Ritonavir Protects Hippocampal Neurons Against Oxidative Stress-Induced Apoptosis

Ritonavir Protects Hippocampal Neurons Against Oxidative Stress-Induced Apoptosis

NeuroToxicology 23 (2002) 301–306 Ritonavir Protects Hippocampal Neurons Against Oxidative Stress-Induced Apoptosis Wenshuai Wan, Paolo B. DePetrillo...

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NeuroToxicology 23 (2002) 301–306

Ritonavir Protects Hippocampal Neurons Against Oxidative Stress-Induced Apoptosis Wenshuai Wan, Paolo B. DePetrillo* Unit of Clinical and Biochemical Pharmacology, Laboratory of Clinical Studies, Division of Intramural Clinical and Biochemical Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 10/3C103, 10 Center Drive MSC 1256, Bethesda, MD 20892-1256, USA Received 19 July 2001; accepted 23 April 2002

Abstract Oxidative stress plays an important role in many neurodegenerative conditions including Alzheimer’s disease and Parkinson’s disease. 4-Hydroxynonenal (HNE), a lipid-soluble aldehydic product of membrane peroxidation, has been known to decrease neuronal survival by impairing Naþ, Kþ, and -ATPase activity. HNE also increases neuronal vulnerability to excitotoxic injury and disrupts homeostasis by activating proteases which mediate the destruction of cellular protein and structure. The present study demonstrated that the hydrophobic HIV protease inhibitor, ritonavir, inhibited HNE-mediated apoptosis in hippocampal primary neurons. In neurons exposed to oxidative stress induced by HNE (1 mM), ritonavir at 100 pM increased cell survival and completely abolished the apoptotic effects of HNE (P < 0.01). Ritonavir and its analogues might have useful cytoprotective effects for use in limiting the natural course of tissue injury after conditions where oxidative stress plays a role. Published by Elsevier Science Inc.

Keywords: 4-Hydroxynonenal; Oxidative stress; Inhibitor; Ritonavir; Hippocampal neuron

INTRODUCTION Reactive oxygen intermediates secreted during oxidative stress disrupt calcium ion homeostasis maintained within the cells. Free radicals such as superoxide anions, hydroxy-radicals, and hydrogen peroxide produced during normal metabolic processes increase oxidative activity (Ishihara et al., 2000). When oxidative intermediates produced during normal metabolism peroxidize the lipid bilayer of neurons, several different aldehydes including malondialdehyde and aldehyde 4-hydroxynonenal (HNE) are released (Mark et al., 1997). This neurotoxin, HNE, decreases neuronal survival by impairing Naþ, Kþ, and -ATPase activity. It also increases neuronal vulnerability to excitotoxic injury. Calcium homeostasis is disrupted when oxidative stress promotes Ca2þ * Corresponding author. Tel.: þ1-301-496-9420; fax: þ1-301-402-0445. E-mail address: [email protected] (P.B. DePetrillo).

0161-813X/02/$ – see front matter Published by Elsevier Science Inc. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 5 7 - 8

influx from the extracellular environment and efflux from the intracellular storage (Mark et al., 1997). The sudden increase in Ca2þ activates proteases, thereby inducing programmed cell death via apoptotic mechanisms. Cultures of isolated hippocampal and cortical neurons from rat fetuses at 18 embryonal day were used to examine mechanisms of apoptotic and necrotic neurodegeneration. Hippocampal pyramidal neurons are highly sensitive to various oxidative insults and neurotoxins including HNE (Keller et al., 1999). Preliminary work in this laboratory indicated that ritonavir, an HIV protease inhibitor, also inhibited calcium-activated proteases (calpains) and exhibited potent cytoprotective effects in PC12 cells permeabilized to calcium (Wan and DePetrillo, 2002). It was therefore hypothesized that ritonavir would be cytoprotective in an oxidative model of neuronal injury and protect hippocampal primary neurons treated with HNE from oxidative stress-induced apoptosis.

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METHODS Supplies and Chemicals Plastic culture dishes were purchased from Mat-Tek Inc. (Ashland, MA). Embryonic rats were obtained from Harlan Sprague–Dawley (Indianapolis, IN). Heat inactivated-fetal bovine serum was purchased from Sigma (St. Louis, MO). Minimum Essential Medium (MEM) was prepared from powder provided by Gibco (Grand Island, NY). B-27 supplemented Neurobasal medium was also purchased from Gibco. Phase-contrast microscope with 35 mm camera was obtained from Zeiss (Thornwood, NY). Hippocampal Primary Neuron Culture The cultures were prepared according to the methods described by Mattson et al. (1995). Hippocampi were obtained from E-18-rat embryos and incubated for 15 min in a solution of 2 mg/ml trypsin in Hank’s Balanced Salt Solution (HBSS) containing 2.4 g/l N[2-hydroxyethyl]piperazine-N0 -[2-ethanesulfonic acid] (HEPES) and 10 mg/l gentamicin, pH 7.2 at a concentration of 2 hippocampi/ml. The hippocampi were then rinsed three times in 10 ml of HBSS and incubated for 5 min in a solution of 1 mg trypsin inhibitor/ml of HBSS. Finally, they were rinsed three times with 10 ml of HBSS. Cells were dissociated by trituration through the narrowed bore of a fire-polished Pasteur pipette and distributed to 35 mm polyethylenimine-coated, plastic culture dishes containing 1 ml of MEMþ. MEMþ consisted of Minimum Essential Medium (MEM) supplemented with 10% heat inactivated-fetal bovine serum, 10 mM sodium bicarbonate, 20 mM KCl, 1 mM pyruvate, 0.5 mM glutamine, 10 g/l glucose, and 10 mg/l gentamicin at pH 7.2. After 4 h incubation at 37 8C in humidified 6% CO2/94% room air, MEMþ was replaced with B-27-supplemented Neurobasal medium. Cultures were maintained in this medium for 7 days until the survival test was performed.

3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES, and 0.01 mg/ml phenol red at pH 7.2. The following concentrations of ritonavir were included in the present study: 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM in Locke’s solution. The final concentration of HNE used for survival test was 1 mM. The effect of ritonavir alone on hippocampal neuron survival was examined at the each of the above concentrations. The cytoprotective effect of ritonavir was evaluated in combination with 1 mM HNE. For the combination of ritonavir and HNE, the culture was pre-treated with ritonavir at least 15 min prior to adding HNE solution. The control cultures without ritonavir or HNE were treated with Locke’s solution containing 10 nM of DMSO. Four dishes were examined for each of the groups in the present study. A phase-contrast inverted Zeiss microscope with attached 35 mm camera was used for photographic imaging. Each of the cultures was photographed at pre-treatment, 24 and 48 h after treatment at the same location marked by a scratched grid on the bottom of the culture dish. Two photos were taken from each of the dishes at different locations. Neuronal survival was quantified by counting the number of neurons on each of the photos. Neuronal viability was identified and determined by morphological criteria: viable neurons had phase-bright somas and intact processes while nonviable neurons had phase-dark, vacuolated somas and fragmented neurites. The survival rate was calculated for each of the dishes by using the number of surviving neurons at the 24 or 48 h time point divided by the number of surviving neurons at pre-treatment from the same dish. Statistical Analysis Mean differences in neuronal survival were explored using parameters derived from an analysis of variance (ANOVA). Post hoc tests were performed using the Student–Newman–Keuls procedure. P  0:05 was accepted as evidence of significant differences between the means of interest.

Hippocampal Neurons Survival Assay The effect of ritonavir on hippocampal neuronal survival against HNE toxicity was evaluated in primary cultures when the cultures were 8 days old. Ritonavir was prepared in dimethysulfoxide (DMSO) at a stock concentration of 10 mM. HNE was freshly prepared in Locke’s solution at the stock concentration of 1 mM. The Locke’s solution consisted of 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2,

RESULTS Hippocampal primary neurons maintained in Locke’s solution (Control Group) showed a slight time-dependent decrease in survival (Fig. 1). Treatment with either HNE, ritonavir, or combination of both significantly affected cell survival in hippocampal primary neurons in a time-dependent manner (treatment, F11;36 ¼ 5:46,

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Fig. 1. Hippocampal primary neuron data. Effect of ritonavir on survival of hippocampal primary neurons against HNE. The height of bars indicates the mean and the vertical line indicates the SEM. Symbol () P < 0:05 as compared to control, ritonavir 1 pM, ritonavir 10 pM, ritonavir 100 pM, and ritonavir 100 pM þ HNE.

P < 0:001; time, F1;36 ¼ 294:48, P < 0:001). Exposure of the cell cultures to HNE at the concentration of 1 mM significantly decreased neuronal survival. After either a 24 or 48 h exposure period, neuronal survival decreased significantly in HNE-treated culture (P < 0:05 as compared to control, ritonavir 1 pM, ritonavir 10 pM, ritonavir 100 pM, and ritonavir 100 pM þ HNE, Fig. 1). Ritonavir alone in culture medium induced concentration-dependent effects on neuronal survival. At lower concentrations of 1–100 pM, ritonavir itself had no effect on cell survival as compared to control cells (Fig. 1). At higher concentrations of 1 nM and particularly 10 nM, ritonavir significantly reduced neuronal survival (P < 0:05 as compared to control, ritonavir 1 pM, ritonavir 10 pM, ritonavir 100 pM, and ritonavir 100 pM þ HNE, Fig. 1). Most importantly, ritonavir was strongly cytoprotective in HNE-treated hippocampal primary neurons (Fig. 1). The cytoprotection was dose-dependent, as ritonavir at the low concentration of 1 pM exhibited no cytoprotective activity against HNE-induced cytotoxicity. However, at the concentration of 10 pM, cytoprotective activity emerged, which increased up to concentrations of 100 pM, where the drug significantly protected hippocampal primary neurons against HNEinduced cytotoxicity (P < 0:05 as compared to HNE, ritonavir 1 pM þ HNE, and ritonavir 10 pM þ HNE). The level of cytoprotection was 100% at this dose level since there was no difference between the ritonavir 100 pM þ HNE and the control group in cell survival measures. At doses higher than 100 pM, the drug exhibited cytotoxicity. Neuronal survival in both ritonavir 1 nM þ HNE and ritonavir 10 nM þ HNE was significantly lower than in the control, ritonavir

100 pM þ HNE, and ritonavir 1–100 pM groups (P < 0:05). Exposure to 10 mM ritonavir resulted in essentially 100% cell death after 24 h from a preliminary experiment prior to the present study. HNE and higher concentrations of ritonavir-induced neuronal apoptosis can be readily observed by phase contrast micrographs. Because of the similarity in morphological changes from HNE or higher concentration of ritonavir, Fig. 2 illustrates only representative micrographs from the control, HNE, ritonavir 10 nM, ritonavir 1 pM þ HNE, and ritonavir 100 pM þ HNE groups at pre-treatment, 24 and 48 h exposure. HNE and higher concentrations of ritonavir caused primary neuron damage as evidenced by neurite fragmentation and cell body vacuolation (as indicated by arrowheads). Ritonavir at a low concentration of 1 pM had no cytoprotective effects in hippocampal neurons exposed to HNE. The majority of the neurons that received only Locke’s medium or ritonavir at the concentration of 100 pM survived and continued to grow. Neurite fragmentation and cell body vacuolization, particularly in cells treated with HNE or higher concentration of ritonavir, are magnified 48 h after treatment in Fig. 3. The representative micrographs taken at high magnification (320) show a closer view of primary neurons that received different treatments.

DISCUSSION The present study demonstrates that ritonavir protects hippocampal primary neurons against oxidative stress-induced apoptosis. Although HNE was selected in the present study for the purpose of inducing apoptosis in primary neurons, this raises the possibility that

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Fig. 2. Cytotoxicity from pre-treatment to 48 h. Phase-contrast micrographs (200) of neurons prior to, 24 and 48 h after treatment with Locke’s solution, ritonavir and/or HNE. Exposure to Locke’s solution had no substantial influence on neuronal survival. Neuronal damage was induced under 1 mM HNE and ritonavir at concentration of 10 nM. Ritonavir at the concentration of 1 pM had no protection against HNE. Neuronal damage was demonstrated by neurite fragmentation and cell body vacuolation (arrowheads indicated) in a time-dependent manner. Ritonavir at the concentration of 100 pM protected neurons against HNE as indicated by fewer cell death after treatment.

ritonavir might also exert cytoprotective effects in cells after diverse types of insults associated with calcium activated protease activation that emerge during oxidative stress. Ritonavir at a concentration of 100 pM produced maximal protection against HNE in hippocampal neu-

rons, suggesting that it is very potent. Ritonavir at a higher concentration increased neuronal death. This dosage-dependent result suggests that ritonavir is neurotoxic at higher doses in this model system. While 1 pM is not enough to significantly increase survival, 1 nM is toxic to cells. Other protease inhibitors such as

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Fig. 3. Dose-dependent inhibition. High magnification (320) of phase-contrast micrographs show neuronal survival status 48 h after treatment with Locke’s solution, HNE, and ritonavir with and without HNE. Exposure to Locke’s solution had no substantial influence on neuronal survival. Neuronal damage was induced under 1 mM HNE and ritonavir at the concentration of 10 nM as demonstrated by neurite fragmentation and cell body vacuolation (arrowheads indicated). Ritonavir at the concentration of 100 pM effectively protected neurons against 1 mM HNE.

calpain inhibitor II have induced cytotoxicity in human acute lymphoblastic leukemic cells and other tumor cells when introduced in high concentrations (Zhu and Uckun, 2000). The cytoprotective effects of ritonavir in this model system should prompt the investigation of its in vivo therapeutic efficacy in animal models for limiting cell death in cellular injuries known to be associated with increased cellular calcium flux. Our results point to promising new indications for ritonavir or its analogues as neuronal protective agents.

ACKNOWLEDGEMENTS The author wishes to thank Dr. Ruiqian Wan for his inspiration and guidance in the hippocampal

experiments and Dr. Mark Mattson who generously donated the hippocampal neuronal cultures used in this work.

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peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid b-peptide. J Neurochem 1997;68: 255–64. Mattson MP, Barger SW, Begley JG, Mark RJ. Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Meth Cell Biol 1995;46:187–216.

Wan W, DePetrillo PB. Ritonavir inhibition of calcium-activated neutral proteases. Biochem Pharmacol 2002;63:1481–4. Zhu DM, Uckun FM. Calpain inhibitor II induces caspasedependent apoptosis in human acute lymphoblastic leukemia and non-Hodgkin’s lymphoma cells as well as some solid tumor cells. Clin Cancer Res 2000;6(6):2456–63.