- Email: [email protected]
Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain
Neuroscience Letters 182 (1994) 188 192
ELSEVIER
HEUROSClENC[ [ETT[R$
Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain P e t e r G a s s a'*, H a n n s j 6 r g
S c h r 6 d e r b, P e t e r P r i o r a, M a r i k a
Kiessling a
alnstitute of Neuropathology, University of Heidelberg, lm Neuenheimer Feld 220, 69120 Heidelberg. Germany bDepartment qf Anatomy. University of Cologne. K61n. Germany
Received 21 September 1994; Revised version received 14 October 1994; Accepted 14 October 1994
Abstract
Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed thoughout all neuronal subpopulations of the CNS but not in nonneuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons. Key words: HSP90; lmmunocytochemistxy; Immunoblotting; Immuno-electron microscopy
Exposure of cells or organisms to heat or other forms of environmental stress leads to the rapid induction of a group of proteins, called 'heat shock' or 'stress proteins' [20]. It has been proposed that the common stimulus for heat shock protein induction is protein denaturation and that one function of inducible members of stress proteins is to prevent or repair denaturation damage [19]. However, several members of the heat shock protein family demonstrate constitutive expression in 'nonstressing' conditions, suggesting that some of these molecules play a role in normal metabolic cellular functions. In general, heat shock proteins are considered to represent molecular chaperones, defined as a family of unrelated classes of proteins that mediate the correct assembly (and disassembly) of other polypeptides, but are not themselves components of the final functional protein structures [5]. The mammalian genome contains three major classes of heat shock proteins, the HSP70s, HSP90s and
*Corresponding author. Fax: (49) (6221) 56,3466. 0304-3940194l$7.00© 1994 Elsevier ScienceB.V, All rights reserved S S D I 0304-3940(94)00794-2
HSP20s, categorized according to the molecular mass of the principle proteins [5]. In the CNS, those o f ~ 7 0 kDa have been most intensively studied, comprising a family of related genes that include constitutively expressed and stress inducible members [5]. The latter have been associated with the development of tolerance to metabolic disturbances in vitro and in vivo, such as hyperthermia or cerebral ischemia [1,9]. In contrast to the HSP70s, little is known about the HSP90s in the CNS. Western blot experiments with cytosolic fractions of whole brain extracts demonstrated the presence of the major 90-kDa heat shock protein HSP90. Furthermore, in situ hybridization studies indicated a generalized expression of HSP90 m R N A in many neuronal populations throughout the brain [8]. Although there are some suggestions of a physiological role of HSP90, e.g., in intracellular transport mechanisms or in regulation of the activities of steroid hormone receptors, the biological function of HSP90 in CNS is largely unknown [15]. As a first step to investigate a putative role of HSP90 in the CNS, it is important to discover its regional distribution and cellular location. Therefore, the present study examined in situ at a cellular
189
P Gass et al./Neuroscience Letters 182 (1994) 188-192
and subcellular level the expression of HSP90 protein in the rat brain. Experiments were performed on male SpragueDawley rats (250-300 g). For Western blots, animals were sacrificed by decapitation under ether anesthesia (n = 4), followed by rapid microdissection of brain tissue (50-100 mg) from hippocampus, somatosensory cortex, striatum and cerebellum. Total protein extracts were gained by boiling in hot lysis buffer (96°C, 4% SDS, 30% glycerol, 5% fl-mercaptoethanol, 10 mM dithiothreitol, 160 mM Tris-HCl, pH 6,8). In an additional series of animals (n = 3), preparation of cytosolic and nuclear protein extracts was performed on whole forebrains by differential centrifugation as described elsewhere [4]. SDS polyacrylamide gel electrophoresis and immunoblot analysis were carried out as previously [7]. A polyclonal antiserum against HSP90 purified from wildtype $49.1 mouse lymphoma cells was produced in a New Zealand rabbit [16]. For immunocytochemistry, animals were sacrificed by decapitation, followed by rapid removal of the brains and freezing on dry ice (n = 6). Coronal 10-/am cryostat sections were incubated in 10% normal swine serum in PBST (PBS containing 0.2% Triton X-100, pH 7.4) for 1 h, followed by the HSP90 antiserum, diluted 1 : 100 in 10% normal swine serum in PBST for 18 h at 4°C. Immunoreactivity (IR) was visualized by the avidin-biotin complex method as previously described [6]. Controls were performed by omitting the primary antibody and always remained immunonegative. A subset of sections was counterstained with hematum. Anatomical structures where identified using a stereotaxic atlas of the rat brain. For immuno-electron microscopy, two animals were perfusion-fixed via the left ventricle using Zamboni's fixative [13]. Immunocytochemistry was performed on coronal 40-/zm vibratome sections as previously described, including visualization of IR with DAB [13]. Thereafter, the sections were osmicated in 2% osmium tetroxide in PBS for 1 h and subsequently dehydrated in a graded series of ethanol and fiat-embedded in durcupan. Regions of interest were mounted onto gelatin capsules, ultrathin sections were prepared and analysed with a Zeiss EM 902 electron microscope. Controls were performed by omitting the primary antibody. Western blot analysis of total protein extracts from different micodissected rat brain areas demonstrated a major immunoreactive band in the range of 90 kDa (Fig. 1A). When loading identical amounts of total protein, about equal levels of IR product were detected in extracts from hippocampus, somatosensory cortex, striatum and cerebellum (Fig. 1A). Using cytosolic and nuclear fractions from whole forebrains, a strong immunoreactive 90-kDa band was found in the cytosolic extracts, however, a significantly weaker band of identical molecular weight was also present in the nuclear fraction (Fig. 1B).
nc
Cx
St
Cb
Cy
Nu
10680-
49-
Fig. 1. Western blot analysisof HSP90 expression in the rat brain. A: using identical amounts of total protein extracts from different micodissectedrat brain areas, aboutequallevelsof HSP90are detected in hippocampus (Hc), somatosensorycortex (Cx), striatum (St) and cerebellum (Cb). B: using cytosolic(Cy) and nuclear (Nu) fractions from wholeforebrains,a strong immunoreactive90-kDa band is present in the cytosolicextracts. A markedlyweaker band of identical molecularweightis also foundin the nuclear fraction.
In immunocytochemical experiments, HSP90 expression was found in virtually all neuronal cell populations of the rat brain (Fig. 2A-F). Within the brain parenchyma, HSP90 expression was restricted to neurons as demonstrated by counterstaining with hemalum (not shown). Only ependyma and choroid plexus also demonstrated distinct HSP90 IR (Fig. 2F). In neurons, IR at light microscopic level was restricted to the perikaryon. Strong labeling was found in all neuronal populations of the hippocampus (Fig. 2A) as well as other areas of the limbic system, such as amygdala complex, piriform, entorhinal, cingulate and olfactory cortex (not shown). Marked neuronal HSP90 expression was also observed in all layers of the neocortex (Fig. 2C), in the striatum (Fig. 2B), thalamus (Fig. 2D) and hypothalamus (not shown) as well as brain stem nuclei (not shown). In the cerebellum, HSP90 was most prominently expressed in Purkinje cells but also in granule cells (Fig. 2E). In addition, distinct HSP90 expression was observed in scattered basket or stellate neurons of the molecular layer (Fig. 2E). At the ultrastructural level, immunoprecipitate was mainly found in neuronal perikarya, mostly associated with the outer membrane of the nuclear envelope and with numerous free polyribosomes (Fig. 3A). In addition, minor IR was observed in dendrites, often associated with the microtubular system, but never with synaptic sites (Fig. 3B). Several neuronal nuclei also contained distinct IR product (Fig. 3C). This finding agrees well with Western blot experiments (see above) that also demonstrated a certain amount of HSP90 in the nuclear compartment. No IR product was observed in glial cells.
190
P. Gass et al./Neuroscience Letters 182 (1994) 188 192
Fig. 2. Regional expression of HSP90 in the rat brain. A: in the hippocampus, all major neuronal subpopulations, i.e., dentate gyrus, hilus, CA1 and CA3 demonstrate marked HSP90 expression. B: in the striatum, HSP90 is found in large, medium-sized and small neurons. C: in the somatosensory cortex, HSP90 IR is observed in numerous neurons in all layers (pial surface = left margin). D: HSP90 expression in the ventrolateral nucleus of the thalamus. E: in the cerebellum, HSP90 IR is most prominent in Purkinje neurons but also found in granule cells and neurons of the molecular layer. F: marked HSP90 expression in neurons of the habenular nuclei and the surrounding thalamic nuclei. In addition, prominent HSP90 IR is found in the ventricular ependymal lining and the choroid plexus (scale bars: A,C,E,F = 300/~m; B,D = 100/lm).
The present study demonstrates that HSP90 is abundantly expressed by many neuronal populations of the rat brain, using a polyclonal antiserum against HSP90 that reacts selectively with a 90-kDa band in whole protein extracts of the rat brain. In Western blot experiments, similar amounts of HSP90 were found in different CNS regions, such as neocortex, hippocampus, striatum and cerebellum (Fig. 1). Similar intensities of HSP90 IR were also found by immunocytochemistry (Fig. 2). In agreement with other organs, HSP90 is mainly localized in the cytoplasm [12]. In contrast to the major form of the HSP70s, HSP72, HSP90 IR at light microscopic level is restricted to the perikaryon and not found in neuronal processes, i.e., dendrites or axons. Immuno-electron microscopy, however, revealed some IR products in dendrites and also in neuronal nuclei. At light and electron microscopic level, HSP90 was not detected in astro- or
oligodendrocytes but distinct expression was found in ependyma and cells of the choroid plexus (Fig. 2F). Recent evidence suggests that members of the HSP90 class act as molecular chaperones in the mechanism of signal transduction by glucocorticoid and other steroid receptors [15]. Steroid receptors are initially localized in the cytoplasm and form complexes with HSP90. This complex formation seems to represent a precondition for receptor activation through specific ligands by allowing it to maintain an activable conformation [15]. After ligand-binding and subsequent HSP90 dissociation, the 'naked' receptor complex is transported to the nucleus where it binds at specific DNA sites. A growing body of evidence indicates that intracellular signal transduction is an important mechanism that converts environmental stimuli to changes of the cellular genetic program. According to current concepts, in the CNS, such changes
P. Gass et al. / Neuroscience Letters 182 (1994) 188-192
191
Fig. 3. Subcellular distribution of HSP90 IR in hippocampal pyramidal cells. A: at the electron microscopic level, HSP90 immunoprecipitate (arrowheads) is mainly found in perikarya, often associated with polyribosomes. B: furthermore, immunoprecipitate is observed in dendrites (here horizontally cut), often associated with microtubules (arrowheads). C: occasionally, immunoprecipitate was also located in neuronal nuclei (M, mitochondrion; Nu, nucleus; A-C: x 39,000).
are a prerequisite for and represent a molecular correlate o f neuronal plasticity, n o t only in physiological but also in pathological conditions. Thus, changes o f neuronal heat shock protein expression have been reported following epilepsy and ischemia and have been postulated to represent adaptive cellular mechanisms to metabolic stress [2,14]. In addition to steroid receptors, H S P 9 0 complexes with a variety o f other cellular proteins, e.g., tyrosine kinases [3], e I F - 2 a l p h a kinase [17] as well as tubulin and actin [10,18]. The functional role o f H S P 9 0 association with these molecules remains to be shown. In vitro, H S P 9 0 can be induced by heat shock [20]. It will be i m p o r t a n t to investigate, whether and by which stimuli H S P 9 0 can be induced in the C N S under in vivo conditions and in which cell populations. Thus, HSP72 is induced in astrocytes following heat shock but in neurons after ischemia and epilepsy [2,11,14]. Similar data for H S P 9 0 are currently n o t available. As basis for further studies, the present study demonstrates the constitutive expression o f H S P 9 0 in widespread C N S neuronal cell populations, suggesting a functional role in the n o r m a l molecular p r o g r a m o f m o s t C N S neurons. We w o u l d like to acknowledge the excellent technical assistance o f S. Hennes, C. Kammerer, A. Jeske and C. H o f f m a n n . Drs. B. Segnitz and U. Gehring ( D e p a r t m e n t o f Biochemistry, University o f Heidelberg, Heidelberg, G e r m a n y ) generously provided the antiserum against HSP90. This w o r k was supported by grants f r o m the Deutsche Forschungsgemeinschaft (SFB 317/A13; Schr 283/8-2). [1] Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817-1820.
[2] Brown, I.R., Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective, J. Neurosci. Res., 27 (1990) 247-255. [3] Brugge, J.S., Erikson, E. and Erikson, R.L., The specific interaction of the Rous sarcoma virus transforming protein, pp60src with two cellular proteins, Cell, 25 (1981) 363-372. [4] Busch, H., Isolation and purification of nuclei. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Academic Press, New York, NY, 1967, pp. 421-448. [5] Ellis, R.J. and Van der Vies, S.M., Molecular chaperons, Annu. Rev. Biochem., 60 (1991) 321-347. [6] Gass, P., Herdegen, T., Bravo, R. and Kiessling, M., Spatiotemporal induction of immediate early genes in the rat brain after limbic seizures: effects of NMDA-receptor antagonist MK-801, Eur. J. Neurosci., 5 (1993) 933-943. [7] Gass, P., Kiessling, M. and Bading, H., Regionally selective stimulation of mitogen activated protein (MAP) kinase tyrosine phosphorylation after generalized seizures in the rat brain, Neurosci. Lea., 162 (1993) 39-42. [8] Izumoto, S. and Herbert, J., Widespread constitutive expression of HSP90 messenger RNA in rat brain, J. Neurosci. Res., 35 (1993) 20-28. [9] Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to ischemia in gerbil hippocampal neurons, J. Cereb. Blood Flow Metab., 11 (1991) 299-307. [10] Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H. and Yahara, I., Two mammalian heat shock proteins, hsp90 and hsp 100, are actin binding proteins, Proc. Natl. Acad. Sci. USA, 83 (1986) 8054-8058. [11] Marini, A.M., Kozuka, M., Lipsky, R.H. and Nowak, T.S., 70kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro: comparison with immunocytochemical localization after hyperthermia in vivo, J. Neurochem., 54 (1990) 1509-1516. [12] McGuire, J.A., Poellinger, L., Wikstr6m, A.C. and Gustaffson, J.A., Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90, J. Steroid Biochem. Mol. Biol., 42 (1992) 813-822. [13] Naas, E., Zilles, K., Gnahn, H., Betz, H., Becker, C.M. and Schr6der, H., Glycine receptor immunoreactivity in rat and human cerebral cortex, Brain Res., 561 (1991) 139-146. [14] Nowak, Jr., T.S., Protein synthesis and heat shock/stress response after ischemia, Cerebrovascular Brain Metab. Rev., 2 (1990) 345366.
192
P Gass et al./Neuroscience Letters 182 (1994) 188-192
[15] Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S. and Yamamoto, K.R., Reduced levels of hsp90 compromise steroid receptor action in vivo, Nature (London), 348 (1990) 166-168. [16] Rexin, M., Busch, W. and Gehring, U., Chemical cross-linking of heteromeric glucocorticoid receptors, Biochemistry, 27 (1988) 5593-5601. [17] Rose, D.W., Wettenhall, R.E.H., Kudlicki, W., Kramer, G. and Hardesty, B., The 90-kilodalton peptide of the heme-regulated elF-2a kinase has sequence similarity with the 90-kilodalton heat schock protein, Biochemistry, 26 (1987) 6583~587.
[18] Sanchez, E.R., Redmont, T., Scherrer, L.C., Bresnick, E.H., Welsh, M.J. and Pratt, W.B., Evidence that the 90-kilodalton heat schock protein is associated with tubulin-containingcomplexes in L cytosol and in intact cells, Mol. Endo., 2 (1988) 756-760. [19] Schlesinger, M.J., Heat shock proteins: the search for functions, J. Cell Biol., 103 (1986) 321-325. [20] Welch, W.J. and Suhan, J.P., Cellular and biochemical events in mammalian cells during and after recovery from physiological stress, J. Cell Biol., 103 (1986) 2035-2052.
ELSEVIER
HEUROSClENC[ [ETT[R$
Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain P e t e r G a s s a'*, H a n n s j 6 r g
S c h r 6 d e r b, P e t e r P r i o r a, M a r i k a
Kiessling a
alnstitute of Neuropathology, University of Heidelberg, lm Neuenheimer Feld 220, 69120 Heidelberg. Germany bDepartment qf Anatomy. University of Cologne. K61n. Germany
Received 21 September 1994; Revised version received 14 October 1994; Accepted 14 October 1994
Abstract
Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed thoughout all neuronal subpopulations of the CNS but not in nonneuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons. Key words: HSP90; lmmunocytochemistxy; Immunoblotting; Immuno-electron microscopy
Exposure of cells or organisms to heat or other forms of environmental stress leads to the rapid induction of a group of proteins, called 'heat shock' or 'stress proteins' [20]. It has been proposed that the common stimulus for heat shock protein induction is protein denaturation and that one function of inducible members of stress proteins is to prevent or repair denaturation damage [19]. However, several members of the heat shock protein family demonstrate constitutive expression in 'nonstressing' conditions, suggesting that some of these molecules play a role in normal metabolic cellular functions. In general, heat shock proteins are considered to represent molecular chaperones, defined as a family of unrelated classes of proteins that mediate the correct assembly (and disassembly) of other polypeptides, but are not themselves components of the final functional protein structures [5]. The mammalian genome contains three major classes of heat shock proteins, the HSP70s, HSP90s and
*Corresponding author. Fax: (49) (6221) 56,3466. 0304-3940194l$7.00© 1994 Elsevier ScienceB.V, All rights reserved S S D I 0304-3940(94)00794-2
HSP20s, categorized according to the molecular mass of the principle proteins [5]. In the CNS, those o f ~ 7 0 kDa have been most intensively studied, comprising a family of related genes that include constitutively expressed and stress inducible members [5]. The latter have been associated with the development of tolerance to metabolic disturbances in vitro and in vivo, such as hyperthermia or cerebral ischemia [1,9]. In contrast to the HSP70s, little is known about the HSP90s in the CNS. Western blot experiments with cytosolic fractions of whole brain extracts demonstrated the presence of the major 90-kDa heat shock protein HSP90. Furthermore, in situ hybridization studies indicated a generalized expression of HSP90 m R N A in many neuronal populations throughout the brain [8]. Although there are some suggestions of a physiological role of HSP90, e.g., in intracellular transport mechanisms or in regulation of the activities of steroid hormone receptors, the biological function of HSP90 in CNS is largely unknown [15]. As a first step to investigate a putative role of HSP90 in the CNS, it is important to discover its regional distribution and cellular location. Therefore, the present study examined in situ at a cellular
189
P Gass et al./Neuroscience Letters 182 (1994) 188-192
and subcellular level the expression of HSP90 protein in the rat brain. Experiments were performed on male SpragueDawley rats (250-300 g). For Western blots, animals were sacrificed by decapitation under ether anesthesia (n = 4), followed by rapid microdissection of brain tissue (50-100 mg) from hippocampus, somatosensory cortex, striatum and cerebellum. Total protein extracts were gained by boiling in hot lysis buffer (96°C, 4% SDS, 30% glycerol, 5% fl-mercaptoethanol, 10 mM dithiothreitol, 160 mM Tris-HCl, pH 6,8). In an additional series of animals (n = 3), preparation of cytosolic and nuclear protein extracts was performed on whole forebrains by differential centrifugation as described elsewhere [4]. SDS polyacrylamide gel electrophoresis and immunoblot analysis were carried out as previously [7]. A polyclonal antiserum against HSP90 purified from wildtype $49.1 mouse lymphoma cells was produced in a New Zealand rabbit [16]. For immunocytochemistry, animals were sacrificed by decapitation, followed by rapid removal of the brains and freezing on dry ice (n = 6). Coronal 10-/am cryostat sections were incubated in 10% normal swine serum in PBST (PBS containing 0.2% Triton X-100, pH 7.4) for 1 h, followed by the HSP90 antiserum, diluted 1 : 100 in 10% normal swine serum in PBST for 18 h at 4°C. Immunoreactivity (IR) was visualized by the avidin-biotin complex method as previously described [6]. Controls were performed by omitting the primary antibody and always remained immunonegative. A subset of sections was counterstained with hematum. Anatomical structures where identified using a stereotaxic atlas of the rat brain. For immuno-electron microscopy, two animals were perfusion-fixed via the left ventricle using Zamboni's fixative [13]. Immunocytochemistry was performed on coronal 40-/zm vibratome sections as previously described, including visualization of IR with DAB [13]. Thereafter, the sections were osmicated in 2% osmium tetroxide in PBS for 1 h and subsequently dehydrated in a graded series of ethanol and fiat-embedded in durcupan. Regions of interest were mounted onto gelatin capsules, ultrathin sections were prepared and analysed with a Zeiss EM 902 electron microscope. Controls were performed by omitting the primary antibody. Western blot analysis of total protein extracts from different micodissected rat brain areas demonstrated a major immunoreactive band in the range of 90 kDa (Fig. 1A). When loading identical amounts of total protein, about equal levels of IR product were detected in extracts from hippocampus, somatosensory cortex, striatum and cerebellum (Fig. 1A). Using cytosolic and nuclear fractions from whole forebrains, a strong immunoreactive 90-kDa band was found in the cytosolic extracts, however, a significantly weaker band of identical molecular weight was also present in the nuclear fraction (Fig. 1B).
nc
Cx
St
Cb
Cy
Nu
10680-
49-
Fig. 1. Western blot analysisof HSP90 expression in the rat brain. A: using identical amounts of total protein extracts from different micodissectedrat brain areas, aboutequallevelsof HSP90are detected in hippocampus (Hc), somatosensorycortex (Cx), striatum (St) and cerebellum (Cb). B: using cytosolic(Cy) and nuclear (Nu) fractions from wholeforebrains,a strong immunoreactive90-kDa band is present in the cytosolicextracts. A markedlyweaker band of identical molecularweightis also foundin the nuclear fraction.
In immunocytochemical experiments, HSP90 expression was found in virtually all neuronal cell populations of the rat brain (Fig. 2A-F). Within the brain parenchyma, HSP90 expression was restricted to neurons as demonstrated by counterstaining with hemalum (not shown). Only ependyma and choroid plexus also demonstrated distinct HSP90 IR (Fig. 2F). In neurons, IR at light microscopic level was restricted to the perikaryon. Strong labeling was found in all neuronal populations of the hippocampus (Fig. 2A) as well as other areas of the limbic system, such as amygdala complex, piriform, entorhinal, cingulate and olfactory cortex (not shown). Marked neuronal HSP90 expression was also observed in all layers of the neocortex (Fig. 2C), in the striatum (Fig. 2B), thalamus (Fig. 2D) and hypothalamus (not shown) as well as brain stem nuclei (not shown). In the cerebellum, HSP90 was most prominently expressed in Purkinje cells but also in granule cells (Fig. 2E). In addition, distinct HSP90 expression was observed in scattered basket or stellate neurons of the molecular layer (Fig. 2E). At the ultrastructural level, immunoprecipitate was mainly found in neuronal perikarya, mostly associated with the outer membrane of the nuclear envelope and with numerous free polyribosomes (Fig. 3A). In addition, minor IR was observed in dendrites, often associated with the microtubular system, but never with synaptic sites (Fig. 3B). Several neuronal nuclei also contained distinct IR product (Fig. 3C). This finding agrees well with Western blot experiments (see above) that also demonstrated a certain amount of HSP90 in the nuclear compartment. No IR product was observed in glial cells.
190
P. Gass et al./Neuroscience Letters 182 (1994) 188 192
Fig. 2. Regional expression of HSP90 in the rat brain. A: in the hippocampus, all major neuronal subpopulations, i.e., dentate gyrus, hilus, CA1 and CA3 demonstrate marked HSP90 expression. B: in the striatum, HSP90 is found in large, medium-sized and small neurons. C: in the somatosensory cortex, HSP90 IR is observed in numerous neurons in all layers (pial surface = left margin). D: HSP90 expression in the ventrolateral nucleus of the thalamus. E: in the cerebellum, HSP90 IR is most prominent in Purkinje neurons but also found in granule cells and neurons of the molecular layer. F: marked HSP90 expression in neurons of the habenular nuclei and the surrounding thalamic nuclei. In addition, prominent HSP90 IR is found in the ventricular ependymal lining and the choroid plexus (scale bars: A,C,E,F = 300/~m; B,D = 100/lm).
The present study demonstrates that HSP90 is abundantly expressed by many neuronal populations of the rat brain, using a polyclonal antiserum against HSP90 that reacts selectively with a 90-kDa band in whole protein extracts of the rat brain. In Western blot experiments, similar amounts of HSP90 were found in different CNS regions, such as neocortex, hippocampus, striatum and cerebellum (Fig. 1). Similar intensities of HSP90 IR were also found by immunocytochemistry (Fig. 2). In agreement with other organs, HSP90 is mainly localized in the cytoplasm [12]. In contrast to the major form of the HSP70s, HSP72, HSP90 IR at light microscopic level is restricted to the perikaryon and not found in neuronal processes, i.e., dendrites or axons. Immuno-electron microscopy, however, revealed some IR products in dendrites and also in neuronal nuclei. At light and electron microscopic level, HSP90 was not detected in astro- or
oligodendrocytes but distinct expression was found in ependyma and cells of the choroid plexus (Fig. 2F). Recent evidence suggests that members of the HSP90 class act as molecular chaperones in the mechanism of signal transduction by glucocorticoid and other steroid receptors [15]. Steroid receptors are initially localized in the cytoplasm and form complexes with HSP90. This complex formation seems to represent a precondition for receptor activation through specific ligands by allowing it to maintain an activable conformation [15]. After ligand-binding and subsequent HSP90 dissociation, the 'naked' receptor complex is transported to the nucleus where it binds at specific DNA sites. A growing body of evidence indicates that intracellular signal transduction is an important mechanism that converts environmental stimuli to changes of the cellular genetic program. According to current concepts, in the CNS, such changes
P. Gass et al. / Neuroscience Letters 182 (1994) 188-192
191
Fig. 3. Subcellular distribution of HSP90 IR in hippocampal pyramidal cells. A: at the electron microscopic level, HSP90 immunoprecipitate (arrowheads) is mainly found in perikarya, often associated with polyribosomes. B: furthermore, immunoprecipitate is observed in dendrites (here horizontally cut), often associated with microtubules (arrowheads). C: occasionally, immunoprecipitate was also located in neuronal nuclei (M, mitochondrion; Nu, nucleus; A-C: x 39,000).
are a prerequisite for and represent a molecular correlate o f neuronal plasticity, n o t only in physiological but also in pathological conditions. Thus, changes o f neuronal heat shock protein expression have been reported following epilepsy and ischemia and have been postulated to represent adaptive cellular mechanisms to metabolic stress [2,14]. In addition to steroid receptors, H S P 9 0 complexes with a variety o f other cellular proteins, e.g., tyrosine kinases [3], e I F - 2 a l p h a kinase [17] as well as tubulin and actin [10,18]. The functional role o f H S P 9 0 association with these molecules remains to be shown. In vitro, H S P 9 0 can be induced by heat shock [20]. It will be i m p o r t a n t to investigate, whether and by which stimuli H S P 9 0 can be induced in the C N S under in vivo conditions and in which cell populations. Thus, HSP72 is induced in astrocytes following heat shock but in neurons after ischemia and epilepsy [2,11,14]. Similar data for H S P 9 0 are currently n o t available. As basis for further studies, the present study demonstrates the constitutive expression o f H S P 9 0 in widespread C N S neuronal cell populations, suggesting a functional role in the n o r m a l molecular p r o g r a m o f m o s t C N S neurons. We w o u l d like to acknowledge the excellent technical assistance o f S. Hennes, C. Kammerer, A. Jeske and C. H o f f m a n n . Drs. B. Segnitz and U. Gehring ( D e p a r t m e n t o f Biochemistry, University o f Heidelberg, Heidelberg, G e r m a n y ) generously provided the antiserum against HSP90. This w o r k was supported by grants f r o m the Deutsche Forschungsgemeinschaft (SFB 317/A13; Schr 283/8-2). [1] Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817-1820.
[2] Brown, I.R., Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective, J. Neurosci. Res., 27 (1990) 247-255. [3] Brugge, J.S., Erikson, E. and Erikson, R.L., The specific interaction of the Rous sarcoma virus transforming protein, pp60src with two cellular proteins, Cell, 25 (1981) 363-372. [4] Busch, H., Isolation and purification of nuclei. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Academic Press, New York, NY, 1967, pp. 421-448. [5] Ellis, R.J. and Van der Vies, S.M., Molecular chaperons, Annu. Rev. Biochem., 60 (1991) 321-347. [6] Gass, P., Herdegen, T., Bravo, R. and Kiessling, M., Spatiotemporal induction of immediate early genes in the rat brain after limbic seizures: effects of NMDA-receptor antagonist MK-801, Eur. J. Neurosci., 5 (1993) 933-943. [7] Gass, P., Kiessling, M. and Bading, H., Regionally selective stimulation of mitogen activated protein (MAP) kinase tyrosine phosphorylation after generalized seizures in the rat brain, Neurosci. Lea., 162 (1993) 39-42. [8] Izumoto, S. and Herbert, J., Widespread constitutive expression of HSP90 messenger RNA in rat brain, J. Neurosci. Res., 35 (1993) 20-28. [9] Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to ischemia in gerbil hippocampal neurons, J. Cereb. Blood Flow Metab., 11 (1991) 299-307. [10] Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H. and Yahara, I., Two mammalian heat shock proteins, hsp90 and hsp 100, are actin binding proteins, Proc. Natl. Acad. Sci. USA, 83 (1986) 8054-8058. [11] Marini, A.M., Kozuka, M., Lipsky, R.H. and Nowak, T.S., 70kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro: comparison with immunocytochemical localization after hyperthermia in vivo, J. Neurochem., 54 (1990) 1509-1516. [12] McGuire, J.A., Poellinger, L., Wikstr6m, A.C. and Gustaffson, J.A., Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90, J. Steroid Biochem. Mol. Biol., 42 (1992) 813-822. [13] Naas, E., Zilles, K., Gnahn, H., Betz, H., Becker, C.M. and Schr6der, H., Glycine receptor immunoreactivity in rat and human cerebral cortex, Brain Res., 561 (1991) 139-146. [14] Nowak, Jr., T.S., Protein synthesis and heat shock/stress response after ischemia, Cerebrovascular Brain Metab. Rev., 2 (1990) 345366.
192
P Gass et al./Neuroscience Letters 182 (1994) 188-192
[15] Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S. and Yamamoto, K.R., Reduced levels of hsp90 compromise steroid receptor action in vivo, Nature (London), 348 (1990) 166-168. [16] Rexin, M., Busch, W. and Gehring, U., Chemical cross-linking of heteromeric glucocorticoid receptors, Biochemistry, 27 (1988) 5593-5601. [17] Rose, D.W., Wettenhall, R.E.H., Kudlicki, W., Kramer, G. and Hardesty, B., The 90-kilodalton peptide of the heme-regulated elF-2a kinase has sequence similarity with the 90-kilodalton heat schock protein, Biochemistry, 26 (1987) 6583~587.
[18] Sanchez, E.R., Redmont, T., Scherrer, L.C., Bresnick, E.H., Welsh, M.J. and Pratt, W.B., Evidence that the 90-kilodalton heat schock protein is associated with tubulin-containingcomplexes in L cytosol and in intact cells, Mol. Endo., 2 (1988) 756-760. [19] Schlesinger, M.J., Heat shock proteins: the search for functions, J. Cell Biol., 103 (1986) 321-325. [20] Welch, W.J. and Suhan, J.P., Cellular and biochemical events in mammalian cells during and after recovery from physiological stress, J. Cell Biol., 103 (1986) 2035-2052.