ASSOCIATION BETWEEN PROTEASE-SPECIFIC PROTEOLYTIC CLEAVAGE OF BREVICAN AND SYNAPTIC LOSS IN THE DENTATE GYRUS OF KAINATE-TREATED RATS W. YUAN, a R. T. MATTHEWS, b J. D. SANDY a;c and P. E. GOTTSCHALL a à a Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, MDC Box 9, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612-4799, USA b Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA c Center for Research in Skeletal Development and Pediatric Orthopedics, Shriners Hospital for Children, Tampa, FL 33612, USA AbstractöProteolytic fragments generated by ADAMTS ( a disintegrin and metalloprotease with thrombo spondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identi¢ed, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEA- VESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS- cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate^putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate. Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by speci¢c matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS- induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: ADAMTS, brevican, chondroitin sulfate proteoglycan, excitotoxicity, kainic acid, antiserum, protease. Brevican (also known as BEHAB, brain-enriched hyal- uronan-binding protein) is a chondroitin sulfate proteo- glycan (CSPG) expressed by astrocytes and neurons (Jaworski et al., 1994, 1999; Seidenbecher et al., 1998) in adult CNS (Milev et al., 1998), where several CSPGs, including brevican, have been identi¢ed in the neuropil and in reticular networks that ensheath perikarya, termed perineuronal nets (PNNs) (Celio and Blumcke, 1994). Brevican has been localized to the synapse, where it surrounds, but is not on or over, synaptic den- sities (Hagihara et al., 1999) and due to its late postnatal expression, brevican may stabilize synapses in mature neural networks (Hock¢eld et al., 1990; Yamaguchi, 2000). Brevican likely exists in a complex with hyaluronic acid and tenascin where its C-terminal lectin domain may interact with sulfoglucuronylglycolipids expressed on the cell surface (Miura et al., 1999) to alter neuronal plasticity (Miura et al., 2001) and glial migration (Zhang et al., 1998a). Brevican is a member of the family of aggregating CSPGs, termed lecticans (Yamaguchi, 2000), which also include aggrecan, versican and neurocan. Proteolytic cleavage and loss of the glycosaminoglycan (GAG)-sub- stituted region of cartilage aggrecan precedes cartilage breakdown in joint diseases such as osteoarthritis (Sandy and Verscharen, 2001). Aggrecan cleavage by the matrix metalloproteinases (MMPs) has been widely studied, however, the principal family of proteases responsible for aggrecan degradation in human cartilage in vivo are the ADAMTSs ( a disintegrin and metallopro- tease with thrombo spondin motifs) (Caterson et al., 1091 *Corresponding author. Tel.: +1-813-974-2543; fax: +1-813-974- 2565. E-mail address: pgottsch@hsc.usf.edu (P. E. Gottschall). Abbreviations: ADAMTS, a disintegrin and metalloprotease with thrombo spondin motifs; ANOVA, analysis of variance; CSPG, chondroitin sulfate proteoglycan; EDTA, ethylenediaminetetra- acetate; GAG, glycosaminoglycan; MMP, matrix metallopro- teinase; PBS, phosphate-bu¡ered saline; PNN, perineuronal net; RT-PCR, reverse transcriptase polymerase chain reaction; SSC, sodium saline citrate. NSC 5771 25-9-02 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 114, No. 4, pp. 1091^1101, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00347-0 0306-4522 / 02 $22.00+0.00