The goal of this project is to characterize the role that cell adhesion molecules play in the development of neuronal polarity. Neurons produce two types of processes, axons and dendrites. Molecules have been identified, such as axonal L1, that localize to one or the other of these processes. It is not known how this differential distribution develops, nor whether it is the result or the cause of the morphological differentiation in these cells. To address this question we investigated whether L1 could influence the mechanisms that underlie axonal or dendritic development. We plated embryonic rat hippocampal cells onto substrate-bound L1, allowed the cells to develop for varying periods of time (a few hours to a few weeks), and then fixed and immunostained them for polarity markers L1, MAP2, and tau. We found that the cells extended multiple, extremely long axons within 12 hours. These axons continued to elongate rapidly without fasciculating and branched in atypical patterns. Dendritic development was significantly suppressed for at least a week. In comparison, sister cultures on poly-L-lysine required at least 24 hours for short axons to develop, but elaborated dendrites shortly thereafter. The cell adhesion molecule, N-cadherin, which is more uniformly distributed over the surface of mature neurons, also affected process outgrowth when used as a substrate. It greatly, and uniformly accelerated the growth of both axons and dendrites. Cells plated on the extracellular matrix component, laminin, showed a selective increase only in axonal growth over that seen on poly-L-lysine. This is consistent with previously published reports. The differential distribution of polarity markers, such as axonal L1, and the dendritic microtubule associated proteins MAP2 and tau, correlated with the accelerated development of polarity. We are continuing to characterize the development of neurons growing on L1 and N-cadherin, looking at other differentially distributed molecules and performing quantitative analyses of process numbers and lengths. We are analyzing the distribution of cytoskeletal elements such as actin and tubulin, and are evaluating the role of protein kinases. This may help us to elucidate the observed effects since both of these are believed to be involved in adhesion molecule signaling. We are also using interference reflection microscopy to visualize the interaction patterns of axonal and dendritic growth cones with various adhesion molecules. This may reveal differences in levels or patterns of adhesion that underlie the effects of these molecules.
