The rigorous evidence summarized above for local protein synthesis in dendrites contrasted strongly with the prevailing assumption that axons lack meaningful levels of translational machinery (Twiss and Fainzilber, 2009). Thus, despite increasing in vitro evidence from culture systems (Donnelly et al., 2010), considerable skepticism remained regarding the in vivo relevance of local protein synthesis in axons. Twiss and colleagues took up the challenge to address this issue in a transgenic mouse model for axonal localization of β-actin (Donnelly et al., 2011; Willis et al., 2011). The β-actin zipcode sequence is one of the most characterized cis-acting elements for axonal targeting of an mRNA (Patel et al., 2012). This element, a conserved bipartite sequence present at the beginning of the β-actin 3′UTR, interacts with the RNA-binding protein ZBP-1 to mediate axonal localization of β-actin (Welshhans and Bassell, 2011). Willis et al. (2011) linked a diffusion-limited GFP reporter with the 3′UTRs of β-actin or of the nonlocalizing γ-actin, and expressed the reporter constructs under control of an injury-activated neuronal promoter in transgenic mice. The GFP-3′UTR-β-actin mice revealed axonal localization of reporter mRNA in adult sensory neurons, and moreover, crush lesion of the nerve enhanced accumulation of the reporter mRNA in injured axons proximal to the lesion site, whereas in contrast GFP-3′-UTR-γ-actin mice did not show any axonal expression (Willis et al., 2011). Donnelly et al. (2011) then assessed endogenous β-actin transcript levels in the transgenic axons and showed that overexpression of GFP-3′UTR-β-actin, but not GFP-3′UTR-γ-actin, in adult sensory neurons caused a reduction in endogenous β-actin levels. The transgenic GFP-3′UTR-β-actin neurons had reduced axon regeneration after nerve injury, and this deficit could be rescued by ZBP1 transfection. A similar regeneration deficit was observed in ZBP+/− heterozygous mice, and the phenotype could be rescued by expression of exogenous ZBP1 (Donnelly et al., 2011). Thus, ZBP1 is required for optimal axon outgrowth following injury in adult sensory neurons, and increases in ZBP1 levels can enhance nerve regeneration. The transgenic β-actin 3′UTR was further found to compete in vivo with other ZBP1 cargo mRNAs such as GAP-43 (Yoo et al., 2013), indicating that the regeneration deficits in the transgenic animals were likely due to reduced axonal localization of other ZBP1 cargos, in addition to β-actin (Fig. 1). In further studies, Twiss and colleagues went on to show that axonal translation of β-actin mRNA primarily supports axon branching, while axonal translation of GAP-43 mRNA supports elongating growth in sensory neurons (Donnelly et al., 2013). Taken together, this series of publications provides strong evidence for local protein synthesis in adult sensory axons of transgenic mice.
Figure 1. Transgenic overexpression of an axon-localizing mRNA affects axonal regeneration (Reproduced with permission from Perry and Fainzilber, EMBO J 2011, 30, 4520–4522, based on the data of Donnelly et al., EMBO J 2011, 30, 4665–4677). A: ZBP1 is required for the axonal localization of β-actin, GAP-43, and likely additional mRNAs. B: Introduction of an exogenous mRNA that competes with endogenous transcripts for binding to ZBP1 reduces the supply of endogenous mRNAs to the axon and attenuates axon regeneration after injury. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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