K. Kuribayashi

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.

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.

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.