AGRIN GENE EXPRESSION

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Identification of the mechanisms that underlie the formation of chemical synapses during development, and their modification in the adult, is of critical importance to understanding nervous system function. Much of what is known about theses processes comes from studies of the neuromuscular junction, where it is believed that the protein agrin, supplied by motor neurons, directs the formation and maintenance of the postsynaptic apparatus. It has recently been established that many neurons in the adult rat CNS also express agrin, raising the possibility that agrin plays a similar role in synapse formation between neurons as it does at the neuromuscular junction. The aim of experiments proposed here is to test the hypothesis that agrin direct the organization and maintenance of neuronal synapses in the CNS. In situ hybridization and polymerase chain- reaction (PCR) techniques will be used to determine the level of agrin mRNA expression in different brain regions during development and the results correlated with the time course of synapse formation. Four alternatively spliced agrin mRNAs are expressed in the brain that encode proteins that differ in their ability to induce postsynaptic specializations in cultured muscle fibers. Since alternative splicing represents a mechanism whereby the levels of agrin proteins with different functional properties might be regulated, changes in the pattern of agrin RNA splicing during development will also be monitored. To determine the cellular origin of specific agrin isoforms, a single cell PCR technique will be employed that allows the agrin mRNA profile of an identified cell to be established and, in the case of a neuron, correlated with its neurotransmitter phenotype. Agrin's location at the neuromuscularjunction was a critical piece of evidence pointing to agrin's role in neuromuscular synaptogenesis. Therefore, in parallel with studies of agrin mRNA, the distribution of agrin protein in the brain and its localization at synapses will be determined, using immunohistochemical and electron microscopy techniques. As a direct test of agrin's role in neuronal synapse formation, the ability of antiagrin antibodies to block organization of neurotransmitter receptors at synapses formed in cell culture between CNS neurons will be examined. Finally, preliminary studies have shown that epileptiform seizures induce marked changes in the levels of agrin mRNA in the brain, suggesting that agrin gene expression may be regulated by synaptic activity. This study will be extended to include examination of the time course of seizure induced changes in agrin mRNA levels and alternative splicing. The experiments outlined here are the first to examine the role of agrin and the regulation of agrin gene expression in the CNS. They will provide insights into the mechanisms of synapse formation between neurons and the cellular changes associated with epilepsy and synaptic plasticity. They can be expected therefore to impact upon the treatment and prevention of epilepsy, and disorders of learning and memory associated with such conditions as Down syndrome and Alzheimer disease.