Polysialic acid limits septal neurite outgrowth on laminin.

Polysialic acid (PSA) is a large carbohydrate found exclusively on the neural cell adhesion molecule (NCAM). In the adult brain, PSA is re-expressed by septal axons sprouting and regenerating in an environment rich in laminin. Using an in vitro model, we tested the possibility that PSA limits septal outgrowth by preventing maximal interactions with a laminin substrate. Our results indicate that PSA removal from primary septal neurons plated on laminin significantly increased neurite outgrowth at 12 h (14%, p<0.05) and 24 h (22%, p<0.01). In contrast, the removal of PSA had no impact on septal neurite outgrowth on poly-D-lysine. PSA did not influence the plating adhesion of septal neurons on laminin or poly-D-lysine, indicating that the increase in neurite outgrowth caused by PSA removal on laminin is not related to the initial attachment of the neurons to this substrate. Neurite length on laminin was significantly reduced by the function-blocking beta1-integrin antibody in the presence of PSA (20% decrease, p<0.05), and following PSA removal (34% decrease compared to neurites treated with endoN and without the beta1-integrin antibody, p<0.01). Importantly, the beta1-integrin antibody completely abolished the neurite outgrowth promoting effect of PSA removal on laminin. The beta1-integrin antibody had no impact on septal neurite length on poly-D-lysine. Taken together, these results indicate that the removal of PSA from septal neurons increases neurite outgrowth on laminin by promoting interactions between beta1-integrin and laminin.

Cell adhesion molecule L1 promotes neurite outgrowth of septal neurons.

To establish if the cell adhesion molecule L1 could promote neurite outgrowth of septal neurons, L1-positive substrates were prepared by genetically modifying 3T3 fibroblasts with a retroviral vector encoding human L1 under the control of a negative tetracycline-regulatory system. In several clones of L1-transfected fibroblasts, L1 expression at the cell surface was prominent and efficiently regulated by doxycycline, a tetracycline analogue. In co-culture of septal neurons and fibroblasts, a two-dimensional fractionator probe provided systematic random sampling of the neurites to be measured. Septal neurons, isolated at embryonic Day 17, were found to express L1 in vitro and to extend significantly longer neurites when plated on L1-expressing fibroblasts compared to control fibroblasts. The neurite outgrowth-promoting effect of L1 was inhibited after a doxycycline treatment, which specifically suppressed L1 expression from the modified fibroblasts. The findings that septal neurons at embryonic Day 17 in vitro express L1 and respond to L1-modulation suggest that this molecule is involved in development of the septohippocampal pathway.