Minocycline blocks nitric oxide-induced neurotoxicity by inhibition p38 MAP kinase in rat cerebellar granule neurons

Neuroscience Letters 315 (2001) 61–64 www.elsevier.com/locate/neulet

Minocycline blocks nitric oxide-induced neurotoxicity by inhibition p38 MAP kinase in rat cerebellar granule neurons Suizhen Lin a, Yuqin Zhang a, Richard Dodel a, Martin R. Farlow b, Steven M. Paul b,c, Yansheng Du a* a

Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Dr, Indianapolis, IN 46202, USA b Neurology, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA Received 16 August 2001; received in revised form 24 September 2001; accepted 25 September 2001

Abstract Minocycline, a semisynthetic second-generation tetracycline, was reported to have neuroprotective effects in models of global and focal cerebral ischemia, the R6/2 mouse model of Huntington disease, as well as glutamate-induced neurotoxicity in mixed neuronal/glial cultures. It was suggested that neuroprotective effects of minocycline resulted from inhibition of microglial/astroglial activation ‘Proc. Natl. Acad. Sci. USA 95 1998 15769’. To determine whether or not minocycline is able to directly protect neurons against injury insults and to delineate its neuroprotective mechanism(s), we treated cultured rat cerebellar granule neurons (CGN) with nitric oxide (NO) in the presence or absence of minocycline. We found that minocycline protected neurons against NO-induced neuronal death in a concentration-dependent fashion. Consistent to other reports, NO was able to induce p38 MAP kinase phosphorylation at 3–6 h and such an induction could be significantly inhibited by minocycline. Furthermore, SB 203580, a p38 MAP kinase inhibitor, almost completely attenuated NO-induced neuronal death of CGN as well. These results suggest that minocycline is able to block NO-induced neurotoxicity in CGN by inhibiting NO-induced phosphorylation of p38 MAP kinase. Our finding may explain the neuroprotective mechanism of minocycline in those neurodegenerative models. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p38 MAP kinase; Minocycline; Cerebellar granule neurons; Nitric oxide; Neurotoxicity

Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory effects. These effects appear completely separate and distinct from its antimicrobial action [6,8,9,14,15]. Minocycline, one of the more brain penetrable of the tetracyclines, has recently been shown to have neuroprotective effects in models of global and focal ischemia [18,19]. The minocycline-induced reduction in infarct size and increased survival of hippocampal neurons following focal or global ischemia, respectively, were accompanied by a reduced expression of interleukin 1b converting enzyme (caspase 1), cyclooxygenase-2 and iNOS mRNA in affected brain regions. Additionally, a recent report by Chen et al. demonstrated that minocycline treatment delays mortality in the R6/2 mouse model of Huntington’s disease, presumably by inhibiting caspase 1 and caspase 3 expression, as well as iNOS activity [3]. The neuro* Corresponding author. Tel.: 11-317-277-2659; fax: 11-317277-6146. E-mail address: [email protected] (Y. Du).

protective effects of minocycline in these papers were assumed to result from the inhibition of microglia activation. Furthermore, most recently, from an in vitro study, it was reported that minocycline-induced neuroprotection against NMDA-induced neurotoxicity results from the inhibition of p38 MAP kinase activity in microglia [16,17]. However, neuroprotective mechanism(s) of this compound in neurons remains unclear. Nitric oxide (NO) plays a critical role in neurodegenerative diseases and cerebral ischemia. It has been suggested that excessive production of NO causes these diseases by destroying neurons. The mechanisms proposed for NOmediated neurotoxicity include inactivation of the mitochondrial respiratory chain [10], S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [13] inhibition of cisaconitase [4] activation of poly (ADP-ribose) synthase, and DNA damage [20], most of which can be mediated by the formation of nitrosocompounds by cellular components. Mitogen-activated protein (MAP) kinase is serine/threo-

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 32 4- 2

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nine kinases that function as critical mediators of signal transduction from cell surface to the nucleus. Members of MAP kinase superfamily include the extracellular signalregulated kinases, the Jun NH2-terminal kinase, and p38 MAP kinase. P38 MAP kinase activated by phosphorylation on Thr-180 and Tyr-182 by a variety of cellular stress. Recently, p38 MAP kinase activity has been implicated in NO- as well as low potassium-induced neuronal apoptosis. Administration of p38 MAP kinase inhibitor significantly enhance neuronal survival in these two models [7] The goal of the present study was to determine whether minocycline can block NO-induced neurotoxicity in cultured cerebellar granule neurons (CGN) and investigated if minocycline induced neuroprotection against NO-induced neurotoxicity through inhibition of p38 MAP kinase phosphorylation. CGN used in this study were prepared from 8-day-old Sprague–Dawley rat pups (Harlan Laboratories, IN) as previously described [5]. Briefly, freshly dissected cerebella were dissociated in the presence of trypsin and DNase I and planted on poly-l-lysine coated dishes. Cells were seeded at a density of 1.5 £ 10 6 cells/ml in basal medium Eagle supplemented with 10% FBS, 25 mM KCl, and gentamicin (0.1 mg/ml). Cytosine arabinoside (10 mM) was added to the culture medium 24 h after initial planting. Viable granule neurons were quantified by counting fluorescein (green) positive cells which results from the fluorescein diacetate (FDA) stained living cells and photographed under UV light microscopy. Values are expressed as a % of control cultures for each experiment and the data is represented as the mean ^ standard error of replicate experiments. Propidium iodide (PI), which interacts with nuclear DNA to produce a red fluorescence, was used to identify dead neurons as previously described [5,12]. Western blot analysis was performed on whole cell extracts (50 mg) which were prepared by lysing cells in RIPA buffer containing 1% Nonidet P-40, 0.1% SDS, 50 mM Tris (pH 8.0), 50 mM NaC1, 0.05% deoxycholate, protease inhibitor (Roche, Indianapolis). Proteins were size fractionated (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a 4–12% polyacrylamide gradient gel and transferred onto nitrocellulose (Hybond N, Amersham, CA, USA). The blots were then probed with polyclonal antibodies specific for phosphorylated and nonphosphorylated p38 MAP kinase (New England Labs, MA) followed by anti-rabbit IgG horseradish peroxidaselinked antibody (Jackson Immuno. Research Laboratories, Inc., PA). Bound antibody was visualized using enhanced chemiluminescence (Amersham, Arlington Heights, IL). In this study, we first investigated the involvement of p38 MAP kinase in NO-induced neuronal death of CGN. As shown in Fig. 1A, exposure of CGN to sodium nitroprusside (SNP, a donor of NO) for 24 h resulted in a concentrationdependent increase in neuronal death with EC50 value of 34 mM. To confirm activation of p38 MAP kinase is responsible for NO-induced neurotoxicity, we pretreated neurons

with SB203580 (1–30 mM), an inhibitor of p38 MAP kinase, for 2 h, followed by a treatment of SNP (50 mM). SB203580 blocks NO-induced neuronal death in a dosedependent manner (Fig. 1B). This result indicated that p38 MAP kinase is indeed involved in the NO-induced neuronal death cascade in CGN. We then determined the effects of minocycline on CGN following SNP treatment. In Fig. 2A, 2 h pretreatment of minocycline (0, 0.1, 0.25, 1, 2.5, 5, 10, 25, 50 mM) resulted in a concentration-dependent increase in neuronal survival against NO-induced neurotoxicity (EC50 ¼ 3.5 mM). Mino-

Fig. 1. (A) SNP induces neuronal death in a concentration-dependent fashion. CGN were exposed to SNP, a donor of NO, at various concentrations for 24 h. Cell viability was determined by staining neurons with FDA/PI. The experiment was repeated three times with similar results. (B) SB203580, a p-38 MAP kinase inhibitor, blocks NO (SNP)-induced toxicity of CGN. Cultured CGN were pretreated with various concentrations of SB203580 (1–30 mM) for 2 h followed by exposed to SNP (50 mM). Cell viability was quantified 24 h later by staining with FDA/PI as described in the test. Bars represent the mean ^ SEM of quadruplicate wells from single experiment repeated several times with similar results. Note the almost completed protection afforded by treatment with SB203580 against NO (*P , 0:05; **P , 0:01; ***P , 0:001; compared with the cultures treated with SNP, ANOVA and then Bonferroni/Dunn).

S. Lin et al. / Neuroscience Letters 315 (2001) 61–64

cycline’s analog, tetracycline, also blocked NO-induced neurotoxicity with similar potency (data not shown). To test whether the neuroprotective effect of minocycline in against NO-induced neurotoxicity are mediated via activation of p38 MAP kinase, we examined the effects of minocycline on the phosphorylation level of p38 MAP kinase. We have found that p38 MAP kinase phosphorylation was

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significantly induced at 3 h by NO and minocycline significantly blocks such an induction without affecting p38 MAP kinase synthesis (Fig. 2B). This data suggest that minocycline is able to block NO-induced neuronal death by inhibiting NO-induced activation of p38 MAP kinase. NO and its synthases, iNOS as well as nNOS, were implicated in the pathogenesis of neurodegenerative diseases [2]

Fig. 2. (A) Minocycline blocks SNP-induced neuronal death in a dose-dependent fashion. Following pretreated with various concentrations of minocycline (0, 0.1, 0.25, 1, 2.5, 5, 10, 25, 50 mM) for 2 h, CGN were exposed to SNP (50 mM) for 24 h. Cell viability was determined by staining neurons with FDA/PI. The experiment was repeated several times with similar results. Neuronal protection was calculated as percentage neuronal protection ¼ (surviving neurons with minocycline in SNP 2 surviving neurons in SNP)/(surviving neurons in control 2 surviving neurons in SNP) £ 100. Data represent the mean ^ SEM (bars) values of triplicate determinations (*P , 0:05; **P , 0:01; ***P , 0:001; compared with the cultures treated with SNP, ANOVA and then Bonferroni/Dunn). (B) Minocycline inhibits SNP-induced p38 MAP kinase activation in CGN. After pretreated with minocycline (10 mM) for 2 h, CGN were exposed to SNP (50 mM) for the indicated times. The lysates were immunoblotted with anti-phospho-p38 MAP kinase and anti-p38 MAP kinase antibody. Note that the increased level of phospho-p38 MAP kinase following SNP treatment is inhibited by minocycline (upper) and no change in protein levels of p38 MAP kinase was observed (lower). Anti-p38 MAP kinase western was used to confirm an equal amount of protein loading in each gel lane and that changes of p38 MAP kinase activity did not result from changes in protein levels of p38 MAP kinase. Similar results were obtained three times.

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and cerebral ischemia [11]. Inhibitors of NO production and n or iNOS- gene knock out conferred significant neuroprotective effects in the animal models of Schizophrenia [1], Parkinson’s disease, Huntington’s disease, and ischemia. The mechanism(s) associated with minocycline’s neuroprotection remains unclear. It was hypothesized that minocycline may block injury-induced neurotoxicity through inhibition of iNOS and caspase 1 overexpression in glial cells. No evidence, however, was provided that minocycline can directly block injury-induced death cascade inside of neurons. In this study, we demonstrated that minocycline was able to block NO-induced neurotoxicity in CGN. To investigate neuroprotective mechanism(s) of minocycline, we used CGN, a pure neuronal culture, to confirm that NO induced neurotoxicity in CGN through p38 MAP kinase phosphorylation as previous reported in a neuroblastoma cell line [7] and minocycline could directly block NOinduced neurotoxicity through inhibiting NO-induced phosphorylation of p38 MAP kinase in the absence of non-neuronal cells. In the present study, the protein levels of p38 MAP kinase were not affected by minocycline. The significance of this study is that we first demonstrated that minocycline is able to directly block NO-induced death cascade inside of neurons and its neuroprotective mechanism is associated with blocking p38 MAP kinase activity. Our findings support minocycline is a potential neuroprotective drug for neurodegenerative diseases since NO and p38 MAP kinase are widely involved in these diseases. Our finding suggest that minocycline confers a neuroprotective effect not just by modulation of inflammatory response in glial cells, but also through directly block NO- or p38 MAP kinase -mediated cellular death cascade inside of neurons. Further studies should be performed to identify minocycline-targeted kinase in the p38 MAP kinase pathway and determine how these kinases regulate neuronal death. Chemically modified tetracyclines, like minocycline, may prove effective in preventing and (or) altering the progression of neurodegenerative diseases. [1] Bujas-Bobanovic, M., Robertson, H.A. and Dursun, S.M., Effects of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester on phencyclidine-induced effects in rats, Eur. J. Pharmacol., 409 (2000) 57–65. [2] Calabrese, V., Bates, T.E. and Stella, A.M., NO synthase and NO-dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance, Neurochem Res., 25 (2000) 1315–1341. [3] Chen, M., Ona, V.O., Li, M., Ferrante, R.J., Fink, K.B., Zhu, S., Bian, J., Guo, L., Farrell, L.A., Hersch, S.M., Hobbs, W., Vonsattel, J.P., Cha, J.H. and Friedlander, R.M., Minocycline inhibits caspase-1and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease, Nat Med., 6 (2000) 797–801. [4] Drapier, J.C., Hirling, H., Wietzerbin, J., Kaldy, P. and Kuhn, L.C., Biosynthesis of nitric oxide activates iron regulatory factor in macrophages, EMBO J., 12 (1993) 3643–3649.

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