SummaryLocal translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

SummaryNascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo β-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal β-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.

The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons.

SummaryRibosome 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.

Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry.

SummaryThe 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) fulfill 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.

These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.

2!SUMMARY Ribosomes are known to be assembled in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Using subcellular proteomics and live-imaging, we show that locally synthesized RPs incorporate into axonal ribosomes in a nucleolus-independent fashion. We revealed that axonal RP translation is regulated through a novel sequence motif, CUIC, that forms a RNA-loop structure in the region immediately upstream of the initiation codon. Inhibition of axonal CUICregulated RP translation leads to defects in local translation activity and axon branching, demonstrating the physiological relevance of the axonal ribosome remodeling. These results indicate that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus. INTRODUCTIONRNA localization and local translation play key roles in the assembly and maintenance of neuronal connections (Campbell and Holt, 2001;Holt and Schuman, 2013;Wu et al., 2005).Recent genome-wide studies on the axonal transcriptome revealed that thousands of mRNAs are localized to the axon. A consistent but unexpected finding of these studies is the robust enrichment of mRNAs that encode ribosomal proteins (RPs), protein components of ribosomes. Axons are long neuronal processes that carry out many vital specific cellular functions far from their cell bodies, including translation, and must therefore maintain their protein synthetic machinery in good order. However, because most eukaryotic ribosome assembly is well known to occur in the nucleolus (Fromont-Racine et al., 2003;Lastick and McConkey, 1976), the physiological function of RP-coding mRNAs in a neuronal subcellular compartment far distant from the nucleus was enigmatic. RP-coding mRNAs have been abundantly detected in axons of a variety of neuron types, such as retinal ganglion cells (RGCs) (Zivraj et al., 2010), sympathetic neurons (Andreassi et al., 2010) and motor ! 5!In this study, we explore intra-ribosomal roles of axonally synthesized RPs using a range of technical approaches including live imaging, in vivo gene knockdown, bioinformatics, nascent protein labeling and mass spectrometry-based proteomics. We found that axonal translation of RPs coordinately peaks at the axon branching stage in RGCs in vivo, and their translation is regulated by a branch-promoting factor, Netrin-1, through a novel loop structure-forming sequence motif, CUIC, that is shared by ~70% of RP-coding mRNAs. Using nascent protein labeling and proteomic mass spectrometry analysis on ribosomes isolated from pure axons, together with live-imaging approaches, we show that axonally synthesized RPs are physically incorporated into axonal ribosomes in a nucleolus-independent fashion. Furthermore, we demonstrate the physiological importance of the axonal ribosome remodeling by showing that inhibition of axonal RP translation leads to a significant decrease in the level of axonal mRNA translation and s...