Decreased Lifespan in the Absence of Expression of the Mitochondrial Small Heat Shock Protein Hsp22 in Drosophila

Aging is a well regulated biological process involving oxidative stress and macromolecular damages. Three main pathways have been shown to influence lifespan, the insulin/insulin-like growth factor-1 pathway, the silent regulator pathway, and the target of rapamycin pathway. Among many proteins influencing lifespan, two transcription factors, FOXO and the heat shock factor, have been shown to be involved in the aging process and in small heat shock proteins (sHsps) expression following stress and during lifespan. We have recently shown that overexpressing the mitochondrial Hsp22 increases Drosophila melanogaster lifespan by 32% and resistance to oxidative stress. Here we show that flies that are not expressing this mitochondrial small Hsp22 have a 40% decrease in lifespan. These flies die faster than their matched control and display a decrease of 30% in locomotor activity compared with controls. The absence of Hsp22 also sensitizes flies to mild stress. These data support a key role of sHsps in aging and underline the importance of mitochondrial sHsps in this process. Aging is a well regulated biological process involving oxidative stress and macromolecular damages. Three main pathways have been shown to influence lifespan, the insulin/insulin-like growth factor-1 pathway, the silent regulator pathway, and the target of rapamycin pathway. Among many proteins influencing lifespan, two transcription factors, FOXO and the heat shock factor, have been shown to be involved in the aging process and in small heat shock proteins (sHsps) expression following stress and during lifespan. We have recently shown that overexpressing the mitochondrial Hsp22 increases Drosophila melanogaster lifespan by 32% and resistance to oxidative stress. Here we show that flies that are not expressing this mitochondrial small Hsp22 have a 40% decrease in lifespan. These flies die faster than their matched control and display a decrease of 30% in locomotor activity compared with controls. The absence of Hsp22 also sensitizes flies to mild stress. These data support a key role of sHsps in aging and underline the importance of mitochondrial sHsps in this process. Aging is a complex biological process involving precisely regulated changes in gene expression (1Pletcher S.D. Macdonald S.J. Marguerie R. Certa U. Stearns S.C. Goldstein D.B. Partridge L. Curr. Biol. 2002; 12: 712-723Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Three main pathways have been proposed to act in lifespan determination: the insulin/IGF-1 1The abbreviations used are: IGF-1, insulin-like growth factor-1; SIR, silent regulator; TOR, target of rapamycin; SOD, superoxide dismutase; Hsp, heat shock protein; sHsp, small Hsp; HSF, heat shock factor; HSE, heat shock element; RNAi, RNA interference. signaling pathway (Refs. 2Kenyon C. Chang J. Gensch E. 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Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1765) Google Scholar) and the target of rapamycin (TOR) signaling pathway (9Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar). Mutations that inhibit the insulin/IGF-1 pathway have been shown to increase lifespan of both Caenorhabditis elegans (2Kenyon C. Chang J. Gensch E. Rudner A. Tabtiang R. Nature. 1993; 366: 461-464Crossref PubMed Scopus (2500) Google Scholar, 3Kimurak D.K. Tissenbaum H.A. Liu Y. Ruvkun G. Science. 1997; 277: 942-946Crossref PubMed Scopus (1725) Google Scholar) and Drosophila melanogaster (4Clancy D.J. Gems D. Harshman L.G. Oldham S. Stocker H. Hafen E. Leevers S.J. Partridge L. Science. 2001; 292: 104-106Crossref PubMed Scopus (1138) Google Scholar, 5Tatar M. Kopelman A. Epstein D. Tu M.P. Yin C.M. Garofalo R.S. 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Among these, overexpression of SOD1 and SOD2 has been reported to be beneficial in D. melanogaster (15Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (695) Google Scholar, 16Sun J. Tower J. Mol. Cell. Biol. 1999; 19: 216-228Crossref PubMed Scopus (391) Google Scholar, 17Sun J. Folk D. Bradley T.J. Tower J. Genetics. 2002; 161: 661-672Crossref PubMed Google Scholar). The overexpression of members of the small HSP family has also been shown to extend longevity in D. melanogaster (18Morrow G. Samson M. Michaud S. Tanguay R.M. FASEB J. 2004; 18: 598-599Crossref PubMed Scopus (254) Google Scholar) and in C. elegans (19Walker G.A. White T.M. McColl G. Jenkins N.L. Babich S. Candido E.P. Johnson T.E. Lithgow G.J. J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B281-B287Crossref PubMed Scopus (90) Google Scholar). The SIR pathway also involves modulation of gene expression but by making large chromosomal regions transcriptionally inactive. Hence, overexpression of SIR proteins extends lifespan by making life-shortening genes inaccessible for the transcriptional machinery (7Kennedy B.K. Gotta M. Sinclair D.A. Mills K. McNabb D.S. Murthy M. Pak S.M. Laroche T. Gasser S.M. Guarente L. Cell. 1997; 89: 381-391Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 8Kaeberlein M. McVeu M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1765) Google Scholar). This pathway has been found to operate in Saccharomyces cerevisiae and in C. elegans and has been shown to promote survival particularly under conditions of scarcity (20Hekimi S. Guarente L. Science. 2003; 299: 1351-1354Crossref PubMed Scopus (377) Google Scholar). Recently a link between the insulin/IGF-1 and the SIR pathways has been established by demonstrating that mammalian SIRT1 deacetylase controls the cellular response to stress by regulating the transcriptional activity of FOXO3 (21Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science. 2004; 303: 2011-2015Crossref PubMed Scopus (2644) Google Scholar) and FOXO4 (22van der Horst A. Tertoolen L.G.J. de Vries-Smits L.M.M. Frye R.A. Medema R.H. Burgering B.M.T. J. Biol. Chem. 2004; 279: 28873-28879Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), two of the four evolutionarily conserved FOXO family members in mammals. Finally, the TOR signaling pathway is involved in cell growth and proliferation and is conserved from Drosophila to human (reviewed in Refs. 23Harris T.E. Lawrence Jr., J.C. Science's STKE. 2003; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/212/re15)PubMed Google Scholar and 24Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1064) Google Scholar). This pathway mediates cell growth in response to nutrient availability by inducing ribosomal protein expression through histone deacetylation (25Rohde J.R. Cardenas M.E. Mol. Cell. Biol. 2003; 23: 629-635Crossref PubMed Scopus (131) Google Scholar). Recently, Kapahi et al. (9Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar) have shown that inhibition of the TOR pathway in D. melanogaster extends lifespan in a manner similar to dietary restriction. Multiple evidence suggests that Hsps, which are molecular chaperones, are also involved in the aging process (Refs. 18Morrow G. Samson M. Michaud S. Tanguay R.M. FASEB J. 2004; 18: 598-599Crossref PubMed Scopus (254) Google Scholar and 26Tatar M. Khazaeli A.A. Curtsinger J.W. Nature. 1997; 390: 30Crossref PubMed Scopus (271) Google Scholar, 27Kurapati R. Passananti H.B. Rose M.R. Tower J. J. Gerontol. A Biol. Sci. Med. Sci. 2000; 55: B552-B559Crossref PubMed Scopus (85) Google Scholar, 28Walker G.A. Lithgow G.J. Aging Cell. 2003; 2: 131-139Crossref PubMed Scopus (242) Google Scholar and reviewed in Refs. 29Minois N. Biogerontology. 2000; 1: 15-29Crossref PubMed Scopus (163) Google Scholar, 30Munoz M.J. Mech. Ageing Dev. 2003; 124: 43-48Crossref PubMed Scopus (46) Google Scholar, 31Morrow G. Tanguay R.M. Semin. Cell Dev. Biol. 2003; 14: 291-299Crossref PubMed Scopus (78) Google Scholar). Expression of sHsps have been shown to be controlled by Daf-16 (14Murphy C.T. McCarroll S.A. Barmann C.I. Fraser A. Kamath R.S. Ahringer J. Li H. Kenyon C. Nature. 2003; 424: 277-283Crossref PubMed Scopus (1705) Google Scholar) and the insulin/IGF-1 pathway in C. elegans (28Walker G.A. Lithgow G.J. Aging Cell. 2003; 2: 131-139Crossref PubMed Scopus (242) Google Scholar). In the same organism, a link between the heat shock factor (HSF) and Daf-16 has been demonstrated in both the insulin/IGF-1 signaling pathway and the heat shock response (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar). Indeed, Hsu et al. (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar) have shown that the presence of HSF is required in daf-2(–) mutants to display their increased longevity phenotype. This study has also revealed the importance of a functional HSF in determination of aging and has identified shsp genes as common targets for HSF and Daf-16 activity. We have recently shown that overexpressing a mitochondrial sHsp (Hsp22) in all cells or specifically in motorneurons using the GAL4/UAS binary system increases D. melanogaster mean lifespan by up to 32% (18Morrow G. Samson M. Michaud S. Tanguay R.M. FASEB J. 2004; 18: 598-599Crossref PubMed Scopus (254) Google Scholar). Moreover, flies displaying an increased longevity maintain their locomotor activity for a longer period and are also more resistant to various stresses. To confirm that this mitochondrial sHsp is important in the aging process of D. melanogaster, we have now examined the longevity phenotype of a strain obtained by P-element jump-out, which does not express Hsp22 in response to stress nor during aging. Our results show that these flies display a decrease in mean lifespan of 40% and are sensitive to stress. These results confirm the key role of mitochondrial sHsps in aging determination. Drosophila Strains, Maintenance, and Longevity Assay—EP(3) 3583-11 (Flybase ID: FBti0023680) and EP(3)3583-18 flies have been obtained by jump-out in a study on P-element local transposition in the male germ line cells of D. melanogaster (33Timakov B. Liu X. Turgut I. Zhang P. Genetics. 2002; 160: 1011-1022PubMed Google Scholar). The genetic background of the two strains is the same except for the newly inserted P-element, which is located at –57 bp and –350 bp of the hsp22 transcription starting site in EP(3)3583-11 and EP(3)3583-18 flies, respectively (Ref. 33Timakov B. Liu X. Turgut I. Zhang P. Genetics. 2002; 160: 1011-1022PubMed Google Scholar; see Fig. 1). Flies were maintained at 25 °C on standard cornmeal/agar medium. Heat shock was performed by incubation for 1 h at 35 °C, the optimal temperature for induction of sHsps, followed by a recovery of 2 h at 25 °C. For longevity experiments, cohorts of male flies (15 per vial) were transferred to fresh medium every 3–4 days and scored for survivors every 2 days. The starting population for each genotype was 210. For analysis, the premortality phase (less than 10% mortality) and the mean lifespan (50% survival) were calculated. For thermal stress resistance assays, 2-day-old flies were kept at 30 or 37 °C until death. PCR Analysis—Genomic DNA was prepared from flies of both genotypes as described in Huang et al. (34Huang A.M. Rehm E.J. Rubin G.M. Sullivan W. Ashburner M. Hawley R.S. Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 429-437Google Scholar). PCR reactions were performed with Pp31 and Pg-A5 primers (200 ng; Ref. 33Timakov B. Liu X. Turgut I. Zhang P. Genetics. 2002; 160: 1011-1022PubMed Google Scholar) to which have been added 100 ng of genomic DNA, 5 μl of 10× buffer number 3 (50 mm Tris-HCl, pH 9.2, 160 mm (NH4)2SO4, 22.5 mm MgCl2, 20% Me2SO and 1% Tween 20), 10 μl of 5 mm dNTP and 2 μl of Taq polymerase (Amersham Biosciences). The reaction mixtures were covered with mineral oil and incubated for 5 min at 95 °C, followed by 30 cycles of 1 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C and a final elongation of 5 min at 72 °C. Negative Geotaxis Assay—The negative geotaxis assay was conducted as described in Morrow et al. (18Morrow G. Samson M. Michaud S. Tanguay R.M. FASEB J. 2004; 18: 598-599Crossref PubMed Scopus (254) Google Scholar). Briefly, flies were transferred in clean empty vials (16 × 125 mm) and allowed to adapt to their environment for 8 s. Flies were than tapped gently to the bottom of the tube, and flies having climbed higher than 7 cm in 8 s were scored. The assay was conducted three times on 105 flies of each cohort. The results are expressed in percentage of flies having climbed above 7 cm in 8 s. Protein Analysis—Protein extracts from flies of each genotype were separated on 12% SDS-PAGE as described in Morrow et al. (35Morrow G. Inaguma Y. Kato K. Tanguay R.M. J. Biol. Chem. 2000; 275: 31204-31210Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Following transfer onto nitrocellulose membranes, Western blots were performed using antibodies specific to Hsp22 (1/5,000; Ref. 35Morrow G. Inaguma Y. Kato K. Tanguay R.M. J. Biol. Chem. 2000; 275: 31204-31210Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), Hsp23 (1/100; Ref. 36Marin R. Valet J.P. Tanguay R.M. Dev. Genet. 1993; 14: 69-77Crossref PubMed Scopus (56) Google Scholar), Hsp26 (1/100; Ref. 36Marin R. Valet J.P. Tanguay R.M. Dev. Genet. 1993; 14: 69-77Crossref PubMed Scopus (56) Google Scholar), Hsp27 (1/100; Ref. 36Marin R. Valet J.P. Tanguay R.M. Dev. Genet. 1993; 14: 69-77Crossref PubMed Scopus (56) Google Scholar), Hsp60 (1/5,000; Ref. 37Laplante A.F. Moulin V. Auger F.A. Landry J. Li H. Morrow G. Tanguay R.M. Germain L. J. Histochem. Cytochem. 1998; 46: 1291-1301Crossref PubMed Scopus (152) Google Scholar), Hsp70 (1/5,000; Ref. 38Tanguay R.M. Wu Y. Khandjian E.W. Dev. Genet. 1993; 14: 112-118Crossref PubMed Scopus (141) Google Scholar) or Hsp83 (1/5,000; Ref. 39Carbajal M.E. Valet J.P. Charest P.M. Tanguay R.M. Eur. J. Cell Biol. 1990; 52: 147-156PubMed Google Scholar) and secondary antibodies coupled to peroxidase (1/20,000, Jackson ImmunoResearch Laboratories). Chemiluminescent detection was done with Western Lightning Chemiluminescence Reagent as described by the manufacturer (PerkinElmer Life Sciences). The EP(3)3583-11 and EP(3)3583-18 flies were obtained from the same starting strain in a study on P-element local transposition (33Timakov B. Liu X. Turgut I. Zhang P. Genetics. 2002; 160: 1011-1022PubMed Google Scholar). The location of the newly inserted P-element was analyzed by PCR amplification using primers specific to the P-element and hsp22 coding sequence. In EP(3)3583-18 flies, the P-element is inserted at position -350 from the hsp22 transcription starting site, and as expected, a PCR amplification with the two primers gives a product at 904 bp, while in EP(3)3583-11 flies, the P-element is inserted at position –57 and the PCR product has 611 bp (Fig. 1B). Heat shock elements (HSEs) are DNA sequences recognized by HSF, the transcription factor responsible for Hsps expression following stress. Studies on hsp22 promoter have revealed that three functional HSEs are required for the proper expression of Hsp22 following stress (40Klemenz R. Gehring W. Mol. Cell. Biol. 1986; 6: PubMed Scopus Google Scholar) and during aging V. Tower J. Dev. Biol. 1999; PubMed Scopus Google Scholar). the P-element in EP(3)3583-11 flies is located between the three HSEs and the on hsp22 promoter (Fig. we performed a heat shock on flies to their of Hsp22 shown in Fig. EP(3)3583-18 flies, which have the of the express a large of Hsp22 following heat while of Hsp22 are in EP(3)3583-11 In during aging EP(3)3583-11 flies show expression of Hsp22 as compared with EP(3)3583-18 The expression of Hsps such as and Hsp83 was the same in the two strains following heat shock and during aging a in HSF activity in the two strains (Fig. The between HSEs and the in the hsp22 promoter of EP(3)3583-11 flies for the absence of Hsp22 expression during aging and following stress. Indeed, to the presence of the P-element, is that the promoter is not optimal or more such as factor, RNA polymerase and HSF to DNA L.S. K. M. Genes Dev. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar) with each Jr., J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). To the key role of Hsp22 in the aging longevity were conducted on EP(3)3583-18 and EP(3)3583-11 EP(3)3583-18 flies have a mean lifespan of EP(3)3583-11 flies that not express Hsp22 have a mean lifespan of days (Fig. which a decrease of In to a EP(3)3583-11 flies in the phase 3 compared with for EP(3)3583-18 Flies not expressing Hsp22 also have a locomotor activity of as by a negative geotaxis on flies compared with for EP(3)3583-18 These results the of Hsp22 in the aging process. The resistance to heat of flies not expressing Hsp22 was also EP(3)3583-11 flies have a mean lifespan of days at 30 °C compared with days for EP(3)3583-18 flies, which a decrease of (Fig. the survival of EP(3)3583-11 flies was not from the survival of EP(3)3583-18 flies at 37 °C h Fig. The results that Hsp22 is a key in the aging process as absence flies lifespan by To this is the that such an is for an In C. shsp has been shown to lifespan of by than (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar). The obtained in the absence of Hsp22 be to (35Morrow G. Inaguma Y. Kato K. Tanguay R.M. J. Biol. Chem. 2000; 275: 31204-31210Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Indeed, are to be sensitive to stress and to aging (reviewed in Ref. R.M. Aging Cell. 2004; PubMed Scopus Google Scholar), and is that the presence of Hsp22 in this to maintain mitochondrial and This be by activity on A. J. J. and R. M. for The of Hsp22 for is also flies are to mild thermal stress such as 30 °C, but we not of the absence of Hsp22 on resistance to 37 °C. This is to the that this stress involves a of to the have been on the aging process Indeed, that specific cell the longevity of the In C. elegans daf-2(–) longevity was to by overexpressing in while were changes was expressed in or cells Ruvkun G. Science. 2000; PubMed Scopus Google Scholar). In D. melanogaster, overexpressing dFOXO in the was to increase lifespan and resistance to M.E. M. Hafen E. Leevers S.J. Partridge L. Science. 2004; PubMed Scopus Google Scholar). et al. D.S. Gersham B. Tu M.-P. Palmer M. Tatar M. Nature. 2004; 429: 562-566Crossref PubMed Scopus (757) Google Scholar) have demonstrated that the of dFOXO in the expression of an insulin-like in of the TOR signaling pathway in the was also to increase lifespan (9Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar). Moreover, HSF has been to have a role in the of longevity in C. elegans (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar, J.F. Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar), and sHsps have been shown to be targets of HSF and Daf-16 (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar). A key role of sHsps in has also been Indeed, shsp the of in C. while for the not have (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar). The the that sHsps are key in the aging process and that mitochondrial sHsps are particularly important as such on lifespan have been obtained with sHsps (32Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar). the of life-promoting genes as important as their expression in specific cell in the determination of these results that sHsps are targets to aging and the of

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