Yasushi Kitaoka

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.

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.

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.

Neuroprotective effect of nitric oxide against NMDA-induced neurotoxicity in the rat retina is associated with tyrosine hydroxylase expression.

N-methyl-D-aspartate (NMDA) may affect dopaminergic cells, which contain tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. To clarify the involvement of TH in the neuroprotective effects of nitric oxide (NO), we investigated whether NMDA alters TH mRNA and TH protein levels and whether NO inhibits NMDA-induced changes in the rat retina. Dopamine levels in the retina were measured by high-performance liquid chromatography (HPLC). Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that intravitreal injection of NMDA caused a significant reduction in TH mRNA levels in the retina. Similarly, Western blot analysis showed that NMDA decreased the production of TH protein. These reductions in TH mRNA and TH protein levels were attenuated by concomitant injection of NOC 18, an NO donor. HPLC analysis showed that NMDA reduced dopamine levels in the retina and that NO attenuated this reduction. Furthermore, morphological analysis showed that NO prevents NMDA-induced neurotoxicity through dopamine D(1) receptors. These results suggest that the neuroprotective effect of NO may be associated with the induction of TH expression and increased levels of dopamine.

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.

Biochemical and morphological characterization of spontaneously hypertensive hyperlipidaemic rats

1. To develop and characterize a new animal model of hypertension and hyperlipidaemia, we cross‐bred spontaneously hypertensive rats (SHR) with spontaneously hyperlipidaemic rats (HLR).

NMDA-induced interleukin-1β expression is mediated by nuclear factor-kappa B p65 in the retina

Transcription factors of the nuclear factor-kappa B (NF-kappaB) p65/RelA may be involved in neuronal cell death. We examined the involvement of NF-kappaB p65 in N-methyl-D-aspartate (NMDA)-induced upregulation of interleukin (IL)-1beta, a proinflammatory cytokine, and subsequent neurotoxicity in the rat retina. Immunohistochemistry showed that IL-1beta is localized not only in glial cells, but also in neurons, especially retinal ganglion cells (RGCs) after intravitreal injection of NMDA. Semi-quantitative real-time PCR showed that NMDA induces an increase in IL-1beta mRNA levels. Preinjection of NF-kappaB p65 antisense oligodeoxynucleotide (AS ODN) ameliorated the NMDA-induced increase in IL-1beta mRNA expression. Western blot analysis showed elevated levels of retinal IL-1beta protein 12 h after intravitreal NMDA injection and this elevation was significantly inhibited by NF-kappaB p65 AS ODN. Neurotracer labeling showed that the inhibition of NF-kappaB p65 by AS ODN or siRNA exerted a protective effect against NMDA-induced RGC loss. IL-1beta siRNA also had a protective effect on RGC number in NMDA-treated eyes. Penetration of AS ODN and siRNA to cells in the RGC layer and inner nuclear layer was confirmed after labeling with rhodamine or Cy3. These results suggest that NF-kappaB p65 may participate in the induction of IL-1beta expression in NMDA-induced retinal neuronal cell death and that the inhibition of NF-kappaB p65 and IL-1beta with the use of AS ODN or siRNA may be a viable neuroprotective strategy for RGC survival.