Energy Thresholds in Brain Mitochondria

Decreases in mitochondrial respiratory chain complex activities have been implicated in neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and Alzheimer's disease. However, the extent to which these decreases cause a disturbance in oxidative phosphorylation and energy homeostasis in the brain is not known. We therefore examined the relative contribution of individual mitochondrial respiratory chain complexes to the control of NAD-linked substrate oxidative phosphorylation in synaptic mitochondria. Titration of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited before causing impairment of mitochondrial energy metabolism. Complex I, III, and IV activities were decreased by approximately 25, 80, and 70%, respectively, before major changes in rates of oxygen consumption and ATP synthesis were observed. These results suggest that, in mitochondria of synaptic origin, complex I activity has a major control of oxidative phosphorylation, such that when a threshold of 25% inhibition is exceeded, energy metabolism is severely impaired, resulting in a reduced synthesis of ATP. Additionally, depletion of glutathione, which has been reported to be a primary event in idiopathic Parkinson's disease, eliminated the complex I threshold in PC12 cells, suggesting that antioxidant status is important in maintaining energy thresholds in mitochondria. The implications of these findings are discussed with respect to neurodegenerative disorders and energy metabolism in the synapse. Decreases in mitochondrial respiratory chain complex activities have been implicated in neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and Alzheimer's disease. However, the extent to which these decreases cause a disturbance in oxidative phosphorylation and energy homeostasis in the brain is not known. We therefore examined the relative contribution of individual mitochondrial respiratory chain complexes to the control of NAD-linked substrate oxidative phosphorylation in synaptic mitochondria. Titration of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited before causing impairment of mitochondrial energy metabolism. Complex I, III, and IV activities were decreased by approximately 25, 80, and 70%, respectively, before major changes in rates of oxygen consumption and ATP synthesis were observed. These results suggest that, in mitochondria of synaptic origin, complex I activity has a major control of oxidative phosphorylation, such that when a threshold of 25% inhibition is exceeded, energy metabolism is severely impaired, resulting in a reduced synthesis of ATP. Additionally, depletion of glutathione, which has been reported to be a primary event in idiopathic Parkinson's disease, eliminated the complex I threshold in PC12 cells, suggesting that antioxidant status is important in maintaining energy thresholds in mitochondria. The implications of these findings are discussed with respect to neurodegenerative disorders and energy metabolism in the synapse. Mitochondria are known to be integrally involved in many cellular mechanisms, such as Ca2+ homeostasis (1Rizutto R. Bastianutto C. Brini M. Murgia M. Pozzan T. J. Cell Biol. 1994; 126: 1183-1194Crossref PubMed Scopus (309) Google Scholar), programmed cell death (2Petit P.X. Susin S.A. Zamzami N. Mignotte B. Kroemer G. 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Neurosci. 1996; 16: 6125-6133Crossref PubMed Google Scholar). Additionally, mitochondrial dysfunction is characteristic of several neurodegenerative disorders (10Beal M.F. Ann. Neurol. 1992; 31: 119-130Crossref PubMed Scopus (896) Google Scholar) and also the aging process (11Ames B.N. Shigenaga M.K. Hagen T.M. Biochim. Biophys. Acta. 1995; 1271: 165-170Crossref PubMed Scopus (340) Google Scholar). Evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on reductions in respiratory chain complex activities in Parkinson's disease (12Schapira A.H.V. Cooper J.M. Dexter D. Jenner P. Clark J.B. Marsden C.D. Lancet. 1989; 8649: 1269Abstract Scopus (1212) Google Scholar, 13Schapira A.H.V. Cooper J.M. Dexter D. Clark J.B Jenner P. Marsden C.D. J. Neurochem. 1990; 54: 823-827Crossref PubMed Scopus (1669) Google Scholar, 14Schapira A.H.V. Mann V.M. Cooper J.M. Dexter D. Daniel S.E. Jenner P Clark J.B. Marsden C.D. J. Neurochem. 1990; 55: 2142-2145Crossref PubMed Scopus (632) Google Scholar), Alzheimer's disease (15Mutisya E.M. Bowling A.C. Beal M.F. J. Neurochem. 1994; 63: 2179-2184Crossref PubMed Scopus (450) Google Scholar), and Huntington's disease (16Brennan Jr., W.A. Bird E.D. Aprille J.R. J. Neurochem. 1985; 44: 1948-1950Crossref PubMed Scopus (225) Google Scholar). Such defects in respiratory chain complex activities in mitochondria are thought to underlie defects in energy metabolism and cellular degeneration (17Bowling A.C. Mutisya E.M. Walker L.C. Price D.L. Cork L.C. Beal M.F. J. Neurochem. 1993; 60: 1964-1967Crossref PubMed Scopus (243) Google Scholar, 18Bowling A.C. Beal M.F. Life Sci. 1995; 56: 1151-1171Crossref PubMed Scopus (319) Google Scholar). Parkinson's disease is characterized by a selective decrease in dopamine in the striatum caused by a degeneration of dopaminergic neurons in the zona compacta of the substantia nigra (19Hornykiewicz O. Fed. Proc. 1973; 32: 183-190PubMed Google Scholar, 20Calne D.B. Langston J.W. Lancet. 1983; 8365: 1457-1459Abstract Scopus (430) Google Scholar). In addition to a reduction in complex I activity in Parkinson's disease, decreased levels of glutathione have also been found in postmortem examination of the substantia nigra (21Perry T.L. Godin D.V. Hansen S. Neurosci. Lett. 1982; 33: 305-310Crossref PubMed Scopus (664) Google Scholar, 22Perry T.L. Yong V.W. Neurosci. Lett. 1986; 67: 269-274Crossref PubMed Scopus (320) Google Scholar, 23Riederer P. Sofic E. Rausch W.D. Schmidt B. Reynolds G.P. Jellinger K. Youdim M.B.H. J. Neurochem. 1989; 52: 515-520Crossref PubMed Scopus (1235) Google Scholar). This suggests an increased oxidative stress involvement in Parkinson's disease, as GSH is present in millimolar concentrations in mammalian cells and is considered to be a major antioxidant in the brain, capable of protecting cells from damage caused by free radicals (24Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5975) Google Scholar). The reduction in GSH levels is believed to be a primary event in Parkinson's disease because in incidental Lewy body disease (thought to be presymptomatic Parkinson's disease) GSH is depleted in the absence of a deficiency in complex I activity (25Jenner, P., Dexter, D. T., Sian, J., Schapira, A. H. V., and Marsden, C. D. (1992) Ann. Neurol.32,(suppl.) S82–S87Google Scholar). In an experimental model of rat brain mitochondria of nonsynaptic origin it was found that thresholds exist (26Davey G.P. Clark J.B. J. Neurochem. 1996; 66: 1617-1624Crossref PubMed Scopus (184) Google Scholar) whereby complex activities need to be reduced by at least 60% before major changes in ATP synthesis and oxygen consumption occur. In this study we examine the relationship between individual respiratory chain complexes and oxidative phosphorylation (rates of respiration and ATP synthesis) in synaptic mitochondria energized with NAD-linked substrates and discuss the consequences for maintenance of energy metabolism in the synapse. In order to observe the consequences of GSH depletion on mitochondrial function, the catecholaminergic PC12 cell line is depleted of GSH, partly imitating that which is thought to occur in idiopathic Parkinson's disease and the effect on the complex I threshold is measured. Chemicals were supplied by either BDH, Dagenham, Essex, UK, or Sigma Chemical Company, Poole, United Kingdom. The Eisai Chemical Company, Tokyo, Japan, supplied ubiquinone-1. The animals used were adult (250 g) male Wistar rats, supplied by B and K Universal, Aldbrough, Hull, UK. Rat pheochromocytoma-derived PC12 cells (27Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4862) Google Scholar) were cultured in Dulbecco's modified Eagle's medium containing 5% horse serum and 5% fetal bovine serum, which was changed every 2–3 days. The cells were incubated in an atmosphere containing 5% CO2 at 37 °C. Glutathione levels were depleted in PC12 cells by the addition of the α-glutamylcysteine synthetase inhibitor,l-buthionine(S,R)-sulfoximine (l-BSO) 1The abbreviations used are: l-BSO,l-buthionine(S,R)-sulfoximine; JO2, oxygen respiration. (10 μm) for 18 h. Synaptic mitochondria were prepared by the method of Lai and Clark (28Lai C.K. Clark J.B. Neuromethods. 1989; 11: 43-97Google Scholar) and were resuspended in isolation medium (320 mm sucrose, 1 mmK+-EDTA, 10 mm Tris-HCl, pH 7.4). Mitochondria routinely had a respiratory control ratio of approximately 5 with glutamate and malate as substrates. Protein concentration was determined by the method of Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as the standard. Oxygen consumption or respiration (JO2) rates in mitochondria were measured using a Clark-type electrode (Yellow Springs Instruments Co., Yellow Springs, OH) fitted into the top of a 250-μl capacity water-jacketed Perspex incubation chamber. An electromagnetic stirrer and bar flea were used to mix the incubation medium. In a typical experiment, mitochondria (0.125 mg) were pre-incubated (2 min, 30 °C) in respiration medium (final volume, 250 μl; 100 mm KCl, 75 mmmannitol, 25 mm sucrose, 10 mm phosphate-Tris, 10 mm Tris-HCl, and 50 μm EDTA, pH 7.4.) containing bovine serum albumin (0.125 mg). Depending on the complex under study, rotenone (0–150 pmol) was used to inhibit complex I, myxothiazol (0–60 pmol) to inhibit complex III, and KCN (0–75 nmol) to inhibit complex IV. After 5 min of incubation with the inhibitor, state 4 respiration was generated with glutamate (10 mm) and malate (5 mm) for 2 min; then state 3 respiration was induced by the addition of ADP (500 μm) and allowed to continue for 3 min before samples were taken for ATP production measurement and for complex activity measurement. Mitochondrial samples were perchloric acid (60% v/v)-extracted and the pH adjusted to 6 with 1m K2HPO4 for ATP analysis. PC12 cell samples were perchloric acid (60% v/v)-extracted, and the pH was adjusted to 2.5 with 5 m NaOH for GSH analysis. ATP and GSH were separated from other nucleotides using isocratic ion-paired reverse-phase high performance liquid chromatography. A Beckman System Gold was used, and the separation performed at 30 °C with a Hichrom S50D2 column (25 cm × 4.6 mm) (Hichrom, Reading, Berkshire, UK). For ATP, the method was based on that of Ingebretsen et al.(30Ingebretsen O.C. Bakken A.M. Segadal L. Farstad M. J. Chromatogr. 1982; 242: 119-126Crossref PubMed Scopus (111) Google Scholar) with UV detection at 254 nm. The mobile phase consisted of 60 mm orthophosphic acid, 2% methanol, and 80 mmtriethylamine (pH 6.0); the flow rate was 1 ml/min. For GSH, the method was based on that of Harvey et al. (31Harvey P.R.C. Ilson R.G. Strasberg S.M. Clin. Chim. Acta. 1989; 180: 203-212Crossref PubMed Scopus (84) Google Scholar) with electrochemical detection (ESA Coulochem electrochemical detector; E1 = 250 mV, E2 = 800 mV). The mobile phase consisted of 10 25 mm pH the flow rate was 1 ml/min. were performed at 30 °C. to samples were and to mitochondrial was Complex I activity was determined using a of the method of al. V.M. a Scholar), and the of at using as the Complex activity was determined using a of the method of et al. V.M. a Scholar), and the of with as the at nm. Complex IV activity was determined by the of at A. Scopus Google Scholar) and was as a order rate control were to the control H. of Scholar, R. J. Biochem. PubMed Scopus Google Scholar). This the control that in a have the of that The control be as the in of a under induced by a in the individual under For oxidative in 1 is the control of the mitochondrial complex under is the rate of of complex activity is the rate of of respiration at concentrations of the complex The present in the with synaptic mitochondria was the decrease in complex activity as the concentration was rates of respiration and ATP synthesis of control The in the of the curves the threshold effect which the activity of these complexes be reduced before oxidative phosphorylation is and decreases in ATP production and oxygen respiration occur. In the of complex I, with rotenone in a relationship between concentration and the activity of that complex of respiration and ATP synthesis in mitochondria energized with NAD-linked substrates and of the control rates and decreased at of rotenone 5 The rates of oxidative and ATP synthesis were as a of the of inhibition of complex I activity and a threshold was generated complex I activity was decreased 25% of the control activity of oxidative phosphorylation decreased at a rate to the rate of complex I I threshold in synaptic mitochondria. The from 1 were used to rates of respiration and ATP synthesis the inhibition of complex I are S.E. of at least bar is the S.E. the of the The effect of complex activity with myxothiazol on the rates of state 3 respiration and ATP synthesis is in myxothiazol concentration was increased to complex activity decreased in a the rates of respiration and ATP synthesis between and of the control and then decreased at of myxothiazol The threshold for complex was that found for complex I was a 100 and of the in rates of respiration and ATP synthesis before this threshold was threshold in synaptic mitochondria. The from 3 were used to rates of respiration and ATP synthesis the inhibition of complex are S.E. of at least bar is the S.E. the of the Complex IV activity was with KCN and the effect on rates of state 3 respiration and ATP synthesis is in the KCN concentration to nmol) caused a decrease in complex IV to approximately of the control The rates of respiration and ATP synthesis decreased this of KCN concentration between and of the control of a threshold showed that complex IV activity be decreased by approximately before a in the rates of respiration and ATP synthesis IV threshold in synaptic mitochondria. The from 5 were used to rates of respiration and ATP synthesis the inhibition of complex IV are S.E. of at least bar is the S.E. the of the were used to the activities of complexes I, III, and IV. The of the respiration inhibition was and as a ratio to the of the complex activity as in the for control under The was for inhibitors and and the control for with complex I the control on oxygen consumption and ATP synthesis in synaptic control in synaptic chain control control for complexes I, III, and IV were as under using the in and in a The control for complexes I, III, and IV were as under using the in and of PC12 cells with for in a decrease in GSH levels PC12 cells, of of PC12 cells, of of complex I activities were from complex I activities and the resulting complex I activities were respiration rates inhibition of complex I oxygen consumption rates were reduced to approximately of the control inhibition in a threshold effect to that found in 2 for synaptic and oxygen consumption rates decreased as a of complex I activity was of GSH in the PC12 cells to of the control the threshold and oxygen consumption decreased in a to the inhibition of complex I We the involvement of respiratory chain complexes in oxidative phosphorylation in synaptic mitochondria that were energized with NAD-linked substrates. the complexes complex I the control for oxygen consumption and ATP thresholds whereby 25, 80, and inhibition of complex I, III, and IV respectively, were before ATP synthesis and respiration were severely Additionally, depletion of GSH the threshold effect for complex I in PC12 cell for the process of in Parkinson's disease. complex I activity was inhibited by approximately 25% was an decrease in rates of respiration and ATP synthesis This is in to the threshold of found for complex I that is present in nonsynaptic mitochondria (26Davey G.P. Clark J.B. J. Neurochem. 1996; 66: 1617-1624Crossref PubMed Scopus (184) Google Scholar). The of this is important in that the reported decrease of in complex I activity in Parkinson's disease (12Schapira A.H.V. Cooper J.M. Dexter D. Jenner P. Clark J.B. Marsden C.D. Lancet. 1989; 8649: 1269Abstract Scopus (1212) Google Scholar, 13Schapira A.H.V. Cooper J.M. Dexter D. Clark J.B Jenner P. Marsden C.D. J. Neurochem. 1990; 54: 823-827Crossref PubMed Scopus (1669) Google Scholar, 14Schapira A.H.V. Mann V.M. Cooper J.M. Dexter D. Daniel S.E. Jenner P Clark J.B. Marsden C.D. J. Neurochem. 1990; 55: 2142-2145Crossref PubMed Scopus (632) Google Scholar) to a inhibition of ATP synthesis and respiration in the synaptic mitochondria model cultured capable of of by mechanisms, the be neurons inhibition of neuronal for of mitochondrial ATP synthesis A. J. Neurochem. 1985; 44: PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, S. 1990; PubMed Scopus Google Scholar), it that a inhibition of mitochondrial oxidative phosphorylation energy homeostasis in the synapse. Synaptic and nonsynaptic mitochondria have respiratory control suggesting major the mitochondrial isolation However, synaptic mitochondria have a complex I activity nonsynaptic mitochondria A. J. G.P. Clark J.B. Neurosci. 1995; PubMed Scopus Google Scholar), therefore of this from the nonsynaptic complex I threshold partly for the complex I threshold in synaptic mitochondria. were also for complexes and IV in synaptic whereby the activities were reduced by and respectively, rates of respiration and ATP synthesis In to the complex I thresholds present in synaptic and nonsynaptic complex and IV thresholds in synaptic mitochondria are to found in nonsynaptic mitochondria (26Davey G.P. Clark J.B. J. Neurochem. 1996; 66: 1617-1624Crossref PubMed Scopus (184) Google Scholar). These threshold are not to brain mitochondria and have been have in rat mitochondria for complex IV R. M. Biochem. J. 1994; PubMed Scopus Google Scholar) and M. T. J. Biol. 1995; Google Scholar), the activity be reduced by approximately and respectively, before major changes in oxidative phosphorylation occur. The results in this study that the threshold for complexes and IV rat brain mitochondria to levels of oxidative phosphorylation complex activities are reduced by to Decreases in complex IV activity of to of the control activities reported in Alzheimer's disease (15Mutisya E.M. Bowling A.C. Beal M.F. J. Neurochem. 1994; 63: 2179-2184Crossref PubMed Scopus (450) Google C. A. S. J.M. J. Neurochem. 1992; PubMed Scopus Google Scholar), Huntington's disease (16Brennan Jr., W.A. Bird E.D. Aprille J.R. J. Neurochem. 1985; 44: 1948-1950Crossref PubMed Scopus (225) Google Scholar), and in (17Bowling A.C. Mutisya E.M. Walker L.C. Price D.L. Cork L.C. Beal M.F. J. Neurochem. 1993; 60: 1964-1967Crossref PubMed Scopus (243) Google Scholar) brain a decrease in respiration and ATP synthesis in the synaptic mitochondria model that a complex IV deficiency not to a reduction in energy have that respiratory chain complexes are involved in the control of mitochondrial respiration and that the of the control of these complexes be on the from which the mitochondria were R. J.M. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, R. S. A. N. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, T. A. S. B. N. A. T. K. Y. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar, B. Biochem. J. 1996; PubMed Scopus Google Scholar). In the of synaptic complex I has a of and is approximately that found in nonsynaptic mitochondria (26Davey G.P. Clark J.B. J. Neurochem. 1996; 66: 1617-1624Crossref PubMed Scopus (184) Google Scholar), suggesting that a of the control of oxidative phosphorylation with complex I in synaptic mitochondria. Additionally, complex and IV of the control of and However, to the for control H. of Scholar), the of the control in is to in the of the complexes the is known to to the control of mitochondrial respiration R. J.M. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, J.M. R. U. G. G. J. L. FEBS Lett. 1983; PubMed Scopus Google Scholar), and R. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, R. Biochim. Biophys. Acta. 1983; PubMed Scopus Google Scholar), M.D. Biochem. J. Google Scholar), and R. J.M. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). Complex I thresholds were in the PC12 cell line and with in of synaptic and nonsynaptic mitochondria. PC12 cells were in which not for a mitochondria were of cell body in oxygen respiration a threshold effect at inhibition of complex I suggesting that PC12 cell mitochondria have a complex I threshold to that found in synaptic and mitochondria. nonsynaptic mitochondria which are of neuronal and cell body origin, have a complex I threshold of synaptic mitochondria have a complex I threshold of suggesting complex I thresholds in mitochondria of of GSH in PC12 cells complex I activity and also the threshold The by which GSH of the complex I threshold in mitochondria is not known. GSH is an antioxidant which mitochondria from A. PubMed Scopus Google Scholar) and when depleted complex I to free depletion of GSH has been to cause and degeneration of brain mitochondria A. J. E. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and a decrease in complex IV activity in brain mitochondrial Clark J.B. Neurochem. 1995; PubMed Scopus Google Scholar). GSH also a in of from the of the J. Neurochem. 1996; 67: PubMed Scopus Google Scholar) and when are with is to the inhibitor, O. J. Neurochem. 1997; PubMed Scopus Google Scholar). These results suggest that, under of GSH mitochondria are to which results in a in energy metabolism. The of a reduced complex I threshold have implications for involved in neuronal degeneration in Parkinson's disease. GSH depletion is a primary event in incidental Lewy body disease (thought to be presymptomatic Parkinson's and as such cause a reduction in complex I threshold in the dopaminergic neurons of the substantia nigra which are in idiopathic Parkinson's disease. This in with a deficiency in dopaminergic neurons from the substantia nigra M. M. J. Neurosci. 1993; PubMed Scopus Google Scholar, M. M. J. Neurosci. 1995; 15: PubMed Google Scholar) in for the selective of the dopamine to In of mitochondrial thresholds exist in of neurons and be involved in specific neuronal death in brain have been in mitochondrial Cell 1986; PubMed Scopus Google J.M. M.D. A. P. Beal M.F. M. R. L.A. M. 1993; PubMed Scopus Google Scholar) and be to the between and the of this of is at the of a respiratory complex then these threshold in mitochondrial metabolism R. M. Biochem. J. 1994; PubMed Scopus Google Scholar, M. T. J. Biol. 1995; Google Scholar). or not respiratory chain complex activities be reduced to oxidative phosphorylation on the of mitochondria to the of brain mitochondria in which complex I thresholds in synaptic mitochondria are to in mitochondria (26Davey G.P. Clark J.B. J. 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