worldwide and the most common cause of irreversible blindness in the world. The only proven treatment for glaucomatous optic neuropathy is lowering the intraocular pressure, achieved with a variety of pharmacological, laser, and surgical approaches. Over the past 2 decades there has been much basic and clinical research into achieving treatment of the underlying optic nerve damage with neuroprotective approaches. However, none has resulted in regulatory approval based on successful phase 3 studies. This chapter discusses the reasons for this "lost in translation" aspect of glaucoma neuroprotection, and outlines issues at the laboratory and clinical trial level that need to be addressed for successful development of neuroprotective therapies.
PMID:27752844 | DOI:10.1007/164_2016_20
Intracellular disulfide reduction by phosphine-borane complexes: Mechanism of action for neuroprotection
Neurochem Int. 2016 Oct;99:24-32. doi: 10.1016/j.neuint.2016.05.014. Epub 2016 Jun 2.
Phosphine-borane complexes are novel cell-permeable drugs that protect neurons from axonal injury in vitro and in vivo. These drugs activate the extracellular signal-regulated kinases 1/2 (ERK1/2) cell survival pathway and are therefore neuroprotective, but do not scavenge superoxide. In order to understand the interaction between superoxide signaling of neuronal death and the action of phosphine-borane complexes, their biochemical activity in cell-free and in vitro assays was studied by electron paramagnetic resonance (EPR) spectrometry and using an intracellular dithiol reporter that becomes fluorescent when its disulfide bond is cleaved. These studies demonstrated that bis(3-propionic acid methyl ester) phenylphosphine-borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine-borane complex (PB2) are potent intracellular disulfide reducing agents which are cell permeable. EPR and pharmacological studies demonstrated reducing activity but not scavenging of superoxide. Given that phosphine-borane complexes reduce cell injury from mitochondrial superoxide generation but do not scavenge superoxide, this implies a mechanism where an intracellular superoxide burst induces downstream formation of protein disulfides. The redox-dependent cleavage of the disulfides is therefore a novel mechanism of neuroprotection.
PMID:27264910 | PMC:PMC5028261 | DOI:10.1016/j.neuint.2016.05.014
Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine
PLoS One. 2015 Dec 18;10(12):e0145270. doi: 10.1371/journal.pone.0145270. eCollection 2015.
PURPOSE: RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.
METHODS: 661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.
RESULTS: Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.
CONCLUSIONS: 661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.
PMID:26684837 | PMC:PMC4684327 | DOI:10.1371/journal.pone.0145270
Reactive Oxygen Species in Mitochondrial Optic Neuropathies: Response
J Neuroophthalmol. 2015 Dec;35(4):446. doi: 10.1097/WNO.0000000000000323.
PMID:26576024 | DOI:10.1097/WNO.0000000000000323
Glaucoma as a neurodegenerative disease
J Neuroophthalmol. 2015 Sep;35 Suppl 1:S22-8. doi: 10.1097/WNO.0000000000000293.
BACKGROUND: The primary pathophysiological feature of glaucoma is a progressive optic neuropathy with characteristic morphological changes of the optic disc and risk factors of age and intraocular pressure. Recently, involvement of other areas of the central nervous system (CNS) beyond the optic nerve has been demonstrated. This article addresses the proposition that glaucoma shares mechanistic and pathophysiologic features with neurodegenerations in the CNS.
METHODS: The literature on CNS alterations in patients with glaucoma is reviewed with particular focus on neuroimaging and pathological studies. A theoretical framework for assessing whether glaucoma is truly a neurodegenerative disease is developed based on the comparison with neurodegenerative and nonneurodegenerative diseases.
RESULTS: Although there is convincing evidence of abnormalities in CNS regions distal to the optic nerve in glaucoma, these are similar to those seen in other disorders of the proximal visual pathways, such as other optic neuropathies or retinal diseases. Similarly, features of glaucoma that are similar to neurodegenerations are also seen in nonneurodegenerative diseases.
CONCLUSIONS: Glaucoma is less likely a primary neurodegeneration affecting the CNS and more likely a primary optic neuropathy with secondary effects in the CNS.
PMID:26274833 | DOI:10.1097/WNO.0000000000000293
Borane-protected phosphines are redox-active radioprotective agents for endothelial cells
Redox Biol. 2015 Dec;6:73-79. doi: 10.1016/j.redox.2015.06.015. Epub 2015 Jun 26.
Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1 µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1 µM vs. 4 mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity.
PMID:26188467 | PMC:PMC4513185 | DOI:10.1016/j.redox.2015.06.015
Superoxide generation explains common features of optic neuropathies associated with cecocentral scotomas
J Neuroophthalmol. 2015 Jun;35(2):152-60. doi: 10.1097/WNO.0000000000000250.
I have presented above a hypothesis that ties together several disparate optic neuropathies, all characterized by a similar clinical presentation. The hypothesis is predicated on the formation of intracellular superoxide within RGCs as a common pathological pathway for the type of cell death that occurs. The anatomical predisposition of the papillomacular bundle to have elevated superoxide levels is tied to the size of the fibers involved, a hypothesis that also implicates the crossing fibers of the chiasm. Much of this work is speculative and is an interpretation of several experimental studies that have been performed to date. Hopefully, this hypothesis will be developed further, and its validity tested in both experimental models and, ultimately, in humans.
PMID:25985432 | PMC:PMC4581581 | DOI:10.1097/WNO.0000000000000250
Optic nerve disease and axon pathophysiology
Int Rev Neurobiol. 2012;105:1-17. doi: 10.1016/B978-0-12-398309-1.00002-0.
Optic neuropathy is the most common cause of irreversible blindness worldwide. Although the most common optic neuropathy is glaucoma, there are also many other optic neuropathies, for example, those associated with multiple sclerosis, giant cell arteritis, ischemia, and many other diseases. In almost all cases, the pathogenesis involves injury to the retinal ganglion cell axon, with consequent somal and axonal degeneration. This chapter reviews the clinical and pathophysiological properties associated with three of the most common optic neuropathies, as well as recent findings in understanding axonal degeneration. It concludes with a status report on therapies for optic nerve disease, including axoprotection, an approach being studied that has the goal of maintaining axonal integrity and function after injury.
PMID:23206593 | DOI:10.1016/B978-0-12-398309-1.00002-0
Solving the lost in translation problem: improving the effectiveness of translational research
Curr Opin Pharmacol. 2013 Feb;13(1):108-14. doi: 10.1016/j.coph.2012.08.005. Epub 2012 Sep 11.
Translational research frequently fails to replicate in the clinic what has been demonstrated in the laboratory. This has been true for neuroprotection in the central nervous system, neuroprotection in glaucoma, as well as many other areas of medicine. Two fundamental reasons for this 'Lost in Translation' problem are the 'Butterfly Effect' (chaotic behavior of many animal models) and the 'Two Cultures' problem (differences between the methodologies for preclinical and clinical research). We propose several strategies to deal with these issues, including the use of ensembles of animal models, adding intraocular pressure lowering to preclinical neuroprotection studies, changing the way in which preclinical research is done, and increasing interactions between the preclinical and clinical teams.
PMID:22980732 | PMC:PMC3529749 | DOI:10.1016/j.coph.2012.08.005
Retrograde and Wallerian axonal degeneration occur synchronously after retinal ganglion cell axotomy
Am J Pathol. 2012 Jul;181(1):62-73. doi: 10.1016/j.ajpath.2012.03.030. Epub 2012 May 26.
Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration.
PMID:22642911 | PMC:PMC3388161 | DOI:10.1016/j.ajpath.2012.03.030
Redox proteomic identification of visual arrestin dimerization in photoreceptor degeneration after photic injury
Invest Ophthalmol Vis Sci. 2012 Jun 26;53(7):3990-8. doi: 10.1167/iovs.11-9321.
PURPOSE: Light-induced oxidative stress is an important risk factor for age-related macular degeneration, but the downstream mediators of photoreceptor and retinal pigment epithelium cell death after photic injury are unknown. Given our previous identification of sulfhydryl/disulfide redox status as a factor in photoreceptor survival, we hypothesized that formation of one or more disulfide-linked homo- or hetero-dimeric proteins might signal photoreceptor death after light-induced injury.
METHODS: Two-dimensional (non-reducing/reducing) gel electrophoresis of Wistar rat retinal homogenates after 10 hours of 10,000 lux (4200°K) light in vivo, followed by mass spectrometry identification of differentially oxidized proteins.
RESULTS: The redox proteomic screen identified homodimers of visual arrestin (Arr1; S antigen) after toxic levels of light injury. Immunoblot analysis revealed a light duration-dependent formation of Arr1 homodimers, as well as other Arr1 oligomers. Immunoprecipitation studies revealed that the dimerization of Arr1 due to photic injury was distinct from association with its physiological binding partners, rhodopsin and enolase1. Systemic delivery of tris(2-carboxyethyl)phosphine, a specific disulfide reductant, both decreased Arr1 dimer formation and protected photoreceptors from light-induced degeneration in vivo.
CONCLUSIONS: These findings suggest a novel arrestin-associated pathway by which oxidative stress could result in cell death, and identify disulfide-dependent dimerization as a potential therapeutic target in retinal degeneration.
PMID:22599583 | PMC:PMC4453105 | DOI:10.1167/iovs.11-9321
High-content neurite development study using optically patterned substrates
PLoS One. 2012;7(4):e35911. doi: 10.1371/journal.pone.0035911. Epub 2012 Apr 26.
The study of neurite guidance in vitro relies on the ability to reproduce the distribution of attractive and repulsive guidance molecules normally expressed in vivo. The identification of subtle variations in the neurite response to changes in the spatial distribution of extracellular molecules can be achieved by monitoring the behavior of cells on protein gradients. To do this, automated high-content screening assays are needed to quantify the morphological changes resulting from growth on gradients of guidance molecules. Here, we present the use of laser-assisted protein adsorption by photobleaching (LAPAP) to allow the fabrication of large-scale substrate-bound laminin-1 gradients to study neurite extension. We produced thousands of gradients of different slopes and analyzed the variations in neurite attraction of neuron-like cells (RGC-5). An image analysis algorithm processed bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the longest neurite.
PMID:22563416 | PMC:PMC3338543 | DOI:10.1371/journal.pone.0035911
Ordering of neuronal apoptosis signaling: a superoxide burst precedes mitochondrial cytochrome c release in a growth factor deprivation model
Apoptosis. 2012 Jun;17(6):591-9. doi: 10.1007/s10495-012-0714-5.
Axonal injury to retinal ganglion cells, a defined central neuron, induces a burst of intracellular superoxide anion that precedes externalization of membrane phosphatidylserine and subsequent apoptotic cell death. Dismutation of superoxide prevents the signal and delays loss of these cells, consistent with superoxide being necessary for transduction of the axotomy signal. However, phosphatidylserine externalization is a relatively late step in apoptosis, and it is possible that the superoxide burst is not an early axotomy signal but rather a result of cytochrome c release from the mitochondrial inner membrane with consequent accumulation of reduced intermediates. Other possibilities are that both superoxide generation and cytochrome c release are induced in parallel by axotomy, or that cytochrome c release potentiates the effect of the superoxide burst. To distinguish these various possibilities, serum-deprived neuronal retinal cells were assayed in vitro for superoxide elevation and release of cytochrome c from mitochondria, and the distribution of these two markers across a large number of cells used to model the temporal ordering of events. Based on this model of factor-dependent cell death, superoxide precedes, and possibly potentiates, cytochrome c release, and thus the former is likely an early signal for certain types of neuronal apoptosis in the central nervous system.
PMID:22411528 | PMC:PMC3464307 | DOI:10.1007/s10495-012-0714-5
Superoxide signaling and cell death in retinal ganglion cell axotomy: effects of metallocorroles
Exp Eye Res. 2012 Apr;97(1):31-5. doi: 10.1016/j.exer.2012.02.006. Epub 2012 Feb 16.
Injury to retinal ganglion cell (RGC) axons within the optic nerve causes apoptosis of the soma. We previously demonstrated that in vivo axotomy causes elevation of superoxide anion within the RGC soma, and that this occurs 1-2 days before annexin-V positivity, a marker of apoptosis. Pegylated superoxide dismutase delivery to the RGC prevents the superoxide elevation and rescues the soma. Together, these results imply that superoxide is an upstream signal for apoptosis after axonal injury in RGCs. We then studied metallocorroles, potent superoxide dismutase mimetics, which we had shown to be neuroprotective in vitro and superoxide scavengers in vivo for RGCs. RGCs were retrograde labeled with the fluorescent dye 4Di-10Asp, and then axotomized by intraorbital optic nerve transection. Iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (Fe(tpfc)(SO(3)H)(2)) (Fe-corrole) was injected intravitreally. Longitudinal imaging of RGCs was performed and the number of surviving RGCs enumerated. There was significantly greater survival of labeled RGCs with Fe-corrole, but the degree of neuroprotection was relatively less than that predicted by their ability to scavenge superoxide-This implies an unexpected complexity in signaling of apoptosis by reactive oxygen species.
PMID:22366296 | PMC:PMC3479435 | DOI:10.1016/j.exer.2012.02.006
Author response: Bayesian estimation of sensitivity of temporal artery biopsies
Invest Ophthalmol Vis Sci. 2012 Feb 1;53(2):586. doi: 10.1167/iovs.11-9361.
PMID:22298152 | DOI:10.1167/iovs.11-9361
Laser-based single-axon transection for high-content axon injury and regeneration studies
PLoS One. 2011;6(11):e26832. doi: 10.1371/journal.pone.0026832. Epub 2011 Nov 2.
The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.
PMID:22073205 | PMC:PMC3206876 | DOI:10.1371/journal.pone.0026832
Clinical evidence for neuroprotection in glaucoma
Am J Ophthalmol. 2011 Nov;152(5):715-6. doi: 10.1016/j.ajo.2011.06.015.
PMID:22017839 | PMC:PMC3298033 | DOI:10.1016/j.ajo.2011.06.015
A cell-permeable phosphine-borane complex delays retinal ganglion cell death after axonal injury through activation of the pro-survival extracellular signal-regulated kinases 1/2 pathway
J Neurochem. 2011 Sep;118(6):1075-86. doi: 10.1111/j.1471-4159.2011.07382.x. Epub 2011 Aug 12.
The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.
PMID:21749374 | PMC:PMC3166386 | DOI:10.1111/j.1471-4159.2011.07382.x
How should patients with indirect traumatic optic neuropathy be treated?
J Neuroophthalmol. 2011 Jun;31(2):169-74. doi: 10.1097/WNO.0b013e31821c9b11.
PMID:21593629 | PMC:PMC6648694 | DOI:10.1097/WNO.0b013e31821c9b11