Heat shock proteins can protect aged human and rodent cells from different stressful stimuli

Mechanisms of Ageing and Development 125 (2004) 201–209

Heat shock proteins can protect aged human and rodent cells from different stressful stimuli Sam Alsbury1 , Konstantina Papageorgiou1 , David S. Latchman∗ Medical Molecular Biology Unit, Institute of Child Health, UCL, 30 Guilford Street, London WC1N 7HX, UK Received 2 October 2003; received in revised form 21 November 2003; accepted 24 November 2003

Abstract Heat shock proteins (hsps) are induced by stressful stimuli and have been shown to protect cells and organs from such stresses both in vitro and in vivo. Because of this, mildly stressful stimuli, sufficient to induce hsp over-expression can protect against a subsequent more severe stress. In cells from aged individuals, however, no hsp induction is observed upon exposure to stress and no protective effect of a mild stress is observed. Here, we show that bypassing the block to hsp induction by artificially over-expressing hsps, can produce a protective effect against a variety of damaging stimuli in cells from aged rats or aged humans, indicating that hsps can have a protective effect in aged cells, provided successful over-expression can be achieved. Hence, hsps over-expression could be of therapeutic benefit in aged individuals if procedures to over-express the hsps can be developed either by devising non-stressful procedures to induce endogenous hsp over-expression or by developing vectors able to efficiently deliver exogenous hsps. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Heat shock proteins; Protection; Ischaemia; Apoptosis; Neurone; Peripheral blood cells

1. Introduction The heat shock proteins (hsps) were originally identified on the basis of their induction by heat or other stresses (for reviews see Lindquist and Craig, 1988; Parsell and Lindquist, 1993). Subsequent experiments over-expressing individual hsps in cultured cells using either transfected plasmids or viral vectors expressing individual hsps demonstrated that these proteins can have a protective effect against a variety of different stress including heat and ischaemia (for review see Latchman, 1998, 2001; Gray et al., 1999). These in vitro experiments were subsequently confirmed using in vitro approaches which demonstrated the protective effect of hsps against cardiac or cerebral ischaemia in transgenic animals over-expressing hsp70 or hsp27 or when individual hsps were delivered to unmodified animals using a viral vector (Marber et al., 1996; Plumier et al., 1996; Radford et al., 1996; Suzuki et al., 1997; Akbar et al., 2003; Kalwy et al., 2003). ∗ Corresponding author. Tel.: +44-20-7905-2611; fax: +44-20-7905-2301. E-mail address: [email protected] (D.S. Latchman). 1 The first two authors contributed equally to this work.

These results indicate a potential therapeutic use of hsps in individuals suffering from cerebral or cardiac ischaemia or in other situations producing cellular damage/death. This would require however, the identification of non-stressful procedures which can induce the endogenous hsp genes or the delivery of exogenous hsp genes with, for example, viral vectors (for discussion see Latchman, 1998). In this regard, it is of particular interest that a mildly stressful procedure, sufficient to produce hsp induction, can protect both cultured cells and specific organs against a subsequent more severe stress (see for example, Currie et al., 1988; Yellon et al., 1992; Marber et al., 1993; Cumming et al., 1996). Most interestingly however, this pre-conditioning phenomenon is not observed in aged animals. Thus, for example, we observed that exposing the intact perfused heart of aged rats to a mild ischaemic stress has no protective effect against a subsequent more severe ischaemic stress whereas the pre-conditioning effect of such a stimulus was readily observed in younger animals (Schulman et al., 2001) and similar results have been obtained by others (Tani et al., 1997; Fenton et al., 2000). This effect is likely to be associated, at least in part, with the failure of stress to induce hsp expression in cells from aged individuals (for review, see Heydari et al., 1994). Thus,

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the induction of HSP70 is reduced in multiple tissues (including heart and adrenal gland) of aged rats exposed to elevated temperature (Kregel et al., 1995); cardiac ischaemia (Nitta et al., 1994); or restraint stress (Blake et al., 1991); compared to the induction observed in young rats. Similar reduced induction is observed when several different cell types obtained from old rats are exposed to thermal stress in vitro (Heydari et al., 1993; Pahlavani et al., 1995). Such effects are also observed in humans with reduced inducibility of HSP70 in response to heat, having been observed in lymphocytes (Deguchi et al., 1988) and skin cells (Muramatsu et al., 1996) obtained from aged individuals. Similarly, late passage human fibroblasts showed reduced induction of HSP70 and HSP90 following exposure to stressful stimuli, such as heat (Luce and Cristofalo, 1992) or amino acid analogues (Liu et al., 1989). In turn, this effect appears to be due to a failure of stress to activate the heat shock transcription factor, HSF-1 which is normally converted to an active form by stressful stimuli and then induces expression of the hsp genes. Thus, although HSF-1 is expressed at normal levels in aged cells, it fails to become activated in response to stressful stimuli in both aged rats (Heydari et al., 1993) and humans (Jurivich et al., 1997) (for review, see Lee et al., 1996). Evidently, since the individuals who would benefit from a potential therapy involving hsps are predominantly elderly, such a therapy must successfully produce hsps over-expression in aged cells. This could involve either identifying procedures that can induce the endogenous hsps genes in aged cells or gene therapy procedures delivering exogenous hsp genes. Before such procedures can be contemplated, however, it is necessary to show that the defect in hsps induction in aged cells is not accompanied by a deficiency in the ability of hsps to protect such cells when successfully induced. To investigate this possibility, we have utilised herpes simplex virus (HSV) vectors to over-express individual hsps in primary cultures of neurones from aged rats and peripheral blood mononuclear cells from aged human individuals and investigated whether they have a protective effect against several different stresses.

2. Materials and methods 2.1. Virus vectors The HSP virus vectors were constructed by inserting HSP cDNAs into the HSV-1 genome under the control of the cytomegalovirus immediate early promoter (Wagstaff et al., 1999).

18 and 20 months (Schulman et al., 2001). The dorsal root ganglia were dissociated chemically using 0.3% collagenase in magnesium and calcium free Hanks balanced salt solution (HBSS), they were then triturated to produce single cells. The cultures were enriched for sensory neurones using a 6% metrizamide density cushion as previously described (Ensor et al., 2001). The neurones were plated on 13 mm glass coverslips coated with poly-d,l-ornithine at a density of 500–1000 cells per coverslip and were maintained in DMEM with 10% foetal calf serum. Twenty-four hours after culturing they were treated with the virus vectors for 1 h in serum-free media after which time, they were returned to DMEM with 10% foetal calf serum. The cells were maintained for a further 24 h to allow expression of the introduced gene before being stressed. 2.3. Isolation of PBMCs Blood samples were taken from five young individuals (average age 32 years) and from three aged individuals (average age 72 years). An appropriate volume of blood was taken into standard 7.2 mg K2 EDTA tubes. The blood was then centrifuged at 1500 rpm for 10 min. The serum was discarded and the rest of the blood was diluted 1:3 with serum-free media (RPMI1640, with 1% Penicillin-Streptomycin, GIBCO BRL). The diluted blood was then layered over 15 ml of lymphoprep solution (Diatrizoate/polysucrose solution, PHARMACIA) and centrifuged at 1500 rpm, for 45 min, at 4 ◦ C, with 0 brake rate. The white blood cell interphase was collected into a fresh Falcon tube with the use of a plastic sterile Pasteur pipette, and then the cells were washed once with serum-free media. The cells were diluted in 5 ml of serum-free media and counted under the light microscope. 2.4. Viral infection of PBMCs An appropriate number of cells (between 105 and 106 ) were taken and placed in a 15 ml Falcon tube. The cells were centrifuged at 1500 rpm for 10 min at 4 ◦ C and the excess media removed by pipetting. An appropriate volume of virus (10–100 ␮l depending on the concentration of virus, MOI = 10) was added and mixed with the cells by gentle pipetting. The cells were incubated for 3 h at 37 ◦ C, 5% CO2 . The cells were then re-suspended in full growth media (RPMI 1640, 10% foetal calf serum, with 1% Penicillin–Streptomycin, GIBCO BRL) and transferred to a 24-well plate (NUNC). The cells were then left overnight at 37 ◦ C, 5% CO2 and the infection rate (GFP assay) and viability (Trypan-blue exclusion assay) of the cells was determined. 2.5. Stressful treatments

2.2. Preparation of sensory neurones Dorsal root ganglia were removed from the spinal column of aged Sprague Dawley rats; all rats were between

Stressful treatments were designed to produce approximately 50% cell death in control samples on the basis of preliminary experiments. Heat shock was carried out at 48 ◦ C

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for 10 min and the cells then allowed to recover for 7 h at 37 ◦ C. Hypoxia was in ischaemic buffer (Esumi et al., 1991) for 4 h in a hypoxic chamber, 5% carbon dioxide, 95% argon, 37 ◦ C. For staurosporine treatment 10 ␮l (1 ␮M) of staurosporine (Sigma, UK) were added to a mixture of 106 PBMCs/ml in serum-free media, which had been cultured overnight at 37 ◦ C/5% CO2 . The cells were incubated for 5 h before detection of apoptosis. 2.6. Neuronal survival The level of survival was measured using light microscopy; intact, phase bright cells were scored as living, cells with broken membranes or a granular appearance were scored as dead or dying. Apoptosis was detected by nick end labelling of DNA 3 ends with dUTP-FITC, using a modified version of the TUNEL method as described by Brar et al., 1999. Positive staining was measured under fluorescent microscopy. 2.7. FACS analysis A cell suspension was prepared from the PBMCs. Cells were mixed with Annexin V PE (Pharmingen) and 7-aminoactinomycin D (7AAD, Sigma) in the presence of 1.8 mM calcium (100 mM Hepes, pH 7.4, 1.5 M NaCl, 50 mM KCl, 10 mM MgCl2 , 1.8 mM CaCl2 ). Cells were incubated at room temperature for 15 min prior to quenching in calcium containing binding buffer and were analysed immediately. A Beckman Coulter XL flow cytometer was used to collect 20,000 events. Staining with annexin V which has a high affinity for the membrane phospholipid phosphatidylserine and in conjunction with the vital dye 7AAD, allows the identification of live cells (annexin −, 7AAD −), early apoptotic cells (annexin +, 7AAD −) necrotic cells (annexin +, 7AAD +) and debris (annexin −, 7AAD +) to give a sensitive measurement of the dynamics of cell death. 2.8. Statistical analysis All results obtained were tested using two-tailed paired t-test, Wilcoxon’s test, unrelated sample t-test and Mann–Whitney test on either Excel or SPSS programmes.

3. Results and discussion We have previously used HSV vectors expressing individual hsps to demonstrate that over-expression of hsp27 or hsp70 could protect neonatal dorsal root ganglion (DRG) neurones from subsequent exposure to either heat shock or simulated ischaemia (Wagstaff et al., 1999). Accordingly, DRG neurones from aged rats were infected with HSV vectors expressing hsp27, hsp70 or hsp56 or a control virus expressing green fluorescent protein (GFP) and then exposed to heat shock.

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Infection with the viruses expressing either hsp27 or hsp70 clearly produced enhanced survival in the aged cells exposed to heat shock compared to the cells infected with control virus expressing only GFP or left uninfected (Fig. 1) (P < 0.05 compared to GFP in each case). In contrast, over-expression of hsp56 had no protective effect. These results exactly parallel those which we previously obtained using the same viral vectors in neonatal DRG neurones (Wagstaff et al., 1999). Hence, over-expression of specific individual hsps can protect DRG neurones from aged animals against thermal stress in exactly the same manner as for younger animals when total cell survival is measured. In neonatal animals, the protective effect of the hsps is due, at least in part, to reduced programmed cell death (apoptosis) (Wagstaff et al., 1999). We therefore, used the TUNEL assay to investigate the mechanisms of protection in aged neurones. As shown in Fig. 2, over-expression of hsp70 resulted in a reduced number of TUNEL positive cells suggesting that hsp70 over-expression was having a protective effect against apoptosis. In contrast, hsp27 over-expression did not produce a reduced number of TUNEL positive cells, compared to infection with the GFP expressing virus, suggesting that it may act by inhibiting necrosis in this case. To determine whether these results could be extended to another form of stress, we examined the effect of hsp over-expression against hypoxia in aged DRGs neurones. As illustrated in Fig. 3, both hsp27 and hsp70 reduced the number of TUNEL positive neurones in cultures exposed to hypoxia compared to the number observed in cultures infected with the GFP expressing virus and this was significant in the case of hsp27 (P < 0.05). Similarly, hsp27 but not hsp70 reduced the number of TUNEL-positive neurones compared to control uninfected cultures exposed to hypoxia. It is clear therefore, that DRG neurones from aged rats can be protected against different stresses by over-expression of hsp27 or hsp70, although the mechanism may differ with different hsps/stresses. These results indicate therefore, that if over-expression of specific hsps can be achieved in aged individuals, this is likely to have a beneficial effect. To test this possibility further, we wished to extend these results to human cells. To do this, we utilised peripheral blood mononuclear cells (PBMCs) from three aged individuals (average age 72 years). In contrast to the case of DRG neurones, the protective effect of hsps delivered with an HSV vector has not previously been tested in PBMCs. We therefore compared the effect of our viruses in PBMCs derived from aged or younger human individuals. As well as viruses expressing GFP, hsp27 or hsp70, we also used a virus expressing a mutant form of the heat shock transcription factor, HSF-1 which is constitutively active and is able to induce multiple hsps in infected cells (Wagstaff et al., 1998). In these experiments assaying total cellular survival (Fig. 4), both hsp27 and the constitutively active form of HSF-1 produced some protective effect against the damaging effects of exposure to staurosporine compared to cells

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50

Percentage survival

40

30

20

10

0 No Virus

GFP

HSP 27

HSP 70

HSP 56

Fig. 1. Total cell survival in uninfected aged DRG neurones or those infected with HSV vectors expressing the indicated protein (control GFP or hsp) and then exposed to heat shock for 10 min at 48 ◦ C. Values are the mean of three independent determinations whose standard error is shown by the bars.

90

80

70

Percent apoptotic cells

60

50

40

30

20

10

0 No Virus

GFP

HSP 27

HSP 70

HSP 56

Fig. 2. Apoptotic cell death in uninfected aged DRG neurones or those infected with HSV vectors expressing the indicated protein (control GFP or hsp) and then exposed to heat shock for 10 min at 48 ◦ C. Values are the mean of four independent determinations whose standard error is shown by the bars.

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80

70

Percent apoptotic cells

60

50

40

30

20

10

0 No Virus

GFP

HSP 27

HSP 70

Fig. 3. Apoptotic cell death in uninfected aged DRG neurones or those infected with HSV vectors expressing the indicated protein (control GFP or hsp) and then exposed to hypoxia for 4 h. Values are the mean of five independent determinations whose standard error is shown by the bars.

Fig. 4. Total cell survival of control and aged human PBMCs either left uninfected or infected with HSV vectors expressing the indicated protein (control GFP or hsp) and then treated with 1 ␮M staurosporine. Values are the mean of three independent determinations whose standard error is shown by the bars.

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Fig. 5. Apoptotic cell death of control and aged human PBMCs either left uninfected or infected with HSV vectors expressing the indicated protein (control GFP or hsp) and then treated with 1 ␮M staurosporine. Values are the mean of three independent determinations whose standard error is shown by the bars.

infected with the GFP expressing virus or uninfected control cells, whereas the hsp70-expressing virus appeared to have no effect. Most importantly, however, a similar protective effect was observed in the PBMCs from younger

and more aged individuals (compare Fig. 4 panels a and b). In all cases, comparison of the protective effect in young and aged PBMCs showed no statistically significant differences. Hence, despite the known lack of hsp inducibility in

Fig. 6. Total cell survival of control and aged human PBMCs either left uninfected or infected with HSV vectors expressing the indicated protein (control GFP or hsp) and exposed to heat shock at 48 ◦ C for 10 min. Values are the mean of three independent determinations whose standard error is shown by the bars.

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Fig. 7. Apoptotic cell death of control and aged human PBMCs either left uninfected or infected with HSV vectors expressing the indicated protein (control GFP or hsp) and exposed to heat shock at 48 ◦ C for 10 min. Values are the mean of three independent determinations whose standard error is shown by the bars.

aged human cells, including PBMCs (Deguchi et al., 1988), their over-expression protects PBMCs from both aged and younger individuals. Similar effects were also observed when we assayed programmed cell death (apoptosis) in the virally infected PBMCs exposed to staurosporine. As with total cell death, both hsp27 and the constitutively active form of HSF-1 reduced apoptosis in the PBMCs from both aged and younger individuals compared to cells infected with the GFP-expressing virus or uninfected control cells (Fig. 5). In contrast, hsp70 did not exhibit any protective effect in either case. We also tested the protective effect of the hsp-expressing viruses in PBMCs exposed to heat shock. Interestingly, in this case smaller protective effects of hsp27 or HSF-1 were observed both in assays of total cell death (Fig. 6) and of apoptosis (Fig. 7) whilst hsp70 over-expression appeared to have a damaging effect. In every case, however, similar protective effects with no statistically significant differences were observed in the PBMCs from aged and younger individuals. Indeed, in several cases better protection was observed in the aged PBMCs compared to the younger PBMCs (compare panels a and b, Figs. 6 and 7). Thus, for example, the constitutively active form of HSF-1 had a protective effect in the aged PBMCs but not in the younger PBMCs in assays of both total cell survival (Fig. 6) and apoptosis (Fig. 7). Hence, as in aged rat neurones, human PBMCs from aged individuals can be protected against two distinct stresses by hsp over-expression. These results indicate therefore, that

the failure of aged cells to be protected by mild stress from a subsequent severe stress is likely to be due entirely to a failure to induce the hsps rather than also involving a lack of protective effect of hsp over-expression. Thus, the protective effect of the hsps may be of therapeutic use in aged individuals if suitable means could be found to enhance their expression in cells from such individuals. This could involve, for example, the identification of non-stressful procedures which can induce the endogenous hsp genes in cells from aged individuals (see Latchman, 1998, for review of this approach in non-aged cells) or the use of HSV or other viral vectors to deliver exogenous hsp genes as has been achieved in young animals (see for example, Kalwy et al., 2003). Interestingly, our data suggest that one way of inducing endogenous hsps for therapeutic benefit in aged cells would be to use a viral vector to deliver a constitutively active form of HSF-1 since this vector produced protection in aged PBMCs in our experiments. This is of particular interest in view of the findings that defective DNA binding-activation of HSF-1 in aged cells is the major cause producing a failure of hsp induction (Fawcett et al., 1994; Lee et al., 1996) whilst HSF-1 activity has been shown to regulate the ageing process in the nematode (Hsu et al., 2003). It should be noted however, that the constitutively active form of HSF-1 used in our studies induces a different range of hsps in different cell types and therefore has only a restricted protective effect in neuronal cells (Wagstaff et al., 1998). Hence, its ability to induce protection would need to be evaluated in each particular situation.

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Whatever the case, it is clear that no defect in the protective effect of hsps exists in aged cells, despite the previously characterised defect in their stress inducibility. Hence, procedures which successfully elevate their expression in aged individuals would have therapeutic potential.

Acknowledgements S.A. and K.P. were supported by research studentships from the BBSRC and the ARC, respectively.

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