Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family q Peter Kabos, a Harry Matundan, b Mandana Zandian, b Cristina Bertolotto, b Michael L. Robinson, c Brian E. Davy, c John S. Yu, a and Richard C. Krueger Jr. b, * a Department of Pediatrics, Division of Molecular and Human Genetics, Children’s Research Institute, The Ohio State University, Columbus, OH, USA b Department of Pediatrics, Division of Neonatology, Cedars-Sinai Medical Center, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA c Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Received 13 April 2004 Available online 6 May 2004 Abstract Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural pre- cursors cultured as neurospheres secrete CSPGs (>30 lg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Aggrecan; Lectican; Phosphacan; Cerebrospinal fluid; Hydrocephalus; LeX/ssea-1; Neurospheres Chondroitin sulfate proteoglycans (CSPGs) are the most prevalent proteoglycans in the developing central nervous system, and have been implicated in mecha- nisms governing neural migration, aggregation, and differentiation [1–9], as well as cytokine regulation [10]. Some CSPGs seem to be constitutively expressed in the CNS, while others have specific developmental expres- sion patterns [4]. For example, the aggrecan/lectican family member versican has been recently shown to be upregulated in oligodendrocyte precursors in mechani- cal injury models of adult rat brain [11]. The roles of specific CSPG gene products in vivo have only begun to be identified, but many CSPG knockouts have little to no recognizable CNS phenotype [12–16]. The events that signal CNS cells to regulate CSPG synthesis are poorly understood. This is equally true in normal development and after injury. Generalized in- creases in glycosaminoglycans have been demonstrated in injured tissues, including the CNS [17–24]. For example, CSPGs and glutamate have been shown to be protective in an additive manner for neonatal retinal ganglion cells undergoing nitric oxide-mediated apoptosis [25]. Aggrecan/lectican family CSPGs and TGF-b2, whose ligand receptor binding is closely regulated by CSPGs, q Abbreviations: BBS, 10 mM borate buffer saline, pH 8.4; BSA, fraction V bovine serum albumin; CSF, cerebrospinal fluid; CSPG, chondroitin sulfate proteoglycan; KC’ased, chondroitinase ABC and keratanase treated; anti-CS4, antibody that recognizes chondroitin-4 sulfate chains on proteoglycans after chondroitinase ABC treatment; anti-C-56, antibody directed against intact CSPGs; HA, hyaluronic acid; PBS, 10 mM phosphate-buffered saline, pH 7.4; anti-ssea-1, antibody against stage-specific embryonic antigen. * Corresponding author. Fax: 1-310-423-2114. E-mail address: richard.krueger@cshs.org (R.C. Krueger Jr.). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.114 Biochemical and Biophysical Research Communications 318 (2004) 955–963 BBRC www.elsevier.com/locate/ybbrc