Axons and nerve terminals are unique subcellular structures of the neuron that play a critical role in the development and maintenance of neural connectivity. One of the central tenets in neuroscience is that the protein constituents of these distal neuronal compartments are synthesized in the nerve cell body and subsequently transported to their ultimate sites of function. Hence, the structure and function of these highly specialized distal domains of the neuron are totally dependent on slow anterograde axoplasmic transport. In contrast to this viewpoint, work in my laboratory focuses on the hypothesis that de novo protein synthesis occurs within microcompartments in the neuron to include the axon and presynaptic nerve terminal. Our studies employ the squid giant axon, which serves as a model invertebrate motor neuron system. Using this model, my colleagues and I have shown that the axon contains a heterogeneous population of approximately 100-200 different mRNAs. These mRNAs are full-length gene transcripts capable of synthesizing protein in a cell-free translation system. We have cloned and characterized several axonal mRNAs that encode B-actin, B-tubulin, spectrin, kinesin, MAP I, neurofilament protein, and enolase. In addition, we have identified several mRNAs that code for novel proteins. The axonal localization of these mRNA species was definitively demonstrated by in situ hybridization histochemistry, and the presence of these sequences in the polysome fraction was established by reverse transcription-PCR methodology. Using biochemical labeling experiments and electron spectroscopic phosphate imaging, we were also able to show that the giant axon contained biologically active polyribosomes. Concurrent with this work, we have demonstrated that protein synthesis occurs in the large presynaptic terminals of squid retinal photoreceptor neurons. This finding was obtained using cell-free translation analysis, high-resolution autoradiography, and electron spectroscopic imaging. Our most recent results suggest that the level of protein synthesis in these presynaptic terminals is effected by calcium ions and, hence, could be regulated by the activity of the terminal itself. Based upon the information gleaned from this invertebrate model system, we have postulated that key elements of the cytomatrix, molecular motors of the axon transport systems, and proteins involved in energy metabolism are locally synthesized in the distal structural and functional domains of the neuron. In the mature neuron, a local system of protein synthesis could contribute significantly to the maintenance and remodeling of axonal architecture, as well as the dynamic properties of the nerve terminal. This system might prove especially important in large asymmetric motor and sensory neurons, where the axon and terminal fields are far removed from the cell body. Currently, my colleagues and I are using differential mRNA display methodology to identify novel constituents of the axonal mRNA population, and we are beginning to explore the mechanisms involved in intracellular trafficking of axonal mRNAs. These latter studies will involve mRNA-protein binding assays, as well as deletion mutation analysis and microinjection of fluorescently labeled mRNAs into isolated squid giant axon preparations. We hope that these investigations will augment our understanding of the molecular mechanisms that play a key role in neuronal development, regeneration, and plasticity.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Intramural Research (Z01)
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U.S. National Institute of Mental Health
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