Doxycycline-regulated over-expression of hsp22 has negative effects on stress resistance and life span in adult Drosophilamelanogaster

Mechanisms of Ageing and Development 125 (2004) 651–663 www.elsevier.com/locate/mechagedev

Doxycycline-regulated over-expression of hsp22 has negative effects on stress resistance and life span in adult Drosophila melanogaster Deepak Bhole1, Michael J. Allikian2, John Tower* Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089 1340, USA Received 22 March 2004; received in revised form 8 August 2004; accepted 9 August 2004 Available online 25 September 2004

Abstract Drosophila hsp22 is a member of the small heat shock proteins family (shsps). The hsp22 is expressed in a tissue-general pattern in response to heat stress and during normal aging, and localizes to the mitochondrial matrix, however, its exact function and targets are unknown. Hsp22 was found to be rapidly induced in response to oxidative stress, indicating that hsp22 is also an oxidative stress response gene. To assay for effects of hsp22, a ubiquitous pattern of hsp22 gene expression was generated in young flies using the ‘‘tet-on’’ doxycycline-regulated promoter system. The hsp22 over-expression made flies more sensitive to heat and oxidative stress, while resistance to coumarin poisoning was not affected. Life span was also reduced, particularly at higher culture temperatures. Members of other hsp families have been shown to feedback-inhibit their own expression by interacting with the heat shock transcription factor (HSF) and preventing binding to the HSEs. Induction of hsp22:lacZ and hsp70:lacZ reporter transgenes in response to acute stress was normal in the presence of hsp22 protein over-expression and in old flies, indicating that the negative effects of hsp22 are downstream of the HSF/HSE pathway and the transcriptional heat shock response. The data demonstrate a specific over-expression phenotype for hsp22 and suggest that hsp22 interacts with heat and oxidative stress resistance pathways. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Aging; ROS; Oxidative stress; Oxidative damage; Mitochondria; Chaperone

1. Introduction In most organisms, heat shock proteins (hsps) are induced in response to protein damage caused by heat and other stresses (Ananthan et al., 1986; Lindquist and Craig, 1988; Morimoto et al., 1997; Parsell and Lindquist, 1993). This heat shock response can result in stress tolerance and protection from stress-induced molecular damage. The hsps have been divided into families based upon molecular weight and sequence homology. The Hsp70, Hsp90 and * Corresponding author. Tel.: +1 213 740 5384; fax: +1 213 740 8631. E-mail address: [email protected] (J. Tower). 1 Present address: Department of Anesthesia, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA. 2 Present address: Department of Medicine, University of Chicago, 5841 S. Maryland, Chicago, IL 60637, USA.

Hsp60 families are molecular chaperones that function in protein folding or refolding and/or facilitate protein translocation across cellular membranes. Their activity is ATP-dependent and sometimes involves co-chaperones. The small heat shock proteins (shsps) are a more diverse group related to each other and to small hsps from other organisms by a conserved a-crystallin domain (Arrigo and Landry, 1994; Haslbeck and Buchner, 2002; Michaud et al., 2002; Van Montfort et al., 2001). Several shsps have been shown to have chaperone-like activity in vitro, however, they may be relatively inefficient chaperones. The mechanism of Drosophila heat shock gene induction by heat stress has been studied in detail (Lis and Wu, 1993; Yost et al., 1990). Heat stress causes trimerization and activation of the constitutively expressed heat shock transcription factor (HSF), which is required for heat shock gene induction (Jedlicka et al., 1997; Westwood et al., 1991).

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HSF binds to the heat shock response elements (HSEs), which are evolutionarily conserved, well-studied promoter elements essential for transcriptional induction during heat stress (Amin et al., 1988; Dudler and Travers, 1984; Holmgren et al., 1981; Topol et al., 1985; Xiao and Lis, 1988). HSF binding to the HSEs results in high-level transcription of the heat shock genes. Deletion and point mutation studies of hsp22 and hsp70 transgenes in Drosophila reveals that the HSEs are also required for the transcriptional induction of these genes in response to aging and in response to oxidative stress (King and Tower, 1999; Wheeler et al., 1999). Several members of the shsp class, Drosophila hsp27 and human hsp27 and aB-crystallin, have each been found to confer increased resistance to heat and oxidative stress when expressed in cultured cells (Arrigo and Landry, 1994; Huot et al., 1991; Landry et al., 1989; Mehlen et al., 1995). Mammalian Hsp27 negatively regulates cell death by interacting with cytochrome c (Bruey et al., 2000). Taken together, these data suggest that shsps generally have beneficial effects, and function in protecting cells from heat and oxidative stress. Several lines of evidence link hsps and the heat shock response with aging and life span regulation in Drosophila and other organisms. In Drosophila, the high level expression of hsp70 in response to a heat stress is prolonged in old flies (Niedzwiecki et al., 1991). This may be due to higher levels of denatured proteins in old flies (Niedzwiecki and Fleming, 1990) and/or increased hsp70 protein stability in old flies (Wheeler et al., 1999). A brief heat shock of 4day-old Drosophila populations causes a period of decreased mortality rates (Khazaeli et al., 1997; Khazaeli et al., 1995). This effect is larger in flies with extras copies of the hsp70 gene thereby showing a beneficial effect of hsp70 on adult survival (Tatar et al., 1997). In contrast, over-expression of hsp70 during development can have negative effects (Feder et al., 1992; Krebs and Feder, 1997a; Krebs and Feder, 1997b; Krebs and Feder, 1998). Genetic selection for increased life span in Drosophila correlates with increased expression of hsp22 and hsp23 in young adults (Kurapati et al., 2000). In Caenorhabditis elegans, mutations of the Daf2/Insulin-like signaling pathway that extend life span also increase hsp gene expression and heat stress resistance, and hsp over-expression can confer extended life span (Hsu et al., 2003; Lithgow et al., 1994; Lithgow et al., 1995; Murphy et al., 2003; Walker and Lithgow, 2003; Walker et al., 2001). Finally, aging is associated with tissue-specific induction of hsps in multiple organisms. The expression of many of the Drosophila hsps is upregulated at the RNA level during normal aging (King and Tower, 1999; Landis et al., 2004; Pletcher et al., 2002; Wheeler et al., 1995; Wheeler et al., 1999; Zou et al., 2000), as well as at the protein level. Hsp22 protein levels increase >150-fold in old flies, and this is among the largest agingrelated increases in gene expression known for eukaryotes (King and Tower, 1999). Hsp22 induction during aging was

observed in all tissues, with particularly high-level expression in nervous and eye tissue. Hsp70 is up-regulated several fold during aging, preferentially in muscle tissue (Wheeler et al., 1995; Wheeler et al., 1999). The time course and level of hsp70 expression is increased by null mutations in either the Sod (cytoplasmic Cu/ZnSOD) or catalase antioxidant genes, suggesting that the up-regulation of hsp70 during aging is at least in part due to oxidative stress. For both hsp22 and hsp70 the increased expression during aging appears to involve both transcriptional and posttranscriptional regulation. Genome-wide assays of gene expression patterns in mammalian muscle, nervous and skin tissues have identified a similar pattern of hsp gene up-regulation during aging that correlates with life span: a tissue-specific up-regulation of a subset of hsps, in particular members of the hsp70 and acrystallin classes (Lee et al., 1999; Lee et al., 2000; Ly et al., 2000). Genome-wide analysis of RNA levels in aging Drosophila reveals that the pattern of gene expression is similar to that produced by oxidative stress (Landis et al., 2004; Pletcher et al., 2002; Zou et al., 2000). The induction of hsps during aging may be a response to oxidative protein damage. In many organisms aging has been found to be associated with the accumulation of ‘‘abnormal’’ proteins that include conformationally altered and inactive enzymes and proteins that are oxidatively damaged (Finch, 1990; Finkel and Holbrook, 2000; Gershon and Gershon, 1970; Stadtman, 1992). It has long been hypothesized that oxidative damage may be a primary cause of aging, and certain data support this hypothesis (Finkel and Holbrook, 2000; Harman, 1956). For example, in Drosophila over-expression of the key antioxidant enzyme superoxide dismutase can significantly extend life span (Parkes et al., 1998; Sun et al., 2002; Sun and Tower, 1999). In humans, a strong correlation has been found between aging-associated neurodegenerative diseases, oxidative damage and the accumulation of abnormal and aggregated proteins. For example, both Alzheimer’s and Parkinson’s diseases involve the accumulation and aggregation of altered protein forms, and have oxidative damage implicated in their etiology (Dunnet and Bjorklund, 1999; Giasson et al., 2000; Selkoe, 1999). In an attempt to generate an hsp22 phenotype, transgenic Drosophila lines were created where the doxycycline (DOX)-regulated ‘‘tet-on’’ promoter system (Bieschke et al., 1998) was used to cause conditional over-expression of hsp22 in young flies, and effects on stress resistance and life span were assayed.

2. Materials and methods 2.1. Drosophila stocks All transgenic Drosophila stocks were generated by P-element based germline transformation (Rubin and

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Spradling, 1982). Specifically, a microinjection technique described by Park and Lim (Park and Lim, 1995) was utilized. Other Drosophila strains used are as described (Lindsley and Zimm, 1992) (Flybase http://flybase.bio.indiana.edu). 2.2. Drosophila culture and life span assays

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2.4. Northern analysis Adult, age-synchronized flies were generated as described above, and maintained +/
DOX for 7 days with transfer to fresh medium every 2 days. RNA extractions were done using Trizol reagent (GibcoBRL) and amount of RNA was quantitated by UV spectrophotometer. Approximately 5–10 mg of RNA denatured using 2  RNA loading buffer (2  TBE, 13% Ficoll, 7 M urea, bromophenol blue) and was loaded onto a native 1.0% agarose gel. The gel was run at 100 V for approximately 1 h and was blotted overnight. RNA markers were loaded for determination of the size of bands (Gibco BRL). The RNA was fixed to the Genescreen nylon membrane (Dupont) using a UV crosslinker (Stratagene). Probes were 32P labeled by random primer labeling using the Prime-It II kit (Stratagene). To determine fold increases/decreases, Northern blots were exposed onto a phosphor screen and results were analyzed using a phosphorimager (ImageQuaNT, Molecular Dynamics).

Drosophila melanogaster were cultured on standard agar/molasses/corn meal/yeast media (Ashburner, 1989). To obtain adult flies of defined age, the indicated hsp22 transgenic lines and Oregon R control strain were crossed to the rtTA(3)E2 stock (Bieschke et al., 1998) and cultured at 25 8C in urine specimen bottles. Prior to eclosion of the majority of pupae, bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. The majority of the males will have mated during this time. The males only were then removed and were designated 1-day-old, and were maintained at 25 8C at 40/ vial in culture vials with food. For the indicated experiments of Fig. 4 the flies were maintained at 29 8C. At 4 days of age the males were split into control and experimental groups of 200 males each, with experimentals (+DOX) placed on culture media supplemented with 250 mg/ml DOX and 60 mg/ml ampicillin. The controls were placed on culture media supplemented with 60 mg/ml ampicillin. These flies were then transferred to fresh food vials every other day. Dead flies were counted at each passage, and the number of vials was progressively reduced to maintain approximately 40 flies/ vial. To calculate mean life spans for the experimental (+DOX) and control (
DOX) cohorts, each fly’s life span was tabulated and their life spans were averaged and the SEM calculated. Means were compared using unpaired, two-sided t-tests.

Adult, age-synchronized flies were generated as described above, and maintained +/
DOX for 7 days with transfer to fresh medium every 2 days. Total adult Drosophila protein was analyzed by Western blot as previously described (King and Tower, 1999). Protein equal to approximately 0.5 flies was loaded per lane. Following transfer to Hybond nitrocellulose membranes (Amersham) the blots were incubated with polyclonal antisera specific for hsp22 (King and Tower, 1999). Bands were detected using the ECL Western detection system (Amersham) and quantitated by phosphorimager (ImageQuaNT, Molecular Dynamics).

2.3. DNA constructs

2.6. b-Galactosidase assays

Constructs for microinjection were created using a derivative of the 7T40 vector (Bieschke et al., 1998) called ‘‘USC 1.0’’ that has unique PstI and EcoRI sites downstream of the tet-on promoter (Allikian et al., 2002). The hsp22 sense construct was made as follows: The hsp22 coding region plus putative polyadenylation signal sequence was amplified using PCR from a plasmid containing the wild type hsp22 gene (King and Tower, 1999), using the following primers. Locations of primers are given relative to the A in the start codon ATG which is designated as +1. hsp22-s(50 ) (
30 to
13): 50 -AGC TCT GCA GGG AAA AAC CCA AGT TACC-30 hsp22-s(30 ) (+729 to +746): 50 -AGC TGA ATT CTG TGT TCC CAA CCC AAC C-30 The resulting PCR product was cloned into the EcoRV site of pBS KS+ (Stratagene) and the insert was then liberated with PstI and EcoRI. The fragment was gel purified and ligated into the PstI and EcoRI sites of USC1.0.

Adult, age-synchronized flies were generated as described above, and maintained +/
DOX with transfer to fresh medium every 2 days until the indicated ages. bGalactosidase enzymatic activity was quantitated in tissue extracts using published procedures (Simon and Lis, 1987; Wheeler et al., 1999). Assays were performed under conditions in which the reaction was linear with regard to time and amount of extract. Data are presented as the means  standard deviation for triplicate samples. Means were compared using unpaired, two-sided t-tests.

2.5. Western blot analysis

2.7. Assay of paraquat toxicity The protocol for paraquat feeding was adapted from published procedures (Humphreys et al., 1996). Adult, agesynchronized flies were generated as described above, and groups of 50 males (10 flies per vial) were maintained on +DOX and
DOX media for 7 days, with transfer to fresh

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medium every 2 days. These males were exposed for 48 h at 25 8C in vials containing one disk of Whatman 3M filter paper saturated with 250 ml of an aqueous solution of paraquat (methyl viologen, Sigma) at the concentrations indicated, in 1% sucrose. Survival was scored as the percent of total flies surviving after a 48 h exposure. Experiments were repeated with essentially identical results. 2.8. Assay of coumarin toxicity Adult, age-synchronized flies were generated as described above, and maintained +/
DOX for 7 days with transfer to fresh medium every 2 days. The males were then transferred to food vials containing the indicated final concentrations of coumarin. These were maintained at 25 8C and serially transferred to fresh food vials containing coumarin every other day. To obtain survival curves, the number of surviving individuals was determined at the time of transfer for each genotype and expressed as a percentage of total sample size. Experiments were repeated with essentially identical results. 2.9. Heat stress test Adult, age-synchronized flies were generated as described above, and maintained +/
DOX for 7 days with transfer to fresh medium every 2 days. The vials were then transferred to 36 8C and the flies were serially transferred to fresh food vials every hour. The number of surviving individuals was determined at the time of each transfer for each genotype to obtain the mean survival time. Experiments were repeated with essentially identical results. 2.10. Statistical analyses For all the life span assays, mean life span and SEM was calculated from tabular survival data. Statistical significance of differences in mean life span was calculated for each experiment using unpaired two-sided t-tests.

3. Results 3.1. Doxycycline-regulated over-expression of hsp22 During heat stress and normal aging, hsp22 appears to be induced in all tissues of the adult fly (King and Tower, 1999). In order to reproduce this ubiquitous expression pattern in the young fly, a conditional system was chosen that yields high-level, tissue-general expression of various transgenes. In the Drosophila ‘‘tet-on’’ system (Bieschke et al., 1998), the rtTA transcriptional activator protein is constitutively expressed in all tissues using the cytoplasmic actin Actin5C promoter (Fig. 1A). One transgenic insertion, called rtTA(3)E2, yields low levels of target construct expression in the absence of DOX, and up to 120-fold induction of

Fig. 1. The tet-on system and conditional hsp22 over-expression. (A) The rtTA transgene encoding the rtTA transcriptional activator protein. rtTA protein is a hybrid of the ‘‘reverse’’ tetracycline repressor regulatory domain and the transcriptional activation domain of the herpes simplex virus protein VP16. (B) hsp22 over-expression target construct tet22. (C) and (D) Northern analyses of each tet22 transgenic line cultured in presence and absence of DOX, as indicated. Ribosomal protein 49 gene (Rp49) was used as loading control. The experiments utilized independently cultured flies and were separated in time by several weeks. (E) Western analysis of selected tet22 lines. Non-transgenic w1118 flies subjected to a very mild heat stress were used as a marker.

various transgenes in the presence of DOX. A target construct, called tet22, was generated with the hsp22 coding sequences and polyadenlylation signal sequences cloned downstream of the tet-on synthetic promoter (Fig. 1B). In the presence of doxycycline (DOX), rtTA will bind to tet operator sequences in the tet-on synthetic promoter and drive expression in all tissues of the fly. DOX itself and 100-

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fold over-expression of E. coli b-galactosidase were previously found to have no detectable effects on Drosophila viability or life span (Bieschke et al., 1998). The site of insertion of transgenes on the chromosome can have significant effects on the magnitude of expression of the tet-on system (‘‘position effects’’) (Bieschke et al., 1998). For this reason, five transgenic lines, tet22(X)20A, tet22(2)23, tet22(3)7, tet22(3)22A and tet22(2)26 were generated containing the tet22 target construct. Each of these transgenic lines was crossed to rtTA to generate progeny containing one copy of each construct. These double transgenic progeny were then cultured +/
DOX for 7 days as adults, and RNA was isolated and Northern analysis was carried out to assay for hsp22 over-expression. Two independent Northern analyses of independently cultured flies gave identical results (Fig. 1C and D). Lines tet22(X)20A, tet22(2)23, tet22(3)22 and tet22(2)26 yielded dramatic increases in hsp22 RNA levels in presence of DOX. In contrast line tet22(3)7 showed a small amount of transcript that was unchanged in the presence or absence of DOX, presumably due to position effects, and was therefore used as one control in the experiments presented below. Western blot analysis using an antisera specific for hsp22 confirmed that hsp22 protein is expressed in correspondence with hsp22 RNA levels (Fig. 1E). All subsequent experiments utilize two experimental strains where hsp22 is overexpressed in the presence of DOX, tet22(X)20A and tet22(2)23, and two control strains where there is no induction of hsp22 in the presence of DOX, tet22(3)7 and Oregon R wild type. The induction of hsp22 in young flies achieved with the tet-on system at this concentration of DOX (100-fold) is less than that normally observed in heat stressed or old flies (150–300-fold (King and Tower, 1999)), and is therefore within the normal physiological range. A key strength of the conditional system is the ability to control for differences in genetic background between strains. Independent transgenic strains containing the same construct will have unavoidable small differences in genetic background. These differences cause variability in life span and stress resistance (Kaiser et al., 1997; Tatar, 1999). Therefore, it was expected that the independent hsp22 overexpression strains would vary in their starting life spans and stress resistance characteristics, and this is observed below. However, the tet-on system allows for the identification of effects specific to transgene over-expression by analyzing any change in life span or stress resistance caused by DOX administration and transgene over-expression. 3.2. Over-expression of hsp22 decreases resistance to heat stress The two control strains and two experimental strains were cultured +/
DOX as young adults, and then subjected to heat shock (Fig. 2). As expected, the lines vary slightly in their starting (
DOX) sensitivity to heat stress. In lines tet22(X)20A and tet22(2)23 hsp22 DOX administration and

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Fig. 2. Survival in response to heat stress. The indicated tet22 transgenic lines were maintained in the presence and absence of DOX and subjected to 36 8C heat stress. Data are presented as mean survival time +/
S.D., and were compared using unpaired, two-sided t-tests.

hsp22 over-expression caused a significant decrease in survival, while in the control strains DOX had no effect. Therefore the ability of the young fly to survive heat stress is reduced by hsp22 over-expression. 3.3. Over-expression of hsp22 decreases resistance to paraquat In vivo paraquat undergoes an NADH-dependent reduction to paraquat radical that reacts rapidly with O2 to generate the reactive oxygen species O2
. The two control strains and two experimental strains were cultured +/
DOX as young adults, and then groups of each were fed various concentrations of paraquat (Fig. 3). As expected, the lines vary slightly in their starting (
DOX) sensitivity to paraquat. In lines tet22(X)20A and tet22(2)23, hsp22 over-expression caused a significant decrease in survival in the presence of increasing concentrations of paraquat, while in the two control strains DOX treatment had no effect. Therefore, the ability of the young fly to survive paraquat is reduced by hsp22 over-expression. 3.4. Over-expression of hsp22 decreases life span The effect of hsp22 over-expression on life span was assayed by measuring the mean life spans of agesynchronized cohorts of flies, with and without induction of the hsp22 transgene by DOX. The experiment was performed at both 25 8C and at 29 8C, culture temperatures typically used in the laboratory. 29 8C is a viable culture temperature for Drosophila, and by itself is not sufficient to induce any detectable heat shock response (King and Tower, 1999; Wheeler et al., 1995). As expected, the starting (
DOX) life spans of the different transgenic strains were found to vary at both temperatures. At 25 8C, where life span is relatively longer, life span was unchanged by DOX administration in experimental line tet22(X)20A (Fig. 4A), and was reduced by 5% in the experimental line tet22(2)23 (Fig. 4B). As expected life span was not affected by DOX

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Fig. 3. Survival in response to paraquat treatment. The indicated tet22 transgenic lines were maintained in the presence and absence of DOX, and groups of flies were fed the indicated paraquat concentrations for 48 h. The percentage of flies surviving was calculated and plotted. (A) hsp22 over-expressing strain tet22(X)A. (B) hsp22 over-expressing strain tet22(2)23. (C) Control strain tet22(3)7. (D) Control strain Oregon R.

feeding in control strain tet22(3)7, where hsp22 expression is unaltered (Fig. 4C). At 29 8C life spans are relatively shorter, and hsp22 over-expression was found to have a larger negative effect. In experimental lines tet22(X)20A and tet22(2)23, over-expression of hsp22 caused a decrease in mean life span of
20.7 and
15.0%, respectively (Fig. 4D and E). In the control strains, tet22(3)7 and Oregon R, there was no significant change in life span (Fig. 4F and G), consistent with previous observations that DOX itself has no detectable effect on life span (Bieschke et al., 1998). Therefore, hsp22 over-expression has a negative effect on life span that is more severe at higher culture temperature. 3.5. Over-expression of hsp22 had no effect on sensitivity to coumarin It was of interest to determine whether hsp22 expression was having a negative effect on specific pathways of stress resistance, or if hsp22 expression was simply resulting in a sick fly that was more sensitive to all toxic insults. To distinguish between these possibilities, a different type of toxic challenge was tested. Coumarin is a natural product used widely in industry as a fragrance enhancer and stabilizer. However, at high concentrations coumarin has toxic effects in both flies and humans by a mechanism that does not appear to involve oxidative stress (Born et al., 2000; Bournias-Vardiabasis and Teplitz, 1982; Bruhlmann et al., 2001; Carlton et al., 1996). To determine the optimum coumarin concentration for dietary administration in adult flies, four groups of adult, Oregon-R wild-type males were

maintained at coumarin concentrations of 1, 2.5, 5 and 10 mM, respectively (Fig. 5). Survival was found to be directly related to dosage, and the intermediate concentration of 5 mM was used for all subsequent experiments. Agesynchronized cohorts of flies of the two control strains and the two experimental strains were cultured +/
DOX in the presence of 5 mM coumarin. There was no detectable difference between the two control (Fig. 6D and E) and two hsp22 over-expressing strains (Fig. 6B and C), indicating that hsp22 does not increase sensitivity to all stresses. Interestingly, DOX itself may have a small effect on resistance to coumarin, as all four strains showed a slight but significant increase in survival +DOX. 3.6. The hsp22 over-expression does not affect the transcriptional induction of heat shock genes Heat shock and oxidative stress cause the transcriptional induction of hsp genes such as hsp70 and hsp22 through activation of the HSF transcription factor, which in turn binds to HSEs in the gene promoters. Several hsps, including hsp70, have been shown to be able to feedback inhibit this pathway in Drosophila, by directly or indirectly inhibiting HSF activity (Marchler and Wu, 2001; Solomon et al., 1991). However, it has not been reported whether acrystallin class hsps, such as hsp22 can have such a feedback effect. It was therefore possible that the decrease in stress resistance and life span observed upon over-expression of hsp22 might be due to negative feedback regulation of the HSF/HSE pathway. Alternatively, hsp22 might have its

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Fig. 4. Life span assay. Age synchronized cohorts of adult males of the indicated tet22 lines were maintained +/
DOX throughout the adult life span. Mean life span was calculated and the percent change caused by DOX is presented along with the P-value calculated using unpaired, two-sided t-tests. (A) hsp22 overexpressing strain tet22(X)20A, 25 8C culture temperature (+2.09%, P = 0.0835). (B) hsp22 over-expressing strain tet22(2)23, 25 8C culture temperature (
5.39%, P = <0.0001). (C) Control strain tet22(3)7, 25 8C culture temperature (
1.04%, P = 0.6114). (D) hsp22 over-expressing strain tet22(X)20A, 29 8C culture temperature (
20.71%, P = <0.0001). (E) hsp22 over-expressing strain tet22(2)23, 29 8C culture temperature (
15.01%, P = <0.0001). (F) Control strain tet22(3)7, 29 8C culture temperature (
2.97%, P = 0.2300). (G) Control strain Oregon R, 29 8C culture temperature (+1.94%, P = 0.3121).

negative effect downstream of the transcriptional stress response. To distinguish between these possibilities, hsp22:LacZ and hsp70:LacZ fusion reporter constructs were assayed for induction by heat and oxidative stress (paraquat), in the presence and absence of hsp22 overexpression. The hsp70 protein and the hsp70:lacZ reporter constructs have previously been shown to be induced in response to heat shock, oxidative stress and aging (Wheeler et al., 1995; Wheeler et al., 1999). The hsp22 protein and the

hsp22:lacZ reporter constructs have previously been shown to be induced by heat shock and aging, but response to oxidative stress was not assayed (King and Tower, 1999). In all situations induction of the hsp:LacZ reporters was shown to be dependent upon the presence of functional HSEs. Triple transgenic lines were created, containing one copy each of a tet22 over-expression construct, the rtTA transactivator, and an hsp:LacZ reporter construct. Three different hsp70:lacZ reporter insertions were assayed for

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Fig. 5. Survival in response to coumarin treatment. (A) Wild-type Oregon R flies were maintained on food supplemented to the indicated coumarin concentrations, and percent survival of the cohorts as a function of time in days is presented. (B)–(E) The indicated tet22 lines were maintained +/
DOX in the presence of 5 mM coumarin. Mean life span was calculated and the percent change caused by coumarin was calculated along with the P-value using unpaired, two-sided t-tests. (B) Line tet22(X)20A (+5.33%, P = 0.0001). (C) Line tet22(2)23 (+7.15%, P = <0.0001). (D) tet22(3)7 (+4.69%, P = 0.0002). (E) Oregon-R (+5.79%, P = <0.0001).

induction by heat stress, in the presence or absence of hsp22 over-expression (Fig. 6A). Consistent with previous results heat stress caused induction of each hsp70:lacZ reporter. DOX and hsp22 over-expression had no consistent effect on lacZ expression. The same experiment was performed using hsp22:lacZ reporters (Fig. 6B). Consistent with previous results heat stress caused a modest induction of each hsp22:lacZ reporter, and again DOX and hsp22 overexpression had no consistent effects. The data suggest that the negative effects of hsp22 over-expression on heat resistance are not due to an inhibition of the transcriptional heat shock response. As hsp22 over-expression decreased resistance to paraquat poisoning, the hsp:LacZ reporters were also assayed for potential induction in response to paraquat, in the presence and absence of hsp22 over-expression. The hsp70 protein and the hsp70:lacZ reporters have previously been shown to be dramatically induced by other in vivo models of oxidative stress, namely ‘‘Cu/ZnSOD’’ and catalase null mutations (Wheeler et al., 1995; Wheeler et al., 1999). The same hsp70:lacZ reporters were assayed for expression in flies that had been fed 20 mm paraquat for

24 h. Under these conditions the hsp70:lacZ reporters exhibited little to no induction by paraquat, and there was no effect of hsp22 on the expression observed (Fig. 7A). The hsp22:lacZ transgenic reporters have not previously been assayed for induction by oxidative stress or paraquat. Interestingly, hsp22:lacZ reporter expression was found to be significantly increased by paraquat, and this expression was not consistently affected by DOX and hsp22 overexpression (Fig. 7B). The data, therefore, demonstrate that the oxidative stress-inducer paraquat causes a larger induction of hsp22:lacZ reporters than it does hsp70:lacZ reporters, and further indicates that hsp22 over-expression has no detectable effect on hsp gene transcription. 3.7. Transcriptional induction of hsp22 and hsp70 reporters is not affected by age As aging is associated with the highest level accumulation of hsp22 (King and Tower, 1999), it affords another opportunity to ask whether hsp22 can feedback inhibit the HSF/HSE pathway. Two hsp70:lacZ reporter lines and two hsp22:lacZ reporter lines were assayed for expression in

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Fig. 6. Effect of hsp22 over-expression on induction of hsp:LacZ reporters by heat stress. Cohorts of flies of Oregon R wild type and cohorts containing the indicated tet22 insertions and the indicated hsp:LacZ reporter insertions were maintained +/
DOX and subjected to heat shock (HS) as indicated. bgalactosidase activity was quantitated in extracts of whole flies and the average +/
S.D. of triplicate samples is presented in bar graphs. (A) hsp70:lacZ and (B) hsp22:lacZ reporters.

Fig. 7. Effect of hsp22 over-expression on induction of hsp:LacZ reporters by paraquat. Cohorts of flies of Oregon R wild type and cohorts containing the indicated tet22 insertions and the indicated hsp:LacZ reporter insertions were maintained +/
DOX and +/
paraquat (PAR) as indicated. b-galactosidase activity was quantitated in extracts of whole flies and the average +/
S.D. of triplicate samples is presented in bar graphs. (A) hsp70:lacZ and (B) hsp22:lacZ reporters.

response to heat stress in young and old animals. The hsp70:lacZ reporters do not contain hsp70 coding sequences and therefore will not exhibit the several fold induction with age that is posttranscriptional (Wheeler et al., 1995; Wheeler et al., 1999). When assayed in extracts of whole flies the expression of the hsp70:lacZ reporters in the absence of heat stress was only slightly higher in old relative to young, consistent with previous results (Wheeler et al., 1995; Wheeler et al., 1999). The hsp70:lacZ reporters were

dramatically induced by heat stress and there was little or no difference in this expression between young and old flies (Fig. 8). An analogous result was obtained with the hsp22:lacZ reporters. As expected hsp22:lacZ reporter expression was greatly increased in old relative to young flies. The induction of the hsp22:lacZ reporters in response to acute heat stress was modest and was not consistently different between young and old flies. The data therefore support the conclusion that aging and the accumulation of

Fig. 8. Expression of hsp:LacZ reporters in young and old flies subjected to heat shock. Age-synchronized cohorts of flies were cultured at 29 8C to generate ‘‘young’’ (4-day-old) and ‘‘old’’ (35-day-old) samples, as indicated. Young and old flies containing the indicated hsp:LacZ reporters and young and old Oregon R wild type were subjected to heat shock (HS) as indicated. b-galactosidase activity was quantitated in extracts of whole flies and the average +/
S.D. of triplicate samples is presented in bar graphs.

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hsp22 has little to no effect on the HSF/HSE pathway and the transcriptional induction of hsps in response to an acute heat stress.

4. Discussion 4.1. The tet-on system yields conditional over-expression of hsp22 in adults Conditional gene expression systems for transgenic Drosophila have in the past relied upon hsp gene promoters and heat stress to initiate and/or maintain transgene expression. Such systems have limitations for the study of hsps, as it is not possible to induce expression of a transgene and study its effects without also inducing some or all of the endogenous hsps. In contrast, the tet-on system allowed for high-level over-expression of hsp22 and the identification of specific phenotypes, without the requirement for a heat stress to initiate or maintain expression. During heat stress and during normal aging hsp22 appears to be expressed at high levels in all tissues (King and Tower, 1999). To best reproduce this ubiquitous expression pattern in young flies, the rtTA transcriptional activator protein was expressed in all cells using the cytoplasmic actin actin5C promoter. In the future it will be of interest to drive conditional expression of hsp22 and other hsps in specific tissues. 4.2. The hsp22 over-expression decreased stress resistance and viability Experimentally causing accumulation of hsp22 in young flies reduced heat stress resistance, oxidative stress resistance and life span. The hsp22 was not simply causing a generalized toxicity or loss of cell function as resistance to coumarin and the transcriptional heat shock response were unaffected. In previous experiments, 100-fold overexpression of a protein not normally found in Drosophila, b-galactosidase (Bieschke et al., 1998) and six-fold over-expression of the highly abundant Drosophila intermediary metabolism enzyme phosphoglucomutase (Allikian et al., 2002) were both found to have no detectable effects on viability or life span—further supporting the specificity of the effects of hsp22 overexpression. In certain situations over-expression of ‘‘abnormal’’ or unfolded proteins can have negative effects, but those situations are characterized by the induction of the transcriptional heat shock response (HSF/ HSE pathway) (Ananthan et al., 1986). As hsp22 overexpression was found to have no detectable effect on the transcriptional heat shock response we conclude that the hsp22 is not simply being expressed in an abnormal or unfolded state. The data, therefore, demonstrate that under certain conditions hsp22 over-expression creates a phenotype characterized by specific deleterious effects on stress resistance and life span.

4.3. Elevated hsp22 levels do not affect the transcriptional induction of hsp genes In several organisms, including Drosophila tissue culture cells, plants and mammals hsp70, hsp90 and hsp40 family members have been shown to be capable offeedback inhibiting the transcriptional heat shock response (Fu et al., 2002; Marchler and Wu, 2001; Mosser et al., 1993; Satyal et al., 1998). Inhibition occurs at the level of the HSF/HSE pathway and appears to involve a complex between the hsps and HSF that holds HSF in the inactive monomeric form. In contrast, the negative effects of hsp22 over-expression appear to result from a different mechanism. Two situations were examined where hsp22 protein levels were high: conditional over-expression in young flies, and the accumulation of hsp22 typical of old flies. In both situations the induction of hsp:LacZ reporters by acute stress was unaffected, indicating that the HSF/HSE pathway was functioning normally. The data suggest that the negative effects of hsp22 over-expression on stress resistance and viability are downstream of the transcriptional heat shock response. The function(s) of Drosophila hsp22 have not been directly examined. However in certain situations related shsps from Drosophila and humans can increase the stress resistance of cells. If Drosophila hsp22 can have similar protective effects it may do so by interacting with other proteins at an optimal stoichiometryproducedbythestressresponse.Wehypothesize that when there is a preferential accumulation of hsp22 this optimal stoichiometry is disrupted, resulting in reduced stress resistance and ultimately reduced life span. 4.4. Induction of hsp22:LacZ and hsp70:lacZ reporters is normal in old flies Inseveral models ofaging,includingreplicative senescence of cultured human fibroblasts and youngversusold rat liver, old cells have been found to have decreased expression of hsp70 genes in response to heat stress (Heydari et al., 2000; Richardson and Holbrook, 1996). For Drosophila, hsp70 gene expression in response to heat stress in old flies has been reported to be either unchanged or slightly reduced when analyzed at the level of protein expression (Blake et al., 1991; Niedzwiecki et al., 1991), and to be unchanged when hsp70: lacZ reporters were analyzed in cells of the antennae (Helfand and Naprta, 1996). In the experiments described here, induction of the hsp22:lacZ and hsp70:LacZ reporters in response to heat and oxidative stress was found to be normal in old flies. As old flies have the highest level of accumulation of hsp22, these experiments support the conclusion that hsp22 does not feedback inhibit the HSE/HSF transcriptional pathway. 4.5. Hsp22 reporters are preferentially induced by oxidative stress In response to the oxidative stress generator paraquat, the hsp22:lacZ reporters were induced to a greater extent than were hsp70:lacZ reporters. The hsp22:lacZ reporters are

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also induced to a greater extent than are hsp70:lacZ reporters in other models of oxidative stress such as 100% oxygen atmosphere (Landis et al., 2004). In contrast heat shock results in an opposite pattern of hsp expression characterized by high-level expression of hsp70 in all tissues, and a smaller accumulation of hsp22. During aging the situation appears most similar to an oxidative stress response, as hsp70 is expressed at low levels specifically in muscle tissue, and hsp22 accumulates to high levels in all tissues (King and Tower, 1999; Wheeler et al., 1995; Wheeler et al., 1999). 4.6. The hsp22 over-expression phenotype may help reveal normal hsp22 functions If hsp22 normally has a positive effect on heat and oxidative stress resistance, then the hsp22 over-expression phenotype is a dominant negative phenotype. As this phenotype is being generated by expression of the wild type hsp22 protein, it suggests that hsp22 may normally function in a complex and that the over-expression disrupts the stoichiometry and function of such hsp22 complexes. The data demonstrate that hsp22 can interact with stress resistance pathways, and this may help in identifying hsp22 partners and targets in the future. Recently, Tanquay and coworkers (Morrow et al., 2004) used the GAL4/UAS system to drive Drosophila hsp22 overexpression during both development and aging. Strikingly, life span and resistance to both heat and paraquat were found to be increased. There are at least three possibilities for the different results obtained in their study. First, the level of hsp22 expression may be critical. Possibly a moderate elevation such as may be generated with GAL4/UAS system is beneficial, while greater increases such as those generated by tet-on system have negative effects. However, so far experiments involving tet-on over-expression of hsp22 at lower levels or for shorter durations of time have not yielded any positive effects on life span (Data not shown). The data suggest that the different results of the two studies are due to some aspect of the different expression systems other than the absolute level of transgene expression achieved. Second, the timing of over-expression during the life cycle is likely to be important. The positive effects were observed with the GAL4/UAS system where over-expression occurs both during development and in young adults. In contrast, the conditional tet-on system implemented here yields expression only in adults. Finally, the GAL4/UAS and tet-on systems have differences in the tissue-specificity of expression that are likely to be important. Each of these models should be testable in the future using a combination of the GAL4/UAS and tet-on systems that yields tissuespecific, doxycycline-inducible expression (Stebbins et al., 2001). Taken together, the data demonstrate that under appropriate conditions hsp22 over-expression can have either positive or negative effects on stress resistance and life span.

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In wild type flies, the highest levels of hsp22 protein are observed at late ages (King and Tower, 1999). An important experiment for the future will be to determine if this accumulation of hsp22 in old flies has beneficial or toxic effects. This should be testable in the future using conditional RNAi technology to inhibit hsp22 expression (Allikian et al., 2002). Based on the results obtained here and by Morrow et al (Morrow et al., 2004), we hypothesize that hsp22 may exhibit antagonistic pleiotropy: regulated expression during development and in young adults may be beneficial while the high-level protein accumulation at late ages is toxic. Consistent with this idea, genetically selected long-lived fly strains have greatly increased hsp22 RNA levels relative to controls in young adults, but not at late ages (Kurapati et al., 2000). The results obtained here with Drosophila may be relevant to other species. The hsp gene expression has been found to be increased during normal aging in mammals, and in a number of human agingrelated disease states (Lee et al., 1999; Ly et al., 2000). The current data suggest that it may be important to determine if the hsps have beneficial or deleterious effects. Acknowledgements M.J.A. was supported by a pre-doctoral training grant from the National Institute on Aging (AG00093). This research was supported by a grant from the Department of Health and Human Services to J.T. (AG11644 and AG11833).

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