Research report3-Nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells
Introduction
Huntington's disease (HD) is an inherited neurological disease characterized by choreiform movements, cognitive impairment, and the loss of neurons in the striata [22], [26], [35], [62]. The mechanism of neuronal degeneration in HD is not clear, although excitatory amino acids have been implicated [6], [19], [20], [38], [39], [54]. Reduction of enzymes of the electron transport chain leading to metabolic impairment was seen in HD postmortem tissues [12], [21], [59]. It has also been shown recently that the transgenic mice for HD have increased 8-oxodG levels in the DNA [8]. A mitochondrial toxin, 3-nitropropionic acid (3-NPA), when injected into rats causes preferential neuronal degeneration in the striatum and produces anatomical changes similar to HD [6], [11], [35]. 3-NPA is an irreversible inhibitor of succinate dehydrogenase, a critical enzyme in complex II of the electron transport chain and Krebs cycle [1], [15]. Previous studies, both in vivo and in vitro, show that 3-NPA exposure induces both necrotic and apoptotic cell death in the striatum and hippocampus [7], [43], [48], [49], [57]. Wang et al. [64] showed that 3-NPA results in generation of ROS, leading to loss of mitochondrial membrane potential and ATP.
Bcl-2 was originally identified as a human lymphoma oncogene. It now represents a family of genes that have the capacity of inhibiting apoptosis induced by several stimuli [10]. Bcl-2 is a multifunctional protein, and there is a growing body of evidence to show that it acts through mitochondria. Several models have been proposed to explain the mechanism of Bcl-2 action. Hockenberry et al. [25] first showed that Bcl-2 acts as an antioxidant to prevent apoptosis. Bcl-2 is known to inhibit the release of cytochrome c, thus inhibiting caspase-3 activation [33], [56], [67]. Mitochondrial membrane permeability transition precedes cell death, which is inhibited by Bcl-2 [61]. Bcl-2 protects the integrity of the mitochondrial oxidative phosphorylation and thus limits the mitochondrial dysfunction induced by various apoptotic stimuli. Transgenic mice overexpressing Bcl-2 are protected from MPTP- and 3-NPA-induced neurotoxicity in mice [9].
Mitochondria play a very important role in the process of apoptosis and also neuronal degeneration. Mitochondrial changes occur very early in apoptosis. Different affectors are known to cause mitochondrial membrane potential dissipation also accompanied by release of cytochrome c [52], [68]. A number of pro-apoptotic secondary messengers require mitochondria to induce caspase or nuclease activation. 3-NPA, being a mitochondrial toxin, causes metabolic impairment and ROS production, leading to mitochondrial damage and apoptosis. Using quantitative polymerase chain reaction (QPCR), we have shown that ROS leads to preferential mitochondrial DNA damage [45], [65], [47] and is found to be a good biomarker of oxidative stress in many different cell types [2], [37], [66] and animal studies [36]. Using QPCR, we also demonstrated that Bcl-2 facilitates rapid recovery from mtDNA damage after oxidative stress [16].
The purpose of this study was twofold. We first investigated whether 3-NPA leads to the production of ROS and mitochondrial DNA damage with subsequent loss of ATP levels in PC12 cells. Secondly, since Bcl-2 protects cells from oxidative damage and is known to localize in the mitochondria, we investigated whether Bcl-2 expression in PC12 cells blocks 3-NPA-induced oxidative DNA damage to the mitochondria. We report here that Bcl-2 overexpression reduced 3-NPA-induced hydrogen peroxide production and completely protected cells from mtDNA damage. Bcl-2 overexpression also maintained higher ATP levels, thus protecting cells from 3-NPA-induced cell death at 48 h. These data indicate that Bcl-2 protects cells from 3-NPA-induced cell death by attenuating hydrogen peroxide production, inhibiting mitochondrial DNA damage, and maintaining higher ATP levels.
Section snippets
Cell culture
Rat pheochromocytoma (PC12) cells transfected with human Bcl-2 or the vector alone was kindly provided by Carl W. Cotman (University of California, Irvine, CA). Both PC12 cells expressing Bcl-2 and vector control transfectants were described previously [27]. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco-BRL, Carlsbad, CA) supplemented with 10% horse serum and 5% heat-inactivated fetal bovine serum. Cells were treated with 3-NPA (Sigma, St. Louis, MO). 3-NPA was dissolved in
Results
The Western blot analysis of antihuman Bcl-2 antibody in Bcl-2-overexpressing PC12 cells shows that the Bcl-2 protein is of 26 kDa molecular weight (data not shown). Flow cytometry analysis using PE-conjugated anti-Bcl-2 antibody shows that ∼80% of the cells express high levels of human Bcl-2 (Fig. 1). 3-NPA is known to induce cell death, both apoptotic and necrotic [43]. To see whether Bcl-2 prevents 3-NPA-induced cell death in PC12 cells, we quantitated the viability of the PC12 cells
Discussion
The present study shows that 3-NPA treatment of PC12 cells leads to a rapid increase in ROS production and mitochondrial DNA damage by 30 min and reduction in ATP levels by 60 min, ultimately leading to cell death at 48 h. We found that overexpression of Bcl-2, an antiapoptotic protein in these cells, leads to significant reduction in the ROS production, maintenance of higher ATP levels, and inhibition of 3-NPA-induced mitochondrial DNA damage after 30 min and cell death at 48 h after 3-NPA (4
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2016, NeuroscienceCitation Excerpt :ATP also typically increases in response to injury (Neary et al., 2005), which can directly injure vulnerable cells, as well as trigger ATP-gated Ca2+ influx (Matute et al., 2007). Reactive species such as cell permeant H2O2 are generated as a consequence of injury both in vivo (Cornelius et al., 2013; O’Hare Doig et al., 2014a) and in vitro (Mandavilli et al., 2005; Ma et al., 2012), and also lead to increased influx through Ca2+ channels (Muralidharan et al., 2016). As such, over-activation of Ca2+ channels and receptors results in an appreciable influx of Ca2+ into cells.