Organization and translation of mRNA in sympathetic axons

Abstract

Many axons carry out the synthesis of macromolecules independent of their cell bodies but the nature, organization and magnitude of axonal protein synthesis remain unclear. We 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 all axons. The specific transcripts for β-actin and actin-depolymerizing factor (ADF) were also present and non-uniformly distributed in axons, with an approximately hundredfold higher concentration in growth cones, branch points and axonal varicosities than in the axon shaft. Immunoprecipitation using specific antibodies indicates that β-actin, ADF and neurofilament protein (NF) are translated in axons independently of cell bodies. Quantification of the distribution of β-actin and ADF mRNAs showed that their ability to enter 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 35S 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. Because axons in our culture conditions contain ∼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.