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Presence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain
Brain Research 816 Ž1999. 99–110
Research report
Presence of molecular chaperones, heat shock cognate ŽHsc . 70 and heat shock proteins ŽHsp . 40, in the postsynaptic structures of rat brain Tatsuo Suzuki a
a, )
, Nobuteru Usuda b , Shigeru Murata c , Ayami Nakazawa b , Kenzo Ohtsuka d , Hiroshi Takagi
Department of Neuroplasticity, Research Center on Aging and Adaptation, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan b Department of Anatomy, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan c Department of Brain Surgery, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan d Laboratory of Experimental Radiology, Aich Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan Accepted 13 October 1998
Abstract The synaptic localization of molecular chaperones, heat shock cognate protein 70 ŽHsc70. and Hsp40, was investigated immunohistochemically in the normal rat brain. Postsynaptic density ŽPSD. fractions contained a constitutive form of HSP70, heat shock cognate protein 70 ŽHsc70 or p73. but not inducible form of HSP70 Žp72.. The immunoreactivities of Hsc70 Žp73. were distributed throughout the rat brain, in neuronal somata, dendrites and axons. Their immunoreactivity in neurons was localized in the cytoplasmic matrix, dendrites, and spines at the electron microscopic level. Presynaptic terminals, but less frequently than postsynaptic staining, were also reactive. Postsynaptic areas immediately beneath the synaptic contact or PSDs were immunoreactive for Hsc70. The Hsp40 was highly concentrated in PSD fractions. The staining of Hsp40 immunoreactivity was punctate and distributed widely in the brain. Hsp40 immunoreactivity was localized in dendritic spines, especially in the subsynaptic web, with weak staining of PSDs at the electron microscopic level. Double immunofluorescent staining and confocal microscopy revealed that Hsc70 and Hsp40 were co-localized on somata and neuronal processes of cultured cerebral neurons, on which synaptophysin immunoreactive spots were scattered. These results suggest that Hsp40 and Hsc70 are co-localized at postsynaptic sites and postsynaptic chaperone activity may be mediated by these two heat shock proteins. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Heat shock protein; Postsynaptic density; Molecular chaperone; Synapse; Synaptic plasticity; Immunohistochemistry
1. Introduction Several polypeptides known as heat- or stress-inducible proteins are expressed in prokaryotes and eukaryotes. They are commonly referred to as heat-shock proteins ŽHsps.. The Hsps are classified into several groups according to their molecular sizes. The expressions of these proteins are induced by the activation of heat shock elements ŽHSE. in the promoter region of their genes. HSP70, a homologue of E. coli DnaK protein, has two major members, Hsp70 Žp72., an inducible form, and the heat shock cognate protein ŽHsc 70, p73., a constitutively expressed form. We refer to the constitutive form of HSP70 as Hsc70 or p73, and the inducible form as p72. We use fully capitalized names to denote the protein family Žex. HSP70. and an ) Corresponding author. Fax: [email protected]
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initial capital letter for specific family members Žex. Hsc70, Hsp40. according to the recent instruction w14x. The general term HSP70 implies both Hsc70 Žp73. and p72. The two proteins are similar with approximately 90% identity in amino acid sequences and mostly indistinguishable biochemical properties except for a slight difference in their electrophoretic mobility w37x. HSP70 is expressed ubiquitously in various tissues, but among adult tissues, only the brain retains high levels of Hsc70 gene expression w13x. The relative levels of Hsp70 mRNA and Hsc70 mRNA are higher in cerebellum than in cerebral cortex and both the transcription and turnover of Hsp70 mRNAs differs between cerebellum and cerebral cortex w30x. The expression of brain HSP70 can be induced by seizure w20x and ischemia w2,41x. Thus, Hsp70 is one of the candidate plasticity-related genes ŽCPGs. w33x. HSP70 has chaperone activity, which facilitate the repair partially denatured proteins after various stress or
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assists correct folding of nascent polypeptides w4x. HSP70s in the yeast endoplasmic reticulum and mitochondria are involved in the translocation and targeting of proteins w6x. Furthermore, HSP70 appears to be involved in the transport of cytoplasmic proteins into nucleus, and shuttles between these two compartments w19,27,28,43,45x. HSP70 does not function by itself in vivo and requires assistance by the DnaJ family proteins. HSP70, with the aid of various kinds of DnaJ-like proteins, fulfills various cellular functions w5,10x. All DnaJ-like proteins contain J-domains, by which the proteins interact with HSP70 and stimulate its ATPase activity. Different DnaJ-like proteins have specialized functions, being coupled to HSP70. Thus, the roles of HSP70 and its target molecules are likely to be determined by the types of partner DnaJ homologs w6x or co-compartmentalization of certain types of DnaJ homologs and HSP70s w44x. Ohtsuka et al. w35x have recently identified a novel DnaJ-like protein, Hsp40, in mammalian and avian cells and showed the co-localization of Hsp40 and HSP70 in HeLa cells and various mammalian cells w17,56x. A detailed localization of HSP70 and its partner chaperones in the brain have not yet been reported. We report here the distribution of Hsc70 and Hsp40 at synaptic sites, and discuss their potential roles in neuronal and synaptic functions.
fraction was prepared by the method of Wu et al. w55x. P1 Žfraction containing nuclei and cell debris., P2 Žcrude mitochondrial fraction., synaptosomal, and mitochondrial fractions were obtained during PSD isolation, and the synaptic plasma membrane ŽSM. fraction was prepared by sucrose gradient centrifugation after hypo-osmotic treatment of the synaptosomal fraction w47x. The soluble fraction was obtained after homogenization in Tris–HCl buffer Ž20 mM, pH 7.4. containing phenylmethylsulfonyl fluoride Ž1 mM., leupeptin Ž10 mM., pepstatin A Ž10 mM., and calyculin A Žphosphatase inhibitor, 10 nM. followed by centrifugation at 100 000 = g for 1 h. HeLa cell proteins were recovered from HeLa cells on culture with or without treatment at 458C for 15 min followed by incubation at 378C for 16 h. All preparations were stored at y808C until use.
2. Materials and methods
Proteins were separated by SDS-PAGE using 10% polyacrylamide gels, then electroblotted onto Immobilon membranes. The membranes were blocked with 5% skimmed milk ŽYukijirushi, Japan. and immunostained using the SuperSignal substrate ŽPierce.. Chemiluminescence was captured and visualized by a CCD video camera system ŽAtto Densitograph Lumino CCD AE-6930.. KO4 antibody, anti-p73 antibody, anti-p72 antibody, anti-Hsp40 antibody, anti-synaptophysin antibody, HRPO-labeled anti-rabbit immunoglobulin antibody, HRPO-labeled antirat immunoglobulin antibody, and HRPO-labeled antimouse immunoglobulin antibody were diluted 1:2000, 1:10 000, 1:2000, 1:2000, 1:2000, 1:20 000, 1:20 000, and 1:5000, respectively.
2.1. Materials Mouse monoclonal anti-p72 antibody ŽRPN1197. was purchased from Amersham; mouse monoclonal anti-synaptophysin antibody ŽMAB368. was from Chemicon; antiPSD-95 antibody was from Transduction Laboratories; anti-rabbit IgG was from Vector Laboratories; Vectastain ABC kit containing anti-rabbit IgG and avidin-biotinylated horseradish peroxidase ŽHRPO. complex was from Vector Laboratories; HRPO-coupled goat anti-rabbit IgG ŽH and L. from Calbiochem.; HRPO-coupled goat anti-mouse IgŽG q A q M. from Cappel; HRPO-coupled goat anti-rat IgGŽH q L. from Chemicon; Fluorescein isothiocyanate ŽFITC.-labeled goat anti-rat IgGŽH q L. and rhodamine isothiocyanate ŽTRITC.-labeled goat anti-rabbit IgGŽH q L. were from Zymed; FITC-labeled goat anti-mouse IgG from Tago; Epok 812 was from Oken ŽTokyo, Japan.; SuperSignal substrate, chemiluminescent detection reagent for Western blotting, was from Pierce; polyvinylidene difluoride ŽPVDF. membranes ŽImmobilon. were from Millipore. Other chemicals were of reagent grade. 2.2. Subcellular fractionation and protein extraction Subcellular fractions of the brain were obtained from Wistar rats Ž6 weeks old, unless otherwise stated.. PSD
2.3. Preparation of antibodies Rabbit polyclonal anti-HSP70 antibody, KO4, rat monoclonal anti-Hsc70 Žp73. antibody, 1B5, and rabbit polyclonal anti-Hsp40 antibody were produced as described previously w16,17,34x. KO4 anti-HSP70 antibody specifically recognizes both p73 and p72 w17,56x. 2.4. Immunoblotting
2.5. Immunohistochemical examination For immunohistochemical examination at the light microscopic level, brains from Wistar rats Žmales, 4 weeks old. were fixed with 4% paraformaldehyde in phosphate buffer ŽPB. Žsodium salt, 100 mM, pH 7.4. by perfusion through left heart ventricle for 15 min and then by immersion for 3 h. After treatment with 0.1 M lysine in the phosphate buffered saline ŽPBS. Žsodium salt, 100 mM, pH 7.4. for 3 h in order to quench free aldehyde groups, they were dehydrated in a series of ethanol and xylene, and embedded in paraffin. After rehydration, the sections were incubated in PBS Ž20 mM. containing 2% normal horse
T. Suzuki et al.r Brain Research 816 (1999) 99–110
serum for 30 min, and anti-HSP70 Ž1:3000 dilution. or anti-Hsp40 antibodies Ž1:1500 dilution. for 12 h. After washing in PBS, they were incubated with biotinylated anti-rabbit IgG for 1 h and with avidin-biotinylated peroxidase complex for 1 h. The reaction product was developed by incubation in 0.05% 3, 3X-diaminobenzidine ŽDAB. tetrahydrochloride and 0.01% hydrogen peroxide in Tris– HCl buffer Ž50 mM, pH 7.5.. They were examined and photomicrographed with a Nikon FXA microscope after counterstaining with 1% methyl green. Some sections were immersed in sodium citrate Ž20 mM, pH 6.0. and boiled for 10 min in a microwave oven after deparaffinization and rehydration. No staining was obtained in the brain tissues following the protocols described above when primary antibodies were omitted or replaced by normal serum. For immunoelectron microscopy, tissues were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PB by perfusion through left heart ventricle for 15 min and by immersion for 3 h. After washing in 0.1 M lysine for 3 h, they were immersed in graded sucrose solutions: 10%, 15% and 20% for 10 h each. Frozen in a mixture of dry ice and n-hexane, 6 mm sections were cut on a cryostat microtome and collected on silane-coated glass slides. After permealizing in 0.1% Triton X-100rPBS, they were treated with the anti-HSP70 Ž1:1500 dilution. or anti-Hsp40 antibodies Ž1:1500 dilution., biotinylated anti-rabbit IgG antibody, and avidin-biotinylated peroxidase by the procedure described above. The reaction product was developed for 15 min in DAB and hydrogen peroxide in PB containing 0.025% cobalt chloride and 0.02% nickel ammonium w1x. They were post-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide in PB and then dehydrated with
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graded ethanol. After embedding in Epok 812, ultra-thin sections of 0.1 mm were cut with a Porter Blum MT2B ultramicrotome. They were examined by a JEOL 1200 EX electron microscope at an accelerating voltage of 100 kV without staining with uranyl acetate or lead citrate w50,51x. No staining was obtained in the brain tissues following the protocol described above when primary antibodies were omitted or replaced by normal serum. Double immunofluorescent staining using combination of rabbit anti-Hsp40 Ž1r100 dilution. antibody and either one of rat anti-p73 Ž1r60 dilution. or mouse anti-synaptophysin Ž1r100 dilution. antibody were followed essentially the protocol described previously w17x. The staining was applied to primary cultured neurons of rat cerebral cortex that were maintained for 25 days from embryonic day 19 ŽE19P25.. Dilutions of the second antibodies were 1:50 for FITC-labeled anti-rat IgG and rhodamine-labeled anti-rabbit IgG and 1:100 for FITC-labeled anti-mouse IgG. Immunoreactivities for p73, Hsp40, and synaptophysin were finally labeled with FITC, rhodamine, and FITC, respectively. Double-stained samples were examined with a laser confocal microscope ŽFluoview, Olympus, Japan.. No staining was obtained in the brain tissues following the protocol described above when primary antibodies were omitted. 2.6. Additional procedures Electrophoresis was performed using the discontinuous SDS-buffer system of Laemmli Ž1970. w23x. Protein was assayed by the method of Lowry et al. Ž1951. w26x with bovine serum albumin as the standard. Proteins were solu-
Fig. 1. Detection of HSP70 and Hsp40 in the PSD fractions prepared from the rat forebrain. PSD proteins Ž15 mg. prepared from rat forebrain and proteins Ž4 mg. of HeLa cells with Žindicated by qH. or without Žindicated by y. treatment at 458C were separated on 10% polyacrylamide gels and Western blotted using KO4 anti-HSP70, anti-p73, anti-p72, and anti-Hsp40 antibodies. Antibodies used are indicated under the gels. Molecular weight standards are shown in kDa on the left.
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bilized for protein assay by boiling in 1 N NaOH for 10 min.
3. Result 3.1. Identification of HSP70 and Hsp40 in the PSD fraction We used three different antibodies to HSP70 and an antibody to Hsp40. The validity of these antibodies has been previously reported w17,56x, and reproduced in Western blots using HeLa cells either nontreated or treated at 458C ŽFig. 1.. All antibodies showed specific bands with proper molecular sizes, and detected increased expression of HSP70 and Hsp40 after heat-treatment of cells except for the 1B5 antibody which recognizes Hsc70, the constitutive form of HSP70. Thus, we judged that the antibodies used were applicable in this study. The presence of chaperone proteins in synaptic structures was tested by Western blotting of proteins in PSD fractions prepared from rat forebrain ŽFig. 1.. There was a single immunoreactive band recognized by KO4 antiHSP70 antibody, which recognizes both p72 ŽHsp70. and p73 ŽHsc70. ŽFig. 1a.. The molecular size of the im-
munoreactive band in PSD fractions was the same as the HSP70 in HeLa cell extracts, which contains both p73 and p72 w17,56x. The HSP70 immunoreactive band was further analyzed by an antibody specific to p73 vs. p72. The band reactive to KO4 antibody in PSD fractions was also reactive to anti-p73 antibody but not to anti-p72 antibody. The band immunoreactive to anti-p72 antibody in PSD fractions was not detected even under conditions that increased sensitivity by prolonging the duration of chemiluminescence more than 20 times the standard measurement. These results suggested that the prominent Hsp in PSDs detected by KO4 anti-HSP70 antibody was p73, the constitutive form of HSP70 ŽHsc70.. This result is consistent with a previous report, in which p70 is not expressed in cells from rodents maintained under normal temperatures w17,56x. Thus, PSDs prepared from unstressed brain contains only the constitutive form of HSP70. There was also a single immunoreactive band recognized by anti-Hsp40 ŽFig. 1b.. The size of this immunoreactive band in PSDs was the same as the Hsp40 in HeLa cell. The distribution of HSP70 and Hsp40 in the PSD fraction was further examined in the fractions prepared from rat cerebral cortex, hippocampus and cerebellum. All PSD fractions examined contained p73 and Hsp40 but not p72 ŽFig. 2..
Fig. 2. Detection of HSP70 and Hsp40 in the PSD fractions prepared from various parts of the rat brain. PSD proteins Ž15 mg. prepared from cerebral cortex ŽCTX., hippocampus ŽHIP. and cerebellum ŽCBLM. of rat and proteins of heat-treated HeLa cells Ž7 mg. were separated on 10% polyacrylamide gel and Western blotted using KO4 anti-HSP70, anti-p73, anti-p72, and anti-Hsp40 antibodies. Fig. 3. Subcellular distribution of HSP70 and Hsp40 in the rat forebrain. Proteins of various subcellular fractions Ž25 mg. and those of heat-treated HeLa cells Ž7 mg. were separated on a 10% polyacrylamide gel and Western blotted as described in the Fig. 2 legend. Sp refers to synaptophysin and S, Syn, and SM to soluble, synaptosomal and synaptic plasma membrane fractions, respectively. The subcellular distribution pattern of PSD-95 verifies the fractionation. Fig. 4. Developmental changes in HSP70 and Hsp40 in the PSD fraction. PSD proteins Ž15 mg. prepared from the rat forebrain at various ages Ž2, 3, 4 and 6 weeks old. and proteins of heat-treated HeLa cells Ž7 mg. were separated by SDS-PAGE Ž10%. and Western blotted as described in the Fig. 2 legend.
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The subcellular distribution of HSP70 and Hsp40 was investigated in various fractions prepared from rat forebrain ŽFig. 3.. The bands immunoreactive to KO4 and anti-p73 antibodies were present with similar abundance in all fractions. There was no immunoreactive p72 in any fraction. The Hsp40 was highly concentrated in the PSD fraction, less abundant in the crude nuclear pellet ŽP1., and was barely detected in the synaptosome and synaptic plasma membrane ŽSM. fractions. The distribution of synaptophysin, a presynaptic marker in the synaptic vesicle membrane, was also compared with that of Hsp40. The distribution of the two proteins was different. Developmental changes were investigated in the HSP70 and Hsp40 content of PSDs prepared from the forebrain ŽFig. 4.. The level of HSP70 and p73 in PSDs 2 weeks after birth was about 50% of adult level and its level did
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not change significantly during 3 to 6 weeks after birth. There was no p72 band in PSDs at any developmental age. The level of Hsp40 in PSD fraction 2 weeks after birth was less than half of the adult level and its level was similar in PSDs during 3 to 6 weeks after birth. Thus, the developmental patterns of these two proteins in PSDs were similar and both proteins increase in amount during the period of synaptogenesis w21,48,50x. 3.2. Immunohistochemical localization of HSP70 in the rat brain at the light microscopic leÕel We used the KO4 anti-HSP70 antibody for immunohistochemical analysis of HSP70 because the KO4 antibody is polyclonal and 1B5 anti-Hsc70 Žp73. antibody is monoclonal. Although the KO4 antibody recognized both p73
Fig. 5. Localization of HSP70 immunoreactivity at the light microscopic level in the cerebral cortex, hippocampus, and cerebellum. Deparaffinized brain tissues were stained with KO4 anti-HSP70 antibody. Ža. Cerebral cortex, Žb. CA1 region of hippocampus, Žc. and Žd. cerebellum. Tissues shown in Ža. – Žc. were treated with microwave Žindicated by q., but that in Žd. was not Žindicated by y., after deparaffinization and rehydration. Somas and dendrites, particularly as seen in Žd., of neuronal cells are positive for the HSP70 immunoreactivity. Bars, 50 mm.
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and p72 in the HeLa cells, the staining revealed by the KO4 antibody in the unstressed normal brain thought to be primarily against the p73 because the normal brain does not contain detectable levels of p72 ŽFigs. 1–4.. The HSP70 immunoreactivity was distributed throughout the rat brain, in neuronal somata, dendrites and axons. Nuclei were also positive, but very weak. Glial cells were also reactive in tissues treated with microwave irradiation after deparaffinization and rehydration, which increased staining sensitivity. Typical examples with or without microwave treatment after deparaffinization and rehydration are shown in Fig. 5. In the neocortex, pyramidal cells were strongly positive for HSP70 in all layers I through VI. In the hippocampus, pyramidal neurons through CA1 to CA4 and granular neurons in the dentate gyrus were stained. Somas and dendrites were clearly stained but nuclei were not in neurons of the cerebral cortex and hippocampus. In the
cerebellum, Purkinje cells were strongly immunoreactive, and the reaction in the granular cell layer was weak ŽFig. 5c.. In specimens without enhancement by microwave irradiation, only Purkinje cell dendrites were immunoreactive and somas of Purkinje cells were not immunoreactive ŽFig. 5d.. 3.3. Immunohistochemical localization of Hsp40 in rat brain at the light microscopic leÕel Hsp40 immunoreactivity in rat brain showed a distribution very similar to that of Hsc70, being distributed throughout the rat brain, in neuronal somata and dendrites. Glial cells were also reactive especially in microwave irradiated tissues. The distribution in the cerebral cortex, hippocampus, and cerebellum are shown in Fig. 6. Cerebral cortex without microwave treatment showed punctate stainings throughout all layers ŽFig. 6b., which suggested that Hsp40
Fig. 6. Localization of Hsp40 immunoreactivity at the light microscopic level in the cerebral cortex, hippocampus, and cerebellum. Deparaffinized brain tissues were stained with anti-Hsp40 antibody. Ža. and Žb. Cerebral cortex, Žc. CA1 region of hippocampus, Žd. cerebellum. Tissues shown in Ža., Žc., and Žd., were treated with microwave Žindicated by q., but that in Žb. was not Žindicated by y., after deparaffinization and rehydration. Bars in Ža., Žc., and Žd., 50 mm. Bar in Žb., 5 mm.
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was concentrated in these restricted but widespread regions in the cerebral cortex. In the hippocampus, pyramidal neurons through CA1 to CA4 and granular neurons in the dentate gyrus were stained but weakly compared with those in the cerebral cortex. In the cerebellum, somas and dendrites of Purkinje cells were weakly stained. Granular neurons and cells in the molecular layer, probably microglia, were strongly immunoreactive.
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3.4. Intracellular localization of HSP70 and Hsp40 at the electron microscopic leÕel The dendritic localization of HSP70 and Hsp40 ŽFigs. 5 and 6., the punctate staining of Hsp40 immunoreactivity ŽFig. 6b., and the presence of Hsc70 and Hsp40 in PSD fractions ŽFig. 1. suggested the localization of these proteins at synaptic sites. These results prompted us to exam-
Fig. 7. Localization of HSP70 immunoreactivity in the synaptic areas at an electron microscopic level. The immunoreactive regions of brain at the light microscopic level, unstained with uranyl acetate or lead citrate, were examined by an electron microscope. Pre and SSW refer to presynaptic terminal and subsynaptic web, respectively. Arrows delineate PSDs. Ža. and Žb. Cerebral cortex, Žc. and Žd. cerebellum. Presynaptic staining is shown in Ža. and postsynaptic in Žb., Žc., and Žd.. Bar, 200 nm.
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Fig. 8. Localization of Hsp40 immunoreactivity in the synaptic areas at an electron microscopic level. The immunoreactive regions of brain at the light microscopic level, unstained with uranyl acetate or lead citrate, were examined by an electron microscope. Pre and SSW refer to presynaptic terminal and subsynaptic web, respectively. Arrows delineate PSDs. Bars, 200 nm.
ine the localization of these two proteins at the electron microscopic level in the cerebral cortex and cerebellum, focusing on their localization at synapses. HSP70 immunoreactivity was localized in the cytoplasmic matrix, and dendrites in neurons of the neocortex and cerebellum.
Postsynaptic structures in the cerebral cortex and cerebellum were stained ŽFig. 7.. All or portions of PSDs in postsynaptic spines were stained. PSDs were immunoreactive but rather weak compared with regions of subsynaptic web. Postsynaptic structures of both axo-somatic and axo-
Fig. 9. Comparison of the distribution of Hsc70 and Hsp40 in cultured cortical neurons by double immunostaining and confocal microscopic examination. Localization in the cultured cerebral neurons Žrat, E19P25. were examined. Hsc70 and Hsp40 were labeled by FITC Ža. and rhodamine Žb., respectively. C is a mixture of the two staining. Bar, 50 mm. Fig. 10. Comparison of the distribution of Hsp40 and synaptophysin by double immunostaining and confocal microscopic examination. Localization in the cultured cerebral neurons Žrat, E19P25. were examined. Synaptophysin and Hsp40 were labeled by FITC Ža. and rhodamine Žb., respectively. C is a mixture of the two staining. Bar, 50 mm.
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dendritic synapses were positive for HSP70. Presynaptic terminals were also stained but less frequently than the postsynaptic structure ŽFig. 7a.. Either one of pre- or
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post-synaptic structure but not both of them in a single synapse was stained for HSP70. More than half of the synapses examined showed postsynaptic staining alone.
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The staining of the nucleus, mitochondria and other cell organella were not evident at the electron microscopic level. The synaptic localization of Hsp40 immunoreactivity in the cerebral cortex was also examined at the electron microscopic level. Postsynaptic structures but not presynaptic structures were stained ŽFig. 8.. PSDs were immunoreactive but rather weak compared to regions of subsynaptic web. Staining patterns of HSP70 and Hsp40 in the spine were similar. 3.5. Comparison of the distribution of Hsc70, Hsp40, and synaptophysin The distribution of Hsc70 and Hsp40 was compared in cultured cortical neurons by double immunofluorescent staining and laser confocal microscopy ŽFig. 9.. Both Hsc70 and Hsp40 were distributed throughout neurons. Glial cells were also reactive to both antibodies. The areas stained by both antibodies were not homogeneous but disconnected and punctate in some regions of neurons. Nearly all of the Hsp40 staining co-localized with that of Hsc70. The distribution of Hsp40 and synaptophysin, a presynaptic marker, was also examined by the same method in cultured neurons ŽFig. 10.. Synaptophysin immunoreactivity was distributed in a patch-like pattern in cultured neurons, to which Hsp40 was localized. Patchy stainings of synaptophysin may represent the localization of presynaptic terminals.
4. Discussion 4.1. Presence of Hsc70 and Hsp40 in the postsynaptic structures The presence of two kinds of chaperone proteins, Hsc70 and Hsp40, in postsynaptic structures was shown in this paper. Hsc70, a constitutive form of HSP70, was localized at synapses, being present both in the pre- and post-synaptic components but more frequently in postsynaptic. The presence of Hsc70 in postsynaptic sites has been suggested previously, however, its presence in vivo was not addressed. Only the presence of an Hsc70 epitope in the PSD fraction w24x and a short peptide homologous to Hsc70 derived from the 68 kDa protein in the PSD fraction have suggested the presence of the molecule in PSDs in vivo w15,54x. Hsc70 was previously known as b-internexin w32x and shares homology with a recently identified PSD protein, a-internexin Žneuron-specific intermediate filament protein. w50x. We also found that an inducible form of HSP70 Žp72. was undetectable in any subcellular compartments in the rat brain under normal unstressed conditions ŽFigs. 1–4..
p72 may be expressed in the brain only under stressed conditions. It should be pointed out that the distribution of Hsc70 Žp73. revealed in this paper was different from that of p72 expression in the adult rat brain induced after transient hypothermia and 6 h of recovery w29x. The difference in the distribution of the two forms of HSP70 suggests different functional roles in the brain for these biochemically similar proteins. Hsp40, which assists the function of HSP70 by stimulating its ATPase activity w11,31x, is also localized to postsynaptic sites. Thus, both Hsc70 and its cooperator Hsp40 are co-localized in the postsynaptic sites. The postsynaptic localization of these two proteins suggests specific roles of these proteins at the sites. The localization of Hsp40 is a marked contrast to the report that the cysteine string protein Žcsp., a mammalian DnaJ homologue w10x, is specifically localized to the presynaptic terminals, especially to synaptic vesicles w7,22x. Csp is believed to be involved in the fusion of synaptic vesicles and presynaptic membrane during neurotransmitter release w7x. These results indicate that two different kinds of DnaJ-like proteins, csp and Hsp40, are differentially localized on opposite sides of the synapse. Thus, Hsc70 with the aid of Hsp40, postsynaptic-specific DnaJ-like proteins, may fulfill a specific role at postsynaptic sites w10,46x. Another HSP, ubiquitin, may also be localized at the postsynaptic sites, because most of PSD proteins are reported to be ubiquitinated w9x. Ubiquitin’s role at postsynaptic sites and the ubiquitination of a number of PSD proteins are not known at present. The localization of HSP90 was not examined in this report, but it is reported to be expressed throughout all neurons in the brain and is expressed predominantly in perikarya, although minor immunoreactivity was observed in dendrites and nuclei w12x. The presence of HSP90 at synaptic sites is currently unknown. 4.2. Are postsynaptic chaperones inÕolÕed in synaptic plasticity? The chaperone activity of HSP70 is related to a variety of cellular functions as described in the introduction. The roleŽs. of the constitutive form of HSP70 Žp73. and inducible form of HSP70 Žp72. in the brain may be different Žsee above., although their biochemical activities are indistinguishable in vitro. The major role of the inducible form of HSP70 may be in the prevention of protein denaturation and following detrimental events in cells exposed to harmful stimuli because the p72 content is rather low until cells are insulted by harmful stimuli. The inducible p72 may be mobilized to sites of harmful cellular events. Many kinds of injuries induce HSP70, probably p72, in the CNS and HSP70 is essential to the neuroprotective effect of heatshock w42x. The inducible form of HSP can also protect neurons from glutamate excitotoxicity w39x.
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Several lines of evidence indicate that HSP70 is related to the expression of synaptic plasticity. Hsc70 can be induced by synaptic activation w20x, and is listed as a CPG w33x. Bip, another member of the HSP70, influences the in vitro phosphorylation of substrate proteins by src kinase w8x, a member of intracellular signaling pathways. Heat shock disrupts long-term memory consolidation in Caenorhabditis elegans, in which HSP20, 70, and 90 family proteins are expressed. In this case, the effect of heat shock response may be through altered protein synthesis w3x. Although most experiments did not discriminate between p72 and p73 in the synaptic plasticity-related events, Kaneko et al. w20x reported that electroconvulsive seizure induced p73 mRNA but not p72. Thus, molecular chaperones, especially Hsc70, at postsynaptic sites could be involved in mechanisms of synaptic plasticity. Considering that Hsc70 is constitutively expressed, the roles of Hsc70rHsp40 could be fundamental for synaptic transmission and its regulation under unstimulated conditions. One of the major roles of the HSP70 which is fundamental for normal synapse is the assistance of forming proper protein conformations by binding to nascent polypeptides w11x. Protein synthesis at postsynaptic sites may be required in unstressed normal neurons, and increased following the development of synaptic plasticity events, such as LTP and the consolidation process of memory w38,52,53x. Thus, it is possible that Hsc70rHsp40 at postsynaptic sites are involved in postsynaptic mechanisms of local protein synthesis, although other functions of HSP70 described above cannot be ruled out. HSP70 is a candidate of messenger from synapse to nucleus ŽMSN. w49x, because accumulating evidence suggests that HSP70 works as a nuclear shuttle. Hsc70 is localized in both the cytoplasm and the nucleus and shuttles between the two compartments w18,25,45x, and members of the HSP70 family recycle between the nucleus and cytoplasm of Xenopus oocytes w28x. Hsc70 is also involved in the nuclear transport of karyophilic proteins w19,36,43x. Hsc interacts with nuclear localization signal ŽNLS.-containing proteins in the cytoplasm before their nuclear import w19x. Hsc70 also interacts with RCC1, which is a chromatin-associated protein required for nuclear transport of proteins w40x. Thus, it is also possible that Hsc70 localized at postsynaptic sites might work as a MSN.
Acknowledgements We thank Dr. P.T. Kelly, University of Texas Medical School, Houston, TX, for his critical reading of the manuscript. This research was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, The Ichiro Kanehara Foundation, and Toyota Physical and Chemical Research Institute.
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w39x G. Rordorf, W.J. Koroshetz, J.V. Bonventre, Heat shock protects cultured neurons from glutamate toxicity, Neuron 7 Ž1991. 1043– 1051. w40x H. Saitoh, M. Dasso, The RCC1 protein interacts with Ran, RanBP1, Hsc70, and a 340-kDa protein in Xenopus extracts, J. Biol. Chem. 270 Ž1995. 10658–10663. w41x S. Sato, K. Abe, J. Kawagoe, M. Aoki, K. Kogure, Isolation of complementary DNAs for heat shock protein ŽHSP. 70 and heat shock cognate protein ŽHSC. 70 genes and the expressions in post-ischaemic gerbil brain, Neurol. Res. 14 Ž1992. 375–380. w42x K. Sato, H. Saito, N. Matsuki, HSP70 is essential to the neuroprotective effect of heat-shock, Brain Res. 740 Ž1996. 117–123. w43x Y. Shi, J.O. Thomas, The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate, Mol. Cell. Biol. 12 Ž1992. 2186–2192. w44x P.A. Silver, J.C. Way, Eukaryotic DnaJ homologs and the specificity of Hsp70 activity, Cell 74 Ž1993. 5–6. w45x G. Simos, E.C. Hurt, Nucleocytoplasmic transport: factors and mechanisms, FEBS Lett. 369 Ž1995. 107–112. w46x K. Sugito, M. Yamane, H. Hattori, Y. Hayashi, I. Tohnai, M. Ueda, N. Tsuchida, K. Ohtsuka, Interaction between Hsp70 and Hsp40, eukaryotic homologues of DnaK and DnaJ, in human cells expressing mutant-type p53, FEBS Lett. 358 Ž1995. 161–164. w47x T. Suzuki, K. Okumura-Noji, R. Tanaka, T. Tada, Rapid translocation of a-subunit of cytosolic Ca2qrcalmodulin-dependent protein kinase II to particulate fraction after decapitation, J. Neurochem. 63 Ž1994. 1529–1537. w48x T. Suzuki, K. Okumura-Noji, E. Nishida, ERK2-type mitogenactivated protein kinase ŽMAPK. and its substrates in postsynaptic density fractions from the rat brain, Neurosci. Res. 22 Ž1995. 277–285. w49x T. Suzuki, Messenger molecules that transmit synaptic information to the nucleus, Neurosci. Res. 25 Ž1996. 1–6. w50x T. Suzuki, S. Mitake, K. Okumura-Noji, H. Shimizu, T. Fujii, T. Tada, Localization of a-internexin in the postsynaptic density of the rat brain, Brain Res. 765 Ž1997. 74–80. w51x T. Suzuki, S. Mitake, K. Okumura-Noji, J.-P. Yang, T. Fujii, T. Okamoto, Presence of both NF-kB-like and Ik B-like immunoreactivities in postsynaptic densities in the rat brain, Neuroreport 8 Ž1997. 2931–2935. w52x H. Tiedge, J. Brosius, Translational machinery in dendrites of hippocampal neurons in culture, J. Neurosci. 16 Ž1996. 7171–7181. w53x E.R. Torre, O. Steward, Demonstration of local protein synthesis within dendrites using a new cell culture system that permits the isolation of living axons and dendrites from their cell bodies, J. Neurosci. 12 Ž1992. 762–772. w54x M.J. Walsh, N. Kuruc, The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing, J. Neurochem. 59 Ž1992. 667–678. w55x K. Wu, R. Carlin, P. Siekevitz, Binding of L-w3 Hx glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain, J. Neurochem. 46 Ž1986. 831–841. w56x M. Yamane, H. Hattori, K. Sugito, Y. Hayashi, I. Tohnai, M. Ueda, K. Nishizawa, K. Ohtsuka, Cotranslocation and co-localization of Hsp40 ŽDnaJ. with HSP70 ŽDnaK. in mammalian cells, Cell. Struct. Funct. 20 Ž1995. 157–166.
Research report
Presence of molecular chaperones, heat shock cognate ŽHsc . 70 and heat shock proteins ŽHsp . 40, in the postsynaptic structures of rat brain Tatsuo Suzuki a
a, )
, Nobuteru Usuda b , Shigeru Murata c , Ayami Nakazawa b , Kenzo Ohtsuka d , Hiroshi Takagi
Department of Neuroplasticity, Research Center on Aging and Adaptation, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan b Department of Anatomy, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan c Department of Brain Surgery, Shinshu UniÕersity School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan d Laboratory of Experimental Radiology, Aich Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan Accepted 13 October 1998
Abstract The synaptic localization of molecular chaperones, heat shock cognate protein 70 ŽHsc70. and Hsp40, was investigated immunohistochemically in the normal rat brain. Postsynaptic density ŽPSD. fractions contained a constitutive form of HSP70, heat shock cognate protein 70 ŽHsc70 or p73. but not inducible form of HSP70 Žp72.. The immunoreactivities of Hsc70 Žp73. were distributed throughout the rat brain, in neuronal somata, dendrites and axons. Their immunoreactivity in neurons was localized in the cytoplasmic matrix, dendrites, and spines at the electron microscopic level. Presynaptic terminals, but less frequently than postsynaptic staining, were also reactive. Postsynaptic areas immediately beneath the synaptic contact or PSDs were immunoreactive for Hsc70. The Hsp40 was highly concentrated in PSD fractions. The staining of Hsp40 immunoreactivity was punctate and distributed widely in the brain. Hsp40 immunoreactivity was localized in dendritic spines, especially in the subsynaptic web, with weak staining of PSDs at the electron microscopic level. Double immunofluorescent staining and confocal microscopy revealed that Hsc70 and Hsp40 were co-localized on somata and neuronal processes of cultured cerebral neurons, on which synaptophysin immunoreactive spots were scattered. These results suggest that Hsp40 and Hsc70 are co-localized at postsynaptic sites and postsynaptic chaperone activity may be mediated by these two heat shock proteins. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Heat shock protein; Postsynaptic density; Molecular chaperone; Synapse; Synaptic plasticity; Immunohistochemistry
1. Introduction Several polypeptides known as heat- or stress-inducible proteins are expressed in prokaryotes and eukaryotes. They are commonly referred to as heat-shock proteins ŽHsps.. The Hsps are classified into several groups according to their molecular sizes. The expressions of these proteins are induced by the activation of heat shock elements ŽHSE. in the promoter region of their genes. HSP70, a homologue of E. coli DnaK protein, has two major members, Hsp70 Žp72., an inducible form, and the heat shock cognate protein ŽHsc 70, p73., a constitutively expressed form. We refer to the constitutive form of HSP70 as Hsc70 or p73, and the inducible form as p72. We use fully capitalized names to denote the protein family Žex. HSP70. and an ) Corresponding author. Fax: [email protected]
q 81-263-37-2725;
E-mail:
initial capital letter for specific family members Žex. Hsc70, Hsp40. according to the recent instruction w14x. The general term HSP70 implies both Hsc70 Žp73. and p72. The two proteins are similar with approximately 90% identity in amino acid sequences and mostly indistinguishable biochemical properties except for a slight difference in their electrophoretic mobility w37x. HSP70 is expressed ubiquitously in various tissues, but among adult tissues, only the brain retains high levels of Hsc70 gene expression w13x. The relative levels of Hsp70 mRNA and Hsc70 mRNA are higher in cerebellum than in cerebral cortex and both the transcription and turnover of Hsp70 mRNAs differs between cerebellum and cerebral cortex w30x. The expression of brain HSP70 can be induced by seizure w20x and ischemia w2,41x. Thus, Hsp70 is one of the candidate plasticity-related genes ŽCPGs. w33x. HSP70 has chaperone activity, which facilitate the repair partially denatured proteins after various stress or
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 0 8 3 - X
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assists correct folding of nascent polypeptides w4x. HSP70s in the yeast endoplasmic reticulum and mitochondria are involved in the translocation and targeting of proteins w6x. Furthermore, HSP70 appears to be involved in the transport of cytoplasmic proteins into nucleus, and shuttles between these two compartments w19,27,28,43,45x. HSP70 does not function by itself in vivo and requires assistance by the DnaJ family proteins. HSP70, with the aid of various kinds of DnaJ-like proteins, fulfills various cellular functions w5,10x. All DnaJ-like proteins contain J-domains, by which the proteins interact with HSP70 and stimulate its ATPase activity. Different DnaJ-like proteins have specialized functions, being coupled to HSP70. Thus, the roles of HSP70 and its target molecules are likely to be determined by the types of partner DnaJ homologs w6x or co-compartmentalization of certain types of DnaJ homologs and HSP70s w44x. Ohtsuka et al. w35x have recently identified a novel DnaJ-like protein, Hsp40, in mammalian and avian cells and showed the co-localization of Hsp40 and HSP70 in HeLa cells and various mammalian cells w17,56x. A detailed localization of HSP70 and its partner chaperones in the brain have not yet been reported. We report here the distribution of Hsc70 and Hsp40 at synaptic sites, and discuss their potential roles in neuronal and synaptic functions.
fraction was prepared by the method of Wu et al. w55x. P1 Žfraction containing nuclei and cell debris., P2 Žcrude mitochondrial fraction., synaptosomal, and mitochondrial fractions were obtained during PSD isolation, and the synaptic plasma membrane ŽSM. fraction was prepared by sucrose gradient centrifugation after hypo-osmotic treatment of the synaptosomal fraction w47x. The soluble fraction was obtained after homogenization in Tris–HCl buffer Ž20 mM, pH 7.4. containing phenylmethylsulfonyl fluoride Ž1 mM., leupeptin Ž10 mM., pepstatin A Ž10 mM., and calyculin A Žphosphatase inhibitor, 10 nM. followed by centrifugation at 100 000 = g for 1 h. HeLa cell proteins were recovered from HeLa cells on culture with or without treatment at 458C for 15 min followed by incubation at 378C for 16 h. All preparations were stored at y808C until use.
2. Materials and methods
Proteins were separated by SDS-PAGE using 10% polyacrylamide gels, then electroblotted onto Immobilon membranes. The membranes were blocked with 5% skimmed milk ŽYukijirushi, Japan. and immunostained using the SuperSignal substrate ŽPierce.. Chemiluminescence was captured and visualized by a CCD video camera system ŽAtto Densitograph Lumino CCD AE-6930.. KO4 antibody, anti-p73 antibody, anti-p72 antibody, anti-Hsp40 antibody, anti-synaptophysin antibody, HRPO-labeled anti-rabbit immunoglobulin antibody, HRPO-labeled antirat immunoglobulin antibody, and HRPO-labeled antimouse immunoglobulin antibody were diluted 1:2000, 1:10 000, 1:2000, 1:2000, 1:2000, 1:20 000, 1:20 000, and 1:5000, respectively.
2.1. Materials Mouse monoclonal anti-p72 antibody ŽRPN1197. was purchased from Amersham; mouse monoclonal anti-synaptophysin antibody ŽMAB368. was from Chemicon; antiPSD-95 antibody was from Transduction Laboratories; anti-rabbit IgG was from Vector Laboratories; Vectastain ABC kit containing anti-rabbit IgG and avidin-biotinylated horseradish peroxidase ŽHRPO. complex was from Vector Laboratories; HRPO-coupled goat anti-rabbit IgG ŽH and L. from Calbiochem.; HRPO-coupled goat anti-mouse IgŽG q A q M. from Cappel; HRPO-coupled goat anti-rat IgGŽH q L. from Chemicon; Fluorescein isothiocyanate ŽFITC.-labeled goat anti-rat IgGŽH q L. and rhodamine isothiocyanate ŽTRITC.-labeled goat anti-rabbit IgGŽH q L. were from Zymed; FITC-labeled goat anti-mouse IgG from Tago; Epok 812 was from Oken ŽTokyo, Japan.; SuperSignal substrate, chemiluminescent detection reagent for Western blotting, was from Pierce; polyvinylidene difluoride ŽPVDF. membranes ŽImmobilon. were from Millipore. Other chemicals were of reagent grade. 2.2. Subcellular fractionation and protein extraction Subcellular fractions of the brain were obtained from Wistar rats Ž6 weeks old, unless otherwise stated.. PSD
2.3. Preparation of antibodies Rabbit polyclonal anti-HSP70 antibody, KO4, rat monoclonal anti-Hsc70 Žp73. antibody, 1B5, and rabbit polyclonal anti-Hsp40 antibody were produced as described previously w16,17,34x. KO4 anti-HSP70 antibody specifically recognizes both p73 and p72 w17,56x. 2.4. Immunoblotting
2.5. Immunohistochemical examination For immunohistochemical examination at the light microscopic level, brains from Wistar rats Žmales, 4 weeks old. were fixed with 4% paraformaldehyde in phosphate buffer ŽPB. Žsodium salt, 100 mM, pH 7.4. by perfusion through left heart ventricle for 15 min and then by immersion for 3 h. After treatment with 0.1 M lysine in the phosphate buffered saline ŽPBS. Žsodium salt, 100 mM, pH 7.4. for 3 h in order to quench free aldehyde groups, they were dehydrated in a series of ethanol and xylene, and embedded in paraffin. After rehydration, the sections were incubated in PBS Ž20 mM. containing 2% normal horse
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serum for 30 min, and anti-HSP70 Ž1:3000 dilution. or anti-Hsp40 antibodies Ž1:1500 dilution. for 12 h. After washing in PBS, they were incubated with biotinylated anti-rabbit IgG for 1 h and with avidin-biotinylated peroxidase complex for 1 h. The reaction product was developed by incubation in 0.05% 3, 3X-diaminobenzidine ŽDAB. tetrahydrochloride and 0.01% hydrogen peroxide in Tris– HCl buffer Ž50 mM, pH 7.5.. They were examined and photomicrographed with a Nikon FXA microscope after counterstaining with 1% methyl green. Some sections were immersed in sodium citrate Ž20 mM, pH 6.0. and boiled for 10 min in a microwave oven after deparaffinization and rehydration. No staining was obtained in the brain tissues following the protocols described above when primary antibodies were omitted or replaced by normal serum. For immunoelectron microscopy, tissues were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PB by perfusion through left heart ventricle for 15 min and by immersion for 3 h. After washing in 0.1 M lysine for 3 h, they were immersed in graded sucrose solutions: 10%, 15% and 20% for 10 h each. Frozen in a mixture of dry ice and n-hexane, 6 mm sections were cut on a cryostat microtome and collected on silane-coated glass slides. After permealizing in 0.1% Triton X-100rPBS, they were treated with the anti-HSP70 Ž1:1500 dilution. or anti-Hsp40 antibodies Ž1:1500 dilution., biotinylated anti-rabbit IgG antibody, and avidin-biotinylated peroxidase by the procedure described above. The reaction product was developed for 15 min in DAB and hydrogen peroxide in PB containing 0.025% cobalt chloride and 0.02% nickel ammonium w1x. They were post-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide in PB and then dehydrated with
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graded ethanol. After embedding in Epok 812, ultra-thin sections of 0.1 mm were cut with a Porter Blum MT2B ultramicrotome. They were examined by a JEOL 1200 EX electron microscope at an accelerating voltage of 100 kV without staining with uranyl acetate or lead citrate w50,51x. No staining was obtained in the brain tissues following the protocol described above when primary antibodies were omitted or replaced by normal serum. Double immunofluorescent staining using combination of rabbit anti-Hsp40 Ž1r100 dilution. antibody and either one of rat anti-p73 Ž1r60 dilution. or mouse anti-synaptophysin Ž1r100 dilution. antibody were followed essentially the protocol described previously w17x. The staining was applied to primary cultured neurons of rat cerebral cortex that were maintained for 25 days from embryonic day 19 ŽE19P25.. Dilutions of the second antibodies were 1:50 for FITC-labeled anti-rat IgG and rhodamine-labeled anti-rabbit IgG and 1:100 for FITC-labeled anti-mouse IgG. Immunoreactivities for p73, Hsp40, and synaptophysin were finally labeled with FITC, rhodamine, and FITC, respectively. Double-stained samples were examined with a laser confocal microscope ŽFluoview, Olympus, Japan.. No staining was obtained in the brain tissues following the protocol described above when primary antibodies were omitted. 2.6. Additional procedures Electrophoresis was performed using the discontinuous SDS-buffer system of Laemmli Ž1970. w23x. Protein was assayed by the method of Lowry et al. Ž1951. w26x with bovine serum albumin as the standard. Proteins were solu-
Fig. 1. Detection of HSP70 and Hsp40 in the PSD fractions prepared from the rat forebrain. PSD proteins Ž15 mg. prepared from rat forebrain and proteins Ž4 mg. of HeLa cells with Žindicated by qH. or without Žindicated by y. treatment at 458C were separated on 10% polyacrylamide gels and Western blotted using KO4 anti-HSP70, anti-p73, anti-p72, and anti-Hsp40 antibodies. Antibodies used are indicated under the gels. Molecular weight standards are shown in kDa on the left.
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bilized for protein assay by boiling in 1 N NaOH for 10 min.
3. Result 3.1. Identification of HSP70 and Hsp40 in the PSD fraction We used three different antibodies to HSP70 and an antibody to Hsp40. The validity of these antibodies has been previously reported w17,56x, and reproduced in Western blots using HeLa cells either nontreated or treated at 458C ŽFig. 1.. All antibodies showed specific bands with proper molecular sizes, and detected increased expression of HSP70 and Hsp40 after heat-treatment of cells except for the 1B5 antibody which recognizes Hsc70, the constitutive form of HSP70. Thus, we judged that the antibodies used were applicable in this study. The presence of chaperone proteins in synaptic structures was tested by Western blotting of proteins in PSD fractions prepared from rat forebrain ŽFig. 1.. There was a single immunoreactive band recognized by KO4 antiHSP70 antibody, which recognizes both p72 ŽHsp70. and p73 ŽHsc70. ŽFig. 1a.. The molecular size of the im-
munoreactive band in PSD fractions was the same as the HSP70 in HeLa cell extracts, which contains both p73 and p72 w17,56x. The HSP70 immunoreactive band was further analyzed by an antibody specific to p73 vs. p72. The band reactive to KO4 antibody in PSD fractions was also reactive to anti-p73 antibody but not to anti-p72 antibody. The band immunoreactive to anti-p72 antibody in PSD fractions was not detected even under conditions that increased sensitivity by prolonging the duration of chemiluminescence more than 20 times the standard measurement. These results suggested that the prominent Hsp in PSDs detected by KO4 anti-HSP70 antibody was p73, the constitutive form of HSP70 ŽHsc70.. This result is consistent with a previous report, in which p70 is not expressed in cells from rodents maintained under normal temperatures w17,56x. Thus, PSDs prepared from unstressed brain contains only the constitutive form of HSP70. There was also a single immunoreactive band recognized by anti-Hsp40 ŽFig. 1b.. The size of this immunoreactive band in PSDs was the same as the Hsp40 in HeLa cell. The distribution of HSP70 and Hsp40 in the PSD fraction was further examined in the fractions prepared from rat cerebral cortex, hippocampus and cerebellum. All PSD fractions examined contained p73 and Hsp40 but not p72 ŽFig. 2..
Fig. 2. Detection of HSP70 and Hsp40 in the PSD fractions prepared from various parts of the rat brain. PSD proteins Ž15 mg. prepared from cerebral cortex ŽCTX., hippocampus ŽHIP. and cerebellum ŽCBLM. of rat and proteins of heat-treated HeLa cells Ž7 mg. were separated on 10% polyacrylamide gel and Western blotted using KO4 anti-HSP70, anti-p73, anti-p72, and anti-Hsp40 antibodies. Fig. 3. Subcellular distribution of HSP70 and Hsp40 in the rat forebrain. Proteins of various subcellular fractions Ž25 mg. and those of heat-treated HeLa cells Ž7 mg. were separated on a 10% polyacrylamide gel and Western blotted as described in the Fig. 2 legend. Sp refers to synaptophysin and S, Syn, and SM to soluble, synaptosomal and synaptic plasma membrane fractions, respectively. The subcellular distribution pattern of PSD-95 verifies the fractionation. Fig. 4. Developmental changes in HSP70 and Hsp40 in the PSD fraction. PSD proteins Ž15 mg. prepared from the rat forebrain at various ages Ž2, 3, 4 and 6 weeks old. and proteins of heat-treated HeLa cells Ž7 mg. were separated by SDS-PAGE Ž10%. and Western blotted as described in the Fig. 2 legend.
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The subcellular distribution of HSP70 and Hsp40 was investigated in various fractions prepared from rat forebrain ŽFig. 3.. The bands immunoreactive to KO4 and anti-p73 antibodies were present with similar abundance in all fractions. There was no immunoreactive p72 in any fraction. The Hsp40 was highly concentrated in the PSD fraction, less abundant in the crude nuclear pellet ŽP1., and was barely detected in the synaptosome and synaptic plasma membrane ŽSM. fractions. The distribution of synaptophysin, a presynaptic marker in the synaptic vesicle membrane, was also compared with that of Hsp40. The distribution of the two proteins was different. Developmental changes were investigated in the HSP70 and Hsp40 content of PSDs prepared from the forebrain ŽFig. 4.. The level of HSP70 and p73 in PSDs 2 weeks after birth was about 50% of adult level and its level did
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not change significantly during 3 to 6 weeks after birth. There was no p72 band in PSDs at any developmental age. The level of Hsp40 in PSD fraction 2 weeks after birth was less than half of the adult level and its level was similar in PSDs during 3 to 6 weeks after birth. Thus, the developmental patterns of these two proteins in PSDs were similar and both proteins increase in amount during the period of synaptogenesis w21,48,50x. 3.2. Immunohistochemical localization of HSP70 in the rat brain at the light microscopic leÕel We used the KO4 anti-HSP70 antibody for immunohistochemical analysis of HSP70 because the KO4 antibody is polyclonal and 1B5 anti-Hsc70 Žp73. antibody is monoclonal. Although the KO4 antibody recognized both p73
Fig. 5. Localization of HSP70 immunoreactivity at the light microscopic level in the cerebral cortex, hippocampus, and cerebellum. Deparaffinized brain tissues were stained with KO4 anti-HSP70 antibody. Ža. Cerebral cortex, Žb. CA1 region of hippocampus, Žc. and Žd. cerebellum. Tissues shown in Ža. – Žc. were treated with microwave Žindicated by q., but that in Žd. was not Žindicated by y., after deparaffinization and rehydration. Somas and dendrites, particularly as seen in Žd., of neuronal cells are positive for the HSP70 immunoreactivity. Bars, 50 mm.
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and p72 in the HeLa cells, the staining revealed by the KO4 antibody in the unstressed normal brain thought to be primarily against the p73 because the normal brain does not contain detectable levels of p72 ŽFigs. 1–4.. The HSP70 immunoreactivity was distributed throughout the rat brain, in neuronal somata, dendrites and axons. Nuclei were also positive, but very weak. Glial cells were also reactive in tissues treated with microwave irradiation after deparaffinization and rehydration, which increased staining sensitivity. Typical examples with or without microwave treatment after deparaffinization and rehydration are shown in Fig. 5. In the neocortex, pyramidal cells were strongly positive for HSP70 in all layers I through VI. In the hippocampus, pyramidal neurons through CA1 to CA4 and granular neurons in the dentate gyrus were stained. Somas and dendrites were clearly stained but nuclei were not in neurons of the cerebral cortex and hippocampus. In the
cerebellum, Purkinje cells were strongly immunoreactive, and the reaction in the granular cell layer was weak ŽFig. 5c.. In specimens without enhancement by microwave irradiation, only Purkinje cell dendrites were immunoreactive and somas of Purkinje cells were not immunoreactive ŽFig. 5d.. 3.3. Immunohistochemical localization of Hsp40 in rat brain at the light microscopic leÕel Hsp40 immunoreactivity in rat brain showed a distribution very similar to that of Hsc70, being distributed throughout the rat brain, in neuronal somata and dendrites. Glial cells were also reactive especially in microwave irradiated tissues. The distribution in the cerebral cortex, hippocampus, and cerebellum are shown in Fig. 6. Cerebral cortex without microwave treatment showed punctate stainings throughout all layers ŽFig. 6b., which suggested that Hsp40
Fig. 6. Localization of Hsp40 immunoreactivity at the light microscopic level in the cerebral cortex, hippocampus, and cerebellum. Deparaffinized brain tissues were stained with anti-Hsp40 antibody. Ža. and Žb. Cerebral cortex, Žc. CA1 region of hippocampus, Žd. cerebellum. Tissues shown in Ža., Žc., and Žd., were treated with microwave Žindicated by q., but that in Žb. was not Žindicated by y., after deparaffinization and rehydration. Bars in Ža., Žc., and Žd., 50 mm. Bar in Žb., 5 mm.
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was concentrated in these restricted but widespread regions in the cerebral cortex. In the hippocampus, pyramidal neurons through CA1 to CA4 and granular neurons in the dentate gyrus were stained but weakly compared with those in the cerebral cortex. In the cerebellum, somas and dendrites of Purkinje cells were weakly stained. Granular neurons and cells in the molecular layer, probably microglia, were strongly immunoreactive.
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3.4. Intracellular localization of HSP70 and Hsp40 at the electron microscopic leÕel The dendritic localization of HSP70 and Hsp40 ŽFigs. 5 and 6., the punctate staining of Hsp40 immunoreactivity ŽFig. 6b., and the presence of Hsc70 and Hsp40 in PSD fractions ŽFig. 1. suggested the localization of these proteins at synaptic sites. These results prompted us to exam-
Fig. 7. Localization of HSP70 immunoreactivity in the synaptic areas at an electron microscopic level. The immunoreactive regions of brain at the light microscopic level, unstained with uranyl acetate or lead citrate, were examined by an electron microscope. Pre and SSW refer to presynaptic terminal and subsynaptic web, respectively. Arrows delineate PSDs. Ža. and Žb. Cerebral cortex, Žc. and Žd. cerebellum. Presynaptic staining is shown in Ža. and postsynaptic in Žb., Žc., and Žd.. Bar, 200 nm.
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Fig. 8. Localization of Hsp40 immunoreactivity in the synaptic areas at an electron microscopic level. The immunoreactive regions of brain at the light microscopic level, unstained with uranyl acetate or lead citrate, were examined by an electron microscope. Pre and SSW refer to presynaptic terminal and subsynaptic web, respectively. Arrows delineate PSDs. Bars, 200 nm.
ine the localization of these two proteins at the electron microscopic level in the cerebral cortex and cerebellum, focusing on their localization at synapses. HSP70 immunoreactivity was localized in the cytoplasmic matrix, and dendrites in neurons of the neocortex and cerebellum.
Postsynaptic structures in the cerebral cortex and cerebellum were stained ŽFig. 7.. All or portions of PSDs in postsynaptic spines were stained. PSDs were immunoreactive but rather weak compared with regions of subsynaptic web. Postsynaptic structures of both axo-somatic and axo-
Fig. 9. Comparison of the distribution of Hsc70 and Hsp40 in cultured cortical neurons by double immunostaining and confocal microscopic examination. Localization in the cultured cerebral neurons Žrat, E19P25. were examined. Hsc70 and Hsp40 were labeled by FITC Ža. and rhodamine Žb., respectively. C is a mixture of the two staining. Bar, 50 mm. Fig. 10. Comparison of the distribution of Hsp40 and synaptophysin by double immunostaining and confocal microscopic examination. Localization in the cultured cerebral neurons Žrat, E19P25. were examined. Synaptophysin and Hsp40 were labeled by FITC Ža. and rhodamine Žb., respectively. C is a mixture of the two staining. Bar, 50 mm.
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dendritic synapses were positive for HSP70. Presynaptic terminals were also stained but less frequently than the postsynaptic structure ŽFig. 7a.. Either one of pre- or
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post-synaptic structure but not both of them in a single synapse was stained for HSP70. More than half of the synapses examined showed postsynaptic staining alone.
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The staining of the nucleus, mitochondria and other cell organella were not evident at the electron microscopic level. The synaptic localization of Hsp40 immunoreactivity in the cerebral cortex was also examined at the electron microscopic level. Postsynaptic structures but not presynaptic structures were stained ŽFig. 8.. PSDs were immunoreactive but rather weak compared to regions of subsynaptic web. Staining patterns of HSP70 and Hsp40 in the spine were similar. 3.5. Comparison of the distribution of Hsc70, Hsp40, and synaptophysin The distribution of Hsc70 and Hsp40 was compared in cultured cortical neurons by double immunofluorescent staining and laser confocal microscopy ŽFig. 9.. Both Hsc70 and Hsp40 were distributed throughout neurons. Glial cells were also reactive to both antibodies. The areas stained by both antibodies were not homogeneous but disconnected and punctate in some regions of neurons. Nearly all of the Hsp40 staining co-localized with that of Hsc70. The distribution of Hsp40 and synaptophysin, a presynaptic marker, was also examined by the same method in cultured neurons ŽFig. 10.. Synaptophysin immunoreactivity was distributed in a patch-like pattern in cultured neurons, to which Hsp40 was localized. Patchy stainings of synaptophysin may represent the localization of presynaptic terminals.
4. Discussion 4.1. Presence of Hsc70 and Hsp40 in the postsynaptic structures The presence of two kinds of chaperone proteins, Hsc70 and Hsp40, in postsynaptic structures was shown in this paper. Hsc70, a constitutive form of HSP70, was localized at synapses, being present both in the pre- and post-synaptic components but more frequently in postsynaptic. The presence of Hsc70 in postsynaptic sites has been suggested previously, however, its presence in vivo was not addressed. Only the presence of an Hsc70 epitope in the PSD fraction w24x and a short peptide homologous to Hsc70 derived from the 68 kDa protein in the PSD fraction have suggested the presence of the molecule in PSDs in vivo w15,54x. Hsc70 was previously known as b-internexin w32x and shares homology with a recently identified PSD protein, a-internexin Žneuron-specific intermediate filament protein. w50x. We also found that an inducible form of HSP70 Žp72. was undetectable in any subcellular compartments in the rat brain under normal unstressed conditions ŽFigs. 1–4..
p72 may be expressed in the brain only under stressed conditions. It should be pointed out that the distribution of Hsc70 Žp73. revealed in this paper was different from that of p72 expression in the adult rat brain induced after transient hypothermia and 6 h of recovery w29x. The difference in the distribution of the two forms of HSP70 suggests different functional roles in the brain for these biochemically similar proteins. Hsp40, which assists the function of HSP70 by stimulating its ATPase activity w11,31x, is also localized to postsynaptic sites. Thus, both Hsc70 and its cooperator Hsp40 are co-localized in the postsynaptic sites. The postsynaptic localization of these two proteins suggests specific roles of these proteins at the sites. The localization of Hsp40 is a marked contrast to the report that the cysteine string protein Žcsp., a mammalian DnaJ homologue w10x, is specifically localized to the presynaptic terminals, especially to synaptic vesicles w7,22x. Csp is believed to be involved in the fusion of synaptic vesicles and presynaptic membrane during neurotransmitter release w7x. These results indicate that two different kinds of DnaJ-like proteins, csp and Hsp40, are differentially localized on opposite sides of the synapse. Thus, Hsc70 with the aid of Hsp40, postsynaptic-specific DnaJ-like proteins, may fulfill a specific role at postsynaptic sites w10,46x. Another HSP, ubiquitin, may also be localized at the postsynaptic sites, because most of PSD proteins are reported to be ubiquitinated w9x. Ubiquitin’s role at postsynaptic sites and the ubiquitination of a number of PSD proteins are not known at present. The localization of HSP90 was not examined in this report, but it is reported to be expressed throughout all neurons in the brain and is expressed predominantly in perikarya, although minor immunoreactivity was observed in dendrites and nuclei w12x. The presence of HSP90 at synaptic sites is currently unknown. 4.2. Are postsynaptic chaperones inÕolÕed in synaptic plasticity? The chaperone activity of HSP70 is related to a variety of cellular functions as described in the introduction. The roleŽs. of the constitutive form of HSP70 Žp73. and inducible form of HSP70 Žp72. in the brain may be different Žsee above., although their biochemical activities are indistinguishable in vitro. The major role of the inducible form of HSP70 may be in the prevention of protein denaturation and following detrimental events in cells exposed to harmful stimuli because the p72 content is rather low until cells are insulted by harmful stimuli. The inducible p72 may be mobilized to sites of harmful cellular events. Many kinds of injuries induce HSP70, probably p72, in the CNS and HSP70 is essential to the neuroprotective effect of heatshock w42x. The inducible form of HSP can also protect neurons from glutamate excitotoxicity w39x.
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Several lines of evidence indicate that HSP70 is related to the expression of synaptic plasticity. Hsc70 can be induced by synaptic activation w20x, and is listed as a CPG w33x. Bip, another member of the HSP70, influences the in vitro phosphorylation of substrate proteins by src kinase w8x, a member of intracellular signaling pathways. Heat shock disrupts long-term memory consolidation in Caenorhabditis elegans, in which HSP20, 70, and 90 family proteins are expressed. In this case, the effect of heat shock response may be through altered protein synthesis w3x. Although most experiments did not discriminate between p72 and p73 in the synaptic plasticity-related events, Kaneko et al. w20x reported that electroconvulsive seizure induced p73 mRNA but not p72. Thus, molecular chaperones, especially Hsc70, at postsynaptic sites could be involved in mechanisms of synaptic plasticity. Considering that Hsc70 is constitutively expressed, the roles of Hsc70rHsp40 could be fundamental for synaptic transmission and its regulation under unstimulated conditions. One of the major roles of the HSP70 which is fundamental for normal synapse is the assistance of forming proper protein conformations by binding to nascent polypeptides w11x. Protein synthesis at postsynaptic sites may be required in unstressed normal neurons, and increased following the development of synaptic plasticity events, such as LTP and the consolidation process of memory w38,52,53x. Thus, it is possible that Hsc70rHsp40 at postsynaptic sites are involved in postsynaptic mechanisms of local protein synthesis, although other functions of HSP70 described above cannot be ruled out. HSP70 is a candidate of messenger from synapse to nucleus ŽMSN. w49x, because accumulating evidence suggests that HSP70 works as a nuclear shuttle. Hsc70 is localized in both the cytoplasm and the nucleus and shuttles between the two compartments w18,25,45x, and members of the HSP70 family recycle between the nucleus and cytoplasm of Xenopus oocytes w28x. Hsc70 is also involved in the nuclear transport of karyophilic proteins w19,36,43x. Hsc interacts with nuclear localization signal ŽNLS.-containing proteins in the cytoplasm before their nuclear import w19x. Hsc70 also interacts with RCC1, which is a chromatin-associated protein required for nuclear transport of proteins w40x. Thus, it is also possible that Hsc70 localized at postsynaptic sites might work as a MSN.
Acknowledgements We thank Dr. P.T. Kelly, University of Texas Medical School, Houston, TX, for his critical reading of the manuscript. This research was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, The Ichiro Kanehara Foundation, and Toyota Physical and Chemical Research Institute.
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