Experimental Gerontology 45 (2010) 611–620
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Gene expression profiling implicates OXPHOS complexes in lifespan extension of flies over-expressing a small mitochondrial chaperone, Hsp22 Hyun-Ju Kim a,1, Geneviève Morrow a,1, J. Timothy Westwood b, Sébastien Michaud a,c, Robert M. Tanguay a,* a
Laboratory of Cell and Developmental Genetics, Department of Medicine, PROTEO, Pav. C.E.-Marchand, 1030 Ave de la Médecine, Université Laval, Québec, Canada G1V 0A6 Canadian Drosophila Microarray Centre, Department of Cell and Systems Biology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6 c Unité de Recherche en Pédiatrie, Centre de recherche du CHUL, CHUQ, 2705 Laurier blvd, Québec, Canada G1V 4G2 b
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Article history: Received 29 October 2009 Received in revised form 16 December 2009 Accepted 17 December 2009 Available online 29 December 2009 Keywords: Hsp22 Aging Lifespan extension Microarray Mitochondria Heat shock proteins OXPHOS complexes
a b s t r a c t Aging is a complex process accompanied by a decreased capacity to tolerate and respond to various stresses. Heat shock proteins as part of cell defense mechanisms are up-regulated following stress. In Drosophila, the mitochondrial Hsp22 is preferentially up-regulated in aged flies. Its over-expression results in an extension of lifespan and an increased resistance to stress. Hsp22 has chaperone-like activity in vitro, but the mechanism(s) by which it increases lifespan in flies are unknown. Genome-wide analysis was performed on long-lived Hsp22+ and control flies to unveil transcriptional changes brought by Hsp22. Transcriptomes obtained at 45 days, 90% and 50% survival were then compared between them to focus more on genes up- or down-regulated in presence of higher levels of hsp22 mRNA. Hsp22+ flies display an upregulation of genes mainly related to mitochondrial energy production and protein biosynthesis, two functions normally down-regulated during aging. Interestingly, among the 26 genes up-regulated in Hsp22+ flies, 7 genes encode for mitochondrial proteins, 5 of which being involved in OXPHOS complexes. Other genes that could influence aging such as CG5002, dGCC185 and GstS1 also displayed a regulation linked to Hsp22 expression. The up-regulation of genes of the OXPHOS system in Hsp22+ flies suggest that mitochondrial homeostasis is at the center of Hsp22 beneficial effects on lifespan. ! 2010 Published by Elsevier Inc.
1. Introduction Aging is a complex process accompanied by a decreased capacity of cells to tolerate and/or respond to various forms of stress. Many theories have been postulated to explain aging (reviewed in Viña et al., 2007). In the free radical theory of aging (Harman, 1956), aging is caused by the accumulation of macromolecular damages induced by toxic reactive oxygen species (ROS). Since mitochondria are the main generators of ROS as a by-product of ATP synthesis (Aguilaniu et al., 2005), they are at the center of the free radical theory of aging (Harman, 1972; Miquel et al., 1980; reviewed in Viña et al., 2007). Although accepted by many, Harman’s theory is still highly debated (Sanz et al., 2006; Muller et al., 2007; Partridge 2009). Genome-wide experiments in different model organisms have identified aging transcriptional profiles shared across species (McCarroll et al., 2004; Smith et al., 2007). Mitochondrial genes including many components of the mitochondrial respiratory chain, ATP synthase complex and citric acid cycle as well as genes involved in ATP-dependent transport such as primary active transporters, * Corresponding author. Tel.: +1 418 656 3339; fax: +1 418 656 5036. E-mail address:
[email protected] (R.M. Tanguay). 1 These authors contributed equally to this work. 0531-5565/$ - see front matter ! 2010 Published by Elsevier Inc. doi:10.1016/j.exger.2009.12.012
ions transporters and ABC transporters are repressed during aging (McCarroll et al., 2004). Interestingly, the repression of these genes seems to be implemented early in adulthood (McCarroll et al., 2004). Several mutants displaying increased longevity in Caenorhabditis elegans and Drosophila melanogaster have been described. Interestingly in addition to having an extended longevity, many of these mutants also displayed increased thermotolerance and resistance to stress (Lin et al., 1998; Kitagawa et al., 2000; Nakamoto et al., 2000; Rogina et al., 2000; Chavous et al., 2001). Heat shock proteins (Hsps) are molecular chaperones that are coordinately expressed in response to stress and have been shown to be major determinants in the acquisition of thermotolerance and stress resistance (Feder and Hofmann, 1999; Verbeke et al., 2001). Along with the antioxidant defense system and the ubiquitin/proteasome machinery, Hsps are part of the cell defense mechanisms to prevent accumulation of protein damages (Ehrnsperger et al., 2000; Hartl and HayerHartl, 2002; Haslbeck, 2002; Walter and Buchner, 2002). Hsps are divided in subfamilies on the basis of their molecular weight and sequence homology; small Hsps (sHsps), Hsp60, Hsp70 and Hsp90/100. In D. melanogaster there are four main sHsps displaying distinct intracellular localization and developmental expression pattern (reviewed in Michaud et al., 2002). One of these, Hsp22, is localized in the mitochondrial matrix (Morrow et al., 2000) and has been shown to be preferentially up-regulated in aged
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flies (King and Tower, 1999). Interestingly, flies selected for their increased longevity display an earlier onset of hsp22 and hsp23 transcription (Kurapati et al., 2000). Recently, we have shown that overexpressing Hsp22 in motor neurons is sufficient to increase lifespan and resistance to oxidative stress by more than 30% (Morrow et al., 2004b). Most importantly, long-lived flies over-expressing Hsp22 maintain their fitness (locomotor activity) for a longer period as revealed by a negative geotaxis assay (Morrow et al., 2004b). As further support for a beneficial role of Hsp22 during aging, flies carrying a P-element preventing normal expression of Hsp22 have a reduced lifespan and resistance to stress (Morrow et al., 2004a). In vitro, Hsp22 shows chaperone-like activity on many model substrates (Morrow et al., 2006; Heikkila et al., 2007), but its function in vivo is still undetermined. We have performed a genome-wide transcriptional analysis on long-lived Hsp22 over-expressing flies and normal-lived control flies to unveil the transcriptional changes related to Hsp22 overexpression and have insights on how Hsp22 might exert its beneficial effect on the aging process in Drosophila. Transcriptomes obtained at 45 days, 90% and 50% survival were then compared between them to focus more on genes up- or down-regulated in presence of higher levels of hsp22 mRNA. This later comparison allowed us to narrow-down the genes linked to hsp22 expression to 26 up-regulated and 9 down-regulated genes. We found that flies over-expressing Hsp22 display an up-regulation of genes normally down-regulated with age. These genes are involved in mitochondrial energy production and protein biosynthesis. The up-regulation of genes of the oxidative-phosphorylation system in Hsp22+ flies suggests that mitochondrial homeostasis is at the center of Hsp22 beneficial effects on lifespan. 2. Materials and methods 2.1. Fly strains, maintenance and longevity The actin5C-Gal4 strain (Flybase ID: FBti0012293) has a P-element insertion containing the actin5C promoter in front of the gal4 coding sequence on the second chromosome, which results in ubiquitous expression of the Gal4 protein. The EP(3)3247 strain (Flybase ID: FBti0011419; Rørth, 1996) contains a UAS-including P-element inserted 643 bp upstream of the hsp22 translation initiation codon (FlyBase Genome Annotators, 2002–2003) in the 30 UTR of hsp67Bb on the third chromosome, and an activation of the UAS by Gal4 results in Hsp22 over-expression (Brand and Perrimon, 1993). Males of siblings obtained from the crossing between actin5C-Gal4/cyo males and EP(3)3247/TM3,sb females were used in the microarray experiments. In the analysis, actin5C-Gal4;EP(3)3247 flies over-expressing Hsp22 (Hsp22+) were compared with two different controls of cyo;EP(3)3247 (control 1) and actin5C-Gal4;TM3,sb (control 2) in parallel at corresponding ages. Flies were maintained at 25 "C on standard cornmeal/agar medium: 0.5% agar, 2.7% yeast bakers dried active, 1.1% sugar, 5.3% cornmeal, 0.4% (v/v) propionic acid, 1.8% (v/v) tegosept. For the longevity experiments, male flies (Hsp22+, control 1 and 2) were collected within 24 h after hatching and placed into tubes (20 flies per tube). Flies were put on fresh food and scored for survivorship every 3–4 days. Fly samples were collected at different chronological ages (day 1 and day 45) and physiological ages (95%, 90% and 50% survival). After collection, the flies were kept at !80 "C until RNA extraction. 2.2. Western blot analysis Protein extracts from whole flies were separated on 12% SDS– PAGE. The SDS polyacrylamide gel was prepared as outlined by
Thomas and Kornberg (1975) with minor modifications in the pH of the running buffer (8.5 instead of 8.8) and in the ratio of acrylamide:bis acrylamide (30:0.8 instead of 30:0.15). These conditions resulted in a better resolution for the sHsps. Following transfer on nitrocellulose membrane, Western blot was performed using a rabbit polyclonal antibody raised against Hsp22 (#36, 1/2500, Morrow et al., 2000) and a peroxidase-conjugated goat-anti-rabbit secondary antibody (1/10,000, Jackson Immuno Research Laboratories, West Grove, PA). Chemiluminescent detection was done using Western Lightning Chemiluminescence reagent (Perkin-Elmer Life Sciences, Boston, MA) according to the manufacturer’s instructions. 2.3. Microarray chips used in the study Microarrays were supplied by the CDMC (Canadian Drosophila Microarray Centre, http://www.flyarrays.com/). The Drosophila 12 K version 1 cDNA based array contains 25,640 spots corresponding to approximately 10,500 genes and various control spots. The cDNAs used to make up the array mainly came from the Berkeley DGC 1.0 and 2.0 UniGene EST set (5900 and 5530 clones respectively), B. Oliver’s and J. Andrew’s non-DGC EST clones (1078 clones), genes from submitted gene lists (572 genes), and several control genes (e.g. LacZ, GFP, Gal4). Further details on the array production can be found in Neal et al. (2003). 2.4. Total RNA extraction, cDNA labeling and microarray hybridization Approximately 200 flies were homogenized with a disposable Tissue Grinder System (VWR International) and total RNA was extracted with Trizol (Invitrogen, Gibco–BRL) as recommended by the manufacturer. RNA was prepared from three biological replicates of each experimental condition. To remove any genomic DNA contamination, total RNA was purified with the MEGAclear™ Kit (Ambion). RNA preparations for Hsp22+ and controls were done pairwise, and equal amounts of RNA (60–80 lg) were used as templates for reverse transcription according to the manufacturer (Invitrogen Life Technologies). Hsp22+ samples were directly labeled with fluorescent dye Cy5-dCTP (NEN) and control samples with Cy3-dCTP (NEN). Hybridization of cDNA probes to the microarrays was performed at 37 "C during 12–18 h using DIG easy hyb buffer (Roche). Details on RNA preparation, cDNA labeling and hybridization can be found in Neal et al. (2003). 2.5. Microarray data processing and analysis Cy5 and Cy3 fluorescence intensities were detected using ScanArray 4000 system (GSI Lumonics/Perkin-Elmer). Scanned 16-bit Tiff images were quantified by GenePix Pro 6.0 software (Axon Instruments). Further procedures for data normalization and filtration were processed by TM4 system (Saeed et al., 2003). Raw data were normalized by the iterative linear regression method as the global mode with ±2.0 standard deviation (SD) range. The spots were filtered with an average background-intensity for each of Cy5 and Cy3. Therefore in this study, the spots with a log2 ratio >0.5 (i.e. over 1.4-fold) were considered as up-regulated and the spots with a log2 ratio
