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Distribution of heat shock protein 108 mRNA in the chicken central nervous system
Neuroscience Letters 283 (2000) 181±184
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Distribution of heat shock protein 108 mRNA in the chicken central nervous system Dong Hoon Shin a, Hyun Joon Kim b, Hwa Young Lee a, Kyung Hoon Lee c, Gye Sun Jeon d, Je Hoon Seo d, Sang Ho Baik d, Sa Sun Cho d, e,* a
Department of Anatomy, Dankook University College of Medicine, Chonan, South Korea Department of Anatomy, Gyeongsang National University College of Medicine, Chinju, South Korea c Department of Anatomy, Sungkyunkwan University College of Medicine, Suwon, South Korea d Department of Anatomy, Seoul National University, College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea e Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea b
Received 10 January 2000; received in revised form 14 February 2000; accepted 16 February 2000
Abstract The constitutive expression of heat shock protein 108 (HSP108) mRNA is mapped in a normal chicken central nervous system using in situ hybridization technique. HSP108 mRNAs were found to be mainly localized in the small neuroglial cells of various regions of the brain, although some neuronal cells also showed positive signals. This tendency is observed to be more marked in the cerebellum; HSP108 signals were not found in the Purkinje cells, but in Bergmann glial cells and oligodendrocytes. Although neuronal cells in the deep cerebellar nuclei and the molecular layer showed occasional HSP108 signals, the expression pattern of HSP108 mRNA is different from homologous HSP90 that is mostly expressed in neurons, but rather similar to that of TfBP immunoreactivity, a new member of the HSP108 family. The constitutive neuroglial localization of HSP108 could suggest that HSP108 may play an important role in the normal metabolism of neuroglial cells in the chicken brain. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: In situ hybridization; Brain; Heat shock protein; Chicken; Bergmann glial cell; Oligodendrocyte
Heat shock protein (HSP) is known to include a series of stress proteins induced by several different stressful conditions such as heat, ischemia, in¯ammation, oxidative stress, and even behavioral and psychological stresses [12,13]. However, the results of several studies suggest that they are not restricted to expression in stress conditions, but rather are also expressed under normal conditions [4,5,7,19]. HSP108, the avian homologue of the mammalian GRP94 family of stress-regulated proteins [6,14], was originally puri®ed from chicken oviduct [11]. Although transferrin binding protein (TfBP), which also belongs to the HSP108 family was extensively studied in our laboratory [1], comparatively little work has been carried out on the expression of HSP108 mRNA in the chicken brain. Thus in the present study, we ®rst utilized riboprobes speci®c to HSP108 mRNA to reveal the constitutional distribution of HSP108 mRNA in the chicken central nervous system (CNS). * Corresponding author. Tel.: 182-2-740-8204; fax: 182-2-7459528. E-mail address: [email protected] (S.S. Cho).
The brains of adult white leghorn chicken were used in this study. After the tissues were sliced into 12 mm sections on a cryostat, the sections were ®xed in 4% (w/v) paraformaldehyde solution and treated with chloroform for the purpose of protein removal. The animals used in this experiment were treated in accordance with the `Principles of Laboratory Animal Care' (NIH publication No. 86-23, revised 1985). Total cellular RNA was extracted from white leghorn chickens aged embryonic day 18 (E18) using the guanidine thiocyanate (GTC) method [17]. HSP108 cDNA was ampli®ed by reverse-transcription polymerase chain reaction (RTPCR) method using the oligonucleotide primers such as 5 0 HSP108 primer: CCA GTT TGG TGT TGG CTT TT and 3 0 HSP108 primer: CCT CCT TTG CTT CCT CCT CT. The design of these PCR primers was based on previous cloning results [3,11], and the predicted size of the ampli®ed chicken HSP108 cDNA was 323bp. The PCR products were cloned into T easy vector (Promega) and sequenced by the dideoxynucleotide chain termination method applying the T7 sequencing kit (Pharmacia). Digoxigenin-11-UTP (Boehringer-Mannheim) labeled antisense HSP108 cRNA probe was
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 96 7- 8
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D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
Table 1 Distribution of HSP108 mRNA in the chicken central nervous system a Forebrain Hyperstriatum ventrale Neostriatum frontale Neostriatum caudale Hyperstriatum accessorium Midbrain Optic tectum SO SGFS SGC SAC SGP
11 11 1 11 11 1 111 11 1 1
Tractus septomesencephalicus Tractus opticus Cerebellum Molecular layer Purkinje cell layer Purkinje cell Bergmann glia Granular layer White matter Medulla oblongata Edinger-Westphal Nu. Nu. of CNVIII
1 1 1 ± 11 1 11 11
a
Five semi-quantitative scale (±, no expression above background density level; 1, ,15% of maximal level; 11, ,30% of maximal level; 111, ,70% of maximal level; 1111, above 70% of maximal level) is based on the quantitation from densitometric measurement. SO, striatum opticum; SGFS, striatum griseum et ®brosum super®ciale; SGC, striatum griseum centrale; SAC, striatum album centrale; SGP, striatum griseum periventricularis; Nu., nucleus; CNVIII, eighth cranial nerve.
generated by transcribing the ApaI (Promega) linearized plasmid with SP6 RNA polymerase (BoehringerMannheim). Hybridization was performed in accordance with the method described earlier [18]. To assess the signals in each region quantitatively, the NIH image program (Scion Image) was used for densitometric measurement. The percent of maximal density level was grouped into six categories ((2) no expression above background density level; (1) ,15% of maximal level; (11) ,30% of maximal level; (111) ,70% of maximal level; (1111) above 70% of maximal level). The nomenclature of the chicken brain areas
involved in this study was based on the atlas of Hodges [8] and Kuenzel and Masson [10]. The HSP108 mRNA was found to be widespread in many regions of the chicken CNS (Table 1). In the forebrain, most of the HSP108 mRNA positive (HSP108 1) signals were localized within the small cells, which seemed to be neuroglial cells judging from their size and shape (Fig. 1A). In the optic tectum, HSP108 1 cells were found in various layers such as the striatum opticum (SO), the striatum griseum et ®brosum super®ciale (SGFS), the striatum griseum centrale (SGC), the striatum album centrale (SAC) and the striatum
Fig. 1. HSP108 mRNA in the chicken forebrain, midbrain and medulla oblongata. HSP108 1 cells were found in the (A) neostriatum caudale (NC). (B) In the optic tectum, HSP108 1 cells were detected in rows along the several layers. SO, striatum opticum; SGFS, striatum griseum et ®brosum super®ciale; SGC, striatum griseum centrale; SAC, striatum album centrale; SGP, striatum griseum periventricularis; VT, ventriculus tecti mesencephali. (C) Magni®ed image of inset of (B). (D) Medulla oblongata. In addition to the neuroglial cells, much larger neurons in the Edinger-Westphal nucleus (EW) also showed HSP108 1 signals. M, Molecular layer; G, granular layer. (E) Magni®ed image of inset of (D). Scale bars, 100 mm.
D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
183
Fig. 2. HSP108 mRNA in the chicken cerebellum. (A) HSP108 1 cells were found in the molecular layer (M), granular layer (G) of the cerebellar folia. In the white matter (W), relatively fewer cells were found HSP108 mRNA positive. (B) Magni®ed image of cerebellar gray matter. Note the unstained Purkinje cell somata (arrowheads) among the intensely stained Bergmann glial cells (arrows). (C) Magni®ed image of cerebellar white matter (W). Note that the small HSP108 1 cells (arrowheads) were profusely observed while large presumptive neurons (arrows) in the deep cerebellar nuclei were occasionally localized. (D) Molecular layer (M) of the cerebellar folia. The small HSP108 1 cells (arrowheads) were found in this region. Scale bars, 100 mm.
griseum periventricularis (SGP) (Fig. 1B,C). Because oligodendrocytes are related intimately to nerve ®bers, along which they form rows or columns [2], HSP108 1 cells were likely to be oligodendrocytes for their alignment in rows within each layer of the optic tectum. However, we could not rule out the possibility that neuronal cells were also present occasionally among the HSP108 1 cells in the forebrain and the optic tectum. In the medulla oblongata, presumptive neuroglial cells were also found with a few HSP108 1 neuronal cells clustered in some nuclei such as Edinger±Westphal nuclues and nucleus of eighth cranial nerve (Fig. 1D,E). In the case of the cerebellum, HSP108 signals were detected in various regions with different intensities (Fig. 2A). In the Purkinje cell layer, HSP108 1 signals were intensely localized to Bergmann glial cells, which were found in proximity to Purkinje cell bodies, while the Purkinje cell itself did not show any HSP108 1 signals (Fig. 2B). In the white matter of the cerebellum, some non-neuro-
nal cells, which showed almost uniform size and were clustered in short rows, were detected mainly in the cerebellar base region and decreased in number and intensity toward the cerebellar folial regions. Judging from these ®ndings, these cells could be tentatively identi®ed as oligodendrocytes as seen in the optic tectum (Fig. 2C). On the other hand, even though they were not as intense, HSP108 1 signals were also detected in neuron-enriched regions such as the deep cerebellar nuclei (Fig. 2C) and the molecular layer (Fig. 2D), in which all the cells were known to be neuronal [8]. However in the granular layer, we could not clearly elucidate the cellular identity of HSP108 1 cells because the neurons of the granular layer were the smallest in the cerebellum and resembled the shape of neuroglial cells [9]. HSPs are known to be highly conserved throughout evolution. Based on their molecular weights and homology, they are classi®ed into ®ve major families, including
184
D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
HSP100s, 90s, 70s, 60s, and 20s. Of these, the chicken HSP108 of the present study showed a strong homology with HSP90 [14]. In previous studies, HSP90 protein and its mRNA were mainly expressed in the neuronal cells of the cerebellum, Purkinje cells and deep cerebellar nuclei. On the contrary, neuroglial cells such as oligodendrocytes and Bergmann glial cells, showed no HSP90 signals [15,16]. However in the present study, we found some differences in the expression of the HSP108 and HSP90 mRNAs. HSP108 1 signals were much more apparent in the neuroglial cells such as Bergmann glial cells and oligodendrocytes, in which no signals were detected by HSP90. Additionally, the Purkinje cells, which were positive for HSP90, displayed no HSP108 signals while adjacent Bergmann glial cells showed intense signals. These results suggest that the distribution of constitutive HSP108 mRNA is somewhat different from that of HSP90; more neuroglial patterns in comparison with HSP90 which showed more neuronal patterns in previous study [15]. In contrast to the expression discrepancies between HSP108 and HSP90 mRNAs, we found some similarities between HSP108 mRNA expression and TfBP immunoreactivity (TfBP-IR), which is known to be a new member of the HSP108 family [6,14]. Our earlier study [1] revealed that TfBP was mainly localized in the oligodendrocytes and Bergmann glial cells of the chicken CNS. Particularly in the cerebellum, TfBP-IR oligodendrocytes were found in abundance in the white matter where myelinated ®bers occurred. In addition, TfBP-IR was also detected within the Bergmann glial cells of the Purkinje cell layer, whereas no TfBP-IR signals were detected in the Purkinje cells themselves [1]. On the contrary, some minor discrepancies were also present between HSP108 mRNA and TfBP-IR. HSP108 mRNA showed the most intense signals in Bergmann glial cells, with less intensity in the granular layer, and the weakest signals in the molecular layer and white matter. However, TfBP-IR signals in the white matter and the granular layer were more intense than those found in Bergmann glial cells and molecular layer. Although we could not explain the exact reason, these discrepancies might be due to the difference of the epitopes between TfBP and HSP108 proteins because TfBP is thought to be formed through posttranslational modi®cation of HSP108 [7]. In summary, we ®rst detected HSP108 mRNA expression in the normal chicken brain and showed that HSP108 mRNA expression was in part similar to the expression of TfBP with a small number of differences. This constitutive expression of HSP108 mRNA could suggest that HSP108 may play some important role in the normal metabolism of the chicken CNS. This study was supported by a grant (1998) from Educa-
tion and Research Foundation, Seoul National University College of Medicine.
[1] Cho, S.S. and Lucas, J.J., Immunohistochemical study with an anti-transferring binding protein serum: a marker for avian oligodendrocytes, Brain Res., 674 (1995) 15±25. [2] Faucett, D.W., A Textbook of Histology, Saunders, Philadelphia, PA, 1986. [3] Forsgren, M., Raden, B., Israelsson, M., Larsson, K. and Heden, L.O., Molecular cloning and characterization of a full-length cDNA clone for human plasminogen, FEBS Lett., 213 (1987) 254±260. [4] Georgopoulos, C. and Welch, W.J., Role of the major heat shock proteins as molecular chaperones, Annu. Rev. Cell Biol., 9 (1993) 601±634. [5] Hartl, F.U., Hlodan, R. and Langer, T., Molecular chaperones in protein folding: the art of avoiding sticky situations, Trends Biochem. Sci., 19 (1994) 20±25. [6] Hayes, G.R., Himpler, B.S., Weiner, K.X. and Lucas, J.J., A chicken transferrin binding protein is heat shock protein 108, Biochem. Biophys. Res. Commun., 200 (1994) 65±70. [7] Hendrick, J.P. and Hartl, F.U., Molecular chaperone functions of heat-shock proteins, Annu. Rev. Biochem., 62 (1993) 349±384. [8] Hodges, R.D., The Histology of the Fowl, Academic Press, London, 1974. [9] Junqueira, L.C., Carneiro, J. and Long, J.A., Basic Histology, Prentice-Hall, Los Altos, 1986. [10] Kuenzel, W.J. and Masson, M., A Stereotaxic Atlas of the Brain of the Chick, Johns Hopkins University Press, Baltimore, MD, 1988. [11] Kulomaa, M.S., Weigel, N.L., Kleinsek, D.A., Beattie, W.G., Conneely, O.M., March, C., Zarucki-Schulz, T., Schrader, W.T. and O'Malley, B.W., Amino acid sequence of a chicken heat shock protein derived from the complementary DNA nucleotide sequence, Biochemistry, 25 (1986) 6244±6251. [12] Lindquist, S. and Craig, E.A., The heat-shock proteins, Annu. Rev. Genet., 22 (1988) 631±677. [13] Morimoto, R.I., Cells in stress: transcriptional activation of heat shock genes, Science, 259 (1993) 1409±1410. [14] Poola, I. and Kiang, J.G., The estrogen-inducible transferrin receptor-like membrane glycoprotein is related to stressregulated proteins, J. Biol. Chem., 269 (1994) 21762±21769. [15] Quraishi, H. and Brown, I.R., Expression of heat shock protein 90 (hsp90) in neural and non-neural tissues of the control and hyperthermic rabbit, Exp. Cell Res., 219 (1995) 358±363. [16] Quraishi, H., Rush, S.J. and Brown, I.R., Expression of mRNA species encoding heat shock protein 90 (hsp90) in control and hyperthermic rabbit brain, J. Neurosci. Res., 43 (1996) 335±345. [17] Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning, Cold Spring Harbor Laboratory Press, New York, 1989. [18] Shin, D.H., Lee, H.Y., Lee, H.W., Kim, H.J., Lee, E., Cho, S.S., Baik, S.H. and Lee, K.H., In situ localization of p53, bcl-2 and bax mRNAs in rat ocular tissue, NeuroReport, 10 (1999) 2165±2167. [19] Stuart, R.A., Cyr, D.M., Craig, E.A. and Neupert, W., Mitochondrial molecular chaperones: their role in protein translocation, Trends Biochem. Sci., 19 (1994) 87±92.
www.elsevier.com/locate/neulet
Distribution of heat shock protein 108 mRNA in the chicken central nervous system Dong Hoon Shin a, Hyun Joon Kim b, Hwa Young Lee a, Kyung Hoon Lee c, Gye Sun Jeon d, Je Hoon Seo d, Sang Ho Baik d, Sa Sun Cho d, e,* a
Department of Anatomy, Dankook University College of Medicine, Chonan, South Korea Department of Anatomy, Gyeongsang National University College of Medicine, Chinju, South Korea c Department of Anatomy, Sungkyunkwan University College of Medicine, Suwon, South Korea d Department of Anatomy, Seoul National University, College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea e Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea b
Received 10 January 2000; received in revised form 14 February 2000; accepted 16 February 2000
Abstract The constitutive expression of heat shock protein 108 (HSP108) mRNA is mapped in a normal chicken central nervous system using in situ hybridization technique. HSP108 mRNAs were found to be mainly localized in the small neuroglial cells of various regions of the brain, although some neuronal cells also showed positive signals. This tendency is observed to be more marked in the cerebellum; HSP108 signals were not found in the Purkinje cells, but in Bergmann glial cells and oligodendrocytes. Although neuronal cells in the deep cerebellar nuclei and the molecular layer showed occasional HSP108 signals, the expression pattern of HSP108 mRNA is different from homologous HSP90 that is mostly expressed in neurons, but rather similar to that of TfBP immunoreactivity, a new member of the HSP108 family. The constitutive neuroglial localization of HSP108 could suggest that HSP108 may play an important role in the normal metabolism of neuroglial cells in the chicken brain. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: In situ hybridization; Brain; Heat shock protein; Chicken; Bergmann glial cell; Oligodendrocyte
Heat shock protein (HSP) is known to include a series of stress proteins induced by several different stressful conditions such as heat, ischemia, in¯ammation, oxidative stress, and even behavioral and psychological stresses [12,13]. However, the results of several studies suggest that they are not restricted to expression in stress conditions, but rather are also expressed under normal conditions [4,5,7,19]. HSP108, the avian homologue of the mammalian GRP94 family of stress-regulated proteins [6,14], was originally puri®ed from chicken oviduct [11]. Although transferrin binding protein (TfBP), which also belongs to the HSP108 family was extensively studied in our laboratory [1], comparatively little work has been carried out on the expression of HSP108 mRNA in the chicken brain. Thus in the present study, we ®rst utilized riboprobes speci®c to HSP108 mRNA to reveal the constitutional distribution of HSP108 mRNA in the chicken central nervous system (CNS). * Corresponding author. Tel.: 182-2-740-8204; fax: 182-2-7459528. E-mail address: [email protected] (S.S. Cho).
The brains of adult white leghorn chicken were used in this study. After the tissues were sliced into 12 mm sections on a cryostat, the sections were ®xed in 4% (w/v) paraformaldehyde solution and treated with chloroform for the purpose of protein removal. The animals used in this experiment were treated in accordance with the `Principles of Laboratory Animal Care' (NIH publication No. 86-23, revised 1985). Total cellular RNA was extracted from white leghorn chickens aged embryonic day 18 (E18) using the guanidine thiocyanate (GTC) method [17]. HSP108 cDNA was ampli®ed by reverse-transcription polymerase chain reaction (RTPCR) method using the oligonucleotide primers such as 5 0 HSP108 primer: CCA GTT TGG TGT TGG CTT TT and 3 0 HSP108 primer: CCT CCT TTG CTT CCT CCT CT. The design of these PCR primers was based on previous cloning results [3,11], and the predicted size of the ampli®ed chicken HSP108 cDNA was 323bp. The PCR products were cloned into T easy vector (Promega) and sequenced by the dideoxynucleotide chain termination method applying the T7 sequencing kit (Pharmacia). Digoxigenin-11-UTP (Boehringer-Mannheim) labeled antisense HSP108 cRNA probe was
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 96 7- 8
182
D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
Table 1 Distribution of HSP108 mRNA in the chicken central nervous system a Forebrain Hyperstriatum ventrale Neostriatum frontale Neostriatum caudale Hyperstriatum accessorium Midbrain Optic tectum SO SGFS SGC SAC SGP
11 11 1 11 11 1 111 11 1 1
Tractus septomesencephalicus Tractus opticus Cerebellum Molecular layer Purkinje cell layer Purkinje cell Bergmann glia Granular layer White matter Medulla oblongata Edinger-Westphal Nu. Nu. of CNVIII
1 1 1 ± 11 1 11 11
a
Five semi-quantitative scale (±, no expression above background density level; 1, ,15% of maximal level; 11, ,30% of maximal level; 111, ,70% of maximal level; 1111, above 70% of maximal level) is based on the quantitation from densitometric measurement. SO, striatum opticum; SGFS, striatum griseum et ®brosum super®ciale; SGC, striatum griseum centrale; SAC, striatum album centrale; SGP, striatum griseum periventricularis; Nu., nucleus; CNVIII, eighth cranial nerve.
generated by transcribing the ApaI (Promega) linearized plasmid with SP6 RNA polymerase (BoehringerMannheim). Hybridization was performed in accordance with the method described earlier [18]. To assess the signals in each region quantitatively, the NIH image program (Scion Image) was used for densitometric measurement. The percent of maximal density level was grouped into six categories ((2) no expression above background density level; (1) ,15% of maximal level; (11) ,30% of maximal level; (111) ,70% of maximal level; (1111) above 70% of maximal level). The nomenclature of the chicken brain areas
involved in this study was based on the atlas of Hodges [8] and Kuenzel and Masson [10]. The HSP108 mRNA was found to be widespread in many regions of the chicken CNS (Table 1). In the forebrain, most of the HSP108 mRNA positive (HSP108 1) signals were localized within the small cells, which seemed to be neuroglial cells judging from their size and shape (Fig. 1A). In the optic tectum, HSP108 1 cells were found in various layers such as the striatum opticum (SO), the striatum griseum et ®brosum super®ciale (SGFS), the striatum griseum centrale (SGC), the striatum album centrale (SAC) and the striatum
Fig. 1. HSP108 mRNA in the chicken forebrain, midbrain and medulla oblongata. HSP108 1 cells were found in the (A) neostriatum caudale (NC). (B) In the optic tectum, HSP108 1 cells were detected in rows along the several layers. SO, striatum opticum; SGFS, striatum griseum et ®brosum super®ciale; SGC, striatum griseum centrale; SAC, striatum album centrale; SGP, striatum griseum periventricularis; VT, ventriculus tecti mesencephali. (C) Magni®ed image of inset of (B). (D) Medulla oblongata. In addition to the neuroglial cells, much larger neurons in the Edinger-Westphal nucleus (EW) also showed HSP108 1 signals. M, Molecular layer; G, granular layer. (E) Magni®ed image of inset of (D). Scale bars, 100 mm.
D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
183
Fig. 2. HSP108 mRNA in the chicken cerebellum. (A) HSP108 1 cells were found in the molecular layer (M), granular layer (G) of the cerebellar folia. In the white matter (W), relatively fewer cells were found HSP108 mRNA positive. (B) Magni®ed image of cerebellar gray matter. Note the unstained Purkinje cell somata (arrowheads) among the intensely stained Bergmann glial cells (arrows). (C) Magni®ed image of cerebellar white matter (W). Note that the small HSP108 1 cells (arrowheads) were profusely observed while large presumptive neurons (arrows) in the deep cerebellar nuclei were occasionally localized. (D) Molecular layer (M) of the cerebellar folia. The small HSP108 1 cells (arrowheads) were found in this region. Scale bars, 100 mm.
griseum periventricularis (SGP) (Fig. 1B,C). Because oligodendrocytes are related intimately to nerve ®bers, along which they form rows or columns [2], HSP108 1 cells were likely to be oligodendrocytes for their alignment in rows within each layer of the optic tectum. However, we could not rule out the possibility that neuronal cells were also present occasionally among the HSP108 1 cells in the forebrain and the optic tectum. In the medulla oblongata, presumptive neuroglial cells were also found with a few HSP108 1 neuronal cells clustered in some nuclei such as Edinger±Westphal nuclues and nucleus of eighth cranial nerve (Fig. 1D,E). In the case of the cerebellum, HSP108 signals were detected in various regions with different intensities (Fig. 2A). In the Purkinje cell layer, HSP108 1 signals were intensely localized to Bergmann glial cells, which were found in proximity to Purkinje cell bodies, while the Purkinje cell itself did not show any HSP108 1 signals (Fig. 2B). In the white matter of the cerebellum, some non-neuro-
nal cells, which showed almost uniform size and were clustered in short rows, were detected mainly in the cerebellar base region and decreased in number and intensity toward the cerebellar folial regions. Judging from these ®ndings, these cells could be tentatively identi®ed as oligodendrocytes as seen in the optic tectum (Fig. 2C). On the other hand, even though they were not as intense, HSP108 1 signals were also detected in neuron-enriched regions such as the deep cerebellar nuclei (Fig. 2C) and the molecular layer (Fig. 2D), in which all the cells were known to be neuronal [8]. However in the granular layer, we could not clearly elucidate the cellular identity of HSP108 1 cells because the neurons of the granular layer were the smallest in the cerebellum and resembled the shape of neuroglial cells [9]. HSPs are known to be highly conserved throughout evolution. Based on their molecular weights and homology, they are classi®ed into ®ve major families, including
184
D.H. Shin et al. / Neuroscience Letters 283 (2000) 181±184
HSP100s, 90s, 70s, 60s, and 20s. Of these, the chicken HSP108 of the present study showed a strong homology with HSP90 [14]. In previous studies, HSP90 protein and its mRNA were mainly expressed in the neuronal cells of the cerebellum, Purkinje cells and deep cerebellar nuclei. On the contrary, neuroglial cells such as oligodendrocytes and Bergmann glial cells, showed no HSP90 signals [15,16]. However in the present study, we found some differences in the expression of the HSP108 and HSP90 mRNAs. HSP108 1 signals were much more apparent in the neuroglial cells such as Bergmann glial cells and oligodendrocytes, in which no signals were detected by HSP90. Additionally, the Purkinje cells, which were positive for HSP90, displayed no HSP108 signals while adjacent Bergmann glial cells showed intense signals. These results suggest that the distribution of constitutive HSP108 mRNA is somewhat different from that of HSP90; more neuroglial patterns in comparison with HSP90 which showed more neuronal patterns in previous study [15]. In contrast to the expression discrepancies between HSP108 and HSP90 mRNAs, we found some similarities between HSP108 mRNA expression and TfBP immunoreactivity (TfBP-IR), which is known to be a new member of the HSP108 family [6,14]. Our earlier study [1] revealed that TfBP was mainly localized in the oligodendrocytes and Bergmann glial cells of the chicken CNS. Particularly in the cerebellum, TfBP-IR oligodendrocytes were found in abundance in the white matter where myelinated ®bers occurred. In addition, TfBP-IR was also detected within the Bergmann glial cells of the Purkinje cell layer, whereas no TfBP-IR signals were detected in the Purkinje cells themselves [1]. On the contrary, some minor discrepancies were also present between HSP108 mRNA and TfBP-IR. HSP108 mRNA showed the most intense signals in Bergmann glial cells, with less intensity in the granular layer, and the weakest signals in the molecular layer and white matter. However, TfBP-IR signals in the white matter and the granular layer were more intense than those found in Bergmann glial cells and molecular layer. Although we could not explain the exact reason, these discrepancies might be due to the difference of the epitopes between TfBP and HSP108 proteins because TfBP is thought to be formed through posttranslational modi®cation of HSP108 [7]. In summary, we ®rst detected HSP108 mRNA expression in the normal chicken brain and showed that HSP108 mRNA expression was in part similar to the expression of TfBP with a small number of differences. This constitutive expression of HSP108 mRNA could suggest that HSP108 may play some important role in the normal metabolism of the chicken CNS. This study was supported by a grant (1998) from Educa-
tion and Research Foundation, Seoul National University College of Medicine.
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