Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons
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.
Section snippets
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 μM) was added to the culture medium 24 h after initial
Results
In this study, we first investigated free radical production in CGN with or without treatment by 6-OHDA. 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
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 (6-OHDA) is, in part, due to the production of reactive oxygen species (ROS) and/or an inhibition of
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Both authors contributed equally to this work.