Vol. 38, No. 111765 Biol. Pharm. Bull. 38, 1765–1771 (2015)
Regular Article
Protective Effects of Everolimus against N-Methyl-D -aspartic Acid-Induced Retinal Damage in Rats Ikumi Hayashi, Yuto Aoki, Daiki Asano, Hiroko Ushikubo, Asami Mori, Kenji Sakamoto, Tsutomu Nakahara,* and Kunio Ishii Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences; 5–9–1 Shirokane, Minato-ku, Tokyo 108–8641, Japan. Received June 4, 2015; accepted August 31, 2015 We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl- D -aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDAinduced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells. Key words excitotoxicity; extracellular signal-regulated kinase (ERK); Müller cell; mammalian target of rapamycin (mTOR); glutamate
Glutamate-induced neurotoxicity is implicated in certain ocular diseases, including diabetic retinopathy and glaucoma.1,2) In many cases, the neurotoxic effect of glutamate is attributed to excessive stimulation of N-methyl-D -aspartic acid (NMDA) receptors.3,4) NMDA receptor-mediated neuronal cell death is associated with an increase in intracellular Ca2+ concentration, leading to activation of Ca2+-dependent enzyme systems, such as neuronal nitric oxide (NO) synthase5,6) and calpains.7,8) In addition to these direct effects on neurons, indirect effects, such as induction of inflammation and degeneration of retinal capillaries, may contribute to NMDA-induced neuronal damage in the retina.9–11) Our recent studies have shown that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), exerts protective effects on NMDA-induced retinal damage in rats.12,13) mTOR is a serine/threonine kinase that signals through two distinct functional mTOR complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2).14) Rapamycin inhibits mTORC1, which prevents phosphorylation of at least 2 well-characterized effectors: the p70S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4E-BP1).15) Inactivation of mTORC1 can cause autophagy.16) This autophagy might contribute to beneficial effects of rapamycin on neurons by enhancing the clearance of harmful protein aggregates.17–20) In addition, rapamycin could prevent neuroinflammatory responses, including activation of microglia.13,21) Multiple mechanisms are likely involved in the neuroprotective effects of rapamycin. In this study, which was designed to expand our previous study,12) we investigated beneficial effects of everolimus, another inhibitor of mTOR, against NMDA-induced retinal neurotoxicity in rats. Activation of the extracellular signal-reg-
ulated kinase (ERK) pathway in the retina might be involved in the neuroprotective effect of rapamycin against NMDAinduced retinal neurotoxicity.12) Therefore, we also examined whether activation of ERK pathway is involved in these neuroprotective effects.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats weighing 220–240 g were maintained on a standard diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum in a room with constant temperature (22±2°C) and humidity (55±5%) and a 12-h light/dark cycle. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research and the Regulations for the Care and Use of Laboratory Animals in Kitasato University adopted by the Institutional Animal Care and Use Committee of Kitasato University. Experimental Procedures We first examined the effects of everolimus on NMDA-induced morphological changes in the retina. Animals were divided into the three groups: NMDA (200 nmol)+vehicle (n=5), NMDA (200 nmol)+everolimus (5 nmol) (n=6), and NMDA (200 nmol)+everolimus (20 nmol) (n=6). Under general anesthesia with 50 mg/kg pentobarbital sodium (Nacalai Tesque, Kyoto, Japan), rats were injected with everolimus (5 or 20 nmol; LC Laboratories, Woburn, MA, U.S.A.) or vehicle (dimethyl sulfoxide (DMSO)), mixed with 200 nmol of NMDA (Nacalai Tesque) in a total volume of 5 µL. Injections were made into the vitreous cavity of one eye.
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
1766
Fig. 1.
Biol. Pharm. Bull.
Vol. 38, No. 11 (2015)
Effects of Everolimus against Retinal Damage 7 d after Intravitreal Injection of NMDA
A: Vehicle (a); NMDA (200 nmol) (b); NMDA (200 nmol)+everolimus (Eve, 5 nmol) (c); NMDA (200 nmol)+Eve (20 nmol) (d). Scale bar=30 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. B: Retinal damage was quantitatively assessed by counting the cell number in the GCL. Data from the treated eye of each animal were normalized to those of the vehicle-treated control eye. Each column with a vertical bar represents the mean±S.E.M. from 5 to 6 animals. * p
Regular Article
Protective Effects of Everolimus against N-Methyl-D -aspartic Acid-Induced Retinal Damage in Rats Ikumi Hayashi, Yuto Aoki, Daiki Asano, Hiroko Ushikubo, Asami Mori, Kenji Sakamoto, Tsutomu Nakahara,* and Kunio Ishii Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences; 5–9–1 Shirokane, Minato-ku, Tokyo 108–8641, Japan. Received June 4, 2015; accepted August 31, 2015 We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl- D -aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDAinduced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells. Key words excitotoxicity; extracellular signal-regulated kinase (ERK); Müller cell; mammalian target of rapamycin (mTOR); glutamate
Glutamate-induced neurotoxicity is implicated in certain ocular diseases, including diabetic retinopathy and glaucoma.1,2) In many cases, the neurotoxic effect of glutamate is attributed to excessive stimulation of N-methyl-D -aspartic acid (NMDA) receptors.3,4) NMDA receptor-mediated neuronal cell death is associated with an increase in intracellular Ca2+ concentration, leading to activation of Ca2+-dependent enzyme systems, such as neuronal nitric oxide (NO) synthase5,6) and calpains.7,8) In addition to these direct effects on neurons, indirect effects, such as induction of inflammation and degeneration of retinal capillaries, may contribute to NMDA-induced neuronal damage in the retina.9–11) Our recent studies have shown that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), exerts protective effects on NMDA-induced retinal damage in rats.12,13) mTOR is a serine/threonine kinase that signals through two distinct functional mTOR complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2).14) Rapamycin inhibits mTORC1, which prevents phosphorylation of at least 2 well-characterized effectors: the p70S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4E-BP1).15) Inactivation of mTORC1 can cause autophagy.16) This autophagy might contribute to beneficial effects of rapamycin on neurons by enhancing the clearance of harmful protein aggregates.17–20) In addition, rapamycin could prevent neuroinflammatory responses, including activation of microglia.13,21) Multiple mechanisms are likely involved in the neuroprotective effects of rapamycin. In this study, which was designed to expand our previous study,12) we investigated beneficial effects of everolimus, another inhibitor of mTOR, against NMDA-induced retinal neurotoxicity in rats. Activation of the extracellular signal-reg-
ulated kinase (ERK) pathway in the retina might be involved in the neuroprotective effect of rapamycin against NMDAinduced retinal neurotoxicity.12) Therefore, we also examined whether activation of ERK pathway is involved in these neuroprotective effects.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats weighing 220–240 g were maintained on a standard diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum in a room with constant temperature (22±2°C) and humidity (55±5%) and a 12-h light/dark cycle. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research and the Regulations for the Care and Use of Laboratory Animals in Kitasato University adopted by the Institutional Animal Care and Use Committee of Kitasato University. Experimental Procedures We first examined the effects of everolimus on NMDA-induced morphological changes in the retina. Animals were divided into the three groups: NMDA (200 nmol)+vehicle (n=5), NMDA (200 nmol)+everolimus (5 nmol) (n=6), and NMDA (200 nmol)+everolimus (20 nmol) (n=6). Under general anesthesia with 50 mg/kg pentobarbital sodium (Nacalai Tesque, Kyoto, Japan), rats were injected with everolimus (5 or 20 nmol; LC Laboratories, Woburn, MA, U.S.A.) or vehicle (dimethyl sulfoxide (DMSO)), mixed with 200 nmol of NMDA (Nacalai Tesque) in a total volume of 5 µL. Injections were made into the vitreous cavity of one eye.
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
1766
Fig. 1.
Biol. Pharm. Bull.
Vol. 38, No. 11 (2015)
Effects of Everolimus against Retinal Damage 7 d after Intravitreal Injection of NMDA
A: Vehicle (a); NMDA (200 nmol) (b); NMDA (200 nmol)+everolimus (Eve, 5 nmol) (c); NMDA (200 nmol)+Eve (20 nmol) (d). Scale bar=30 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. B: Retinal damage was quantitatively assessed by counting the cell number in the GCL. Data from the treated eye of each animal were normalized to those of the vehicle-treated control eye. Each column with a vertical bar represents the mean±S.E.M. from 5 to 6 animals. * p
