Proteasome Inhibition Alters Neural Mitochondrial Homeostasis and Mitochondria Turnover
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m313579200
ISSN1083-351X
AutoresPatrick G. Sullivan, Natasa Dragicevic, Jian-Hong Deng, Yidong Bai, Edgardo Dimayuga, Qunxing Ding, Qinghua Chen, Annadora J. Bruce‐Keller, Jeffrey N. Keller,
Tópico(s)Autophagy in Disease and Therapy
ResumoInhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system. Inhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system. The proteasome is a large multicatalytic protease that is responsible for the majority of overall intracellular protein degradation (1Goldberg A.L. Akopian T.N. Kisselev A.F. Lee D.H. Rohrwild M. Biol. Chem. 1997; 378: 131-140PubMed Google Scholar, 2Davies K.J. Biochimie (Paris). 2001; 83: 301-310Crossref PubMed Scopus (720) Google Scholar, 3Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3352) Google Scholar). Increasing evidence suggests that proteasome inhibition occurs in a wide array of neurodegenerative conditions (4Lopez-Salon M. Morelli L. Castano E.M. Soto E.F. Pasquini J.M. J. Neurosci. Res. 2000; 62: 302-310Crossref PubMed Scopus (203) Google Scholar, 5Keller J.N. Huang F.F. Zhu H. Yu J. Ho Y.S. Kindy M.S. J. Cereb. Blood Flow Metab. 2000; 20: 1467-1473Crossref PubMed Scopus (130) Google Scholar, 6Keller J.N. Hanni K.B. Markesbery W.R. J. Neurochem. 2000; 75: 436-439Crossref PubMed Scopus (694) Google Scholar, 7Ding Q. Keller J.N. Free Radic. Biol. Med. 2001; 31: 574-584Crossref PubMed Scopus (110) Google Scholar, 8McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (553) Google Scholar) as well as normal aging (9Keller J.N. Gee J. Ding Q. Ageing Res. Rev. 2002; 1: 279-293Crossref PubMed Scopus (207) Google Scholar) with inhibition of proteasome activity sufficient to induce multiple and diverse effects on intracellular homeostasis (1Goldberg A.L. Akopian T.N. Kisselev A.F. Lee D.H. Rohrwild M. Biol. Chem. 1997; 378: 131-140PubMed Google Scholar, 7Ding Q. Keller J.N. Free Radic. Biol. Med. 2001; 31: 574-584Crossref PubMed Scopus (110) Google Scholar, 10Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar). In particular, severe pharmacological impairment of proteasome activity has been demonstrated to potently induce neuronal apoptosis in vitro (11Keller J.N. Markesbery W.R. J. Neurosci. Res. 2000; 61: 436-442Crossref PubMed Scopus (51) Google Scholar, 12Pasquini L.A. Besio-Moreno M. Adamo A.M. Pasquini J.M. Soto E.F. J. Neurosci. Res. 2000; 59: 601-611Crossref PubMed Scopus (80) Google Scholar, 13Qiu J.H. Asai A. Chi S. Saito N. Hamada H. Kirino T. J. Neurosci. 2000; 20: 259-265Crossref PubMed Google Scholar, 14Lee M.H. Hyun D.H. Jenner P. Halliwell B. J. Neurochem. 2001; 78: 32-41Crossref PubMed Scopus (120) Google Scholar, 15Rideout H.J. Wang Q. Park D.S. Stephanis L. J. Neurosci. 2003; (in press)Google Scholar). Although increasing evidence suggests that proteasome inhibition plays a direct role in mediating neurodegenerative and neuropathological processes, at present the mechanism(s) responsible for inducing the neurotoxicity associated with proteasome inhibition has not been fully elucidated. To survive, cells must continually generate energy through either the mitochondria-dependent mechanisms or mitochondrial-independent mechanisms such as glycolysis. Within mitochondria, energy is produced as the result of electrons flowing down the electron transport system (ETS). 1The abbreviations used are: ETS, electron transport system; ROS, reactive oxygen species; FCCP, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone; H2DCFDA, dichlorodihydrofluorescein diacetate; CYTb, apocytochrome b; COI, COII, and COIII, subunits I, II, and III of cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5 and ND6, subunits 1, 2, 3, 4, 4L, 5, and 6 of NADH dehydrogenase; A6 and A8, subunits 6 and 8 of the H+-ATPase. Impairments in ETS are associated with increased formation of reactive oxygen species (ROS) and decreased energy production (16Albers D.S. Beal M.F. J. Neural Transm. Suppl. 2000; 59: 133-154PubMed Google Scholar, 17Manfredi G. Beal M.F. Brain Pathol. 2000; 10: 462-472Crossref PubMed Scopus (98) Google Scholar, 18Schon E.A. Manfredi G. J. Clin. Investig. 2003; 111: 303-312Crossref PubMed Scopus (308) Google Scholar), which are both believed to directly contribute to neurotoxicity in a wide range of neurodegenerative conditions (19Cooper J.M. Schapira A.H. J. Bioenerg. Biomembr. 1997; 29: 175-183Crossref PubMed Scopus (57) Google Scholar, 20Greenamyre J.T. MacKenzie G. Peng T.I. Stephans S.E. Biochem. Soc. Symp. 1999; 66: 85-97Crossref PubMed Scopus (231) Google Scholar, 21Beal M.F. Trends Neurosci. 2000; 23: 298-304Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Interestingly, numerous neurodegenerative conditions associated with mitochondria dysfunction are also known to have significant levels of proteasome inhibition, thus raising the possibility that proteasome inhibition may play a direct role in inducing the observed mitochondrial dysfunction. However, at the present time, a direct role for proteasome inhibition mediating mitochondrial dysfunction in neural cells has not been reported. We have recently generated a clonal line of human SH-SY5Y cells that allows for the analysis of the cellular and molecular alterations that occur following low level proteasome inhibition (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). These cells possess neuropathology relevant to aging and age-related disease (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) yet remain fully viable for multiple passages, thus allowing for neurochemical analysis to be conducted without the potentially confounding issues surrounding other models that possess rapid and widespread apoptotic cell death. In this study, we utilized this clonal line to determine the effects of low level proteasome inhibition on mitochondrial homeostasis. Together, these data demonstrate the ability of proteasome inhibition to directly alter multiple aspects of neural mitochondrial homeostasis and alter lysosomal-mediated degradation of mitochondria, demonstrating a possible role for proteasome inhibition serving as a direct mediator of neural mitochondrial dysfunction. Reagents—All of the cell culture medium, serum, and antibiotics were purchased from Invitrogen. dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes (Eugene, OR). The MG-115 and ATP assay kit were purchased from Calbiochem, and all of the other reagents and chemicals were purchased from Sigma. Establishment of Clonal Lines—Neural SH-SY5Y cells were obtained from the ATCC and propagated as described previously (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar, 24Ding Q. Lewis J.J. Strum K.M. Dimayuga E. Bruce-Keller A.J. Dunn J.C. Keller J.N. J. Biol. Chem. 2002; 277: 13935-13942Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 25Sitte N. Huber M. Grune T. Ladhoff A. Doecke W.D. Von Zglinicki T. Davies K.J. FASEB J. 2000; 14: 1490-1498Crossref PubMed Google Scholar). To establish individual clonal lines following chronic exposure, neural SH-SY5Y cells were maintained in normal growth medium containing 100 nm MG115 with individual clones selected and characterized as described previously (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). The medium was replaced weekly, and fresh MG115 was added each week. Control cultures consisted of sister SH-SY5Y cells that were propagated alongside MG115-exposed cultures for the duration of the selection period. Cells of fewer than 25 passages were utilized for all of the described studies. Isolation of Mitochondria—All of the mitochondrial procedures were conducted using established methodologies that have been published by our group in previous studies (26Sullivan P.G. Geiger J.D. Mattson M.P. Scheff S.W. Ann. Neurol. 2000; 48: 723-729Crossref PubMed Scopus (232) Google Scholar, 27Sullivan P.G. Dube C. Dorenbos K. Steward O. Baram T.Z. Ann. Neurol. 2003; 53: 711-717Crossref PubMed Scopus (214) Google Scholar). On each day of experimentation, semiconfluent cell cultures from 75-cm2 flasks were isolated using 0.25% trypsin with cells from at least six flasks pooled to generate a single sample of either wild type or clonal cells. Cells were utilized for mitochondrial assays immediately following their isolation with wild type and clonal lines analyzed side by side on the same day. Cells from at least five separate preparations obtained on 5 separate days were utilized to generate data in the present report. Following the addition of trypsin, the cells were pelleted by centrifugation at 300 × g for 5 min at 4 °C. All of the subsequent steps were preformed at on ice or at 4 °C. The resulting pellet was then resuspended in 0.5 ml of mitochondrial isolation buffer (215 mm mannitol, 75 mm sucrose, 0.1% bovine serum albumin, 1 mm EGTA, 20 mm HEPES, pH 7.2), and the plasma membranes were ruptured by nitrogen decompression (Parr Cell Disruption Bomb) at 1000 p.s.i. for 5 min as described previously (27Sullivan P.G. Dube C. Dorenbos K. Steward O. Baram T.Z. Ann. Neurol. 2003; 53: 711-717Crossref PubMed Scopus (214) Google Scholar). The mitochondria were then purified by differential centrifugation at 1300 × g for 5 min to pellet unbroken cells and the nuclei. The supernatant was then centrifuged at 13,000 × g for 10 min to pellet the mitochondria. The pellet was resuspended in EGTA-free isolation buffer and centrifuged at 10,000 × g for 10 min. The resulting pellet was resuspended in EGTA-free isolation buffer at a concentration of ∼10 mg/ml. Analysis of Mitochondrial Respiration and ATP Levels—Mitochondrial respiration was assessed using a miniature Clark-type electrode in a sealed, thermostated, and continuously stirred chamber as described previously (26Sullivan P.G. Geiger J.D. Mattson M.P. Scheff S.W. Ann. Neurol. 2000; 48: 723-729Crossref PubMed Scopus (232) Google Scholar, 27Sullivan P.G. Dube C. Dorenbos K. Steward O. Baram T.Z. Ann. Neurol. 2003; 53: 711-717Crossref PubMed Scopus (214) Google Scholar). Mitochondria were added to the chamber to yield a final protein concentration of 1 mg/ml in respiration buffer (215 mm mannitol, 75 mm sucrose, 2 mm MgCl2, 2.5 mm inorganic phosphates, 0.1% bovine serum albumin, 20 mm HEPES, pH 7.2). State II respiration was initiated by the addition of pyruvate and malate. State III respiration was initiated by the addition of 150 nm ADP followed by the addition of oligomycin (1 μg/ml) to induce State IV respiration. The mitochondrial uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP; 1 μm) was added to the chamber to induce maximum State V respiration (complex I-driven). The complex I inhibitor rotenone (1 μm) was added to the chamber followed by the addition of succinate (10 mm) to allow for the quantification of State V complex II-driven respiration. Data are presented as nmolO2/min/mg mitochondrial protein. Cellular ATP levels were determined using a commercially available ATP assay kit (Calbiochem) according to manufacturer's instructions. Analysis of Mitochondrial Reactive Oxygen Species—Mitochondrial ROS production was assessed using the ROS indicator H2DCFDA as described previously (26Sullivan P.G. Geiger J.D. Mattson M.P. Scheff S.W. Ann. Neurol. 2000; 48: 723-729Crossref PubMed Scopus (232) Google Scholar, 27Sullivan P.G. Dube C. Dorenbos K. Steward O. Baram T.Z. Ann. Neurol. 2003; 53: 711-717Crossref PubMed Scopus (214) Google Scholar). 50 μg of isolated mitochondria was incubated in a total volume of 100 μl of respiration buffer at 37 °C for 15 min in the presence of 10 μm H2DCFDA, which was made fresh before each use. The relative amounts of mitochondrial ROS produced were measured using a fluorometric plate reader (excitation 490 nm, emission 526 nm). Controls included the addition of FCCP to inhibit membrane potential-dependent ROS production (minimum ROS production) and oligomycin to maximize membrane potential (maximum ROS production). In each experiment, the mitochondrial-independent ROS production was accounted for by subtracting out the fluorescence intensity measured in control wells in which no mitochondria were added. All of the assays for mitochondrial experiments were run in replicates of 4–8/assay, and the mean data were used for analysis. Data are expressed as raw fluorescence units. Analysis of Mitochondrial Protein Levels—To analyze mitochondrial protein synthesis, pulse-labeling experiments with [35S]methionine were carried out as described previously (28Bai Y. Attardi G. EMBO J. 1998; 17: 4848-4858Crossref PubMed Scopus (167) Google Scholar). Samples of either control or clone 6 cells (2 × 106 cells) were plated onto 10-cm2 dishes, incubated overnight, and then washed with methionine-free Dulbecco's modified Eagle's medium followed by a 7-min incubation at 37 °C in 4 ml of the same medium containing 50 μg/ml cytoplasmic translation inhibitor emetine. Thereafter, [35S]methionine (0.4 mCi/plate) was added and the cells were incubated for 1 h. To test the stability of the mitochondrial translation products, pulse-chase labeling experiments were performed. Cell plating and labeling were carried out as described above with the exception that emetine was replaced with cycloheximide, a reversible cytosolic protein synthesis inhibitor, and incubation time with [35S]methionine was extended to 2 h. After the labeling, the cells were washed and subjected to a 22-h chase period in complete and unlabeled medium in the absence of cycloheximide. The labeled cells were trypsinized, washed, and lysed in 1% SDS. Samples containing 50 μg of protein were electrophoresed through an SDS-polyacrylamide gel (15–20% exponential gradient). The intensities of the bands were quantified by phosphorimaging analysis. The ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 are subunits of NADH dehydrogenase; CYTb is apocytochrome b; COI, COII, and COIII are subunits of cytochrome c oxidase; and A6 and A8 are subunits of the H+-ATP synthase. Analysis of Lipofuscin Levels—Analysis of lipofuscin levels was determined by quantifying the amount of cellular autofluorescence as described previously (29Holtsberg F.W. Steiner M.R. Bruce-Keller A.J. Keller J.N. Mattson M.P. Moyers J.C. Steiner S.M. J. Neurosci. Res. 1998; 53: 685-696Crossref PubMed Scopus (37) Google Scholar). Electron Microscopy—Analysis of mitochondria by electron microscopy was conducted as described previously (25Sitte N. Huber M. Grune T. Ladhoff A. Doecke W.D. Von Zglinicki T. Davies K.J. FASEB J. 2000; 14: 1490-1498Crossref PubMed Google Scholar) with some minor modifications. Following experimental treatment, cells were rinsed in ice-cold 0.1 m Sorenson's buffer (pH 7.4) followed by 30-min fixation in 3.5% glutaraldehyde, 0.1 m Sorenson's solution (v/v) followed by a 30-min incubation in 1% osmium tetroxide. Cells were then subjected to dehydration via incubations in increasing concentrations of ethanol and embedded in Eponate 12 resin. The tissue was then sectioned (60 nm), placed into copper grids, and imaged using a Philips CM100 EM (Philips Electron Optics, Eindhoven, The Netherlands). Data was collected from at least 100 cells for each condition. Analysis of Neural Survival—Cell viability was determined by Hoechts 33258 staining as described previously (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar, 24Ding Q. Lewis J.J. Strum K.M. Dimayuga E. Bruce-Keller A.J. Dunn J.C. Keller J.N. J. Biol. Chem. 2002; 277: 13935-13942Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 30Wallace D.C. Shoffner J.M. Trounce I. Brown M.D. Ballinger S.W. Corral-Debrinski M. Horton T. Jun A.S. Lott M.T. Biochim. Biophys. Acta. 1998; 1271: 141-151Crossref Scopus (207) Google Scholar). For each time point, at least eight cultures from two separate experiments were utilized with at least 200 cells counted for each time point. Statistical Analysis—Statistical significance was determined using Student's t test with a p value of < 0.05 required for significance. Clonal Cell Lines Exhibit Altered Mitochondrial Function—To determine the effects of prolonged low level proteasome inhibition on neural mitochondria homeostasis, we conducted studies using our recently developed clonal SH-SY5Y cell line (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). These cells have been extensively characterized in previous studies (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) and are known to possess a small but significant decrease in proteasome-mediated protein degradation (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) and to be a useful model for the analysis of the long term neurotoxicity associated with proteasome inhibition. Mitochondrial respiration is the most sensitive and reliable method available to measure mitochondrial bioenergetics; therefore, we assessed mitochondrial oxygen consumption in control and clonal cell populations of equal passage number. As illustrated in Fig. 1A, oxygen consumption driven by the NADH-linked substrates pyruvate and malate (complex I-driven) was not significantly different between control or clonal cells (clone 6) when measured in the presence of ADP (State III) or when the ATP synthase was inhibited (State IV; oligomycin present). However, when maximum respiration was assessed (State V; FCCP present), oxygen consumption was significantly reduced in the mitochondria isolated from the clone 6 cells (Fig. 1A). State V respiration was significantly reduced in clone 6-derived mitochondria regardless of the whether the mitochondria were utilizing complex I (Fig. 1A) or complex II (succinate) to drive respiration (Fig. 1B). Mitochondrial ROS production is a byproduct of mitochondrial respiration but can be dramatically increased in response to inefficient ETS function. Therefore, we next sought to determine whether the alterations in mitochondrial respiration, observed in the cells undergoing inhibition of proteasome activity, altered the amount of mitochondrial-derived ROS. Mitochondria isolated from clone 6 cells were observed to possess a significant increase in mitochondrial-derived ROS (21% increase) as compared with control cultures measured during State IV complex I-driven respiration (Fig. 2). Clonal Cells Possess Decreased Levels of Mitochondrial-derived Proteins of the Electron Transport Chain—In order for the electron transport chain to function properly, the mitochondria must produce 13 different mtDNA-encoded proteins (18Schon E.A. Manfredi G. J. Clin. Investig. 2003; 111: 303-312Crossref PubMed Scopus (308) Google Scholar, 31Wei Y.H. Lu C.Y. Lee H.C. Pang C.Y. Ma Y.S. Ann. N. Y. Acad. Sci. 1998; 854: 155-170Crossref PubMed Scopus (225) Google Scholar, 32Blass J.P. Sheu R.K. Cedarbaum J.M. Rev. Neurol. (Paris). 1988; 144: 543-563PubMed Google Scholar). These proteins are transcribed and translated within the mitochondria and do not therefore require mitochondrial protein import. Seven of these proteins are identified as ND1, ND2, ND3, ND4, ND4L, ND5, and ND6, which are subunits of NADH dehydrogenase. The mtDNA-encoded protein CYTb is also known as apocytochrome b, whereas COI, COII, and COIII are subunits of cytochrome c oxidase. The mtDNA-encoded proteins A6 and A8 are subunits of the H+-ATP synthase. To determine whether the loss of complex I and complex II activity in clone 6 cells may be due in part to alterations in the amount of mtDNA-encoded protein production, we conducted studies analyzing the synthesis and degradation of each of these mtDNA-encoded proteins. These pulse-chase studies were conducted using conditions that allowed for the selective analysis of intramitochondrial protein synthesis (28Bai Y. Attardi G. EMBO J. 1998; 17: 4848-4858Crossref PubMed Scopus (167) Google Scholar) and demonstrated the presence of 13 major protein bands that correspond to the 13 mtDNA-encoded proteins in both the control and clonal cells (Fig. 3). The banding pattern and intensity in these gels were identical to previously published reports using this method (28Bai Y. Attardi G. EMBO J. 1998; 17: 4848-4858Crossref PubMed Scopus (167) Google Scholar). The overall mitochondrial protein synthesis was decreased by 64.8% in clone 6 cells (Fig. 3) as compared with control cells with all of the 13 proteins showing a dramatic decrease in expression. Interestingly, pulse-chase experiments revealed that the level of mtDNA-encoded protein was further decreased by 81.4% in clone 6 cells (Fig. 3). Taken together, these data indicate that chronic low level proteasome inhibition decreases the amount of mtDNA-encoded protein synthesis with proteins that are able to be translated within the mitochondria, exhibiting decreased stability. Neural Cells Undergoing Proteasome Inhibition Are More Dependent on Glycolysis—To maintain homeostasis, neural cells primarily rely on energy produced from either the mitochondria or the result of mitochondria-independent mechanisms such as glycolysis. Because no significant alteration in cellular ATP levels was observed between cultures of normal and clone 6 cells (data not shown), we sought to determine whether clone 6 cells maintained cellular energy levels through an increased dependence on glycolysis. In this set of experiments, neural cells were maintained in complete medium or switched to a glucose-free medium that contained serum and all of the other media components other than glucose. Normal cells that underwent cell death within 48 h of transfer to no glucose medium (Fig. 4) with clone 6 cells were observed to undergo significantly higher levels of cell death than control cells (Fig. 4). Cell death in this model was primarily associated with nuclear condensation and not with nuclear fragmentation (Fig. 4), suggesting that cell toxicity may be mediated by a necrotic and not an apoptotic pathway. It is important to point out that the increased levels of neural death in clone 6 cells occurs despite the fact that these cells are more resistant to both serum withdrawal-induced and hydrogen peroxide-induced neural death (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; 86: 489-497Crossref PubMed Scopus (121) Google Scholar, 23Ding Q. Bruce-Keller A.J. Chen Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). Neural Cells Undergoing Proteasome Inhibition Possess Morphologically Normal Mitochondria but Possess Evidence of Altered Mitochondrial Turnover—To determine whether the mitochondrial alterations observed in our clonal cells were simply because of alterations in mitochondrial integrity, we conducted studies using electron microscopy. In these studies we analyzed hundreds of individual cells and observed that proteasome inhibition did not alter the size or shape of mitochondria in clone 6 cells (Fig. 5) and was not associated with a loss or swelling of christae within mitochondria (Fig. 5). The mitochondria population in the control and clonal cell lines was essentially uniform with no significant alteration in the intracellular localization or number of mitochondria observed between the control and clonal cells (data not shown). Despite such similarities, it was consistently noted that the mitochondria of proteasome inhibitor-treated cells were nearly always located in close proximity to dense core lysosomes (Fig. 5). Because these lysosomes could be indicators of lysosomal-mediated degradation of mitochondria, we conducted more in depth analysis to determine whether direct evidence for an up-regulation in lysosomal-mediated degradation of mitochondria could be observed in cells undergoing proteasome inhibition. Although no evidence of lysosomal-mediated degradation of mitochondria was ever observed in our control cells (data not shown), our clonal cell lines were routinely observed to contain large autophagosomes (Fig. 6), indicative of increased lysosomal activation. Surprisingly, whole or even partial mitochondria (Fig. 6) could often be found in these autophagosome and lysosomal structures, thus demonstrating direct evidence for increased levels of lysosomal-mediated mitochondria turnover. Interestingly, clone 6 cells possessed significantly higher levels of lipofuscin (Fig. 7), an intracellular aggregate composed primarily of protein, lipids, and carbohydrates. Because lipofuscin is believed to be derived in part from the incomplete degradation of mitochondria (33Brunk U.T. Terman A. Free Radic. Biol. Med. 2002; 33: 611-619Crossref PubMed Scopus (696) Google Scholar), these data may indicate that the ability of clone 6 cells to remove mitochondria by macroautophagy is somehow impaired or insufficient to remove damaged or unwanted mitochondria. Additionally, studies have suggested that lipofuscin may be a potent inhibitor of macroautophagy-mediated protei
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