The ultimate goals of this work are to gain insights into the molecular mechanisms that regulate synapse formation and function. As synapses are the structures that enable neuronal communication, information' regarding molecules involved in the regulation of these processes may have a fundamental significance for the understanding of higher brain functions such as behavior, learning and memory. It is also possible that defective regulation, or the introduction of mutations in these molecules, may lead to aberrant functioning of the brain, as in the case of mental health disorders, or may initiate neurodegenerative processes similar to those observed in Alzheimer's disease. This proposal focuses on agrin, a molecule that participates in the differentiation of developing and regenerating neuromuscular junctions (nmj). Since many agrin isoforms have been described, and since agrin expression has been detected in various tissues including the brain, I am interested in the following two aspects: first, to study the developmental onset of agrin expression along with the tissue specificity of the known isoforms; and second, to determine the physiological role of agrin.
The first aim will be investigated by different approaches, using a series of molecular biological techniques. Agrin gene expression will be examined by Northern blotting using RNAs obtained from various tissues at different developmental stages. in order to identify and localize the RNA transcripts of the individual agrin-isoform expressed, PCR and in situ hybridizations techniques will be used. The analyses of the expression and localization of the agrin-isoform polypeptides will be performed by Western blotting and immunohistochemical techniques. For this purpose, we will produce polyclonal antisera using synthetic peptides specific for each agrin isoform. The results of these experiments may reveal whether agrin-isoform expression correlates with particular molecular, morphological or physiological changes that occur during nmj differentiation, or if agrin isoforms participate in the differentiation of other types of synapses. This correlation will be suggestive of additional agrin functions. To investigate the physiological role of agrin, we are creating two sets of agrin mutant mice by homologous recombination techniques. The first mutation suppresses agrin expression, and the second prevents the formation of some agrin isoforms. Depending on the phenotype of these mutations, it may be possible to analyze the developmental and functional relevance of agrin and agrin-isoforms in synaptogenesis. It is also possible that these mutated mice will exhibit behavioral abnormalities or develop neurodegenerative phenotypes.
