Tumor protein Tctp regulates axon development in the embryonic visual system.

The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.This work was supported by Fundação para a Ciência e Tecnologia (C.G.R.; fellowship SFRH/BD/33891/2009), Sir Edward Youde Memorial Fund, Croucher Foundation, Cambridge Commonwealth–European & International Trust (H.W.), Gates Cambridge Scholarship (J.Q.L.), and a Wellcome Trust Programme Grant (C.E.H.; grant 085314/Z/08/Z).This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.13106

A Protocol for Single-Molecule Translation Imaging in Xenopus Retinal Ganglion Cells

Single-molecule translation imaging (SMTI) is a straightforward technique for the direct quantification of local protein synthesis. The protein of interest is fused to a fast-folding and fast-bleaching fluorescent protein, allowing one to monitor the appearance of individual fluorescence events after photobleaching of pre-existing proteins in the cell under investigation. The translation of individual molecules is then indicated by photon bursts of sub-second length that appear over a dark background. The method thus shares attributes with fluorescence recovery after photobleaching (FRAP) microscopy. Resulting datasets are similar to those generated by localization-based super-resolution microscopy techniques and can be used both to generate density maps of local protein production and to quantify the kinetics of local synthesis. The detailed protocol described in this chapter uses a Venus-β-actin fusion construct to visualize and measure the β-actin mRNA translational activity in Xenopus retinal ganglion cell growth cones upon Netrin-1 stimulation, which can be readily adapted for detecting translation events of other mRNAs in various cell types

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that Cytoplasmic FMR1-Interacting Proteins (CYFIPs) fulfil non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2’s function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics and optic tract sorting.This work was supported by Wellcome Trust Programme Grants ( 085314 , C.E.H.), Wellcome Trust Investigator Award (SIA 100329/Z/12/Z , W.A.H.), European Research Council Advanced Grant ( 322817 , C.E.H.), EMBO Long Term Fellowship ( ALTF849-2013 , J.-M.C.), Cambridge Trust, Croucher Foundation, and Sir Edward Youde Memorial Fund (H.H.W.)

Cue-Polarized Transport of β-actin mRNA Depends on 3′UTR and Microtubules in Live Growth Cones

Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4–7 min window. This polarized mRNA trafficking depended on the 3′ untranslated region (UTR) since mRNA-Δ3′UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated “worm-like” trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3′UTR is essential for this cue-induced regulation

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons.

Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.This work was supported by Wellcome Trust Grants (085314/Z/08/Z and 203249/Z/16/Z) to C.E.H. and (100329/Z/12/Z) to W.A.H., European Research Council Advanced Grant (322817) to C.E.H., Champalimaud Vision Award to C.E.H. and by the Netherlands Organization for Scientific Research (NWO Rubicon 019.161LW.033) to M.K. CFK acknowledges funding from the UK Engineering and Physical Sciences Research Council, EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council, MRC (grants MR/K015850/1 and MR/K02292X/1) and Infinitus (China) Ltd

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring

Cdk5 Phosphorylates a Component of the HDAC Complex and Regulates Histone Acetylation during Neuronal Cell Death

Cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is critical for regulating neural development and neuronal survival. Dysregulation of Cdk5 is associated with abnormal expression of cell cycle-related proteins during neuronal apoptosis. We have previously found that p35, a Cdk5 activator, interacts with mSds3, an integral component of the histone deacetylase complex in vitro, suggesting a functional role of Cdk5 in gene regulation through modulation of chromatin integrity. In this study, we further demonstrate that Cdk5-dependent phosphorylation of mSds3 at Ser228 occurs in mouse brain nuclei. The expression of mSds3 protein and its interaction with Cdk5 activators is developmentally regulated in the mouse brain. Importantly, our findings suggest that the ability of Cdk5 to regulate activity deprivation-induced apoptosis of cerebellar granule neurons is likely mediated by the regulation of histone acetylation. Suppression of Cdk5 not only attenuates the induction of histone H3 acetylation and the aberrant upregulation of cyclin proteins in neurons after activity deprivation, but also results in protection of neurons against apoptotic cell death. Taken together, our findings suggest that Cdk5 regulates neuronal survival by precise epigenetic control through modulation of histone acetylation

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring

Cue-Polarized Transport of β-actin mRNA Depends on 3'UTR and Microtubules in Live Growth Cones.

Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4-7 min window. This polarized mRNA trafficking depended on the 3' untranslated region (UTR) since mRNA-Δ3'UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated "worm-like" trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3'UTR is essential for this cue-induced regulation