Subcellular Compartmentilization of Neuronal Gene Expres

National Institute Of Mental Health

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Abstract

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. 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 testing working hypotheses generated empiracally from these invertebrate studies in the mammalian nervous system. This work employs rat primary sympathetic neurons cultured in the Campenot compartimentalized microchamber system. Preliminary data indicate that, like the squid, mammalian axons and presynaptic nerve terminals contain a heterogeneous population of mRNAs and that the local protein synthetic system plays a key role in the function and viability of the local mitochondrial population. We hope that these investigations will augment our understanding of the molecular mechanisms that play a key role in neuronal development, regeneration, and plasticity.