Histone H2B induces retinal ganglion cell death through toll-like receptor 4 in the vitreous of acute primary angle closure patients.

Acute primary angle closure (APAC) is a disease of ophthalmic urgency; lack of treatment can lead to blindness. Even after adequate treatment for APAC, subsequent elevated acute intraocular pressure induces severe neuronal damage which can result in secondary glaucomatous optic neuropathy (GON). Damage-associated molecular patterns (DAMPs) are released from damaged and dead neuronal cells, which induce secondary inflammatory changes and further tissue damage. Our hypothesis is that histone H2B (H2B), which is one of the DAMPs, is released from damaged cells in the development of GON after APAC treatment. Intravitreal injection of H2B induces neuronal cell death through toll-like receptor 4 (TLR4) expression, following the upregulation of inflammatory cytokine mRNAs and phosphorylation of mitogen activated protein kinases (MAPKs). Knockdown of TLR4 caused a reduction of H2B neurotoxicity in damaged cells through TLR4 signaling. Significantly increased H2B was observed in the vitreous cells of APAC patients. In addition, enhanced H2B protein correlated with decreased ganglion cell analysis and retinal ganglion cell (RGC) layer thinning, which indicates the effect of H2B on RGCs. Our data from clinical and animal studies show the involvement of H2B-TLR4 pathways in the development of GON after APAC treatment providing new insight for the mechanism of RGC degeneration.

Axonal protection by thioredoxin-1 with inhibition of interleukin-1ß in TNF-induced optic nerve degeneration.

Interleukin (IL)-1ß, a proinflammatory cytokine, is a key mediator in several acute and chronic neurological diseases. Thioredoxin-1 (TRX1) acts as an antioxidant and plays a protective role in certain neurons. We examined whether exogenous TRX1 exerts axonal protection and affects IL-1ß levels in tumor necrosis factor (TNF)-induced optic nerve degeneration in rats. Immunoblot analysis showed that IL-1ß was upregulated in the optic nerve after intravitreal injection of TNF. Treatment with recombinant human (rh) TRX1 exerted substantial protective effects against TNF-induced axonal loss. The increase in the IL-1ß level in the optic nerve was abolished by rhTRX1. Treatment with rhTRX1 also significantly inhibited increased glial fibrillary acidic protein (GFAP) levels induced by TNF. Immunohistochemical analysis showed substantial colocalization of IL-1ß and GFAP in the optic nerve after TNF injection. These results suggest that IL-1ß is upregulated in astrocytes in the optic nerve after TNF injection and that exogenous rhTRX1 exerts axonal protection with an inhibitory effect on IL-1ß.

Axonal protection by short-term hyperglycemia with involvement of autophagy in TNF-induced optic nerve degeneration.

Previous reports showed that short-term hyperglycemia protects optic nerve axons in a rat experimental hypertensive glaucoma model. In this study, we investigated whether short-term hyperglycemia prevents tumor necrosis factor (TNF)-induced optic nerve degeneration in rats and examined the role of autophagy in this axon change process. In phosphate-buffered saline (PBS)-treated rat eyes, no significant difference in axon number between the normoglycemic (NG) and streptozotocin (STZ)-induced hyperglycemic (HG) groups was seen at 2 weeks. Substantial degenerative changes in the axons were noted 2 weeks after intravitreal injection of TNF in the NG group. However, the HG group showed significant protective effects on axons against TNF-induced optic nerve degeneration compared with the NG group. This protective effect was significantly inhibited by 3-methyladenine (3-MA), an autophagy inhibitor. Immunoblot analysis showed that the LC3-II level in the optic nerve was increased in the HG group compared with the NG group. Increased p62 protein levels in the optic nerve after TNF injection was observed in the NG group, and this increase was inhibited in the HG group. Electron microscopy showed that autophagosomes were increased in optic nerve axons in the HG group. Immunohistochemical study showed that LC3 was colocalized with nerve fibers in the retina and optic nerve in both the NG and HG groups. Short-term hyperglycemia protects axons against TNF-induced optic nerve degeneration. This axonal-protective effect may be associated with autophagy machinery.

The Effect of Rebamipide on Ocular Surface Disorders Induced by Latanoprost and Timolol in Glaucoma Patients.

Purpose. To examine the efficacy of ophthalmic rebamipide suspensions on ocular surface disorders induced by antiglaucoma eye drops. Patients and Methods. Forty eyes of 40 patients receiving latanoprost (0.005%) and timolol (0.5%) were included in this randomized prospective study. The patients were randomly divided into two groups (n = 20): the rebamipide-treated group and control group. Changes in intraocular pressure, tear film break-up time (TBUT), and corneal epithelial barrier function were evaluated at baseline, 4 weeks, and 8 weeks after rebamipide administration. Furthermore, superficial punctate keratopathy severity was evaluated by scoring the lesion area and density. Results. There was no significant difference in intraocular pressure before and after rebamipide treatment. However, corneal epithelial barrier function improved significantly 4 and 8 weeks after rebamipide treatment. TBUT was partially, but significantly, increased (P = 0.02) 8 weeks after rebamipide treatment, whereas no significant change was observed at 4 weeks. Additionally, a significant decrease in area and density of keratopathy was observed 8 weeks after rebamipide treatment but not at 4 weeks. The control group showed no significant difference compared to baseline. Conclusions. Our data suggests that rebamipide treatment may reduce the occurrence of drug-induced ocular surface disorder.

Axonal protection by brimonidine with modulation of p62 expression in TNF-induced optic nerve degeneration.

PURPOSE: The p62, also called sequestosome 1 (SQSTM1), plays a crucial role in tumor necrosis factor (TNF)-induced optic nerve degeneration. Brimonidine has been shown to have protective effects on retinal ganglion cell bodies, although its role in their axons remains to be examined. We determined whether brimonidine modulates axonal loss induced by TNF and affects the expression of p62 in the optic nerve. METHODS: Experiments were performed on adult male Wistar rats that received an intravitreal injection of 10 ng TNF alone or simultaneous injection of TNF and 2, 20, or 200 pmol of brimonidine tartrate. The expression of p62 in the optic nerve was examined by immunoblot analysis. The effects of brimonidine on axons were evaluated by counting axon numbers 2 weeks after intravitreal injection. RESULTS: Intravitreal injection of brimonidine exerted substantial axonal protection against TNF-induced optic nerve degeneration. Immunoblot analysis showed that p62 was upregulated in the optic nerve after intravitreal injection of TNF, and that this increase was completely inhibited by brimonidine. Treatment with brimonidine alone also significantly decreased p62 protein levels in the optic nerve compared with the basal level. CONCLUSIONS: These results suggest that the modulation of p62 levels in the optic nerve by brimonidine may be involved partly in its axonal protection.

Autophagy in axonal degeneration in glaucomatous optic neuropathy.

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.

Proteomic study of retinal proteins associated with transcorneal electric stimulation in rats.

Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina.

Axonal protection by modulation of p62 expression in TNF-induced optic nerve degeneration.

p62, which is also called sequestosome 1 (SQSTM1), plays a critical role in neuronal cell death. However, the role of p62 in axonal degeneration remains unclear. We evaluated whether the modulation of p62 expression may affect axonal loss in tumor necrosis factor (TNF)-induced optic nerve degeneration. Immunoblot analysis showed that p62 was upregulated in the optic nerve after intravitreal injection of TNF. Treatment with p62 small interfering RNA (siRNA) exerted a partial but significant protective effect against TNF-induced axonal loss. Rapamycin exerted substantial axonal protection after TNF injection. We found that the increase in p62 was significantly inhibited by p62 siRNA. Treatment with rapamycin also significantly inhibited increased p62 protein levels induced by TNF. These results suggest that the upregulation of p62 may be involved in TNF-induced axonal degeneration and that decreased p62 levels may lead to axonal protection.

Axonal protection via modulation of the amyloidogenic pathway in tumor necrosis factor-induced optic neuropathy.

PURPOSE: To examine the changes in and localization of phosphorylated presenilin1 (p-PS1) and amyloid precursor protein (APP) in the optic nerve after intravitreal injection of TNF and to investigate the role of γ-secretase in the cleavage of APP in optic nerve degeneration. METHODS: Groups of rats were euthanatized at 1 or 2 weeks after intravitreal injection of TNF. Levels of p-PS1 protein in the optic nerve were determined by immunoblotting and immunohistochemistry. The localization of APP was determined by immunohistochemistry, and its downstream cleavage was determined by immunoprecipitation using 6E10 antibody followed by immunoblotting with an APP intracellular domain (AICD) antibody. The effect of a γ-secretase inhibitor on TNF-induced optic nerve degeneration was determined by counting the number of axons. RESULTS: p-PS1 was increased in the optic nerve after TNF injection and was found to colocalize with vimentin and glial fibrillary acidic protein, markers of astrocytes. Immunoprecipitation using 6E10 antibody followed by immunoblotting with AICD antibody revealed an increase in γ-secretase activation in the optic nerve after TNF injection, which was inhibited by treatment with the γ-secretase inhibitor. Moreover, γ-secretase inhibition significantly prevented the loss of axons in the optic nerve after TNF injection. CONCLUSIONS: The increase in p-PS1 and activation of γ-secretase in the optic nerve may be associated with TNF-induced axonal degeneration. Modulation of γ-secretase activity may be useful for the treatment of TNF-related optic neuropathy.

[Effects of selective laser trabeculoplasty treatment in steroid-induced glaucoma].

PURPOSE: To evaluate the effectiveness of selective laser trabeculoplasty (SLT) on steroid-induced glaucoma. METHODS: The study included 46 eyes of 41 subjects who were followed up for at least 12 months after SLT. The included 10 eyes with steroid-induced glaucoma, 16 eyes with primary open angle glaucoma (POAG), 10 eyes with pseudoexfoliation glaucoma (PEX.G) and 10 eyes with mixed glaucoma (Mixed. G). The range of the SLT laser was 360 degrees. Intraocular pressure (IOP) before and after SLT, and cumulative survival rate after SLT were determined. RESULTS: Significant decreases in IOP were observed after SLT in the steroid-induced glaucoma group, the POAG group and the PEX.G group. At 12 months after SLT, preoperation IOP decreased by 35.9% (29.9 +/- 7.5 mmHg to 17.9 +/- 2.2 mmHg) in the steroid-induced glaucoma group, 13.2% (20.0 +/- 3.0 mmHg to 17.3 +/- 3.1 mmHg) in the POAG group, 10.7% (21.1 +/- 4.0 mmHg to 18.1 +/- 4.1 mmHg) in the PEX.G group and 6.9% (21.3 +/- 1.9 mmHg to 19.9 +/- 3.4 mmHg) in the Mixed.G group. Cumulative survival rates were 80%, 56.3%, 50.0%, 40.0% in the steroid-induced glaucoma, POAG, PEX.G, and Mixed. G groups, respectively, at 12 months after SLT (Logrank test, p = 0.467). CONCLUSION: These data suggest that SLT increased IOP reduction rates for steroid-induced glaucoma more than for any other group.

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury.

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).

Molecular mechanisms of retinal ganglion cell degeneration in glaucoma and future prospects for cell body and axonal protection.

Glaucoma, which affects more than 70 million people worldwide, is a heterogeneous group of disorders with a resultant common denominator; optic neuropathy, eventually leading to irreversible blindness. The clinical manifestations of primary open-angle glaucoma (POAG), the most common subtype of glaucoma, include excavation of the optic disc and progressive loss of visual field. Axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies are observed in glaucoma, in which the reduction of intraocular pressure (IOP) is known to slow progression of the disease. A pattern of localized retinal nerve fiber layer (RNFL) defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. The mechanisms of degeneration of neuronal cell bodies and their axons may differ. In this review, we addressed the molecular mechanisms of cell body death and axonal degeneration in glaucoma and proposed axonal protection in addition to cell body protection. The concept of axonal protection may become a new therapeutic strategy to prevent further axonal degeneration or revive dying axons in patients with preperimetric glaucoma. Further study will be needed to clarify whether the combination therapy of axonal protection and cell body protection will have greater protective effects in early or progressive glaucomatous optic neuropathy (GON).

17ß-estradiol prevents reduction of retinal phosphorylated 14-3-3 zeta protein levels following a neurotoxic insult.

Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron, although its mechanism of action remains to be elucidated. In this study, we found that the levels of 14-3-3 zeta mRNA and phosphorylated and total 14-3-3 zeta proteins were significantly decreased in the rat retina after intravitreal injection of N-methyl-d-aspartate (NMDA). 17ß-E2 implantation significantly inhibited NMDA-induced decreases in phosphorylated but not in total 14-3-3 zeta protein levels in the retina. There was a decrease in both phosphorylated and total 14-3-3 protein levels in RGC-5 cells, a retinal ganglion cell line, after glutamate and buthionine sulfoximine (BSO) exposure, and 17ß-E2 treatment significantly inhibited only the decrease in phosphorylated but not in total 14-3-3 zeta protein levels. The cell viability assay showed substantial cell death after glutamate and BSO exposure and that 17ß-E2 treatment significantly protects against this cell death. 17ß-E2 treatment also significantly increased the level of phosphorylated 14-3-3 protein in RGC-5 cells without other treatments. These results suggest that a decrease in 14-3-3 zeta expression may be associated with retinal neurotoxicity induced by NMDA or the combination of glutamate and BSO. The regulation of 14-3-3 zeta phosphorylation is one possible mechanism of the protective effect of 17ß-E2 in the retina.

Protective effect of thalidomide against N-methyl-D-aspartate-induced retinal neurotoxicity.

Thalidomide, an inhibitor of tumor necrosis factor-α (TNF-α) production, has been indicated to be useful for many inflammatory and oncogenic diseases. In the present study, we examined whether thalidomide (50 mg/kg/day, p.o.) has a protective effect against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity in rats. A morphometric analysis showed that systemic administration of thalidomide protects neural cells in the ganglion cell layer (GCL) in a dose-dependent manner and significantly decreases the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in GCL and in the inner nuclear layer (INL). ELISA showed that thalidomide significantly suppressed the elevation of TNF-α 6 and 24 hr after an NMDA injection. Western blot analysis revealed a significant increase in nuclear factor-κB (NF-κB) p65 level in the retinas treated with NMDA at 24 hr after the injection, but not at 6 or 72 hr. Furthermore, an increase in p-JNK and p-p38 levels was also observed in the retina after NMDA injection. Thalidomide suppressed the increased expressions of NF-κB p65, p-JNK, and p-p38 after NMDA injection. Immunohistochemical analysis showed that thalidomide attenuated NF-κB p65 immunoreactivity in the GCL induced by NMDA treatment. In the NMDA-treated group, translocation of NF-κB p65 from the cytoplasm to the nucleus was detected in TUNEL-positive cells exposed to NMDA treatment. These results suggest new indications for thalidomide against neurodegenerative diseases.

Axonal protection by 17ß-estradiol through thioredoxin-1 in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron. However, most studies examined cell body protection, and the role of 17ß-E2 in axonal degeneration of retinal ganglion cells (RGC) remains unclear. In this study, we showed the presence of thioredoxin-1 (Trx1) in the optic nerve axons and found that the levels of Trx1 protein were significantly decreased in isolated RGC and the optic nerve after intravitreal injection of TNF, which was shown previously to induce optic nerve degeneration and subsequent loss of RGC. These changes were concomitant with disorganization of the microtubules with neurofilament accumulation, which were blocked by 17ß-E2 implantation. 17ß-E2 treatment also totally abolished TNF-induced decreases in Trx1 protein levels in isolated RGC and the optic nerve. The induction of Trx1 by 17ß-E2 in the optic nerve was significantly inhibited by simultaneous injection of Trx1 small interfering RNA (siRNA) with TNF. Up-regulation of Trx1 by 17ß-E2 in RGC-5 cells was prevented by Trx1 siRNA treatment. 17ß-E2 significantly prevented TNF-induced axonal loss, and this axonal-protective effect was inhibited by intravitreal injection of Trx1 siRNA. This finding was also supported by the quantification of microtubules and neurofilaments. These results suggest that a Trx1 decrease in RGC bodies and their axons may be associated with TNF-induced optic nerve axonal degeneration. Axonal protection by 17ß-E2 may be related to its regulatory effect on Trx1 induction.

Modulation of mitochondria in the axon and soma of retinal ganglion cells in a rat glaucoma model.

Mitochondrial abnormality has been implicated in various models of retinal ganglion cell (RGC) degeneration. We investigated modulation of mitochondrial membrane permeability and apoptosis-inducing factor (AIF) translocation in a rat experimental glaucoma model. A decrease in MitoTracker-labeled mitochondria around the lamina area of the optic nerve was observed in the glaucomatous eye. Immunoblot analysis for axonal motor proteins showed that a significant decrease in kinesin 1 and myosin Va levels in the glaucomatous optic nerve. A significant decrease in mitochondrial thioredoxin 2 (Trx2) level was observed in the optic nerve after intraocular pressure (IOP) elevation. Translocation of AIF from the mitochondria to the axoplasm and nucleus was observed in the axon and cell body, respectively. Trx2 over-expression in the mitochondrial membrane of RGC-5 cells inhibited AIF translocation, resulting in cytoprotective effect against neurotoxicity induced by TNF-α/buthionine sulfoximine treatment. In vivo transfection was performed with EGFP-Trx2 plasmid and electroporation. Over-expression of Trx2 in the retina and optic nerve indicated the protective effect against high IOP induced axonal degeneration. Thus, the decreased mitochondrial membrane potential and subsequent AIF translocation were involved in the glaucomatous neurodegeneration. Furthermore, modulation of mitochondria through the inhibition of AIF translocation may become a new treatment strategy for neurodegenerative disease, such as glaucoma.

Kinesin-1 and degenerative changes in optic nerve axons in NMDA-induced neurotoxicity.

We examined the histologic findings of optic nerve axons and changes in kinesin-1, which is involved in axonal flow, in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in rats. Substantial degenerative changes visualized as black profiles and pale large axons were observed 72h after NMDA injection, but those degenerative changes were not apparent in axons 12 and 24h after injection. Morphometric analysis showed a significant, approximately 40% reduction in the number of axons 72h after NMDA injection. Immunohistochemical study showed that there was a recognizable loss of neurofilament-immunopositive dots, but myelin basic protein immunostaining was unchanged 72h after NMDA injection. Western blot analysis showed early elevation of kinesin-1 (KIF5B) protein levels in the retina 24 and 72h after NMDA injection. Conversely, significant decreases in KIF5B protein levels in the optic nerve were seen during the same time course. Immunohistochemical study also showed that there was a reduction in KIF5B immunoreactivity in axons, but neurofilament immunostaining was unchanged 24h after NMDA injection. These findings suggest that the intravitreal injection of NMDA causes neurofilament loss without myelin alteration in the early stage. The depletion of kinesin-1 precedes axonal degeneration of the optic nerve in NMDA-induced neurotoxicity.

Thioredoxins 1 and 2 protect retinal ganglion cells from pharmacologically induced oxidative stress, optic nerve transection and ocular hypertension.

Oxidative damage has been implicated in retinal ganglion cell (RGC) death after optic nerve transection (ONT) and during glaucomatous neuropathy. Here, we analyzed the expression and cell protective role of thioredoxins (TRX), key regulators of the cellular redox state, in RGCs damaged by pharmacologically induced oxidative stress, ONT and elevated intraocular pressure (IOP). The endogenous level of thioredoxin-1 (TRX1) and thioredoxin-2 (TRX2) in RGCs after axotomy and in RGC-5 cells after glutamate/buthionine sulfoximine (BSO) treatment showed upregulation of TRX2, whereas no significant change was observed in TRX1 expression. The increased level TRX-interacting protein (TXNIP) in the retinas was observed 2 and 5 weeks after IOP elevation. TRX1 level was decreased at 2 weeks and more prominently at 5 weeks after IOP increase. No change in TRX2 levels in response to IOP change was observed. Overexpression of TRX1 and TRX2 in RGC-5 treated with glutamate/BSO increased the cell survival by 2- and 3-fold 24 and 48 h after treatment, respectively. Overexpression of these proteins in the retina increased the survival of RGCs by 35 and 135% 7 and 14 days after ONT, respectively. In hypertensive eyes, RGC loss was approximately 27% 5 weeks after IOP elevation compared to control. TRX1 and TRX2 overexpression preserved approximately 45 and 37% of RGCs, respectively, that were destined to die due to IOP increase.

Axonal and cell body protection by nicotinamide adenine dinucleotide in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies have demonstrated the crucial role of nicotinamide adenine dinucleotide (NAD) biosynthesis in axonal protection of motor neurons, but the role of nicotinamide mononucleotide adenylyltransferase 1 and NAD in optic nerve degeneration is unclear. Intravitreal injection of tumor necrosis factor (TNF) induces optic nerve degeneration and subsequent loss of retinal ganglion cells. We found that the levels of nicotinamide mononucleotide adenylyltransferase 1 mRNA and protein and of NAD were significantly decreased in the optic nerve after intravitreal injection of TNF in rats. The concomitant disorganization of microtubules with vacuoles and neurofilament accumulations in the axons were blocked by exogenous NAD treatment. Nicotinamide adenine dinucleotide also prevented TNF-induced axonal loss and delayed retinal ganglion cell loss 2 months after TNF injection. Microglia identified by immunohistochemistry were increased in the optic nerves after TNF injection; this increase was inhibited by NAD treatment. These results suggest that axonal nicotinamide mononucleotide adenylyltransferase 1 and NAD declines are associated with TNF-induced optic nerve axonal degeneration and that axonal protection of NAD may be related to its inhibitory effect on microglial activation.

The role of alphaA- and alphaB-crystallins in the survival of retinal ganglion cells after optic nerve axotomy.

PURPOSE: Stress-induced crystallin expression is commonly viewed as activation of the cell survival mechanism. The authors analyzed the expression of alphaA- and alphaB-crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival. METHODS: ONT was performed on adult Wistar rats. Quantitative and spatial expression were examined with Western blot analysis and immunohistochemistry, respectively. Electroporation was used to deliver alphaA and alphaB expression constructs to RGCs. Cell-protective effects of alphaA and alphaB overexpression after ONT were determined by RGC density analysis. RESULTS: Expression of alphaA and alphaB in the retina was observed predominantly in the ganglion cell layer, where most crystallin-positive cells were colocalized with RGCs. Levels of alphaA and alphaB proteins after ONT were decreased 1.6-fold. The effect of alphaA and alphaB overexpression on RGC survival was evaluated 7 and 14 days after axotomy. At day 7 after ONT, 1426 +/- 70 and 1418 +/- 81 RGCs/mm(2) were present in retinas electroporated with alphaA and alphaB expression constructs, respectively, compared with 1010 +/- 121 RGCs/mm(2) in sham-transfected or 1016 +/- 88 RGCs/mm(2) in nontransfected retinas. Numbers of surviving RGCs at 14 days were 389 +/- 57 and 353.57 +/- 60 cells/mm(2) after alphaA and alphaB transfection, respectively, compared with 198 +/- 29 cells/mm(2) after transfection with the vector alone or 206 +/- 60 cells/mm(2) in nontransfected retinas. CONCLUSIONS: Increases of approximately 95% and 75% in RGC survival mediated by alphaA and alphaB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of alphaA and alphaB overexpression was approximately 40%.