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Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie
Neuroscience Letters 420 (2007) 138–143
Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie Jae-Kwang Jin a , Yeo-Jung Na a , Joon-Ho Song b , Hong-Gu Joo c , Seungjoon Kim c , Jae-Il Kim d , Eun-Kyoung Choi a , Richard I. Carp d , Yong-Sun Kim a , Taekyun Shin c,∗ a
Ilsong Institute of Life Science, Hallym University Medical Center, Anyang, Kyonggi-do 431-060, South Korea Department of Neurosurgery, Hallym University Medical Center, Anyang, Kyonggi-do 431-060, South Korea c Department of Veterinary Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, South Korea d New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA
b
Received 20 February 2007; received in revised form 21 April 2007; accepted 23 April 2007
Abstract To investigate the involvement of galectin-3 in the process of neurodegeneration in prion diseases, the expression and cellular localization of galectin-3 in the brain were studied in scrapie, a mouse model of prion disease. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses showed that the expression of galectin-3 protein and mRNA was induced in scrapie-affected brains, particularly at the time when the abnormal prion protein PrPSc began to accumulate in the brains. Immunohistochemically, immunostaining for galectin-3 was found mainly in B4-isolectin-positive cells (presumably activated microglia/macrophages), but not in astrocytes. Galectin-3 immunoreactivity was localized mainly in areas of PrPSc accumulation and neuronal death in scrapie-infected brains. These findings suggest that the expression of galectin-3 by activated microglia/macrophages in prion disease correlates with abnormal prion protein accumulation. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Galectin-3; Macrophage; Microglia; Scrapie; Prion disease
Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cows are well studied prion diseases. The etiological agent of some prion diseases is PrPSc , which is the abnormal isoform of the normal cellular protein PrPC [13]. In scrapie, this abnormal prion protein may play a role in the pathogenesis of the glial cell reaction, and the activation of microglia has been proposed to play an important role in the initial neuropathological changes [2]. During the degenerative process in the brains of prion disease models, microglial cells increase expression in a variety of activation molecules, including cytokines [23]. Galectins, which belong to a growing family of galactoside-binding animal lectins, are composed of one or two carbohydrate-recognition domains (CRDs) of approximately 130 amino acids [10,16]. Of the 14 galectin members, the expres-
∗ Corresponding author at: Department of Veterinary Medicine, Cheju National University, Jeju 690-756, South Korea. Tel.: +82 64 754 3363; fax: +82 64 756 3354. E-mail address: [email protected] (T. Shin).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.04.069
sion of galectin-3 has been extensively documented in activated macrophages and in cells that are involved in central nervous system (CNS) inflammation [1,3]. Recently, galectin-3 has been shown to play a pivotal role not only in diverse physiological functions, such as cell growth, apoptosis, and mRNA splicing, but also in pathological processes as an inflammatory mediator [1]. In addition, galectin-3 plays a role in regulating metastasisrelated functions, which include angiogenesis [12], adhesion to the extracellular matrix [7], integrin expression [21], and extravasation [4]. Although galectin-3 expression under pathological conditions of the CNS remains poorly understood, it is strongly associated with the activation of microglia in mouse brains in a model of experimental autoimmune disease [14]. In this regard, the upregulation of galectin-3 in the brain may play a role in the pathology of neurodegenerative CNS disorders, including prion diseases. Studies have shown that galectin-3 mRNA levels are upregulated after prion infections [11,15], but it is not known how galectin-3 is involved in the neuropathology of prion diseases, specifically in scrapie. Therefore, we investigated whether any change occurs in the expression of galectin-3 during the developmental stage of
J.-K. Jin et al. / Neuroscience Letters 420 (2007) 138–143
scrapie and determined the possible association of galectin-3 and PrPSc deposition in diverse regions of the scrapie-infected brain. Eighty C57BL mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The ME7 scrapie strain was kindly provided by Alan Dickinson of the Neuropathogenesis Unit (Edinburgh, UK). Half of the animals (n = 40) were inoculated intracerebrally with 30 l of 1% (w/v) brain homogenate in 0.01 M phosphate-buffered saline (PBS) prepared from ME7infected C57BL mice. Control mice (n = 40) were injected with 30 l of 1% (w/v) homogenate of normal mouse brain and harvested at the same age as the scrapie-positive mice. The animals were euthanized with 16.5% urethane at 60–160 days postinfection (dpi) with the ME7 scrapie strain. The mice were perfused transcardially with PBS followed by 4% paraformaldehyde in PBS. The brains were removed immediately, post-fixed in the same fixative for 2 h at room temperature (RT), rinsed with PBS, dehydrated with ethanol, and embedded in paraffin. For the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, unfixed brains were removed immediately from anesthetized mice and stored at −70 ◦ C until analysis. Total RNA samples from whole mouse brains (sampled at 160 dpi) were extracted with TRIzol reagent (Gibco-BRL, Rockville, MD), according to the manufacturer’s instructions. RT-PCR was performed as follows. The cDNA was synthesized from 2 g of total RNA by reverse transcription using AMV reverse transcriptase (Promega, Madison, WI) and oligo (dT) primer. After incubation for 1 h at 42 ◦ C, the samples were heat-inactivated and kept at 4 ◦ C until used. A 5-l aliquot of the cDNA of each sample was used for PCR with galectin-3specific primers. The PCR conditions consisted of an initial denaturation step at 94 ◦ C for 2 min, then 30 cycles at 94 ◦ C for 1 min, 54 ◦ C for 1 min, and 72 ◦ C for 1.5 min, with a final 6-min extension at 72 ◦ C. The PCR primers used were 5 CAGACAGCTTTTCGCTTAAC-3 (galectin-3 sense) and 5 ACTGTCTTTCTTCCTTTCCC-3 (galectin-3 antisense), and 5 -TGGTATCGTGGAAGGACTCATGAC-3 (GAPDH sense) and 5 -ATGCCAGTGAGCTTCCCGTTCAGC-3 (GAPDH antisense). Total brain lysates from control and scrapie-infected mice (sampled at 60–160 dpi) were obtained by homogenization in lysis buffer [40 mM Tris (pH 8.0), 0.1% Nonidet P-40, 120 mM NaCl, 10 g/ml leupeptin] in the presence of protease inhibitors (10 g/ml leupeptin, 2 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Western blotting was performed as described previously [5]. Briefly, the membranes were incubated overnight at 4 ◦ C with rat monoclonal anti-galectin-3 (M3/38 hybridoma) or mouse monoclonal anti-PrP 10E4 (kindly provided by Dr. Richard Rubenstein, New York State Institute for Basic Research, Staten Island, NY) [6]. After washing three times in TBS buffer containing 0.05% Tween-20, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Pierce, Rockford, IL) or HRP-conjugated anti-rat IgG (Pierce). The protein bands were detected by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). To detect PrPSc , brain homogenates
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Fig. 1. RT-PCR and Western blot analyses of galectin-3 from the brains of control and scrapie-infected mice 160 days postinoculation with the normal inoculum or scrapie strain. Galectin-3 mRNA (A) and protein (B) were significantly upregulated in the brains of scrapie-infected mice. (**)p < 0.01 vs. control levels. Each experiment was repeated at least three times, and similar results were obtained in each experiment. GAPDH and anti--actin were used as controls. Melanoma (B16F10) cell lysate was used as a positive control for galectin-3.
Fig. 2. Western blot analysis of galectin-3 protein expression at various stages during the scrapie incubation period (sampled at 60–160 dpi). For the PrPSc analysis, the samples were treated without or with PK (100 g/ml) before SDSPAGE. Note that the expression of galectin-3 and PrPSc started to increase at 100 dpi. Each experiment was performed at least three times, with similar results; -actin was used as a control.
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(50 g protein) were treated with proteinase K (PK, 100 g/ml) and then incubated for 30 min at 37 ◦ C before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunohistochemical staining was performed as described previously [5]. Briefly, the 6-m-thick sections were exposed to normal donkey serum, and then incubated with anti-galectin-3 (1:5000) overnight at 4 ◦ C for PrPSc staining. Next, the sections were exposed to PK (10 g/ml) for 10 min at RT, and then incubated with anti-PrP 10E4 overnight at 4 ◦ C. Then, the sections were washed in PBS, and treated sequentially with biotinylated anti-rat and anti-mouse immunoglobulin, followed by peroxidase-labeled avidin, and developed with diaminoben-
zidine (Vector ABC Elite kit; Vector Laboratories, Burlingame, CA). For double staining, the sections were incubated with 10% normal donkey serum in PBS (pH 7.4) for 1 h, followed by anti-galectin-3 antibody overnight at 4 ◦ C and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG (1:100) for 1 h at RT. Then, the sections were washed, blocked with 10% normal goat serum in PBS for 1 h at RT, incubated with rabbit polyclonal anti-GFAP (Dako, Copenhagen, Denmark) antibody overnight at 4 ◦ C, and then washed. The final step involved incubation with Lissamine rhodamine (LRSC)-conjugated donkey anti-rabbit IgG (1:100). For microglia and macrophage staining, the lectin GSA was optimized by incubating the sections in
Fig. 3. Immunohistochemical localization of galectin-3 and PrPSc in control and scrapie-infected brains. Galectin-3 immunoreactivity was observed in the striatum (B), hippocampus (E), midbrain (H), and cerebellum (K) in the brains of the scrapie-infected mice, but not in the brains of controls (A, D, G, J). PrPSc deposits were also observed in these regions [striatum (C), hippocampus (F), midbrain (I), and cerebellum (L)]. Scale bar, 10 m.
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0.5 mg/ml trypsin in 0.05 M TBS containing 1 mM CaCl2 (pH 7.6) for 5 min at 37 ◦ C. Biotinylated lectin Griffonia simplicifolia (GSA B4-isolectin; Sigma, St. Louis, MO; 10 mg/ml TBS) was then added for 1 h at RT, followed by tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptavidin (Zymed, San Francisco, CA). The immunofluorescence-stained specimens were examined under a laser confocal microscope (LSM 510; Zeiss, Oberkochen, Germany). Quantitative results are expressed as the mean ± standard deviation (S.D.). One hundred and sixty days after the intracerebral injection of either ME7 or normal brain homogenate, we examined the galectin-3 mRNA levels in control and scrapie-infected mouse brains using RT–PCR. As shown in Fig. 1A, galectin3 mRNA was expressed strongly (over three folds) in ME7 scrapie-infected mice, but only weakly in control mice. Since galectin-3 gene expression was observed in the scrapie-infected brains at 160 dpi, the levels of galectin-3 protein expression were examined in total brain lysates from control and scrapieinfected brains using Western blot analysis. Galectin-3 protein was expressed in the scrapie-infected brains, but not in the control brains (Fig. 1B). The approximate molecular weight of galectin-3 was found to be 30 kDa. The change in galectin-3 protein expression was examined in total lysates of scrapie-infected brains using Western blot analysis during the various scrapie developmental stages (sampled at 60–160 dpi). As shown in Fig. 2, galectin-3 protein was
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weakly expressed in the scrapie-infected brain at 80 and 100 dpi and was upregulated gradually with progression of the disease. Similarly, PrPSc accumulation was low by 100 dpi, but significantly increased in the scrapie-infected brain from 120 dpi on Fig. 2. To examine the cellular distribution of galectin-3, sections from control and scrapie-infected mouse brains were analyzed immunohistochemically. Galectin-3 immunoreactivity was very slight in the control brains (Fig. 3A, D, G, I), but was strong in the hippocampal formation (Fig. 3E) and midbrain (Fig. 3H) in the brains of scrapie-infected mice. As shown in Fig. 3, galectin3 immunoreactivity was more intense in both the hippocampus and midbrain, as compared to other regions, such as the striatum (Fig. 3B) and cerebellum (Fig. 3K). PrPSc deposits were also observed in the regions showing galectin-3 immunoreactivity (Fig. 3C, F, I, L). To determine the cell phenotype of galectin-3 expressing cells in the scrapie-infected brain, we immunostained brain sections with either GSA B4-isolectin as a marker for microglia or antiGFAP as a marker for astrocytes. As shown in Fig. 4, galectin-3 immunoreactivity was present mainly in B4-isolectin-positive microglia, and not in GFAP-positive astrocytes in the midbrains of scrapie-infected mouse brains. The major findings of this report are that galectin-3 is expressed in activated microglia/macrophages during the neurodegenerative process of scrapie and that it is expressed
Fig. 4. Confocal micrographs of scrapie-infected brain sections were analyzed for galectin-3, GFAP, and GSA B4-isolectin immunoreactivity. Confocal image of double-immunostaining of galectin-3 and GSA B4-isolectin showed that B4-isolectin-positive cells (B; arrowheads) were positive for galectin-3 (A; arrows). The merged image is shown in (C). Galectin-3-positive cells (D; arrows) were not GFAP-positive (E; arrowheads). The merged image is shown in (F). Scale bar, 20 m.
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in diverse regions, including the hippocampus, where PrPSc is mainly found during prion infection. Recently, the functional roles of galectin-3 in the CNS have begun to emerge [14], and the mechanisms of galectin-3 induction in the brain have been investigated extensively [20]. Our results suggest that galectin-3 plays an important role in the pathogenic mechanisms in the neurodegenerative process of scrapie. Little is known about the functions of activated microglia in scrapie, although one of the important features of scrapie pathology is that glial cells are activated in close proximity to the pathological lesions [22,23]. In contrast, several studies have focused on the activation of astrocytes. Based on our current study, we postulate that the galectin-3 expression seen in the activated microglia/macrophages of the scrapie-affected region exhibiting intense extracellular PrPSc accumulation is associated with the neurodegeneration observed in scrapie. Since galectin-3 upregulation may play an important role in the phagocytosis of microorganisms and apoptotic cells [17], increased expression of galectin-3 in TSE-affected brains may also be involved in the phagocytosis of damaged cells resulting from PrPSc neurotoxicity. This concept is supported by the observation that prion peptide (106–126) enhances the phagocytic function of microglia [19]. In addition, Le et al. [8] reported that the prion peptide enhances monocyte chemotaxis. Although the exact mechanisms underlying phagocytosis and chemotaxis by microglia/macrophages in prion diseases remain unresolved, it has been postulated that galectin-3 expression in these cells is involved in the mediation of phagocytosis and chemotaxis in regions of injury. Furthermore, galectin3 is a chemoattractant for monocytes and macrophages, and it is more potent than the chemokine monocyte chemoattractant protein-1 [17]. As reported previously [18], galectin-3 has a chemotactic effect that in turn induces the activation of chemokine receptors. In this regard, previous reports from our laboratory have provided evidence that chemokine receptors, including CCR5, are activated in mice that are infected with scrapie [9]. From these observations, we propose that galectin-3-positive cells in the damaged region enhance chemotactic function in the prion animal model. In this model, PrPSc formation started at 100 dpi, when microglial activation occurred [2]. Furthermore, the galectin-3 expression was correlated with PrPSc deposition at the same stage of disease pathology. In conclusion, we postulate that galectin-3 expression by activated microglia/macrophages after PrPSc accumulation plays a role in the subsequent neurodegeneration in murine scrapie. Further experiments using a transgenic animal model, such as galectin-3 knockout mice, are required to fully understand the specific role of galectin-3 in the pathogenesis of scrapie. Acknowledgments This work was supported by a program of the Basic Atomic Energy Research Institute (BAERI), which is a part of the
Nuclear R&D Programs funded by the Ministry of Science & Technology (MOST) and the MRC program of MOST/KOSEF (R13-2005-022-01002),Korea. References [1] J. Almkvist, A. Karlsson, Galectins as inflammatory mediators, Glycoconj. J. 19 (2004) 575–581. [2] A. Giese, D.R. Brown, M.H. Groschup, C. Feldmann, I. Haist, H.A. Kretzschmar, Role of microglia in neuronal cell death in prion disease, Brain Pathol. 8 (1998) 449–457. [3] R.C. Hughes, Secretion of the galectin family of mammalian carbohydrate-binding proteins, Biochim. Biophys. Acta 1473 (1999) 172– 185. [4] H. Inohara, A. Raz, Identification of human melanoma cellular and secreted ligands for galectin-3, Biochem. Biophys. Res. Commun. 201 (1994) 1366–1375. [5] J.K. Jin, Y.J. Na, C. Moon, H. Kim, M. Ahn, Y.S. Kim, T. Shin, Increased expression of osteopontin in the brain with scrapie infection, Brain Res. 1072 (2006) 227–233. [6] J.I. Kim, C. Wang, S. Kuizon, J. Xu, D. Barengolts, P.C. Gray, R. Rubenstein, Simple and specific detection of abnormal prion protein by a magnetic bead-based immunoassay coupled with laser-induced fluorescence spectrofluorometry, J. Neuroimmunol. 158 (2005) 112–119. [7] I. Kuwabara, F.T. Liu, Galectin-3 promotes adhesion of human neutrophils to laminin, J. Immunol. 156 (1996) 3939–3944. [8] Y. Le, H. Yazawa, W. Gong, Z. Yu, V.J. Ferrans, P.M. Murphy, J.M. Wang, The neurotoxic prion peptide fragment PrP106-126 is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1, J. Immunol. 166 (2001) 1448–1451. [9] H.P. Lee, Y.C. Jun, J.K. Choi, J.I. Kim, R.I. Carp, Y.S. Kim, The expression of RANTES and chemokine receptors in the brains of scrapie-infected mice, J. Neuroimmunol. 158 (2005) 26–33. [10] F.T. Liu, R.J. Patterson, J.L. Wang, Intracellular functions of galectins, Biochim. Biophys. Acta 1572 (2002) 263–273. [11] S.W. Mok, K.M. Thelen, C. Riemer, T. Bamme, S. Gultner, D. Lutjohann, M. Baier, Simvastatin prolongs survival times in prion infections of the central nervous system, Biochem. Biophys. Res. Commun. 348 (2006) 697–702. [12] P. Nangia-Makker, Y. Honjo, R. Sarvis, S. Akahani, V. Hogan, K.J. Pienta, A. Raz, Galectin-3 induces endothelial cell morphogenesis and angiogenesis, Am. J. Pathol. 156 (2000) 899–909. [13] S.B. Prusiner, Molecular biology of prion diseases, Science 252 (1991) 1515–1522. [14] F. Reichert, S. Rotshenker, Galectin-3/MAC-2 in experimental allergic encephalomyelitis, Exp. Neurol. 160 (1999) 508–514. [15] C. Riemer, S. Neidhold, M. Burwinkel, A. Schwarz, J. Schultz, J. Kratzshmar, U. Monning, M. Baier, Gene expression profiling of scrapieinfected brain tissue, Biochem. Biophys. Res. Commun. 323 (2004) 556– 564. [16] N. Rubinstein, J.M. Ilarregui, M.A. Toscano, G.A. Rabinovich, The role of galectins in the initiation, amplification and resolution of the inflammatory response, Tissue Antigens 64 (2004) 1–12. [17] H. Sano, D.K. Hsu, J.R. Apgar, L. Yu, B.B. Sharma, I. Kuwabara, S. Izui, F.T. Liu, Critical role of galectin-3 in phagocytosis by macrophages, J. Clin. Invest. 112 (2003) 389–397. [18] H. Sano, D.K. Hsu, L. Yu, J.R. Apgar, I. Kuwabara, S. Izui, F.T. Liu, Human galectin-3 is a novel chemoattractant for monocytes and macrophages, J. Immunol. 165 (2003) 2156–2164. [19] V. Silei, C. Fabrizi, G. Venturini, M. Salmona, O. Bugiani, F. Tagliavini, G.M. Lauro, Activation of microglial cells by PrP and -amyloid fragments raises intracellular calcium through L-type voltage-sensitive calcium channels, Brain Res. 818 (1999) 168–170. [20] M. Walther, S. Kuklinski, P. Pesheva, O. Guntinas-Lichius, D.N. Angelov, W.F. Neiss, H.A. Asou, R. Probstmeier, Galectin-3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy, J. Neurosci. Res. 61 (2000) 430–435.
J.-K. Jin et al. / Neuroscience Letters 420 (2007) 138–143 [21] P.R. Warfield, P.N. Makker, A. Raz, J. Ochieng, Adhesion of human breast carcinoma to extracellular matrix proteins is modulated by galectin-3, Invasion Metastasis 17 (1997) 101–112. [22] A.E. Williams, A.M. Van Dam, W.K.H. Man-A-Hing, F. Berkenbosch, P. Eikelenboom, H. Fraser, Cytokines,prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice, Brain Res. 654 (1994) 200–206.
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[23] A.E. Williams, A.M. Van Dam, D. Ritchie, P. Eikelenboom, H. Fraser, Immunocytochemical appearance of cytokines, prostaglandin E2 and lipocortin-1 in the CNS during the incubation period of murine scrapie correlates with progressive PrP accumulations, Brain Res. 754 (1997) 171–180.
Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie Jae-Kwang Jin a , Yeo-Jung Na a , Joon-Ho Song b , Hong-Gu Joo c , Seungjoon Kim c , Jae-Il Kim d , Eun-Kyoung Choi a , Richard I. Carp d , Yong-Sun Kim a , Taekyun Shin c,∗ a
Ilsong Institute of Life Science, Hallym University Medical Center, Anyang, Kyonggi-do 431-060, South Korea Department of Neurosurgery, Hallym University Medical Center, Anyang, Kyonggi-do 431-060, South Korea c Department of Veterinary Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, South Korea d New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA
b
Received 20 February 2007; received in revised form 21 April 2007; accepted 23 April 2007
Abstract To investigate the involvement of galectin-3 in the process of neurodegeneration in prion diseases, the expression and cellular localization of galectin-3 in the brain were studied in scrapie, a mouse model of prion disease. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses showed that the expression of galectin-3 protein and mRNA was induced in scrapie-affected brains, particularly at the time when the abnormal prion protein PrPSc began to accumulate in the brains. Immunohistochemically, immunostaining for galectin-3 was found mainly in B4-isolectin-positive cells (presumably activated microglia/macrophages), but not in astrocytes. Galectin-3 immunoreactivity was localized mainly in areas of PrPSc accumulation and neuronal death in scrapie-infected brains. These findings suggest that the expression of galectin-3 by activated microglia/macrophages in prion disease correlates with abnormal prion protein accumulation. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Galectin-3; Macrophage; Microglia; Scrapie; Prion disease
Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cows are well studied prion diseases. The etiological agent of some prion diseases is PrPSc , which is the abnormal isoform of the normal cellular protein PrPC [13]. In scrapie, this abnormal prion protein may play a role in the pathogenesis of the glial cell reaction, and the activation of microglia has been proposed to play an important role in the initial neuropathological changes [2]. During the degenerative process in the brains of prion disease models, microglial cells increase expression in a variety of activation molecules, including cytokines [23]. Galectins, which belong to a growing family of galactoside-binding animal lectins, are composed of one or two carbohydrate-recognition domains (CRDs) of approximately 130 amino acids [10,16]. Of the 14 galectin members, the expres-
∗ Corresponding author at: Department of Veterinary Medicine, Cheju National University, Jeju 690-756, South Korea. Tel.: +82 64 754 3363; fax: +82 64 756 3354. E-mail address: [email protected] (T. Shin).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.04.069
sion of galectin-3 has been extensively documented in activated macrophages and in cells that are involved in central nervous system (CNS) inflammation [1,3]. Recently, galectin-3 has been shown to play a pivotal role not only in diverse physiological functions, such as cell growth, apoptosis, and mRNA splicing, but also in pathological processes as an inflammatory mediator [1]. In addition, galectin-3 plays a role in regulating metastasisrelated functions, which include angiogenesis [12], adhesion to the extracellular matrix [7], integrin expression [21], and extravasation [4]. Although galectin-3 expression under pathological conditions of the CNS remains poorly understood, it is strongly associated with the activation of microglia in mouse brains in a model of experimental autoimmune disease [14]. In this regard, the upregulation of galectin-3 in the brain may play a role in the pathology of neurodegenerative CNS disorders, including prion diseases. Studies have shown that galectin-3 mRNA levels are upregulated after prion infections [11,15], but it is not known how galectin-3 is involved in the neuropathology of prion diseases, specifically in scrapie. Therefore, we investigated whether any change occurs in the expression of galectin-3 during the developmental stage of
J.-K. Jin et al. / Neuroscience Letters 420 (2007) 138–143
scrapie and determined the possible association of galectin-3 and PrPSc deposition in diverse regions of the scrapie-infected brain. Eighty C57BL mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The ME7 scrapie strain was kindly provided by Alan Dickinson of the Neuropathogenesis Unit (Edinburgh, UK). Half of the animals (n = 40) were inoculated intracerebrally with 30 l of 1% (w/v) brain homogenate in 0.01 M phosphate-buffered saline (PBS) prepared from ME7infected C57BL mice. Control mice (n = 40) were injected with 30 l of 1% (w/v) homogenate of normal mouse brain and harvested at the same age as the scrapie-positive mice. The animals were euthanized with 16.5% urethane at 60–160 days postinfection (dpi) with the ME7 scrapie strain. The mice were perfused transcardially with PBS followed by 4% paraformaldehyde in PBS. The brains were removed immediately, post-fixed in the same fixative for 2 h at room temperature (RT), rinsed with PBS, dehydrated with ethanol, and embedded in paraffin. For the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, unfixed brains were removed immediately from anesthetized mice and stored at −70 ◦ C until analysis. Total RNA samples from whole mouse brains (sampled at 160 dpi) were extracted with TRIzol reagent (Gibco-BRL, Rockville, MD), according to the manufacturer’s instructions. RT-PCR was performed as follows. The cDNA was synthesized from 2 g of total RNA by reverse transcription using AMV reverse transcriptase (Promega, Madison, WI) and oligo (dT) primer. After incubation for 1 h at 42 ◦ C, the samples were heat-inactivated and kept at 4 ◦ C until used. A 5-l aliquot of the cDNA of each sample was used for PCR with galectin-3specific primers. The PCR conditions consisted of an initial denaturation step at 94 ◦ C for 2 min, then 30 cycles at 94 ◦ C for 1 min, 54 ◦ C for 1 min, and 72 ◦ C for 1.5 min, with a final 6-min extension at 72 ◦ C. The PCR primers used were 5 CAGACAGCTTTTCGCTTAAC-3 (galectin-3 sense) and 5 ACTGTCTTTCTTCCTTTCCC-3 (galectin-3 antisense), and 5 -TGGTATCGTGGAAGGACTCATGAC-3 (GAPDH sense) and 5 -ATGCCAGTGAGCTTCCCGTTCAGC-3 (GAPDH antisense). Total brain lysates from control and scrapie-infected mice (sampled at 60–160 dpi) were obtained by homogenization in lysis buffer [40 mM Tris (pH 8.0), 0.1% Nonidet P-40, 120 mM NaCl, 10 g/ml leupeptin] in the presence of protease inhibitors (10 g/ml leupeptin, 2 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Western blotting was performed as described previously [5]. Briefly, the membranes were incubated overnight at 4 ◦ C with rat monoclonal anti-galectin-3 (M3/38 hybridoma) or mouse monoclonal anti-PrP 10E4 (kindly provided by Dr. Richard Rubenstein, New York State Institute for Basic Research, Staten Island, NY) [6]. After washing three times in TBS buffer containing 0.05% Tween-20, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Pierce, Rockford, IL) or HRP-conjugated anti-rat IgG (Pierce). The protein bands were detected by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). To detect PrPSc , brain homogenates
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Fig. 1. RT-PCR and Western blot analyses of galectin-3 from the brains of control and scrapie-infected mice 160 days postinoculation with the normal inoculum or scrapie strain. Galectin-3 mRNA (A) and protein (B) were significantly upregulated in the brains of scrapie-infected mice. (**)p < 0.01 vs. control levels. Each experiment was repeated at least three times, and similar results were obtained in each experiment. GAPDH and anti--actin were used as controls. Melanoma (B16F10) cell lysate was used as a positive control for galectin-3.
Fig. 2. Western blot analysis of galectin-3 protein expression at various stages during the scrapie incubation period (sampled at 60–160 dpi). For the PrPSc analysis, the samples were treated without or with PK (100 g/ml) before SDSPAGE. Note that the expression of galectin-3 and PrPSc started to increase at 100 dpi. Each experiment was performed at least three times, with similar results; -actin was used as a control.
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(50 g protein) were treated with proteinase K (PK, 100 g/ml) and then incubated for 30 min at 37 ◦ C before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunohistochemical staining was performed as described previously [5]. Briefly, the 6-m-thick sections were exposed to normal donkey serum, and then incubated with anti-galectin-3 (1:5000) overnight at 4 ◦ C for PrPSc staining. Next, the sections were exposed to PK (10 g/ml) for 10 min at RT, and then incubated with anti-PrP 10E4 overnight at 4 ◦ C. Then, the sections were washed in PBS, and treated sequentially with biotinylated anti-rat and anti-mouse immunoglobulin, followed by peroxidase-labeled avidin, and developed with diaminoben-
zidine (Vector ABC Elite kit; Vector Laboratories, Burlingame, CA). For double staining, the sections were incubated with 10% normal donkey serum in PBS (pH 7.4) for 1 h, followed by anti-galectin-3 antibody overnight at 4 ◦ C and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG (1:100) for 1 h at RT. Then, the sections were washed, blocked with 10% normal goat serum in PBS for 1 h at RT, incubated with rabbit polyclonal anti-GFAP (Dako, Copenhagen, Denmark) antibody overnight at 4 ◦ C, and then washed. The final step involved incubation with Lissamine rhodamine (LRSC)-conjugated donkey anti-rabbit IgG (1:100). For microglia and macrophage staining, the lectin GSA was optimized by incubating the sections in
Fig. 3. Immunohistochemical localization of galectin-3 and PrPSc in control and scrapie-infected brains. Galectin-3 immunoreactivity was observed in the striatum (B), hippocampus (E), midbrain (H), and cerebellum (K) in the brains of the scrapie-infected mice, but not in the brains of controls (A, D, G, J). PrPSc deposits were also observed in these regions [striatum (C), hippocampus (F), midbrain (I), and cerebellum (L)]. Scale bar, 10 m.
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0.5 mg/ml trypsin in 0.05 M TBS containing 1 mM CaCl2 (pH 7.6) for 5 min at 37 ◦ C. Biotinylated lectin Griffonia simplicifolia (GSA B4-isolectin; Sigma, St. Louis, MO; 10 mg/ml TBS) was then added for 1 h at RT, followed by tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptavidin (Zymed, San Francisco, CA). The immunofluorescence-stained specimens were examined under a laser confocal microscope (LSM 510; Zeiss, Oberkochen, Germany). Quantitative results are expressed as the mean ± standard deviation (S.D.). One hundred and sixty days after the intracerebral injection of either ME7 or normal brain homogenate, we examined the galectin-3 mRNA levels in control and scrapie-infected mouse brains using RT–PCR. As shown in Fig. 1A, galectin3 mRNA was expressed strongly (over three folds) in ME7 scrapie-infected mice, but only weakly in control mice. Since galectin-3 gene expression was observed in the scrapie-infected brains at 160 dpi, the levels of galectin-3 protein expression were examined in total brain lysates from control and scrapieinfected brains using Western blot analysis. Galectin-3 protein was expressed in the scrapie-infected brains, but not in the control brains (Fig. 1B). The approximate molecular weight of galectin-3 was found to be 30 kDa. The change in galectin-3 protein expression was examined in total lysates of scrapie-infected brains using Western blot analysis during the various scrapie developmental stages (sampled at 60–160 dpi). As shown in Fig. 2, galectin-3 protein was
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weakly expressed in the scrapie-infected brain at 80 and 100 dpi and was upregulated gradually with progression of the disease. Similarly, PrPSc accumulation was low by 100 dpi, but significantly increased in the scrapie-infected brain from 120 dpi on Fig. 2. To examine the cellular distribution of galectin-3, sections from control and scrapie-infected mouse brains were analyzed immunohistochemically. Galectin-3 immunoreactivity was very slight in the control brains (Fig. 3A, D, G, I), but was strong in the hippocampal formation (Fig. 3E) and midbrain (Fig. 3H) in the brains of scrapie-infected mice. As shown in Fig. 3, galectin3 immunoreactivity was more intense in both the hippocampus and midbrain, as compared to other regions, such as the striatum (Fig. 3B) and cerebellum (Fig. 3K). PrPSc deposits were also observed in the regions showing galectin-3 immunoreactivity (Fig. 3C, F, I, L). To determine the cell phenotype of galectin-3 expressing cells in the scrapie-infected brain, we immunostained brain sections with either GSA B4-isolectin as a marker for microglia or antiGFAP as a marker for astrocytes. As shown in Fig. 4, galectin-3 immunoreactivity was present mainly in B4-isolectin-positive microglia, and not in GFAP-positive astrocytes in the midbrains of scrapie-infected mouse brains. The major findings of this report are that galectin-3 is expressed in activated microglia/macrophages during the neurodegenerative process of scrapie and that it is expressed
Fig. 4. Confocal micrographs of scrapie-infected brain sections were analyzed for galectin-3, GFAP, and GSA B4-isolectin immunoreactivity. Confocal image of double-immunostaining of galectin-3 and GSA B4-isolectin showed that B4-isolectin-positive cells (B; arrowheads) were positive for galectin-3 (A; arrows). The merged image is shown in (C). Galectin-3-positive cells (D; arrows) were not GFAP-positive (E; arrowheads). The merged image is shown in (F). Scale bar, 20 m.
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in diverse regions, including the hippocampus, where PrPSc is mainly found during prion infection. Recently, the functional roles of galectin-3 in the CNS have begun to emerge [14], and the mechanisms of galectin-3 induction in the brain have been investigated extensively [20]. Our results suggest that galectin-3 plays an important role in the pathogenic mechanisms in the neurodegenerative process of scrapie. Little is known about the functions of activated microglia in scrapie, although one of the important features of scrapie pathology is that glial cells are activated in close proximity to the pathological lesions [22,23]. In contrast, several studies have focused on the activation of astrocytes. Based on our current study, we postulate that the galectin-3 expression seen in the activated microglia/macrophages of the scrapie-affected region exhibiting intense extracellular PrPSc accumulation is associated with the neurodegeneration observed in scrapie. Since galectin-3 upregulation may play an important role in the phagocytosis of microorganisms and apoptotic cells [17], increased expression of galectin-3 in TSE-affected brains may also be involved in the phagocytosis of damaged cells resulting from PrPSc neurotoxicity. This concept is supported by the observation that prion peptide (106–126) enhances the phagocytic function of microglia [19]. In addition, Le et al. [8] reported that the prion peptide enhances monocyte chemotaxis. Although the exact mechanisms underlying phagocytosis and chemotaxis by microglia/macrophages in prion diseases remain unresolved, it has been postulated that galectin-3 expression in these cells is involved in the mediation of phagocytosis and chemotaxis in regions of injury. Furthermore, galectin3 is a chemoattractant for monocytes and macrophages, and it is more potent than the chemokine monocyte chemoattractant protein-1 [17]. As reported previously [18], galectin-3 has a chemotactic effect that in turn induces the activation of chemokine receptors. In this regard, previous reports from our laboratory have provided evidence that chemokine receptors, including CCR5, are activated in mice that are infected with scrapie [9]. From these observations, we propose that galectin-3-positive cells in the damaged region enhance chemotactic function in the prion animal model. In this model, PrPSc formation started at 100 dpi, when microglial activation occurred [2]. Furthermore, the galectin-3 expression was correlated with PrPSc deposition at the same stage of disease pathology. In conclusion, we postulate that galectin-3 expression by activated microglia/macrophages after PrPSc accumulation plays a role in the subsequent neurodegeneration in murine scrapie. Further experiments using a transgenic animal model, such as galectin-3 knockout mice, are required to fully understand the specific role of galectin-3 in the pathogenesis of scrapie. Acknowledgments This work was supported by a program of the Basic Atomic Energy Research Institute (BAERI), which is a part of the
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