Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo

Glaucoma is a major cause of vision impairment, which arises from the sustained and progressive apoptosis of retinal ganglion cells (RGC), with ocular hypertension being a major risk or co-morbidity factor. Because RGC death often continues after normalization of ocular hypertension, growth factor-mediated protection of compromised neurons may be useful. However, the therapeutic use of nerve growth factor (NGF) has not proven effective at delaying RGC death in glaucoma. We postulated that one cause for the failure of NGF may be related to its binding to two receptors, TrkA and p75. These receptors have distinct cellular distribution in the retina and in neurons they induce complex and sometimes opposing activities. Here, we show in an in vivo therapeutic model of glaucoma that a selective agonist of the pro-survival TrkA receptor was effective at preventing RGC death. RGC loss was fully prevented by combining the selective agonist of TrkA with intraocular pressure-lowering drugs. In contrast, neither NGF nor an antagonist of the pro-apoptotic p75 receptor protected RGCs. These results further a neurotrophic rationale for glaucoma.     

The Expression Patterns of Nogo-A,Myelin Associated Glycoprotein and Oligodendrocyte Myelin Glycoprotein in the Retina After Ocular Hypertension

Nogo-A, a major myelin inhibitory protein, inhibits axon growth and synaptic function in the central nervous system. Glaucoma is a progressive neuropathy as a result of retinal ganglion cell (RGC) death. Synaptic degeneration is thought to be an early pathology of neurodegeneration in glaucoma and precedes RGC loss. Here experimental ocular hypertension model was induced in adult rats with laser coagulation of the episcleral and limbal veins. The expression of Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) in the retina was investigated using immunohistochemistry and Western blotting. We found that Nogo-A was expressed in the RGCs and upregulated after the induction of ocular hypertension. OMgp was only expressed in the inner plexiform layer. There was no MAG expression in the retina. Our data provided, for the first time, the expression patterns of three myelin proteins in the adult retina and suggested an important role of Nogo-A in the RGC death and synaptic degeneration in glaucoma.     

Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.     

Changes in retinal expression of neurotrophins and neurotrophin receptors induced by ocular hypertension

Open angle glaucoma is defined as a progressive and time-dependent death of retinal ganglion cells concomitant with high intraocular pressure, leading to loss of visual field. Because neurotrophins are a family of growth factors that support neuronal survival, we hypothesized that quantitative and qualitative changes in neurotrophins or their receptors may take place early in ocular hypertension, preceding extensive cell death and clinical features of glaucoma. We present molecular, biochemical, and phenotypic evidence that significant neurotrophic changes occur in retina, which correlate temporally with retinal ganglion cell death. After 7 days of ocular hypertension there is a transient up-regulation of retinal NGF, while its receptor TrkA is up-regulated in a sustained fashion in retinal neurons. After 28 days of ocular hypertension there is sustained up-regulation of retinal BDNF, but its receptor TrkB remains unchanged. Throughout, NT-3 levels remain unchanged but there is an early and sustained increase of its receptor TrkC in Müller cells but not in retinal ganglion cells. These newly synthesized glial TrkC receptors are truncated, kinase-dead isoforms. Expression of retinal p75 also increases late at day 28. Asymmetric up-regulation of neurotrophins and neurotrophin receptors may preclude efficient neurotrophic rescue of RGCs from apoptosis. A possible rationale for therapeutic intervention with Trk receptor agonists and p75 receptor antagonists is proposed.     

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.     

Rescue of Retinal Function by BDNF in a Mouse Model of Glaucoma

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.     

Glaucoma Alters the Circadian Timing System

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (∼50–70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.     

The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase

Glutamate neurotoxicity has been postulated to play a prominent role in glaucoma. In this study the possible roles of two subunits of glutamate receptors during the early phase of retinal ganglion cell (RGC) loss in a rat chronic ocular hypertension (COH) model were investigated. COH was induced by applying argon laser to the episcleral and limbal veins of the right eye of rats, the observation times were at 4, 14 and 28 days after the first laser. RGCs were retrogradely labeled by putting Fluoro-Gold (FG) on the surface of both side superior colliculus. Immunohistochemical staining using specific antibodies against N-methyl-d-aspartate receptor 1 (NR1) or glutamate receptor 2/3 (GluR2/3) was performed on the retinal sections of normal and COH eyes. Fluorescent images were captured using confocal laser scanning microscope and the number of NR1 and GluR2/3 labeled cells were counted and cell size was measured using Stereo Investigator. During the observation period, the numbers of NR1 and GluR2/3 positive RGCs in the RGC layer were reduced parallel to the loss of RGC. The dramatic loss of GluR2/3 immunoreactive neurons occurred starting immediately after the first laser to 4 days while the dramatic loss of NR1 immunoreactive neurons occurred from 14 to 28 days after the first laser. Size difference was detected in NR1 immunoreactive RGCs, large ones were more sensitive to the high ocular pressure. These results suggest that both NR1 and GluR2/3 are involved in the mediation of RGC death in the early stage of COH.     

Synaptic Degeneration of Retinal Ganglion Cells in a Rat Ocular Hypertension Glaucoma Model

Aims Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs) and their axons. Little is known of the synaptic degeneration involved in the pathophysiology of glaucoma. Here we used an experimental ocular hypertension model in rats to investigate this issue. Methods Elevated intraocular pressure (IOP) was induced by laser coagulation of the episcleral and limbal veins. RGCs were retrogradely labeled with Fluoro-Gold (FG). The c-fos protein was used as a neuronal connectivity marker. Expression of c-fos in the retinas was investigated by immunohistochemistry at 5 days and 2 weeks after the induction of ocular hypertension. Both surviving RGCs as revealed by retrograde FG-labeled and c-fos-labeled RGCs were counted. Results The c-fos protein was mainly expressed in the nuclei and nucleoli of cells in the ganglion cell layer and inner nuclear layer in the normal retina. We also confirmed that c-fos was also expressed in the nuclei and nucleoli of RGCs retrogradely labeled with FG. There was no significant RGC loss at 5 days but about 13% RGC loss at 2 weeks after the induction of ocular hypertension. The number of RGCs expressing c-fos was significantly lower in the experimental animals at both 5 days and 2 weeks than normal. Conclusion Our study suggests that there is synaptic disconnection for RGCs after ocular hypertension and it may precede the cell death in the early stage. It may provide insight into novel therapeutic strategies to slow the progress of glaucoma. Qing-ling Fu and Xin Li contributed equally to this work.     

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.     

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.     

Pigment Epithelium-Derived Factor Released by Müller Glial Cells Exerts Neuroprotective Effects on Retinal Ganglion Cells

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.     

Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL⁺ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.     

Hippocampal CA3 calcineurin activity participates in depressive-like behavior in rats

The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.     

Activation of autophagy induces retinal ganglion cell death in a chronic hypertensive glaucoma model

Autophagy is reported to have important roles in relation to regulated cell death pathways and neurodegeneration. This study used chronic hypertensive glaucoma rat model to investigate whether the autophagy pathway has a role in the apoptosis of retinal ganglion cells (RGCs) after chronic intraocular pressure (IOP) elevation. Under electron microscopy, autophagosomes were markedly accumulated in the dendrites and cytoplasm of RGCs after IOP elevation. Western blot analysis showed that LC3-II/LC3-I and beclin-1 were upregulated throughout the 8-weeks period after IOP elevation. The pattern of LC3 immunostaining showed autophagy activation in the cytoplasm of RGCs to increase and peak at 4 weeks after IOP elevation. Most of these LC3B-positive RGCs underwent apoptosis by terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling, and inhibition of autophagy with 3-methyladenine decreased RGC apoptosis. The activated pattern shows that autophagy is initially activated in the dendrites of the RGCs, but, thereafter autophagy is mainly activated in the cytoplasm of RGCs. This may show that autophagy is differently regulated in different compartments of the neuron. This present study showed that autophgy is activated in RGCs and has a role in autophagic cell death after chronic IOP elevation.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

IL-17A injury to retinal ganglion cells is mediated by retinal Müller cells in diabetic retinopathy

Diabetic retinopathy (DR), the most common and serious ocular complication, recently has been perceived as a neurovascular inflammatory disease. However, role of adaptive immune inflammation driven by T lymphocytes in DR is not yet well elucidated. Therefore, this study aimed to clarify the role of interleukin (IL)-17A, a proinflammatory cytokine mainly produced by T lymphocytes, in retinal pathophysiology particularly in retinal neuronal death during DR process. Ins2^Akita (Akita) diabetic mice 12 weeks after the onset of diabetes were used as a DR model. IL-17A-deficient diabetic mice were obtained by hybridization of IL-17A-knockout (IL-17A-KO) mouse with Akita mouse. Primarily cultured retinal Müller cells (RMCs) and retinal ganglion cells (RGCs) were treated with IL-17A in high-glucose (HG) condition. A transwell coculture of RGCs and RMCs whose IL-17 receptor A (IL-17RA) gene had been silenced with IL-17RA-shRNA was exposed to IL-17A in HG condition and the cocultured RGCs were assessed on their survival. Diabetic mice manifested increased retinal microvascular lesions, RMC activation and dysfunction, as well as RGC apoptosis. IL-17A-KO diabetic mice showed reduced retinal microvascular impairments, RMC abnormalities, and RGC apoptosis compared with diabetic mice. RMCs expressed IL-17RA. IL-17A exacerbated HG-induced RMC activation and dysfunction in vitro and silencing IL-17RA gene in RMCs abolished the IL-17A deleterious effects. In contrast, RGCs did not express IL-17RA and IL-17A did not further alter HG-induced RGC death. Notably, IL-17A aggravated HG-induced RGC death in the presence of intact RMCs but not in the presence of RMCs in which IL-17RA gene had been knocked down. These findings establish that IL-17A is actively involved in DR pathophysiology and particularly by RMC mediation it promotes RGC death. Collectively, we propose that antagonizing IL-17RA on RMCs may prevent retinal neuronal death and thereby slow down DR progression.Subject terms: Cell death, Medical research     

Structural and functional neuroprotection in glaucoma: role of galantamine-mediated activation of muscarinic acetylcholine receptors

Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision due to glaucoma is caused by the selective death of retinal ganglion cells (RGCs). Treatments for glaucoma, limited to drugs or surgery to lower intraocular pressure (IOP), are insufficient. Therefore, a pressing medical need exists for more effective therapies to prevent vision loss in glaucoma patients. In this in vivo study, we demonstrate that systemic administration of galantamine, an acetylcholinesterase inhibitor, promotes protection of RGC soma and axons in a rat glaucoma model. Functional deficits caused by high IOP, assessed by recording visual evoked potentials from the superior colliculus, were improved by galantamine. These effects were not related to a reduction in IOP because galantamine did not change the pressure in glaucomatous eyes and it promoted neuronal survival after optic nerve axotomy, a pressure-independent model of RGC death. Importantly, we demonstrate that galantamine-induced ganglion cell survival occurred by activation of types M1 and M4 muscarinic acetylcholine receptors, while nicotinic receptors were not involved. These data provide the first evidence of the clinical potential of galantamine as neuroprotectant for glaucoma and other optic neuropathies, and identify muscarinic receptors as potential therapeutic targets for preventing vision loss in these blinding diseases.     

Serum and Antibodies of Glaucoma Patients Lead to Changes in the Proteome,Especially Cell Regulatory Proteins,in Retinal Cells

Purpose

Previous studies show significantly specifically changed autoantibody reactions against retinal antigens in the serum of glaucoma and ocular hypertension (OHT) patients in comparison to healthy people. As pathogenesis of glaucoma still is unknown the aim of this study was to analyze if the serum and antibodies of glaucoma patients interact with neuroretinal cells.

Methods

R28 cells were incubated with serum of patients suffering from primary open angle glaucoma (POAG), normal tension glaucoma (NTG) or OHT, POAG serum after antibody removal and serum from healthy people for 48 h under a normal or an elevated pressure of 15000 Pa (112 mmHg). RGC5 cells were additionally incubated with POAG antibodies under a normal pressure. Protein profiles of the R28 cells were measured with Seldi-Tof-MS, protein identification was performed with Maldi-TofTof-MS. Protein analysis of the RGC5 cells was performed with ESI-Orbitrap MS. Statistical analysis including multivariate statistics, variance component analysis as well as calculating Mahalanobis distances was performed.

Results

Highly significant changes of the complex protein profiles after incubation with glaucoma and OHT serum in comparison to healthy serum were detected, showing specific changes in the cells (e.g. Protein at 9192 Da (p<0.001)). The variance component analysis showed an effect of the serum of 59% on the cells. The pressure had an effect of 11% on the cells. Antibody removal led to significantly changed cell reactions (p<0.03). Furthermore, the incubation with POAG serum and its antibodies led to pro-apoptotic changes of proteins in the cells.

Conclusions

These studies show that the serum and the antibodies of glaucoma patients significantly change protein expressions involved in cell regulatory processes in neuroretinal cells. These could lead to a higher vulnerability of retinal cells towards stress factors such as an elevated IOP and eventually could lead to an increased apoptosis of the cells as in glaucoma.