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Originally published In Press as doi:10.1074/jbc.M313579200 on January 22, 2004

J. Biol. Chem., Vol. 279, Issue 20, 20699-20707, May 14, 2004
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Proteasome Inhibition Alters Neural Mitochondrial Homeostasis and Mitochondria Turnover*

Patrick G. Sullivan{ddagger}§, Natasa B. Dragicevic{ddagger}§, Jian-Hong Deng¶, Yidong Bai¶||, Edgardo Dimayuga{ddagger}, Qunxing Ding{ddagger}, Qinghua Chen**, Annadora J. Bruce-Keller{ddagger}, and Jeffrey N. Keller{ddagger}¶**{ddagger}{ddagger}

From the {ddagger}Department of Anatomy and Neurobiology, University of Kentucky, §Spinal Cord and Brain Injury Research Center, University of Kentucky, **Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536, and the Department of Cellular and Structural Biology, University of Texas Health Sciences Center, San Antonio, Texas 78229

Received for publication, December 11, 2003 , and in revised form, January 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The proteasome is a large multicatalytic protease that is responsible for the majority of overall intracellular protein degradation (1–3). Increasing evidence suggests that proteasome inhibition occurs in a wide array of neurodegenerative conditions (4–8) as well as normal aging (9) with inhibition of proteasome activity sufficient to induce multiple and diverse effects on intracellular homeostasis (1, 7, 10). In particular, severe pharmacological impairment of proteasome activity has been demonstrated to potently induce neuronal apoptosis in vitro (11–15). 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).1 Impairments in ETS are associated with increased formation of reactive oxygen species (ROS) and decreased energy production (16–18), which are both believed to directly contribute to neurotoxicity in a wide range of neurodegenerative conditions (19–21). 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 (22, 23). These cells possess neuropathology relevant to aging and age-related disease (22, 23) 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (22–25). 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 (22, 23). 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 (26, 27). 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 x 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 (27). The mitochondria were then purified by differential centrifugation at 1300 x g for 5 min to pellet unbroken cells and the nuclei. The supernatant was then centrifuged at 13,000 x g for 10 min to pellet the mitochondria. The pellet was resuspended in EGTA-free isolation buffer and centrifuged at 10,000 x 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 (26, 27). 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 (26, 27). 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 (28). Samples of either control or clone 6 cells (2 x 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 (29).

Electron Microscopy—Analysis of mitochondria by electron microscopy was conducted as described previously (25) 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 (22–24, 30). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (22, 23). These cells have been extensively characterized in previous studies (22, 23) and are known to possess a small but significant decrease in proteasome-mediated protein degradation (22, 23) 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).



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  		FIG. 1.
Proteasome inhibition alters mitochondrial bioenergetics. Mitochondria were isolated from control (cont) or a clonal line of human SH-SY5Y cells that have undergone continuous low level proteasome inhibition (clone 6, C6). Mitochondrial respiration was quantified by measuring oxygen consumption (nmols/min/mg) as detailed under "Experimental Procedures." Panel A demonstrates that when utilizing complex I substrates C6, mitochondrial oxygen consumption is not significantly different from control cells when measured in the presence of ADP (State III) or when the ATP synthase is inhibited (State IV) by the addition of oligomycin. In contrast, the induction of maximum respiration (State V) by the addition of the uncoupler FCCP clearly demonstrates that oxygen consumption is significantly reduced in C6 mitochondria. This reduction in maximum ETS capacity (State V respiration) was apparent regardless of the whether the mitochondria were utilizing complex I or complex II (panel B) substrates. Bars represent group means + S.E., * indicates p < 0.05.

 
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).



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  		FIG. 2.
Mitochondrial ROS is elevated in clonal cells with chronic low level proteasome inhibition. ROS production was measured in mitochondria isolated from neural cells exposed to chronic low level proteasome inhibition (clone 6, C6) and wild type cells of equal passage number (cont) using the fluorescent indicator dichlorodihydrofluorescein (DCF). It was demonstrated that C6 mitochondrial ROS production was significantly increased compared with control cells when utilizing complex I substrates to drive the ETS. Bars represent group means ± S.E. * indicates p < 0.05.

 
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 (18, 31, 32). 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 (28) 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 (28). 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.



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  		FIG. 3.
Intramitochondrial protein synthesis is altered in clonal cells. Electrophoretic patterns of SDS lysates were analyzed from wild type (N) and a clonal line of neural cells exposed to chronic low level proteasome inhibition (C6). These studies demonstrated the presence of 13 major protein bands that correspond to the 13 mtDNA-encoded proteins in both the control and clonal cells (A) with the banding pattern in these gels identical to previously published reports using this method (28). Analysis consisted of pulse-labeling under conditions that allow for the selective analysis of intramitochondrial protein synthesis (28). The data from two separate pulse-labeling (B) and two separate pulse-chase (C) experiments are presented in graph form. *, p < 0.05 compared with C6 cultures.

 
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 (22, 23).



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  		FIG. 4.
Clonal cells are more vulnerable to glucose deprivation. Neural cells exposed to chronic low level proteasome inhibition (C6) and wild type cells of equal passage number (N) were examined for cell viability 48 h following placement in complete medium (+ Glucose) or complete medium lacking only glucose (– Glucose). Under basal conditions in complete medium (+glucose), the nuclei of N (A) and C6 cells (C) appear smooth and oval. Within 48 h of transfer to –glucose medium, non-viable nuclei are still not widespread in N cultures (B) but are clearly evident in C6 cultures (D) (see arrows). The data from multiple cultures are quantified and expressed as the mean ± S.E. (E). *, p < 0.05 compared with +glucose medium; **, p < 0.05 compared with N cultures in –glucose medium.

 
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).



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  		FIG. 5.
Mitochondria in clonal cells appear morphologically normal. Control cells and neural cells exposed to chronic low level proteasome inhibition (clone 6) were analyzed by electron microscopy. Mitochondrion from control cells (A) and cells subjected to chronic low level proteasome inhibition (B) appeared morphologically similar, possessing a similar size, shape, and intact christae. Mitochondria (M) in neural cells subject to chronic low level proteasome inhibition were consistently surrounded by dense core lysosomes (B, white arrows). Data are representative of results from over 100 cells. Bars indicate 500 nm.

 
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 (33), 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 protein degradation (33, 34), again consistent with clone 6 cells possessing impaired macroautophagy-induced mitochondrial turnover.



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  		FIG. 6.
Clonal cells exhibit evidence of increased mitochondrial turnover. Control cells and neural cells exposed to chronic low level proteasome inhibition (clone 6) were analyzed by electron microscopy. Control cells were never observed to possess evidence of lysosomal-mediated degradation of mitochondria (M) (data not shown) with clonal cell lines undergoing proteasome inhibition consistently possessing large autophagic bodies (A) and dense core lysosomes (dark arrows), consistent with elevated levels of lysosomal-mediated degradation of mitochondria. Remnants of mitochondria are observed in the debris located within the autophagic bodies (white arrow) and dense core lysosomes. Data are representative of results from over 100 cells. Bar indicates 1 µm.

 



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  		FIG. 7.
Clonal cells possess increased levels of lipofuscin. Control cells and neural cells exposed to chronic low level proteasome inhibition (clone 6, C6) were analyzed for lipofuscin content by quantifying the amount of cellular autofluorescence. Data are provided as fluorescence intensity per 100,000 cells and are presented are the mean ± S.E. from six separate cultures. *, p < 0.02 compared with control cultures.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that mitochondria isolated from clone 6 cells have a reduced ETS reserve as indicated by reduced oxygen consumption when the ETS is uncoupled from the ATP synthase, a commonly utilized measure of maximum ETS oxygen consumption (26, 27). This reduction in maximum respiration was measured in mitochondria-utilizing complex I substrates as well as complex II substrates. Together, these data indicate that a loss of complex activity most probably has occurred downstream of complex I because even when it is bypassed by using complex II substrates, respiration is still impaired in mitochondria from cells undergoing inhibition of proteasome activity. In future studies it will be important to determine the exact site in the ETS responsible for the loss of ETS reserve observed in the clonal cell line.

The loss of both complex I and complex II activity was associated with a significant decrease in the level of mtDNA-encoded proteins. Because of the severity by which mtDNA-encoded proteins are decreased in clone 6 cells and the fact that all 13 of the proteins were decreased to a similar extent, it is highly probable that the loss of mtDNA-encoded proteins plays a direct role in the observed loss of complex I and complex II function. Numerous studies have demonstrated the vital role mtDNA-encoded proteins play in maintaining function of the ETS (18, 31, 32). It is important to point out that the decrease in mtDNA-encoded protein may not necessarily serve as the initiating event that triggers the first decreases in neural mitochondrial ETS function. For example, initial impairments in mitochondrial function may occur as the result of alterations in cytosolic calcium or ROS levels or the activation of cell death pathways. In future studies, it will be important to determine the potential contribution of such events to the mitochondrial dysfunction that occurs following inhibition of proteasome activity.

The underlying basis for the decreased translation and increased turnover of mitochondrial proteins in the present study is unclear and probably multifactorial. At the present time, we believe that the most probable cause of both of these events is the increased levels of mitochondrial ROS generated in cells subject to proteasome inhibition. Mild protein oxidation is known to potently induce protein instability with the slight elevations in mitochondrial ROS generation observed in this study sufficient to induce mild protein oxidation. Additionally, gene array studies revealed that no significant alteration the expression of any mitochondrial proteases including the LON-ATP-dependent protease and caseinolytic protease were observed in our clone 6 cell line (23). Such data suggest that it is increased protein instability and not an increase in protease expression or activation that contributes to increased levels of protein turnover observed in the present study. Similarly, continually elevated levels of mitochondrial ROS would be expected to alter mtDNA in a manner that ultimately impairs the rate at which mtDNA encoded proteins are produced. Together, these processes would be expected to proceed in a feed-forward fashion to ultimately induce the severe inhibition of maximal complex I and complex II inhibition that was observed in this study.

ROS production was significantly increased in mitochondria from clone 6 cells as compared with control cells of equal passage number. Based on our analysis of mitochondria bioenergetics, we would anticipate that the increased ROS generation is mediated by the electron loss from the ETS. Such a hypothesis is supported by the numerous studies indicating that uncoupling of the ETS results in both electron loss and increased mitochondrial ROS production. Once released from the ETS these electrons would be expected to react with and reduce molecular oxygen, generating the superoxide anion. It is important to note that we assessed ROS formation in mitochondria utilizing complex I substrates and in the presence of oligomycin (State IV) where we found no difference in respiration between the cell lines. By locking the mitochondria in State IV, we maximize the membrane potential and inhibit the ETS by blocking the flow of protons through the ATP synthase. The fact that the mitochondria from our clonal cells produced more ROS under these conditions implicates the presence of intramitochondrial damage to components of the ETS in these cells. However, we cannot rule out potential differences in mitochondria antioxidant enzymes as another potential underlying mechanism.

The data in this report are the first studies to our knowledge to demonstrate the ability of proteasome inhibition to decrease complex I and complex II activity in neural cells. Mitochondrial function is known to be inhibited in multiple neurodegenerative conditions (19, 21, 32–36) as well as normal aging (30, 31). Although numerous studies have demonstrated that inhibition of these two complexes is sufficient to induce neuron death (17, 20), thus solidifying a role for complex I and complex II inhibition as mediators of neurotoxicity in a variety of disorders, the identification of what factor(s) mediate mitochondrial dysfunction has proved to be elusive. The data within this report provide experimental support for proteasome inhibition serving as a direct mediator of complex I and complex II inhibition in neural cells. It is interesting to note that the ability of proteasome inhibition to alter complex I and complex II was not dependent on the presence of severe and acutely toxic proteasome inhibition (22, 23). As such, these data indicate that even a low level of proteasome inhibition, if sustained, is sufficient to induce profound alterations in mitochondrial ETS activity.

Recent studies from our laboratory have demonstrated the ability of increased heat shock protein expression to ameliorate the acute toxicity of proteasome inhibition (24). This neuroprotection was associated with an attenuation of mitochondrial-derived ROS production (24). The ability of an individual heat shock protein, heat shock protein 40 (HSP40), to attenuate mitochondrial ROS production suggests that increased levels of protein aggregation and/or a decrease in mitochondrial protein import may play a causal role in the mitochondrial dysfunction induced by proteasome inhibition. For example, previous studies have demonstrated that increased expression of HSP40 alone is sufficient to decrease protein aggregation (37) and it is well established that heat shock proteins play an important role in both the intracellular trafficking of proteins to the mitochondria as well as mitochondrial protein import itself (39, 40).

In this study, clonal lines were more sensitive to glucose deprivation than control cultures (Fig. 4), even though previous studies have demonstrated that the clonal lines used in this study were more resistant to both hydrogen peroxide treatment and serum-withdrawal toxicity (22, 23). Based on the observed impairments in maximal complex I and complex II activity, it is understandable why clonal lines used in this report are more sensitive to impairments in glycolysis. No difference in oxidative phosphorylation capacity was observed in our clonal cells, even though a significant difference in the maximum ETS reserve and basal ROS production was demonstrated. These differences could reduce the ability of cells undergoing proteasome inhibition to maintain a sufficiently high ATP:ADP ratio, which is necessary to maintain cellular homeostasis in response to stressors that increase cellular energy consumption. Together, these data suggest that neurons undergoing low level proteasome inhibition are not more vulnerable to oxidative and apoptotic injuries, but are selectively vulnerable to cellular stressors that induce dramatic fluxes in available energy.

In our study, neural cells exposed to proteasome inhibition routinely possessed mitochondria that were surrounded by lysosomes, suggesting that elevated levels of mitochondria turnover occurs in neural cells undergoing proteasome inhibition. More direct evidence for increased lysosomal-mediated degradation of mitochondria was obtained by directly demonstrating mitochondria as being present in the intralysosomal debris of autophagolysosomes. It is again important to note that these cells were completely viable and were actually more resistant to both oxidative and apoptotic injury (22, 23). As such, the degradation of mitochondria in these cells appears to be a protective and beneficial event, at least in the short term. These data also indicate that proteasome inhibition may be an initiator of increased macroautophagy in neural cells mediated by the effects of proteasome inhibition on mitochondrial homeostasis. It will be important in future studies to determine what mitochondrial alterations may be responsible for triggering macroautophagy in neural cells and elucidating whether dysfunctional mitochondria are preferentially degraded following the induction of macroautophagy in neural cells.

Cells subjected to proteasome inhibition possessed significantly elevated levels of lipofuscin. Because lipofuscin is believed to be generated through incomplete lysosomal-mediated degradation of mitochondria (33, 34), these data suggest that macroautophagy may be insufficient for the continual removal of damaged mitochondria or, perhaps more likely, may become impaired during the prolonged induction of macroautophagy that occurs following proteasome inhibition. The probable role of impaired macroautophagy-mediated degradation of mitochondria is based on previous studies in which we demonstrated that while clone 6 cells exhibit increased levels of macroautophagy activation, the amount of macroautophagy-mediated protein degradation may ultimately be impaired in clone 6 cells (22). Although the cause of this impairment has not been elucidated, previous reports (33, 34) have indicated that lipofuscin may serve as a potent inhibitor of macroautophagy-induced protein degradation, primarily by its ability to accumulate within lysosomes, sequester lysosomal proteases, and generate ROS through the release of trace metals. As such, the increased levels of lipofuscin observed in this study may serve as an inhibitor of macroautophagy-mediated proteolysis. Age-related increases in lipofuscin occur in nearly all of the cells with neurons in the brain previously demonstrated to accumulate extremely high levels of lipofuscin during normal aging (33, 34). Although the cause of such increases has remained unclear, the data presented in this report indicate a possible role for proteasome inhibition and the mitochondrial dysfunction that results from proteasome inhibition, serving as potent inducers of neural lipofuscin accumulation.

Recent studies have suggested that impairment of mitochondrial function may serve as an initiating event for the development of later pathological features (40, 41). It should be noted that the cells utilized in this report have been shown previously to possess multiple pathological features present in the aging brain such as Alzheimer's and Parkinson's Diseases. For example, these cells have been demonstrated to contain elevated levels of protein oxidation and protein aggregation (22, 23). Because each of these pathological features coincides with the presence of severe mitochondrial disturbances (Fig. 1, 2, 3), our data further strengthen the existence of a functional relationship between mitochondrial disturbances and the generation of neural pathology. Perhaps more importantly, because all of these events were established by the induction of a chronic low level proteasome inhibition, our data suggest that mild and sustained impairments in proteasome activity are sufficient to trigger both mitochondrial dysfunction and the generation of neuropathology.

Taken together, the data within this report and our previously published studies allow for the generation of a centralized working model to explain the role of proteasome inhibition as a mediator of mitochondrial dysfunction and altered mitochondrial turnover (Fig. 8). In this model, low level proteasome inhibition occurs in the brain as the result of aging or some specific stressor (7, 9). The initiation of low level proteasome inhibition then directly induces alterations in gene expression, increased protein oxidation, and increased protein aggregation. Each of these events, over time promotes a mild uncoupling or impairment in mitochondrial ETS. As the result of this impairment, there is an enhanced loss of electrons from the ETS, resulting in an increased level of mitochondrial ROS generation, which then promotes increased protein oxidation and increased protein instability within the mitochondria. Ultimately, this cycle continues until there is an increased number of dysfunctional mitochondria within the neural cells. To prevent a large deleterious accumulation of dysfunctional mitochondria, neural cells induce a macroautophagy response, which is designed to degrade dysfunctional mitochondria and allow for the generation of new mitochondria. Although mitochondria are degraded as the result of macroautophagy in the short term, increased levels of lipofuscin accumulate as the result of incomplete proteolysis, which over time results in a more severe inhibition of macroautophagy-induced protein degradation. Given enough time, this feed-forward pathway results in a continual and progressive increase in lipofuscin accumulation and decreased mitochondrial turnover. Ultimately, each of these events promotes the manifestation of neurophysiological and neuropathological alterations associated with neurons in aging and age-related neuropathological disorders. Further elucidation of the causes and effects of proteasome inhibition in the brain are therefore likely to contribute significantly to the understanding of aging and age-related disease in the brain.



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  		FIG. 8.
Effects of proteasome inhibition on mitochondrial homeostasis. Based on our experimental findings and previous findings from other studies, a schematic diagram of the effects of proteasome inhibition on mitochondrial homeostasis and the possible causes of those effects can be illustrated. Following the initiation of low level proteasome inhibition, there is known to be a dramatic alteration in global gene expression and increased levels of protein oxidation and protein aggregation. Each of these events probably plays a direct role in altering maximal mitochondrial function, increasing the loss of electrons from the ETS, and directly contributes to increased mitochondrial ROS formation. The increase in mitochondrial ROS formation then increases protein oxidation and alters protein stability within the mitochondria, directly contributing to further compromises in mitochondrial function. To remove these defective mitochondria and respond to the increased levels of protein oxidation/aggregation, neural cells undergo an induction of macroautophagy. However, because of the elevated levels of lipofuscin, the ability of macroautophagy to turnover defective mitochondria is impaired, resulting in an accumulation of defective mitochondria. This feed-forward cycle is continued, ultimately resulting in compromised neural function and further accumulation of lipofuscin.

 

   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Supported by the United Mitochondrial Disease Foundation and Ellison Medical Foundation New Scholar in Aging Grant AG-NS-0183-02. Back

{ddagger}{ddagger} Supported by American Heart Association and National Institutes of Health Grants AG08437 and AG005119. To whom correspondence should be addressed: University of Kentucky, 205 Sanders-Brown Bldg., Lexington, KY 40536-0230. Tel.: 859-257-1412; Fax: 859-323-2866; E-mail: Jnkell0{at}pop.uky.edu.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Mary Gail Engel for assistance with electron microscopy, Dr. Ana Cuervo for helpful discussions, and Dr. William R. Markesbery for continual support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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