Effects of enhancing mitochondrial oxidative phosphorylation with reducing equivalents and ubiquinone on 1-methyl-4-phenylpyridinium toxicity and complex I–IV damage in neuroblastoma cells
Introduction
The pathogenesis of Parkinson’s disease (PD) involves targeted degeneration of nigrostriatal dopaminergic neurons. An extensive body of research suggests that mitochondrial mutations or environmental exposure to mitochondrial toxins such as herbicides and pesticides may play a predominant role in disease etiology [1], [2]. In neurons, the loss of mitochondrial function can lead to abnormal glucose oxidation and a loss of energy (ATP) derived through OXPHOS. Moreover, abnormal glucose oxidation patterns are inherent to several degenerative CNS diseases such as Alzheimer’s disease, dementia, schizophrenia and other psychoses [3].
In PD, the dysfunction of NADH-ubiquinone oxidoreductase (complex I) has been the focus of many studies. Mitochondrial DNA mutations and biochemical defects, specific to mitochondrial complex I, were manifested in confirmed PD cases [1], [4], [5]. In vivo, chronic exposure to rotenone and specific complex I inhibitors, can reproduce the biochemical and pathological features of PD. Furthermore, the administration of complex I inhibitors can enhance the formation of localized alpha-synuclein aggregates, increase reactive oxygen species, stimulate apoptosis and prompt targeted destruction of dopaminergic nigral neurons [6], [7], [8]. The neurotoxin MPTP causes PD pathology in humans and primates, through the direct action of MPP+ on complex I inhibition [9]. Similarly, endogenously produced toxins structurally similar to MPP+ such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline [10], N-methyl-(R)-salsolinol [11], [12] and 5-S-cysteinyldopamine/7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid [13] are believed to mediate targeted nigral damage through complex I inhibition.
While the effects of MPP+ on complex I have been the focus of many studies, there are several conceptual concerns observed. First, is the reported high concentrations of MPP+ required to moderately inhibit complex I activity, up to 10 mM [14], [15], [16], indicating its weakness as an inhibitor. These findings suggest that MPP+ may contribute to mitochondrial dysfunction through another mechanism. Second, Co-Q10 is currently under clinical investigation for therapeutic use in PD [17]. However, the effects of Co-Q10 on complex I, with regards to MPP+ are not clearly understood. Complex I transfers electrons, from NADH to Co-Q10, and translocates protons across the inner mitochondria, indicating its role is downstream to the catalytic activity of complex I, the target of MPP+. Likewise, pilot studies in our lab, have indicated that Co-Q10 does not appear to increase the Vmax or reduce the Km of complex I, but exerts kinetic effects on complex II. However, complex II is not a known target of MPP+. Other studies have reported similar intrinsic affinity of Co-Q10 for complex II activity rather than complex I [18]. Lastly, the cytoprotective effects of glucose against MPP+ in neuroblastoma cells reportedly occur through sustaining anaerobic glycolysis, without reversing mitochondrial impairment as demonstrated by a sustained block in cell O2 consumption [19]. However, increased viability is observed during glucose protection against MPP+ using MTT [20]. MTT detects viability by measuring mitochondrial NADH oxidoreductase (complex I) activity, a synonymous target of MPP+. However, an increase of MTT cleavage during cytoprotection with glucose is observed even when mitochondrial function is completely blocked by MPP+ [19], [20], [21]. These findings suggest that MPP+ inhibits the mitochondria downstream to complex I, or that MTT detects viability primarily through cytosolic dehydrogenase enzymes. Furthermore, the incongruent nature of MTT and MPP+ on complex I, questions the validity of this method for in vitro toxicology models, where ATP can be produced by anaerobic substrate level phosphorylation. Therefore, the current investigation was designed to elucidate the specific effects of MPP+ on complexes I–IV activity in isolated mitochondria and whole cells. In addition, the cytoprotective role of ergogenic compounds against MPP+ toxicity through aerobic and anaerobic survival responses were examined.
Section snippets
Materials
Murine brain neuroblastoma cells (N-2A cells) were obtained from American Type Culture Collection. DMEM, l-glutamine, fetal bovine serum-heat inactivated (FBS), HBSS, PBS and penicillin/streptomycin were supplied by Fischer Scientific, Mediatech. MPP+, H2SO4, coenzyme Q10 and all other chemicals and supplies were purchased from Sigma Chemical Co.
Cell culture
N-2A cells exhibit neuronal brain morphology, display vast neurite extensions and adhere to plastic. Moreover, these cells are vulnerable to MPP+,
MPP+ toxicity
The effects of MPP+ at various concentrations (0, 0.01, 0.05, 0.1, 1, 5 and 10 mM) were evaluated to determine the effects on cell viability and mitochondrial respiration (Fig. 1A). The data presented in Fig. 1A show a decline in both parameters measured. Toxicity of MPP+ (500 μM) corresponded to accelerated glucose utilization and depletion, resulting in exhaustion of energy supplies in a glucose-limited environment (Fig. 1B). The following set of experiments corroborate that rapid consumption
Discussion
Aerobic glucose oxidation through mitochondrial OXPHOS requires the channeling of reducing equivalents to the ETC for synthesis of ATP. The ETC consists of five mitochondrial enzyme complexes located on the inner mitochondrial membrane. Complexes I–IV transfer protons through redox reactions with functional requirements including supply of nicotinamide (NADH), flavins (FMN, FAD), Co-Q10, non-heme-iron copper proteins and cytochromes [28]. The results in this study indicate that MPP+ exerts
Acknowledgements
The authors would like to acknowledge the support of the National Institutes of Health grant (RR03020) to this research investigation.
References (66)
Glucose/mitochondria in neurological conditions
Int. Rev. Neurobiol.
(2002)- et al.
Mitochondrial polymorphisms significantly reduce the risk of Parkinson disease
J. Hum. Genet.
(2003) - et al.
Dopamine-derived endogenous N-methyl-(R)-salsolinol: its role in Parkinson’s disease
Neurotoxicol. Teratol.
(2002) - et al.
Inhibition of complex I by isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Biochem. Pharmacol.
(1995) - et al.
Coenzyme Q10 supplementation provides mild symptomatic benefit in patients with Parkinson’s disease
Neurosci. Lett.
(2003) - et al.
d-(+)-Glucose rescue against 1-methyl-4-phenylpyridinium toxicity through anaerobic glycolysis in neuroblastoma cells
Brain Res.
(2003) - et al.
1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) is toxic to dopaminergic neuroblastoma SH-SY5Y cells via impairment of cellular energy metabolism
Brain Res.
(2000) - et al.
Pyruvic acid cytoprotection against 1-methyl-4-phenylpyridinium, 6-hydroxydopamine and hydrogen peroxide toxicities
Neurosci. Lett.
(2003) - et al.
Neurotoxicity of MPTP and MPP+ in vitro: characterization using specific cell lines
Brain Res.
(1988) - et al.
Development of a high throughput in vitro toxicity screen predictive of high acute in vivo toxic potential
Toxicol. in Vitro
(2001)
Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminal chemiluminescence
Anal. Biochem.
Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells
Mech. Ageing Dev.
Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid
Neurochem. Int.
Protein measurement with Folin phenol reagent
J. Biol. Chem.
A dual effect of 1-methyl-4-phenylpyridinium (MPP+)-analogs on the respiratory chain of bovine heart mitochondria
Arch Biochem. Biophys.
Decreased expression of the NADH: ubiquinone oxidoreductase (complex I) subunit 4 in 1-methyl-4-phenylpyridinium-treated human neuroblastoma SH-SY5Y cells
Neurosci. Lett.
MPP (+) causes inhibition of cellular energy supply in cerebellar granule cells
Neurotoxicology
Inhibition of mitochondrial succinate oxidation—similarities and differences between N-methylated beta-carbolines and MPP+
Arch. Biochem. Biophys.
MPP (+)-induced neurotoxicity in mouse is age-dependent: evidenced by the selective inhibition of complexes of electron transport
Brain Res.
Respiratory chain abnormalities in skeletal muscle from patients with Parkinson’s disease
J. Neurol. Sci.
Biochemical evaluations in skeletal muscles of primates with MPTP Parkinson-like syndrome
Pharmacol. Res.
Riboflavin-responsive complex I deficiency
Biochim. Biophys. Acta
Deviant energetic metabolism of glycolytic cancer cells
Biochimie
l-Deprenyl prevents the cell hypoxia induced by dopaminergic neurotoxins, MPP(+) and beta-carbolinium: a microdialysis study in rats
Neurosci. Lett.
The role of glycolysis and gluconeogenesis in the cytoprotection of neuroblastoma cells against 1-methyl 4-phenylpyridinium ion toxicity
Neurotoxicology
Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of Parkinson’s disease
Biol. Signals Recept.
Chronic systemic pesticide exposure reproduces features of Parkinson’s disease
Nat. Neurosci.
The role of mitochondria in Parkinson’s disease
Biol. Chem.
Complex I and Parkinson’s disease
IUBMB Life
Environment, mitochondria, and Parkinson’s disease
Neuroscientist
An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage
J. Neurosci.
MPTP-induced Parkinsonism in human and non-human primates—clinical and experimental aspects
Acta Neurol. Scand. Suppl.
Tetrahydroisoquinoline derivatives as possible Parkinson’s disease-inducing substances
Yakugaku Zasshi
Cited by (19)
Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells
2018, Molecular and Cellular NeuroscienceCitation Excerpt :The protective effect exerted by NGB overexpression against MPP+ toxicity was almost totally abrogated by pre-treatment of cell cultures with agents capable of perturbating microdomains. In details, our results showed that i) NGB overexpression preserves activity of the complex only when mitochondrial raft-like microdomains are intact, since NGB overexpression failed to protect the activity of complex IV in purified mitochondria directly treated with the lipid rafts disruptor methyl-beta-cyclodextrin and that ii) MPP+ hinders the functionality of the complex IV, in line with the results obtained by Mazzio and Soliman (2004) in a neuroblastoma cell line. These findings confirm those by Watanabe et al. (2012), who reported that human NGB is present in plasma membrane lipid rafts during oxidative stress and that lipid rafts are crucial for neuroprotection by NGB.
Hypothalamic-pituitary-thyroid axis hormones stimulate mitochondrial function and biogenesis in human hair follicles
2014, Journal of Investigative DermatologyCitation Excerpt :The qRT–PCR in ORS keratinocytes for SOD2 and catalase was performed as previously described in Giesen et al., 2011 (further details, Supplementary Text S1, S6 online). Complex I and IV activity were analyzed in HF homogenates as described by (Poeggeler et al., 2010a, 2010b), according to the protocols of Mazzio and Soliman, 2004, Dabbeni-Sala et al., 2001, and Rustin et al., 1994. Both experiments were performed with eight HFs each and were repeated multiple times, with HFs from six different subjects.
Whole genome expression profile in neuroblastoma cells exposed to 1-methyl-4-phenylpyridine
2012, NeuroToxicologyCitation Excerpt :Many of the earlier studies examining the effects of MPP+, involved assays that monitored the oxidation of NADH/NAD+-linked substrates in the TCA cycle on intact mitochondria, demonstrating significant losses to state 3 and 4 respiration; events parallel to the loss of complex I (Mizuno, 1989; Suzuki et al., 1990). Since then, a number of studies, including our work on intact mitochondria, demonstrate that MPP+ is not only an inhibitor of complex I, but also cytochrome oxidase (complex IV), with the latter being parallel to loss of cell respiration (Mazzio and Soliman, 2004; Steyn et al., 2005; Sundar Boyalla et al., 2011). If complex I was the only molecular target of MPP+, then fueling energy equivalents through complex II could overcome the loss of OXPHOS, however results from our lab show that not to be the case, suggesting overriding damages occur downstream to complex I.
Supramolecular organization of protein complexes in the mitochondrial inner membrane
2009, Biochimica et Biophysica Acta - Molecular Cell ResearchMeasurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2α) cells, a simple and rapid in situ assay to investigate mitochondrial toxins
2005, Journal of Biochemical and Biophysical MethodsMetabolic response to the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) in LDH-A/B double-knockout LS174T colon cancer cells
2021, Cancer Genomics and Proteomics