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. JING b and C.-F. ZHOU a à a 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 b Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China AbstractöCystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extra- cellular 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¡erenta- tion. 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 entorhinaldea¡erentationandthatcystatinCmaybeinvolvedintheastroglia-mediatedneuralplasticityeventsinthehippo- campus following perforant path transections. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dea¡erentation, protease inhibitor, astrocyte, in situ hybridization, immunohistochemistry. An important model system for studying the neural plas- ticity after injury in the brain of adult animals has been the hippocampus following destruction of its principal extrinsic input from the entorhinal cortex. The projec- tions 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 an- terograde 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- 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 technol- ogy aimed to identify the genes that may contribute to the plasticity, we separated a set of genes whose expres- sion 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 pro- tease 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 intra- molecular 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 pro- tease activity, cystatin C has been reported to be involved in a broad spectrum of biological events asso- ciated with tissue remodeling, including tumor metastasis and invasion (Coulibaly et al., 1999; Cox et al., 1999), 289 *Corresponding author. Tel.: +86-21-64370080; fax: +86-21- 64332445. E-mail address: cfzhou@server.shcnc.ac.cn (C.-F. Zhou). Abbreviations: DG, dentate gyrus; EDTA, ethylenediaminetetra- acetate; 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-molecu- lare. NSC 5535 24-5-02 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 112, No. 2, pp. 289^298, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00083-0 0306-4522/02 $22.00+0.00