We study cellular and molecular mechanisms involved in development of neurons of the central nervous system and the formation of central synapses, utilizing cell culture, microscopy and molecular techniques. Agrin, a proteoglycan secreted by motoneurons, is required for postsynaptic differentiation in skeletal muscle. A transmembrane form of agrin is widely expressed in the central nervous system but its functions are not well understood, although there is evidence for its involvement in neurite outgrowth as well as synapse formation and function. Our current studies focus on the trafficking and functions of transmembrane agrin in hippocampal neurons. We previously compared the targeting of full-length and truncated forms of agrin-GFP in hippocampal neurons in order to determine the sequences involved in targeting to the axonal growth cone. We found that the N-terminal moiety of agrin is required for targeting to the axon growth cone, with a contribution from the N-terminal cytoplasmic tail. The C-terminal moiety is not sufficient for targeting to the axon growth cone but may contain additional targeting information. We are continuing to examine the roles of different agrin domains in its trafficking. We previously found that the expression of the transmembrane form of agrin in skeletal muscle and other cultured cells, including hippocampal neurons, induces the formation of filopodia. We have now found that that the N-terminal moiety of transmembrane agrin is sufficient to induce filopodia formation, although not as effectively as full-length agrin. We are investigating the roles of agrin?s glycosaminoglycan side chains, which are attached to the N-terminal moiety, and the possible role of an interaction of agrin with fibroblast growth factor or its receptor in filopodia induction. We are testing the role of endogenous transmembrane agrin in the regulation of neuronal filopodia. We have found that suppression of agrin expression in hippocampal neurons by small inhibitory RNA results in a reduction in the number of filopodia. This suggests that transmembrane agrin positively regulates the formation or stability of filopodia. We will use time-lapse studies of living neurons to distinguish effects on formation versus stabilization of filopodia.
