Chondroitin sulfate E promotes neurite outgrowth of rat embryonic day 18 hippocampal neurons Albrecht M. Clement a,1 , Kazuyuki Sugahara b , Andreas Faissner a, c, * a Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany b Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku 20, Kobe 658±8558, Japan c Laboratoire de Neurobiologie du De  veloppement et de la Re  ge  ne Âration, UPR 1352, Centre de Neurochimie du CNRS, et Universite  Louis Pasteur, 5, rue Blaise Pascal, F-67084 Strasbourg, France Received 12 March 1999; received in revised form 1 May 1999; accepted 1 May 1999 Abstract In light of controversial reports concerning the effects of chondroitin sulfates on neurite outgrowth, several glycosa- minoglycans belonging to this structural class were compared with regard to their in¯uence on axon formation by embryonic day 18 hippocampal neurons. In these studies, chondroitin sulfate A (CS-A), CS-B and CS-C proved weak or inef®cient in the neurite outgrowth promotion assay. As expected, CS-D stimulated both the fraction of neurite bearing neurons and the length of their processes. This effect could be neutralized by the monoclonal antibody (mAb) 473HD. In contrast, CS-E enacted a dramatic promotion of neurite outgrowth. This effect persisted in the presence of mAb 473HD, consistent with the observation that this antibody did not react with CS-E in glycosaminoglycan transfer and blotting techniques. We conclude that CSE contains a novel glycosaminoglycan based neurite outgrowth promoting motif, which is distinct from other known activities. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurite outgrowth; Chondroitin sulfate; Hippocampal neurons; Monoclonal antibody Axon growth and guidance are of crucial importance for the formation of interneuronal connections during develop- ment of the nervous system. In adult tissues, the modi®cation of axonal projections by local sprouting and pruning processes seems required for plasticity of the central nervous system (CNS) [24]. Conversely, prevention of sprouting is thought to play a pivotal role in the inhibition of CNS axon tract regeneration after lesion. Several gene families have been implicated in axon growth and guidance, for example the immunoglobulin- and cadherin-superfamilies of cell adhesion molecules [4,21], extracellular matrix glycopro- teins and the corresponding integrin receptors [18], the chemotactic netrins and their receptors [13,23] and inhibitory cues such as semaphorins and the ephrin ligands of Eph- kinase receptors [2,20]. Several examples illustrate that chondroitin sulfate proteoglycans exert inhibitory in¯uences on axon outgrowth in the context of astroglial scar formation, or the construction of transient glial boundaries of neural tissues [6,9,17]. The application of chondroitin sulfate- degrading enzymes has resulted in the modi®cation of axon growth trajectories in vivo and in vitro, presumably by redu- cing inhibitory properties of chondroitin sulfate-expressing structures [3,7]. However, several studies have illustrated neurite outgrowth promoting effects of core proteins or chon- droitin sulfate constituents. For example, chondroitin sulfate B (CS-B) selectively stimulates dendritic growth of embryo- nic day 16 (E16) cortical neurons and the DSD-1-epitope which depends on CS-D and sulfation promotes neurite formation by E18 hippocampal neurons [5,16]. The latter was identi®ed with the monoclonal antibody (mAb) 473HD and localized in discrete regions of the central nervous system with extensive axon outgrowth [8,12]. These seemingly opposite results could re¯ect structural heterogeneity of complex chondroitin sulfate polymers, or a lineage-dependence of neuronal responses [10]. In order to advance our understanding of the effects of chondroitin sulfate glycosaminoglycans on neurite outgrowth, several structural types were compared in a neurite outgrowth assay. Embryonic day 18 hippocampal neurons were cultivated at low density in de®ned media on coverslips conditioned with poly-dl-ornithine (PORN) (15 mg/ml) and subse- quently coated with chondroitin sulfates at 5 mg/ml uronic Neuroscience Letters 269 (1999) 125±128 0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(99)00432-2 * Corresponding author. Tel.: 133-3-88-456-651; fax: 133-3- 88-411-780. E-mail address: faissner@neurochem.u-strasbg.fr (A. Faissner) 1 Current address: Ludwig Institute for Cancer Research, UCSD, 9500 Gilman Drive, La Jolla, CA 92093±0660, USA.