Primary Retinal Ganglion Cells Research Articles

Mitochondrial inhibition of retinal Müller cells alter glutamate homeostasis and their ability to sustain retinal ganglion cells

PurposeGlia‐neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing neurons with energy sources. In this study, we evaluated consequences of mitochondrial inhibition on retinal Müller cells and their ability to sustain RGCs.MethodsThe human Müller glial cell line, MIO‐M1, as well as mono‐ and co‐cultures of primary C56Bl/6 mouse Müller cells and RGCs, was used as cellular models. Cells were treated with 10 uM antimycin A to inhibit mitochondrial function. Changes in glutamate uptake in Müller cells were examined by kinetic assays with 3H‐l‐glutamate. Cell viability was evaluated by LDH assays. Regulations in gene and protein expression were evaluated by qPCR and western blotting.ResultsMitochondrial inhibition significantly reduced protein‐ and mRNA expression of the major glutamate transporter, EAAT1, in Müller cells. Moreover, mitochondrial inhibition significantly decreased Müller cell glutamate uptake. Mitochondrial inhibition solely did not effect cell viability neither in Müller cells nor in RGC. However, simultaneous mitochondrial inhibition and starvation significantly decreased survival of both cell types. The protective effect of Müller cells in co‐cultures of primary RGCs and MCs were attenuated during mitochondrial inhibition.ConclusionsInhibition of mitochondrial function alter the neuroprotective characteristics of Müller cells by decreasing the expression of glutamate transporter and functional reduction of glutamate uptake. Furthermore, the impaired mitochondrial activity may affect the ability of Müller cells to maintain a cellular homeostasis in such way that their ability to protect RGCs may to suffer.

Müller cells increase survival of retinal ganglion cells – a coculture model of primary retinal ganglion cells and primary Müller cells

PurposeMüller cells are considered to be vital in the maintenance of retinal ganglion cells (RGCs), and since RGCs are essential to maintain the neuronal function of the retina, a functioning symbiotic partnership between Müller cells and RGCs is fundamental. The present study evaluates glia‐neuron interactions in a coculture model of primary Müller cells and primary RGCs.MethodsTo investigate the Müller cell‐RGC interaction we developed a coculture model, in which primary Müller cells from mice were grown in inserts on top of pure primary RGC cultures likewise from mice. The impact of 24 h of starvation on the ability of Müller cells to protect RGCs was evaluated. Moreover, changes in glutamate uptake were studied in Müller cells as well as cell viability in response to starvation.ResultsThe presence of Müller cells significantly increased the survival of RGCs during normal conditions as well as during 24 h of pre‐starvation. Glutamate uptake capacity was significantly increased during starvation along with an increased Vmax. Starvation induced significantly reduced cell viability both in Müller cells and RGCs with a significantly greater reduction of cell viability in Müller cells.ConclusionsThe present study reveals an increased survival of RGCs in presence of Müller cells and herewith we confirm Müller cells as being key players in RGC homeostasis. Moreover, we verify the coculture model as being a unique approach to study the glia‐neuron partnership in inner retinal diseases.

Avian axons undergo Wallerian degeneration after injury and stress.

The integrity of long axons is essential for neural communication. Unfortunately, relatively minor stress to a neuron can cause extensive loss of this integrity. Axon degeneration is the cell-intrinsic program that actively deconstructs an axon after injury or damage. Although ultrastructural examination has revealed signs of axon degeneration in vivo, the occurrence and progression of axon degeneration in avian species have not yet been documented in vitro. Here, we use a novel cell culture system with primary embryonic zebra finch retinal ganglion cells to interrogate the properties of avian axon degeneration. First, we establish that both axotomy and a chemically induced injury (taxol and vincristine) are sufficient to initiate degeneration. These events are dependent on a late influx of calcium. In addition, as in mammals, the NAD pathway is involved, since a decrease in NMN with FK866 can reduce degeneration. Importantly, these retinal ganglion cell axons were sensitive to a pressure-induced injury, which may mimic the effect of high intraocular pressure associated with glaucoma. We have demonstrated that avian neurons undergo Wallerian degeneration in response to both physical and chemical injury. Subsequent avian studies will investigate whether blocking the degeneration pathway can protect individuals from neurodegenerative disease.

Puerarin Attenuates N-Methyl-D-aspartic Acid-induced Apoptosis and Retinal Ganglion Cell Damage Through the JNK/p38 MAPK Pathway.

To explore the protective effect of puerarin on N-methyl-D-aspartic acid (NMDA)-induced retinal ganglion cells (RGCs) injury and its underlying mechanism. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. NMDA was used to mimic the glutamate activation, cell apoptosis, reactive oxygen species (ROS), malondialdehyde levels, SOD and NO production, nNOS and iNOS expression, as well as caspase-3 activity, Bcl-2, and Bax expression in the RGCs were analyzed by ELISA, RT-PCR, and Western blotting. A rat model of retinal injury was used to detect the protective effect of puerarin. Puerarin protected against NMDA-induced RGCs injury in a dose-dependent manner. Compared with the NMDA-treated group, puerarin pretreatment significantly reduced ROS and malondialdehyde levels, promoted SOD and NO production, and downregulated nNOS and iNOS expression in the RGCs. Mechanism analysis showed that pretreatment with puerarin could effectively offset the increase of Bax expression and caspase-3 activity brought by NMDA, and promote Bcl-2 expression in the RGCs. Puerarin pretreatment also effectively inhibited NMDA-induced JNK and p38 phosphorylation in the RGCs, whereas pretreatment with either JNK agonist anisomycin or p38 agonist P79350 could significantly compensate the effects caused by puerarin. Furthermore, puerarin prevented RGCs loss in the retinal injury induced by intravitreal NMDA in a rat model. The present results of this study demonstrated that puerarin protected against NMDA-induced apoptosis and RGCs damage through the JNK/p38 MAPK pathway.

Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

Abstract The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non‐coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up‐regulated in the retinas, Muller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Muller cell activation, and RGC survival in vivo and in vitro . MALAT1‐CREB binding maintains CREB phosphorylation by inhibiting PP2A‐mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling.

A Self-Assembling Injectable Biomimetic Microenvironment Encourages Retinal Ganglion Cell Axon Extension in Vitro.

Sensory-somatic nervous system neurons, such as retinal ganglion cells (RGCs), are typically thought to be incapable of regenerating. However, it is now known that these cells may be stimulated to regenerate by providing them with a growth permissive environment. We have engineered an injectable microenvironment designed to provide growth-stimulating cues for RGC culture. Upon gelation, this injectable material not only self-assembles into laminar sheets, similar to retinal organization, but also possesses a storage modulus comparable to that of retinal tissue. Primary rat RGCs were grown, stained, and imaged in this three-dimensional scaffold. We were able to show that RGCs grown in this retina-like structure exhibited characteristic long, prominent axons. In addition, RGCs showed a consistent increase in average axon length and neurite-bearing ratio over the 7 day culture period, indicating this scaffold is capable of supporting substantial RGC axon extension.

Nonlinear computations shaping temporal processing of precortical vision.

Computations performed by the visual pathway are constructed by neural circuits distributed over multiple stages of processing, and thus it is challenging to determine how different stages contribute on the basis of recordings from single areas. In the current article, we address this problem in the lateral geniculate nucleus (LGN), using experiments combined with nonlinear modeling capable of isolating various circuit contributions. We recorded cat LGN neurons presented with temporally modulated spots of various sizes, which drove temporally precise LGN responses. We utilized simultaneously recorded S-potentials, corresponding to the primary retinal ganglion cell (RGC) input to each LGN cell, to distinguish the computations underlying temporal precision in the retina from those in the LGN. Nonlinear models with excitatory and delayed suppressive terms were sufficient to explain temporal precision in the LGN, and we found that models of the S-potentials were nearly identical, although with a lower threshold. To determine whether additional influences shaped the response at the level of the LGN, we extended this model to use the S-potential input in combination with stimulus-driven terms to predict the LGN response. We found that the S-potential input "explained away" the major excitatory and delayed suppressive terms responsible for temporal patterning of LGN spike trains but revealed additional contributions, largely PULL suppression, to the LGN response. Using this novel combination of recordings and modeling, we were thus able to dissect multiple circuit contributions to LGN temporal responses across retina and LGN, and set the foundation for targeted study of each stage.

A localized autophagic filter prevents entry of mitochondria carrying pathogenic Opa1 mutations in retinal ganglion cell axons

Mitochondria are dynamic organelles that are endowed by a complex fission/fusion machinery. Fission is regulated by the Dynamin related protein 1 (DRP1), which translocates to mitochondria by calcineurin dependent dephosphorylation (Yoon et al., 2001;Smirnova et al., 2001a;Cereghetti et al., 2008a). The two Mitofusins orchestrate the fusion of the outer mitochondrial membrane (Santel and Fuller, 2001a;Legros et al., 2002a;Chen et al., 2003a;Santel et al., 2003a), while Optic Atrophy 1 (OPA1), a dynamin related-protein, cooperates with MFN1 in fusion of the inner mitchondrial membrane (Cipolat et al., 2004b). OPA1 has a key role in regulating the shape of mitochondrial cristae, pleomorphic structures of the inner mitochondrial membrane. In particular, OPA1 is localized at the level of cristae junctions, forming oligomers that regulate the opening of the cristae and thus, the release of cytochrome c, a soluble factor involved in apoptosis (Cipolat et al., 2006b;Frezza et al., 2006a). Recently, it has been shown that OPA1 has an additional role in the assembly of respiratory chain supercomplexes by modulating the ultrastructure of the inner mitochondrial membrane (Cogliati et al., 2013). Opa1 is mutated in autosomal dominant optic atrophy (ADOA), the most common of inherited optic neuropathy, caused by the selective loss of retinal ganglion cells (RGCs). The pathophysiology of ADOA is still unknown. Evidences from in vitro studies on neuronal cultures reveal mitochondrial abnormalities in morphology and function: mitochondria appear fragmented, improperly accumulated in the soma, and with impaired potential and Ca2+ buffer capacity (Kamei et al., 2005;Dayanithi et al., 2010;Bertholet et al., 2013;Kushnareva et al., 2013). In neurons, mitochondria are transported along microtubules in sites of high energy demand as Ranvier Nodes and synapses, where they buffer Ca2+ and produce ATP to sustain neuronal activity (Sheng and Cai, 2012;Itoh et al., 2013). Moreover, many neurodegenerative diseases, as Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis, present a strong relationship between dysfunctional mitochondrial and autophagy (Schapira et al., 1990;Betarbet et al., 2000;Nixon et al., 2005;Sasaki et al., 2005;Magrane et al., 2009;Yao et al., 2009;Chinta et al., 2010). The degradation of dysfunctional mitochondria trough autophagy is a process called mitophagy. Fragmented and stationary mitochondria are easily engulfed by a double-membrane vesicle, the autophagosome, and subsequently degraded (Twig et al., 2010). Depolarization of mitochondria triggers the activation of the Pink1/Parkin pathway (Greene et al., 2003;Park et al., 2006;Narendra et al., 2008;Matsui et al., 2013), which add ubiquitin chains on proteins of the outer mitochondria membrane targeting mitochondria to the autophagosome (Gegg et al., 2010;Geisler et al., 2010;Ziviani et al., 2010a;Chan et al., 2011). In 2007, Davies and collegues have demostransted a central role of autophagy in ADOA. In particular, ultrastructural analysis revealed increased autophagic vesicles preceding optic atrophy in an ADOA mouse model (Davies et al., 2007;White et al., 2009). RGCs are the adequate model to study ADOA pathology. These neurons are enriched in mitochondria in the proximal segment of the axon, that has high energy demand to sustain action potentials (Carelli et al., 2004). OPA1 inactivation could be particularly important in RGCs, which have a poor glycolitic activity and thus depend almost exclusively on the energy provided by mitochondria. Therefore, the aim of this thesis was to study the impact of dysfunctional mitochondria carrying Opa1 pathogenic mutants in primary RGCs. In this model, the expression of Opa1 mutants caused mitochondrial dysfunction, including fragmentation, depolarization and immobility. Indeed, this phenotype is representative of mitochondria targeted to autophagy. Live imaging data revealed that mitochondria accumulated in proximity of the axon, where they were actively degraded by autophagy that prevented their entry into the axons. Therefore, we modulated autophagy using different inhibitors and when autophagy was blocked, mitochondria expressing pathogenic Opa1 mutants redistributed in axons. Moreover, RGCs were less sensitive to apoptotic inducers. This phenotype could be due by two different/concomitant mechanisms. Pharmacological and genetic evidences support both a role for a Ca2+-calcineurin and one for AMP/AMPK in mitochondrial accumulation at the axonal hillock and degradation by autophagy. Interestingly, blockage of calcineurin reduced RGCs sensitivity to apoptosis. In conclusion, we demonstrated that OPA1 dysfunction induces abnormal autophagy close to the axonal hillock, where dysfunctional mitochondria are actively degraded, and this mechanism could be fundamental in the pathogenesis of ADOA.

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.

Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

Crocin Upregulates CX3CR1 Expression by Suppressing NF-κB/YY1 Signaling and Inhibiting Lipopolysaccharide-Induced Microglial Activation.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Microglial activation has been shown to be deleterious to RGCs and may participate in the progression of glaucoma. Crocin, one of the major active ingredients in saffron, has been found to inhibit microglial activation. However, the mechanism remains unclear. The aim of this study was to investigate whether crocin can inhibit lipopolysaccharide (LPS)-induced microglial activation and to clarify the mechanisms involved. The influence of crocin on primary RGCs and LPS-stimulated BV2 microglial cells survival was determined by the MTT and lactate dehydrogenase assays, or by flow cytometry. BV2 cells were pretreated with various concentrations of crocin for 2h followed by 1μg/mL LPS stimulation. Microglial markers and pro-inflammatory mediators were assessed by real-time PCR, western blot and ELISA. Furthermore, CX3CR1 expression was detected and the underlying mechanism was examined. The concentrations of crocin ranged from 0.1 to 1μM, and did not show any cytotoxicity in RGC and BV2 cells. After crocin pretreatment, the expression of microglial markers (CD11b and Iba-1) and pro-inflammatory mediators (iNOS, COX-2, IL-1β, and TNF-α) induced by LPS were significantly decreased in a dose-dependent manner. Additionally, CX3CR1 expression was remarkably increased by crocin via the suppression of NF-κB/Yin Yang 1 (YY1) signaling in BV2 cells. In conclusion, crocin effectively suppresses microglial activation and upregulates CX3CR1 expression by suppressing NF-κB/YY1 signaling.

Cigarette Smoke Extract Causes Injury in Primary Retinal Ganglion Cells via Apoptosis and Autophagy

ABSTRACTAims: To investigate whether tobacco smoke directly injures retinal ganglion cells (RGCs) and to evaluate the mechanisms of cell death.Methods: Primary rat RGCs were harvested from 3- or 4-day-old newborn rats and exposed to cigarette smoke extract (CSE). Cell viability was determined by adenosine 5ˊ-triphosphate (ATP) assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and real-time reverse transcription polymerase chain reaction (RT-PCR) for Bcl-2 family. Autophagy was also assessed by Western immunoblots for light chain (LC) 3B.Results: When the primary RGCs were exposed to CSE for 2 h, cell viability decreased in a dose-dependent manner, as measured by ATP assay. In the presence of 0.05% CSE, the RGC viability was 77.68% ± 7.60% compared to the control cells; in the presence of 1.0% CSE, viability was 47.48% ± 2.56% of the control cells. As determined by TUNEL, CSE increased the apoptotic RGCs in a dose-dependent manner. In the presence of 0.05% CSE, the apoptosis was 26.55% ± 1.97% of the control cells; in the presence of 2.5% CSE, it was 41.07% ± 3.75% of the control cells. When apoptosis was evaluated using real-time RT-PCR, exposure to 0.05% CSE resulted in significantly increased expression of Bad, Bax, Bcl-2, and Bcl-XL mRNA. When autophagy was assessed by Western immunoblots, exposure to 0.05% CSE significantly increased the expression of LC3B II.Conclusions: Our data suggest that CSE directly injures primary RGCs, and both cell death mechanisms of apoptosis and autophagy seem to be related to this CSE-induced RGC damage.

Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non‐coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up‐regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro. MALAT1‐CREB binding maintains CREB phosphorylation by inhibiting PP2A‐mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling.

Multipronged approach to identify and validate a novel upstream regulator of Sncg in mouse retinal ganglion cells.

Loss of retinal ganglion cells (RGCs) is one of the hallmarks of retinal neurodegenerative diseases, glaucoma being one of the most common. Mechanistic studies on RGCs are hindered by the lack of sufficient primary cells and consensus regarding their signature markers. Recently, γ-synuclein (SNCG) has been shown to be highly expressed in the somas and axons of RGCs. In various mouse models of glaucoma, downregulation of Sncg gene expression correlates with RGC loss. To investigate the role of Sncg in RGCs, we used a novel systems genetics approach to identify a gene that modulates Sncg expression, followed by confirmatory studies in both healthy and diseased retinae. We found that chromosome 1 harbors an expression quantitative trait locus that modulates Sncg expression in the mouse retina, and identified the prefoldin-2 (PFDN2) gene as the candidate upstream modulator of Sncg expression. Our immunohistochemical analyses revealed similar expression patterns in both mouse and human healthy retinae, with PFDN2 colocalizing with SNCG in RGCs and their axons. In contrast, in retinae from glaucoma subjects, SNCG levels were significantly reduced, although PFDN2 levels were maintained. Using a novel flow cytometry-based RGC isolation method, we obtained viable populations of murine RGCs. Knocking down Pfdn2 expression in primary murine RGCs significantly reduced Sncg expression, confirming that Pfdn2 regulates Sncg expression in murine RGCs. Gene Ontology analysis indicated shared mitochondrial function associated with Sncg and Pfdn2. These data solidify the relationship between Sncg and Pfdn2 in RGCs, and provide a novel mechanism for maintaining RGC health.

Glia-Neuron Interactions in the Retina Can Be Studied in Cocultures of Müller Cells and Retinal Ganglion Cells.

Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller cells on RGC survival. To investigate the Müller cell/RGC interactions we developed a coculture model, in which primary Müller cells were grown in inserts on top of pure primary RGC cultures. The impact of starvation and mitochondrial inhibition on the Müller cell ability to protect RGCs was studied. Moreover, the ability of Müller cells to remove glutamate from the extracellular space was investigated. RGC survival was evaluated by cell viability assays and glutamate uptake was assessed by kinetic uptake assays. We demonstrated a significantly increased RGC survival in presence of untreated and prestarved Müller cells. Additionally, prestarved Müller cells significantly increased RGC survival after mitochondrial inhibition. Finally, we revealed a significantly increased ability to take up glutamate in starved Müller cells. Overall, our study confirms essential roles of Müller cells in RGC survival. We suggest that targeting Müller cell function could have potential for future treatment strategies to prevent blinding neurodegenerative retinal diseases.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Fasudil, a Rho-Associated Protein Kinase Inhibitor, Attenuates Traumatic Retinal Nerve Injury in Rabbits.

Inhibition of RhoA/Rock could promote axon growth and alleviate optic nerve injury. However, the role of RhoA/Rock in the traumatic retinal nerve in vivo was not completely clear. In this study, we established a rabbit model of traumatic retinal nerve injury, and primary retinal ganglion cells (RGCs) were isolated and cultured under hypoxia-hypoglycemia condition that was mock to the microenvironment in the injured retinas in vivo. The Rock inhibitor fasudil was used to treat primary RGCs and ear vein injected into the model rabbits in vivo. RhoA/Rock signaling was activated in the injured optic nerve in rabbits. Western blotting analysis showed that RhoA/Rock signaling in the retina was activated during the traumatic optic neuropathy. Data on gene expression examination and Annexin V/PI dual staining combined with flow cytometry analysis displayed that fasudil injection reduced expression of Rho/Rock and apoptotic genes, as well as the apoptosis of RGCs in traumatic retinal nerve injury in vitro and in vivo. Moreover, fasudil injection reduced expression of Rho/Rock and apoptotic genes, as well as the apoptosis of RGCs in the rabbits with traumatic retinal nerve injury in vivo. In conclusion, fasudil treatment could significantly reduce the apoptosis of RGCs and relieved retinal nerve injury in vitro and in vivo.

Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

Endoplasmic reticulum stress-regulated CXCR3 pathway mediates inflammation and neuronal injury in acute glaucoma.

Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.

Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.

Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I/R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type retinal ganglion cells, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species. The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited reactive oxygen species increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2 knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I/R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. Oxidative stress is thought to be an important mediator of retinal ganglion cell death in ischemia-reperfusion injury. We found that the transcription factor NF-E2-related factor 2 (Nrf2), a major regulator of oxidative stress, is an important endogenous neuroprotective molecule in retinal ganglion cells in ischemia-reperfusion, exerting a cell-autonomous protective effect. The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) reduces neurodegeneration following ischemia-reperfusion in an Nrf2-dependent fashion. This suggests that Nrf2-activating drugs including triterpenoids could be a therapeutic strategy for retinal neuroprotection.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4–7 Sprague–Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 μM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 μM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 μM) or the combination of s-AMPA and cyclothiazide (50 μM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.