Elsevier

Biochemical Pharmacology

Volume 67, Issue 6, 15 March 2004, Pages 1167-1184
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2003.11.016Get rights and content

Abstract

The effects of increasing mitochondrial oxidative phosphorylation (OXPHOS), by enhancing electron transport chain components, were evaluated on 1-methyl-4-phenylpyridinium (MPP+) toxicity in brain neuroblastoma cells. Although glucose is a direct energy source, ultimately nicotinamide and flavin reducing equivalents fuel ATP produced through OXPHOS. The findings indicate that cell respiration/mitochondrial O2 consumption (MOC) (in cells not treated with MPP+) is not controlled by the supply of glucose, coenzyme Q10 (Co-Q10), NADH+, NAD or nicotinic acid. In contrast, MOC in whole cells is highly regulated by the supply of flavins: riboflavin, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), where cell respiration reached up to 410% of controls. In isolated mitochondria, FAD and FMN drastically increased complex I rate of reaction (1300%) and (450%), respectively, having no effects on complex II or III. MPP+ reduced MOC in whole cells in a dose-dependent manner. In isolated mitochondria, MPP+ exerted mild inhibition at complex I, negligible effects on complexes II–III, and extensive inhibition of complex IV. Kinetic analysis of complex I revealed that MPP+ was competitive with NADH, and partially reversible by FAD and FMN. Co-Q10 potentiated complex II (∼200%), but not complex I or III. Despite positive influence of flavins and Co-Q10 on complexes I–II function, neither protected against MPP+ toxicity, indicating inhibition of complex IV as the predominant target. The nicotinamides and glucose prevented MPP+ toxicity by fueling anaerobic glycolysis, evident by accumulation of lactate in the absence of MOC. The data also define a clear anomaly of neuroblastoma, indicating a preference for anaerobic conditions, and an adverse response to aerobic. An increase in CO2, CO2/O2 ratio, mitochondrial inhibition or O2 deprivation was not directly toxic, but activated metabolism through glycolysis prompting depletion of glucose and starvation. In conclusion, the results of this study indicate that the mechanism of action for MPP+ involves the inhibition of complexes I and IV, leading to impaired OXPHOS and MOC. Moreover, the results also indicate that flavin derivatives control the rate of complex I and more specifically complex IV, leading to impaired OXPHOS and MOC.

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)

  • B. Naslund et al.

    Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminal chemiluminescence

    Anal. Biochem.

    (1991)
  • J. Zhang et al.

    Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells

    Mech. Ageing Dev.

    (2002)
  • A.M. Brusque et al.

    Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

    Neurochem. Int.

    (2002)
  • O.J. Lowry et al.

    Protein measurement with Folin phenol reagent

    J. Biol. Chem.

    (1951)
  • E. Hasegawa et al.

    A dual effect of 1-methyl-4-phenylpyridinium (MPP+)-analogs on the respiratory chain of bovine heart mitochondria

    Arch Biochem. Biophys.

    (1997)
  • K.J. Conn et al.

    Decreased expression of the NADH: ubiquinone oxidoreductase (complex I) subunit 4 in 1-methyl-4-phenylpyridinium-treated human neuroblastoma SH-SY5Y cells

    Neurosci. Lett.

    (2001)
  • R.A. Gonzalez-Polo et al.

    MPP (+) causes inhibition of cellular energy supply in cerebellar granule cells

    Neurotoxicology

    (2003)
  • J.Z. Fields et al.

    Inhibition of mitochondrial succinate oxidation—similarities and differences between N-methylated beta-carbolines and MPP+

    Arch. Biochem. Biophys.

    (1992)
  • V.G. Desai et al.

    MPP (+)-induced neurotoxicity in mouse is age-dependent: evidenced by the selective inhibition of complexes of electron transport

    Brain Res.

    (1996)
  • L.A. Bindoff et al.

    Respiratory chain abnormalities in skeletal muscle from patients with Parkinson’s disease

    J. Neurol. Sci.

    (1991)
  • O. Pastoris et al.

    Biochemical evaluations in skeletal muscles of primates with MPTP Parkinson-like syndrome

    Pharmacol. Res.

    (1995)
  • H.R. Scholte et al.

    Riboflavin-responsive complex I deficiency

    Biochim. Biophys. Acta

    (1995)
  • L.G. Baggetto

    Deviant energetic metabolism of glycolytic cancer cells

    Biochimie

    (1992)
  • K. Matsubara et al.

    l-Deprenyl prevents the cell hypoxia induced by dopaminergic neurotoxins, MPP(+) and beta-carbolinium: a microdialysis study in rats

    Neurosci. Lett.

    (2001)
  • E. Mazzio et al.

    The role of glycolysis and gluconeogenesis in the cytoprotection of neuroblastoma cells against 1-methyl 4-phenylpyridinium ion toxicity

    Neurotoxicology

    (2003)
  • M. Ebadi et al.

    Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of Parkinson’s disease

    Biol. Signals Recept.

    (2001)
  • R. Betarbet et al.

    Chronic systemic pesticide exposure reproduces features of Parkinson’s disease

    Nat. Neurosci.

    (2000)
  • S. Kosel et al.

    The role of mitochondria in Parkinson’s disease

    Biol. Chem.

    (1999)
  • J.T. Greenamyre et al.

    Complex I and Parkinson’s disease

    IUBMB Life

    (2001)
  • T.B. Sherer et al.

    Environment, mitochondria, and Parkinson’s disease

    Neuroscientist

    (2002)
  • T.B. Sherer et al.

    An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage

    J. Neurosci.

    (2002)
  • J.W. Langston et al.

    MPTP-induced Parkinsonism in human and non-human primates—clinical and experimental aspects

    Acta Neurol. Scand. Suppl.

    (1984)
  • Y. Kotake

    Tetrahydroisoquinoline derivatives as possible Parkinson’s disease-inducing substances

    Yakugaku Zasshi

    (2002)
  • 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 Neuroscience
      Citation 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 Dermatology
      Citation 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, NeuroToxicology
      Citation 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 Research
    View all citing articles on Scopus
    View full text