Association of Heat Shock Proteins andNeuronal Membrane Components WithLipid Rafts From the Rat Brain

Sheng Chen, Damanpreet Bawa, Shintaro Besshoh, James W. Gurd,and Ian R. Brown*

Centre for the Neurobiology of Stress, University of Toronto at Scarborough, Toronto, Ontario, Canada

Lipid rafts are specialized plasma membrane microdo-mains enriched in cholesterol and sphingolipids that serveas major assembly and sorting platforms for signal trans-duction complexes. Constitutively expressed heat shockproteins Hsp90, Hsc70, Hsp60, and Hsp40 and a rangeof neurotransmitter receptors are present in lipid rafts iso-lated from rat forebrain and cerebellum. Depletion of cho-lesterol dissociates these proteins from lipid rafts. Afterhyperthermic stress, flotillin-1, a lipid raft marker protein,does not show major change in levels. Stress-inducibleHsp70 is detected in lipid rafts at 1 hr posthyperthermia,with the peak levels attained at 24 hr, suggesting thatHsp70 may play roles in maintaining the stability of lipidraft-associated signal transduction complexes followingneural stress. VVC 2005 Wiley-Liss, Inc.

Key words: heat shock proteins; lipid raft; hyperthermia

Heat shock proteins (Hsps) are a family of highlyconserved proteins found in all organisms. In unstressedcells, constitutively expressed heat shock proteins (Hscs)act as molecular chaperones to assist in the proper folding,assembly, and intercellular trafficking of newly synthesizedproteins (Lindquist and Craig, 1988; Becker and Craig,1994). After stress, specific Hsps are up-regulated andinvolved in cellular repair and protective mechanisms. Forexample, they bind to proteins that have been denaturedby stressful stimuli and assist in their refolding (Lindquistand Craig, 1988; Becker and Craig, 1994; Hartl, 1996).Hsps are classified into several groups according to theirmolecular size, such as Hsp90, Hsp70, Hsp60, Hsp40, andlow-molecular-weight Hsps (Hightower and Hendershot,1997). The HSP70 family comprises stress-inducibleHsp70 and constitutively expressed Hsc70. Hsp90, Hsc70,Hsp60, and Hsp40 are constitutively expressed in themammalian brain (D’Souza and Brown, 1998; Tanakaet al., 2002). Compared with nonneural tissues, brain tis-sue shows higher levels of Hsp90 and Hsc70 (Manzerraet al., 1997). In response to a range a stressful stimuli,including hyperthermia and ischemia, the mammalianbrain demonstrates a rapid and intense induction of Hsp70(Mayer and Brown, 1994; Brown and Sharp, 1999). Theneuroprotective effects of Hsp70 have been shown bothin vitro and in vivo. A conditioning thermal stress, which

is sufficient to induce Hsp70, or transgenic overexpressionof Hsp70, increases neural cell survival following sub-sequent insults (Tytell et al., 1994; Plumier et al., 1997;Yenari et al., 1998).

Hsps have been shown to be involved in severalprocesses during synaptic transmission. For example,Hsc70 is essential for the uncoating of synaptic vesicles atpresynaptic terminals (Morgan et al., 2001), and Hsp90 isinvolved in the synaptic cycling of AMPA receptors(Gerges et al., 2004). Previous work in our laboratory sug-gests Hsps may exert protective effects at the synapse.Constitutively expressed Hsp90, Hsc70, and Hsp60 werefound associated with synaptosomes isolated from thebrains of unstressed rats (Bechtold et al., 2000). Afterhyperthermia, inducible Hsp70 localized to both presy-naptic and postsynaptic elements in the rat brain (Bechtoldet al., 2000). By using a macropatch electrode to recordsynaptic activity at individual visualized synaptic boutons,our collaborative studies show that prior mild heat treat-ment of Drosophila larvae protected synaptic transmissionat higher test temperatures, and the time course of thissynaptic protection paralleled the induction profile ofHsp70 (Karunanithi et al., 1999). In addition, selectiveoverexpression of Hsp70 in transgenic animals enhancedthe level of synaptic protection (Karunanithi et al., 2002).

Lipid rafts, also termed detergent-insoluble glycolipidfractions or detergent-resistant membranes, are enriched incholesterol and sphingolipids and form a distinct, liquid-ordered phase in the lipid bilayer of membranes (Brownand Rose, 1992; Brown and London, 1998). Lipid raftsalso contain specific subpopulations of membrane pro-teins, which are often in the form of glycosylphosphatidy-linositol-anchored proteins and specific tyrosine kinases

Contract grant sponsor: National Science and Engineering Research

Council of Canada.

*Correspondence to: Dr. Ian R. Brown, Centre for the Neurobiology of

Stress, University of Toronto at Scarborough, 1265 Military Trail, Tor-

onto, Ontario, Canada M1C 1A4. E-mail: ibrown@utsc.utoronto.ca

Received 17 March 2005; Revised 29 April 2005; Accepted 29 April

2005

Published online 9 June 2005 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.20575

Journal of Neuroscience Research 81:522–529 (2005)

' 2005 Wiley-Liss, Inc.

(Brown and Rose, 1992; Brown and London, 1998).Lipid rafts have been shown to be important for signalingprocesses in basophils and mast cells and for T cell activa-tion (Simons and Toomre, 2000). As a result, lipid raftshave been suggested to serve as major assembly and sortingplatforms for signal transduction complexes (Simons andToomre, 2000).

The brain is enriched in lipid rafts, as more than 1% oftotal brain protein is recovered in a lipid raft fraction,whereas less than 0.1% of total protein is associated withlipid raft isolated from nonneural tissues (Maekawa et al.,2003). Several neurotransmitter receptors have been shownto associate with lipid rafts isolated from brain tissues, forexample, nicotinic acetylcholine receptors (Bruses et al.,2001), g-aminobutyric acid type B (GABAB) receptors(Becher et al., 2001), ionotropic AMPA receptors (GluR1,GluR2/4), and N-methyl-D-aspartate (NMDA) receptors(Suzuki et al., 2001; Hering et al., 2003; Besshoh et al.,2005). It has been suggested that lipid rafts are critical for themaintenance of the stability of synapses and dendritic spines(Hering et al., 2003). Suzuki (2002) has proposed that lipidrafts are an important compartment of the synapse.

Little is known about the association of Hsps withlipid rafts in the brain. In the present study, we demon-strate that constitutively expressed Hsps are present,along with various neurotransmitter receptors, in lipidrafts isolated from the P2 membrane fraction of rat brain,suggesting that they may play roles in normal lipid raftfunctions. After hyperthermia, stress-inducible Hsp70 isdetected in lipid rafts, where it could facilitate therefolding of lipid raft-associated proteins that are suscep-tible to stress-induced denaturation.

MATERIALS AND METHODS

Reagents and Antibodies

Methyl-b-cyclodextrin (MCD) was obtained from Sigma(St. Louis, MO). The following antibodies were used: mouse anti-Hsp90 monoclonal antibody (gift from Dr. A.C. Wikstrom),mouse anti-Hsp60 monoclonal antibody (gift form Dr. Gupta),mouse anti-b-Naþ-Kþ-ATPase antibody and rabbit anti-NR2Aantibody, rat anti-Hsc70 monoclonal antibody (Stressgen, Vancou-ver, British Columbia, Canada; SPA815), rabbit anti-Hsp70 poly-clonal antibody (Stressgen; SPA812), rabbit anti-Hsp40 polyclonalantibody (Stressgen; SPA400), mouse anti-flotillin-1 monoclonalantibody (BD Biosciences, San Jose, CA; 610820), mouse anti-NR1 antibody (BD Biosciences; 556308), rabbit anti-GluR6/7polyclonal antibody (Upstate, Lake Placid, NY; 06-309), mouseanti-GluR1 monoclonal antibody (Santa Cruz Biotechnology,Santa Cruz, CA; sc-13152), mouse anti-GluR2/4 monoclonalantibody (Chemicon, Temecula, CA; MAB396), mouse anti-PSD95 monoclonal antibody (Affinity Bioreagent; MA1-046).

Treatment of Animals

All procedures using animals were approved by the Ani-mal Care Committee of the University of Toronto and werein accordance with the guidelines established by the CanadianCouncil on Animal Care. The body temperature of adult maleWistar rats (42 days old) was raised 3.58C above normal

(388C) by using a dry air incubator set at 458C. Body temper-ature of the animal was monitored with a rectal thermistorprobe and maintained at the elevated temperature for 1 hr.After incubation, the rats were placed at room temperaturefor recovery until they were killed.

Preparation of Lipid Rafts

Animals were decapitated, and brains were removed andseparated into forebrain and cerebellum. Tissue was thenimmediately frozen on dry ice and kept at –708C until use.Forebrains and cerebella were homogenized in ice-cold bufferA (0.32 M sucrose, 1 mM NaCHO3, 1 mM MgCl2, 0.5 mMCaCl2, 1 mM NaF, 0.1 mM sodium orthovanadate, 20 mMp-NPP, 20 mM b-glycerophosphate, and 5 mg/ml leupetin,antipain, and aprotinin). After centrifugation at 700g for 5 minat 48C, the supernatant was spun at 15,000g for 13 min at48C. The pellet was washed with ice-cold buffer B (0.32 Msucrose, 1 mM NaHCO3, 1 mM NaF, 0.1 mM sodiumorthovanadate, 20 mM b-glycerophosphate, and 5 mg/ml leu-petin, antipain, and aprotinin) and spun at 15,000g for 15min. The pellet (termed P2), representing a mixture of neuro-nal and glial membranes, was then homogenized in buffer C(40% sucrose, 1 mM NaHCO3, 1 mM NaF, 0.1 mM sodiumorthovanadate, 20 mM b-glycerophosphate, and 5 mg/ml ofleupetin, antipain, and aprotinin) and protein concentrationdetermined with the Bio-Rad (Hercules, CA) protein assaykit. Five milligrams of P2 membrane fraction protein wereadjusted to 5 mg/ml with buffer C, and Triton X-100 wasadded to a final detergent concentration of 0.5% (v/v) and aprotein:detergent ratio of 1:1. The membrane fraction wasthen extracted with rotation for 20 min at 48C, followed by10 min of incubation on ice. The extract was placed into anultraclear centrifuge tube (14 � 95 mm; Beckman) and over-laid with 5 ml 30% sucrose and 5 ml 5% sucrose. After centri-fugation at 200,000g (40,000 rpm; SW40 Ti rotor; Beckman)for 18 hr at 48C, 1-ml fractions were collected from the topto the bottom of the gradient. The detergent-insoluble pelletat the base of the gradient was resuspended in 1 ml buffer B.

For the MCD cholesterol-depletion experiment, 5 mgforebrain P2 membrane fraction protein were mixed with100 ml of 500 mM of MCD, and the volume was adjusted to1 ml (protein concentration 5 mg/ml) and rotated at 48C for30 min. The Triton X-100 extraction was then performed asdescribed above.

Protein and Total Cholesterol Assay

Protein concentration was determined with the Bio-Rad protein assay kit. Total cholesterol concentrations weredetermined with the Wako Cholesterol E assay kit, an in vitroenzymatic colorimetric method for the quantitative determina-tion of total cholesterol.

Western Blotting

Samples were solubilized by boiling in 2� sodiumdodecyl sulfate (SDS) loading buffer (8 M urea, 2% SDS, 2%2-mercaptoethanol, 20% glycerol), separated by SDS-PAGEwith 5% stacking gel and 10% or 12% separating gel, andtransferred to nitrocellulose membrane. After blocking for

Heat Shock Proteins in Rat Brain Lipid Rafts 523

1 hr with 5% fat-free milk powder, blots were incubatedovernight at room temperature with primary antibodies di-luted 1:5,000 for Hsp90, 1:50,000 for Hsc70, 1:10,000 forHsp70, 1:10,000 for Hsp60, 1:10,000 for Hsp40, 1:10,000for NR2A, 1:3,000 for NR1, 1:3,000 for PSD95, 1:500 forflotillin-1, 1:500 for b-Naþ-Kþ-ATPase, 1:250 for GluR1,1:3,000 for GluR2/4, and 1:1,000 for GluR6/7. Immunoactivitywas visualized with ECL Western blot detection reagents(Amersham, Arlington Heights, IL).

RESULTS

Effects of Hyperthermia on Membrane-AssociatedProteins in the Rat Brain

As shown in Figure 1, Western blotting detected arange of neurotransmitter receptors in a P2 membranefraction isolated from the unstressed adult rat forebrainand cerebellum. The constitutively expressed heat shockproteins Hsp90, Hsc70, Hsp60, and Hsp40 were alsodetected in the membrane fraction in addition to the lipidraft marker flotillin-1. After elevation of body tempera-ture, levels of several neurotransmitter receptors and con-stitutively expressed Hsps did not show major changes;however, an increase in stress-inducible Hsp70 wasobserved. In agreement with previous reports, forebrainshowed higher levels of NR1, NR2A, and PSD95 com-pared with cerebellum (Brose et al., 1993; Wang et al.,1995; Fukaya et al., 1999). An increase in levels of GluR1was noted in both brain regions and a decrease in Hsc70 atthe 5 hr to 24 hr time points in the cerebellum.

Association of Neurotransmitter Receptors andHsps With Isolated Lipid Rafts

Lipid rafts were next isolated from the P2 membranefraction of adult rat forebrain and cerebellum on the basisof their insolubility in Triton X-100 at 48C and their abil-ity to float in density gradients (Brown and Rose, 1992).The lipid raft-containing fraction was tracked by enrich-ment of cholesterol and the protein flotillin-1, both lipidraft markers (Dermine et al., 2001). As shown inFigure 2A,C, cholesterol and flotillin-1 were enriched infraction 6 of sucrose gradient. This fraction was alsoenriched in protein (Fig. 2B). In contrast, b-Naþ-Kþ-ATPase, a negative marker of lipid rafts (Becher et al.,2001), was excluded from fraction 6 and localized at thebottom of the gradient, where the majority of the proteinsolubilized by Triton X-100 was found (Fig. 2B,C).

Lipid rafts localized in fraction 6 of the sucrose gra-dients were next analyzed for the presence of neurotrans-mitter receptors. As shown in Figure 2C, postsynapticproteins such as the NMDA receptors (NR2A, NR1) andPSD95, a major scaffold protein that interacts withNMDA receptors, were abundantly present in the lipidraft fraction. AMPA receptors (GluR1, GluR2/4) andkainate receptors (GluR6/7) were also associated with thelipid raft fraction. These observations are consistent withprevious reports (Suzuki et al., 2001; Hering et al., 2003;Bessoh et al., 2005). In addition to neurotransmitterreceptors, our result demonstrate that the constitutively

expressed heat shock proteins Hsp90, Hsc70, Hsp60, andHsp40 are associated with lipid rafts isolated from the ratbrain (Fig. 2C). The glial marker glial fibrillary acidic pro-tein (GFAP) was not detected in the lipid raft fraction(data not shown).

Cholesterol is known to be a component essentialfor the structural stability of lipid rafts (Brown and Rose,1992; Brown and London, 1998). We therefore investi-gated the effect of cholesterol depletion on lipid raft-asso-ciated proteins. MCD, a carbohydrate polymer withpockets for binding cholesterol, shows high affinity forcholesterol and can remove cholesterol from membraneswithout binding or inserting into membrane (Klein et al.,1995; Simons and Toomre, 2000). In the MCD choles-terol depletion experiment, the forebrain P2 membranefraction was treated with 50 mM MCD prior to TritonX-100 extraction and centrifugation. As shown inFigure 2A, after MCD treatment, the cholesterol peakshifted from fraction 6 to the bottom of the sucrose gra-dient, as did the protein that was present in fraction 6

Fig. 1. Western blot analysis of neurotransmitter receptors and heat shockproteins in a rat brain P2 membrane fraction in control animals (C) and at1, 5, 15, 24, and 48 hr posthyperthermia. Induction of stress-inducedHsp70 was observed. Flotillin-1 is a lipid raft marker protein. Comparableresults were obtained in three separate experiments.

524 Chen et al.

Fig. 2. Analysis of sucrose gradients of forebrain lipid raft preparations with or without MCDtreatment, which depletes cholesterol. Lipid rafts were prepared as described in Materials andMethods. Sucrose gradients were harvested in 1-ml fractions (fraction 1, top of gradient; fractionP, pellet fraction). A: Distribution of cholesterol in sucrose gradients. B: Distribution of protein insucrose gradients. C,D: Western blot analysis of sucrose gradient fractions prepared with or with-out MCD treatment. Comparable results were obtained in three separate experiments.

Heat Shock Proteins in Rat Brain Lipid Rafts 525

(Fig. 2B). The lipid raft marker flotillin-1 and the associ-ated constitutively expressed Hsps and neurotransmitterreceptors were also removed from fraction 6 by the cho-lesterol depletion. The MCD treatment experiment dem-onstrates that the presence of the neurotransmitter recep-tors and constitutively expressed Hsps in fraction 6 wasdependent on the presence of cholesterol, consistent withtheir association with lipid rafts.

Effects of Hyperthermia on the Associationof Hsps With Lipid Rafts

Lipid raft fractions were next isolated from forebrainand cerebellum P2 membrane fractions following hyper-thermia. During the time course of the thermal stress, lipidrafts in both the forebrain and the cerebellum appeared tobe preserved, in that the protein yield of lipid rafts did notshow significant change (Fig. 3), and the levels of the lipidraft marker flotillin-1 remained constant at up to 48 hrposthyperthermia (Fig. 4, bottom). Within 1 hr of hyper-thermia, stress-induced Hsp70 was detected in lipid raftsisolated from the cerebellum (Fig. 4) and increased pro-gressively to 24 hr posthyperthermia before decreasing atthe 48 hr time point (Fig. 4). Hsp70 was also noted at24 hr posthyperthermia in lipid rafts isolated from theforebrain, but the signal was less intense than that observedin the cerebellum (Fig. 4). Our previous results haveshown that the level of stress-induced Hsp70 in cerebel-lum is higher than that observed in forebrain (Manzerraet al., 1997). Constitutively expressed Hsp90 and Hsp40did not show major changes in lipid rafts from either theforebrain or the cerebellum during the time course of thethermal stress nor were major changes observed for PSD-

95 or a range of neurotransmitter receptors (Fig. 4).Hsp60 levels in the forebrain decreased over the timecourse of hyperthermia, whereas Hsc70 levels increased at1 hr in the cerebellum.

DISCUSSION

Hsps are composed of constitutively expressed mem-bers that are present in unstressed cells and induciblemembers that are expressed in response to stressful stimuli(Hartl, 1996). Hsp90, Hsc70, Hsp60, and Hsp40 are themajor constitutively expressed Hsps. Hsp90 is one of themost abundant cytosolic proteins and plays vital roles inthe maturation of signal transduction proteins such assteroid hormone receptor and protein kinase (Csermelyet al., 1998). Hsc70 is localized to the cytoplasm and actsas a molecular chaperone in protein folding and traffick-ing. After stress, there is a transient translocation of Hsc70from the cytoplasm to the nucleus (Manzerra et al., 1993).Hsp60 is a mitochondrial matrix protein required for fold-ing and assembly of polypeptides imported into the mito-chondria (Ostermann et al., 1989). In addition, Hsp60 is

Fig. 3. Lipid raft protein yield from forebrain and cerebellum follow-ing hyperthermia. Five milligams of protein of membranes from fore-brain and cerebellum were used for lipid raft preparation. No signifi-cant difference in lipid raft protein yield was observed in tripletexperiments carried out at individual time points. Results areexpressed as mean 6 SD.

Fig. 4. Time course analysis of lipid raft-associated proteins followingwhole-body hyperthermia. Lipid raft fractions from forebrain andcerebellum at different time points posthyperthermia were analyzedby Western blotting. Stress-inducible Hsp70 appeared in lipid rafts,peaking at 24 hr posthyperthermia. Comparable results were obtainedin three separate experiments.

526 Chen et al.

involved in immune responses to infectious diseases andautoimmune disorders (Zugel and Kaufmann, 1999).Hsp40 is closely associated with Hsc70 and is consideredas a cochaperone of Hsc70 in the process of protein fold-ing (Ohtsuka and Hata, 2000). A range of Hsps localizesto the cell membrane (Arispe and De Maio, 2000; Kovaret al., 2004; Cicconi et al., 2004). In the brain, Hsp90,Hsc70, and Hsp60 are found in a synaptic membrane frac-tion isolated by biochemical methods (Bechtold et al.,2000). Hsc70 and Hsp40 have also been visually localizedto synaptic structure by immunoelectron microscopy(Suzuki et al., 1999; Bechtold et al., 2000).

Lipid rafts are distinct plasma membrane microdo-mains. They are assembled in the Golgi and subsequentlydelivered to the plasma membrane (Simons and Toomre,2000). Recently, it has been proposed that lipid raftsmight be an important platform for delivery of Hsp70 tothe cell membrane. In cultured human intestinal Caco-2cells, Hsp70 was found in lipid rafts of plasma mem-branes, and the Hsp70 delivery pathway was not blockedby classical secretory pathway blockers but was affectedby the raft-disrupting drug MCD (Broquet et al., 2003).

In the present study, we isolated lipid rafts from ratforebrain and cerebellum, and we provide evidence thatconstitutively expressed Hsp90, Hsc70, Hsp60, Hsp40,and a range of neurotransmitter receptors are associatedwith lipid rafts. Becher et al. (2001) have reported thatNaþ-Kþ-ATPase is not present in lipid rafts. The obser-vation that Naþ-Kþ-ATPase is excluded from brain lipidrafts demonstrated the stringency of our isolation proce-dure. In addition, treatment of the forebrain P2 mem-brane with MCD disrupted lipid rafts and dissociatedHsps and known raft-associated proteins from the iso-lated lipid rafts.

Analysis of T-cell activation suggests that lipid raftscould rapidly recruit other signaling complexes intofunctional assemblies following appropriate stimulation,leading to the formation of the immunological synapse:a contact zone between the antigen-presenting cell andthe T cell (Langlet et al., 2000). In the central nervoussystem, communication between neurons through synap-tic contacts is a neural-specific process. Lipid rafts havebeen shown to be involved in synapse formation duringneural development and also in synaptic signal transduc-tion. Lipid raft signaling and recruitment of proteins viap42/p44 MAP kinase are essential for chemotropic guid-ance of nerve cone growth (Guirland et al., 2004). Inaddition, lipid rafts are important in the maintenance ofsynapses, dendritic spines, and surface AMPA receptors(Hering et al., 2003). Given the dynamics of proteininteractions in the lipid raft microenvironment duringthe process of signal transduction, it is reasonable thatHsps are required as protein chaperones to ensure theproper folding status of lipid raft-associated proteins. Infavor of this point of view, Hsc70 has been shown to beassociated with the postsynaptic NMDA receptor proteincomplex (Husi et al., 2000), and Hsp90 is thought tohave roles in the continuous synaptic cycling of AMPAreceptors (Gerges et al., 2004).

To investigate the effect of hyperthermia on lipidrafts, we elevated the body temperature of rats by 3.58Cabove normal for 1 hr. Hyperthermia has cytotoxic effectson cells and can induce cell death by inducing denatura-tion of cytoplasmic and membrane proteins and inhibitingDNA, RNA, and protein synthesis (Hildebrandt et al.,2002). In addition, hyperthermia affects fluidity and stabil-ity of cellular membranes and impedes the function oftransmembranal transport proteins and surface receptors(Hildebrandt et al., 2002).

Lipid rafts were isolated from forebrain and cere-bellum at different times following hyperthermia. Theprotein yield of lipid rafts was not affected by hyperther-mia. Western blotting indicated that levels of flotillin-1,a specific lipid raft marker, and several neurotransmitterreceptors did not show major changes during the timecourse of hyperthermia. This suggests that lipid rafts arepreserved after hyperthermic stress.

In response to temperature increase and otherstresses, cells exhibit an induction of Hsp70, which playsroles in cellular repair and protective mechanisms (Beckerand Craig, 1994). The induction of Hsp70 in the nervoussystem following hyperthermia and other stresses has beenwidely studied (Brown and Sharp, 1999). Hsp70 appearsto play a role in neuroprotective phenomena, as intra-ocular injection of purified Hsp70 confers protectionagainst stress-induced cell death in the retinal system(Tytell et al., 1993). In addition, neurons in transgenicanimals overexpressing Hsp70 are protected from stressfulstimuli (Plumier et al., 1997). During stressful conditions,it is important that the functionality of the synapse is pre-served to prevent the breakdown of the neural communi-cation system. Our laboratory’s collaborative studies haveextended the neural protective effects of Hsp70 to synapticfunction. A connection between the heat shock responseand preservation of synaptic transmission under stressfulconditions has been established, in that prior heat shock ofDrosophila larvae, sufficient to induce Hsp70, or transgenicoverexpression of Hsp70 in Drosophila, protected synapticperformance at high test temperatures (Karunanithi et al.,1999; Karunanithi et al., 2002). Hyperthermia also in-duced Hsp70 to localize to synaptic elements in the ratbrain (Bechtold et al., 2000).

In the time-course analysis of stress-inducible Hsp70in lipid rafts, we found that, within 1 hr of hyperthermia,stress-induced Hsp70 was detected in lipid rafts isolatedfrom the cerebellum. Levels of this stress protein increasedto 24 hr posthyperthermia and then decreased at the 48 hrtime point. Hsp70 was also noted at 24 hr posthyperther-mia in lipid rafts isolated from the forebrain. The presenceof Hsp70 in the lipid rafts could correct hyperthermia-induced conformational damage to proteins within thelipid raft microenvironment. The resultant preservation oflipid raft structure and function could underlie the neuro-protective effects of Hsp70.

In summary, we have demonstrated that constitu-tively expressed Hsp90, Hsc70, Hsp60, and Hsp40 areassociated with lipid rafts isolated from rat forebrain andcerebellum. These constitutively expressed Hsps may

Heat Shock Proteins in Rat Brain Lipid Rafts 527

participate in the normal functions of lipid rafts in neuralevents. After hyperthermic stress, the presence of stress-inducible Hsp70 in lipid rafts could play important rolesin preserving the functional stability of lipid rafts andtheir associated signal transduction complexes.

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