The recently discovered pathway whereby axoplasmic proteins in Aplysia neurons are transported retrogradely through the axon to the cell body and then into the nucleus provides a unique connection between the axonal periphery ad the protein synthesizing machinery in the cell body. We hypothesize that this pathway provides a vital link in the process of neuronal plasticity in which axons and terminals undergo structural changes in response to injury or environmental clues. The long-term goals of this project are to explore the mechanism and functions of the retrograde transport/nuclear import pathway. Our immediate focus is on injury and we have evidence that one of the proteins that uses this pathway is an injury signal. Access to both the retrograde transport and nuclear import processes is via a short sequence of basic amino acids comprising a signal peptide (sp). An antibody to the sp recognizes several proteins in both the axoplasm and nucleus of Aplysia neurons. The most abundant of these, sp83, is a glycoprotein that contains single O-linked N-Acetylglucosamine moieties. Sp83 accumulates behind a ligation on peripheral nerves and may be constituitively transported to the nucleus. Another protein, Sp97, is retrogradely transported after axon crush and behaves like an injury signal. Both proteins exist in soluble and membrane-associated forms in the axon. We will use affinity chromatography to purify both proteins from the Aplysia nervous system and will obtain a partial sequence to generate polyclonal antibodies. The antibodies are used to determine the distribution of sp83 and sp97 in the nervous system and as specific probes of t heir function. To study transport/import, the purified proteins are injected directly into axons of Aplysia neurons growing in vitro and their fat in the cell is monitored by confocal microscopy. We will also test the hypotheses that it is the membrane-associated form of the protein that is transported and that the transition from soluble to membrane-associated is regulated by phosphorylation. In addition, the possibility that the sp- proteins are retrogradely transported on the surface of vesicles will be examined by EM immunocytochemistry, as will the possible role of dynein as the retrograde motor. Two proteins (SPRs) in axoplasm recognize the sp and may couple sp-proteins to both the transport and import machinery. This idea is tested by purifying the SPRs and injecting them into the axon and cell body to see if they enter the nucleus. Aplysia sensory neurons undergo changes in their electrical properties after injury. We will see if injecting sp97 into noninjured sensory neurons can elicit these changes. Interestingly, the giant neuron R2 has a different response to injury, however. Sp97 will be injected into this cell to test the idea that unique neurons have unique injury signals. A similar series of experiments are carried out to see if sp83 regulates axon growth in vitro and in vivo. If we are correct, that the transport/import pathway in neurons conveys signals to the nucleus that regulate growth and plasticity, it would have profound affects on our understanding of how peripheral events influence the neuronal phenotype. Moreover, identification of an injury signal would have important implications for therapeutic intervention after nerve injury.
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