Proteasome Inhibition Alters Neural Mitochondrial Homeostasis and Mitochondria Turnover

Abstract

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. 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; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). cells 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; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) fully for multiple thus for analysis to conducted the potentially that and In this study, we this clonal line to the effects of low level proteasome inhibition on mitochondrial homeostasis. these data demonstrate the of proteasome inhibition to directly multiple aspects of neural mitochondrial homeostasis and lysosomal-mediated degradation of mitochondria, a role for proteasome inhibition as a direct of neural mitochondrial dysfunction. of the and were dichlorodihydrofluorescein The and were and of the and were of SH-SY5Y cells were the and as (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar, Q. Dimayuga E. Bruce-Keller A.J. Keller J.N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, N. M. T. A. T. K.J. J. 2000; PubMed Google Scholar). To clonal following neural SH-SY5Y cells were in normal with and as (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). The and each cultures of SH-SY5Y cells that were cultures for the of the of were for of the of of the mitochondrial were conducted that have been by in 2000; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). each of cultures were with cells at to generate a of either or clonal were for mitochondrial following with and clonal by on the at on were to generate data in the present the of the cells were by at for at of the were at on or at The in of mitochondrial and the were by Cell at for as 2003; PubMed Scopus Google Scholar). The mitochondria were by at for to cells and the The at for to the The in and at for The in at a of of and a in a and as 2000; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). Mitochondria were to the to a protein of in II by the of and III by the of by the of to induce The mitochondrial carbonyl cyanide 4-trifluoromethoxy to the to induce The complex I to the by the of to for the of complex are as mitochondrial levels were a to of production the as 2000; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). of mitochondria in a of of at for in the of H2DCFDA, which each The of mitochondrial produced were a the of to production and to In each the mitochondrial-independent production for by the in in which mitochondria were of the for mitochondrial were in of and the data were used for are as of mitochondrial protein with were as G. J. 1998; PubMed Scopus Google Scholar). of either or 6 cells were and with by a at in of the and the cells were for To the of the mitochondrial were Cell and were as with the that with a protein and with to the the cells were and to a in complete and in the of The cells were and in of protein were through The of the were by The ND1, ND2, ND3, ND4, ND4L, and are subunits of NADH dehydrogenase; is apocytochrome b; COI, COII, and are subunits of cytochrome c oxidase; and A6 and are subunits of the of of lipofuscin levels by the of cellular as Bruce-Keller A.J. Keller J.N. J. Neurosci. Res. 1998; PubMed Scopus Google Scholar). of mitochondria by electron conducted as N. M. T. A. T. K.J. J. 2000; PubMed Google Scholar) with cells were in by in by a in were to in increasing of and in The and a The at cells for each of Neural by as (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar, Q. Dimayuga E. Bruce-Keller A.J. Keller J.N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. M. T. 1998; Scopus (207) Google Scholar). each at cultures were with at cells for each with a of for Cell the effects of low level proteasome inhibition on neural mitochondria we conducted recently clonal SH-SY5Y line (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). cells have been in (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) and are known to a significant in protein degradation (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar) and to a for the analysis of the neurotoxicity associated with proteasome inhibition. is the and to mitochondrial we mitochondrial oxygen in and clonal of equal passage number. in oxygen by the and not between or clonal cells in the of or the However, oxygen reduced in the mitochondria the 6 cells reduced in mitochondria of the the mitochondria were complex I or complex II to production is a of mitochondrial dramatically increased in to ETS we to the alterations in mitochondrial observed in the cells inhibition of proteasome the of Mitochondria 6 cells were observed to a significant in as compared with cultures complex of of the order for the electron transport chain to the mitochondria must E.A. Manfredi G. J. Clin. Investig. 2003; 111: 303-312Crossref PubMed Scopus (308) Google Scholar, Lee Y.S. N. 1998; PubMed Scopus Google Scholar, J.M. Rev. (Paris). Google Scholar). are and within the mitochondria and not mitochondrial protein of these are as ND1, ND2, ND3, ND4, ND4L, and ND6, which are subunits of NADH The protein is also known as apocytochrome whereas COI, COII, and are subunits of cytochrome c The A6 and are subunits of the To the loss of complex I and complex II activity in 6 cells may in to alterations in the of protein production, we conducted the and degradation of each of these were conducted conditions that for the analysis of intramitochondrial protein G. J. 1998; PubMed Scopus Google Scholar) and demonstrated the of protein that to the in both the and clonal cells The and in these were to this G. J. 1998; PubMed Scopus Google Scholar). The overall mitochondrial protein decreased by in 6 cells as compared with cells with of the a in Interestingly, that the level of protein decreased by in 6 cells Taken together, these data that low level proteasome inhibition the of protein with that are to within the mitochondria, decreased Neural Proteasome Inhibition on neural cells on energy produced either the mitochondria or the result of mechanisms such as glycolysis. significant in cellular levels observed between cultures of normal and 6 cells not we to 6 cells cellular energy levels through increased dependence on glycolysis. In this of neural cells were in complete or to a that and of the cells that within of to with 6 cells were observed to levels of cells Cell in this associated with and not with that may mediated by a and not is to that the increased levels of neural in 6 cells occurs the that these cells are more to both and neural (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). Neural Proteasome Inhibition Mitochondria of the mitochondrial alterations observed in clonal cells were of alterations in mitochondrial we conducted electron In these we of cells and observed that proteasome inhibition not the or of mitochondria in 6 cells and not associated with a loss or of within mitochondria The mitochondria in the and clonal with significant in the intracellular or of mitochondria observed between the and clonal cells not Despite such similarities, consistently that the mitochondria of proteasome cells were located in close proximity to lysosomes these lysosomes of lysosomal-mediated degradation of mitochondria, we conducted more in analysis to direct evidence for in lysosomal-mediated degradation of mitochondria observed in cells proteasome inhibition. Although evidence of lysosomal-mediated degradation of mitochondria observed in cells not clonal were observed to large of increased or mitochondria in these and thus direct evidence for increased levels of lysosomal-mediated mitochondria Interestingly, 6 cells possessed levels of lipofuscin intracellular of and lipofuscin is believed to in the degradation of mitochondria A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar), these data may that the of 6 cells to mitochondria by is or to or have that lipofuscin may a of protein degradation A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. Biochem. 2002; PubMed Scopus Google Scholar), with 6 cells mitochondrial cells increased levels of cells and neural cells exposed to low level proteasome inhibition were for lipofuscin by the of cellular are as cells and are are the S.E. compared with demonstrate that mitochondria 6 cells have a reduced ETS as by reduced oxygen the ETS is the a of ETS oxygen 2000; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). in in complex I as well as complex II these data that a loss of complex activity has of complex I is by complex II is in mitochondria cells inhibition of proteasome In to the in the ETS responsible for the loss of ETS observed in the clonal The loss of both complex I and complex II activity associated with a significant in the level of of the by which are decreased in 6 cells and the that of the were decreased to a similar is that the loss of plays a direct role in the observed loss of complex I and complex II have demonstrated the role play in of the ETS E.A. Manfredi G. J. Clin. Investig. 2003; 111: 303-312Crossref PubMed Scopus (308) Google Scholar, Lee Y.S. N. 1998; PubMed Scopus Google Scholar, J.M. Rev. (Paris). Google Scholar). is to that the in protein may not as the that the in neural mitochondrial ETS impairments in mitochondrial may occur as the result of alterations in or levels or the of In to the of such to the mitochondrial dysfunction that occurs following inhibition of proteasome The for the decreased and increased turnover of mitochondrial in the present is and the present time, we that the of both of these is the increased levels of mitochondrial generated in cells subject to proteasome inhibition. protein is known to potently induce protein with the in mitochondrial observed in this sufficient to induce protein array that significant the of mitochondrial including the protease and protease were observed in 6 line Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). data suggest that is increased protein and not in protease or that to increased levels of protein turnover observed in the present continually levels of mitochondrial to in a that the at which are these to in a to induce the severe inhibition of maximal complex I and complex II inhibition that observed in this production increased in mitochondria 6 cells as compared with cells of equal passage number. on analysis of mitochondria we that the increased is mediated by the electron loss the a is by the numerous that of the ETS in both electron loss and increased mitochondrial the ETS these electrons to with and molecular the is to that we formation in mitochondria complex I and in the of we in between the the mitochondria in we the and the ETS by the of through the The that the mitochondria clonal cells produced more under these conditions the of intramitochondrial to of the ETS in these However, we in mitochondria as The data in this are the to to demonstrate the of proteasome inhibition to complex I and complex II activity in neural is known to in multiple neurodegenerative conditions (19Cooper J.M. Schapira A.H. J. Bioenerg. Biomembr. 1997; 29: 175-183Crossref PubMed Scopus (57) Google Scholar, 21Beal M.F. Trends Neurosci. 2000; 23: 298-304Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, J.M. Rev. (Paris). Google Scholar, A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. Biochem. 2002; PubMed Scopus Google Scholar, J. Neural Transm. 1998; PubMed Scopus Google Scholar, G. A. N. 2003; PubMed Scopus Google Scholar) as well as normal aging J.M. M. T. 1998; Scopus (207) Google Scholar, Lee Y.S. N. 1998; PubMed Scopus Google Scholar). Although numerous have demonstrated that inhibition of these is sufficient to induce G. Beal M.F. Brain Pathol. 2000; 10: 462-472Crossref PubMed Scopus (98) 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), thus a role for complex I and complex II inhibition as of neurotoxicity in a variety of the of mitochondrial dysfunction has to The data within this for proteasome inhibition as a direct of complex I and complex II inhibition in neural is to that the of proteasome inhibition to complex I and complex II not on the of severe and proteasome inhibition (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). these data that a low level of proteasome inhibition, is sufficient to induce alterations in mitochondrial ETS have demonstrated the of increased protein to the of proteasome inhibition Q. Dimayuga E. Bruce-Keller A.J. Keller J.N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). associated with of production Q. Dimayuga E. Bruce-Keller A.J. Keller J.N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of protein to mitochondrial production suggests that increased levels of protein a in mitochondrial protein may play a role in the mitochondrial dysfunction by proteasome inhibition. have demonstrated that increased of is sufficient to protein J. Neurosci. 1999; PubMed Google Scholar, Martin H. T. N. 1999; PubMed Scopus Google Scholar) and is well that play role in both the intracellular of to the mitochondria as well as mitochondrial protein P. N. N. Rev. Biochem. Biol. 2001; PubMed Scopus Google Scholar, G. G. Res. 2003; PubMed Scopus Google Scholar). In this study, clonal were more to cultures have demonstrated that the clonal used in this were more to both and (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). on the observed impairments in maximal complex I and complex II is clonal used in this are more to impairments in glycolysis. in observed in clonal a significant in the ETS and basal production the of cells proteasome inhibition to a which is to cellular homeostasis in to that cellular energy these data suggest that low level proteasome inhibition are not more to and are to cellular that induce in In study, neural cells exposed to proteasome inhibition possessed mitochondria that were by that levels of mitochondria turnover occurs in neural cells proteasome inhibition. direct evidence for increased lysosomal-mediated degradation of mitochondria by directly mitochondria as present in the debris of is to that these cells were and were more to both and (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). the degradation of mitochondria in these cells to a and at in the data also that proteasome inhibition may of increased in neural cells mediated by the effects of proteasome inhibition on mitochondrial homeostasis. in to mitochondrial alterations may responsible for in neural cells and mitochondria are following the of in neural to proteasome inhibition possessed levels of lipofuscin is believed to generated through lysosomal-mediated degradation of mitochondria A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. Biochem. 2002; PubMed Scopus Google Scholar), these data suggest that may for the of mitochondria more may the of that occurs following proteasome inhibition. The role of degradation of mitochondria is on in which we demonstrated that 6 cells increased levels of the of protein degradation may in 6 cells (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar). Although the of this impairment has not been A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. Biochem. 2002; PubMed Scopus Google Scholar) have that lipofuscin may as a of protein by to within and generate through the of the increased levels of lipofuscin observed in this may as of in lipofuscin occur in of the cells with in the demonstrated to levels of lipofuscin normal aging A. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. Biochem. 2002; PubMed Scopus Google Scholar). Although the of such has the data in this a role for proteasome inhibition and the mitochondrial dysfunction that proteasome inhibition, as of neural lipofuscin have that impairment of mitochondrial may as for the of features G. G. Res. 2003; PubMed Scopus Google Scholar, G. A. A. G. J. Neurosci. Res. 2002; PubMed Scopus Google Scholar). that the cells in this have been to multiple features present in the aging such as Alzheimer's and Parkinson's these cells have been demonstrated to levels of protein and protein (22Ding Q. Dimayuga E. Martin S. Bruce-Keller A.J. Nukala V. Cuervo A.M. Keller J.N. J. Neurochem. 2003; PubMed Scopus Google Scholar, Q. Bruce-Keller A.J. Q. Keller J.N. Free Radic. Biol. Med. 2003; (in press)Google Scholar). each of these features with the of severe mitochondrial 1, 2, data the of a relationship between mitochondrial and the of neural more of these were by the of a low level proteasome inhibition, data suggest that and impairments in proteasome activity are sufficient to both mitochondrial dysfunction and the of Taken together, the data within this and for the of a to the role of proteasome inhibition as a of mitochondrial dysfunction and mitochondrial turnover In this low level proteasome inhibition occurs in the as the result of aging or specific Q. Keller J.N. Free Radic. Biol. Med. 2001; 31: 574-584Crossref PubMed Scopus (110) Google Scholar, J.N. Gee J. Ding Q. Ageing Res. Rev. 2002; 1: 279-293Crossref PubMed Scopus (207) Google Scholar). The of low level proteasome inhibition directly alterations in increased protein and increased protein of these a or impairment in mitochondrial the result of this is loss of electrons the ETS, in increased level of mitochondrial which increased protein and increased protein within the this is increased of mitochondria within the neural To a large of mitochondria, neural cells induce a which is to mitochondria and for the of Although mitochondria are as the result of in the increased levels of lipofuscin as the result of which in a more severe inhibition of protein time, this in a and in lipofuscin and decreased mitochondrial each of these the of and neuropathological alterations associated with in aging and age-related neuropathological of the and effects of proteasome inhibition in the are to contribute to the understanding of aging and age-related disease in the We for with electron Cuervo for and Markesbery for