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Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons
Life Sciences 72 (2003) 1635 – 1641 www.elsevier.com/locate/lifescie
Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons Suizhen Lin a,1, Xing Wei a,1, Yong Xu b, Chong Yan a, Richard Dodel c, Yuqin Zhang a, Junyi Liu d, James E. Klaunig b, Martin Farlow a, Yansheng Du a,* a
Department of Neurology, Indiana University School of Medicine, 975 West Walnut Street, Rm 457, Indianapolis, IN 46202, USA b Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Department of Neurology, Rheinische Friedrich-Wilhelms-University, Bonn, Germany d School of Pharmaceutical Education, Peking University, Beijing, China Received 13 August 2002; accepted 30 October 2002
Abstract Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Anti-oxidants by inhibiting free radical generation, protect neurons against 6OHDA-induced neurotoxicity. In this study, we investigated whether or not minocycline, a neuroprotective compound, could directly protect neurons against 6-OHDA-induced neurotoxicity and inhibit 6-OHDA-induced free radical production in cultured rat cerebellar granule neurons (CGN). We now report that exposure of CGN to 6-OHDA (100 AM) resulted in a significant increase in free radical production with death of 86% of CGN. Pretreatment with minocycline (10 AM) for 2 h prevented 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, minocycline also attenuated H2O2-induced neurotoxicity. Our results suggest that minocycline blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation in CGN. Both the antioxidative and neuroprotective effects of minocycline may be beneficial in the therapy of Parkinson’s disease and other neurodegenerative diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Minocycline; Cerebellar granule neurons; 6-ODHA; Free radicals; Neurotoxicity
* Corresponding author. Tel.: +1-317-278-0220; fax: +1-317-274-3587. E-mail address: [email protected] (Y. Du). 1 Both authors contributed equally to this work. 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(02)02442-6
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Introduction 6-hydroxydopamine (6-OHDA) is a neurotoxin specific for catecholamine neurons in both the central and peripheral nervous systems. Recently, cultured rat cerebellar granule neurons (CGN) have been demonstrated to be another useful in vitro model for studying the mechanism of 6-OHDA-induced neurotoxicity [4,8]. It has been hypothesized that 6-OHDA induces neuronal death possibly via uncoupling mitochondrial oxidative phosphorylation resulting in energy deprivation [9]. Alternatively, 6-OHDA-induced neurotoxicity has been correlated with this compound’s rapid auto-oxidize at neutral pH producing hydrogen peroxide, hydroxyl and superoxide radicals [13,16]. Quinones formed during the autooxidation of 6-OHDA may undergo covalent binding with nucleophilic groups of macromolecules such as -SH, -NH2, -OH possibly further contributing to 6-OHDA-induced neurotoxicity [18]. Although the exact mechanisms by which 6-OHDA via free radical production induces neurotoxicity are not fully understood, lipid peroxidation, membrane protein damage and amino acid modification caused by free radicals, alone or in combination, may be the events which eventually lead to neuronal cell death. Minocycline is a second-generation tetracycline which exerts anti-inflammatory effects that are completely separate and distinct from its antimicrobial actions [19]. Clinical studies have shown that minocycline and related tetracyclines appear to be useful for treating both rheumatoid arthritis and osteoarthritis through their anti-inflammatory activity [19]. In addition, tetracyclines have also been reported to have a number of biological and pharmacological actions including an ability to inhibit matrix metalloproteases, superoxide production from neutrophils, and most recently, iNOS expression in rat brains, human cartilage, and murine macrophages [10,23]. Recently, minocycline has been reported to exert neuroprotective effects in animal models of global and focal ischemia [23,24], Huntington’s disease [2], amyotrophic lateral sclerosis [25], and Parkinson disease [5,11,22]. We and others have reported that minocycline could effectively block NMDA- and NO-induced neurotoxicity of neurons via inhibition of p38 MAP kinase activation [5,21]. Since minocycline has also been shown to be an anti-oxidant [15], in this study, we investigated whether or not minocycline could directly protect neurons against 6-OHDA- and H2O2- induced neurotoxicity and inhibit 6-OHDA-induced free radical production.
Methods CGN used in this study were prepared from 8-day-old Sprague–Dawley rat pups (Harlan Laboratories, IN) as previously described [4,5]. 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 1.5 106 cells/ml in basal medium Eagle supplemented with 10% FBS, 25 mM KCl, and gentamicin (0.1 mg/ml). Cytosine arabinoside (10 AM) was added to the culture medium 24 h after initial planting. Viable granule neurons were quantified by counting fluorescein (green) positive cells which resulted from fluorescein diacetate (FDA) staining living cells and were photographed under UV light microscopy. Values were expressed as a % of control cultures for each experiment and the data is represented as the mean F 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 has previously been described [4,5].
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A modified method of Floyd et al. [7] was used to analyze free radical generation. Briefly, the CGN were pretreated with minocycline for 2 hours followed by a challenge with 6-OHDA for an additional two hours, and then loaded with 10 mM of salicylic acid as the spin trap agent. At 0.5 hour after adding with salicylic acid, the reaction was terminated by adding 10% of trichloroacetic acid. The cell membrane was broken down using a 550 Sonic Dismenbrator (Fisher Sci.) After centrifuging at 10,000 g for 10 min, the contents of 2,3-, and 2,5-DHBA in the supernatant were analyzed in HPLC-EC. The HPLC system consisted of a Waters 600s controller and 616 pump with a Waters 717 Autoinjector (4 jC) controlled by a Waters Millennium 2010 software package in an IBM 486 computer. An ESA coulochem II Detector (ESA, Inc. Chelmsford, MA) was set for channel one at + 100 mV and channel two at 250 mV for 2,3-, and 2,5-DHBA analysis. A series of two Waters Nova–Pak C18 columns, 4, 8 100 mm, in a Radial– Pak cartridge guarded by a Nova–Pak C18 Guard–Pak Insert were eluted by 5% methanol in 50 mM sodium citrate, pH 4.5, at 1 ml/min flow rate. Results were represented as pmole/hour in 106 cells.
Results In this study, we first investigated free radical production in CGN with or without treatment by 6OHDA. As shown in Table 1, exposure of CGN to 6-OHDA for 2 h resulted in a significant increase in free radical production. We then examined the effects of minocycline on 6-OHDA-induced free radical production and found that 6-OHDA-induced free radical production was significantly inhibited by minocycline (Table 1). This data suggests that minocycline was able to block 6-OHDA-induced free radical production. Since it has been previously suggested that blockage of free radical generation induced by 6-OHDA could protect neurons against 6-OHDA-induced neurotoxicity [1,14], we pretreated neurons with minocycline (10 and 50 AM) for 2 h, followed by a treatment with 6-OHDA (100 AM). Minocycline significantly blocked 6-OHDA-induced neuronal death in a dose-dependent manner (Fig. 1). Twenty-four H exposure of CGN with 100 AM 6-OHDA resulted in significant neuronal death, whereas neurons that received minocycline pretreatment showed increased neuronal viability ranging from 14% of control (no minocycline treatment) to 68% and 81% of control (p < 0.05) following minocycline pretreatment (10 and 50 AM), respectively. Furthermore, to confirm minocycline’s neuroprotection results from its antioxidative effects, we pretreated CGN with minocycline (10 AM) for 2 h followed by a treatment with H2O2 Table 1 Minocycline blocks 6-OHDA-induced free radical production in CGN Treatments
No.
2,3- and 2,5-DHBA (pmole/hour in 1 106 cells)
Control M 6-OHDA M + 6-OHDA
3 3 4 6
1.74 1.77 2.08 1.90
F F F F
0.07 0.03 0.04a 0.12b
Treatment by 6-OHDA (100 AM) for 2 hours significantly increased free radical generation in CGN compared to non-treated controls. While minocycline (M, 10 AM) alone did not affect basal level of free radicals, minocycline significantly decreased 6-OHDA-induced free radical production in CGN. a p < 0.05, compared to controls (ANOVA followed by a post-hoc t – test). b p < 0.05, compared to 6-OHDA (ANOVA followed by a post-hoc t – test).
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Fig. 1. Minocycline blocks 6-OHDA induce neuronal death of CGN in a dose-dependant manner. Following pretreatment of 0, 10 and 50 AM minocycline for 2 h, CGN were exposed to 6-OHDA (100 AM) for 24 h. Cell viability was determined by staining neurons with FDA/PI. Bars represent the mean F SEM of quadruplicate wells from single experiments repeated three times with similar results. (***p < 0.001; compared with the cultures treated with 6-OHDA, as analyzed by ANOVA followed by a post-hoc t – test).
Fig. 2. Minocycline attenuates H2O2-induce neuronal death of CGN. Following pretreatment of 0 and 10 AM minocycline for 2 h, CGN were exposed to H2O2 (10 AM) for 24 h. Cell viability was determined by staining neurons with FDA/PI. Bars represent the mean F SEM of triplicate wells from single experiments repeated three times with similar results. (*p < 0.05; compared with the cultures treated with H2O2, as analyzed by ANOVA followed by a post-hoc t – test).
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(10 AM) (Fig. 2). Minocycline significantly blocked H2O2-induced neurotoxicity. Since it is well known that 6-OHDA-induced neurotoxicity could be blocked by antioxidants including Vitamin E and C (data not shown, [13,16,20]), our results suggest that minocycline blocks the 6-OHDA-induced neuronal death cascade in CGN, possibly via inhibiting 6-OHDA-induced free radical generation. Discussion The catecholamine analogue 6-hydroxydopamine (6-OHDA) causes specific degeneration of substantia nigra neurons in rodents and primates [3,12,17] as well as cell death in cell lines and primary cultures of mesencephalic cells. Recently, we and others have found CGN are also a good in-vitro model to study 6-OHDA-induced neurotoxicity [4,5,8]. Neurotoxicity induced by 6-hydroxydopamine (6OHDA) is, in part, due to the production of reactive oxygen species (ROS) and/or an inhibition of mitochondrial function [9,13,14]. In previous studies, it has been observed that antioxidant compounds were able to delay or inhibit 6-OHDA induced cell death, arguing that generation of ROS may be an important step in 6-OHDA toxicity [3,13]. In vivo, antioxidants have conferred significant neuroprotective effects in the 6-OHDA animal model [1,6]. Minocycline can protect neurons against several types of insult-induced neurotoxicities, however, the mechanism(s) associated with minocycline’s neuroprotective effects remains unclear. In this study, we demonstrated that minocycline was able to block 6-OHDA-induced neurotoxicity in CGN. To investigate neuroprotective mechanism(s) of minocycline, we used CGN, in a pure neuronal culture, to confirm that 6-OHDA-induced neurotoxicity with induced free radical generation as previous reported in other neuronal systems could be directly blocked by minocycline. Although we could not confirm minocycline protects neurons against 6OHDA-induced neurotoxicity solely through blockage of free radical generation, we, at least, could provide data showing minocycline exerts antioxidant properties which may be involved in the neuroprotection afforded by minocycline. However, we cannot rule out the possibility that minocycline’s neuroprotective effects resulted from an inhibition of the 6-OHDA uptake process. The significance of this paper is that we first demonstrated that minocycline is able to directly block 6-OHDA-induced neuronal death and that this drug’s neuroprotective mechanisms are both antioxidant and associated with blocking free radical production. Our findings support minocycline as a potential neuroprotective drug for Parkinson disease since minocycline can protect neurons against both MPTP as well as 6-OHDA-induced neurotoxicity [5,11,22]. Our findings further suggest that minocycline confers neuroprotective effects not just by modulation of inflammatory response in glial cells or by directly blocking the p38 MAP kinase -mediated cellular death cascade inside of neurons, but also through inhibiting free radical generation, although relative higher concentrations are required to exert antioxidant effects. Further studies will be needed to determine whether minocycline can more effectively protect dopaminergic neurons against 6-OHDA-induced neurotoxicity in vivo after higher brain levels are achieved and to determine if inhibition of free radical generation is the predominant mechanism in minocycline-induced neuroprotection against 6-OHDA neurotoxicity. References [1] Bensadoun JC, Mirochnitchenko O, Inouye M, Aebischer P, Zurn AD. Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice. The European Journal of Neuroscience 1998;10:3231 – 6.
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[2] Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, Bian J, Guo L, Farrell LA, Hersch SM, Hobbs W, Vonsattel JP, Cha JH, Friedlander RM. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nature Medicine 2000;6:797 – 801. [3] Carvey PM, Lin DH, Faselis CJ, Notermann JK, Ling ZD. Loss of striatal DA innervation increases striatal trophic activity directed at DA neurons in culture. Experimental Neurology 1996;140:184 – 97. [4] Dodel RC, Du Y, Bales KR, Ling Z, Carvey PM, Paul SM. Caspase-3-like proteases and 6-hydroxydopamine induced neuronal cell death. Brain Research Molecular Brain Research 1999;64:141 – 8. [5] Du Y, Ma M, Lin SZ, Gao F, Dodel RC, Bales KR, Triarhou LC, Chernet E, Perry K, Nelson DLG, Luecke S, Phebus LA, Bymaster FP, Paul SM. Minocycline Prevents Nigrostriatal Dopaminergic Neurodegeneration in the MPTP Model of Parkinson Disease. Proceedings of the National Academy of Sciences of the United States of America 2001;98:14669 – 74. [6] Ferger B, Teismann P, Mierau J. The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study. Brain Research 2000;883:216 – 23. [7] Floyd RA, Watson JJ, Wong PK. Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products. Journal of Biochemical and Biophysical Methods 1984;10:221 – 35. [8] Garber-Goldsman CB, Marshall KC, Bielajew C. 6-Hydroxydopamine treatment enhances excitation of cultured cerebellar neurons by glutamate. Canadian Journal of Physiology and Pharmacology 1986;64:1335 – 9. [9] Glinka Y, Tipton KF, Youdim MB. Nature of inhibition of mitochondrial respiratory complex I by 6-Hydroxydopamine. Journal of Neurochemistry 1996;66:2004 – 10. [10] Golub LM, Ramamurthy NS, Llavaneras A, Ryan ME, Lee HM, Liu Y, Bain S, Sorsa T. A chemically modified nonantimicrobial tetracycline (CMT- 8) inhibits gingival matrix metalloproteinases, periodontal breakdown, and extra-oral bone loss in ovariectomized rats. Annals of the New York Academy of Sciences 1999;878:290 – 310. [11] He Y, Appel S, Le W. Minocycline inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum. Brain Res 2001;909:187 – 93. [12] Kostrzewa R, Jacobwitz D. Acute effects of 6-hydroxydopa on central monoaminergic neurons. European Journal of Pharmacology 1973;21:70 – 80. [13] Kumar R, Agarwal AK, Seth PK. Free radical-generated neurotoxicity of 6-hydroxydopamine. Journal of Neurochemistry 1995;64:1703 – 7. [14] Mendez-Alvarez E, Soto-Otero R, Hermida-Ameijeiras A, Lopez-Martin ME, Labandeira-Garcia JL. Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants. Free Radical Biology and Medicine 2001;31:986 – 98. [15] Miyachi Y, Yoshioka A, Imamura S, Niwa Y. Effect of antibiotics on the generation of reactive oxygen species. The Journal of Investigative Dermatology 1986;86:449 – 53. [16] Napolitano A, Crescenzi O, Pezzella A, Prota G. Generation of the neurotoxin 6-hydroxydopamine by peroxidase/H2O2 oxidation of dopamine. Journal of Medicinal Chemistry 1995;38:917 – 22. [17] Onn SP, Berger TW, Stricker EM, Zigmond MJ. Effects of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: histochemical and neurochemical analysis. Brain Research 1986;376:8 – 19. [18] Pezzella A, d’Ischia M, Napolitano A, Misuraca G, Prota G. Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: a possible contributory mechanism for neuronal degeneration in Parkinson’s disease. Journal of Medicinal Chemistry 1997;40:2211 – 6. [19] Ryan ME, Ashley RA. How do tetracyclines work? Advances in Dental Research 1998;12:149 – 51. [20] Tiffany-Castiglioni E, Saneto RP, Proctor PH, Perez-Polo JR. Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line. Biochemical Pharmacology 1982;31:181 – 8. [21] Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 2001;21:2580 – 8. [22] Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 2002;22:1763 – 71. [23] Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proceedings of the National Academy of Sciences of the United States of America 1998;95:15769 – 74. [24] Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J. A tetracycline derivative, minocycline, reduces
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inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proceedings of the National Academy of Sciences of the United States of America 1999;96:13496 – 500. [25] Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu du C, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander RM. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 2002;417(6884):74 – 8.
Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons Suizhen Lin a,1, Xing Wei a,1, Yong Xu b, Chong Yan a, Richard Dodel c, Yuqin Zhang a, Junyi Liu d, James E. Klaunig b, Martin Farlow a, Yansheng Du a,* a
Department of Neurology, Indiana University School of Medicine, 975 West Walnut Street, Rm 457, Indianapolis, IN 46202, USA b Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Department of Neurology, Rheinische Friedrich-Wilhelms-University, Bonn, Germany d School of Pharmaceutical Education, Peking University, Beijing, China Received 13 August 2002; accepted 30 October 2002
Abstract Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Anti-oxidants by inhibiting free radical generation, protect neurons against 6OHDA-induced neurotoxicity. In this study, we investigated whether or not minocycline, a neuroprotective compound, could directly protect neurons against 6-OHDA-induced neurotoxicity and inhibit 6-OHDA-induced free radical production in cultured rat cerebellar granule neurons (CGN). We now report that exposure of CGN to 6-OHDA (100 AM) resulted in a significant increase in free radical production with death of 86% of CGN. Pretreatment with minocycline (10 AM) for 2 h prevented 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, minocycline also attenuated H2O2-induced neurotoxicity. Our results suggest that minocycline blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation in CGN. Both the antioxidative and neuroprotective effects of minocycline may be beneficial in the therapy of Parkinson’s disease and other neurodegenerative diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Minocycline; Cerebellar granule neurons; 6-ODHA; Free radicals; Neurotoxicity
* Corresponding author. Tel.: +1-317-278-0220; fax: +1-317-274-3587. E-mail address: [email protected] (Y. Du). 1 Both authors contributed equally to this work. 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(02)02442-6
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Introduction 6-hydroxydopamine (6-OHDA) is a neurotoxin specific for catecholamine neurons in both the central and peripheral nervous systems. Recently, cultured rat cerebellar granule neurons (CGN) have been demonstrated to be another useful in vitro model for studying the mechanism of 6-OHDA-induced neurotoxicity [4,8]. It has been hypothesized that 6-OHDA induces neuronal death possibly via uncoupling mitochondrial oxidative phosphorylation resulting in energy deprivation [9]. Alternatively, 6-OHDA-induced neurotoxicity has been correlated with this compound’s rapid auto-oxidize at neutral pH producing hydrogen peroxide, hydroxyl and superoxide radicals [13,16]. Quinones formed during the autooxidation of 6-OHDA may undergo covalent binding with nucleophilic groups of macromolecules such as -SH, -NH2, -OH possibly further contributing to 6-OHDA-induced neurotoxicity [18]. Although the exact mechanisms by which 6-OHDA via free radical production induces neurotoxicity are not fully understood, lipid peroxidation, membrane protein damage and amino acid modification caused by free radicals, alone or in combination, may be the events which eventually lead to neuronal cell death. Minocycline is a second-generation tetracycline which exerts anti-inflammatory effects that are completely separate and distinct from its antimicrobial actions [19]. Clinical studies have shown that minocycline and related tetracyclines appear to be useful for treating both rheumatoid arthritis and osteoarthritis through their anti-inflammatory activity [19]. In addition, tetracyclines have also been reported to have a number of biological and pharmacological actions including an ability to inhibit matrix metalloproteases, superoxide production from neutrophils, and most recently, iNOS expression in rat brains, human cartilage, and murine macrophages [10,23]. Recently, minocycline has been reported to exert neuroprotective effects in animal models of global and focal ischemia [23,24], Huntington’s disease [2], amyotrophic lateral sclerosis [25], and Parkinson disease [5,11,22]. We and others have reported that minocycline could effectively block NMDA- and NO-induced neurotoxicity of neurons via inhibition of p38 MAP kinase activation [5,21]. Since minocycline has also been shown to be an anti-oxidant [15], in this study, we investigated whether or not minocycline could directly protect neurons against 6-OHDA- and H2O2- induced neurotoxicity and inhibit 6-OHDA-induced free radical production.
Methods CGN used in this study were prepared from 8-day-old Sprague–Dawley rat pups (Harlan Laboratories, IN) as previously described [4,5]. 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 1.5 106 cells/ml in basal medium Eagle supplemented with 10% FBS, 25 mM KCl, and gentamicin (0.1 mg/ml). Cytosine arabinoside (10 AM) was added to the culture medium 24 h after initial planting. Viable granule neurons were quantified by counting fluorescein (green) positive cells which resulted from fluorescein diacetate (FDA) staining living cells and were photographed under UV light microscopy. Values were expressed as a % of control cultures for each experiment and the data is represented as the mean F 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 has previously been described [4,5].
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A modified method of Floyd et al. [7] was used to analyze free radical generation. Briefly, the CGN were pretreated with minocycline for 2 hours followed by a challenge with 6-OHDA for an additional two hours, and then loaded with 10 mM of salicylic acid as the spin trap agent. At 0.5 hour after adding with salicylic acid, the reaction was terminated by adding 10% of trichloroacetic acid. The cell membrane was broken down using a 550 Sonic Dismenbrator (Fisher Sci.) After centrifuging at 10,000 g for 10 min, the contents of 2,3-, and 2,5-DHBA in the supernatant were analyzed in HPLC-EC. The HPLC system consisted of a Waters 600s controller and 616 pump with a Waters 717 Autoinjector (4 jC) controlled by a Waters Millennium 2010 software package in an IBM 486 computer. An ESA coulochem II Detector (ESA, Inc. Chelmsford, MA) was set for channel one at + 100 mV and channel two at 250 mV for 2,3-, and 2,5-DHBA analysis. A series of two Waters Nova–Pak C18 columns, 4, 8 100 mm, in a Radial– Pak cartridge guarded by a Nova–Pak C18 Guard–Pak Insert were eluted by 5% methanol in 50 mM sodium citrate, pH 4.5, at 1 ml/min flow rate. Results were represented as pmole/hour in 106 cells.
Results In this study, we first investigated free radical production in CGN with or without treatment by 6OHDA. As shown in Table 1, exposure of CGN to 6-OHDA for 2 h resulted in a significant increase in free radical production. We then examined the effects of minocycline on 6-OHDA-induced free radical production and found that 6-OHDA-induced free radical production was significantly inhibited by minocycline (Table 1). This data suggests that minocycline was able to block 6-OHDA-induced free radical production. Since it has been previously suggested that blockage of free radical generation induced by 6-OHDA could protect neurons against 6-OHDA-induced neurotoxicity [1,14], we pretreated neurons with minocycline (10 and 50 AM) for 2 h, followed by a treatment with 6-OHDA (100 AM). Minocycline significantly blocked 6-OHDA-induced neuronal death in a dose-dependent manner (Fig. 1). Twenty-four H exposure of CGN with 100 AM 6-OHDA resulted in significant neuronal death, whereas neurons that received minocycline pretreatment showed increased neuronal viability ranging from 14% of control (no minocycline treatment) to 68% and 81% of control (p < 0.05) following minocycline pretreatment (10 and 50 AM), respectively. Furthermore, to confirm minocycline’s neuroprotection results from its antioxidative effects, we pretreated CGN with minocycline (10 AM) for 2 h followed by a treatment with H2O2 Table 1 Minocycline blocks 6-OHDA-induced free radical production in CGN Treatments
No.
2,3- and 2,5-DHBA (pmole/hour in 1 106 cells)
Control M 6-OHDA M + 6-OHDA
3 3 4 6
1.74 1.77 2.08 1.90
F F F F
0.07 0.03 0.04a 0.12b
Treatment by 6-OHDA (100 AM) for 2 hours significantly increased free radical generation in CGN compared to non-treated controls. While minocycline (M, 10 AM) alone did not affect basal level of free radicals, minocycline significantly decreased 6-OHDA-induced free radical production in CGN. a p < 0.05, compared to controls (ANOVA followed by a post-hoc t – test). b p < 0.05, compared to 6-OHDA (ANOVA followed by a post-hoc t – test).
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Fig. 1. Minocycline blocks 6-OHDA induce neuronal death of CGN in a dose-dependant manner. Following pretreatment of 0, 10 and 50 AM minocycline for 2 h, CGN were exposed to 6-OHDA (100 AM) for 24 h. Cell viability was determined by staining neurons with FDA/PI. Bars represent the mean F SEM of quadruplicate wells from single experiments repeated three times with similar results. (***p < 0.001; compared with the cultures treated with 6-OHDA, as analyzed by ANOVA followed by a post-hoc t – test).
Fig. 2. Minocycline attenuates H2O2-induce neuronal death of CGN. Following pretreatment of 0 and 10 AM minocycline for 2 h, CGN were exposed to H2O2 (10 AM) for 24 h. Cell viability was determined by staining neurons with FDA/PI. Bars represent the mean F SEM of triplicate wells from single experiments repeated three times with similar results. (*p < 0.05; compared with the cultures treated with H2O2, as analyzed by ANOVA followed by a post-hoc t – test).
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(10 AM) (Fig. 2). Minocycline significantly blocked H2O2-induced neurotoxicity. Since it is well known that 6-OHDA-induced neurotoxicity could be blocked by antioxidants including Vitamin E and C (data not shown, [13,16,20]), our results suggest that minocycline blocks the 6-OHDA-induced neuronal death cascade in CGN, possibly via inhibiting 6-OHDA-induced free radical generation. Discussion The catecholamine analogue 6-hydroxydopamine (6-OHDA) causes specific degeneration of substantia nigra neurons in rodents and primates [3,12,17] as well as cell death in cell lines and primary cultures of mesencephalic cells. Recently, we and others have found CGN are also a good in-vitro model to study 6-OHDA-induced neurotoxicity [4,5,8]. Neurotoxicity induced by 6-hydroxydopamine (6OHDA) is, in part, due to the production of reactive oxygen species (ROS) and/or an inhibition of mitochondrial function [9,13,14]. In previous studies, it has been observed that antioxidant compounds were able to delay or inhibit 6-OHDA induced cell death, arguing that generation of ROS may be an important step in 6-OHDA toxicity [3,13]. In vivo, antioxidants have conferred significant neuroprotective effects in the 6-OHDA animal model [1,6]. Minocycline can protect neurons against several types of insult-induced neurotoxicities, however, the mechanism(s) associated with minocycline’s neuroprotective effects remains unclear. In this study, we demonstrated that minocycline was able to block 6-OHDA-induced neurotoxicity in CGN. To investigate neuroprotective mechanism(s) of minocycline, we used CGN, in a pure neuronal culture, to confirm that 6-OHDA-induced neurotoxicity with induced free radical generation as previous reported in other neuronal systems could be directly blocked by minocycline. Although we could not confirm minocycline protects neurons against 6OHDA-induced neurotoxicity solely through blockage of free radical generation, we, at least, could provide data showing minocycline exerts antioxidant properties which may be involved in the neuroprotection afforded by minocycline. However, we cannot rule out the possibility that minocycline’s neuroprotective effects resulted from an inhibition of the 6-OHDA uptake process. The significance of this paper is that we first demonstrated that minocycline is able to directly block 6-OHDA-induced neuronal death and that this drug’s neuroprotective mechanisms are both antioxidant and associated with blocking free radical production. Our findings support minocycline as a potential neuroprotective drug for Parkinson disease since minocycline can protect neurons against both MPTP as well as 6-OHDA-induced neurotoxicity [5,11,22]. Our findings further suggest that minocycline confers neuroprotective effects not just by modulation of inflammatory response in glial cells or by directly blocking the p38 MAP kinase -mediated cellular death cascade inside of neurons, but also through inhibiting free radical generation, although relative higher concentrations are required to exert antioxidant effects. Further studies will be needed to determine whether minocycline can more effectively protect dopaminergic neurons against 6-OHDA-induced neurotoxicity in vivo after higher brain levels are achieved and to determine if inhibition of free radical generation is the predominant mechanism in minocycline-induced neuroprotection against 6-OHDA neurotoxicity. References [1] Bensadoun JC, Mirochnitchenko O, Inouye M, Aebischer P, Zurn AD. Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice. The European Journal of Neuroscience 1998;10:3231 – 6.
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