The form and physiology of the dendrites of nerve cells determine many aspects of neuronal function. Perhaps the most remarkable aspect of dendrites is their complexity; individual neurons of the CNS are contacted by tens of thousands of individual synapses, mostly upon dendrites, and each synaptic site must have the molecular machinery that Is appropriate for the particular synapse. Intradendritic transport of materials synthesized in the cell body must play a prominent role in controlling the formation of dendrites during development, in regulating their potential for modification in response to neuronal activity, and in determining their capacity for reinnervation following neural injury. An especially important aspect of dendrites that may contribute to the construction and maintenance of the many synaptic sites is the selective localization of polyribosomes beneath synapses. The mRNA and ribosomes comprising this machinery must be transported from the neuronal cell body. The paucity of information concerning dendritic transport of RNA represents a major gap in our understanding of these crucial aspects of dendritic function. The present project evaluates the mechanisms of dendritic transport of RNA in hippocampal neurons maintained in culture. Our previous studies have revealed that dendrites possess a system for the selective transport of recently synthesized RNA that is not present in axons. This transport occurs at a rate comparable to slow axonal transport, and the recently synthesized RNA that is transported is bound to the dendritic cytoskeleton. Preliminary studies suggest that there is a selectivity in mRNA routing in neurons such that some mRNAs are transported into dendrites, while others are restricted to the cell body. By evaluating the migration of recently synthesized glycoproteins, we also found that dendrites possess a rapid transport system. Moreover, we made the unexpected discovery that dendrites possess the machinery for local glycosylation of protein. The experiments of the present proposal follow-up on these observations. We will: 1) Evaluate the nature of the interaction between RNA that is transported into dendrites and the dendritic cytoskeleton; 2) Compare the dendritic transport of mRNA and mRNA; 3) Identify the types of mRNA that are transported into dendrites and the types that are retained in the soma; 4) Identify the molecular signals that determine which mRNAs are transported into dendrites and which are retained in the cell body; 5) Evaluate whether recently synthesized RNA accumulates at synaptic sites as would be predicted by the selective localization of polyribosomes beneath synapses: 6) Evaluate the partitioning of RNA between cell body and dendritic domains by studying dendritic transport in the hippocampal formation 7) Examine the extent of synthesis and glycosylation of proteins within dendrites; and 8) Identify the organelles responsible for protein glycosylation within dendrites. It is hoped that this research program will provide a complete picture of the mechanisms of mRNA sorting and transport in nerve cells, and reveal the relevance of local protein synthesis and posttranslational processing to the overall metabolic economy of the neuron.
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