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Up-regulation of cystatin C expression in the murine hippocampus following perforant path transections
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Neuroscience Vol. 112, No. 2, pp. 289^298, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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UP-REGULATION OF CYSTATIN C EXPRESSION IN THE MURINE HIPPOCAMPUS FOLLOWING PERFORANT PATH TRANSECTIONS G.-X. YING,a C. HUANG,a Z.-H. JIANG,a X. LIU,a N.-H. JINGb and C.-F. ZHOUa a
b
Key Laboratory of Neurobiology, Shanghai Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, PR China
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China
Abstract1Cystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extracellular proteins. These inhibitors are strongly implicated in a variety of pathological processes such as tumor metastasis and many degenerating CNS disorders. Here we report the expression of cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner with a signi¢cant increase at 7 and 15 days post-lesion (219% and 185% of control, respectively) in the rat hippocampus after entorhinal dea¡erentation. In situ hybridization and immunohistochemistry con¢rmed the time-dependent up-regulation of both cystatin C mRNA and protein expressions in a mouse model which initiated at 3 days post-lesion, reached maximal levels 7^15 days post-lesion, and remained slightly elevated by day 30 post-lesion. The modulation of cystatin C expression was observed to occur speci¢cally in the entorhinally denervated zones: the stratum lacunosum-moleculare of the hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either a combination of in situ hybridization for cystatin C with immunohistochemistry for glial ¢brillary acidic protein or double immuno£uorescence staining for both proteins in mouse hippocampus at 7 and 15 days post-lesion revealed that most cystatin C-expressing cells are astrocytes. From these results we suggest that the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinal dea¡erentation and that cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dea¡erentation, protease inhibitor, astrocyte, in situ hybridization, immunohistochemistry.
pus (for review, see Deller and Frotscher, 1997). Although a number of molecules have been shown to be involved in the process, the mechanisms underlying it are incompletely understood (for review, see Deller and Frotscher, 1997; Turner et al., 1998). In a di¡erential screening using cDNA array technology aimed to identify the genes that may contribute to the plasticity, we separated a set of genes whose expression levels are altered in the rat hippocampus following PP transections, one of which is cystatin C (Ying et al., 2001). Cystatin C is a member of type II cysteine protease inhibitors of the cystatin superfamily with high a⁄nity for papain, dipeptidyl peptidase and cathepsins B, H, L and S (Barrett et al., 1984, 1986; Abrahamson et al., 1986; Hiltke et al., 1999). It is initially synthesized as a pre-protein with a secretory signal sequence of 26 amino acids, and the mature active form contains 120 amino acids in a single polypeptide chain with two intramolecular disul¢de bridges (Abrahamson et al., 1987). Cystatin C is widely distributed in almost all tissues of vertebrates and is secreted by many kinds of cell types into a number of body £uids (Abrahamson et al., 1986, 1990). Serving to regulate the endogenous cysteine protease activity, cystatin C has been reported to be involved in a broad spectrum of biological events associated with tissue remodeling, including tumor metastasis and invasion (Coulibaly et al., 1999; Cox et al., 1999),
An important model system for studying the neural plasticity after injury in the brain of adult animals has been the hippocampus following destruction of its principal extrinsic input from the entorhinal cortex. The projections from the stellate and pyramidal neurons of layer II and III of the entorhinal cortex mainly project to the outer molecular layers (OML) of the dentate gyrus (DG) and the stratum lacunosum-moleculare (SLM) of the hippocampus via the perforant path (PP) (Steward, 1976; Amaral and Witter, 1989). Following entorhinal cortex lesion, the neural plasticity events, including anterograde terminal degeneration (Matthews et al., 1976a; Jensen et al., 1994), axonal sprouting (Lynch et al., 1972), terminal proliferation and synaptogenesis (Matthews et al., 1976b; Lee et al., 1977; Steward and Vinsant, 1983; Steward, 1992), occur leading to the structural reorganization in the dea¡erented hippocam-
*Corresponding author. Tel.: +86-21-64370080; fax: +86-2164332445. E-mail address: [email protected] (C.-F. Zhou). Abbreviations : DG, dentate gyrus; EDTA, ethylenediaminetetraacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase ; GFAP, glial ¢brillary acidic protein; OML, outer molecular layer; PBS, phosphate-bu¡ered saline; PP, perforant path; SDS, sodium dodecylsulfate; SLM, stratum lacunosum-moleculare. 289
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embryo implantation (Afonso et al., 1997), bone matrix remodeling (Lerner and Grubb, 1992) and the development of many degenerating disorders of the CNS such as Alzheimer’s disease (Uchida et al., 1997; Levy et al., 2001). It has also been reported that cystatin C promotes cell proliferation in vitro and in vivo (Tavera et al., 1992; Taupin et al., 2000). Moreover, many protease inhibitors together with their target enzymes have been shown to regulate neuronal di¡erentiation and migration as well as neurite growth of several neural cell types (Gibson et al., 1984; Hawkins and Seeds, 1989; Friedman and Seeds, 1995). Therefore, the present knowledge of cystatin C likely supports the suggestion that this molecule may play an important role in neural plasticity in the hippocampus after entorhinal cortex lesion. In the present study we analyzed the expression of cystatin C mRNA and protein in the murine hippocampus after PP transections, and possible roles of cystatin C on neural plasticity in the dea¡erented hippocampus are subsequently discussed.
EXPERIMENTAL PROCEDURES
Animals and surgery Adult female Sprague^Dawley rats (body weight 220^250 g) and ICR mice (20^25 g), supplied by the Shanghai Center for Laboratory Animals (Shanghai, China) and housed under standard laboratory conditions, were used in the present study. All procedures were performed in agreement with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Shanghai Institutes for Biological Sciences, Shanghai, Institutional Animal Care and Use Committee. All e¡orts were made to minimize animal su¡ering and the number of animals used. Rats were used for northern blot analysis and mice for in situ hybridization and immunohistochemistry. The procedure for performing rat bilateral (n = 15) and mouse unilateral (n = 20) PP transections has been previously described elsewhere (Zhou et al., 1998). In brief, under deep anesthesia by sodium pentobarbital (40 mg/kg body weight), the animal was positioned in a stereotaxic apparatus, two trenches were made by drilling the occipital skull and the perforant paths were manually transected completely with a curved silver microknife. At 3, 7, 15 or 30 days post-lesion, the rats (n = 3 for each group) with bilateral PP lesions as well as normal controls (n = 3) were decapitated under deep anesthesia and the brains were quickly removed. The hippocampus was carefully dissected out, snap frozen in liquid nitrogen and stored at 380‡C until used for northern blot. For in situ hybridization and immunohistochemistry, normal mice were used as controls (n = 3) and the mice with unilateral PP lesions were allowed to survive for 3, 7, 15 or 30 days following PP transections (n = 5 for each group). The animal was deeply anesthetized with an overdose of sodium pentobarbital and transcardially perfused with ice-cold ¢xative of 4% paraformaldehyde in 0.1 M phosphate-bu¡ered saline (PBS), pH 7.4. The brain was dissected out, post-¢xed for 4 h at 4 ‡C, incubated in 30% sucrose to sink, and cut coronally at 30 Wm on a cryostat. Northern blot analysis Total RNA was extracted from the frozen rat hippocampi using the acid guanidinium isothiocyanate^phenol method (Chomczynski and Sacchi, 1987), electrophoresed (20 Wg per lane) on a 1% formaldehyde agarose gel, blotted onto nylon membrane, and cross-linked by ultraviolet ¢xation. DNA
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probes for northern hybridization were synthesized from fulllength mouse cystatin C cDNA (GenBank accession number M59470) by using the random-primed DNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). After pre-hybridization in hybridization bu¡er (15% formamide, 40 mM NaPO4 pH 7.2, 10 mM EDTA, 7% sodium dodecylsulfate (SDS), 1UDenhardt’s, 100 Wg/ml salmon sperm DNA), the ¢lters were hybridized with 32 P-labeled cDNA probes (0.5^2U107 c.p.m./ml) in the same bu¡er at 65‡C overnight. Hybridized membranes were sequentially washed to high stringency (40 mM NaPO4 , 1 mM EDTA, 1% SDS, 65‡C) and exposed to ¢lm (Kodak). To control the total RNA amount loaded in each lane, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal standard in each hybridization. The hybridization signals were quanti¢ed over the optical densities using the GDS8000 system with software Gel work 3.01 (UVP), and normalized by GAPDH signal. Results are expressed as mean T S.E.M. from three animals and statistical di¡erences were assessed using a two-tailed Student’s t-test with signi¢cance set at P 6 0.05. In situ hybridization histochemistry The mouse cystatin C cDNA used in the present study corresponded to the published sequences (GenBank accession number M59470). The cystatin C cDNA cloned in vector pBluescript II SK3 was linearized, and digoxigenin-labeled antisense and sense riboprobes were synthesized using an in vitro transcription kit (Roche). The 670-bp-long cRNA probes were further subjected to alkaline hydrolysis for 20 min at 65‡C in hydrolysis bu¡er (80 mM NaHCO3 , 120 mM Na2 CO3 ), and ¢nally dissolved in distilled water at 100 ng/Wl. Coronal sections from di¡erent groups of mice were sampled and processed simultaneously to ensure the same conditions for all hybridizations. Sections were washed twice in 2USSC (20USSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0), treated with 0.1 N HCl for 10 min, washed once in deionized water, then incubated in 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) for 10 min. After a brief wash with 2USSC, the sections were pre-hybridized for 3 h at 50‡C in the hybridization bu¡er containing 50% deionized formamide, 10% dextran sulfate, 10 mM Tris^HCl pH 7.5, 600 mM NaCl, 0.25% SDS, 1UDenhardt’s and 400 Wg/ml salmon sperm DNA, and hybridized in the same bu¡er containing 0.2 Wg/ml of either antisense or sense cystatin C probe at 50‡C overnight. Following hybridization, sections were rinsed brie£y in 4USSC at room temperature, washed in 50% deionized formamide/2USSC for 30 min at 55‡C. After a 10-min wash with RNase bu¡er containing 10 mM Tris^HCl, 0.5 M NaCl, the unbound RNA probes were digested with 20 Wg/ml RNase A in the same bu¡er for 30 min at 37‡C. The sections were then washed in RNase bu¡er for 10 min, followed by two washes in 2USSC for 15 min at 50‡C, 1USSC for 2U15 min at 50‡C, then 0.1USSC for 4U15 min at 37‡C. After several rinses in Tris-bu¡ered saline (0.15 M NaCl, 0.1 M Tris, pH 7.5), the sections were incubated in 1% blocking solution (Roche) for 30 min, followed by incubation for 2 h with alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (1:1000, Roche) at room temperature. After several washes with 50 mM MgCl2 in Tris-bu¡ered saline pH 9.5, the phosphatase reaction was performed using 4-nitroblue tetrazolium (0.35 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.18 mg/ml) as substrates. The color reaction was developed in the dark until satisfying results were obtained. Some of the developed sections were subsequently processed for glial ¢brillary acidic protein (GFAP) immunohistochemistry (see below) to determine whether the cystatin C gene-expressing cells are mainly astrocytes that have proliferated and hypertrophied in the hippocampus in response to entorhinal dea¡erentation (Rose et al., 1976; Jensen et al., 1994; Hailer et al., 1999). Immunohistochemistry The mouse sections were washed three times in 0.1 M PBS, incubated for 1 h with 0.3% Triton X-100 and 10% normal goat
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serum, then incubated at 4‡C overnight either with a mixture of primary mouse antibody to GFAP (monoclonal, 1:200, Sigma, St. Louis, MO, USA) and rabbit anti-human cystatin C antibody (polyclonal, 1:2000, Dakopatts, Glostrup, Denmark), or with one of the two antibodies alone diluted in PBS containing 1% goat serum. For the double labeling of cystatin C and GFAP proteins, the primary antibody bindings were directly visualized with a mixture of £uorescein-conjugated goat antirabbit and Texas Red-conjugated goat anti-mouse antibodies (1:200, Dako) after several washes in PBS. For separate GFAP or cystatin C immunoreaction, the sections were rinsed in PBS several times, incubated with biotinylated goat antimouse (1:200, Dako) antibody or biotinylated goat anti-rabbit antibody (1:200, Vector Laboratories, Burlingame, CA, USA), respectively, washed with PBS, reacted with avidin^biotin horseradish peroxidase solution (standard ABC Elite kit, Vector), and visualized with 0.003% H2 O2 and 0.03% 3,3-diaminobenzidine tetrahydrochloride in 0.05 M Tris^HCl pH 7.6. Confocal microscopy (Leica TCS-NT) was used for the analysis of the double immuno£uorescence staining.
RESULTS
The timing of cystatin C mRNA expression in rat hippocampus after perforant path transections Northern blot hybridization was employed to analyze the expression levels of cystatin C mRNA in the rat hippocampus at 3, 7, 15 and 30 days following PP transections. It was observed that the rat cystatin C gene was expressed as a single transcript of about 0.7^0.8 kb as shown by previous studies (Solem et al., 1990) and the expression was regulated in a transient manner (Fig. 1A, B). There was only a small alteration of hybridization signal of cystatin C mRNA in the dea¡erented hippocampus at 3 days post-lesion compared with that of normal control (120% of control, no statistical di¡erence). However, by 7 days post-lesion, the hybridization signal showed a massive increase with a value of 219% of control, reaching the maximal level among all time points examined. By 15 days post-lesion, the elevation of cystatin C mRNA expression was still prominent and comparable to that at 7 days post-lesion with a value of 185% of control. Quantitative analysis (Fig. 1B) revealed that the e¡ect of PP transections on hippocampal cystatin C mRNA expression at 7 and 15 days post-lesion was statistically signi¢cant (P 6 0.05). By 30 days post-lesion, the last time point examined, the cystatin C mRNA signal in the dea¡erented hippocampus declined to nearly normal level with a small elevation (113% of control, no statistical di¡erence). It is of interest that the regulation of cystatin C mRNA expression in the hippocampus following PP transection occurs exactly at the time of the structural reorganization in the tissue subjected to the same lesions (for review, see Deller and Frotscher, 1997). Layer-speci¢c up-regulation of cystatin C mRNA and protein in mouse hippocampus after perforant path transections To examine the regional distribution of cystatin C transcripts in the dea¡erented hippocampus, we performed an in situ hybridization analysis with mouse
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Fig. 1. Northern blot analysis of cystatin C mRNA expression in the hippocampus of control (c in A and B) and lesioned rats at 3, 7, 15 and 30 days following PP transections. (A) Photograph shows a transient increase of cystatin C mRNA expression (upper panel) in the hippocampus after lesions compared to control (c). GAPDH (lower panel) is used as an internal standard to control the amount of total RNA loaded in each lane. (B) Northern signals in A are quanti¢ed by densitometry. The values are expressed as control percentage and represent a ratio of cystatin C signal density at various times post-lesion divided by that measured on control after normalization with the GAPDH signal. Values given are mean T S.E.M. for three animals at each post-lesion time point. Note the signi¢cant increase of cystatin C mRNA expression in the hippocampus at 7 and 15 days after PP transections with values of 219% and 187% of control, respectively. *P 6 0.05, two-tailed Student’s t-test.
material to localize the cystatin C mRNA expression. In agreement with previous studies (Cole et al., 1989; Olsson et al., 2000), our in situ hybridization in normal control animals revealed that cystatin C mRNA was present in the choroid plexus and many regions of the mouse brain, including the cerebral cortex, hippocampus, hypothalamus and cerebellum (data not shown). In the hippocampus, the signal for cystatin C mRNA labeling was observed sparsely throughout all sub¢elds except for the SLM and sub-granular zone where more labeled cells were identi¢ed (data not shown). In the mouse hippocampus ipsilateral to PP transections, cystatin C mRNA expression in the hippocampal SLM and the dentate OML, the PP projection zones, appeared to increase at 3 days post-lesion and abundantly increased at 7 and 15 days post-lesion, but recovered at 30 days post-lesion to the level similar to that at 3 days post-lesion (Fig. 2B, D, F, H). The overall increase of the labeling in the denervated areas can be ascribed to the elevated cystatin C mRNA labeling intensity within individual cells and the increased number of cystatin C mRNA-expressing cells. In areas other than the PP terminal zones, i.e. the stratum radiatum and
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Fig. 2.
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pyramidal cell layer of the hippocampus, and the inner molecular layer and granular cell layer of the DG, there were no detectable alterations of cystatin C mRNA expression induced by PP transections (Fig. 2B, D, F, H). In the mouse hippocampus contralateral to PP lesions, more cystatin C mRNA-expressing cells were visualized in the SLM and OML only at 7 and 15 days post-lesion (Fig. 2C, E) but not other time points examined (Fig. 2A, G) compared with cystatin C mRNA expression in the same areas of the normal control, even if this elevation was much less than the PP cut side (Fig. 2D, F). In the parallel control experiments, sections hybridized with the sense probe were completely devoid of signals at all samples (data not show). The in situ hybridization study of the cystatin C mRNA expression in the hippocampus following PP transections revealed that the regulation of cystatin C transcripts had a temporal pattern very similar to that observed by northern analysis with maximum expression between 7 and 15 days post-lesion. To further analyze whether cystatin C protein expression in the dea¡erented hippocampus also undergoes regulation after PP transections, immunohistochemistry for mouse cystatin C protein was performed using a polyclonal rabbit anti-human cystatin C antibody that had been successfully used to react with the murine antigen in previous studies (Palm et al., 1995; Miyake et al., 1996). The immunostaining patterns in the dea¡erented hippocampus were spatially coincident with in situ hybridization, i.e. the up-regulation of cystatin C immunostaining occurred only in the hippocampal SLM and the dentate OML, but not in the other layers such as stratum radiatum of the hippocampus and the inner molecular layer of the DG (Fig. 3). The post-lesion time-dependent alteration of cystatin C immunostaining was also identical to that of in situ hybridization with the strongest immunostaining at 7 and 15 days post-lesion (Fig. 3D, F). This result suggests that entorhinal dea¡erentation regulates not only the transcription of the cystatin C gene but also the translation of cystatin C transcripts. Compared with in situ hybridization, cystatin C immumostaining revealed more clearly the cytological morphology of the positively stained cells in the denervated zones, which likely showed the morphological characteristics of astrocytes (Fig. 3). Cystatin C-expressing cells are mainly astrocytes To further con¢rm that the cystatin C mRNA- and protein-expressing cells in the hippocampus at 7 and 15
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days following PP lesions are reactive astrocytes, we used double labeling of either a combination of in situ hybridization for cystatin C mRNA with immunohistochemistry for GFAP protein or double immuno£uorescence staining for both proteins on the mouse model. In the double labeling of cystatin C mRNA and GFAP protein, the purple-blue cystatin C mRNA signals were primarily con¢ned to the cytoplasm of astrocytes, while the brown GFAP immunostaining located mainly in the cell processes (Fig. 4A, B). It was observed that the great majority of cystatin C mRNA-positive cells in the SLM and OML were GFAP-positive and all GFAP-immunoreactive cells were cystatin C-positive (Fig. 4A). Consistently, confocal microscopy analysis of double immuno£uorescence staining clearly veri¢ed that most cystatin C-positive cells in the denervated hippocampal SLM and the dentate OML were GFAP-positive cells, and a few of them showed GFAP-negative which would be microglia (Fig. 4C^E). It was noticed that at high magni¢cation the cystatin C signals were unevenly localized in a dot/lump-like shape in the somata and processes of GFAP-positive cells (Fig. 4E), indicating that cystatin C protein probably binds to/exists in certain intracellular organelles in situ.
DISCUSSION
In this study, we found the spatiotemporally restricted up-regulation of both cystatin C mRNA and protein in the hippocampal SLM and the dentate OML after PP transections, which strongly suggests that the expression of cystatin C is induced by entorhinal dea¡erentation. It is well known that there are about 5% of entorhinal projections to the hippocampus originating from the contrary side (Steward et al., 1976). Correspondingly, a small, but evident elevation of cystatin C mRNA expression was demonstrated in the hippocampus contralateral to PP transections at 7 and 15 days post-lesion, which would be the result of the loss of the crossed entorhinal projections. As one of the most abundant endogenous cysteine protease inhibitors in mammalian tissues, cystatin C has been shown to be involved in many CNS diseases or injuries. For instance, following transient forebrain ischemia cystatin C immunoreaction is transiently up-regulated in the rat degenerating hippocampal CA1 neurons but not in viable neurons (Palm et al., 1995). Elevation of cystatin C expression in the facial nucleus following facial nerve axotomy has also been observed (Miyake et al., 1996). Immunochemical analysis of brains of patients with Alzheimer’s disease revealed
Fig. 2. In situ hybridization of cystatin C mRNA in the mouse hippocampus showing more intense dot-like hybridization products in the OML and SLM in the hippocampus ipsilateral (right side) than contralateral (left side) to PP transections with the di¡erence in the expression between two sides being less, but notable at 3 (A, B) and 30 (G, H) days, and most prominent at 7 (C, D) and 15 days (E, F) after lesions. Note that the cystatin C mRNA expression in the SLM and OML in the lesion-contralateral hippocampus at 7 (C) and 15 (E) days post-lesion appears to be up-regulated in comparison with the same zones of the normal control (data not shown) and the lesion-contralateral hippocampus at 3 (A) and 30 (G) days postlesion, and that there is no di¡erence in cystatin C mRNA expression between the corresponding non-entorhinal projection zones, i.e. stratum pyramidale (SP), stratum radiatum (SR), inner molecular layer (IML) and stratum granulare (SG), of left and right hippocampus of the same lesioned animals. Scale bar = 200 Wm.
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Fig. 3. Immunostaining of cystatin C in the mouse hippocampus after unilateral PP transections. At 3 days post-lesion (A, B), there is a slight increase of cystatin C staining in the SLM and OML of the hippocampus ipsilateral to perforant path transections (B) compared with that of the corresponding areas in the contralateral side (A). The elevation of cystatin C immunostaining in the ipsilateral SLM and OML is abundant at 7 days post-lesion (D) and this up-regulation remains prominent at 15 days post-lesion (F) compared with the staining in the contralateral hippocampus (C, E). Note that the cystatin C-positive cells appear to have the morphological characteristics of astrocytes and that there is no di¡erence in staining between the corresponding non-entorhinal projection zones of the ipsilateral and contralateral sides. Abbreviations as in Fig. 2. Scale bar = 150 Wm.
that cystatin C is accumulated in the senile plaque amyloid deposits in the hippocampus (Uchida et al., 1997) and colocalized with amyloid L protein in the vascular amyloid deposits of the brain (Levy et al., 2001). Our observation that entorhinal dea¡erentation-induced upregulation of cystatin C in the hippocampus again sug-
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gests that cystatin C may exert important roles in various neuropathologies (Palm et al., 1995; Levy et al., 2001). The activation of astrocytes in the hippocampus following entorhinal cortex lesion is characterized by their proliferation, hypertrophy, and expression increase for GFAP and vimentin in PP projection zones, which starts
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Fig. 4. Double labeling of cystatin C and GFAP in the mouse hippocampus following PP transections. (A, B) Double labeling of cystatin C by in situ hybridization and GFAP by immunohistochemistry in the hippocampus at 7 days post-lesion. Almost all cystatin C-positive cells are GFAP-immunoreactive, while all GFAP-labeled cells are cystatin C-positive in the SLM and OML (A). The framed area in A is shown in B at higher magni¢cation, showing several double-labeled astrocytes with purple-blue cystatin C mRNA signal in the somata and brown GFAP protein staining in the processes. (C^E) Confocal images of double immuno£uorescence labeling of cystatin C (C, green) and GFAP (D, red) in the hippocampus at 15 days post-lesion, showing that most cystatin C-labeled cells are GFAP-positive and all GFAP-positive cells are cystatin C-immunopositive in the SLM and OML with several representative double-labeled cells indicated by arrows (E). Few cells positive for cystatin C but negative for GFAP in these areas are also found. Note the uneven dot-shaped immunostaining of cystatin C in the somata and processes of astrocytes (C, E). Abbreviations as in Fig. 2. Scale bar = 50 Wm (A), 10 Wm (B), 40 Wm (C, D), 20 Wm (E).
at 2 days post-lesion, reaches the maximum around 6^7 days post-lesion, and subsides after about 21 days postlesion (Rose et al., 1976; Gall et al., 1979; Steward et al., 1993; Jensen et al., 1994; Krohn et al., 1995; Baldwin
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and Sche¡, 1997; Hailer et al., 1999). Cystatin C upregulation by astrocytes demonstrated in this study is almost simultaneous with astrocyte activation in the dea¡erented PP terminal zones. Therefore, cystatin C
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expression represents an additional feature of the activated astrocytes in the hippocampus in response to deafferentation. The signal that initiates the target area-speci¢c elevation of cystatin C expression by reactivated astrocytes is unclear at present. However, it has been reported that transforming growth factor L induces cystatin C mRNA expression in the astrocyte precursor cells in vitro (Solem et al., 1990), and its transcripts in the dea¡erented hippocampus are up-regulated in the activated microglia between 4 and 10 days after entorhinal cortex lesion (Morgan et al., 1993), a period of time when cystatin C expression is enhanced. Therefore, the microglia transforming growth factor L might regulate cystatin C expression in astrocytes in situ. Astrocytes activated by dea¡erentation have been reported to produce a wide variety of extracellular matrix molecules such as laminin (Tian et al., 1997), tenascin (Deller et al., 1997), chondroitin sulfate proteoglycan (Haas et al., 1999; Thon et al., 2000) and heparin a⁄n regulatory peptide (Poulsen et al., 2000), all of which e¡ect the axonal regeneration and growth in the CNS correlating with the lesion-induced neural plasticity in the dea¡erented hippocampus. Recent studies have revealed that cathepsins are e¡ective extracellular matrix protein-degrading proteases (Mason et al., 1986; Xin et al., 1992; Bromme et al., 1996; Petanceska et al., 1996) and that many of them are up-regulated in the hippocampus following entorhinal cortex lesion (Petanceska et al., 1996). It is plausible, therefore, that in the dea¡erented hippocampus the up-regulated cystatin C modulates the activities of cathepsins that are concomitantly elevated such that the extracellular matrix molecules produced by the activated astrocytes would not be degraded, a postulation supported by an in vitro study which showed that cysteine protease inhibitors decrease the matrix degradation induced by cathepsins (Corticchiato et al., 1992). The extracellular matrix remodeling function of cystatin C has been widely demonstrated in kinds of in vivo biological processes such as tumor metastasis (Coulibaly et al., 1999; Cox et al., 1999) and embryo placentation (Afonso et al., 1997). In addition to protecting the extracellular matrix molecules from proteolysis, cystatin C may also contribute to the turnover of various cytokines and neurotrophic factors including interleukin, ¢broblast growth factor, brain-derived neurotrophic factor and nerve growth factor released by activated glial cells in the hippocampus after entorhinal cortex lesion (for review, see Deller and Frotscher, 1997; Turner et al., 1998). A case in point for the defense e¡ect of cystatin C on cytokines is that cathepsin inhibitors increase the residence time of interleukin-6 in HepG2 cells by
interfering with the lysosomal processing of this molecule (Peppard and Knap, 1999). Moreover, cystatin C has been reported to inhibit the proteolysis of neuropeptide substance P that improves neuronal survival and neurite growth in vitro (Aghajanyan et al., 1988), implying that cystatin C protects neurotransmitters favorable for neural growth. Taken together, it is probable that the upregulated cystatin C may facilitate the establishment of an axonal growth-promoting environment by protecting neurite growth-associated molecules from degradation by cysteine proteases in the dea¡erented hippocampus. Very interestingly, cystatin C has been reported to play an important role in cell proliferation and growth. In vitro experiments showed that chicken egg cystatin prompts the proliferation of mouse 3T3 ¢broblasts (Sun, 1989) and that native rat cystatin C evokes the proliferation of glomerular mesangial cells in an autocrine manner (Tavera et al., 1992). The tumor metastasis decrease in cystatin C-de¢cient mice was also reported to be associated with reduced tumor cell growth (Huh et al., 1999). Taupin et al. (2000) have recently reported that the N-glycosylated cystatin C is required for ¢broblast growth factor 2 to induce the proliferation of neural stem cells in vivo or in vitro. Thus the spatiotemporal overlapping of cystatin C up-regulation and astrocyte proliferation in the hippocampus following PP transections raises the possibility that increased cystatin C promotes the proliferation of astrocytes in an autocrine/paracrine manner.
CONCLUSIONS
In the present study, we have demonstrated alterations of the expression of cystatin C mRNA and protein in the terminal zones of entorhino-hippocampal projections after their lesions by transection. A dramatic transient up-regulation of both cystatin C mRNA and protein was observed in the reactive astrocytes of the deafferented hippocampal SLM and the dentate OML. Concomitant with the activation of astrocytes, the regulation of cystatin C expression is proposed to be involved in various astrocytic responses to PP transections that are associated with the lesion-induced neural plasticity in the hippocampus.
Acknowledgements&This work was supported by the National Basic Research Program (G 1999054005), National Natural Science Foundation (39930090, 39870248) of China, and Life Science Special Fund of the CAS supported by the Ministry of Finance.
REFERENCES
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Expression of vimentin increases in the hippocampus and cerebral cortex after entorhinal cortex lesioning and in response to transforming growth factor beta 1. J. Neuroimmunol. 56, 53^63. Lee, K.S., Stanford, E.J., Cotman, C.W., Lynch, G., 1977. Ultrastructural evidence for bouton proliferation in the partially dea¡erented dentate gyrus of the adult rat. Exp. Brain Res. 29, 475^485. Lerner, U.H., Grubb, A., 1992. Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy. J. Bone Miner. Res. 7, 433^440. Levy, E., Sastre, M., Kumar, A., Gallo, G., Piccardo, P., Ghetti, B., Tagliavini, F., 2001. Codeposition of cystatin C with amyloid-beta protein in the brain of Alzheimer disease patients. J. Neuropathol. Exp. Neurol. 60, 94^104. Lynch, G., Matthews, D.A., Mosko, S., Parks, T., Cotman, C.W., 1972. 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Neuroscience Vol. 112, No. 2, pp. 289^298, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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UP-REGULATION OF CYSTATIN C EXPRESSION IN THE MURINE HIPPOCAMPUS FOLLOWING PERFORANT PATH TRANSECTIONS G.-X. YING,a C. HUANG,a Z.-H. JIANG,a X. LIU,a N.-H. JINGb and C.-F. ZHOUa a
b
Key Laboratory of Neurobiology, Shanghai Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, PR China
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China
Abstract1Cystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extracellular proteins. These inhibitors are strongly implicated in a variety of pathological processes such as tumor metastasis and many degenerating CNS disorders. Here we report the expression of cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner with a signi¢cant increase at 7 and 15 days post-lesion (219% and 185% of control, respectively) in the rat hippocampus after entorhinal dea¡erentation. In situ hybridization and immunohistochemistry con¢rmed the time-dependent up-regulation of both cystatin C mRNA and protein expressions in a mouse model which initiated at 3 days post-lesion, reached maximal levels 7^15 days post-lesion, and remained slightly elevated by day 30 post-lesion. The modulation of cystatin C expression was observed to occur speci¢cally in the entorhinally denervated zones: the stratum lacunosum-moleculare of the hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either a combination of in situ hybridization for cystatin C with immunohistochemistry for glial ¢brillary acidic protein or double immuno£uorescence staining for both proteins in mouse hippocampus at 7 and 15 days post-lesion revealed that most cystatin C-expressing cells are astrocytes. From these results we suggest that the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinal dea¡erentation and that cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dea¡erentation, protease inhibitor, astrocyte, in situ hybridization, immunohistochemistry.
pus (for review, see Deller and Frotscher, 1997). Although a number of molecules have been shown to be involved in the process, the mechanisms underlying it are incompletely understood (for review, see Deller and Frotscher, 1997; Turner et al., 1998). In a di¡erential screening using cDNA array technology aimed to identify the genes that may contribute to the plasticity, we separated a set of genes whose expression levels are altered in the rat hippocampus following PP transections, one of which is cystatin C (Ying et al., 2001). Cystatin C is a member of type II cysteine protease inhibitors of the cystatin superfamily with high a⁄nity for papain, dipeptidyl peptidase and cathepsins B, H, L and S (Barrett et al., 1984, 1986; Abrahamson et al., 1986; Hiltke et al., 1999). It is initially synthesized as a pre-protein with a secretory signal sequence of 26 amino acids, and the mature active form contains 120 amino acids in a single polypeptide chain with two intramolecular disul¢de bridges (Abrahamson et al., 1987). Cystatin C is widely distributed in almost all tissues of vertebrates and is secreted by many kinds of cell types into a number of body £uids (Abrahamson et al., 1986, 1990). Serving to regulate the endogenous cysteine protease activity, cystatin C has been reported to be involved in a broad spectrum of biological events associated with tissue remodeling, including tumor metastasis and invasion (Coulibaly et al., 1999; Cox et al., 1999),
An important model system for studying the neural plasticity after injury in the brain of adult animals has been the hippocampus following destruction of its principal extrinsic input from the entorhinal cortex. The projections from the stellate and pyramidal neurons of layer II and III of the entorhinal cortex mainly project to the outer molecular layers (OML) of the dentate gyrus (DG) and the stratum lacunosum-moleculare (SLM) of the hippocampus via the perforant path (PP) (Steward, 1976; Amaral and Witter, 1989). Following entorhinal cortex lesion, the neural plasticity events, including anterograde terminal degeneration (Matthews et al., 1976a; Jensen et al., 1994), axonal sprouting (Lynch et al., 1972), terminal proliferation and synaptogenesis (Matthews et al., 1976b; Lee et al., 1977; Steward and Vinsant, 1983; Steward, 1992), occur leading to the structural reorganization in the dea¡erented hippocam-
*Corresponding author. Tel.: +86-21-64370080; fax: +86-2164332445. E-mail address: [email protected] (C.-F. Zhou). Abbreviations : DG, dentate gyrus; EDTA, ethylenediaminetetraacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase ; GFAP, glial ¢brillary acidic protein; OML, outer molecular layer; PBS, phosphate-bu¡ered saline; PP, perforant path; SDS, sodium dodecylsulfate; SLM, stratum lacunosum-moleculare. 289
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embryo implantation (Afonso et al., 1997), bone matrix remodeling (Lerner and Grubb, 1992) and the development of many degenerating disorders of the CNS such as Alzheimer’s disease (Uchida et al., 1997; Levy et al., 2001). It has also been reported that cystatin C promotes cell proliferation in vitro and in vivo (Tavera et al., 1992; Taupin et al., 2000). Moreover, many protease inhibitors together with their target enzymes have been shown to regulate neuronal di¡erentiation and migration as well as neurite growth of several neural cell types (Gibson et al., 1984; Hawkins and Seeds, 1989; Friedman and Seeds, 1995). Therefore, the present knowledge of cystatin C likely supports the suggestion that this molecule may play an important role in neural plasticity in the hippocampus after entorhinal cortex lesion. In the present study we analyzed the expression of cystatin C mRNA and protein in the murine hippocampus after PP transections, and possible roles of cystatin C on neural plasticity in the dea¡erented hippocampus are subsequently discussed.
EXPERIMENTAL PROCEDURES
Animals and surgery Adult female Sprague^Dawley rats (body weight 220^250 g) and ICR mice (20^25 g), supplied by the Shanghai Center for Laboratory Animals (Shanghai, China) and housed under standard laboratory conditions, were used in the present study. All procedures were performed in agreement with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Shanghai Institutes for Biological Sciences, Shanghai, Institutional Animal Care and Use Committee. All e¡orts were made to minimize animal su¡ering and the number of animals used. Rats were used for northern blot analysis and mice for in situ hybridization and immunohistochemistry. The procedure for performing rat bilateral (n = 15) and mouse unilateral (n = 20) PP transections has been previously described elsewhere (Zhou et al., 1998). In brief, under deep anesthesia by sodium pentobarbital (40 mg/kg body weight), the animal was positioned in a stereotaxic apparatus, two trenches were made by drilling the occipital skull and the perforant paths were manually transected completely with a curved silver microknife. At 3, 7, 15 or 30 days post-lesion, the rats (n = 3 for each group) with bilateral PP lesions as well as normal controls (n = 3) were decapitated under deep anesthesia and the brains were quickly removed. The hippocampus was carefully dissected out, snap frozen in liquid nitrogen and stored at 380‡C until used for northern blot. For in situ hybridization and immunohistochemistry, normal mice were used as controls (n = 3) and the mice with unilateral PP lesions were allowed to survive for 3, 7, 15 or 30 days following PP transections (n = 5 for each group). The animal was deeply anesthetized with an overdose of sodium pentobarbital and transcardially perfused with ice-cold ¢xative of 4% paraformaldehyde in 0.1 M phosphate-bu¡ered saline (PBS), pH 7.4. The brain was dissected out, post-¢xed for 4 h at 4 ‡C, incubated in 30% sucrose to sink, and cut coronally at 30 Wm on a cryostat. Northern blot analysis Total RNA was extracted from the frozen rat hippocampi using the acid guanidinium isothiocyanate^phenol method (Chomczynski and Sacchi, 1987), electrophoresed (20 Wg per lane) on a 1% formaldehyde agarose gel, blotted onto nylon membrane, and cross-linked by ultraviolet ¢xation. DNA
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probes for northern hybridization were synthesized from fulllength mouse cystatin C cDNA (GenBank accession number M59470) by using the random-primed DNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). After pre-hybridization in hybridization bu¡er (15% formamide, 40 mM NaPO4 pH 7.2, 10 mM EDTA, 7% sodium dodecylsulfate (SDS), 1UDenhardt’s, 100 Wg/ml salmon sperm DNA), the ¢lters were hybridized with 32 P-labeled cDNA probes (0.5^2U107 c.p.m./ml) in the same bu¡er at 65‡C overnight. Hybridized membranes were sequentially washed to high stringency (40 mM NaPO4 , 1 mM EDTA, 1% SDS, 65‡C) and exposed to ¢lm (Kodak). To control the total RNA amount loaded in each lane, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal standard in each hybridization. The hybridization signals were quanti¢ed over the optical densities using the GDS8000 system with software Gel work 3.01 (UVP), and normalized by GAPDH signal. Results are expressed as mean T S.E.M. from three animals and statistical di¡erences were assessed using a two-tailed Student’s t-test with signi¢cance set at P 6 0.05. In situ hybridization histochemistry The mouse cystatin C cDNA used in the present study corresponded to the published sequences (GenBank accession number M59470). The cystatin C cDNA cloned in vector pBluescript II SK3 was linearized, and digoxigenin-labeled antisense and sense riboprobes were synthesized using an in vitro transcription kit (Roche). The 670-bp-long cRNA probes were further subjected to alkaline hydrolysis for 20 min at 65‡C in hydrolysis bu¡er (80 mM NaHCO3 , 120 mM Na2 CO3 ), and ¢nally dissolved in distilled water at 100 ng/Wl. Coronal sections from di¡erent groups of mice were sampled and processed simultaneously to ensure the same conditions for all hybridizations. Sections were washed twice in 2USSC (20USSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0), treated with 0.1 N HCl for 10 min, washed once in deionized water, then incubated in 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) for 10 min. After a brief wash with 2USSC, the sections were pre-hybridized for 3 h at 50‡C in the hybridization bu¡er containing 50% deionized formamide, 10% dextran sulfate, 10 mM Tris^HCl pH 7.5, 600 mM NaCl, 0.25% SDS, 1UDenhardt’s and 400 Wg/ml salmon sperm DNA, and hybridized in the same bu¡er containing 0.2 Wg/ml of either antisense or sense cystatin C probe at 50‡C overnight. Following hybridization, sections were rinsed brie£y in 4USSC at room temperature, washed in 50% deionized formamide/2USSC for 30 min at 55‡C. After a 10-min wash with RNase bu¡er containing 10 mM Tris^HCl, 0.5 M NaCl, the unbound RNA probes were digested with 20 Wg/ml RNase A in the same bu¡er for 30 min at 37‡C. The sections were then washed in RNase bu¡er for 10 min, followed by two washes in 2USSC for 15 min at 50‡C, 1USSC for 2U15 min at 50‡C, then 0.1USSC for 4U15 min at 37‡C. After several rinses in Tris-bu¡ered saline (0.15 M NaCl, 0.1 M Tris, pH 7.5), the sections were incubated in 1% blocking solution (Roche) for 30 min, followed by incubation for 2 h with alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (1:1000, Roche) at room temperature. After several washes with 50 mM MgCl2 in Tris-bu¡ered saline pH 9.5, the phosphatase reaction was performed using 4-nitroblue tetrazolium (0.35 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.18 mg/ml) as substrates. The color reaction was developed in the dark until satisfying results were obtained. Some of the developed sections were subsequently processed for glial ¢brillary acidic protein (GFAP) immunohistochemistry (see below) to determine whether the cystatin C gene-expressing cells are mainly astrocytes that have proliferated and hypertrophied in the hippocampus in response to entorhinal dea¡erentation (Rose et al., 1976; Jensen et al., 1994; Hailer et al., 1999). Immunohistochemistry The mouse sections were washed three times in 0.1 M PBS, incubated for 1 h with 0.3% Triton X-100 and 10% normal goat
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serum, then incubated at 4‡C overnight either with a mixture of primary mouse antibody to GFAP (monoclonal, 1:200, Sigma, St. Louis, MO, USA) and rabbit anti-human cystatin C antibody (polyclonal, 1:2000, Dakopatts, Glostrup, Denmark), or with one of the two antibodies alone diluted in PBS containing 1% goat serum. For the double labeling of cystatin C and GFAP proteins, the primary antibody bindings were directly visualized with a mixture of £uorescein-conjugated goat antirabbit and Texas Red-conjugated goat anti-mouse antibodies (1:200, Dako) after several washes in PBS. For separate GFAP or cystatin C immunoreaction, the sections were rinsed in PBS several times, incubated with biotinylated goat antimouse (1:200, Dako) antibody or biotinylated goat anti-rabbit antibody (1:200, Vector Laboratories, Burlingame, CA, USA), respectively, washed with PBS, reacted with avidin^biotin horseradish peroxidase solution (standard ABC Elite kit, Vector), and visualized with 0.003% H2 O2 and 0.03% 3,3-diaminobenzidine tetrahydrochloride in 0.05 M Tris^HCl pH 7.6. Confocal microscopy (Leica TCS-NT) was used for the analysis of the double immuno£uorescence staining.
RESULTS
The timing of cystatin C mRNA expression in rat hippocampus after perforant path transections Northern blot hybridization was employed to analyze the expression levels of cystatin C mRNA in the rat hippocampus at 3, 7, 15 and 30 days following PP transections. It was observed that the rat cystatin C gene was expressed as a single transcript of about 0.7^0.8 kb as shown by previous studies (Solem et al., 1990) and the expression was regulated in a transient manner (Fig. 1A, B). There was only a small alteration of hybridization signal of cystatin C mRNA in the dea¡erented hippocampus at 3 days post-lesion compared with that of normal control (120% of control, no statistical di¡erence). However, by 7 days post-lesion, the hybridization signal showed a massive increase with a value of 219% of control, reaching the maximal level among all time points examined. By 15 days post-lesion, the elevation of cystatin C mRNA expression was still prominent and comparable to that at 7 days post-lesion with a value of 185% of control. Quantitative analysis (Fig. 1B) revealed that the e¡ect of PP transections on hippocampal cystatin C mRNA expression at 7 and 15 days post-lesion was statistically signi¢cant (P 6 0.05). By 30 days post-lesion, the last time point examined, the cystatin C mRNA signal in the dea¡erented hippocampus declined to nearly normal level with a small elevation (113% of control, no statistical di¡erence). It is of interest that the regulation of cystatin C mRNA expression in the hippocampus following PP transection occurs exactly at the time of the structural reorganization in the tissue subjected to the same lesions (for review, see Deller and Frotscher, 1997). Layer-speci¢c up-regulation of cystatin C mRNA and protein in mouse hippocampus after perforant path transections To examine the regional distribution of cystatin C transcripts in the dea¡erented hippocampus, we performed an in situ hybridization analysis with mouse
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Fig. 1. Northern blot analysis of cystatin C mRNA expression in the hippocampus of control (c in A and B) and lesioned rats at 3, 7, 15 and 30 days following PP transections. (A) Photograph shows a transient increase of cystatin C mRNA expression (upper panel) in the hippocampus after lesions compared to control (c). GAPDH (lower panel) is used as an internal standard to control the amount of total RNA loaded in each lane. (B) Northern signals in A are quanti¢ed by densitometry. The values are expressed as control percentage and represent a ratio of cystatin C signal density at various times post-lesion divided by that measured on control after normalization with the GAPDH signal. Values given are mean T S.E.M. for three animals at each post-lesion time point. Note the signi¢cant increase of cystatin C mRNA expression in the hippocampus at 7 and 15 days after PP transections with values of 219% and 187% of control, respectively. *P 6 0.05, two-tailed Student’s t-test.
material to localize the cystatin C mRNA expression. In agreement with previous studies (Cole et al., 1989; Olsson et al., 2000), our in situ hybridization in normal control animals revealed that cystatin C mRNA was present in the choroid plexus and many regions of the mouse brain, including the cerebral cortex, hippocampus, hypothalamus and cerebellum (data not shown). In the hippocampus, the signal for cystatin C mRNA labeling was observed sparsely throughout all sub¢elds except for the SLM and sub-granular zone where more labeled cells were identi¢ed (data not shown). In the mouse hippocampus ipsilateral to PP transections, cystatin C mRNA expression in the hippocampal SLM and the dentate OML, the PP projection zones, appeared to increase at 3 days post-lesion and abundantly increased at 7 and 15 days post-lesion, but recovered at 30 days post-lesion to the level similar to that at 3 days post-lesion (Fig. 2B, D, F, H). The overall increase of the labeling in the denervated areas can be ascribed to the elevated cystatin C mRNA labeling intensity within individual cells and the increased number of cystatin C mRNA-expressing cells. In areas other than the PP terminal zones, i.e. the stratum radiatum and
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Fig. 2.
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pyramidal cell layer of the hippocampus, and the inner molecular layer and granular cell layer of the DG, there were no detectable alterations of cystatin C mRNA expression induced by PP transections (Fig. 2B, D, F, H). In the mouse hippocampus contralateral to PP lesions, more cystatin C mRNA-expressing cells were visualized in the SLM and OML only at 7 and 15 days post-lesion (Fig. 2C, E) but not other time points examined (Fig. 2A, G) compared with cystatin C mRNA expression in the same areas of the normal control, even if this elevation was much less than the PP cut side (Fig. 2D, F). In the parallel control experiments, sections hybridized with the sense probe were completely devoid of signals at all samples (data not show). The in situ hybridization study of the cystatin C mRNA expression in the hippocampus following PP transections revealed that the regulation of cystatin C transcripts had a temporal pattern very similar to that observed by northern analysis with maximum expression between 7 and 15 days post-lesion. To further analyze whether cystatin C protein expression in the dea¡erented hippocampus also undergoes regulation after PP transections, immunohistochemistry for mouse cystatin C protein was performed using a polyclonal rabbit anti-human cystatin C antibody that had been successfully used to react with the murine antigen in previous studies (Palm et al., 1995; Miyake et al., 1996). The immunostaining patterns in the dea¡erented hippocampus were spatially coincident with in situ hybridization, i.e. the up-regulation of cystatin C immunostaining occurred only in the hippocampal SLM and the dentate OML, but not in the other layers such as stratum radiatum of the hippocampus and the inner molecular layer of the DG (Fig. 3). The post-lesion time-dependent alteration of cystatin C immunostaining was also identical to that of in situ hybridization with the strongest immunostaining at 7 and 15 days post-lesion (Fig. 3D, F). This result suggests that entorhinal dea¡erentation regulates not only the transcription of the cystatin C gene but also the translation of cystatin C transcripts. Compared with in situ hybridization, cystatin C immumostaining revealed more clearly the cytological morphology of the positively stained cells in the denervated zones, which likely showed the morphological characteristics of astrocytes (Fig. 3). Cystatin C-expressing cells are mainly astrocytes To further con¢rm that the cystatin C mRNA- and protein-expressing cells in the hippocampus at 7 and 15
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days following PP lesions are reactive astrocytes, we used double labeling of either a combination of in situ hybridization for cystatin C mRNA with immunohistochemistry for GFAP protein or double immuno£uorescence staining for both proteins on the mouse model. In the double labeling of cystatin C mRNA and GFAP protein, the purple-blue cystatin C mRNA signals were primarily con¢ned to the cytoplasm of astrocytes, while the brown GFAP immunostaining located mainly in the cell processes (Fig. 4A, B). It was observed that the great majority of cystatin C mRNA-positive cells in the SLM and OML were GFAP-positive and all GFAP-immunoreactive cells were cystatin C-positive (Fig. 4A). Consistently, confocal microscopy analysis of double immuno£uorescence staining clearly veri¢ed that most cystatin C-positive cells in the denervated hippocampal SLM and the dentate OML were GFAP-positive cells, and a few of them showed GFAP-negative which would be microglia (Fig. 4C^E). It was noticed that at high magni¢cation the cystatin C signals were unevenly localized in a dot/lump-like shape in the somata and processes of GFAP-positive cells (Fig. 4E), indicating that cystatin C protein probably binds to/exists in certain intracellular organelles in situ.
DISCUSSION
In this study, we found the spatiotemporally restricted up-regulation of both cystatin C mRNA and protein in the hippocampal SLM and the dentate OML after PP transections, which strongly suggests that the expression of cystatin C is induced by entorhinal dea¡erentation. It is well known that there are about 5% of entorhinal projections to the hippocampus originating from the contrary side (Steward et al., 1976). Correspondingly, a small, but evident elevation of cystatin C mRNA expression was demonstrated in the hippocampus contralateral to PP transections at 7 and 15 days post-lesion, which would be the result of the loss of the crossed entorhinal projections. As one of the most abundant endogenous cysteine protease inhibitors in mammalian tissues, cystatin C has been shown to be involved in many CNS diseases or injuries. For instance, following transient forebrain ischemia cystatin C immunoreaction is transiently up-regulated in the rat degenerating hippocampal CA1 neurons but not in viable neurons (Palm et al., 1995). Elevation of cystatin C expression in the facial nucleus following facial nerve axotomy has also been observed (Miyake et al., 1996). Immunochemical analysis of brains of patients with Alzheimer’s disease revealed
Fig. 2. In situ hybridization of cystatin C mRNA in the mouse hippocampus showing more intense dot-like hybridization products in the OML and SLM in the hippocampus ipsilateral (right side) than contralateral (left side) to PP transections with the di¡erence in the expression between two sides being less, but notable at 3 (A, B) and 30 (G, H) days, and most prominent at 7 (C, D) and 15 days (E, F) after lesions. Note that the cystatin C mRNA expression in the SLM and OML in the lesion-contralateral hippocampus at 7 (C) and 15 (E) days post-lesion appears to be up-regulated in comparison with the same zones of the normal control (data not shown) and the lesion-contralateral hippocampus at 3 (A) and 30 (G) days postlesion, and that there is no di¡erence in cystatin C mRNA expression between the corresponding non-entorhinal projection zones, i.e. stratum pyramidale (SP), stratum radiatum (SR), inner molecular layer (IML) and stratum granulare (SG), of left and right hippocampus of the same lesioned animals. Scale bar = 200 Wm.
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Fig. 3. Immunostaining of cystatin C in the mouse hippocampus after unilateral PP transections. At 3 days post-lesion (A, B), there is a slight increase of cystatin C staining in the SLM and OML of the hippocampus ipsilateral to perforant path transections (B) compared with that of the corresponding areas in the contralateral side (A). The elevation of cystatin C immunostaining in the ipsilateral SLM and OML is abundant at 7 days post-lesion (D) and this up-regulation remains prominent at 15 days post-lesion (F) compared with the staining in the contralateral hippocampus (C, E). Note that the cystatin C-positive cells appear to have the morphological characteristics of astrocytes and that there is no di¡erence in staining between the corresponding non-entorhinal projection zones of the ipsilateral and contralateral sides. Abbreviations as in Fig. 2. Scale bar = 150 Wm.
that cystatin C is accumulated in the senile plaque amyloid deposits in the hippocampus (Uchida et al., 1997) and colocalized with amyloid L protein in the vascular amyloid deposits of the brain (Levy et al., 2001). Our observation that entorhinal dea¡erentation-induced upregulation of cystatin C in the hippocampus again sug-
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gests that cystatin C may exert important roles in various neuropathologies (Palm et al., 1995; Levy et al., 2001). The activation of astrocytes in the hippocampus following entorhinal cortex lesion is characterized by their proliferation, hypertrophy, and expression increase for GFAP and vimentin in PP projection zones, which starts
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Fig. 4. Double labeling of cystatin C and GFAP in the mouse hippocampus following PP transections. (A, B) Double labeling of cystatin C by in situ hybridization and GFAP by immunohistochemistry in the hippocampus at 7 days post-lesion. Almost all cystatin C-positive cells are GFAP-immunoreactive, while all GFAP-labeled cells are cystatin C-positive in the SLM and OML (A). The framed area in A is shown in B at higher magni¢cation, showing several double-labeled astrocytes with purple-blue cystatin C mRNA signal in the somata and brown GFAP protein staining in the processes. (C^E) Confocal images of double immuno£uorescence labeling of cystatin C (C, green) and GFAP (D, red) in the hippocampus at 15 days post-lesion, showing that most cystatin C-labeled cells are GFAP-positive and all GFAP-positive cells are cystatin C-immunopositive in the SLM and OML with several representative double-labeled cells indicated by arrows (E). Few cells positive for cystatin C but negative for GFAP in these areas are also found. Note the uneven dot-shaped immunostaining of cystatin C in the somata and processes of astrocytes (C, E). Abbreviations as in Fig. 2. Scale bar = 50 Wm (A), 10 Wm (B), 40 Wm (C, D), 20 Wm (E).
at 2 days post-lesion, reaches the maximum around 6^7 days post-lesion, and subsides after about 21 days postlesion (Rose et al., 1976; Gall et al., 1979; Steward et al., 1993; Jensen et al., 1994; Krohn et al., 1995; Baldwin
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and Sche¡, 1997; Hailer et al., 1999). Cystatin C upregulation by astrocytes demonstrated in this study is almost simultaneous with astrocyte activation in the dea¡erented PP terminal zones. Therefore, cystatin C
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expression represents an additional feature of the activated astrocytes in the hippocampus in response to deafferentation. The signal that initiates the target area-speci¢c elevation of cystatin C expression by reactivated astrocytes is unclear at present. However, it has been reported that transforming growth factor L induces cystatin C mRNA expression in the astrocyte precursor cells in vitro (Solem et al., 1990), and its transcripts in the dea¡erented hippocampus are up-regulated in the activated microglia between 4 and 10 days after entorhinal cortex lesion (Morgan et al., 1993), a period of time when cystatin C expression is enhanced. Therefore, the microglia transforming growth factor L might regulate cystatin C expression in astrocytes in situ. Astrocytes activated by dea¡erentation have been reported to produce a wide variety of extracellular matrix molecules such as laminin (Tian et al., 1997), tenascin (Deller et al., 1997), chondroitin sulfate proteoglycan (Haas et al., 1999; Thon et al., 2000) and heparin a⁄n regulatory peptide (Poulsen et al., 2000), all of which e¡ect the axonal regeneration and growth in the CNS correlating with the lesion-induced neural plasticity in the dea¡erented hippocampus. Recent studies have revealed that cathepsins are e¡ective extracellular matrix protein-degrading proteases (Mason et al., 1986; Xin et al., 1992; Bromme et al., 1996; Petanceska et al., 1996) and that many of them are up-regulated in the hippocampus following entorhinal cortex lesion (Petanceska et al., 1996). It is plausible, therefore, that in the dea¡erented hippocampus the up-regulated cystatin C modulates the activities of cathepsins that are concomitantly elevated such that the extracellular matrix molecules produced by the activated astrocytes would not be degraded, a postulation supported by an in vitro study which showed that cysteine protease inhibitors decrease the matrix degradation induced by cathepsins (Corticchiato et al., 1992). The extracellular matrix remodeling function of cystatin C has been widely demonstrated in kinds of in vivo biological processes such as tumor metastasis (Coulibaly et al., 1999; Cox et al., 1999) and embryo placentation (Afonso et al., 1997). In addition to protecting the extracellular matrix molecules from proteolysis, cystatin C may also contribute to the turnover of various cytokines and neurotrophic factors including interleukin, ¢broblast growth factor, brain-derived neurotrophic factor and nerve growth factor released by activated glial cells in the hippocampus after entorhinal cortex lesion (for review, see Deller and Frotscher, 1997; Turner et al., 1998). A case in point for the defense e¡ect of cystatin C on cytokines is that cathepsin inhibitors increase the residence time of interleukin-6 in HepG2 cells by
interfering with the lysosomal processing of this molecule (Peppard and Knap, 1999). Moreover, cystatin C has been reported to inhibit the proteolysis of neuropeptide substance P that improves neuronal survival and neurite growth in vitro (Aghajanyan et al., 1988), implying that cystatin C protects neurotransmitters favorable for neural growth. Taken together, it is probable that the upregulated cystatin C may facilitate the establishment of an axonal growth-promoting environment by protecting neurite growth-associated molecules from degradation by cysteine proteases in the dea¡erented hippocampus. Very interestingly, cystatin C has been reported to play an important role in cell proliferation and growth. In vitro experiments showed that chicken egg cystatin prompts the proliferation of mouse 3T3 ¢broblasts (Sun, 1989) and that native rat cystatin C evokes the proliferation of glomerular mesangial cells in an autocrine manner (Tavera et al., 1992). The tumor metastasis decrease in cystatin C-de¢cient mice was also reported to be associated with reduced tumor cell growth (Huh et al., 1999). Taupin et al. (2000) have recently reported that the N-glycosylated cystatin C is required for ¢broblast growth factor 2 to induce the proliferation of neural stem cells in vivo or in vitro. Thus the spatiotemporal overlapping of cystatin C up-regulation and astrocyte proliferation in the hippocampus following PP transections raises the possibility that increased cystatin C promotes the proliferation of astrocytes in an autocrine/paracrine manner.
CONCLUSIONS
In the present study, we have demonstrated alterations of the expression of cystatin C mRNA and protein in the terminal zones of entorhino-hippocampal projections after their lesions by transection. A dramatic transient up-regulation of both cystatin C mRNA and protein was observed in the reactive astrocytes of the deafferented hippocampal SLM and the dentate OML. Concomitant with the activation of astrocytes, the regulation of cystatin C expression is proposed to be involved in various astrocytic responses to PP transections that are associated with the lesion-induced neural plasticity in the hippocampus.
Acknowledgements&This work was supported by the National Basic Research Program (G 1999054005), National Natural Science Foundation (39930090, 39870248) of China, and Life Science Special Fund of the CAS supported by the Ministry of Finance.
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