Organization and translation of mRNA in sympathetic axons

Sunkyung Lee, Purdue University

Abstract

The synthesis of macromolecules in neurons has long been thought to be restricted to the cell body. Yet, neurons maintain axonal processes that reach considerable distance from cell body, and contain most of the neuron's mass. The mass transport of material from the cell body to the axon, termed slow axonal transport, supplies proteins to the axon, but whether slow axonal transport is sufficient to fulfill the whole protein requirement of the axon has recently been questioned, and the likelihood of mRNA localization and translation in the axon has also been suggested. There is some evidence that axons can carry out the synthesis of macromolecules independent of their cell bodies, but the nature, organization, and magnitude of axonal protein synthesis remain unclear. I have examined these features in axons of chick sympathetic neurons in cell culture. In situ hybridization showed that poly(A) mRNA is abundant and non-uniformly distributed in nearly 100% of axons. The specific transcripts for β-actin and actin depolymerizing factor (ADF) were also present and non-uniformly distributed in axons, with an approximately 100-fold higher concentration in growth cones, branch points, and axonal varicosities than in the axon shaft. Immunoprecipitation using specific antibody indicates that β-actin and ADF, as well as neurofilament protein (NF) are synthesized in axons without cell bodies. Quantification of the distribution of β-actin and ADF mRNA showed that their entry into the axon was likely to be a property of the neuron as a whole rather than of individual axons. To compare the distribution of axonally-translated protein to that of mRNA, we performed 35 S metabolic labeling with axons separated from their cell bodies. Axonally-synthesized proteins were distributed throughout the axons, and their synthesis was inhibited by cycloheximide but not by chloramphenicol. Proteins translated mainly or exclusively in axons or cell bodies were both detected by metabolic labeling. Axons separated from their cell bodies synthesized up to 5% as much protein in a 3-hour-period as did intact neurons. Since axons in our culture conditions contain approximately 50% of the non-nuclear volume of the neurons, we estimate that axoplasm of sympathetic neurons has a protein synthetic capacity per unit volume equal to 10% that of cell body cytoplasm.

Degree

Ph.D.

Advisors

Hollenbeck, Purdue University.

Subject Area

Neurology|Cellular biology

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