Molecular mechanisms behind mRNA localization in axons.

Messenger RNA (mRNA) localization allows spatiotemporal regulation of the proteome at the subcellular level. This is observed in the axons of neurons, where mRNA localization is involved in regulating neuronal development and function by orchestrating rapid adaptive responses to extracellular cues and the maintenance of axonal homeostasis through local translation. Here, we provide an overview of the key findings that have broadened our knowledge regarding how specific mRNAs are trafficked and localize to axons. In particular, we review transcriptomic studies investigating mRNA content in axons and the molecular principles underpinning how these mRNAs arrived there, including cis-acting mRNA sequences and trans-acting proteins playing a role. Further, we discuss evidence that links defective axonal mRNA localization and pathological outcomes

Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro.

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology.This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.12385

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

Dynamic Axonal Translation in Developing and Mature Visual Circuits.

Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.This work was supported by Wellcome Trust Programme Grant (085314/Z/08/Z), European Research Council Advanced Grant (322817) to CEH , Cambridge Wellcome Trust PhD programme in Developmental Biology (PMAG/406; BT-B), Gates Cambridge Scholarship (JQL), Basic Science Research Program (2013R1A1A1009625 & 2014K2A7A1036305), Biomedical Technology Development Program (2013M3A9D5072551), & Brain Research Program (2015M3C7A1028396) funded through the NRF by the Korean government (MSIP), Yonsei University Future-leading Research Initiative of 2015 (2015-22-0095), and a faculty research grant from Yonsei University College of Medicine for 2013 (6-2013-0064-2-1) to HJ.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Cell Press

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local 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

Towards understanding the mechanisms behind mRNA localisation in growing axons

During embryonic nervous system assembly, mRNA localisation is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localisation patterns across the axon. Little is known, however, about how mRNAs move in axons, or how these patterns are generated. Here, I develop a method for live single molecule imaging of identified endogenous mRNA within Xenopus laevis retinal ganglion cell axons using molecular beacon technology coupled with highly inclined and laminated optical sheet (HILO) microscopy. By combining quantitative single molecule imaging with biophysical motion models, I show that β-actin mRNA travels mainly as single copies, and exhibits different motion type frequencies in different axonal sub-compartments. I find that β-actin mRNA density is four-fold enriched in the growth cone central domain compared to the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modelling it is further demonstrated that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone. Distinctive features within the mRNA have been shown to drive localisation and subsequently translation at specified subcellular regions through recognition by different RNA binding proteins. I employed fluorescent recovery after photobleaching (FRAP) in a screen to investigate whether short sequence motifs that were common to axonally translated mRNAs are sufficient to promote axonal translation. I further use this technique to show that the 5’ untranslated regions (UTRs) of two alternative isoforms of the Acot7 gene generate stark differences in local translation within growing axons. Through in situ hybridization, I demonstrate that these translational profiles result from differences in axonal mRNA localisation. Finally, I probe the relative structural and sequence requirements necessary for 5’UTR mediated axonal Acot7 mRNA localisation. Together, these results provide insight into the intrinsic mRNA features necessary for axonal localisation and the subsequent trafficking mechanisms that drive mRNA enrichment to the axon tip of neurons during development.Wellcome Trust PhD Programme in Developmental Biolog

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

Single-molecule analysis of endogenous β-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons.

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that β-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that β-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments