Mechanisms of Central Nervous System Neuron Development

Heart, Lung, And Blood Institute

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Abstract

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. [unreadable] [unreadable] Agrin, a proteoglycan secreted by motor neurons, 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 functions of transmembrane agrin in hippocampal neurons. [unreadable] [unreadable] We previously used siRNA suppression of agrin expression to demonstrate that endogenous agrin positively regulates the filopodia of developing hippocampal neurons in culture by increasing their stability and rate of initiation. We also 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 and that the N-terminal extracellular moiety of transmembrane agrin is required to do this in hippocampal neurons and cell lines. Moreover we have shown that the glycosaminoglycan (GAG) side chains, which are attached to the N-terminal moiety, play an important role in filopodia induction in both cell lines and neurons. We now have shown that the GAG chains, particularly the heparan sulfate GAG chains, are important for activation of the Rho-family GTP-binding proteins Rac1 and Cdc42, which in turn are required for the induction of filopodia-like processes in cell lines. [unreadable] [unreadable] We now have generated a defective lentiviral vector for siRNA suppression of agrin expression. We have infected hippocampal neurons with this vector during the period of rapid synapse formation and shown that suppression of agrin expression results in decreased numbers of filopodia as well as decreased formation of synapes on neuronal dendrites. These results suggest that agrin positively regulates synapse formation, possibly through its effects on dendritic filopodia, which are believed to be involved in synaptogenesis.