Retinal Ganglion Cells In Retina Research Articles

Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis

Crystallin βb2 (crybb2) is upregulated in regenerating retinas and in various pathological conditions of the retina, including uveoretinitis. However, the role of crybb2 in this disease is largely unknown. Therefore, we used recombinant crybb2 (rcrybb2) as intravitreal treatment of B10.RIII mice prior to immunization with human interphotoreceptor retinoid-binding protein peptide 161–180 (hIRBPp161-180) in complete Freund’s adjuvant (CFA) and concomitant injection of pertussis toxin (PTX) to induce experimental autoimmune uveoretinitis (EAU). In naïve mice, more beta III-tubulin (TUBB3) + and RNA-binding protein with multiple splicing (RBPMS) + cells were found in the ganglion cell layer of the retina than in EAU eyes, suggesting a loss of retinal ganglion cells (RGC) during the development of EAU. At the same time, the number of glial fibrillary acidic protein (GFAP) + cells increased in EAU eyes. RGCs were better protected in EAU eyes treated with rcrybb2, while the number of GFAP+ cells decreased. However, in retinal flatmounts, both retinal ganglion cells and retinal endothelial cells stained positive for TUBB3, indicating that TUBB3 is present in naïve B10.RIII mouse eyes not exclusive to RGCs. A significant decline in the number of RBPMS-positive retinal ganglion cells was observed in retinal flatmounts from EAU retinas in comparison to naïve retinas or EAU retinas with intravitreal rcrybb2 treatment. Whereas no significant decrease in TUBB3 levels was detected using Western blot and RT-qPCR, GFAP level, as a marker for astrocytes, increased in EAU mice compared to naïve mice. Level of Bax and Bcl2 in the retina was altered by treatment, suggesting better cell survival and inhibition of apoptosis. Furthermore, our histologic observations of the eyes showed no change in the incidence and severity of EAU, nor was the immune response affected by intravitreal rcrybb2 treatment. Taken together, these results suggest that intravitreal injection of rcrybb2 reduces retinal RGC death during the course of EAU, independent of local or systemic autoimmune responses. In the future, treating posterior uveitis with rcrybb2 to protect RGCs may offer a promising novel therapeutic strategy.

Self-Assembly Hypoxic and ROS Dual Response Nano Prodrug as a New Therapeutic Approach for Glaucoma Treatments.

Glaucoma is an irreversible blinding eye disease characterized by retinal ganglion cell (RGC) death.Previous studies have demonstrated that protecting mitochondria and activating the CaMKII/CREB signaling pathway can effectively protect RGC and axon. However, currently treatments are often unsatisfactory, and the pathogenesis of glaucoma requires further elucidation. In this study, a ROS-responsive dual drug conjugate (OLN monomer) is first designed that simultaneously bonds nicotinamide and oleic acid. The conjugate self-assembled into nanoparticles (uhOLN-NPs) through the aggregation of multiple micelles and possesses ROS scavenging capability. Then, a polymer with a hypoxic response function is designed, which encapsulates uhOLN-NPs to form nanoparticles with hypoxic and ROS responses (HOLN-NPs). Under hypoxia in RGCs, the azo bond of HOLN-NPs breaks and releases uhOLN-NPs. Meanwhile, under high ROS conditions, the thioketone bond broke, leading to the dissociation of nano-prodrug. The released nicotinamide and oleic acid co-scavenge ROS and activate the CaMKII/CREB pathway, protecting mitochondria in RGCs. HOLN-NPs exhibit a significantly superior protective effect on R28 cells in glutamate models of glaucoma. The accumulation of HOLN-NPs in retinal RGCs lead to significant inhibition of RGC apoptosis and axonal damage in vivo. Notably, HOLN-NPs provide a new therapeutic approach for patients with neurodegenerative disease.

GSK872 and necrostatin-1 protect retinal ganglion cells against necroptosis through inhibition of RIP1/RIP3/MLKL pathway in glutamate-induced retinal excitotoxic model of glaucoma

BackgroundGlaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-d-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear.MethodsThe glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting.ResultsWe found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1β (IL-1β), was markedly inhibited.ConclusionsOur findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.

Acteoside protects retinal ganglion cells from experimental glaucoma by activating the PI3K/AKT signaling pathway via caveolin 1 upregulation.

BackgroundGlaucoma is the second leading cause of blindness in the world and is characterized by optic neuropathy and degeneration of retinal ganglion cells (RGCs). Our preliminary research found that acteoside can inhibit autophagy-induced apoptosis of RGCs via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. However, it is unclear how acteoside activates the PI3K/AKT signaling pathway to prevents RGCs autophagic apoptosis.MethodsAnimal and cell models were used in this study. Hematoxylin-eosin staining revealed pathological histology of retinas. The number of RGCs in retinas was counted using immunofluorescence. Malondialdehyde and superoxide dismutase were determined using enzyme-linked immunosorbent assay kits. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were used to detect cell apoptosis. The reactive oxygen species was determined by the Flow cytometry. The proteins were determined by Western blot.ResultsThe results showed that acteoside treatment significantly reduced RGC loss, oxidative stress, and autophagy, thereby preventing glaucoma exacerbation. Acteoside reversed caveolin 1 (Cav1) expression and PI3K/AKT signaling activation, according to Western blot results. Cav1 knockdown also reversed acteoside’s effects on RGC loss, PI3K/AKT signaling pathway activation, autophagy and oxidative stress. Notably, 3-methyladenine, a PI3K inhibitor, reversed the effects of acteoside and Cav1 overexpression on RGC loss, oxidative stress, and autophagy.ConclusionsThese finding imply that acteoside alleviates RGC loss and oxidative stress by activating of the PI3K/AKT signaling pathway by upregulating Cav1.

Attenuated Herpes simplex virus 1 vector expressing oncomodulin effectively allieviates mechanical optic nerve injury in rats

To evaluate the efficacy and safety of attenuated Herpes simplex virus 1 (HSV-1) vector expressing oncomodulin (OCM) for treatment of mechanical optic nerve injury in rats. The proliferation characteristics and OCM expression of the recombinant HSV-1 vector (1716-OCM) was assessed in cultured Vero cells. Twelve-week-old SD rats were randomly divided into control group, 1716-OCM injection group and wild-type virus corneal infection group, and at 7, 14, 30 and 60 days post-infection (3 rats in each group at each time point), the expressions of OCM and HSV-1 structural protein gB in the retina and the hypothalamus of the rats were detected using immunofluorescence assay. Another 20 rats were randomized into sham operation group, PBS treatment group, 1716-OCM infection group and 1716-OCM infection with cAMP sensitization group (n=5), and in the latter 3 groups, rat models of optic nerve injury models were established followed by intravitreal injection of PBS, 1716-OCM or cAMP as indicated. At 45 days after the treatments, the rats were examined for visual electrophysiological function using FVEP method, and the number of retinal ganglion cells (RGCs) and the expression of myelin basic protein in the optic nerve were detected using immunofluorescence assay. The recombinant 1716-OCM vector was capable of mediating effective expression of OCM in Vero cells in vitro, but its proliferation rate was much lower than that of the wild-type virus. In SD rats, the recombinant virus could mediate the expression of OCM in the RGC layer and choroid layer of the eyes without inducing significant structural damage of the eyes as compared with the wild-type virus. In rat models of optic nerve injury, 1716-OCM combined with cAMP significantly promoted the survival of retinal RGCs (P= 0.007) and inhibited demyelination of the optic nerve (P=0.03) as compared with the mock treatment. FVEP analysis showed that 1716-OCM combined with cAMP significantly promoted the recovery of the peak amplitude of ΔN1-P1 in the rats (P < 0.0001). Attenuated recombinant 1716-OCM vector can mediate OCM expression in the retina of rats, and in rat models of mechanical optic nerve injury, intravitreal injection of 1716-OCM combined with cAMP can effectively alleviate optic nerve injuries.

7,8-Dihydroxiflavone Protects Adult Rat Axotomized Retinal Ganglion Cells through MAPK/ERK and PI3K/AKT Activation.

We analyze the 7,8-dihydroxyflavone (DHF)/TrkB signaling activation of two main intracellular pathways, mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol 3 kinase (PI3K)/AKT, in the neuroprotection of axotomized retinal ganglion cells (RGCs). Methods: Adult albino Sprague-Dawley rats received left intraorbital optic nerve transection (IONT) and were divided in two groups. One group received daily intraperitoneal DHF (5 mg/kg) and another vehicle (1%DMSO in 0.9%NaCl) from one day before IONT until processing. Additional intact rats were employed as control (n = 4). At 1, 3 or 7 days (d) after IONT, phosphorylated (p)AKT, p-MAPK, and non-phosphorylated AKT and MAPK expression levels were analyzed in the retina by Western blotting (n = 4/group). Radial sections were also immunodetected for the above-mentioned proteins, and for Brn3a and vimentin to identify RGCs and Müller cells (MCs), respectively (n = 3/group). Results: IONT induced increased levels of p-MAPK and MAPK at 3d in DHF- or vehicle-treated retinas and at 7d in DHF-treated retinas. IONT induced a fast decrease in AKT in retinas treated with DHF or vehicle, with higher levels of phosphorylation in DHF-treated retinas at 7d. In intact retinas and vehicle-treated groups, no p-MAPK or MAPK expression in RGCs was observed. In DHF- treated retinas p-MAPK and MAPK were expressed in the ganglion cell layer and in the RGC nuclei 3 and 7d after IONT. AKT was observed in intact and axotomized RGCs, but the signal intensity of p-AKT was stronger in DHF-treated retinas. Finally, MCs expressed higher quantities of both MAPK and AKT at 3d in both DHF- and vehicle-treated retinas, and at 7d the phosphorylation of p-MAPK was higher in DHF-treated groups. Conclusions: Phosphorylation and increased levels of AKT and MAPK through MCs and RGCs in retinas after DHF-treatment may be responsible for the increased and long-lasting RGC protection afforded by DHF after IONT.

Resveratrol protects the retina from I/R injury by inhibiting RGCS apoptosis, glial activation and expression of inflammatory factors

Purpose: To study the protective effect of resveratrol against retinal injury induced by ischemiareperfusion (I/R), and the underlying mechanism action.Methods: Retinal I/R injury was established in 72 healthy male SD rats. The rats were assigned to 3 groups: control, model and resveratrol groups, with 24 rats in each group. Pathological changes in retina were determined using H&amp;E staining. Retinal ganglion cells (RGCs) were counted usingfluorescence gold retrograde staining. Western blotting was used to assay the expressions of caspase- 3, Bax, Bcl-2, GFAP, COX-2 and iNOS. The expressions of COX-2 and iNOS were measured by immunofluorescence.Results: The retina in the control group was dense and ordered, and its morphology was normal. In contrast, the retina in the model group was thinner, with loose cells and disordered structure. In the resveratrol group, the retina was thicker, the structure was orderly, and the cells were dense. Relative to control, the population of RGCs in model rat retina was significantly reduced, and the expressions of Bcl-2, Bax, caspase-3, GFAP, COX-2 and iNOS were significantly upregulated (p &lt; 0.05). In the resveratrol group, the number of RGCs in the retina was markedly elevated, relative to model rats, and the expressions of Bcl-2, Bax, caspase-3, GFAP, COX-2 and iNOS were significantly decreased (p &lt; 0.05).Conclusion: Resveratrol protects the retina from I/R injury in rats by inhibiting RGCs apoptosis, glial activation and expressions of inflammatory factors. Thus, this compound may be potentially suitable for the management of retina damage.&#x0D; Keywords: Resveratrol, RGCs apoptosis, Glial activation, Inflammatory factors, I/R injury

Sulforaphane alleviates retinal ganglion cell death and inflammation by suppressing NLRP3 inflammasome activation in a rat model of retinal ischemia/reperfusion injury.

This study aims to study the potentials of sulforaphane (SFN) against retinal ischemia/reperfusion (I/R) injury. A rat retinal I/R injury method was established. Retinal thickness change and retinal ganglion cell (RGC) death were determined using hematoxylin and eosin (H&E) staining and Fluoro-Gold (FG) labeling. The inflammatory cytokines production and microglia activation were evaluated by using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and enzyme-linked immunosorbent assay (ELISA). Knockdown NLRP3 was performed, and the according changes of retinal RGCs were assessed. SFN administration significantly inhibited I/R and caused retinal thickness change and prevented RGCs death in retinal I/R model. SFN suppressed inflammatory cytokines production, microglia activation, and inflammasome activation. In accordance, NLRP3 knockdown presented the similar inhibitory effect on I/R rats. This study demonstrates that SFN prevents RGCs death and acts as a potent neuroprotective modulator in retinal I/R injury, which may be associated with inhibition of the NLRP3 inflammasome activation.

Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-d-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.

Rapamycin mediates mTOR signaling in reactive astrocytes and reduces retinal ganglion cell loss

Damage and loss of retinal ganglion cells (RGCs) can cause visual impairment. The underlying molecular mechanisms that mediate RGC death in ischemic retinal diseases are still unclear. In this study, we sought to understand the neuroprotective effect of rapamycin, the selective inhibitor of mTORC1, on RGC survival and the cellular mechanics that mediate this effect. Recent studies have reported that the epidermal growth factor (EGF) receptor shows an increase in expression in astrocytes after injury, and this receptor can promote their transformation into reactive astrocytes. Our results, along with previous works from others, show the colocalization of phosphor-EGF receptors with the astrocyte marker glial fibrillary acidic proteins in reactive astrocytes in the injured retina. In our in vitro studies, using primary astrocyte cultures of the optic nerve head of rats, showed that rapamycin significantly blocked EGF-induced mTOR signaling mainly through the PI3K/Akt pathway in primary astrocytes, but not through the MAPK/Erk pathway. Additionally, rapamycin dramatically inhibited the activation of mTOR signaling in our ratinal ischemia-reperfusion (I/R) injury model in vivo. Astrocyte activation was assessed by immunostaining retinal flat mounts or cross sections with antibody against GFAP, and we also used western blots to detect the expression of GFAP. Taken together, these results revealed that rapamycin decreases the activation of astrocytes after retinal ischemia-reperfusion injury. Furthermore, rapamycin can improve retinal RGC survival in rats during I/R, as detected by FluoroGold labeling. Our data reveals the neuroprotective effects of rapamycin in an experimental retina injury model, possibly through decreasing glial-dependent intracellular signaling mechanisms for suppressing apoptosis of RGCs. Our study also presents an approach to targeting reactive astrocytes for the treatment of optic neurodegenerations.

Neuroprotective effect of Qinggan Lishui formula on retinal ganglion cell apoptosis in a microbead-induced rat chronic glaucoma model

OBJECTIVETo investigate a possible mechanism for protective effects of a decoction of the Qinggan Lishui formula (QF) on retinal ganglion cells (RGCs) in a rat model of microbead-induced chronic intraocular hypertension (COH). METHODSThe COH model was generated by injecting microbeads (superparamagnetic iron oxide) into the anterior chamber of rat eyes. QF was given by intragastric administration (gavage) once daily at a dose of 6.2 g/kg until day 28, following microbead injection. Cholera toxin B subunit (CTB) retrograde labeling and immunohistochemistry were used to evaluate changes in the number of RGCs in the retina. Terminal dUTP nick end labeling (TUNEL) staining was used to assess apoptotic changes in RGCs. RESULTSMicrobead injection induced a steady increase in intraocular pressure (IOP) of rats. Elevated IOP resulted in a progressive reduction in the number of CTB-labeled RGCs, 2-4 weeks after microbead injection. QF administration may moderately reduce IOP in the rat COH model and attenuate reduction of the number of CTB-labeled RGCs in COH rats. Furthermore, elevated IOP resulted in a progressive increase in the number of TUNEL-positive RGCs, 2–4 weeks after microbead injection, suggestive of an increase in the extent of RGC apoptosis. There was a significant reduction in the number of TUNEL-positive signals in QF-treated COH retinas, compared with untreated COH retinas. CONCLUSIONQF decoction may provide a protective effect for RGCs in COH retinas by reducing RGC loss; these effects may be mediated by inhibition of RGC apoptosis.

The Nrf2 Signaling in Retinal Ganglion Cells under Oxidative Stress in Ocular Neurodegenerative Diseases.

Retinal ganglion cells (RGCs) are one of the important cell types affected in many ocular neurodegenerative diseases. Oxidative stress is considered to be involved in retinal RGCs death in ocular neurodegenerative diseases. More and more attention has been focused on studying the agents that may have neuroprotective effects. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key nuclear transcription factor for the systemic antioxidant defense system. This review elucidates the underlying mechanism of the Nrf2-mediated neuroprotective effects on RGCs in ocular neurodegenerative diseases, such as diabetic retinopathy and retinal ischemia-reperfusion injury. Several Nrf2 inducers that shield RGCs from oxidative stress-induced neurodegeneration via regulating Nrf2 signaling are discussed.

A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice

Src knockout mice show no detectable abnormalities in central nervous system (CNS) post-mitotic neurons, likely reflecting functional compensation by other Src family kinases. Cdk1- or Cdk5-dependent Ser75 phosphorylation in the amino-terminal Unique domain of Src, which shares no homology with other Src family kinases, regulates the stability of active Src. To clarify the roles of Src Ser75 phosphorylation in CNS neurons, we established two types of mutant mice with mutations in Src: phospho-mimicking Ser75Asp (SD) and non-phosphorylatable Ser75Ala (SA). In ageing SD/SD mice, retinal ganglion cell (RGC) number in whole retinas was significantly lower than that in young SD/SD mice in the absence of inflammation and elevated intraocular pressure, resembling the pathogenesis of progressive optic neuropathy. By contrast, SA/SA mice and wild-type (WT) mice exhibited no age-related RGC loss. The age-related retinal RGC number reduction was greater in the peripheral rather than the mid-peripheral region of the retina in SD/SD mice. Furthermore, Rho-associated kinase activity in whole retinas of ageing SD/SD mice was significantly higher than that in young SD/SD mice. These results suggest that Src regulates RGC survival during ageing in a manner that depends on Ser75 phosphorylation.

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H2O2 and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H2O2-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H2O2-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H2O2-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H2O2 induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.

Cell type-specific expression of FoxP2 in the ferret and mouse retina

Although the anatomical and physiological properties of subtypes of retinal ganglion cells (RGCs) have been extensively investigated, their molecular properties are still unclear. Here, we examined the expression patterns of FoxP2 in the retina of ferrets and mice. We found that FoxP2 was expressed in small subsets of neurons in the adult ferret retina. FoxP2-positive neurons in the ganglion cell layer were divided into two groups. Large FoxP2-positive neurons expressed Brn3a and were retrogradely labeled with cholera toxin subunit B injected into the optic nerve, indicating that they are RGCs. The soma size and the projection pattern of FoxP2-positive RGCs were consistent with those of X cells. Because we previously reported that FoxP2 was selectively expressed in X cells in the ferret lateral geniculate nucleus (LGN), our findings indicate that FoxP2 is specifically expressed in the parvocellular pathway from the retina to the LGN. Small FoxP2-positive neurons were positive for GAD65/67, suggesting that they are GABAergic amacrine cells. Most Foxp2-positive cells were RGCs in the adult mouse retina. Dendritic morphological analyses suggested that Foxp2-positive RGCs included direction-selective RGCs in mice. Thus, our findings suggest that FoxP2 is expressed in specific subtypes of RGCs in the retina of ferrets and mice.

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.

Restoring Light Sensitivity in Blind Retinae using Third Generation Photopharmacology

More than 300 million people worldwide are suffering from blindness or impaired vision. Although the photoreceptor cells often undergo complete degeneration, the remains of the retinal circuitry are often not affected. In order to restore light sensitivity to blind retinae, optochemical tools targeting receptors natively expressed in retinal cells provide a new photopharmacological strategy without the need of any genetic manipulation. Photochromic ligands (PCL) are chemically modified in such a way that they enable optical control of neuronal activity by acting as selective photoswitchable receptor ligands or blockers of voltage-gated ion channels (VGIC). It has previously been shown that photoswitchable potassium channel blockers, e.g. AAQ, or DENAQ (Polosukhina et al. 2012 and Tochitsky et al. 2014, respectively), are able to restore light sensitivity to a retina from genetically blind mice. However, the aforementioned molecules harbor some difficulties in the application to the retina. We now developed Red-DAD, a third-generation PCL targeting VGICs. In multi-electrode array and whole-cell patch-clamp experiments we could demonstrate that Red-DAD is able to trigger retinal ganglion cell (RGC) spiking in a light-dependent fashion in blind mice. This molecule exhibits several features making it superior to previous PCLs for the application in vision restoration. The compound is not permanently charged and also red-shifted, i.e. one can switch with blue or white light. Additionally, pharmacological approaches reveal that Red-DAD targets bipolar cells (BPs) rather than retinal RGCs, thereby utilizing the intrinsic processing circuits of the retina. Taken together, Red-DAD is a very promising next-generation molecule for restoring light sensitivity in blind retinae.

Celastrol supports survival of retinal ganglion cells injured by optic nerve crush

The present study evaluates the effect of celastrol on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). Celastrol, a quinine methide triterpene extracted from the perennial vine Tripterygium wilfordii (Celastraceae), has been identified as a potential neuroprotective candidate in a comprehensive drug screen against various neurodegenerative diseases. Two weeks after ONC, the average density of remaining RGCs in retinas of animals treated with daily intraperitoneal (i.p.) injections of celastrol (1mg/kg) was approximately 1332cells/mm2, or 40.8% of the Celastrol/Control group. In retinas of the Vehicle/ONC group about 381RGCs/mm2 were counted, which is 9.6% of the total number of RGCs in the DMSO/Control group. This corresponds to approximately a 250% increase in RGC survival mediated by celastrol treatment compared to Vehicle/ONC group. Furthermore, the average RGC number in retinas of ONC animals treated with a single intravitreal injection of 1mg/kg or 5mg/kg of celastrol was increased by approximately 80% (760RGCs/mm2) and 78% (753RGCs/mm2), respectively, compared to Vehicle/ONC controls (422cells/mm2). Injection of 0.2mg/kg of celastrol had no significant effect on cell survival, with the average number of RGCs being 514cells/mm2 in celastrol-treated animals versus 422cells/mm2 in controls. The expression levels of Hsp70, Hsf1, Hsf2, HO-1 and TNF-alpha in the retina were analyzed to evaluate the roles of these proteins in the celastrol-mediated protection of injured RGCs. No statistically significant change in HO-1, Hsf1 and Hsp70 levels was seen in animals with ONC. An approximately 2 fold increase in Hsf2 level was observed in celastrol-treated animals with or without injury. Hsf2 level was also increased 1.8 fold in DMSO-treated animals with ONC injury compared to DMSO-treated animals with no injury suggesting that Hsf2 induction has an injury-induced component. Expression of TNF-alpha in retinas of celastrol-treated uninjured and ONC animals was reduced by approximately 2 and 1.5 fold compared to vehicle treated animals, respectively. The observed results suggest that mechanisms underlying celastrol׳s RGC protective effect are associated with inhibition of TNF-alpha-mediated cell death.

Crystallins in Retinal Ganglion Cell Survival and Regeneration

Crystallins are heterogeneous proteins classified into alpha, beta, and gamma families. Although crystallins were first identified as the major structural components of the ocular lens with a principal function to maintain lens transparency, further studies have demonstrated the expression of these proteins in a wide variety of tissues and cell types. Alpha crystallins (alpha A and alpha B) share significant homology with small heat shock proteins and have chaperone-like properties, including the ability to bind and prevent the precipitation of denatured proteins and to increase cellular resistance to stress-induced apoptosis. Stress-induced upregulation of crystallin expression is a commonly observed phenomenon and viewed as a cellular response mechanism against environmental and metabolic insults. However, several studies reported downregulation of crystallin gene expression in various models of glaucomatous nerodegeneration suggesting that that the decreased levels of crystallins may affect the survival properties of retinal ganglion cells (RGCs) and thus, be associated with their degeneration. This hypothesis was corroborated by increased survival of axotomized RGCs in retinas overexpressing alpha A or alpha B crystallins. In addition to RGC protective functions of alpha crystallins, beta and gamma crystallins were implicated in RGC axonal regeneration. These findings demonstrate the importance of crystallin genes in RGC survival and regeneration and further in-depth studies are necessary to better understand the mechanisms underlying the functions of these proteins in healthy RGCs as well as during glaucomatous neurodegeneration, which in turn could help in designing new therapeutic strategies to preserve or regenerate these cells.

Stanniocalcin-1 Protects Retinal Ganglion Cells by Inhibiting Apoptosis and Oxidative Damage

Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.