Stabilization of primary cilia reduces abortive cell cycle re-entry to protect injured adult CNS neurons from apoptosis.

Abortive cell cycle (ACC) re-entry of apoptotic neurons is a recently characterized phenomenon that occurs after central nervous system (CNS) injury or over the course of CNS disease. Consequently, inhibiting cell cycle progression is neuroprotective in numerous CNS pathology models. Primary cilia are ubiquitous, centriole-based cellular organelles that prevent cell cycling, but their ability to modulate abortive cell cycle has not been described. Here, we show that neuronal cilia are ablated in-vitro and in-vivo following injury by hypoxia or optic nerve transection (ONT), respectively. Furthermore, forced cilia resorption sensitized neurons to these injuries and enhanced cell death. In contrast, pharmacological inhibition or shRNA knockdown of the proteins that disassemble the cilia increased neuron survival and decreased the phosphorylation of retinoblastoma (Rb), a master switch for cell cycle re-entry. Our findings show that the stabilization of neuronal primary cilia inhibits, at least transiently, apoptotic cell cycling, which has implications for future therapeutic strategies that halt or slow the progression of neurodegenerative diseases and acute CNS injuries.

MMP Inhibition Preserves Integrin Ligation and FAK Activation to Induce Survival and Regeneration in RGCs Following Optic Nerve Damage.

Purpose: Integrin adherence to the extracellular matrix (ECM) is essential for retinal ganglion cell (RGC) survival: damage causes production and release of ECM degrading matrix metalloproteinases (MMPs) that disrupt integrin ligation, leading to RGC death. The interplay of MMPs, integrins, and focal adhesion kinase (FAK) was studied in RGCs after optic nerve injury. Methods: Optic nerve transection and optic nerve crush were used to study RGC survival and regeneration, respectively. Treatments were administered intravitreally or into the cut end of the optic nerve. RGC survival was assessed by fluorescence or confocal microscopy; cell counting, peptide levels, and localization were assessed by Western blot and immunohistochemistry. Results: MMP-9 was most strongly increased and localized to RGCs after injury. Pan-MMP, MMP-2/-9, and MMP-3 inhibition all significantly enhanced RGC survival and increased RGC axon regeneration. FAK activation was decreased at 4 days postaxotomy, when apoptosis begins. FAK inhibition reduced RGC survival and abrogated the neuroprotective effects of MMP inhibition, whereas FAK activation increased RGC survival despite MMP activation. Integrin ligation with CD29 antibody or glycine-arginine-glycine-aspatate-serine (GRGDS) peptide increased RGC survival after axotomy. Conclusions: ECM-integrin ligation promotes RGC survival and axon regeneration via FAK activation.

Neurosurgical Modeling of Retinal Ischemia-Reperfusion Injury.

BACKGROUND: A reliable model of ischemia-reperfusion is required to evaluate the efficacy and safety of neuroprotective therapies for stroke. We present a novel reproducible pterygopalatine-ophthalmic artery ligation model of ischemia-reperfusion injury in the retina. METHODS: Rats were subjected to ophthalmic artery/meningeal sheath ligation (OAML-standard method) or clamping of the pterygopalatine-ophthalmic artery (OAC-new method) for 30 minutes. Retinal ganglion cell (RGC) survival was assessed by prelabeling with FluoroGold (FG) (Santa Cruz Biotechnology, CA, USA) and RNA-binding protein with multiple splicing (RBPMS) at 14 days after ischemia, and all results were compared with a sham group (n = 7 in each group). RESULTS: RGC density in the normal-uninjured (FG-labeled) group was 2111 ± 38 cells/mm2 (mean ± standard error of mean) and that in the RBPMS-labeled group was 2142 ± 35 cells/mm2. The OAML procedure significantly reduced RGC density to 738 ± 23 cells/mm2 and 780 ± 41 cells/mm2 (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. Similarly, OAC reduced RGC survival to 782 ± 19 cells/mm2 and 813 ± 22 cells/mm2 (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. RGC survival was similar following OAC and OAML models, suggesting that both induce comparable levels of damage. However, RGC survival in the OAC model was found to have less dispersion than OAML-induced ischemia. CONCLUSIONS: These results suggest that the OAC procedure is a reliable reproduction of ischemia-reperfusion injury that mimics the effects of ophthalmic artery occlusion in humans and provides a useful research model for testing manipulations directed against pathways involved in RGC ischemic degeneration.

Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells.

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.

What can we learn about stroke from retinal ischemia models?

Retinal ischemia is a very useful model to study the impact of various cell death pathways, such as apoptosis and necrosis, in the ischemic retina. However, it is important to note that the retina is formed as an outpouching of the diencephalon and is part of the central nervous system. As such, the cell death pathways initiated in response to ischemic damage in the retina reflect those found in other areas of the central nervous system undergoing similar trauma. The retina is also more accessible than other areas of the central nervous system, thus making it a simpler model to work with and study. By utilizing the retinal model, we can greatly increase our knowledge of the cell death processes initiated by ischemia which lead to degeneration in the central nervous system. This paper examines work that has been done so far to characterize various aspects of cell death in the retinal ischemia model, such as various pathways which are activated, and the role neurotrophic factors, and discusses how these are relevant to the treatment of ischemic damage in both the retina and the greater central nervous system.

Quantitative retinal protein analysis after optic nerve transection reveals a neuroprotective role for hepatoma-derived growth factor on injured retinal ganglion cells.

PURPOSE: Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment and can be modeled by optic nerve transection, which causes the death of 90% of RGCs within 14 days postaxotomy. We performed a proteomic study to identify and quantify proteins in the rat retina after optic nerve transection. Our goal was to isolate potential targets for therapeutic intervention to prevent RGC degeneration. METHODS: iTRAQ proteomics was used to analyze adult rat retinas at 1, 3, 4, 7, 14, and 21 days postaxotomy. Hepatoma-derived growth factor (HDGF), a target identified by iTRAQ, was delivered by intraocular injections. Wortmannin or PD98059 were coadministered with HDGF to determine if the protective effects of HDGF are dependent on PI3 kinase or MAP kinase activity, respectively. RESULTS: At a false-discovery rate of 5%, 216 proteins were identified by iTRAQ proteomics, 71 of which showed changes in expression (<0.7× or >1.3×) at one time point after injury: 52 proteins had expression peaks, whereas 19 showed downward expression spikes. Levels of GAPDH did not change after axotomy. Among these differentially expressed proteins was HDGF. HDGF delivery significantly increased RGC survival compared with control treatments, and increased Akt phosphorylation in the retina at 24 hours after intraocular injection. RGC rescue by HDGF was dependent on both MAP kinase and PI3 kinase activity in the retina. CONCLUSIONS: We have identified numerous proteins that are differentially regulated at key time points after axotomy, and how the temporal profiles of their expression parallel RGC death. Using these data, we showed that HDGF is a potent neuroprotective factor for injured adult RGCs.

Gene therapy for traumatic central nervous system injury and stroke using an engineered zinc finger protein that upregulates VEGF-A.

Recent studies have identified anti-apoptotic functions for vascular endothelial growth factor (VEGF) in the central nervous system (CNS). However, VEGF therapy has been hampered by a tendency to promote vascular permeability, edema, and inflammation. Recently, engineered zinc finger proteins (ZFPs) that upregulate multiple forms of VEGF in their natural biological ratios, have been developed to overcome these negative side effects. We used retinal trauma and ischemia models, and a cortical pial strip ischemia model to determine if VEGF upregulating ZFPs are neuroprotective in the adult CNS. Optic nerve transection and ophthalmic artery ligation lead to the apoptotic degeneration of retinal ganglion cells (RGCs) and are, respectively, two highly reproducible models for CNS trauma or ischemia. Adeno-associated vectors (AAV) vectors encoding VEGF-ZFPs (AAV-VEGF-ZFP) significantly increased RGC survival by ∼twofold at 14 days after optic nerve transection or ophthalmic artery ligation. Furthermore, AAV-VEGF-ZFP enhanced recovery of the pupillary light reflex. RECA-1 immunostaining demonstrated no appreciable differences between retinas treated with AAV-VEGF-ZFP and controls, suggesting that AAV-VEGF-ZFP treatment did not affect retinal vasculature. Following pial strip of the forelimb motor cortex, brains treated with an adenovirus encoding VEGF ZFPs (AdV-ZFP) showed higher neuronal survival, accelerated wound contraction, and reduced lesion volume between 1 and 6 weeks after injury. Behavioral testing using the cylinder test for vertical exploration showed that AdV-VEGF-ZFP treatment enhanced contralateral forelimb function within the first 2 weeks after injury. Our results indicate that VEGF ZFP therapy is neuroprotective following traumatic injury or stroke in the adult mammalian CNS.

Involvement of caspase-6 and caspase-8 in neuronal apoptosis and the regenerative failure of injured retinal ganglion cells.

To promote functional recovery after CNS injuries, it is crucial to develop strategies that enhance both neuronal survival and regeneration. Here, we report that caspase-6 is upregulated in injured retinal ganglion cells and that its inhibition promotes both survival and regeneration in these adult CNS neurons. Treatment of rat retinal whole mounts with Z-VEID-FMK, a selective inhibitor of caspase-6, enhanced ganglion cell survival. Moreover, retinal explants treated with this drug extended neurites on myelin. We also show that caspase-6 inhibition resulted in improved ganglion cell survival and robust axonal regeneration following optic nerve injury in adult rats. The effects of Z-VEID-FMK were similar to other caspase inhibitory peptides including Z-LEHD-FMK and Z-VAD-FMK. In searching for downstream effectors for caspase-6, we identified caspase-8, whose expression pattern resembled that of caspase-6 in the injured eye. We then showed that caspase-8 is activated downstream of caspase-6 in the injured adult retina. Furthermore, we investigated the role of caspase-8 in RGC apoptosis and regenerative failure both in vitro and in vivo. We observed that caspase-8 inhibition by Z-IETD-FMK promoted survival and regeneration to an extent similar to that obtained with caspase-6 inhibition. Our results indicate that caspase-6 and caspase-8 are components of a cellular pathway that prevents neuronal survival and regeneration in the adult mammalian CNS.

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush.

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ∼ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-ß4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-ß4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-ß4 significantly increased RGC survival by ∼ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.

Optic nerve transection: a model of adult neuron apoptosis in the central nervous system.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS (1-4). This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs (siRNAs), plasmids, or viral vectors to the cut end of the optic nerve (5-7) or injecting vectors into their target, the superior colliculus (8). This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy (9-11). RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for experimental manipulations directed against pathways involved in apoptosis.

Methods for experimental manipulations after optic nerve transection in the Mammalian CNS.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS (1-4). This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells (5-12). The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site (6, 7, 13-15). Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve (16-19) or injecting vectors into their target the superior colliculus (10). This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.