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.
Glaucoma and the brain: trans-synaptic degeneration, structural change and implications for neuroprotection.
Surv Ophthalmol. 2017 Oct 03;:
Authors: Lawlor M, Danesh-Meyer H, Levin LA, Davagnanam I, De Vita E, Plant GT
A recent hypothesis to enter the literature suggests that glaucoma is a neuro-degenerative disease. The basis for this has been the finding of central nervous system (CNS) changes in glaucoma patients on histology and neuro-imaging. It is known that retinal ganglion cell (RGC) pathology of any cause leads to anterograde and retrograde RGC degeneration, as well as trans-synaptic (trans-neuronal) 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 CNS pathology. This has critical implications for neuroprotective and regenerative treatment strategies, and our basic understanding of glaucoma.
PMID: 28986311 [PubMed - as supplied by publisher]
The Academic-Industrial Complexity: Failure to Launch.
Trends Pharmacol Sci. 2017 Oct 27;:
Authors: Levin LA, Behar-Cohen F
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 [PubMed - as supplied by publisher]
Cobalamin-Associated Superoxide Scavenging in Neuronal Cells Is a Potential Mechanism for Vitamin B12-Deprivation Optic Neuropathy.
Am J Pathol. 2017 Oct 13;:
Authors: Chan W, Almasieh M, Catrinescu MM, Levin LA
Chronic deficiency of vitamin B12 is the only nutritional deficiency definitively proven 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 redox-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 [PubMed - as supplied by publisher]
Neuroprotection in Glaucoma: Animal Models and Clinical Trials.
Annu Rev Vis Sci. 2017 Jul 21;:
Authors: Almasieh M, Levin LA
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. Expected final online publication date for the Annual Review of Vision Science Volume 3 is September 15, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
PMID: 28731838 [PubMed - as supplied by publisher]
Polyester-Based Microdisc Systems for Sustained Release of Neuroprotective Phosphine-Borane Complexes.
Pharm Dev Technol. 2017 May 19;:1-32
Authors: Janus DA, Lieven CJ, Crowe ME, Levin LA
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 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. Sustained release could enable translation to a clinical neuroprotective strategy for chronic diseases such as glaucoma.
PMID: 28524719 [PubMed - as supplied by publisher]
Axonal Degeneration in Retinal Ganglion Cells is Associated with a Membrane Polarity-Sensitive Redox Process.
J Neurosci. 2017 Mar 08;:
Authors: Almasieh M, Catrinescu MM, Binan L, Costantino S, Levin LA
Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (Wld(S)) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight for 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 Wld(S) 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 Wld(S) RGCs, compared to wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, significantly slowed the onset and velocity of phosphatidylserine externalization in wild-type axons, replicating the Wld(S) phenotype, while extracellular redox modulation reversed the Wld(S) 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 STATEMENTAxonal 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 (Wld(S)) axons, and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in Wld(S) axons. Together, these results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.
PMID: 28275163 [PubMed - as supplied by publisher]
Intracellular disulfide reduction by phosphine-borane complexes: Mechanism of action for neuroprotection.
Neurochem Int. 2016 Oct;99:24-32
Authors: Niemuth NJ, Thompson AF, Crowe ME, Lieven CJ, Levin LA
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 [PubMed - in process]
Detection and measurement of clinically meaningful visual field progression in clinical trials for glaucoma.
Prog Retin Eye Res. 2016 Oct 20;:
Authors: De Moraes CG, Liebmann JM, Levin LA
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 [PubMed - as supplied by publisher]
Translational Pharmacology in Glaucoma Neuroprotection.
Handb Exp Pharmacol. 2016 Oct 18;
Authors: Levin LA
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 [PubMed - as supplied by publisher]
Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.
PLoS One. 2015;10(12):e0145270
Authors: Thompson AF, Crowe ME, Lieven CJ, Levin LA
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 [PubMed - in process]
Solving the lost in translation problem: improving the effectiveness of translational research.
Curr Opin Pharmacol. 2013 Feb;13(1):108-14
Authors: Ergorul C, Levin LA
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 [PubMed - indexed for MEDLINE]
Retrograde and Wallerian axonal degeneration occur synchronously after retinal ganglion cell axotomy.
Am J Pathol. 2012 Jul;181(1):62-73
Authors: Kanamori A, Catrinescu MM, Belisle JM, Costantino S, Levin LA
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 [PubMed - indexed for MEDLINE]
Redox proteomic identification of visual arrestin dimerization in photoreceptor degeneration after photic injury.
Invest Ophthalmol Vis Sci. 2012 Jun;53(7):3990-8
Authors: Lieven CJ, Ribich JD, Crowe ME, Levin LA
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 [PubMed - indexed for MEDLINE]
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
Authors: Lieven CJ, Thurber KA, Levin EJ, Levin LA
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 [PubMed - indexed for MEDLINE]
Superoxide signaling and cell death in retinal ganglion cell axotomy: effects of metallocorroles.
Exp Eye Res. 2012 Apr;97(1):31-5
Authors: Catrinescu MM, Chan W, Mahammed A, Gross Z, Levin LA
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 [PubMed - indexed for MEDLINE]
Optic nerve disease and axon pathophysiology.
Int Rev Neurobiol. 2012;105:1-17
Authors: Ghaffarieh A, Levin LA
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 [PubMed - indexed for MEDLINE]
High-content neurite development study using optically patterned substrates.
PLoS One. 2012;7(4):e35911
Authors: Bélisle JM, Levin LA, Costantino S
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 [PubMed - indexed for MEDLINE]
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
Authors: Almasieh M, Lieven CJ, Levin LA, Di Polo A
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 [PubMed - indexed for MEDLINE]
Laser-based single-axon transection for high-content axon injury and regeneration studies.
PLoS One. 2011;6(11):e26832
Authors: Kunik D, Dion C, Ozaki T, Levin LA, Costantino S
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 [PubMed - indexed for MEDLINE]
Superoxide is an associated signal for apoptosis in axonal injury.
Brain. 2010 Sep;133(9):2612-25
Authors: Kanamori A, Catrinescu MM, Kanamori N, Mears KA, Beaubien R, Levin LA
Optic neuropathy is the leading cause of irreversible blindness, and a paradigm for central nervous system axonal disease. The primary event is damage to retinal ganglion cell axons, with subsequent death of the cell body by apoptosis. Trials of neuroprotection for these and other neuronal diseases have mostly failed, primarily because mechanisms of neuroprotection in animals do not necessarily translate to humans. We developed a methodology for imaging an intracellular transduction pathway that signals neuronal death in the living animal. Using longitudinal confocal scanning multilaser ophthalmoscopy, we identified the production of superoxide within retrograde-labelled rat retinal ganglion cells after optic nerve transection. Superoxide was visualized by real-time imaging of its reaction product with intravitreally administered hydroethidine and confirmed by differential spectroscopy of the specific product 2-hydroxyethidium. Retinal ganglion cell superoxide increased within 24 h after axotomy, peaking at 4 days, and was not observed in contralateral untransected eyes. The superoxide signal preceded phosphatidylserine externalization, indicating that superoxide generation was an early event and preceded apoptosis. Intravitreal pegylated superoxide dismutase blocked superoxide generation after axotomy and delayed retinal ganglion cell death. Together, these results are consistent with superoxide being an upstream signal for retinal ganglion cell apoptosis after optic nerve injury. Early detection of axonal injury with superoxide could serve as a predictive biomarker for patients with optic neuropathy.
PMID: 20495185 [PubMed - indexed for MEDLINE]
Neuroprotection against superoxide anion radical by metallocorroles in cellular and murine models of optic neuropathy.
J Neurochem. 2010 Jul;114(2):488-98
Authors: Kanamori A, Catrinescu MM, Mahammed A, Gross Z, Levin LA
Corroles are tetrapyrrolic macrocycles that have come under increased attention because of their unique capabilities for oxidation catalysis, reduction catalysis, and biomedical applications. Corrole-metal complexes (metallocorroles) can decompose certain reactive oxygen species (ROS), similar to metalloporphyrins. We investigated whether Fe-, Mn-, and Ga-corroles have neuroprotective effects on neurons and correlated this with superoxide scavenging activity in vitro and in vivo. Apoptosis was induced in retinal ganglion cell-5 neuronal precursor cells by serum deprivation. Cell death was measured with sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate and calcein-AM/propidium iodide assays. Fe- and Mn-corroles, but not the non-redox-active Ga-corrole used as control, reduced RGC-5 cell death after serum deprivation. Serum deprivation caused increased levels of intracellular superoxide, detected by an increase in the fluorescence intensity of 2-hydroxyethidium, and this was blocked by Fe- and Mn-corroles, but not Ga-corrole. In vivo real-time confocal imaging of retinas after optic nerve transection assessed the superoxide production within individual rat retinal ganglion cells. Fe- and Mn-corroles, but not Ga-corrole, scavenged neuronal superoxide in vivo. Given that the neuroprotective activity of metallocorroles correlated with superoxide scavenging activity, Fe- and Mn-corroles could be candidate drugs for delaying neuronal death after axonal injury in optic neuropathies, such as glaucoma.
PMID: 20456018 [PubMed - indexed for MEDLINE]
Serial multifocal electroretinograms during long-term elevation and reduction of intraocular pressure in non-human primates.
Doc Ophthalmol. 2010 Jun;120(3):273-89
Authors: Nork TM, Kim CB, Heatley GA, Kaufman PL, Lucarelli MJ, Levin LA, Ver Hoeve JN
The purpose of this study was to evaluate the relationship between elevations of intraocular pressure (IOP) and the multifocal electroretinogram (mfERG) in non-human primates. Experimental glaucoma was induced in 4 rhesus and 4 cynomolgus monkeys by laser trabecular meshwork destruction (LTD) in one eye. To evaluate the contribution of ganglion cells to mfERG changes, one monkey of each species had previously underwent unilateral optic nerve transection (ONT). After >or=44 weeks of elevation, the IOP was reduced by trabeculectomy in 2 non-transected animals. In the intact (non-transected) animals, there was an increase in the amplitude of the early mfERG waveforms (N1 and P1) of the first-order kernel (K1) throughout the period of IOP elevation in all of the rhesus, but not all of the cynomolgus monkeys. A species difference was also present as a decrease of the second-order kernel, first slice (K2.1) in all of the cynomolgus monkeys but only in 1 of the rhesus monkeys (the 1 with the ONT). Similar IOP effects on the mfERG were seen in the ONT animals. Surgical lowering of IOP resulted in a return of the elevated K1 amplitudes to baseline levels. However, the depressed K2.1 RMS in the cynomolgus monkeys did not recover. These results demonstrate species-specific changes in cone-driven retinal function during periods of elevated IOP. These IOP-related effects can occur in the absence of retinal ganglion cells and may be reversible.
PMID: 20422254 [PubMed - indexed for MEDLINE]
Overexpression of Bcl-2 in vascular endothelium inhibits the microvascular lesions of diabetic retinopathy.
Am J Pathol. 2010 May;176(5):2550-8
Authors: Kern TS, Du Y, Miller CM, Hatala DA, Levin LA
Recent studies on the pathogenesis of diabetic retinopathy have focused on correcting adverse biochemical alterations, but there have been fewer efforts to enhance prosurvival pathways. Bcl-2 is the archetypal member of a group of antiapoptotic proteins. In this study, we investigated the ability of overexpressing Bcl-2 in vascular endothelium to protect against early stages of diabetic retinopathy. Transgenic mice overexpressing Bcl-2 regulated by the pre-proendothelin promoter were generated, resulting in increased endothelial Bcl-2. Diabetes was induced with streptozotocin, and mice were sacrificed at 2 months of study to measure superoxide generation, leukostasis, and immunohistochemistry, and at 7 months to assess retinal histopathology. Diabetes of 2 months duration caused a significant decrease in expression of Bcl-2 in retina, upregulation of Bax in whole retina and isolated retinal microvessels, and increased generation of retinal superoxide and leukostasis. Seven months of diabetes caused a significant increase in the number of degenerate (acellular) capillaries in diabetic animals. Furthermore, overexpression of Bcl-2 in the vascular endothelium inhibited the diabetes-induced degeneration of retinal capillaries and aberrant superoxide generation, but had no effect on Bax expression or leukostasis. Therefore, overexpression of Bcl-2 in endothelial cells inhibits the capillary degeneration that is characteristic of the early stages of diabetic retinopathy, and this effect seems likely to involve inhibition of oxidative stress.
PMID: 20363911 [PubMed - indexed for MEDLINE]
In vivo imaging of retinal ganglion cell axons within the nerve fiber layer.
Invest Ophthalmol Vis Sci. 2010 Apr;51(4):2011-8
Authors: Kanamori A, Catrinescu MM, Traistaru M, Beaubien R, Levin LA
Purpose. Optic nerve injury causes loss of retinal ganglion cells (RGCs) and their axons. The reduction in RGC counts over time in axonal injury is well studied, but the correlation with the timing of anterograde and retrograde axonal degeneration is less clear. The authors longitudinally imaged RGC axons stained with a chloromethyl derivative of fluorescein diacetate (CMFDA) in live rats after optic nerve injury. Methods. Optic nerves were transected. Three days later CMFDA was intravitreously injected. Confocal scanning laser ophthalmoscopy was performed daily, and mean fluorescence intensity and the number of CMFDA bundles were calculated. RGC soma survival was studied after retrograde fluorescence labeling. Retinal nerve fiber layer (RNFL) thickness was evaluated histologically. Results. CMFDA-positive RGC axon bundles could be imaged in vivo. Axons lost 68% +/- 29% of their fluorescence by 7 days after transection compared with 25% +/- 21% in nontransected eyes. The number of labeled axon bundles decreased by 61% +/- 28% at 7 days after transection compared with 26% +/- 9% in nontransected eyes. The number of retrograde-labeled RGCs detected in vivo declined by 53% at 7 days and by 76% at 14 days after transection. RGC soma and CMFDA axon counts decreased most rapidly between 5 and 7 days after transection. Histologic examination demonstrated a reduction in RNFL thickness 7 days after transection. Conclusions. Intravitreal CMFDA can be used to longitudinally monitor RGC axons within the RNFL in vivo. Imaging the disappearance of retrograde-labeled RGC somas and axons indicates that axonal and somal degeneration occur in parallel after axotomy.
PMID: 19797216 [PubMed - indexed for MEDLINE]
Neuronal differentiation by analogs of staurosporine.
Neurochem Int. 2010 Mar;56(4):554-60
Authors: Thompson AF, Levin LA
RGC-5 cells are transformed cells that express several surface markers characteristic of neuronal precursor cells, but resemble glial cells morphologically and divide in culture. When treated with the apoptosis-inducing agent staurosporine, RGC-5 cells assume a neuronal morphology, extend neurites, stop dividing, and express ion channels without acute signs of apoptosis. This differentiation with staurosporine is similar to what has been described for certain other neuronal cell lines, and occurs by a mechanism not yet understood. Inhibition of several kinases known to be inhibited by staurosporine fails to differentiate RGC-5 cells, and examination of the kinome associated with staurosporine-dependent differentiation has been unhelpful so far. To better understand the mechanism of staurosporine-mediated differentiation of neuronal precursor cells, we studied the effects of the following structurally similar molecules on differentiation of neuronal and non-neuronal cell lines, comparing them to staurosporine: 9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-, methyl ester, (9S,10R,12R)-(K252a), (5R,6S,8S)-6-hydroxy-5-methyl-13-oxo-6,7,8,13,14,15-hexahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacene-6-carboxylic acid (K252b), staurosporine aglycone (K252c), 7-hydroxystaurosporine (UCN-01), and 4'-N-benzoylstaurosporine (PKC-412). Morphological differentiation, indicated by neurite extension and somal rounding, was quantitatively assessed with NeuronJ. We found that the critical structural component for differentiation in RGC-5 cells is a basic amine adjacent to an accessible methoxy group at the 3' carbon. Given that UCN-01 and similar compounds are potent anti-cancer drugs, examination of molecules that share similar structural features may yield insights into the design of other drugs for differentiation.
PMID: 20043966 [PubMed - indexed for MEDLINE]
Effectiveness of Novel Borane-Phosphine Complexes In Inhibiting Cell Death Depends on the Source of Superoxide Production Induced by Blockade of Mitochondrial Electron Transport.
ACS Chem Neurosci. 2010 Feb 17;1(2):95-103
Authors: Seidler EA, Lieven CJ, Thompson AF, Levin LA
Central neurons undergo cell death after axotomy. One of the signaling pathways for this process is oxidative modification of one or more critical sulfhydryls in association with superoxide generation within mitochondria. Agents that reduce oxidized sulfhydryls are neuroprotective of axotomized retinal ganglion cells, and we hypothesized that this occurs via reversal of the effects of mitochondrial-produced superoxide. To study this, we measured the ability of the novel borane-phosphine complex drugs bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester)diphenylphosphine borane complex (PB2) to inhibit the death of neuron-like RGC-5 cells induced by perturbation of the mitochondrial electron transport chain. We found that borane-phosphine complexes prevent neuronal cell death from superoxide produced by the redox-cycling agent menadione and the complex III inhibitor antimycin A, which produce superoxide towards the cytoplasm and matrix, but not the complex I inhibitor rotenone, which produces superoxide in the matrix alone. The ability of these disulfide reductants to prevent cell death may be predicted by the topology of superoxide production with respect to the mitochondrial matrix and extramitochondrial space.
PMID: 20532184 [PubMed - as supplied by publisher]
Cell-autonomous generation of mitochondrial superoxide is a signal for cell death in differentiated neuronal precursor cells.
Brain Res. 2010 Jan 8;1306:142-8
Authors: Scott CJ, Seidler EA, Levin LA
The vast majority of optic neuropathies result from retinal ganglion cell (RGC) axonal injury. This induces cell death and is associated with a burst of mitochondria-generated superoxide within the soma. It is unclear whether there is a clear causal relationship between superoxide generation and cell death. To determine whether mitochondrial-generated superoxide can cause cell-autonomous death signaling, we knocked down SOD2 in a pure population of RGC-5 cells, a neuronal precursor cell line that can be differentiated to resemble retinal ganglion cells. RGC-5 cells were differentiated and transfected with siRNA for SOD2 or a scramble control. Viability, superoxide production, cytotoxic RNA transfection efficiency, and measurement of SOD2 protein levels by immunoblotting were assayed at varying times after transfection. SOD2 knockdown increased intracellular superoxide levels and cell death was presumed triggered from knockdown. This was amplified when extramitochondrial superoxide was elevated with the redox cycling agent menadione. Dysregulation of mitochondrial superoxide in differentiated RGC-5 cells is likely a potent signal for cell death, consistent with a role of this reactive oxygen species in apoptosis signaling after axonal injury.
PMID: 19819231 [PubMed - indexed for MEDLINE]