Non-Linear Effects of Cycloheximide in Glutamate-Treated Cultured Rat Cerebellar Neurons

NeuroToxicology 23 (2002) 307±312

Non-Linear Effects of Cycloheximide in Glutamate-Treated Cultured Rat Cerebellar Neurons$ Diane Marotta1, Ann Marini2, Krishna Banaudha2, Susan Maharaj3, John Ives1,4, Craig R. Morrissette5, Wayne B. Jonas1,* 1

Samueli Institute for Informational Biology, Program on Neuroprotection and Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA 2 Department of Neurology and Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA 3 Department of Environmental and Toxicologic Pathology, Armed Forces Institute of Pathology, Washington, DC 20306, USA 4 Department of Neuropharmacology and Molecular Biology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA 5 Department of Statistics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA Received 15 January 2002; accepted 14 May 2002

Abstract Multiple cell types and organisms across a wide array of phyla and a variety of toxins demonstrate non-linear dose responses to low-level chemical exposures with high doses inhibiting cellular function and low doses stimulating function. We tested whether such non-linear responses to low and ultra-low dose N-methyl-D-aspartate (NMDA), 1-methyl-4-phenylpyridinium (MPP‡) or cycloheximide moderated toxic glutamate exposure in cultured cerebellar granule cells. Neurons were incubated over 72 h with successive NMDA, MPP‡ iodide or cycloheximide additions producing speci®ed low (10 5, 10 7, 10 9, 10 11, and 10 13 M) and ultra-low (10 27, 10 29, 10 63, and 10 65 M) concentrations. Subsequently these neuronal cells were exposed to a 50% excitotoxic concentration of glutamate for 24 h. Neuronal viability was signi®cantly reduced in neurons treated with micromolar (10 5 M) cycloheximide whereas viability was enhanced in neurons treated with an ultra-low dose exposure of 10 27 M cycloheximide. Neither NMDA nor MPP‡ elicited harmful or protective responses. This is the ®rst report demonstrating non-linear dose±response effects of cycloheximide in low and ultra-low concentration ranges. # 2002 Elsevier Science Inc. All rights reserved.

Keywords: Cycloheximide; Excitotoxicity; Hormesis; N-Methyl-D-aspartate; 1-Methyl-4-phenylpyridinium iodide

INTRODUCTION

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This research was conducted according to the principles set forth in the National Institutes of Health (NIH) Publication No. 8523, Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act of 1986, as amended. The views, opinions and assertions expressed in this article are those of the authors and do not reflect official policy of the Department of Defense, the Department of Health and Human Services or the US Government. * Corresponding author. Present address: Samueli Institute, 121 South Saint, Asaph Street, Alexandria, VA 22314, USA. Tel.: ‡1-703-535-6750; fax: ‡1-703-535-6752. E-mail address: [email protected] (W.B. Jonas).

Non-linear dose±response effects are a common observation in toxicology and pharmacology, but are rarely examined for their potential utility. We have previously found that low and ultra-low doses of glutamate will paradoxically protect against glutamate toxicity in neuronal cultures (Jonas et al., 2001). Our laboratory is now screening other cellular toxins at low and ultra-low doses for their potential to protect against

0161-813X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 5 8 - X

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high-dose glutamate. We report here the results from studies with cycloheximide, N-methyl-D-aspartate (NMDA), and 1-methyl-4-phenylpyridinium (MPP‡). Cycloheximide has been employed as a tool to study basic mechanisms of apoptosis (Kaplan and Miller, 2000), survival (Marini and Paul, 1992), synaptic plasticity (Barea-Rodriguez et al., 2000) and neurotransmitter release (Auld et al., 2001) in neuronal systems. Although cycloheximide has been used to probe basic mechanisms of neuronal cell death and survival, there is no information on cycloheximide as a neurotoxin or neuroprotectant. The role of N-methyl-Daspartate (NMDA) receptors in glutamate-mediated excitotoxicity has been studied in rat cerebellar granule cells in vitro. Over activation of NMDA receptors results in neuronal cell death (Novelli et al., 1988; Marini and Paul, 1992). In addition, intrinsic survival pathways may exist in neurons as protective mechanisms against NMDA receptor-mediated excitotoxicity. The toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is converted to MPP‡ and destroys the nigrostriatal dopaminergic neurons in the brain of humans and other primates. One promising strategy for neuroprotection is the use of low doses of chemicals to enhance cell tolerance and recovery. It is known that high doses of toxic chemicals will inhibit and kill biological systems, while low doses frequently stimulate those systems, a phenomenon known as hormesis (Calabrese and Baldwin, 1998, 2001). The phenomenon of low dose induced tolerance has been observed in a variety of systems, including the brain (Blondeau et al., 2001). Due to both recent reports of ultra-low dose effects (Oberbaum et al., 1992, 2001) and increased interest at the National Institutes of Health in alternative and homeopathic medicine, we extended our research to include both low and ultra-low doses. We have found that high dilutions of glutamate protect against glutamate toxicity in vitro (Jonas et al., 2001) and can reduce infarct size by over 40% in an ischemia model in rats (Jonas et al., 1999). In this study, three neurotoxins, cycloheximide, MPP‡, and NMDA, were chosen for study based upon their distinct mechanisms of action. Cycloheximide is a well-known inhibitor of protein translation whereas NMDA mediates its excitotoxic actions through NMDA glutamate receptor subtypes (Marini and Paul, 1992). The chemical neurotoxin, MPTP is converted to MPP‡ and destroys the nigrostriatal dopaminergic neurons in the brain producing a Parkinsonian syndrome (Burns et al., 1983) from enhanced hydroxyl radical formation (Obata, 1999). This leads to apop-

tosis as a ®nal common pathway of neuronal death (Dipasquale et al., 1991). The potential neuroprotective effects of these toxins were based, in part, on our previous work suggesting that low-dose NMDA protected vulnerable neurons against the excitotoxic actions of glutamate via a brain-derived neurotrophic factor autocrine loop (Marini and Paul, 1992; Marini et al., 1998). In addition, low-dose cycloheximide has been shown to reduce apoptosis in the brain (Lemaine et al., 1999). In contrast, a neuroprotective role has yet to be demonstrated for MPP‡. We investigated whether cultured rat cerebellar cells pretreated with low and ultra-low doses of cycloheximide, NMDA, or MPP‡ iodide could protect those neurons against an excitotoxic glutamate concentration. MATERIALS AND METHODS Chemicals NMDA and MPP‡ iodide were purchased from Research Biochemicals (Natick, MA). Glutamate, cycloheximide and 3-[4,5-dimethylthiozol-2-yl]-2,5diphenyltetrazolium bromide (MTT) were obtained from Sigma±Aldrich (St. Louis, MO). Cell Cultures Primary cerebellar neuronal cultures were prepared from postnatal day 8 Sprague±Dawley rat pups as described previously (Marini and Paul, 1992). Following trituration, cells were plated at a seeding density of 1:8  106 cells/ml in Nunc 35 mm dishes pre-coated with poly-L-lysine. Cultures were maintained in Basal Medium Eagle (BME) (with Earle's salts, without Lglutamine) supplemented with 10% heat inactivated fetal calf serum, 2 mM glutamine, 25 mM KCl, and were kept in a humidi®ed 95% air/5% CO2 incubator at 37 8C. After 24 h, cytosine arabinoside (10 mM) was added to inhibit non-neuronal elements. Neurons were used in experiments at the times indicated below. About 95% of the cultured cells are granule cell neurons by day 8 in vitro (Marini et al., 1989). Low, Ultra-Low Concentration and Control Preparations All dilutions were prepared in 24 ml glass vials in the following manner. Reverse osmosis deionized (Barnstead) sterile-®ltered water, pH 7.4, was the solvent used to generate dilutions. The starting solutions were:

D. Marotta et al. / NeuroToxicology 23 (2002) 307±312

cycloheximide, NMDA, and MPP‡ iodide at 10 mg/ ml, 200 and 60 mM, respectively, buffered to pH 7.4 with NaOH. Manual shaking to a height of 12 in. at 120 succussions per minute for 1 min vigorously mixed this solution, after which one part was added to nine parts solvent to produce the initial dilution. Using this initial dilution, a second dilution was prepared in a like manner. All subsequent dilutions consisted of one part of the previous dilution to 99 parts solvent, with succussion between each dilution. Controls consisted of water that had been succussed 120 times per minute for 1 min. Solutions were prepared in sterile conditions avoiding direct intense light, and were stored at room temperature, in the dark, wrapped in aluminum foil. Neuroprotection Experiments Four low (10 5, 10 7, 10 9, and 10 11 M) and two ultra-low (10 27 and 10 63 M) cycloheximide concentrations were investigated. In addition, the effects of NMDA and MPP‡ iodide against neurotoxicity were evaluated at four low concentrations (10 7, 10 9, 10 11, and 10 13 M) and two ultra-low (10 29 and 10 65 M) concentrations. On day 5, 6, and 7 in vitro neurons were incubated with successive additions totaling the respective concentration over a 72 h period prior to the addition of an excitotoxic concentration of glutamate (100 mM) on day 8. Cell viability was determined 24 h later (day 9) as described below. Typically, 3±6 dishes were used for each treatment on each experimental day. Each experimental condition was replicated in between 40 and 50 dishes for each toxin, over the course of 8±10 neuronal cell preparations. Vehicle controls consisted of the addition of water prepared as described above followed by either the addition of 100 mM glutamate (glutamate) or an equal volume of water (vehicle). Exposure concentrations are one log below the applied concentration due to the dilution effect of the media and data is expressed in terms of exposed concentrations. Viability Assessments Following 24 h exposure to glutamate (100 mM), cells were quantitatively assessed for damage using the MTT assay in a manner similar to that described by Lin and Long (1996). However, the ®nal concentration of MTT was 0.3 mg/ml in our experimental protocol, followed by incubation at 37 8C for 1.5 h in a humidi®ed 95% air/5% CO2 incubator. Additionally, the dye was solubilized in 0.1 N HCl in 70% isopropanol. Values were expressed relative to the negative (water)

309

control cells run in parallel and the percent neuronal viability was calculated. Sample Purity Inductively coupled Plasma±Optical Emission Spectrometry (ICP-OES; Perkin-Elmer Optima 3000) was used to test the dilutions for purity. Calibration, sample blanks, standards (0.1±30 ppm; ppm ˆ mg=l), standard reference water solutions and spiked samples (i.e. samples containing a known amount of an analyte) were used for quality assurance. The concentrations of 29 elements (Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Si, Sn, Sr, Ti, Tl, V, Zn) were determined. Data Analysis Because the sensitivity of cerebellar neuronal cells displays seasonal variability, our data was analyzed using a random block design, with time and concentration selected. The average absorbance values of replicates within trials for both samples and the respective glutamate control were normalized to the vehicle control run in parallel, and percent viabilities were calculated. This normalization process allowed treated samples to be compared directly against the positive control and adjusted for the baseline variability of the model. Sample size estimates for detection of effects were estimated from the mean square error (MSE) in the analysis of variance. For each of the toxins, the respective percent viabilities were then calculated over multiple experimental sets. Differences in cell viability between treatment groups and the glutamate control were then generated using two-way analysis of variance (ANOVA) followed by one-sided Dunnett's ttests, which adjust for multiple comparison. This allowed a quantitative estimate of the capability of the respective drug concentration only to elicit protective or toxic responses. RESULTS Dilutions were found to contain zero or negligible amounts of most elements. Values are typically !0.1 ppm, the concentration of the lowest standard. Mean Ca, K, Na, and Si concentrations were 10.68, 0.15, 4.66, and 15.55 ppm, respectively. The NaOH used to pH adjust the solutions explains the elevated Na concentrations, while the relatively high Si levels arise from the glass vials used to generate and store the

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Fig. 1. (A) Effect of low and ultra-low dose cycloheximide pretreatment on glutamate toxicity in cultured rat cerebellar neurons. A 24 h exposure to glutamate (100 mM) reduced neuronal viability by approximately 50%. Neurons were pretreated with the indicated cycloheximide concentrations prior to the addition of an excitotoxic glutamate concentration. Percent control ˆ ((surviving neurons in toxin or water ‡ glutamate)/(surviving neurons in water))  100. Values represent the mean  S:E:M: of the percent vehicle control of a total of approximately 45 dishes from 10 neuronal preparations as outlined in Section 2. It should be noted that neurons represent about 95% of the total population (5% astrocytes). Thus, the reduction in viability is strictly neuronal. P  0:05 and P < 0:001; (B) effect of low and ultra-low concentrations of NMDA on glutamate toxicity in cultured rat cerebellar neurons. Values represent the mean  S:E:M: of the percent vehicle control of a total of about 45 dishes from 10 neuronal preparations as outlined in Section 2; (C) effect of low and ultralow concentrations of MPP‡ iodide on glutamate toxicity in cultured rat cerebellar neurons. Values represent the mean  S:E:M: of the percent vehicle control of a total of approximately 45 dishes from 8 neuronal preparations as outlined in Section 2.

dilutions. Comparable levels were found in both test and control samples. Cell viability of cerebellar neuronal cells pretreated with cycloheximide, NMDA, or MPP‡ iodide prior to a toxic glutamate concentration (100 mM) is depicted in Fig. 1. Pretreatment of cerebellar granule cells with 10 5 M (10 mM) cycloheximide decreased neuronal survival by 18.2% (P < 0:001), whereas a cycloheximide concentration of 10 27 M enhanced neuronal viability by about 5% (P  0:05) compared to glutamate alone. A neuroprotective trend is suggested at the higher cycloheximide dilutions (10 9 to 10 63 M) as displayed in Fig. 1A. Neither low (10 7, 10 9, 10 11, and 10 13 M) nor ultra-low (10 29 and 10 65 M) NMDA (Fig. 1B) or MPP‡ iodide (Fig. 1C) pretreatments displayed signi®cant harmful or protective effects against glutamate excitotoxicity. The standard error of the mean (S.E.M.) for the vehicle control group, prior to normalization, was approximately 3% in the MPP‡ iodide control and 4% in the cycloheximide and NMDA controls. Table 1 displays the neu-

roprotective capacities of cycloheximide, NMDA and MPP‡ iodide for the statistical parameters indicated. DISCUSSION We show that micromolar cycloheximide enhances the excitotoxic effects of glutamate acting on NMDA receptors. This concentration is well within the range used in the literature to probe mechanisms of action in vitro (Auld et al., 2001; Gao et al., 1995) and in vivo (Oretti et al., 1996). We also show that an ultra-low cycloheximide concentration can protect vulnerable neurons against a subsequent glutamate insult. Our high dilution ®nding is consistent with previous reports demonstrating that pretreatment with ultra-low levels of glutamate protect against subsequent injury (Jonas et al., 1999, 2001). Although the degree of protection from cycloheximide is small compared to more traditional receptor antagonists, the magnitude of protection is comparable with that observed by other low dose

D. Marotta et al. / NeuroToxicology 23 (2002) 307±312

311

Table 1 Comparison of the neuroprotective capacities of cycloheximide, MPP‡ iodide and NMDA 10 Cycloheximide Mean viability (%) Neuroprotectionb

5

M**

31.0 18.2 10

7

M

10

7

M

45.6 3.6 10

9

10

9

M

52.2 3.0 M

10

11

10

11

M

51.4 2.2 M

10

13

10

27

M*

53.7 4.5 M

10

29

10

63

M

53.0 3.8 M

10

65

Controla 49.2 ±

M

MPP‡ iodide Mean viability (%) Neuroprotectionb

51.6 1.1

51.5 1.2

52.7 0.0

51.2 1.5

50.7 2.0

50.1 2.6

52.7 ±

NMDA Mean viability (%) Neuroprotectionb

55.4 0.8

54.0 0.6

53.6 1.0

53.8 0.8

53.5 1.1

56.0 1.4

54.6 ±

a

Depicts positive control pretreated with succussed water and subsequently exposed to 100 mM glutamate. Represents the viability difference obtained by subtracting positive control from treated. The S.E.M.'s for each drug at the various concentrations are derived from the analysis of variance estimate of the sigma-squared. Based on the ANOVA assumption of equal variances, the S.E.M.'s for cycloheximide, MPP‡ and NMDA are 1.374, 0.8624, and 0.9778, respectively. Cerebellar granule cells were prepared as previously described (Marini and Paul, 1992) and were pretreated on day in vitro 5, 6, and 7 with successive additions of the respective concentrations of cycloheximide, NMDA or MPP‡ iodide for 72 h. On day in vitro 8 they were exposed to an excitotoxic glutamate concentration (100 mM). Cell viability was determined 24 h later using the MTT assay (Lin and Long, 1996). * P < 0:05. ** P < 0:001. b

toxins (Weigant et al., 1997). Protection induced by pretreatment with low dose toxins is an example of the phenomenon of pre-conditioning. Cardiac cells that have been pre-exposed to brief intermittent periods of ischemia develop tolerance to moderate ischemia, and have increased stress protein production (Elliot et al., 1996). Thus ultra-low concentration effects may be another example of pre-conditioning. Schwartz et al. (2000) postulate that the high dilution phenomenon occurs through a process of systemic memory resonance in which information can be stored and transmitted by a dynamic network, such as a solvent. The potential for dynamic networks to store information is deduced from mathematical analysis of interactive recurrent feedback loops. Although these recurrent feedback loops are routinely used to explain memory storage in neuronal networks, the systemic memory model suggests information storage can be generalized to other complex dynamic systems. Our data does not allow us to speculate on whether this or other mechanisms occur and, for the moment, we would suggest that others attempt replication of our ®ndings to determine their generalizability.

NMDA nor MPP‡ elicited protective or toxic responses. Micromolar cycloheximide enhances neuronal cell death mediated by glutamate whereas ultralow dose cycloheximide demonstrates mild neuroprotection. This ultra-low dose effect may be attributable, at least in part, to systemic memory resonance involving interactive recurrent feedback loops. Because cycloheximide is used widely for studying mechanisms of neurotransmitter release and apoptosis mediated by glutamate and other excitotoxins, investigators should be cognizant of the non-linear effects of cycloheximide on neurons.

CONCLUSIONS

REFERENCES

Our results show that cycloheximide produces a nonlinear dose±response effect on glutamate toxicity at low and ultra-low concentrations and that neither

ACKNOWLEDGEMENTS We thank Dr. Kip Hartman and Ms. Maggie Meitzler for their valuable comments on this manuscript. Additionally, we recognize Ms. Cindy Crawford for her assistance in the preparation of this manuscript. This study was supported, in part, by NIH NCCAM grant # AT 00178-01 and the Samueli Institute for Information Biology.

Auld DS, Mennicken F, Quirion R. Nerve growth factor rapidly induces prolonged acetylcholine release from cultured basal forebrain neurons: differentiation between neuromodulatory and neurotrophic influences. J Neurosci 2001;15:3375±82.

312

D. Marotta et al. / NeuroToxicology 23 (2002) 307±312

Barea-Rodriguez EJ, Rivera DT, Jaffe DB, Martinez JL. Protein synthesis inhibition blocks the induction of mossy fiber longterm potentiation in vivo. J Neurosci 2000;15:8528±32. Blondeau N, Widmann C, Lazdunski M, Heurteaux C. Activation of the nuclear factor-kB is a key event in brain tolerance. J Neurosci 2001;21:4668±77. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of Parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci USA 1983;80:4546±50. Calabrese EJ, Baldwin LA. A general classification of U-shaped dose±response relationships in toxicology and their mechanistic foundations. Hum Exp Toxicol 1998;17:353±64. Calabrese EJ, Baldwin LA. Hormesis: a generalizable and unifying hypothesis. Crit Rev Toxicol 2001;31:353±424. Dipasquale B, Marini AM, Youle RJ. Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons. Biochem Biophys Res Commun 1991;181:1442±8. Elliot G, Cornerford M, Smith J, Zhao L. Myocardial ischemia/ reperfusion protection using monophosphoryl lipid a is abrogated by the ATP-sensitive potassium channel blocker, glibenclamide. Cardiovasc Res 1996;32:1071±80. Gao XM, Margolis RL, Leeds P, Hough C, Post RM, Chuang D. Carbamazepine induction of apoptosis in cultured cerebellar neurons: effects of N-methyl-D-aspartate, aurintricarboxylic acid and cycloheximide. Brain Res 1995;703:63±71. Jonas WB, Lin Y, Tortella F. Neuroprotection from glutamate toxicity with ultra-low dose glutamate. Neuroreport 2001;12: 335±9. Jonas WB, Lin Y, Williams A, Tortella R, Tuma R. Treatment of experimental stroke with low dose glutamate and homeopathic Arnica Montana. Perfusion 1999;12:452±62. Kaplan DR, Miller FD. Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 2000;10:381±91. Lemaine C, Andreau K, Souvannavong V, Adam A. Specific dual effect of cycloheximide on B lymphocyte apoptosis: involvement of Cpp32/caspace-3. Biochem Pharmacol 1999;58:85±93.

Lin Y, Long JB. Acute or prolonged exposure to 1aminocyclopropanecarboxylic acid protects spinal neurons against NMDA toxicity. Eur J Pharmacol 1996;318:491±6. Marini AM, Paul SM. N-Methyl-D-asparate receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis. Proc Natl Acad Sci USA 1992;89:6555± 9. Marini AM, Rabin SJ, Lipsky RH, Mocchetti I. Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate. J Biol Chem 1998;273: 29394±9. Marini AM, Schwartz JP, Kopin IJ. The neurotoxicity of 1-methyl4-phenylpyridinium in cultured cerebellar granule cells. J Neurosci 1989;9:3665±72. Novelli A, Reilly JA, Lysko PG, Henneberry RC. Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res 1988;451:205± 12. Obata T. Reserpine prevents hydroxyl radical formation by MPP‡ in rat striatum. Brain Res 1999;15:205±12. Oberbaum M, Markovits R, Weisman Z, Kalinkevits A, Bentwich Z. Wound healing by homeopathic silica dilutions in mice. Harefuah 1992;123:79±82. Oberbaum M, Yaniv I, Ben-Gal Y, Stein J, Ben-Zvi N, Freedman LS, Branski D. A randomized, controlled clinical trial of the homeopathic medication TRAUMEEL S in the treatment of chemotherapy-induced stomatitis in children undergoing stemcell transplantation. Cancer 2001;92:684±90. Oretti R, Bano S, Morgan CJ, Badawy, Bonner AA, Buckland P, McGuffin P. Prevention by cycloheximide of the audiogenic seizures and tryptophan metabolic disturbances of ethanol withdrawal in rats. Alcohol 1996;31:243±7 Schwartz GER, Russek LGS, Bell IR, Riley D. Plausibility of homeopathy and conventional chemical therapy: the systemic memory resonance hypothesis. Med Hyp 2000;54:634±7. Weigant FAC, Van Rijn J, van Wijk R. Enhancement of the stress response by minute amounts of cadmium in sensitized Reuber H35 hepatoma cells. Toxicology 1997;116:27±37.