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Heat Shock Proteins Hsp27 and Hsp32 Localize to Synaptic Sites in the Rat Cerebellum Following Hyperthermia

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Heat Shock Proteins Hsp27 and Hsp32 Localize to Synaptic Sites in the Rat Cerebellum Following Hyperthermia Molecular Brain Research 75Ž. 2000 309±320 www.elsevier.comrlocaterbres Research report Heat shock proteins Hsp27 and Hsp32 localize to synaptic sites in the rat cerebellum following hyperthermia David A. Bechtold, Ian R. Brown ) Department of Zoology, UniÕersity of Toronto at Scarborough, 1265 Military Trail, West Hill, Toronto, Ontario, Canada M1C 1A4 Accepted 10 November 1999 Abstract Stressful stimuli activate the heat shockŽ. stress response in which a set of heat shock proteins Ž. hsps is induced, which play roles in cellular repair and protective mechanisms. Most studies in the mammalian nervous system have focused on Hsp70, however, the present investigation targets other members of the induced set, namely Hsp27 and Hsp32. In response to hyperthermia, these hsps are strongly induced in Bergmann glial cells in the rat brain and transported into their radial fibers, which project into the `synaptic-enriched' molecular layer of the cerebellum. Using subcellular fractionation and immunoelectron microscopy, hyperthermia-induced Hsp27 and Hsp32 were detected in synaptic elements and in perisynaptic glial processes. These results suggest that stress-induced Hsp27 and Hsp32 may contribute to repair and protective mechanisms at the synapse. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Hsp70; Hsp27; Stress response; Synapse; Hyperthermia; HO-1 1. Introduction that overexpression of either Hsp27 or Hsp32 protects cells from subsequent stresswx 26,27,31,41,46,59 . In the present In response to a range of stressful stimuli, including investigation, we have therefore examined the expression hyperthermia and ischemia, the mammalian brain demon- patterns of Hsp27 and Hsp32 in the rat brain in response to strates a rapid and intense induction of the heat shock hyperthermia and also determined whether these hsps lo- protein, Hsp70wx 6,40,54 . The neuroprotective effects of calize to synaptic sites following stress. These studies have Hsp70 have been demonstrated both in vitro and in vivo focused on the cerebellum since its laminar structure facili- wx2,30,60,66,67 and it is well-established that a condition- tates the identification of cell types and zones enriched in ing thermal stress increases neuronal survival during sub- synaptic termini are clearly defined. sequent insultswx 3,8,34,53 . Recently, the protective effects of prior heat shock have been extended to neural specific phenomena such as synaptic function. For example, in collaborative studies, we have demonstrated that mild heat 2. Materials and methods treatment of Drosophila larvae facilitates synaptic trans- mission at high test temperatureswx 20 . While Hsp70 is a major protein induced by cellular 2.1. Treatment of animals stress, it is not the only heat shock proteinŽ. hsp which is up-regulated during activation of the heat shockŽ. stress The body temperature of male Wistar ratsŽ. 30-days-old response. A set of hsps is induced which includes the low was raised 3.58C above normalŽ. 388C using a dry air molecular weight Hsp27 and also Hsp32, the stress-respon- incubator set to 428C. Body temperature of the animals sive form of heme oxygenasewx 12 . Studies have shown was monitored with a rectal thermistor probe and main- tained at the elevated temperature for 1 h. Following incubation, the rats were placed at room temperature until time of sacrifice. At 30 days of age, the rats had achieved ) Corresponding author. Fax: q1-416-287-7642; e-mail: adult brain structure and synaptogenesis was essentially [email protected] complete. 0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328XŽ. 99 00323-X 310 D.A. Bechtold, I.R. BrownrMolecular Brain Research 75() 2000 309±320 2.2. Immunocytochemistry Ž.S2b was centrifuged at 45,000 rpm for 1 h and provided microsomalŽ. pellet and cell soluble Ž supernatant . frac- tions. The P2 pellet was homogenized in 0.05 mM CaCl . Rats were anesthetized with sodium pentobarbital and 2 After 20 min at 48C, the homogenate was incubated with perfused intracardially with 0.1 M phosphate-buffered succinate and INT for 30 min at 378C. Membrane fractions salineŽ. PBS , pH 7.4, followed by 4% paraformaldehyde in were separated on a discontinuous sucrose gradient by PBS. Brain tissue was removed and fixed overnight in 4% centrifugation at 26,000 rpm for 90 min and harvested as paraformaldehyde at 48C. Tissue was then equilibrated follows: light membranesŽ. LM at the 0.8r1.0 M sucrose with 20% sucrose in PBS, mounted in OCT embedding interface and synaptic membranesŽ. SMs from the 1.0r1.2 compoundŽ. Miles , and frozen at y708C. Cryostat sections M sucrose interface. Synaptic junctionsŽ. SJs were isolated Ž.20 mm were collected on gelatin-coated microscope slides by the extraction of the SM fraction with Triton X-100 and air-dried for 3 h. Following rehydration in PBS-T Ž.0.5% vrv . Mitochondria were collected from the pellet bufferŽ 0.1 M PBS, pH 7.4, 0.2% Triton X-100, 0.1% of the sucrose gradient spin and washed in ice-cold ace- BSA. , tissue sections were blocked in 5% goat serum in tone to remove INT. PBS-T for 1 h. Sections were incubated overnight in primary antibody diluted to 1:25,000 for Hsp27Ž Stress- Gen, SPA-801.Ž , 1:7500 for Hsp32 gift from P.R. Ortiz de 2.4. Gel electrophoresis and Western blot analysis Montellano, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA. , and 1:25,000 for Hsp70wx 58Ž gift from R. Tanguay, Laboratory of Protein samples were separated by electrophoresis on Cellular and Developmental Genetics, Universite Laval, 15% SDS polyacrylamide gels with a 5% stacking gel r Ste-Foy, Quebec. in PBS-T. After washing, sections were using a Tris Tricine buffer system. Following protein incubated in biotinylated anti-rabbit IgG diluted in block- transfer, nitrocellulose blots were stained with Ponceau S ing bufferŽ. 1:400 for 90 min. Brain sections were pro- to ensure equal protein loading. Blots were blocked for 5 h cessed using the Vectastain Elite ABC kitŽ. Vector Labs in 5% Carnation milk powder in TBSTŽ 10 mM Tris, 0.25 X and immunoreaction was visualized using 3,3 -diamino- M NaCl, 0.05% Tween, pH 7.5. , then incubated 14±16 h benzidineŽ. DAB . Sections were dehydrated in ethanol, with either Hsp27 or Hsp32 antibody diluted 1:10,000 in cleared in xylene, and coverslipped for inspection and 1% purified BSA in TBST containing 0.02% sodium azide. photography. Immunocytochemical results shown are rep- After washing, blots were incubated for 2 h with anti-rab- resentative of four separate animal trials. Omission of bit IgGŽ. Sigma diluted 1:10,000 in 1% BSA in TBST. either the primary or secondary antibody resulted in clear Immunoreactivity was visualized using ECL detection sections with no disposition of reaction product. reagentsŽ. Amersham, RPN 2106 . Subcellular fractions were collected from a minimum of three separate trials and Western blots shown are representative of repeat experi- ments on each set of fractions. 2.3. Isolation of subcellular fractions Animals were sacrificed by decapitation and brains 2.5. Immunoelectron microscopy were removed and separated into forebrain and cerebellum. Tissue was then rinsed with ice-cold saline to remove Rats were perfused as above using 3.5% glutaraldehyde excess blood and subcellular fractions were prepared as in PBS. Brain tissue was removed, cut into 3 mm slabs and follows: Tissue was subfractionated according to the fixed for an additional 2 h at 48C. Tissue was then method of Gurd et al.wx 15 . Briefly, brainsŽ 14±18 rats per immersed in 0.1% sodium borohydride in PBS for 30 min. preparation. were homogenized in 0.32 M sucrose contain- After extensive washes, 40 mm sections were cut using a ing 1 mM MgCl2 and centrifuged twice for 5 min at 3200 vibratome. Cerebellar sections were equilibrated to 30% rpm. The supernatantŽ. S1 was centrifuged at 13,000 rpm sucrose and quick-frozen in isopentane cooled to y708C for 15 min resulting in S2 and P2. The S2 supernatant was to increase antibody penetration. Non-specific immunore- spun at 12,000 rpm for 10 min. The resulting supernatant action was blocked by incubating the tissue sections for 90 Fig. 1. Time course analysis of Hsp27 expression in the rat cerebellum following hyperthermia. Hsp27 was not detected in the cerebellum in the absence of stressŽ. Panel A ; however, immunoreactive staining of microvasculature was observed 5 h after hyperthermiaŽ. Panel B . The inset in Panel B shows a higher magnification of the induced Hsp27 signal in the microvasculature. By 15 hŽ. Panel C , pronounced Hsp27-immunoreactivity was evident in the Purkinje cellular layerŽ. p . Hsp27 was localized to the parallel arrays of Bergmann glial fibers lying within the molecular layerŽ. m 24 h after hyperthermia Ž.Panel D, arrowheads . dwm, deep white matter; g, granule cell layer; m, molecular layer; p, Purkinje cellular layer. Bars250 mm. D.A. Bechtold, I.R. BrownrMolecular Brain Research 75() 2000 309±320 311 312 D.A. Bechtold, I.R. BrownrMolecular Brain Research 75() 2000 309±320 min with 10% goat serum in PBS. Primary antibodies were In response to hyperthermia, Hsp32, the stress-respon- diluted in blocking buffer at 1:7500 for Hsp70, 1:7500 for sive form of heme oxygenase, showed a similar pattern of Hsp25, and 1:3000 for Hsp32 and incubated with the tissue induction compared to Hsp27 in Bergmann glial cells and sections for 24±36 h at 48C. Sections were washed and their radial fibersŽ. Figs. 2 and 4B . However, Hsp32 immersed in biotinylated anti-rabbit IgG diluted 1:800 in induction in these cells was earlier in onsetŽ i.e., prominent blocking buffer for 2.5 h.
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