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Heat shock proteins and aging in Drosophila melanogaster
Seminars in Cell & Developmental Biology 14 (2003) 291–299
Heat shock proteins and aging in Drosophila melanogaster Geneviève Morrow, Robert M. Tanguay∗ Laboratory of Cellular and Developmental Genetics, Department of Medicine and CREFSIP, Université Laval, Pavillon C.E. Marchand, Ste-Foy, Que., Canada G1K 7P4
Abstract Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination. © 2003 Elsevier Ltd. All rights reserved. Keywords: Aging; Oxidative stress; Chaperones; Drosophila; sHsp
1. Introduction Aging is a complex process involving hereditary, environmental and life style factors [1,2]. According to the free radical theory of aging, lifespan is determined by the ability of organisms to cope with random somatic damages induced by reactive oxygen species (ROS), the natural by-products of energy metabolism [3]. Superoxide anions and hydroxyl radicals are highly reactive and thought to be responsible for damages to DNA, lipids and proteins [4,5]. As a result, cellular membrane functions are altered [6,7], mitochondrial DNA is preferentially mutated [8,9] telomeric DNA is less stable [10,11] and proteins are subjected to covalent modifications which can alter their functions [12,13]. Therefore, an imbalance between ROS production and removal will lead to the accumulation of cellular damages and subsequent lifespan shortening [3,14]. Model organisms such as Caenorhabditis elegans and Drosophila melanogaster have been used to try to understand the mechanisms underlying the aging process. Recent data from these systems have been shown to apply to mammals implying a conservation of aging mechanisms [15,16]. Mutants displaying increased longevity in both model organisms have been obtained and analyzed for differences in gene expression during the aging process [17–23]. Interestingly in addition to having an extended longevity, many of these mutants displayed increased thermotolerance and resistance ∗ Corresponding
author. Tel.: +1-418-656-3339; fax: +1-418-656-7176. E-mail address: [email protected] (R.M. Tanguay).
1084-9521/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2003.09.023
to stress [17,18,21]. Heat shock proteins (Hsps) are molecular chaperones that are coordinately expressed following stress and have been shown to be major determinants in the acquisition of thermotolerance and stress resistance [24,25]. 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 [26–29]. As such, Hsps could be important regulators of the aging process. This view takes support from observations on the modulation of Hsp expression during Drosophila lifespan and points to a beneficial effect of some Hsps on aging. 2. Hsps are involved in multiple cellular processes Hsps are divided in many subfamilies on the basis of their molecular weight and the sequence homology of their members. In Drosophila, where the Hsps were originally discovered, there is Hsp83, a homologue of the Hsp90/100 family found in mammals, the Hsp/Hsc70 complex, Hsp60 and the family of small Hsps (sHsps). Many Hsps have been shown to act as molecular chaperones surveying nascent protein from their release from ribosomes to their trafficking across cell membrane to their final destination [27,29]. In vitro, many Hsps also prevent heat-induced aggregation of proteins and help proper refolding of partially denatured proteins to their native state [26,28]. In addition to their rather general and unspecific chaperone function during de novo synthesis of polypeptides or under stress conditions, Hsps are also involved in diverse specific cellular processes. For
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Table 1 Functions of Drosophila melanogaster Hsps Families
Genes in Drosophila
Functions
References
sHSP
hsp22, hsp23, hsp26, hsp27, hsp67Ba, hsp67Bb, hsp67Bc, l2efl, CG14207, CG4461, CG7409, CG13133
Thermotolerance Protection against apoptosis
[41,103,104]
HSP60
hsp60, hsp60B, hsp64
Involved in spermatid individualization process Folding of nascent proteins
[37,105–109]
HSP70
hsp70, hsp68, hsc-1, hsc-2, hsc-3, hsc-4, hsc-5
Folding of nascent proteins
[38,86,109–112]
Thermotolerance Involved in clathrin-mediated endocytosis HSP90/100
hsp83
example in yeast, Hsp70 and Hsp104 play a role in snRNP assembly under normal conditions and in their maintenance in heat-stressed cells, providing a splicing tolerance essential for the functioning of the mRNA splicing machinery under stress conditions [30]. Hsp90 is required in various processes ranging from signaling of numerous receptors (steroid, tyrosine kinase) [31,32] and proteins (Raf) [33], telomerase [34] and centrosome assembly [35] to spermatogenesis [36]. Many specialized functions of Hsps have been unveiled by studies in Drosophila (Table 1). Indeed, Hsp60B, one of the three members of the Hsp60 family, has been shown to be required for the spermatid individualization process in male flies [37] and the Hsp70 cognate member Hsc-4 is involved in clathrin-mediated endocytosis [38]. In addition to its well-documented function in receptor activity, Hsp90 also acts as a capacitor of morphological evolution buffering genetic variations [39]. The fly genome encodes 12 putative small Hsps, four of which have been more extensively studied as they display distinctive features [40,41]. While Hsp22 is located in the mitochondrial matrix, Hsp23 and Hsp26 are cytoplasmic and Hsp27 is found in the nucleus [40–42]. Following exposure to environmental stresses such as heat shock, these four main sHsps are coordinately upregulated. However, during normal development and throughout lifespan, each sHsp shows a distinct stage- and cell-specific pattern of expression without coordination [40,41].
3. Cell- and stage-specific expression of Hsps during Drosophila development and lifespan As the expression pattern of shsp genes during embryonic development and metamorphosis has been previously reviewed [40,41], we will here summarize certain aspects of this expression during embryogenesis and adulthood that are pertinent to lifespan. Small Hsps display tissue- and stage-specific embryonic expression. Hsp26, Hsp27 and Hsp83 are the first Hsps to accumulate in early embryos as a
Ensure proper centrosome function Maintain of signal transduction pathways and microtubule effectors Capacitor of morphological evolution
[31–33,36,39,113]
result of the translation of their respective maternal mRNAs [43,44]. The zygotic expression of Hsp23 begins at stage 11 and is restricted to the central nervous system (CNS) where this small Hsp is successively expressed in MP2 neuronal cells, in ventral unpaired median (VUM) cells at stage 13 and in dorsal midline glia (MG) during late embryogenesis (Michaud and Tanguay, in preparation). In stage 11 embryos, there is a restricted accumulation of hsp22 mRNA in metameric ectodermal patches [45], but the corresponding protein has yet to be detected. Other sHsp mRNAs such as l(2)efl [46] and hsp67Bc [47] have also been found to be expressed throughout embryogenesis and larval stages until the beginning of pupation. Another evidence for the tight regulation of sHsps expression during development is illustrated by the hsp67Bb gene. Transcription of this gene gives rise to two mRNA products of different lengths, the shortest (560 bp) being expressed during embryogenesis while the longest (780 bp) is restricted to the male germline [48]. During the larval–pupal metamorphosis, the expression of sHsps is regulated by the steroid molting hormone ecdysone and other enhancer elements. The sHsp mRNAs appear in pulses related to those of the molting hormone [49–54]. Hsp23 accumulates in pupae but is almost totally absent from 1-week-old adult flies [55] except in the fat body at Day 1 and in gonads, where it is specifically expressed in cyst and epithelial cells in addition to spermatid bundles [56]. The stress-induction of Hsp23 is also regulated in a cell-specific manner in some tissues such as the eye where its expression is restricted to a single cell type of the ommatidium, the cone cells while Hsp27 is induced in all cells including the cone, pigment and photoreceptor cells [57]. Hsp26 and Hsp27 are also found in gonads and in the CNS where they display distinct levels of expression [56,58]. Table 2 summarizes the present data on the different cell types where each of the small Hsps is expressed during Drosophila development. Like during embryogenesis and morphogenesis, Hsps expression during aging is closely regulated and the heat-shock response following exposure to stress is also modified.
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Table 2 Small Hsps of Drosophila are expressed in different cell-types throughout development sHsp
mRNA/ protein
Developmental stages
References
Embryonic
Larval
Adult
Hsp22 Hsp23
mRNA Protein
Metameric ectodermal patches CNS (MP2, VUM, MG cells)
Absent Present
Head and thorax Gonads, fat body, CNS
Hsp26
Protein
CNS, male gonads
CNS, gonads
Hsp27
Protein
Ubiquitous
Male gonads, CNS, ventral ganglion CNS, gonads
[45,61] Michaud and Tanguay (in preparation), [40,50,55,58] [56,58,114]
CNS, gonads
[58,115,116]
Fleming et al. [59] were the first to report that the heat-shock response of young and old flies displayed striking differences in terms of Hsps expression. Young and old flies were submitted to heat shock and allowed to recover in presence of filters saturated with [35 S]methionine for 2 h. Flies were then homogenized and loaded on two-dimensional gel electrophoresis. In young flies, the heat shock treatment resulted in the induction of 14 new proteins whereas in old flies, 50 new proteins were found. Proteins common to the two groups were synthesized at a higher rate in old flies. This clear difference between the response of young and old flies suggests an increased sensitivity to environmental stress. This sensitivity is probably due to accumulation of damaged proteins since young flies fed with canavanine, an arginine analogue used to mimic accumulation of damaged proteins, displayed the same set of induced proteins as old flies [59,60]. The sequence requirement for Hsp22 and Hsp70 expression during aging has also been partially defined by using Hsp22:lacZ and Hsp70:lacZ fusion proteins [61,62]. In both cases the presence of three functional heat shock elements (HSE) has been shown to be required for upregulation of expression during aging. Since the hsp22 promoter is preferentially induced during aging comparatively to other hsp genes, it has been suggested that a specific trans-acting factor other than the heat shock factor (HSF) that binds specifically to HSE could be responsible for aging-dependent induction [61,63]. However the existence of such an aging specific trans-acting factor remains to be proven in Drosophila. Recent data obtained in organisms displaying extended longevity phenotypes are in favor of an endocrine regulation of aging featuring ecdysone and insulin mediated signals [16,64]. According to studies in C. elegans, DAF-16/FOXO is the most probable trans-acting factor that acts in concert with HSF to upregulate sHsps expression during aging [65]. Indeed, it was shown that both Daf-16 and Hsf-1 trigger sHsps expression in the insulin/IGF system and after heat shock [65]. Moreover, consensus sequences for Daf-16 were found in most shsp genes of C. elegans [66]. A gene homologous to FOXO has recently been described in Drosophila [67] but its effect on aging remains to be tested in this organism. In addition to displaying a modified stress response, old flies have an altered expression pattern of Hsps under normal
conditions comparatively to young flies. In a genome-wide study aimed at elucidating transcriptional changes accompanying the aging process in Drosophila melanogaster, the expression of Hsp26 and an Hsp60-like protein have been respectively shown to be down and upregulated [19]. An upregulation of shsp genes, ROS detoxifying enzymes and proteins involved in the antimicrobial response has also been observed in a similar study conducted in C. elegans mutants displaying increased longevity [66]. Thus because Hsps are ubiquitous and play a role in a wide variety of cellular processes by interacting with many different proteins, Macario and Macario [68] have suggested that a chaperone failure would have widespread consequences and might be an intrinsic component of the aging process. Further indirect evidence for a role of the stress response in the aging process stems from the findings that, in many species, aging is associated with a reduced heat-shock response [25,69].
4. The stress response in the aging process In attempts to understand the molecular mechanism of aging in D. melanogaster, genome-wide studies have been performed. By characterizing RNA transcript levels of the whole genome of Drosophila, Pletcher et al. [70] found that nearly 23% of the genome varies in transcripts representation with age. Among them, mRNAs for cytochrome P450s, antibacterial and several proteins involved in the stress response were upregulated [70]. In a similar study, aimed at comparing changes in gene transcripts during aging and following oxidative stress, genes of the reproductive system were found to be downregulated while genes involved in detoxification and chaperoning were upregulated in aging flies [19]. Genes associated with immunity response, microtubule organization and muscle function were also shown to be important in lifespan determination [71]. Comparing the data obtained from transcript analysis during aging in different organism reveals common features of aging among eukaryotes. Hence, while stress and inflammatory response genes are upregulated, energy metabolism and growth genes are downregulated [19,66,71–75]. Therefore, aging does not appear to be associated with a widespread disregulation of
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gene expression but represents a state of precisely regulated changes [70,75–77]. Consistent with the upregulation of stress response genes during Drosophila lifespan, hsp22 and hsp23 mRNAs and Hsp70 protein were demonstrated to be upregulated in aging flies [61,63]. The increase in Hsp70 expression with age is restricted to flight and leg muscles in the absence of heat-shock as assessed by western blotting and in studies of transgenic flies containing a reporter Hsp70--galactosidase construct [63]. The increase in reporter activity is restricted to these specific tissues as it was not detectable by western blot on whole fly extracts (Fig. 1). The expression profile of sHsps throughout lifespan has also been analyzed by northern blots. A preferential induction of hsp22 mRNA was observed during aging [61]. Interestingly, while hsp22 transcripts were upregulated by 60-fold in the head and 16-fold in thorax of 35 days old comparatively to 6 days old flies, the level of hsp26 and hsp27 transcripts remained constant over this period [61]. This preferential induction of hsp22 in aging is delayed at the protein level as demonstrated by western blotting on whole fly extracts (Fig. 1). Indeed, the level of Hsp22 is higher at 90 days of age than at 35. The level of hsp23 mRNA is upregulated by five-fold in flies thorax between Days 6 and 35 [61], but this is not visible on whole fly extracts (Fig. 1). Interestingly, tissues displaying the preferential upregulation of hsp genes either have an increased sensitivity to stress (like neurons [78]) or are submitted to intensive stress (like flying muscles cells [79]). As mentioned before, old flies have a different stress response than young flies [59]. Consistent with an increased sensitivity of old flies to environmental stresses, Minois and Le Bourg [80] have noted that old flies were more sensitive
Fig. 1. Profile of Hsps expression throughout Drosophila lifespan. Wild-type flies of different ages were homogenized and proteins separated on a 12% SDS–polyacrylamide gel. (A) Coomassie blue staining. (B) Western blotting was performed with anti-Hsp70 #799 (1/5000), anti-Hsp60 #37 (1/10,000), anti-Hsp23 #7B12 (1/100) or anti-Hsp22 #36 (1/10,000) and the corresponding secondary antibody coupled with peroxydase.
to starvation, desiccation, heat and cold stress. This points again to the accumulation of damaged proteins during aging since, as discussed above, young flies fed with canavanine display the same set of induced proteins as old flies [59,60].
Fig. 2. The GAL4/UAS system used in the gene search strategy of Seong et al. [89]. Flies having a P-element insertion containing the yeast GAL4 coding sequence are crossed with different fly lines having a P-element insertion containing UAS sequences at both ends. These GAL4-binding sequences are directed to express sequence flanking the P-element. In the progeny kept at 30 ◦ C, the promoter of hsp70 is on and induces the expression of GAL4, which than binds to UAS sequences and activates transcription of gene nearby the insertion (in the example, gene X).
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5. sHsps have beneficial effect on the aging process The first evidence that Hsps could have a beneficial effect on aging was suggested from mild stress experiments, which activate the stress response without causing cellular damages [81]. Giving repeated mild heat stresses has been shown to increase Hsps expression and allow subsequent cell survival to otherwise lethal conditions in addition to extending the organism longevity [81–83]. Intriguingly, all these studies failed to demonstrate a correlation between Hsp70 expression and the increase of lifespan by mild stress [83–85], suggesting that the effect is due to the general stress response rather than to the specific expression of Hsp70 [86]. The effect of overexpressing Hsp70 on longevity in Drosophila also failed to demonstrate a protective effect against aging injuries. Indeed, no increase in the mean lifespan (50% survival) of transgenic flies carrying varying
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numbers of the heat-inducible gene hsp70 and submitted to heat pulse was observed [87,88]. However, flies heat-treated for up to 30 min at 36 ◦ C displayed 10–30% increase in survival in the 2 weeks following the treatment [87]. In order to explain this beneficial effect, Tatar et al. [87] have suggested that Hsp70 could refold damaged proteins and/or interact with other stress-response mechanisms such as with superoxide dismutase enzymes. While the overexpression of Hsp70 failed to substantially increase lifespan, overexpressing the small Hsps seems to be more advantageous. The sole overexpression of Hsp26 in all cells leads to a 15% increase in Drosophila mean lifespan [89]. This phenotype was obtained in a study aimed at identify genes crucial for extension of longevity by mean of a gene search system. This system takes advantage of the GAL4/UAS system, which allows a tight control of targetted gene expression (Fig. 2). In these experiments of Seong et al.
Fig. 3. Hsf and Daf-16 act together to promote lifespan. In C. elegans, under normal growth conditions, Daf-2 signaling results in the phosphorylation of Daf-16 and its subsequent sequestration in the cytoplasm. The involvement of Daf-2 signaling in the inactivation of Hsf have been suggested by Hsu et al. [65] but is not yet clear. In long-lived mutants for daf-2, the phosphorylation of Daf-16 is inhibited resulting in its translocation to the nucleus where it activates the expression of proteins promoting life together with Hsf [65,66]. Daf-16 could regulate the expression of a set of genes on its own (sod-3, mtl-1) while it could act in concert with Hsf to upregulate shsp expression and other genes. Hsf could also be responsible of the transcription of other genes such as hsp70 [65,66].
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[89] GAL4 expression is driven by an hsp70 promoter and activates UAS sequences of another P-element, which has been inserted randomly. As the UAS sequence is directed to express sequences flanking the P-element, the extension of longevity can be inferred to the overexpression of the gene nearby the P-element. These genes can then be identified by gene rescue. The hsp26 gene was isolated among 25 cDNA obtained from flies displaying increased longevity. Preliminary results in our laboratory also argue for a beneficial effect of sHsps overexpression on aging. While overexpressing Hsp22 ubiquitously using an actin driver resulted in over 30% increase in mean lifespan, overexpressing Hsp23 led to an increase similar (∼15%) to the one reported for Hsp26 (data not shown). Moreover, Hsp22 appears to act on early aging events as the 30% increase in Hsp22 overexpressing flies occurs in their pre-mortality phase (<10% mortality). The 15% increase with Hsp23 was not observed in the pre-mortality phase suggesting that sHsps can affect the aging process by different pathways. As Hsp22, Hsp23 and Hsp26 have distinct intracellular localization (Hsp22 is mitochondrial [42] while Hsp23 and Hsp26 are cytoplasmic [41]), their different efficiency to increase lifespan could reflect the difference in alteration of cell compartment during aging. Interestingly, the upregulation of two mitochondrial Hsps (Hsp22 and a Hsp60-like protein) during aging in D. melanogaster [19,61] is in agreement with the hypothesis that mitochondrial proteins are particularly important in the aging process likely because of their sensitivity to oxidative stress during aging [90,91]. This also supports the idea that mitochondria are key targets of the aging process [9]. As the energy factory of cells, mitochondria play a vital role in many processes such as development, morphogenesis and aging. In addition it is now evident that mitochondria are a key element of the apoptotic process [92–94] that also has influence on aging processes [95,96]. The results obtained with the overexpression of sHsps argue in favor of an increase in protein damages during aging since sHsps are molecular chaperones preventing protein aggregation and maintaining misfolded protein in a refoldable state [28]. A beneficial role of sHsps on the aging process has also been reported in the worm C. elegans, in which the insulin-like signaling pathway known to be a key determinant of lifespan [97,98] has been demonstrated to control the expression of Hsp16 [99]. Walker and Lithgow [99] have shown that introducing extra copies of the gene encoding Hsp16 was sufficient to confer thermotolerance and increase worms longevity. Moreover, they demonstrated that the DAF-16 transcription factor, which is regulated by the insulin-like signaling pathway [97], was required for maximal expression of the sHsp. To assess that Hsp16 was involved in lifespan extension, Hsu et al. [65] performed RNAi experiments targeting the sHsp. As expected, worms submitted to Hsp16 RNAi displayed decreased lifespan as compared to wild-type worms [65]. In a similarly designed experiment, Hsf, which is known to be responsible for the heat-induction of Hsps expression, has been shown to be
involved in the insulin/IGF-1 system [65]. Fig. 3 illustrates a model to link Hsps and the insulin/IGF-1 system to aging. Small Hsps could exert their beneficial effects by preventing aggregation of damaged/oxidized proteins [65].
6. Conclusion Hsps may not be simple unspecific molecular chaperones as their presence is required for the maintenance of specific cellular functions during stress but also through normal lifespan. Modulating the heat-shock response could have many advantages, notably in the treatment or prevention of misfolded diseases. Arguing in that sense, recent work in Drosophila models of Parkinson’s and Poly(Q) diseases have demonstrated the beneficial effect of overexpressing Hsp70 on aggregates toxicity [100–102]. Using sHsp RNAi methods in C. elegans, a beneficial role of sHsps in the onset delay of Poly(Q) diseases has also been demonstrated [65].
Acknowledgements Work in the author’s laboratory was supported by a grant from the Canadian Institutes of Health Research (CIHR) to R.M.T. and a studentship to G.M.
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Heat shock proteins and aging in Drosophila melanogaster Geneviève Morrow, Robert M. Tanguay∗ Laboratory of Cellular and Developmental Genetics, Department of Medicine and CREFSIP, Université Laval, Pavillon C.E. Marchand, Ste-Foy, Que., Canada G1K 7P4
Abstract Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination. © 2003 Elsevier Ltd. All rights reserved. Keywords: Aging; Oxidative stress; Chaperones; Drosophila; sHsp
1. Introduction Aging is a complex process involving hereditary, environmental and life style factors [1,2]. According to the free radical theory of aging, lifespan is determined by the ability of organisms to cope with random somatic damages induced by reactive oxygen species (ROS), the natural by-products of energy metabolism [3]. Superoxide anions and hydroxyl radicals are highly reactive and thought to be responsible for damages to DNA, lipids and proteins [4,5]. As a result, cellular membrane functions are altered [6,7], mitochondrial DNA is preferentially mutated [8,9] telomeric DNA is less stable [10,11] and proteins are subjected to covalent modifications which can alter their functions [12,13]. Therefore, an imbalance between ROS production and removal will lead to the accumulation of cellular damages and subsequent lifespan shortening [3,14]. Model organisms such as Caenorhabditis elegans and Drosophila melanogaster have been used to try to understand the mechanisms underlying the aging process. Recent data from these systems have been shown to apply to mammals implying a conservation of aging mechanisms [15,16]. Mutants displaying increased longevity in both model organisms have been obtained and analyzed for differences in gene expression during the aging process [17–23]. Interestingly in addition to having an extended longevity, many of these mutants displayed increased thermotolerance and resistance ∗ Corresponding
author. Tel.: +1-418-656-3339; fax: +1-418-656-7176. E-mail address: [email protected] (R.M. Tanguay).
1084-9521/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2003.09.023
to stress [17,18,21]. Heat shock proteins (Hsps) are molecular chaperones that are coordinately expressed following stress and have been shown to be major determinants in the acquisition of thermotolerance and stress resistance [24,25]. 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 [26–29]. As such, Hsps could be important regulators of the aging process. This view takes support from observations on the modulation of Hsp expression during Drosophila lifespan and points to a beneficial effect of some Hsps on aging. 2. Hsps are involved in multiple cellular processes Hsps are divided in many subfamilies on the basis of their molecular weight and the sequence homology of their members. In Drosophila, where the Hsps were originally discovered, there is Hsp83, a homologue of the Hsp90/100 family found in mammals, the Hsp/Hsc70 complex, Hsp60 and the family of small Hsps (sHsps). Many Hsps have been shown to act as molecular chaperones surveying nascent protein from their release from ribosomes to their trafficking across cell membrane to their final destination [27,29]. In vitro, many Hsps also prevent heat-induced aggregation of proteins and help proper refolding of partially denatured proteins to their native state [26,28]. In addition to their rather general and unspecific chaperone function during de novo synthesis of polypeptides or under stress conditions, Hsps are also involved in diverse specific cellular processes. For
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Table 1 Functions of Drosophila melanogaster Hsps Families
Genes in Drosophila
Functions
References
sHSP
hsp22, hsp23, hsp26, hsp27, hsp67Ba, hsp67Bb, hsp67Bc, l2efl, CG14207, CG4461, CG7409, CG13133
Thermotolerance Protection against apoptosis
[41,103,104]
HSP60
hsp60, hsp60B, hsp64
Involved in spermatid individualization process Folding of nascent proteins
[37,105–109]
HSP70
hsp70, hsp68, hsc-1, hsc-2, hsc-3, hsc-4, hsc-5
Folding of nascent proteins
[38,86,109–112]
Thermotolerance Involved in clathrin-mediated endocytosis HSP90/100
hsp83
example in yeast, Hsp70 and Hsp104 play a role in snRNP assembly under normal conditions and in their maintenance in heat-stressed cells, providing a splicing tolerance essential for the functioning of the mRNA splicing machinery under stress conditions [30]. Hsp90 is required in various processes ranging from signaling of numerous receptors (steroid, tyrosine kinase) [31,32] and proteins (Raf) [33], telomerase [34] and centrosome assembly [35] to spermatogenesis [36]. Many specialized functions of Hsps have been unveiled by studies in Drosophila (Table 1). Indeed, Hsp60B, one of the three members of the Hsp60 family, has been shown to be required for the spermatid individualization process in male flies [37] and the Hsp70 cognate member Hsc-4 is involved in clathrin-mediated endocytosis [38]. In addition to its well-documented function in receptor activity, Hsp90 also acts as a capacitor of morphological evolution buffering genetic variations [39]. The fly genome encodes 12 putative small Hsps, four of which have been more extensively studied as they display distinctive features [40,41]. While Hsp22 is located in the mitochondrial matrix, Hsp23 and Hsp26 are cytoplasmic and Hsp27 is found in the nucleus [40–42]. Following exposure to environmental stresses such as heat shock, these four main sHsps are coordinately upregulated. However, during normal development and throughout lifespan, each sHsp shows a distinct stage- and cell-specific pattern of expression without coordination [40,41].
3. Cell- and stage-specific expression of Hsps during Drosophila development and lifespan As the expression pattern of shsp genes during embryonic development and metamorphosis has been previously reviewed [40,41], we will here summarize certain aspects of this expression during embryogenesis and adulthood that are pertinent to lifespan. Small Hsps display tissue- and stage-specific embryonic expression. Hsp26, Hsp27 and Hsp83 are the first Hsps to accumulate in early embryos as a
Ensure proper centrosome function Maintain of signal transduction pathways and microtubule effectors Capacitor of morphological evolution
[31–33,36,39,113]
result of the translation of their respective maternal mRNAs [43,44]. The zygotic expression of Hsp23 begins at stage 11 and is restricted to the central nervous system (CNS) where this small Hsp is successively expressed in MP2 neuronal cells, in ventral unpaired median (VUM) cells at stage 13 and in dorsal midline glia (MG) during late embryogenesis (Michaud and Tanguay, in preparation). In stage 11 embryos, there is a restricted accumulation of hsp22 mRNA in metameric ectodermal patches [45], but the corresponding protein has yet to be detected. Other sHsp mRNAs such as l(2)efl [46] and hsp67Bc [47] have also been found to be expressed throughout embryogenesis and larval stages until the beginning of pupation. Another evidence for the tight regulation of sHsps expression during development is illustrated by the hsp67Bb gene. Transcription of this gene gives rise to two mRNA products of different lengths, the shortest (560 bp) being expressed during embryogenesis while the longest (780 bp) is restricted to the male germline [48]. During the larval–pupal metamorphosis, the expression of sHsps is regulated by the steroid molting hormone ecdysone and other enhancer elements. The sHsp mRNAs appear in pulses related to those of the molting hormone [49–54]. Hsp23 accumulates in pupae but is almost totally absent from 1-week-old adult flies [55] except in the fat body at Day 1 and in gonads, where it is specifically expressed in cyst and epithelial cells in addition to spermatid bundles [56]. The stress-induction of Hsp23 is also regulated in a cell-specific manner in some tissues such as the eye where its expression is restricted to a single cell type of the ommatidium, the cone cells while Hsp27 is induced in all cells including the cone, pigment and photoreceptor cells [57]. Hsp26 and Hsp27 are also found in gonads and in the CNS where they display distinct levels of expression [56,58]. Table 2 summarizes the present data on the different cell types where each of the small Hsps is expressed during Drosophila development. Like during embryogenesis and morphogenesis, Hsps expression during aging is closely regulated and the heat-shock response following exposure to stress is also modified.
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Table 2 Small Hsps of Drosophila are expressed in different cell-types throughout development sHsp
mRNA/ protein
Developmental stages
References
Embryonic
Larval
Adult
Hsp22 Hsp23
mRNA Protein
Metameric ectodermal patches CNS (MP2, VUM, MG cells)
Absent Present
Head and thorax Gonads, fat body, CNS
Hsp26
Protein
CNS, male gonads
CNS, gonads
Hsp27
Protein
Ubiquitous
Male gonads, CNS, ventral ganglion CNS, gonads
[45,61] Michaud and Tanguay (in preparation), [40,50,55,58] [56,58,114]
CNS, gonads
[58,115,116]
Fleming et al. [59] were the first to report that the heat-shock response of young and old flies displayed striking differences in terms of Hsps expression. Young and old flies were submitted to heat shock and allowed to recover in presence of filters saturated with [35 S]methionine for 2 h. Flies were then homogenized and loaded on two-dimensional gel electrophoresis. In young flies, the heat shock treatment resulted in the induction of 14 new proteins whereas in old flies, 50 new proteins were found. Proteins common to the two groups were synthesized at a higher rate in old flies. This clear difference between the response of young and old flies suggests an increased sensitivity to environmental stress. This sensitivity is probably due to accumulation of damaged proteins since young flies fed with canavanine, an arginine analogue used to mimic accumulation of damaged proteins, displayed the same set of induced proteins as old flies [59,60]. The sequence requirement for Hsp22 and Hsp70 expression during aging has also been partially defined by using Hsp22:lacZ and Hsp70:lacZ fusion proteins [61,62]. In both cases the presence of three functional heat shock elements (HSE) has been shown to be required for upregulation of expression during aging. Since the hsp22 promoter is preferentially induced during aging comparatively to other hsp genes, it has been suggested that a specific trans-acting factor other than the heat shock factor (HSF) that binds specifically to HSE could be responsible for aging-dependent induction [61,63]. However the existence of such an aging specific trans-acting factor remains to be proven in Drosophila. Recent data obtained in organisms displaying extended longevity phenotypes are in favor of an endocrine regulation of aging featuring ecdysone and insulin mediated signals [16,64]. According to studies in C. elegans, DAF-16/FOXO is the most probable trans-acting factor that acts in concert with HSF to upregulate sHsps expression during aging [65]. Indeed, it was shown that both Daf-16 and Hsf-1 trigger sHsps expression in the insulin/IGF system and after heat shock [65]. Moreover, consensus sequences for Daf-16 were found in most shsp genes of C. elegans [66]. A gene homologous to FOXO has recently been described in Drosophila [67] but its effect on aging remains to be tested in this organism. In addition to displaying a modified stress response, old flies have an altered expression pattern of Hsps under normal
conditions comparatively to young flies. In a genome-wide study aimed at elucidating transcriptional changes accompanying the aging process in Drosophila melanogaster, the expression of Hsp26 and an Hsp60-like protein have been respectively shown to be down and upregulated [19]. An upregulation of shsp genes, ROS detoxifying enzymes and proteins involved in the antimicrobial response has also been observed in a similar study conducted in C. elegans mutants displaying increased longevity [66]. Thus because Hsps are ubiquitous and play a role in a wide variety of cellular processes by interacting with many different proteins, Macario and Macario [68] have suggested that a chaperone failure would have widespread consequences and might be an intrinsic component of the aging process. Further indirect evidence for a role of the stress response in the aging process stems from the findings that, in many species, aging is associated with a reduced heat-shock response [25,69].
4. The stress response in the aging process In attempts to understand the molecular mechanism of aging in D. melanogaster, genome-wide studies have been performed. By characterizing RNA transcript levels of the whole genome of Drosophila, Pletcher et al. [70] found that nearly 23% of the genome varies in transcripts representation with age. Among them, mRNAs for cytochrome P450s, antibacterial and several proteins involved in the stress response were upregulated [70]. In a similar study, aimed at comparing changes in gene transcripts during aging and following oxidative stress, genes of the reproductive system were found to be downregulated while genes involved in detoxification and chaperoning were upregulated in aging flies [19]. Genes associated with immunity response, microtubule organization and muscle function were also shown to be important in lifespan determination [71]. Comparing the data obtained from transcript analysis during aging in different organism reveals common features of aging among eukaryotes. Hence, while stress and inflammatory response genes are upregulated, energy metabolism and growth genes are downregulated [19,66,71–75]. Therefore, aging does not appear to be associated with a widespread disregulation of
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gene expression but represents a state of precisely regulated changes [70,75–77]. Consistent with the upregulation of stress response genes during Drosophila lifespan, hsp22 and hsp23 mRNAs and Hsp70 protein were demonstrated to be upregulated in aging flies [61,63]. The increase in Hsp70 expression with age is restricted to flight and leg muscles in the absence of heat-shock as assessed by western blotting and in studies of transgenic flies containing a reporter Hsp70--galactosidase construct [63]. The increase in reporter activity is restricted to these specific tissues as it was not detectable by western blot on whole fly extracts (Fig. 1). The expression profile of sHsps throughout lifespan has also been analyzed by northern blots. A preferential induction of hsp22 mRNA was observed during aging [61]. Interestingly, while hsp22 transcripts were upregulated by 60-fold in the head and 16-fold in thorax of 35 days old comparatively to 6 days old flies, the level of hsp26 and hsp27 transcripts remained constant over this period [61]. This preferential induction of hsp22 in aging is delayed at the protein level as demonstrated by western blotting on whole fly extracts (Fig. 1). Indeed, the level of Hsp22 is higher at 90 days of age than at 35. The level of hsp23 mRNA is upregulated by five-fold in flies thorax between Days 6 and 35 [61], but this is not visible on whole fly extracts (Fig. 1). Interestingly, tissues displaying the preferential upregulation of hsp genes either have an increased sensitivity to stress (like neurons [78]) or are submitted to intensive stress (like flying muscles cells [79]). As mentioned before, old flies have a different stress response than young flies [59]. Consistent with an increased sensitivity of old flies to environmental stresses, Minois and Le Bourg [80] have noted that old flies were more sensitive
Fig. 1. Profile of Hsps expression throughout Drosophila lifespan. Wild-type flies of different ages were homogenized and proteins separated on a 12% SDS–polyacrylamide gel. (A) Coomassie blue staining. (B) Western blotting was performed with anti-Hsp70 #799 (1/5000), anti-Hsp60 #37 (1/10,000), anti-Hsp23 #7B12 (1/100) or anti-Hsp22 #36 (1/10,000) and the corresponding secondary antibody coupled with peroxydase.
to starvation, desiccation, heat and cold stress. This points again to the accumulation of damaged proteins during aging since, as discussed above, young flies fed with canavanine display the same set of induced proteins as old flies [59,60].
Fig. 2. The GAL4/UAS system used in the gene search strategy of Seong et al. [89]. Flies having a P-element insertion containing the yeast GAL4 coding sequence are crossed with different fly lines having a P-element insertion containing UAS sequences at both ends. These GAL4-binding sequences are directed to express sequence flanking the P-element. In the progeny kept at 30 ◦ C, the promoter of hsp70 is on and induces the expression of GAL4, which than binds to UAS sequences and activates transcription of gene nearby the insertion (in the example, gene X).
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5. sHsps have beneficial effect on the aging process The first evidence that Hsps could have a beneficial effect on aging was suggested from mild stress experiments, which activate the stress response without causing cellular damages [81]. Giving repeated mild heat stresses has been shown to increase Hsps expression and allow subsequent cell survival to otherwise lethal conditions in addition to extending the organism longevity [81–83]. Intriguingly, all these studies failed to demonstrate a correlation between Hsp70 expression and the increase of lifespan by mild stress [83–85], suggesting that the effect is due to the general stress response rather than to the specific expression of Hsp70 [86]. The effect of overexpressing Hsp70 on longevity in Drosophila also failed to demonstrate a protective effect against aging injuries. Indeed, no increase in the mean lifespan (50% survival) of transgenic flies carrying varying
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numbers of the heat-inducible gene hsp70 and submitted to heat pulse was observed [87,88]. However, flies heat-treated for up to 30 min at 36 ◦ C displayed 10–30% increase in survival in the 2 weeks following the treatment [87]. In order to explain this beneficial effect, Tatar et al. [87] have suggested that Hsp70 could refold damaged proteins and/or interact with other stress-response mechanisms such as with superoxide dismutase enzymes. While the overexpression of Hsp70 failed to substantially increase lifespan, overexpressing the small Hsps seems to be more advantageous. The sole overexpression of Hsp26 in all cells leads to a 15% increase in Drosophila mean lifespan [89]. This phenotype was obtained in a study aimed at identify genes crucial for extension of longevity by mean of a gene search system. This system takes advantage of the GAL4/UAS system, which allows a tight control of targetted gene expression (Fig. 2). In these experiments of Seong et al.
Fig. 3. Hsf and Daf-16 act together to promote lifespan. In C. elegans, under normal growth conditions, Daf-2 signaling results in the phosphorylation of Daf-16 and its subsequent sequestration in the cytoplasm. The involvement of Daf-2 signaling in the inactivation of Hsf have been suggested by Hsu et al. [65] but is not yet clear. In long-lived mutants for daf-2, the phosphorylation of Daf-16 is inhibited resulting in its translocation to the nucleus where it activates the expression of proteins promoting life together with Hsf [65,66]. Daf-16 could regulate the expression of a set of genes on its own (sod-3, mtl-1) while it could act in concert with Hsf to upregulate shsp expression and other genes. Hsf could also be responsible of the transcription of other genes such as hsp70 [65,66].
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[89] GAL4 expression is driven by an hsp70 promoter and activates UAS sequences of another P-element, which has been inserted randomly. As the UAS sequence is directed to express sequences flanking the P-element, the extension of longevity can be inferred to the overexpression of the gene nearby the P-element. These genes can then be identified by gene rescue. The hsp26 gene was isolated among 25 cDNA obtained from flies displaying increased longevity. Preliminary results in our laboratory also argue for a beneficial effect of sHsps overexpression on aging. While overexpressing Hsp22 ubiquitously using an actin driver resulted in over 30% increase in mean lifespan, overexpressing Hsp23 led to an increase similar (∼15%) to the one reported for Hsp26 (data not shown). Moreover, Hsp22 appears to act on early aging events as the 30% increase in Hsp22 overexpressing flies occurs in their pre-mortality phase (<10% mortality). The 15% increase with Hsp23 was not observed in the pre-mortality phase suggesting that sHsps can affect the aging process by different pathways. As Hsp22, Hsp23 and Hsp26 have distinct intracellular localization (Hsp22 is mitochondrial [42] while Hsp23 and Hsp26 are cytoplasmic [41]), their different efficiency to increase lifespan could reflect the difference in alteration of cell compartment during aging. Interestingly, the upregulation of two mitochondrial Hsps (Hsp22 and a Hsp60-like protein) during aging in D. melanogaster [19,61] is in agreement with the hypothesis that mitochondrial proteins are particularly important in the aging process likely because of their sensitivity to oxidative stress during aging [90,91]. This also supports the idea that mitochondria are key targets of the aging process [9]. As the energy factory of cells, mitochondria play a vital role in many processes such as development, morphogenesis and aging. In addition it is now evident that mitochondria are a key element of the apoptotic process [92–94] that also has influence on aging processes [95,96]. The results obtained with the overexpression of sHsps argue in favor of an increase in protein damages during aging since sHsps are molecular chaperones preventing protein aggregation and maintaining misfolded protein in a refoldable state [28]. A beneficial role of sHsps on the aging process has also been reported in the worm C. elegans, in which the insulin-like signaling pathway known to be a key determinant of lifespan [97,98] has been demonstrated to control the expression of Hsp16 [99]. Walker and Lithgow [99] have shown that introducing extra copies of the gene encoding Hsp16 was sufficient to confer thermotolerance and increase worms longevity. Moreover, they demonstrated that the DAF-16 transcription factor, which is regulated by the insulin-like signaling pathway [97], was required for maximal expression of the sHsp. To assess that Hsp16 was involved in lifespan extension, Hsu et al. [65] performed RNAi experiments targeting the sHsp. As expected, worms submitted to Hsp16 RNAi displayed decreased lifespan as compared to wild-type worms [65]. In a similarly designed experiment, Hsf, which is known to be responsible for the heat-induction of Hsps expression, has been shown to be
involved in the insulin/IGF-1 system [65]. Fig. 3 illustrates a model to link Hsps and the insulin/IGF-1 system to aging. Small Hsps could exert their beneficial effects by preventing aggregation of damaged/oxidized proteins [65].
6. Conclusion Hsps may not be simple unspecific molecular chaperones as their presence is required for the maintenance of specific cellular functions during stress but also through normal lifespan. Modulating the heat-shock response could have many advantages, notably in the treatment or prevention of misfolded diseases. Arguing in that sense, recent work in Drosophila models of Parkinson’s and Poly(Q) diseases have demonstrated the beneficial effect of overexpressing Hsp70 on aggregates toxicity [100–102]. Using sHsp RNAi methods in C. elegans, a beneficial role of sHsps in the onset delay of Poly(Q) diseases has also been demonstrated [65].
Acknowledgements Work in the author’s laboratory was supported by a grant from the Canadian Institutes of Health Research (CIHR) to R.M.T. and a studentship to G.M.
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