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.

Trafficking Guidance Receptors

“…It is currently unknown whether guidance cues or receptors are secreted from Golgi outposts. The possibility that axons also contain some secretory compartments and might synthesize membrane proteins locally is currently only supported by scarce data, but future work will bring more light to this tantalizing prospect (Hengst and Jaffrey 2007;Lee and Hollenbeck 2003;Merianda et al 2009;Willis et al 2005;Yao et al 2006). Recent data suggest that the transport pathways and machinery for axonal and dendritic cargos are distinct.…”

Differential Transport and Local Translation of Cytoskeletal, Injury-Response, and Neurodegeneration Protein mRNAs in Axons

“…Evidence for protein synthesis in axons was initially provided from studies in invertebrates (Capano et al, 1987;Giuditta et al, 1991;Davis et al, 1992;Chun et al, 1996;van Minnen et al, 1997). Subsequent work showed that developing vertebrate axons contain mRNAs and actively synthesize proteins (Olink-Coux and Hollenbeck, 1996;Bassell et al, 1998;Eng et al, 1999;Zhang et al, 1999;Aronov et al, 2001;Campbell and Holt, 2001; Lee and Hollenbeck, 2003). Several lines of evidence indicate that mammals retain the capacity for axonal protein synthesis as they mature (Koenig et al, 2000;Zheng et al, 2001;Hanz et al, 2003).…”

“…Lee and Hollenbeck (2003) recently estimated that axonal protein synthesis represents 5% of total neuronal protein synthesis. At present, the list of proteins locally synthesized in vertebrate axons is limited to ␤-actin, ␣-tubulin, low molecular weight neurofilament, Tau, actindepolymerizing factor (ADF), EphA2, and Importin ␤1 (OlinkCoux and Hollenbeck, 1996;Bassell et al, 1998;Eng et al, 1999;Zheng et al, 2001;Hanz et al, 2003;Lee and Hollenbeck, 2003). Including mRNAs identified in vertebrate axons, such as neurohormones and other neurofilament subunits, approximately doubles the list of proteins that are potentially synthesized in axons (Piper and Holt, 2004).…”

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

“…The first one relies on restricted localization of mRNAs that encode cytosolic proteins, allowing local protein translation, thus creating differential protein activity and generating cell asymmetry and polarity (Bashirullah et al, 1998). This is, for instance, the case of actin mRNA localization in the nerve terminal of neurons (Lee and Hollenbeck, 2003), oskar localization at the posterior pole of Drosophila oocytes (Ephrussi et al, 1991), and ash1 localization in dividing yeast cells (Long et al, 1997). On the other hand, the mechanism ensuring the polarized deposition of transmembrane or secreted proteins is thought, in mammalian cells, to be achieved by posttranslational sorting events in the trans-Golgi network (TGN).…”