Yasunari Munemasa

Involvement of RhoA and possible neuroprotective effect of fasudil, a Rho kinase inhibitor, in NMDA-induced neurotoxicity in the rat retina

RhoA, a key protein involved in cytoskeleton regulation modulating neurogenesis and neural plasticity, has been implicated in a variety of cellular functions including the modulation of N-methyl-D-aspartate (NMDA) receptor activity. We examined its possible involvement in NMDA-induced excitotoxicity in the retina, and evaluated the neuroprotective effect of fasudil, a Rho kinase inhibitor, in this model of neurotoxicity. RhoA protein levels in NMDA-treated retinas were assessed by Western blot analysis and localized by immunohistochemistry. Fasudil (10(-6)-10(-4) M together with 4 x 10(-2) M NMDA) was given intravitreally and its effect was evaluated by counting the number of cells in the ganglion cell layer (GCL), measuring the thickness of the inner plexiform layer (IPL), and measuring retinal Thy-1 mRNA levels at 5 days after injection. Western blot analysis showed a transient increase in the level of retinal RhoA and ROCKII proteins at 1 day after NMDA injection, and that this increment was significantly prevented by simultaneous injection of fasudil. Immunohistochemistry showed that NMDA induced a substantial increase in RhoA immunoreactivity in the GCL and the IPL. Fasudil injection reduced cell loss in the GCL and the reduction in IPL thickness after NMDA injection. The reduction in Thy-1 mRNA levels by NMDA was also significantly attenuated by concomitant injection of fasudil. These results suggest that RhoA and ROCKII are upregulated and may be involved in NMDA-induced retinal neurotoxicity, and that fasudil is neuroprotective against glutamate-related excitotoxicity.

Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma.

We investigated the neuroprotective effect of thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) which play critical roles in the regulation of oxidative stress on retinal ganglion cells (RGCs) in a rat glaucoma model. Expression of Trx1 and Trx2 and Trx-interacting protein (Txnip) was observed in the RGC layer (GCL), nerve fiber layer and inner nuclear layer. Txnip-, Trx1- and Trx2-expressing cells in the GCL were primarily colocalized with RGCs. The increased Txnip protein level was observed 2 and 5 weeks after glaucoma induction. Trx1 level decreased 2 weeks after glaucoma induction and more prominently after 5 weeks. No change in Trx2 levels was detected. The effects of Trx1 and Trx2 overexpression on RGC survival were evaluated 5 weeks after glaucoma induction. In nontransfected and EGFP-transfected (used as a negative control) retinas, RGC loss was approximately 27% compared with control. The loss of RGCs in Trx1- and Trx2- transfected retinas was approximately 15 and 17%, respectively. Thus, Trx1 and Trx2 preserved 45 and 37% of cells, respectively that were destined to die in glaucomatous retinas. The results of this study provide evidence for the involvement of oxidative stress in RGC degeneration in experimental glaucoma and point to potential strategies to reduce its impact.

Activation of autophagy in retinal ganglion cells

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell‐protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy‐related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule‐associated protein 1 light chain 3–II (LC3‐II) and beclin‐1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was up‐regulated 1.5‐ to 1.8‐fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6‐fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6‐, 2.7‐, and 1.7‐fold increases in LC3‐II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin‐1 was 1.7‐fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3‐methyladenine, and Wortmannin in RGC‐5 cells under serum‐deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC‐5 cells maintained under conditions of serum deprivation.

Protective effect of thioredoxins 1 and 2 in retinal ganglion cells after optic nerve transection and oxidative stress.

PURPOSE Oxidative stress has been implicated in retinal ganglion cell (RGC) death pathways after optic nerve transection (ONT) and during glaucomatous neuropathy. The authors investigated the expression and cell-protective roles of thioredoxins (cytosolic Trx1 and mitochondrial Trx2), important regulators of the cellular redox state, on RGCs after ONT and pharmacologic oxidative stress induction. METHODS ONT was performed on adult Wistar rats. Trx1 and Trx2 quantitative and spatial expression were examined with Western blot and immunohistochemistry, respectively. Electroporation and calcium phosphate-mediated procedures were used to deliver Trx1 and Trx2 expression constructs to RGCs in vivo and to cultured RGC-5 cells, respectively. Cell-protective effects of Trx1 and Trx2 overexpression on RGCs after ONT and on RGC-5 cells treated with glutamate/buthionine sulfoximine (BSO) were determined by RGC density analysis and cell viability assay, respectively. RESULTS Upregulation of Trx1 and Trx2 was observed in RGCs at different times after ONT and in RGC-5 cells after glutamate/BSO treatment. Trx1 and Trx2 overexpression in RGC-5 cells increased their survival rate by approximately twofold and threefold 24 and 48 hours after glutamate/BSO treatment, respectively. A neuroprotective effect of Trx1 and Trx2 overexpression on RGCs was also observed in vivo; the survival rate of RGCs was increased by 35% and 135%, respectively, 1 and 2 weeks after ONT. CONCLUSIONS These findings provide evidence for in vitro and in vivo cell-protective effects of Trx1 and Trx2 on RGCs against oxidative stress-induced neurodegeneration.

The Role of αA- and αB-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%.

Contribution of mitogen-activated protein kinases to NMDA-induced neurotoxicity in the rat retina

We examined the contributions of the mitogen-activated protein kinases (MAPKs) family [extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK)] to N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Detection of apoptotic cell death in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining began 6 h after intravitreal NMDA (100 nmol) injection and continued to increase thereafter. Western blot analysis showed that phosphorylated MAPKs (p-MAPKs) were expressed in the retina following a temporal manner: maximal expression of phosphorylated ERK (p-ERK) at 1 h, maximal expression of phosphorylated p38 (p-p38) at 6 h, and beginning of phosphorylated JNK (p-JNK) significant increase at 6 h after injection. An immunohistochemical/TUNEL co-localization study showed that p-JNK- and p-p38-positive cells in the RGCL were frequently TUNEL-positive, whereas few p-ERK-positive cells were TUNEL-positive. Moreover, co-injection of inhibitors for JNK (0.2 nmol SP600125) and/or p38 (2.0 nmol SB203580) with NMDA was effective in ameliorating NMDA-induced apoptotic cell loss in the RGCL 12 h after injection, as shown by TUNEL-positive cell counts. These inhibitors also protected the inner retina as shown by morphometric studies such as cell counts in the RGCL and measurement of the IPL thickness 7 days after injection. On the other hand, an ERK inhibitor (2.0 nmol U0126) did not suppress NMDA-induced cell death in the RGCL nor thinning of the IPL. These findings suggest that JNK and p38 are proapoptotic in NMDA-induced cell death in the RGCL, but not ERK.

Calcium/calmodulin-dependent protein kinase II regulates the phosphorylation of CREB in NMDA-induced retinal neurotoxicity.

We examined the role of the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and cyclic AMP-response element binding protein (CREB) in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed early elevation of phosphorylated CaMKII (p-CaMKII) protein levels and subsequential elevation of phosphorylated CREB (p-CREB) protein after NMDA injection. Immunohistochemistry showed that p-CaMKII was colocalized with Thy-1-positive retinal ganglion cells (RGCs) after NMDA injection. The increase in the p-CaMKII protein level was significantly inhibited by the preinjection of CaMKII small interfering RNA (siRNA), whereas negative control siRNA did not affect. Moreover, the increase in the p-CREB protein level after NMDA injection was also prevented by preinjection of CaMKII siRNA. In addition, our morphometric study of neurotracer retrograde labeling and Thy-1-positive cells showed that CaMKII siRNA significantly accelerated NMDA-induced RGC loss. Furthermore, the prevention of CREB binding by CRE decoy oligonucleotide also exacerbated RGC loss. These results suggest that the activation of CaMKII may regulate CREB phosphorylation and that the transient phosphorylation of CaMKII and CREB may be a neuroprotective response against NMDA-induced neurotoxicity.

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

J. Neurochem. (2010) 115, 1508–1519.

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).

Neuroprotective effect of atrial natriuretic peptide against NMDA-induced neurotoxicity in the rat retina

Atrial natriuretic peptide (ANP) can regulate aqueous humor production in the eye and has recently been suggested to play some functional roles in the retina. It has also been reported that ANP increases tyrosine hydroxylase (TH) mRNA levels and intracellular dopamine levels in PC12 cells. The effect of ANP on TH levels and the role of ANP in retinal excitotoxicity remain unknown. In this study, we investigated the effects of ANP on TH expression and dopamine levels in rat retina after intravitreal injection of NMDA. Immunohistochemistry localized natriuretic peptide receptor-A (NPRA) in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in the rat retina. Quantitative real-time PCR and Western blot analysis showed a dramatic reduction in retinal TH levels 5 days after NMDA injection, while ANP, at a concentration of 10(-4) M, ameliorated this reduction in TH mRNA and TH protein levels. High-performance liquid chromatography (HPLC) analysis showed that NMDA reduced dopamine levels in the retina, and that ANP attenuated this reduction. Moreover, morphological analysis showed that ANP ameliorated NMDA-induced neurotoxicity through NPRA. The ameliorative effect of ANP was inhibited by a dopamine D(1) receptor antagonist. These results suggest that ANP may have a neuroprotective effect through possible involvement of dopamine induction.