Mutant Cu/Zn-Superoxide Dismutase Proteins Have Altered Solubility and Interact with Heat Shock/Stress Proteins in Models of Amyotrophic Lateral Sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and αB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1·HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins. Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and αB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1·HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins. Amyotrophic lateral sclerosis (ALS) 1The abbreviations used are: ALSamyotrophic lateral sclerosisHSPheat shock proteinSOD-1Cu/Zn-superoxide dismutase is an adult-onset neurodegenerative disease characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord. Approximately 10% of ALS cases are familial, and about 20% of these familial cases are caused by dominantly inherited mutations in the gene encoding the enzyme Cu/Zn-superoxide dismutase (SOD-1) (1Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.-X. Rahmani Z. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J.J. Herzfeldt B. Van den Bergh R. Hung W.-Y. Bird T. Deng G. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar). Evidence indicates that some “toxic gain of function” is responsible for motor neuron loss rather than a decreased ability of mutant SOD-1 to carry out its primary enzymatic function, dismutation of superoxide to hydrogen peroxide. Most SOD-1 mutant proteins have significant enzymatic activity, and any impairment relative to wild-type does not correlate with clinical severity of the disease (2Bowling A.C. Barkowski E.E. McKenna-Yasek D. Sapp P. Horvitz H.R. Beal M.F. Brown Jr., R.H. J. Neurochem. 1995; 64: 2366-2369Crossref PubMed Scopus (117) Google Scholar, 3Ratovitski T. Corson L.B. Strain J. Wong P. Cleveland D.W. Culotta V.C. Borchelt D.R. Hum. Mol. Genet. 1999; 8: 1451-1460Crossref PubMed Scopus (148) Google Scholar). SOD-1 knockout mice do not develop motor neuron disease (4Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Crossref PubMed Scopus (1051) Google Scholar), whereas mice expressing mutant SOD-1 transgenes develop an ALS-like phenotype (5Gurney M.E. N. Engl. J. Med. 1994; 331: 1721-1722Crossref PubMed Scopus (197) Google Scholar, 6Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Abstract Full Text PDF PubMed Scopus (1258) Google Scholar, 7Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar). amyotrophic lateral sclerosis heat shock protein Cu/Zn-superoxide dismutase Over 60 different mutations have been identified and associated with ALS (reviewed in Ref. 8Wong P.C. Rothstein J.D. Price D.L. Curr. Opin. Neurobiol. 1998; 8: 791-799Crossref PubMed Scopus (63) Google Scholar). The distribution of such a large number of disease-causing mutations throughout all exons of the SOD-1gene suggests that altered protein conformation may contribute to the toxic gain of function. Additional evidence comes from x-ray crystallography showing that conserved interactions within the protein that determine the conformation of the active channel are altered in mutant SOD-1 proteins (9Deng H.-X. Hentati A. Tainer J.A. Iqbal Z. Cayabyab A. Hung W.-Y. Getzoff E.D. Hu P. Herzfeldt B. Roos R.P. Warner C. Deng G. Soriano E. Smyth C. Parge H.E. Ahmed A. Roses A.D. Hallewell R.A. Pericak-Vance M.A. Siddique T. Science. 1993; 261: 1047-1051Crossref PubMed Scopus (1360) Google Scholar). How this contributes to toxicity is not clear. Improperly folded SOD-1 enzyme could allow greater access of abnormal substrates to the active site; could enhance other known enzymatic functions of SOD-1, such as catalysis of protein nitration; or could alter metal binding, all of which would ultimately lead to increased oxidative damage (reviewed in Refs. 10Bromberg M.B. Curr. Opin. Neurol. 1999; 12: 581-588Crossref PubMed Scopus (28) Google Scholar, 11Cleveland D.W. Neuron. 1999; 24: 515-520Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 12Morrison B.M. Morrison J.H. Brain Res. Rev. 1999; 29: 121-135Crossref PubMed Scopus (75) Google Scholar). Alternatively, proteins with altered conformation could form insoluble precipitates or participate in abnormal protein-protein interactions unless prevented from doing so by association with chaperone proteins. Thus, depletion of chaperones could result in formation of mutant SOD-1 aggregates, as well as compromising cellular chaperoning function in general (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). Mutant SOD-1 proteins appear to be degraded by the proteasome, a pathway generally involved in proteolysis of denatured or misfolded protein (14Hoffman E.K. Wilcox H.M. Scott R.W. Siman R. J. Neurol. Sci. 1996; 139: 15-20Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 15Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (513) Google Scholar). Moreover, several mutant SOD-1 proteins have a tendency to precipitate in solution relative to wild-type (16Okado-Matsumoto A. Myint T. Fujii J. Taniguchi N. Free Radic. Res. 2000; 33: 65-73Crossref PubMed Scopus (23) Google Scholar). Proteinaceous aggregates of SOD-1 have been found in the cytoplasm of cultured primary motor neurons expressing several different SOD-1 mutants (17Durham H.D. Roy J. Dong L. Figlewicz D.A. J. Neuropathol. Exp. Neurol. 1997; 56: 523-530Crossref PubMed Scopus (303) Google Scholar), in motor neurons and astrocytes of mice expressing mutant SOD-1 transgenes (7Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar, 18Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Crossref PubMed Scopus (1001) Google Scholar), in familial ALS patients at autopsy (19Shibata N. Hirano A. Kobayashi M. Siddique T. Deng H.X. Hung W.Y. Kato T. Asayama K. J. Neuropathol. Exp. Neurol. 1996; 55: 481-490Crossref PubMed Scopus (252) Google Scholar, 20Kato S. Shimoda M. Watanabe Y. Nakashima K. Takahashi K. Ohama E. J. Neuropathol. Exp. Neurol. 1996; 55: 1089-1101Crossref PubMed Scopus (103) Google Scholar), and in transfected cell lines (15Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (513) Google Scholar, 21Koide T. Igarashi S. Kikugawa K. Nakano R. Inuzuka T. Yamada M. Takahashi H. Tsuji S. Neurosci. Lett. 1998; 257: 29-32Crossref PubMed Scopus (33) Google Scholar). In cultured motor neurons, coexpression of HSP70 and mutant SOD-1 expression vectors considerably reduced formation of SOD-containing aggregates and prolonged viability (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). Mutant SOD-1-transfected NIH3T3 cell lines showed up-regulation of HSP70, HSP25, and αB-crystallin compared with untransfected cells or cells transfected with the wild-type SOD-1 construct (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). Heat shock proteins could protect cells from mutant SOD-1 by direct interaction, thus preventing aggregation and facilitating degradation, or indirectly by protecting cells from downstream consequences of some other toxic property. In this study, we have demonstrated that mutant SOD-1 coimmunoprecipitates with HSP70, HSP40, and αB-crystallin. Furthermore, a portion of mutant SOD-1 was detergent-insoluble in three different experimental models of familial ALS: NIH3T3 cell lines stably or transiently transfected with mutant SOD-1 constructs, spinal cord cultures derived from G93A transgenic embryonic spinal cords, and lumbar spinal cord from adult G93A transgenic mice. The following primary antibodies were used: Rabbit anti-SOD-1 antibodies (SOD-100 diluted 1:500 and SOD-101 diluted 1:250, Stressgen Biotechnologies Corp., Victoria, British Columbia, Canada) (SOD-100 reacts with both human and mouse SOD-1 but favors human SOD-1; SOD-101 also reacts with both human and mouse SOD-1 but favors mouse SOD-1); rabbit anti-αB-crystallin (SPA-223) (1:250) and rabbit anti-HSP40 (SPA-400) (1:1000) antibodies from Stressgen Biotechnologies Corp.; mouse monoclonal HSP70 antibody (W27 catalog number SC-24, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:250) (may cross-react with HSC70); mouse monoclonal actin antibody, clone C4 (691001) (1:1000) and rabbit polyclonal tubulin antibody (650951) (1:500) from ICN Biomedicals Inc. (Irvine, CA). Secondary antibodies used were goat anti-rabbit horseradish peroxidase (P0399, Dako Corp., Mississauga, ON) (1:3000) and sheep anti-mouse horseradish peroxidase (515035062, Jackson ImmunoResearch Laboratories, Willow Grove, PA) (1:10,000). cDNA encoding human HSP70 was a kind gift from D. Mosser and was subcloned into pcDNA3. NIH3T3 cell lines stably expressing humanSOD-1 gene constructs (wild-type or with G93A or G41S mutations) have been described previously (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). Cultures were maintained under standard conditions in minimum essential medium supplemented with 3.7 g of NaHCO3, 5 g of dextrose, and 10% fetal bovine serum. Stable cell lines were supplemented with G418 (Life Technologies, Inc.) (250 for G41S and G93A lines and for the wild-type NIH3T3 cells were to at a of and transfected with the construct to the standard for (Life Technologies, extracts were lines of mice transgenic for G93A mutant human SOD-1, and as well as of mice transgenic for wild-type human SOD-1, were maintained in our were from The Jackson were by the and the of the mice develop in the and at of mice develop in the and at of Cultures were from mouse as previously described J. S. Dong L. Figlewicz D.A. Durham H.D. J. Neurosci. 1998; 18: PubMed Google that spinal from transgenic mouse were and at a of were for the of human SOD-1 transgenes the by The Jackson NIH3T3 cell lines and spinal cord cultures were in containing and and to and detergent-insoluble were three in and in 10% and The lumbar of mouse spinal cord was in of and at g for at of were with and in of of were by protein proteins were to were at in in primary and antibodies were for at proteins were the NIH3T3 cell lines were in of containing and for and for at the of 70 of cells were at and were total cell the was of SOD-1 antibody of HSP70 antibody, 5 of antibody, or 5 of αB-crystallin antibody was to of protein and at a complexes were of protein by a at for the were with in of and for 5 The were of the was a and proteins were by The tendency of mutant SOD-1 proteins to form aggregates in cultured cells and spinal cord that in compared with wild-type NIH3T3 cell lines stably expressing human G41S or G93A mutant SOD-1, established for previous (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar), were used to the solubility of wild-type and mutant SOD-1 in and detergent-insoluble were from these cell as well as from untransfected NIH3T3 and to The endogenous wild-type and mutant human SOD-1 proteins were by A with SOD-1 antibody which a for human SOD-1 relative to is in SOD-1 and wild-type human SOD-1 were only in the not in the fraction. In G41S and G93A mutant human SOD-1 were in both of were the relative to to of wild-type and of mutant SOD-1 in the to relative and for of was that about of total cellular mutant SOD-1 was detergent-insoluble in this was with the relative of the total of and were the not T. Corson L.B. Strain J. Wong P. Cleveland D.W. Culotta V.C. Borchelt D.R. Hum. Mol. Genet. 1999; 8: 1451-1460Crossref PubMed Scopus (148) Google that several different mutant SOD-1 proteins in cells by were in the was NIH3T3 cells transiently transfected with the G93A mutant SOD-1 in mutant human SOD-1, but not SOD-1, was in the detergent-insoluble by with antibody and was to to about 10% of total cellular mutant The of mouse SOD-1 in the was a SOD-1 antibody with greater for mouse SOD-1 the in with antibody also to SOD-1 in detergent-insoluble of any cell Whereas cell lines are models for the of mutant protein in these cells do not in primary by the the solubility of G93A mutant SOD-1 in primary spinal cord cultures from G93A and wild-type human SOD-1 transgenic mouse and in lumbar spinal cord from transgenic mice our in transfected NIH3T3 cells in that both and detergent-insoluble of G93A SOD-1 were In extracts of primary but not lumbar spinal a small of transgenic wild-type SOD-1 was in the detergent-insoluble but this was considerably than the of mutant protein mouse SOD-1 was not in the detergent-insoluble of mutant human SOD-1 from lumbar spinal cord of G93A SOD-1 transgenic mice is spinal from wild-type and G93A SOD-1 transgenic mice were in containing is the distribution of human and mouse SOD-1 in the and and with protein were with antibody to a protein that to both and detergent-insoluble In to different of cells of heat proteins. NIH3T3 cells were transfected with G41S or G93A mutant human SOD-1, of the cells cells that were to and were as of heat shock proteins as compared with and untransfected cells (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). Furthermore, coexpression of HSP70 and mutant SOD-1 expression vectors primary cultured motor neurons from toxicity of several different SOD-1 formation of SOD-containing aggregates and viability (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). determine heat shock proteins protect cells from mutant SOD-1 by direct protein-protein interaction, were NIH3T3 cell lines expressing mutant SOD-1 have endogenous of heat shock proteins (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar), this was for this total cell extracts were with antibody to SOD-1 HSP70 was from both G93A and G41S expressing cell lines but not from untransfected NIH3T3 or lines expressing wild-type human SOD-1 5 of total cell extracts with antibody to HSP70 G93A and G41S mutant SOD-1 but not wild-type human or endogenous SOD-1 5 The were the was used to interactions not SOD-1 complexes were in the detergent-insoluble fraction. of anti-SOD-1 or antibodies were used for the of HSP70 or mutant SOD-1, from was compared with the from total cell was not to of HSP70 in of cell lines expressing mutant SOD-1; HSP70 was by antibody from of both mutant cell lines 5 and was in both and detergent-insoluble of 5 G41S and G93A mutant SOD-1 were to both and and were from by the antibody The in 5 also our previous that HSP70 is in cell lines expressing mutant association of HSP70 with wild-type human SOD-1 was A and this could have been to the of HSP70 in this cell this cells stably expressing wild-type human SOD-1 were transiently transfected with expression of HSP70, by of total cell with antibody to HSP70, wild-type human SOD-1 with HSP70 not from 5 is that HSP70 was not in of untransfected or wild-type human SOD-1 expressing NIH3T3 Thus, HSP70 and wild-type SOD-1 proteins are in G41S and cell a of both HSP70 and mutant SOD-1 are How of the detergent-insoluble complexes of HSP70 and mutant SOD-1 be from these The interaction of mutant SOD-1 with stress proteins was not to SOD-1 but wild-type human endogenous SOD-1, were total cell extracts of NIH3T3 cell lines were by antibodies to in with HSP70, to complexes of and wild-type human SOD-1 was not to of was in both and of all cell lines were were out antibody to αB-crystallin. G41S and G93A SOD-1 but not wild-type human or endogenous SOD-1, were In to heat shock or to toxic cells of heat proteins. function is to the of folded proteins by to and of the proteins or by facilitating by D.A. S. A. C. The of Heat and Scholar, 1998; Google Scholar, Curr. Opin. 2000; PubMed Scopus Google Scholar). have evidence that heat shock proteins a in protecting cells from the toxicity of mutant SOD-1 proteins associated with human familial of HSP70 prolonged viability of cultured motor neurons expressing mutant SOD-1 proteins and reduced the formation of cytoplasmic aggregates (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). S. D. A. and H. D. of NIH3T3 cells expressing different SOD-1 G41S and with up-regulation of heat shock proteins (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). HSP70, HSP40, and αB-crystallin were with mutant SOD-1 proteins from mutant SOD-1-transfected cell lines the that heat shock proteins protect at in by direct association with mutant protein. SOD-1, a cytoplasmic is in However, a small percentage of mutant human SOD-1 protein was in the detergent-insoluble of protein extracts from NIH3T3 spinal cord cultures from of G93A mutant SOD-1 transgenic and lumbar spinal cord of adult G93A transgenic mice. mutant SOD-1 proteins can form with wild-type protein of different D.R. M. Wong P.C. Lee M.K. Sisodia S.S. Price D.L. Cleveland D.W. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the endogenous SOD-1 was not found in the detergent-insoluble of with protein and with antibody this HSP70 and mutant SOD-1 were from total cell extracts of the mutant SOD-1 transfected NIH3T3 cell whereas only were from the that SOD-1 complexes were predominantly of heat shock to the detergent-insoluble under a of heat oxidative and with R.P. M. J. PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). all detergent-insoluble mutant SOD-1 was with HSP70 be from the of mutant SOD-1 is to alter solubility or to of a small of total cellular mutant SOD-1 at was found in the in which the complexes were suggests the that altered conformation by the may not be of heat shock proteins would be by preventing protein-protein interactions facilitating cells may be different from spinal cord cells in familial detergent-insoluble mutant SOD-1 also was in spinal cord cultures from of G93A mutant SOD-1 transgenic mice and from lumbar spinal cord of adult G93A transgenic mice. significant expression of HSP70 was not in spinal cord (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar), are under to expression of heat shock proteins and interaction with mutant SOD-1 in these a of mutant SOD-1 is detergent-insoluble in the transfected NIH3T3 cell large aggregates are by in these cells (13Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Crossref PubMed Scopus (223) Google Scholar). However, in the by (15Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (513) Google Scholar), formation of large aggregates of mutant SOD-1 was in transfected cells by with The of mutant SOD-1 and association with heat shock proteins in our could an mutant SOD-1 and protein into may form heat shock proteins are to mutant protein and its in could result from as in the by (15Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. 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However, this does not that aggregates The of aggregates may the of the cell to a sufficient stress to general chaperoning function in the thus its to a of other and which ultimately contribute to cellular and to other motor neuron altered protein of heat shock proteins and mutant protein within aggregates in neurodegenerative caused by of (reviewed in Ref. N. Engl. J. Med. 1999; PubMed Scopus Google Scholar). is a motor neuron disease from in the gene Nature. PubMed Scopus Google Scholar). of heat shock proteins HSP40, and in or cell models of spinal reduced both formation of aggregates and toxicity D.L. K. M. M.A. Hum. Mol. Genet. 1999; 8: PubMed Scopus Google Scholar, Y. A. M. M. M. K. G. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). distribution and altered of the and the of were found in cells with aggregates of Thus, both of chaperones and proteolysis could contribute to D.L. K. M. M.A. Hum. Mol. Genet. 1999; 8: PubMed Scopus Google Scholar). 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