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Heat shock protein 90 and its cochaperone, p23, are markedly increased in the aged gerbil hippocampus
Experimental Gerontology 46 (2011) 768–772
Contents lists available at ScienceDirect
Experimental Gerontology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ex p g e r o
Short report
Heat shock protein 90 and its cochaperone, p23, are markedly increased in the aged gerbil hippocampus Choong Hyun Lee a, Joon Ha Park b, Jung Hoon Choi c, Ki-Yeon Yoo d, Pan Dong Ryu a,⁎, Moo-Ho Won b,⁎⁎ a
Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, 200-701, South Korea Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Oral Anatomy, College of Dentistry, Gangneung-Wonju National University, Gangneung 210-702, South Korea b c
a r t i c l e
i n f o
Article history: Received 6 March 2011 Received in revised form 19 April 2011 Accepted 6 May 2011 Available online 13 May 2011 Section Editor: Christian Humpel Keywords: Heat shock proteins Chaperone Aging Hippocampus proper Dentate gyrus
a b s t r a c t In the present study, we compared HSP90 and its co-chaperone, p23, immunoreactivity and their protein levels in the hippocampus between adult (postnatal month 6) and aged (postnatal month 24) gerbils using immunohistochemistry and western blot analysis. HSP90 immunoreactivity was markedly increased in pyramidal cells in the hippocampus proper and in polymorphic cells in the dentate gyrus of the aged group compared to the adult group. p23 immunoreactivity was slightly increased in pyramidal cells of the hippocampus proper and in granule cells of the dentate gyrus in the aged group. In addition, HSP90 and p23 protein levels in the aged hippocampus were much higher than the adult hippocampus. These results indicate that HSP90 and p23 immunoreactivity and protein levels in the hippocampus are distinctively increased in the aged gerbils compared to the adult gerbils. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Aging is closely related to neurobiological, neuropathological and neurochemical alterations in the central nervous system (ErrajiBenchekroun et al., 2005; Lister and Barnes, 2009). Brain aging can lead to various changes in structural plasticity, oxidative stress, DNA repair ability, calcium regulation and neuroinflammatory function (Godbout and Johnson, 2006; Thibault et al., 1998; Wei and Lee, 2002). In addition, aging in cellular functions is also related to the accumulation of misfolded proteins and failure of chaperoning systems (Macario and Conway de Macario, 2002). Among various brain regions, the hippocampus has been well known as a most vulnerable and sensitive region to aging process (Casadesus et al., 2005; Jacobson et al., 2008; Rosenzweig and Barnes, 2003). Heat shock proteins (HSPs) are highly induced following exposure to cellular stress, and HSPs are also expressed in unstressed cells (Sonna et al., 2002; Welch, 1992). One of main functions of HSPs is well known as molecular chaperones that guide protein folding, unfolding, assembly
⁎ Corresponding author. Tel.: + 82 2 880 1254. ⁎⁎ Corresponding author. Tel.: + 82 33 250 8891; fax: + 82 33 256 1614. E-mail addresses: [email protected] (P.D. Ryu), [email protected] (M.-H. Won). 0531-5565/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2011.05.002
and so on (Gething and Sambrook, 1992; Welch, 1992). HSPs are divided into major families, such as HSP90, HSP70 and HSP60 (Jolly and Morimoto, 2000). Among them, HSP90 has molecular chaperoning activity and plays roles in protein refolding and signal transduction (Imai et al., 2003; Wiech et al., 1992). In HSP90 chaperone activity, the formation of dynamic complex is required with other chaperones, co-chaperones and other binding proteins (Smith et al., 1995). Among co-chaperones, p23 is a small protein, closely involved in HSP90 chaperone activity (Pearl and Prodromou, 2006; Sullivan et al., 2002). The interaction between HSP90 and p23 requires ATP and occurs in the late stage of chaperone cycle (Sullivan et al., 2002). In addition, p23 is known to act as a chaperone in absence of HSP90 (Bose et al., 1996; Oxelmark et al., 2003). Although some studies have been focused on the relationship between the modulation of HSP90 and protein misfolding diseases (Auluck et al., 2002; Dickey et al., 2007; Waza et al., 2005), few studies on changes in HSP90 in the hippocampus during normal aging have been reported (Ghi et al., 2009). In addition, although HSP90 and p23 have been well known as their chaperoning activity, it has not clearly elucidated how both HSP90 and p23 are changed in the increase of misfolded proteins and modification of chaperoning systems with age yet, especially in the aged rodent brain. In the present study, therefore, we compared HSP90 and its co-chaperone, p23, immunoreactivity and their protein levels in the hippocampus between adult and aged gerbils.
C.H. Lee et al. / Experimental Gerontology 46 (2011) 768–772 Table 1 Semi-quantifications of HSP90 and p23 immunostaining intensity in the hippocampus between adult and aged gerbils. Postnatal month (PM)
HSP90
p23
Pyramidal cells Non-pyramidal cells Granule cells Polymorphic cells Pyramidal cells Non-pyramidal cells Granule cells Polymorphic cells
PM 6
PM 24
± + − + ± + ± +
++ + ± ++ + + + +
The intensity of immunostaining was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥ 200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively.
2. Materials and methods 2.1. Experimental animals We used male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chuncheon, South Korea, at postnatal month 6 (PM 6) and PM 24 as adult and aged group, respectively. The animals (n = 14 at each age) were housed in a conventional state under adequate temperature (23 °C) and humidity
769
(60%) control with a 12-h light/12-h dark cycle, and provided with free access to food and water. The procedures for handling and caring for animals adhered to the guidelines that are in compliance with the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996), and were approved by the Institutional Animal Care and Use Committee of Hallym's Medical Center. All of the experiments were conducted in a way as to minimize the number of animals used and the suffering caused by the procedures used in the present study. 2.2. Immunohistochemistry The animals (n = 7 per group) were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphatebuffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and postfixed with the same solution for 6 h. The tissues were cryoprotected by infiltration with 30% sucrose overnight. The brain tissues were then frozen and sectioned with a cryostat at 30 μm, and consecutive sections were collected in six-well plates containing 0.1 M PBS. To ensure that immunohistochemical data were comparable between groups, the free-floating sections were carefully processed under the same conditions. The sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. They were next incubated with diluted mouse anti-HSP90 (1:100, Abcam, Cambridge, UK) or mouse anti-p23 (1:100, Abcam) overnight at room temperature and subsequently
Fig. 1. HSP90 immunohistochemistry in the CA1 region (A and B), CA2/3 region (C and D) and dentate gyrus (E and F) at PM 6 (A, C, and E) and PM 24 (B, D, and F). In the CA1 and CA2/3 regions at PM 24, HSP90 immunoreactivity in pyramidal cells (asterisks) of the stratum pyramidale (SP) is markedly increased compared to the PM 6 group; however, HSP90 immunoreactivity in non-pyramidal cells (arrows) is not increased. In the dentate gyrus, HSP90 immunoreaction is hardly detected in granule cells (asterisks) of the granule cell layer (GCL) in the PM 6 group; however, HSP90 immunoreactivity is increased at PM 24. Polymorphic cells (arrows) at PM 24 is much stronger than the PM 6 group. SO, stratum oriens; SR, stratum radiatum; ML, molecular layer; PL, polymorphic layer. Scale bar = 100 μm.
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C.H. Lee et al. / Experimental Gerontology 46 (2011) 768–772
Fig. 2. Immunohistochemistry for p23 in the CA1 region (A and B), CA2/3 region (C and D) and dentate gyrus (E and F) at PM 6 (A, C, and E) and PM 24 (B, D, and F). In the CA1-3 regions at PM 24, p23 immunoreactivity in pyramidal cells (asterisks) of the stratum pyramidale (SP) is higher than the PM 6 group; however, the immunoreactivity in nonpyramidal cells (arrows) is not changed. In the dentate gyrus, p23 immunoreactivity in the granule cells (asterisk) is weak at PM 6 group, and the immunoreactivity is slightly increased at PM 24. p23 immunoreactivity in polymorphic cells (arrows) at PM 24 is not altered. SO, stratum oriens; SR, stratum radiatum; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar = 100 μm.
exposed to biotinylated goat anti-mouse IgG (1:200, Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector). Then, the sections were visualized by staining with 3,3′-diaminobenzidine in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration the sections were mounted with Canada balsam (Kanto, Tokyo, Japan). A negative control test was carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining. The negative control resulted in the absence of immunoreactivity in all structures. Eight sections per animal were selected to quantitatively analyze HSP90 and p23 immunoreactivity, respectively. HSP90 and p23 immunoreactivity was graded: Digital images of the hippocampal subregions were captured with an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss,
Fig. 3. Western blot analysis of HSP90 and p23 in the gerbil hippocampus derived from the PM 6 (adult, n = 7) and PM 24 (aged, n = 7) groups.
Germany) connected to a PC monitor. Semi-quantification of the immunostaining intensity of HSP90 and p23 was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). The mean intensity of HSP90 and p23 immunostaining in each immunoreactive structure was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively. 2.3. Western blot analysis To confirm change in HSP90 and p23 levels in the hippocampus between groups, animals at each ages (n = 7) were used for western blot analysis. After sacrificing animals, the hippocampus was removed. The tissues were then homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol bis (2-aminoethyl ether)-N,N, N′,N′ tetraacetic acid (pH 8.0), 0.2% Nonidet P-40, 10 mM ethylendiamine tetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol (DTT). After centrifugation at 10,000 g, the protein level in the supernatants was determined using a Micro BCA protein assay kit with bovine serum albumin as a standard (Pierce Chemical, Rockford, IL). Aliquots containing 20 μg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM
C.H. Lee et al. / Experimental Gerontology 46 (2011) 768–772
DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. The aliquots were then loaded onto a 12% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Corp, East Hills, NY). To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, followed by incubation with mouse anti-HSP90 (1:1,000, Abcam) or mouse anti-p23 antiserum (1:1000, Abcam), peroxidase-conjugated goat anti-mouse IgG (Sigma) and an ECL kit (Pierce Chemical). 3. Results 3.1. HSP90 and p23 immunoreactivity Weak HSP90 immunoreactivity was observed in pyramidal cells in the stratum pyramidale (SP) of the hippocampus proper (CA1-3 regions) in the PM 6 group (Table 1, Fig. 1A and C). At PM 24, HSP90 immunoreactivity in pyramidal cells is distinctively increased compared to the PM 6 group (Table 1, Fig. 1B and D). Non-pyramidal cells in the strata oriens (SO) and radiatum (SR) showed moderate HSP90 immunoreactivity, and the immunoreactivity was not changed at PM 24 (Table 1, Fig. 1A–D). HSP90 immunoreactivity in granule cells in the granule cell layer (GCL) of the dentate gyrus was hardly detected at PM 6 (Table 1, Fig. 1E); In the PM 24 group, weak HSP90 immunoreactivity was detected in the GCL (Table 1, Fig. 1F). Moderate HSP90 immunoreactivity was detected in polymorphic cells in the polymorphic layer (PL) of the dentate gyrus, and the immunoreactivity was strong at PM 24 (Table 1, Fig. 1E and F). The change pattern of p23 immunoreactivity in pyramidal and non-pyramidal cells in the CA1-3 regions of both the PM 6 and 24 groups was similar to the change pattern of HSP90 immunoreactivity (Table 1, Fig. 2A–D). Granule cells in the GCL of the PM 6 group showed weak p23 immunoreactivity, and the cells in the PM 24 group moderated p23 immunoreactivity (Table 1, Fig. 2E and F). Polymorphic cells in PL of both the PM 6 and 24 groups showed moderate p23 immunoreactivity (Table 1, Fig. 2E and F). 3.2. HSP90 and p23 protein levels In western blot study, we found that changes in HSP and p23 levels in the hippocampus of both the adult and aged groups were generally similar to the immunohistochemical changes. HSP90 and p23 protein levels were increased in the PM 24 group compared to the PM 6 group (Fig. 3). 4. Discussion In the present study, we used Mongolian gerbils as an experimental animal. The Mongolian gerbil has a relatively short lifespan, and it is genetically homogeneous. It has been known that the mean survival lifespan of male gerbils is about 110 weeks, and the oldest lifespan is about 200 weeks (Troup et al., 1969). The Mongolian gerbil is also known to have a lot of unique physiological, behavioral and biological attributes, which are analogous to similar phenomena in humans (Cheal, 1986). Especially, auditory failure, loss of perception, cholesterol and bile acid metabolism, and thermoregulation in old gerbils are analogous to those in aged humans rather than rats and mice. Therefore, it has been suggested that the Mongolian gerbil is unique and a suitable model for research on aging, which could offer valuable information for gerontology (Cheal, 1986). In this study, we compared HSP90 immunoreactivity and its protein levels in the gerbil hippocampus. HSP90 immunoreactivity in the hippocampus proper and dentate gyrus of the aged group was much higher than the adult group. We also found that HSP90 protein level in the aged groups was significantly increased compared to the adult group. This result is supported by a previous study, which
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showed that HSP90 protein levels and genes were increased gradually in the dog hippocampus with age (Ghi et al., 2009). Another previous study has reported that basal HSP90 levels are significantly increased in blood cells of the elderly human subjects (Njemini et al., 2007). They suggested that higher levels of HSP90 might be due to the higher load of damaged proteins. p23, an important factor for HSP90 chaperone activity, is known to be necessary for perinatal survival and particularly for the final fetal stages of lung and skin development and maturation (Grad et al., 2006; Sullivan et al., 2002). In this study, we also observed that, like the results of HSP90 immunoreactivity and protein levels, both p23 immunoreactivity and protein level in the hippocampus of the aged group were increased compared to the adult group. It is hard to discuss why p23 immunoreactivity and protein level were increased in the aged hippocampus, because there is no study on age-related changes of p23 in the central nervous system. However, it can be postulated that the increase of p23 may be associated with the increase of damaged proteins and the requirement of chaperone activity in the aged hippocampus, as is the case in the increase of HSP90 in the aged hippocampus. In conclusion, our present study indicates that HSP90 and p23 immunoreactivity and its protein levels in the gerbil hippocampus are higher in the aged group rather than in the adult group. These elevations of HSP90 and p23 may reflect an increase of chaperone activity, which is closely related to increase of damaged proteins in the hippocampus with age. Acknowledgements This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2009-351-1E00001). References Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M., 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295, 865–868. Bose, S., Weikl, T., Bugl, H., Buchner, J., 1996. Chaperone function of Hsp90-associated proteins. Science 274, 1715–1717. Casadesus, G., Shukitt-Hale, B., Stellwagen, H.M., Smith, M.A., Rabin, B.M., Joseph, J.A., 2005. Hippocampal neurogenesis and PSA-NCAM expression following exposure to 56Fe particles mimics that seen during aging in rats. Exp. Gerontol. 40, 249–254. Cheal, M.L., 1986. The gerbil: a unique model for research on aging. Exp. Aging Res. 12, 3–21. Dickey, C.A., Kamal, A., Lundgren, K., Klosak, N., Bailey, R.M., Dunmore, J., Ash, P., Shoraka, S., Zlatkovic, J., Eckman, C.B., Patterson, C., Dickson, D.W., Nahman Jr., N.S., Hutton, M., Burrows, F., Petrucelli, L., 2007. The high-affinity HSP90–CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J. Clin. Invest. 117, 648–658. Erraji-Benchekroun, L., Underwood, M.D., Arango, V., Galfalvy, H., Pavlidis, P., Smyrniotopoulos, P., Mann, J.J., Sibille, E., 2005. Molecular aging in human prefrontal cortex is selective and continuous throughout adult life. Biol. Psychiatry 57, 549–558. Gething, M.J., Sambrook, J., 1992. Protein folding in the cell. Nature 355, 33–45. Ghi, P., Di Brisco, F., Dallorto, D., Osella, M.C., Orsetti, M., 2009. Age-related modifications of egr1 expression and ubiquitin–proteasome components in pet dog hippocampus. Mech. Ageing Dev. 130, 320–327. Godbout, J.P., Johnson, R.W., 2006. Age and neuroinflammation: a lifetime of psychoneuroimmune consequences. Neurol. Clin. 24, 521–538. Grad, I., McKee, T.A., Ludwig, S.M., Hoyle, G.W., Ruiz, P., Wurst, W., Floss, T., Miller III, C.A., Picard, D., 2006. The Hsp90 cochaperone p23 is essential for perinatal survival. Mol. Cell. Biol. 26, 8976–8983. Imai, J., Maruya, M., Yashiroda, H., Yahara, I., Tanaka, K., 2003. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 22, 3557–3567. Jacobson, L., Zhang, R., Elliffe, D., Chen, K.F., Mathai, S., McCarthy, D., Waldvogel, H., Guan, J., 2008. Correlation of cellular changes and spatial memory during aging in rats. Exp. Gerontol. 43, 929–938. Jolly, C., Morimoto, R.I., 2000. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J. Natl. Cancer Inst. 92, 1564–1572. Lister, J.P., Barnes, C.A., 2009. Neurobiological changes in the hippocampus during normative aging. Arch. Neurol. 66, 829–833. Macario, A.J., Conway de Macario, E., 2002. Sick chaperones and ageing: a perspective. Ageing Res. Rev. 1, 295–311.
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Njemini, R., Lambert, M., Demanet, C., Kooijman, R., Mets, T., 2007. Basal and infectioninduced levels of heat shock proteins in human aging. Biogerontology 8, 353–364. Oxelmark, E., Knoblauch, R., Arnal, S., Su, L.F., Schapira, M., Garabedian, M.J., 2003. Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface involved in estrogen receptor signaling. J. Biol. Chem. 278, 36547–36555. Pearl, L.H., Prodromou, C., 2006. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu. Rev. Biochem. 75, 271–294. Rosenzweig, E.S., Barnes, C.A., 2003. Impact of aging on hippocampal function: plasticity, network dynamics, and cognition. Prog. Neurobiol. 69, 143–179. Smith, D.F., Whitesell, L., Nair, S.C., Chen, S., Prapapanich, V., Rimerman, R.A., 1995. Progesterone receptor structure and function altered by geldanamycin, an hsp90binding agent. Mol. Cell. Biol. 15, 6804–6812. Sonna, L.A., Fujita, J., Gaffin, S.L., Lilly, C.M., 2002. Invited review: effects of heat and cold stress on mammalian gene expression. J. Appl. Physiol. 92, 1725–1742. Sullivan, W.P., Owen, B.A., Toft, D.O., 2002. The influence of ATP and p23 on the conformation of hsp90. J. Biol. Chem. 277, 45942–45948.
Thibault, O., Porter, N.M., Chen, K.C., Blalock, E.M., Kaminker, P.G., Clodfelter, G.V., Brewer, L.D., Landfield, P.W., 1998. Calcium dysregulation in neuronal aging and Alzheimer's disease: history and new directions. Cell Calcium 24, 417–433. Troup, G.M., Smith, G.S., Walford, R.L., 1969. Life span, chronologic disease patterns, and age-related changes in relative spleen weights for the Mongolian gerbil (Meriones unguiculatus). Exp. Gerontol. 4, 139–143. Waza, M., Adachi, H., Katsuno, M., Minamiyama, M., Sang, C., Tanaka, F., Inukai, A., Doyu, M., Sobue, G., 2005. 17-AAG, an Hsp90 inhibitor, ameliorates polyglutaminemediated motor neuron degeneration. Nat. Med. 11, 1088–1095. Wei, Y.H., Lee, H.C., 2002. Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp. Biol. Med. (Maywood) 227, 671–682. Welch, W.J., 1992. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72, 1063–1081. Wiech, H., Buchner, J., Zimmermann, R., Jakob, U., 1992. Hsp90 chaperones protein folding in vitro. Nature 358, 169–170.
Contents lists available at ScienceDirect
Experimental Gerontology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ex p g e r o
Short report
Heat shock protein 90 and its cochaperone, p23, are markedly increased in the aged gerbil hippocampus Choong Hyun Lee a, Joon Ha Park b, Jung Hoon Choi c, Ki-Yeon Yoo d, Pan Dong Ryu a,⁎, Moo-Ho Won b,⁎⁎ a
Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, 200-701, South Korea Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, South Korea d Department of Oral Anatomy, College of Dentistry, Gangneung-Wonju National University, Gangneung 210-702, South Korea b c
a r t i c l e
i n f o
Article history: Received 6 March 2011 Received in revised form 19 April 2011 Accepted 6 May 2011 Available online 13 May 2011 Section Editor: Christian Humpel Keywords: Heat shock proteins Chaperone Aging Hippocampus proper Dentate gyrus
a b s t r a c t In the present study, we compared HSP90 and its co-chaperone, p23, immunoreactivity and their protein levels in the hippocampus between adult (postnatal month 6) and aged (postnatal month 24) gerbils using immunohistochemistry and western blot analysis. HSP90 immunoreactivity was markedly increased in pyramidal cells in the hippocampus proper and in polymorphic cells in the dentate gyrus of the aged group compared to the adult group. p23 immunoreactivity was slightly increased in pyramidal cells of the hippocampus proper and in granule cells of the dentate gyrus in the aged group. In addition, HSP90 and p23 protein levels in the aged hippocampus were much higher than the adult hippocampus. These results indicate that HSP90 and p23 immunoreactivity and protein levels in the hippocampus are distinctively increased in the aged gerbils compared to the adult gerbils. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Aging is closely related to neurobiological, neuropathological and neurochemical alterations in the central nervous system (ErrajiBenchekroun et al., 2005; Lister and Barnes, 2009). Brain aging can lead to various changes in structural plasticity, oxidative stress, DNA repair ability, calcium regulation and neuroinflammatory function (Godbout and Johnson, 2006; Thibault et al., 1998; Wei and Lee, 2002). In addition, aging in cellular functions is also related to the accumulation of misfolded proteins and failure of chaperoning systems (Macario and Conway de Macario, 2002). Among various brain regions, the hippocampus has been well known as a most vulnerable and sensitive region to aging process (Casadesus et al., 2005; Jacobson et al., 2008; Rosenzweig and Barnes, 2003). Heat shock proteins (HSPs) are highly induced following exposure to cellular stress, and HSPs are also expressed in unstressed cells (Sonna et al., 2002; Welch, 1992). One of main functions of HSPs is well known as molecular chaperones that guide protein folding, unfolding, assembly
⁎ Corresponding author. Tel.: + 82 2 880 1254. ⁎⁎ Corresponding author. Tel.: + 82 33 250 8891; fax: + 82 33 256 1614. E-mail addresses: [email protected] (P.D. Ryu), [email protected] (M.-H. Won). 0531-5565/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2011.05.002
and so on (Gething and Sambrook, 1992; Welch, 1992). HSPs are divided into major families, such as HSP90, HSP70 and HSP60 (Jolly and Morimoto, 2000). Among them, HSP90 has molecular chaperoning activity and plays roles in protein refolding and signal transduction (Imai et al., 2003; Wiech et al., 1992). In HSP90 chaperone activity, the formation of dynamic complex is required with other chaperones, co-chaperones and other binding proteins (Smith et al., 1995). Among co-chaperones, p23 is a small protein, closely involved in HSP90 chaperone activity (Pearl and Prodromou, 2006; Sullivan et al., 2002). The interaction between HSP90 and p23 requires ATP and occurs in the late stage of chaperone cycle (Sullivan et al., 2002). In addition, p23 is known to act as a chaperone in absence of HSP90 (Bose et al., 1996; Oxelmark et al., 2003). Although some studies have been focused on the relationship between the modulation of HSP90 and protein misfolding diseases (Auluck et al., 2002; Dickey et al., 2007; Waza et al., 2005), few studies on changes in HSP90 in the hippocampus during normal aging have been reported (Ghi et al., 2009). In addition, although HSP90 and p23 have been well known as their chaperoning activity, it has not clearly elucidated how both HSP90 and p23 are changed in the increase of misfolded proteins and modification of chaperoning systems with age yet, especially in the aged rodent brain. In the present study, therefore, we compared HSP90 and its co-chaperone, p23, immunoreactivity and their protein levels in the hippocampus between adult and aged gerbils.
C.H. Lee et al. / Experimental Gerontology 46 (2011) 768–772 Table 1 Semi-quantifications of HSP90 and p23 immunostaining intensity in the hippocampus between adult and aged gerbils. Postnatal month (PM)
HSP90
p23
Pyramidal cells Non-pyramidal cells Granule cells Polymorphic cells Pyramidal cells Non-pyramidal cells Granule cells Polymorphic cells
PM 6
PM 24
± + − + ± + ± +
++ + ± ++ + + + +
The intensity of immunostaining was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥ 200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively.
2. Materials and methods 2.1. Experimental animals We used male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chuncheon, South Korea, at postnatal month 6 (PM 6) and PM 24 as adult and aged group, respectively. The animals (n = 14 at each age) were housed in a conventional state under adequate temperature (23 °C) and humidity
769
(60%) control with a 12-h light/12-h dark cycle, and provided with free access to food and water. The procedures for handling and caring for animals adhered to the guidelines that are in compliance with the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996), and were approved by the Institutional Animal Care and Use Committee of Hallym's Medical Center. All of the experiments were conducted in a way as to minimize the number of animals used and the suffering caused by the procedures used in the present study. 2.2. Immunohistochemistry The animals (n = 7 per group) were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphatebuffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and postfixed with the same solution for 6 h. The tissues were cryoprotected by infiltration with 30% sucrose overnight. The brain tissues were then frozen and sectioned with a cryostat at 30 μm, and consecutive sections were collected in six-well plates containing 0.1 M PBS. To ensure that immunohistochemical data were comparable between groups, the free-floating sections were carefully processed under the same conditions. The sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. They were next incubated with diluted mouse anti-HSP90 (1:100, Abcam, Cambridge, UK) or mouse anti-p23 (1:100, Abcam) overnight at room temperature and subsequently
Fig. 1. HSP90 immunohistochemistry in the CA1 region (A and B), CA2/3 region (C and D) and dentate gyrus (E and F) at PM 6 (A, C, and E) and PM 24 (B, D, and F). In the CA1 and CA2/3 regions at PM 24, HSP90 immunoreactivity in pyramidal cells (asterisks) of the stratum pyramidale (SP) is markedly increased compared to the PM 6 group; however, HSP90 immunoreactivity in non-pyramidal cells (arrows) is not increased. In the dentate gyrus, HSP90 immunoreaction is hardly detected in granule cells (asterisks) of the granule cell layer (GCL) in the PM 6 group; however, HSP90 immunoreactivity is increased at PM 24. Polymorphic cells (arrows) at PM 24 is much stronger than the PM 6 group. SO, stratum oriens; SR, stratum radiatum; ML, molecular layer; PL, polymorphic layer. Scale bar = 100 μm.
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Fig. 2. Immunohistochemistry for p23 in the CA1 region (A and B), CA2/3 region (C and D) and dentate gyrus (E and F) at PM 6 (A, C, and E) and PM 24 (B, D, and F). In the CA1-3 regions at PM 24, p23 immunoreactivity in pyramidal cells (asterisks) of the stratum pyramidale (SP) is higher than the PM 6 group; however, the immunoreactivity in nonpyramidal cells (arrows) is not changed. In the dentate gyrus, p23 immunoreactivity in the granule cells (asterisk) is weak at PM 6 group, and the immunoreactivity is slightly increased at PM 24. p23 immunoreactivity in polymorphic cells (arrows) at PM 24 is not altered. SO, stratum oriens; SR, stratum radiatum; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar = 100 μm.
exposed to biotinylated goat anti-mouse IgG (1:200, Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector). Then, the sections were visualized by staining with 3,3′-diaminobenzidine in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration the sections were mounted with Canada balsam (Kanto, Tokyo, Japan). A negative control test was carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining. The negative control resulted in the absence of immunoreactivity in all structures. Eight sections per animal were selected to quantitatively analyze HSP90 and p23 immunoreactivity, respectively. HSP90 and p23 immunoreactivity was graded: Digital images of the hippocampal subregions were captured with an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss,
Fig. 3. Western blot analysis of HSP90 and p23 in the gerbil hippocampus derived from the PM 6 (adult, n = 7) and PM 24 (aged, n = 7) groups.
Germany) connected to a PC monitor. Semi-quantification of the immunostaining intensity of HSP90 and p23 was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). The mean intensity of HSP90 and p23 immunostaining in each immunoreactive structure was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively. 2.3. Western blot analysis To confirm change in HSP90 and p23 levels in the hippocampus between groups, animals at each ages (n = 7) were used for western blot analysis. After sacrificing animals, the hippocampus was removed. The tissues were then homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol bis (2-aminoethyl ether)-N,N, N′,N′ tetraacetic acid (pH 8.0), 0.2% Nonidet P-40, 10 mM ethylendiamine tetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol (DTT). After centrifugation at 10,000 g, the protein level in the supernatants was determined using a Micro BCA protein assay kit with bovine serum albumin as a standard (Pierce Chemical, Rockford, IL). Aliquots containing 20 μg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM
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DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. The aliquots were then loaded onto a 12% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Corp, East Hills, NY). To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, followed by incubation with mouse anti-HSP90 (1:1,000, Abcam) or mouse anti-p23 antiserum (1:1000, Abcam), peroxidase-conjugated goat anti-mouse IgG (Sigma) and an ECL kit (Pierce Chemical). 3. Results 3.1. HSP90 and p23 immunoreactivity Weak HSP90 immunoreactivity was observed in pyramidal cells in the stratum pyramidale (SP) of the hippocampus proper (CA1-3 regions) in the PM 6 group (Table 1, Fig. 1A and C). At PM 24, HSP90 immunoreactivity in pyramidal cells is distinctively increased compared to the PM 6 group (Table 1, Fig. 1B and D). Non-pyramidal cells in the strata oriens (SO) and radiatum (SR) showed moderate HSP90 immunoreactivity, and the immunoreactivity was not changed at PM 24 (Table 1, Fig. 1A–D). HSP90 immunoreactivity in granule cells in the granule cell layer (GCL) of the dentate gyrus was hardly detected at PM 6 (Table 1, Fig. 1E); In the PM 24 group, weak HSP90 immunoreactivity was detected in the GCL (Table 1, Fig. 1F). Moderate HSP90 immunoreactivity was detected in polymorphic cells in the polymorphic layer (PL) of the dentate gyrus, and the immunoreactivity was strong at PM 24 (Table 1, Fig. 1E and F). The change pattern of p23 immunoreactivity in pyramidal and non-pyramidal cells in the CA1-3 regions of both the PM 6 and 24 groups was similar to the change pattern of HSP90 immunoreactivity (Table 1, Fig. 2A–D). Granule cells in the GCL of the PM 6 group showed weak p23 immunoreactivity, and the cells in the PM 24 group moderated p23 immunoreactivity (Table 1, Fig. 2E and F). Polymorphic cells in PL of both the PM 6 and 24 groups showed moderate p23 immunoreactivity (Table 1, Fig. 2E and F). 3.2. HSP90 and p23 protein levels In western blot study, we found that changes in HSP and p23 levels in the hippocampus of both the adult and aged groups were generally similar to the immunohistochemical changes. HSP90 and p23 protein levels were increased in the PM 24 group compared to the PM 6 group (Fig. 3). 4. Discussion In the present study, we used Mongolian gerbils as an experimental animal. The Mongolian gerbil has a relatively short lifespan, and it is genetically homogeneous. It has been known that the mean survival lifespan of male gerbils is about 110 weeks, and the oldest lifespan is about 200 weeks (Troup et al., 1969). The Mongolian gerbil is also known to have a lot of unique physiological, behavioral and biological attributes, which are analogous to similar phenomena in humans (Cheal, 1986). Especially, auditory failure, loss of perception, cholesterol and bile acid metabolism, and thermoregulation in old gerbils are analogous to those in aged humans rather than rats and mice. Therefore, it has been suggested that the Mongolian gerbil is unique and a suitable model for research on aging, which could offer valuable information for gerontology (Cheal, 1986). In this study, we compared HSP90 immunoreactivity and its protein levels in the gerbil hippocampus. HSP90 immunoreactivity in the hippocampus proper and dentate gyrus of the aged group was much higher than the adult group. We also found that HSP90 protein level in the aged groups was significantly increased compared to the adult group. This result is supported by a previous study, which
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showed that HSP90 protein levels and genes were increased gradually in the dog hippocampus with age (Ghi et al., 2009). Another previous study has reported that basal HSP90 levels are significantly increased in blood cells of the elderly human subjects (Njemini et al., 2007). They suggested that higher levels of HSP90 might be due to the higher load of damaged proteins. p23, an important factor for HSP90 chaperone activity, is known to be necessary for perinatal survival and particularly for the final fetal stages of lung and skin development and maturation (Grad et al., 2006; Sullivan et al., 2002). In this study, we also observed that, like the results of HSP90 immunoreactivity and protein levels, both p23 immunoreactivity and protein level in the hippocampus of the aged group were increased compared to the adult group. It is hard to discuss why p23 immunoreactivity and protein level were increased in the aged hippocampus, because there is no study on age-related changes of p23 in the central nervous system. However, it can be postulated that the increase of p23 may be associated with the increase of damaged proteins and the requirement of chaperone activity in the aged hippocampus, as is the case in the increase of HSP90 in the aged hippocampus. In conclusion, our present study indicates that HSP90 and p23 immunoreactivity and its protein levels in the gerbil hippocampus are higher in the aged group rather than in the adult group. These elevations of HSP90 and p23 may reflect an increase of chaperone activity, which is closely related to increase of damaged proteins in the hippocampus with age. Acknowledgements This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2009-351-1E00001). References Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M., 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295, 865–868. Bose, S., Weikl, T., Bugl, H., Buchner, J., 1996. Chaperone function of Hsp90-associated proteins. Science 274, 1715–1717. Casadesus, G., Shukitt-Hale, B., Stellwagen, H.M., Smith, M.A., Rabin, B.M., Joseph, J.A., 2005. Hippocampal neurogenesis and PSA-NCAM expression following exposure to 56Fe particles mimics that seen during aging in rats. Exp. Gerontol. 40, 249–254. Cheal, M.L., 1986. The gerbil: a unique model for research on aging. Exp. Aging Res. 12, 3–21. Dickey, C.A., Kamal, A., Lundgren, K., Klosak, N., Bailey, R.M., Dunmore, J., Ash, P., Shoraka, S., Zlatkovic, J., Eckman, C.B., Patterson, C., Dickson, D.W., Nahman Jr., N.S., Hutton, M., Burrows, F., Petrucelli, L., 2007. The high-affinity HSP90–CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J. Clin. Invest. 117, 648–658. Erraji-Benchekroun, L., Underwood, M.D., Arango, V., Galfalvy, H., Pavlidis, P., Smyrniotopoulos, P., Mann, J.J., Sibille, E., 2005. Molecular aging in human prefrontal cortex is selective and continuous throughout adult life. Biol. Psychiatry 57, 549–558. Gething, M.J., Sambrook, J., 1992. Protein folding in the cell. Nature 355, 33–45. Ghi, P., Di Brisco, F., Dallorto, D., Osella, M.C., Orsetti, M., 2009. Age-related modifications of egr1 expression and ubiquitin–proteasome components in pet dog hippocampus. Mech. Ageing Dev. 130, 320–327. Godbout, J.P., Johnson, R.W., 2006. Age and neuroinflammation: a lifetime of psychoneuroimmune consequences. Neurol. Clin. 24, 521–538. Grad, I., McKee, T.A., Ludwig, S.M., Hoyle, G.W., Ruiz, P., Wurst, W., Floss, T., Miller III, C.A., Picard, D., 2006. The Hsp90 cochaperone p23 is essential for perinatal survival. Mol. Cell. Biol. 26, 8976–8983. Imai, J., Maruya, M., Yashiroda, H., Yahara, I., Tanaka, K., 2003. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 22, 3557–3567. Jacobson, L., Zhang, R., Elliffe, D., Chen, K.F., Mathai, S., McCarthy, D., Waldvogel, H., Guan, J., 2008. Correlation of cellular changes and spatial memory during aging in rats. Exp. Gerontol. 43, 929–938. Jolly, C., Morimoto, R.I., 2000. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J. Natl. Cancer Inst. 92, 1564–1572. Lister, J.P., Barnes, C.A., 2009. Neurobiological changes in the hippocampus during normative aging. Arch. Neurol. 66, 829–833. Macario, A.J., Conway de Macario, E., 2002. Sick chaperones and ageing: a perspective. Ageing Res. Rev. 1, 295–311.
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