Prevented Retinal Ganglion Cells Loss Research Articles

HLA-Homozygous iPSC-Derived Mesenchymal Stem Cells Rescue Rotenone-Induced Experimental Leber's Hereditary Optic Neuropathy-like Models In Vitro and In Vivo.

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy, yet the sourcing, quality, and invasive methods of MSCs impede their mass production and quality control. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) can be infinitely expanded, providing advantages over conventional MSCs in terms of meeting unmet clinical demands. The potential of MSC therapy for Leber's hereditary optic neuropathy (LHON) remains uncertain. In this study, we used HLA-homozygous induced pluripotent stem cells to generate iMSCs using a defined protocol, and we examined their therapeutic potential in rotenone-induced LHON-like models in vitro and in vivo. The iMSCs did not cause any tumorigenic incidence or inflammation-related lesions after intravitreal transplantation, and they remained viable for at least nine days in the mouse recipient's eyes. In addition, iMSCs exhibited significant efficacy in safeguarding retinal ganglion cells (RGCs) from rotenone-induced cytotoxicity in vitro, and they ameliorated CGL+IPL layer thinning and RGC loss in vivo. Optical coherence tomography (OCT) and an electroretinogram demonstrated that iMSCs not only prevented RGC loss and impairments to the retinal architecture, but they also improved retinal electrophysiology performance. The generation of iMSCs via the HLA homozygosity of iPSCs offers a compelling avenue for overcoming the current limitations of MSC-based therapies. The results underscore the potential of iMSCs when addressing retinal disorders, and they highlight their clinical significance, offering renewed hope for individuals affected by LHON and other inherited retinal conditions.

Schwann cell–derived extracellular vesicles as a potential therapy for retinal ganglion cell degeneration

Optic neuropathy is the leading cause of irreversible blindness and is characterized by progressive degeneration of retinal ganglion cells (RGCs). Several studies have demonstrated that transplantation of Schwann cells (SCs) is a promising candidate therapy for optic neuropathy and that intravitreally transplanted cells exert their effect via paracrine actions. Extracellular vesicle (EV)-based therapies are increasingly recognized as a potential strategy for cell replacement therapy. In this study, we aimed to investigate the neuroprotective and regenerative effects of SC-EVs following optic nerve injury. We found that SC-EVs were internalized by RGCs in vitro and in vivo without any transfection reagents. Intriguingly, SC-EVs significantly enhanced the survival and axonal growth of primary RGCs in a coculture system. In a rat optic nerve crush model, SC-EVs mitigated RGC degeneration, prevented RGC loss, and preserved the thickness of the ganglion cell complex, as demonstrated by the statistically significant improvement in RGC counts and thickness measurements. Mechanistically, SC-EVs activated the cAMP-response element binding protein (CREB) signaling pathway and regulated reactive gliosis in ONC rats, which is crucial for RGC protection and axonal regeneration. These findings provide novel insights into the neuroprotective and regenerative properties of SC-EVs, suggesting their potential as a cell-free therapeutic strategy and natural biomaterials for neurodegenerative diseases of the central nervous system.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

Targeted delivery of LM22A-4 by cubosomes protects retinal ganglion cells in an experimental glaucoma model

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. Statement of significanceRecent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.

Rescue the retina after the ischemic injury by polymer-mediated intracellular superoxide dismutase delivery

Oxidative stress is a hallmark of the pathophysiogenesis of retinal ischemia. The direct delivery of antioxidant enzymes such as superoxide dismutase (SOD) into retinal cells provides a promising option for the down-regulation of oxidative stress in retinal ischemia, however, efficient intracellular protein delivery remains a major challenge for this application. Here, a boronic acid-rich polymer was used for the intracellular delivery of SOD both in vitro and in vivo. The polymer assembled with SOD into uniform nanoparticles with high binding affinity, and transported the cargo protein into several cell lines with maintained bioactivity and low cytotoxicity. We investigated the intraocular biodistribution, therapeutic efficacy and safety of the SOD nanoformulation in a retinal ischemia/reperfusion (I/R) injury model. After intravitreal injection, the nanoparticles rapidly diffused through the vitreous and penetrated into retinal ganglion cells (RGCs). Compared to free SOD, the nanoformulation exhibited much enhanced therapeutic efficacy with reduced RGC apoptosis and protected retinal function. Enzymatic results confirmed that the SOD nanoformulation reduced malondialdehyde expression and increased glutathione level in the ocular tissues, and thereby down-regulated oxidative stress and prevented RGC loss. Overall, this work offers a new therapeutic option for the treatment of retinal ischemic disorders by direct delivery of antioxidant proteins.

Targeted Delivery of Lm22a-4 by Cubosomes Protects Retinal Ganglion Cells in an Experimental Glaucoma Model

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of glaucoma, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss in a mouse model of acute IOP elevation. These results provide evidence that cubosomes-based LM22A-4 delivery may be a useful targeted approached to prevent the progression of RGCs loss in glaucoma.

Neuronal Epac1 mediates retinal neurodegeneration in mouse models of ocular hypertension

Progressive loss of retinal ganglion cells (RGCs) leads to irreversible visual deficits in glaucoma. Here, we found that the level of cyclic AMP and the activity and expression of its mediator Epac1 were increased in retinas of two mouse models of ocular hypertension. Genetic depletion of Epac1 significantly attenuated ocular hypertension-induced detrimental effects in the retina, including vascular inflammation, neuronal apoptosis and necroptosis, thinning of ganglion cell complex layer, RGC loss, and retinal neuronal dysfunction. With bone marrow transplantation and various Epac1 conditional knockout mice, we further demonstrated that Epac1 in retinal neuronal cells (especially RGCs) was responsible for their death. Consistently, pharmacologic inhibition of Epac activity prevented RGC loss. Moreover, in vitro study on primary RGCs showed that Epac1 activation was sufficient to induce RGC death, which was mechanistically mediated by CaMKII activation. Taken together, these findings indicate that neuronal Epac1 plays a critical role in retinal neurodegeneration and suggest that Epac1 could be considered a target for neuroprotection in glaucoma.

Loss-of-function mutation of serine racemase attenuates retinal ganglion cell loss in diabetic mice

Consistent results suggest the promoting roles of serine racemase (SR)/D-serine in retinal neurodegeneration in diabetic retinopathy (DR). However, the direct evidence connecting SR deficiency with retinal neuroprotection in genetic model of diabetes mellitus has not been reported. In this investigation, we explore the effect of absence of functional SR on the degeneration of retinal ganglion cells (RGCs) with a diabetic murine model, Ins2Akita mice. We established a murine strain with double mutation, termed Ins2Akita-Srr, by mating heterozygous Ins2Akita mice with homozygous Srrochre269 mice. Ins2Akita retained less RGC in posterior, middle, and peripheral retinae than the counterpart from non-diabetic sibling mice at the age of five or seven months. Ins2Akita-Srr mice retained more RGC in middle and peripheral--but not in posterior-- retinae than the counterpart from Ins2Akita sibling mice at the age of five months. By contrast, at the age of seven months, Ins2Akita-Srr mice contained more RGC in peripheral, middle, and posterior retinae than the counterpart from Ins2Akita. RGCs were identified with retrograde labeling in vivo or with immunolabeling against a RGC-specific transcription factor, Brn3a, in retinal flat mounts. Correspondingly, the aqueous humor of Ins2Akita-Srr contained less amount of D-serine than sibling Ins2Akita mice. Thus, SR deficiency significantly prevented RGC loss in diabetic mice. We conclude that D-serine is a critical factor in the degeneration of RGC in DR. Targeting SR expression or activity may be a strategy for ameliorating RGC loss in DR.

Targeting HDAC3 Activity with RGFP966 Protects Against Retinal Ganglion Cell Nuclear Atrophy and Apoptosis After Optic Nerve Injury.

HDAC3 regulates nuclear atrophy as an early response to axonal injury in retinal ganglion cells (RGCs) following optic nerve crush (ONC). Since conditional knockout of Hdac3 prevents nuclear atrophy post ONC, HDAC3 selective inhibition with RGFP966 through localized and systemic dosing of RGFP966 is necessary for application to acute and chronic models of optic nerve injury. C57BL/6 mice were injected intravitreally with 1-10 μM RGFP966 immediately following ONC, and retinas were analyzed at 5, 7, and 14 days for metrics of nuclear atrophy and cell loss. Mice were similarly assessed after intraperitoneal (IP) injections with RGFP966 doses of 2-10 mg/kg, and eyes were harvested at 5, 14, and 28 days after ONC. H&E and BrdU staining were used to analyze toxicity to off-target tissues after 14 days of daily treatment with RGFP966. A single intravitreal injection of RGFP966 prevented histone deacetylation, heterochromatin formation, apoptosis, and DNA damage at 5 and 7 days post ONC. After IP injection, RGFP966 bioavailability in the retina reached peak concentration within 1 h after injection and then rapidly declined. A single IP injection of 2-10 mg/kg RGFP966, significantly prevented histone deacetylation. Repeated IP injections of 2 mg/kg RGFP966 over the course of 2 and 4 weeks post ONC prevented RGC loss. There were no significant toxic or antiproliferative effects to off-target tissues in mice treated daily for 14 days with RGFP966. Inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents apoptosis-related histone deacetylation and attenuates RGC loss after acute optic nerve injury.

Neurotrophin-conjugated nanoparticles prevent retina damage induced by oxidative stress

Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retina, the very short half-life of NTs, and the need for multiple injections. We demonstrate that NTs can be conjugated to magnetic nanoparticles (MNPs), which act as smart drug carriers. This combines the advantages of the self-localization of the drug in the retina and drug protection from fast degradation. We tested the nerve growth factor and brain-derived neurotrophic factor by comparing the neuroprotection of free versus conjugated proteins in a model of RGC loss induced by oxidative stress. Histological data demonstrated that the conjugated proteins totally prevented RGC loss, in sharp contrast to the equivalent dose of free proteins, which had no effect. The overall data suggest that the nanoscale MNP-protein hybrid is an excellent tool in implementing ocular drug delivery strategies for neuroprotection and therapy.

An intraocular drug delivery system using targeted nanocarriers attenuates retinal ganglion cell degeneration

Glaucoma is a common blinding disease characterized by loss of retinal ganglion cells (RGCs). To date, there is no clinically available treatment directly targeting RGCs. We aim to develop an RGC-targeted intraocular drug delivery system using unimolecular micelle nanoparticles (unimNPs) to prevent RGC loss. The unimNPs were formed by single/individual multi-arm star amphiphilic block copolymer poly(amidoamine)–polyvalerolactone–poly(ethylene glycol) (PAMAM–PVL–PEG). While the hydrophobic PAMAM–PVL core can encapsulate hydrophobic drugs, the hydrophilic PEG shell provides excellent water dispersity. We conjugated unimNPs with the cholera toxin B domain (CTB) for RGC-targeting and with Cy5.5 for unimNP-tracing. To exploit RGC-protective sigma-1 receptor (S1R), we loaded unimNPs with an endogenous S1R agonist dehydroepiandrosterone (DHEA) as an FDA-approved model drug. These unimNPs produced a steady DHEA release in vitro for over two months at pH7.4. We then co-injected (mice, intraocular) unimNPs with the glutamate analog N-methyl-d-aspartate (NMDA), which is excito-toxic and induces RGC death. The CTB-conjugated unimNPs (i.e., targeted NPs) accumulated at the RGC layer and effectively preserved RGCs at least for 14days, whereas the unimNPs without CTB (i.e., non-targeted NPs) showed neither accumulation at nor protection of NMDA-treated RGCs. Consistent with S1R functions, targeted NPs relative to non-targeted NPs showed markedly better inhibitory effects on apoptosis and oxidative/inflammatory stresses in the RGC layer. Hence, the DHEA-loaded, CTB-conjugated unimNPs represent an RGC/S1R dual-targeted nanoplatform that generates an efficacious template for further development of a sustainable intraocular drug delivery system to protect RGCs, which may be applicable to treatments directed at glaucomatous pathology.

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.

Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway.

Background:Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival.Methods:RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis.Results:Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group.Conclusions:The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy.

Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.

BackgroundElevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).MethodsRetinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.ResultsWe report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.ConclusionsThis work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma

Glaucoma is conventionally defined as a chronic optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Although glaucoma is often associated with elevated intraocular pressure (IOP), significant IOP reduction does not prevent progression of the disease in some glaucoma patients. Thus, exploring IOP-independent mechanisms of RGC loss is important. We describe chronic systemic administration of aldosterone and evaluate its effect on RGCs in rat. Aldosterone was administered via an osmotic minipump that was implanted subcutaneously into the mid-scapular region. Although systemic administration of aldosterone caused RGC loss associated with thinning of the retinal nerve fiber layer without elevated IOP, the other cell layers appeared to be unaffected. After chronic administration of aldosterone, RGC loss was observed at 2 weeks in the peripheral retina and at 4 weeks in the central retina. However, administration of mineralocorticoid receptor blocker prevented RGC loss. These results demonstrate aldosterone is a critical mediator of RGC loss that is independent of IOP. We believe this rat normal-tension glaucoma (NTG) animal model not only offers a powerful system for investigating the mechanism of neurodegeneration in NTG, but can also be used to develop therapies directed at IOP-independent mechanisms of RGC loss.

Inhibition of voltage‐gated Ca2+ channels by sst4 stimulation is mediated by Gβγ and PKC activation

Healthcare System, Los Angeles, CA Stimulation of somatostatin subtype‐4 receptors (sst4) in isolated retinal ganglion cells (RGCs) inhibits Ca2+ channel current (ICa). Reducing intracellular Ca2+ is known to be neuroprotective; thus, stimulating sst4 may prevent RGC loss in trauma and disease. The aim of this study was to determine the signaling pathways involved in sst4 stimulation leading to suppression of ICa in RGCs. Isolated RGCs were prepared using a modified Thy‐1 immunopanning procedure. Electrophysiology experiments were performed using whole‐cell patch clamp to isolate ICa. L‐803,087 (sst4 agonist, L‐803) inhibited ICa by 40.4% and reduced calcium conductance (2.1 vs. 1.4 nS; p<0.05). Pretreatment of cells with pertussis toxin did not prevent the action of L‐803 (34.7%). Pre‐pulse facilitation did not reverse the inhibitory effects of L‐803 on ICa (11.4 vs. 9.7 %). However, pharmacologic inhibition of Gβγ prevented ICa suppression by L‐803 (23.0%, p<0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration‐dependent effect in preventing the action of L‐803 on ICa (1 μM GFX, 34.3%; 5 μM GFX, 18.4%, p<0.05). This suggests that sst4 stimulation modulates RGC Ca2+ channels via Gβγ and PKC activation. Thus, modulating these signaling pathways may provide unique therapeutic targets to reduce intracellular Ca2+ levels in RGCs. Support from CIHR‐NSHRF RPP, NIH.

Deficiency in Bim, Bid and Bbc3 (Puma) do not prevent axonal injury induced death

Dear Editor, BAX activation is a critical step in the apoptotic pathway in neurons. BAX activation is multistep process that can be driven by a subset of other pro-apoptotic Bcl-2 family members, specifically the BH3-only proteins BIM, BBC3 (PUMA), and BID.1 In fact BAX activation appears to require at least one of these pro-apoptotic chaperones during development and after many apoptotic insults.2 Retinal ganglion cells (RGCs) die by a BAX-dependent mechanism in development and after axonal injury,3 a key insult in many neurodegenerations. During development BAX-dependent death of RGCs requires BBC3 (Harder and Libby4 and Supplementary Figure 1A); however, the molecules controlling BAX activation after axonal injury are unknown. We have previously shown that single deficiency in Bim or Bbc3 lessen early RGC death after axonal injury.4, 5 However, by 5 days after injury, RGC death is no longer different in Bbc3-deficient mice and there is a significant number of apoptotic RGCs in Bim-deficient mice (Supplementary Figure 1B). These data show that both BIM and BBC3 are involved in BAX activation in axonally injured RGCs, but are not solely responsible for RGC death. As BIM and BBC3 appear to regulate BAX activation in axonally injured RGCs, we tested whether deficiency in both Bim and Bbc3 phenocopied Bax deficiency and prevented RGC loss after axonal injury. The amount of protection in Bim Bbc3 double knockouts was additive and significant compared with wildtype and single mutants as judged by the number of RGCs surviving 21 days after injury (Supplementary Figure 1C). However, apoptotic cell death persisted in the Bim Bbc3 double mutants after axonal injury, which led to significant RGC loss over time (P<0.001; Supplementary Figure 1C). BID has been implicated in neuronal death after many types of insults;2 therefore, it is possible that it is contributing to RGC death in the absence of BIM and BBC3. Bid deficiency alone did not alter the rate or amount of RGC death after injury (Supplementary Figures 1B and C). Bid deficiency was also not additive in the context of Bim deficiency or Bbc3 deficiency (Supplementary Figures 1B and C). In order to test whether removing the major direct activators of BAX was necessary to prevent RGC loss, mice deficient in Bim, Bbc3, and Bid were generated. Triple deficiency did not prevent RGC death after axonal injury and did not increase the long term survival of RGCs compared with the protection of Bim Bbc3 combined deficiency (64±3 and 61±4% of control, respectively; Supplementary Figure 1C). However, triple deficiency did reduce the rate of RGC death compared with Bim Bbc3 double-deficient animals suggesting that BID can have a role in RGC death after axonal injury (Supplementary Figure 1B). Thus, in RGCs axonal injury induces cell death independently of BIM, BBC3, and BID despite the requirement for BAX. RGC death in the absence of BIM, BBC3, and BID is consistent with observations suggesting that BAX (and BAK) could be activated independently of these BH3-only proteins.6 BH3-only proteins with a lower affinity for BAX have been implicated in neuronal death, such as HRK, BMF, and NOXA after neuronal trauma.7 These molecules may be less efficient at killing cells due to their primary function of antagonizing pro-survival Bcl-2 family members. Interestingly, lowering levels of pro-survival Bcl-2 family members alone can lead to apoptosis.8 Necroptotic cell death is also dependent on BAX9 and observed in injured neurons, but may not require BIM, BBC3, and BID. Finally, it is possible that BAX activation could be occurring independently of the Bcl-2 family (e.g. Chipuk et al.10). Therefore, further defining the mechanism of BAX activation may be important in understanding cell death in neuronal degenerations or after neuronal trauma.

A Potential Neuroprotective Role of Apolipoprotein E-containing Lipoproteins through Low Density Lipoprotein Receptor-related Protein 1 in Normal Tension Glaucoma

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3β, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.