Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth. We have found that the region of tenascin-C containing only alternately spliced fibronectin type-III repeat D, called fnD, when used by itself, dramatically increases neurite outgrowth in culture. We used overlapping synthetic peptides to localize the neurite outgrowth-promoting site within fnD to a 15 amino acid sequence, called D5. An antibody against D5 blocked promotion of neurite outgrowth by fnD as well as tenascin-C, indicating that this peptide sequence is functional in the context of the native molecule. Further testing of shorter synthetic peptides restricted the neurite outgrowth-promoting site to eight amino acids, VFDNFVLK. Of these, "FD" and "FV" are conserved in tenascin-C sequences derived from all the species available in the GenBank. To investigate the hypothesis that FD and FV are critical for the interaction with neurons, we tested a recombinant fnD protein and synthetic peptides with alterations in FD and/or FV. These molecules did not facilitate process extension, suggesting that the conserved amino acids are required for formation of the active site in fnD. We next investigated whether VFDNFVLK could be used as a reagent to overcome the neurite outgrowth inhibitory properties of chondroitin sulfate proteoglycans, the major inhibitory molecules in the glial scar. The peptide significantly enhanced outgrowth on proteoglycans and was more effective than laminin-1, L1-Fc, or intact tenascin-C, thus demonstrating the potential applicability of tenascin-C regions as therapeutic reagents.

Tenascin-C has been implicated in regulation of both neurite outgrowth and neurite guidance. We have shown previously that a particular region of tenascin-C has powerful neurite outgrowth-promoting actions in vitro. This region consists of the alternatively spliced fibronectin type-III (FN-III) repeats A-D and is abbreviated fnA-D. The purpose of this study was to investigate whether fnA-D also provides neurite guidance cues and whether the same or different sequences mediate outgrowth and guidance. We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that neurites demonstrated a strong preference for fnA-D when given a choice at a poly-L-lysine-fnA-D interface, even when fnA-D was intermingled with otherwise repellant molecules. Furthermore, neurites preferred cells that overexpressed the largest but not the smallest tenascin-C splice variant when given a choice between control cells and cells transfected with tenascin-C. The permissive guidance cues of large tenascin-C expressed by cells were mapped to fnA-D. Using a combination of recombinant proteins corresponding to specific alternatively spliced FN-III domains and monoclonal antibodies against neurite outgrowth-promoting sites, we demonstrated that neurite outgrowth and guidance were facilitated by distinct sequences within fnA-D. Hence, neurite outgrowth and neurite guidance mediated by the alternatively spliced region of tenascin-C are separable events that can be independently regulated.