Synthesis of β-Tubulin, Actin, and Other Proteins in Axons of Sympathetic Neurons in Compartmented Cultures

Abstract

The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [35S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [35S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43-55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and beta-tubulin. Axons synthesized <1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75-85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.

Progressive Motor Neuronopathy: A Critical Role of the Tubulin Chaperone TBCE in Axonal Tubulin Routing from the Golgi Apparatus

“…In neurons, tubulins are mainly synthesized in the cell body (Eng et al, 1999), and most of them are rapidly routed into the axon (Campenot et al, 1996). Our in vivo observations in pmn mice showed that microtubule loss starts in the distal axon and progresses retrogradely.…”

“…Studies in cultured neurons have shown that axonal tubulins are mainly synthesized in the cell soma (Eng et al, 1999) and transported into the distal axon within 2 d after synthesis (Campenot et al, 1996). Metabolic labeling experiments in compartmentalized cultures had provided evidence that this process depends on the Golgi apparatus (Campenot et al, 2003).…”

How does an axon grow?

“…Ribosomes and mRNAs have been localized to mammalian axons, suggesting at least a possible role for axonal translation (Tennyson 1970;Bassell et al 1998; Koenig et al 2000), as had been found previ-ously for dendrites (for review, see Job and Eberwine 2001). Furthermore, severed axons locally synthesize such axonal structural building blocks as neurofilaments, tubulin, and actin (Eng et al 1999;Zheng et al 2001). Intriguingly, axonal localization may be specific for axon growth-related processes-only ␤-actin mRNA is enriched in neurites and growth cones, corresponding to ␤-actin protein's localization to distal growth cones and filipodia of actively growing neurites in vitro (Bassell et al 1998) and in vivo (Weinberger et al 1996).…”

Functional and Selective RNA Interference in Developing Axons and Growth Cones

“…Axons and growth cones contain polyadenylated mRNAs and ribosomes (Kleiman et al, 1994;Olink-Coux and Hollenbeck, 1996;Steward, 2002), and isolated axons are capable of translating endogenous (Eng et al, 1999) and heterologous (Brittis et al, 2002) mRNAs into proteins. mRNAs encoding proteins such as ␤-actin and RhoA have been identified in developing axons (Olink-Coux and Hollenbeck, 1996;Bassell et al, 1998;Wu et al, 2005).…”