Characterization of Ribosomal Plaque Domains in Axons

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The axon serves as a long line of communication that makes up the 'hard wiring' of the nervous system. Its long term maintenance and potential for structural plasticity depends on a continuous supply of proteins. It was generally assumed that all axoplasmic proteins were synthesized in the cell body and transported at slow rates to the axon. The reasoning was based on an apparent lack of axoplasmic ribosomes, the ultrastructural correlates of a protein synthesizing machinery, despite metabolic and biochemical evidence to the contrary. Recently, periodic small discrete ribosome-containing domains were documented in the periphery of axoplasm isolated from myelinated axons. The research proposal is concerned with testing the hypothesis that "periaxoplasmic ribosomal domains" serve as centers of protein synthesis, which supply immediate surrounding regions with essential proteins, such as actin. Experiments will be performed in the Mauthner neuron, which is a large identifiable cell in the goldfish brain stem that projects its myelinated axon along the full length of spinal cord. They will determine (1) whether a cDNA construct that codes for a fusion protein, comprised of actin and green fluorescent protein (GFP), in which the latter serves as a reporter, will result in the transport of the mRNA from the cell body to the axon, (2) localization of the mRNA in periaxoplasmic ribosomal domains and (3) synthesis and subsequent distribution of the fusion protein product to surrounding axoplasm. Additional experiments will test the hypothesis in rabbit myelinated motor axons, in which sites of initial metabolic uptake of [35S]methionine along axons will be analyzed and related to the localization of periaxoplasmic ribosomal domains. Subsequent time-dependent distribution of radioactive proteins to surrounding axoplasm will be mapped, and analysis of radiolabeled proteins will also be undertaken. A complementary issue is whether any membrane proteins are also synthesized in the axon compartment, for which signal recognition particles (SRPs) would be required. Experiments are proposed to analyze (1) for the occurrence of SRPs, (2) for the presence and localization of SNAP-25 mRNA, which codes for a membrane protein involved in transmitter release, and (3) for glycosylation, a process of cotranslationally adding sugars to newly synthesized membrane proteins. Specifically, the experiments will address an important question as to whether periaxoplasmic ribosomal domains are metabolically active in synthesizing proteins for local utilization in the axon compartment. More generally, however, the findings will add substantially to a better fundamental understanding of the complex biology of the axon.