Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs

Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of "young" (pathfinding) versus "old" (target-arriving) Xenopus growth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that them RNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal. Copyright © 2010 the authors

Functional Complexity of the Axonal Growth Cone: A Proteomic Analysis

The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions

The expression and activity of β-catenin in the thalamus and its projections to the cerebral cortex in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>The mammalian thalamus relays sensory information from the periphery to the cerebral cortex for cognitive processing via the thalamocortical tract. The thalamocortical tract forms during embryonic development controlled by mechanisms that are not fully understood. β-catenin is a nuclear and cytosolic protein that transduces signals from secreted signaling molecules to regulate both cell motility via the cytoskeleton and gene expression in the nucleus. In this study we tested whether β-catenin is likely to play a role in thalamocortical connectivity by examining its expression and activity in developing thalamic neurons and their axons.</p> <p>Results</p> <p>At embryonic day (E)15.5, the time when thalamocortical axonal projections are forming, we found that the thalamus is a site of particularly high β-catenin mRNA and protein expression. As well as being expressed at high levels in thalamic cell bodies, β-catenin protein is enriched in the axons and growth cones of thalamic axons and its growth cone concentration is sensitive to Netrin-1. Using mice carrying the β-catenin reporter <it>BAT-gal </it>we find high levels of reporter activity in the thalamus. Further, Netrin-1 induces <it>BAT-gal </it>reporter expression and upregulates levels of endogenous transcripts encoding β-actin and L1 proteins in cultured thalamic cells. We found that β-catenin mRNA is enriched in thalamic axons and its 3'UTR is phylogenetically conserved and is able to direct heterologous mRNAs along the thalamic axon, where they can be translated.</p> <p>Conclusion</p> <p>We provide evidence that β-catenin protein is likely to be an important player in thalamocortcial development. It is abundant both in the nucleus and in the growth cones of post-mitotic thalamic cells during the development of thalamocortical connectivity and β-catenin mRNA is targeted to thalamic axons and growth cones where it could potentially be translated. β-catenin is involved in transducing the Netrin-1 signal to thalamic cells suggesting a mechanism by which Netrin-1 guides thalamocortical development.</p

Remote control of gene function by local translation

The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function

The RNA-binding protein MARTA2 regulates dendritic targeting of MAP2 mRNAs in rat neurons.

Dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2) in neurons involves a cis-acting dendritic targeting element. Two rat brain proteins, MARTA1 and MARTA2, bind to the cis-element with both high affinity and specificity. In this study, affinity-purified MARTA2 was identified as orthologue of human far-upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant-negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule based motor KIF5 and inhibition of KIF5 but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans-acting factor involved in KIF5c-mediated dendritic targeting of MAP2 mRNAs

Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization

mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development

Intra-axonal Synthesis of SNAP25 Is Required for the Formation of Presynaptic Terminals

Localized protein synthesis is a mechanism for developing axons to react acutely and in a spatially restricted manner to extracellular signals. As such, it is important for many aspects of axonal development, but its role in the formation of presynapses remains poorly understood. We found that the induced assembly of presynaptic terminals required local protein synthesis. Newly synthesized proteins were detectable at nascent presynapses within 15 min of inducing synapse formation in isolated axons. The transcript for the t-SNARE protein SNAP25, which is required for the fusion of synaptic vesicles with the plasma membrane, was recruited to presynaptic sites and locally translated. Inhibition of intra-axonal SNAP25 synthesis affected the clustering of SNAP25 and other presynaptic proteins and interfered with the release of synaptic vesicles from presynaptic sites. This study reveals a critical role for the axonal synthesis of SNAP25 in the assembly of presynaptic terminals.Portuguese Foundation for Science and Technology (FCT) in the context of the FCT funded University of Minho MD/PhD Program (SFRH/BD/52322/2013). J.C.M. was supported by the National Institutes of Health (F31GM116617). U.H. was supported by the NIH (R01MH096702), the BrightFocus Foundation (A2015093S), and the Irma T. Hirschl Trus

Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

Local protein synthesis plays a key role in regulating stimulus-induced responses in dendrites and axons. Recent genome-wide studies have revealed that thousands of different transcripts reside in these distal neuronal compartments, but identifying those with functionally significant roles presents a challenge. We performed an unbiased screen to look for stimulus-induced, protein synthesis-dependent changes in the proteome ofXenopus retinal ganglion cell (RGC) axons. The intermediate filament protein lamin B2 (LB2), normally associated with the nuclear membrane, was identified as an unexpected major target. Axonal ribosome immunoprecipitation confirmed translation of lb2 mRNA in vivo. Inhibition of lb2 mRNA translation in axons in vivo does not affect guidance but causes axonal degeneration. Axonal LB2 associates with mitochondria, and LB2-deficient axons exhibit mitochondrial dysfunction and defects in axonal transport. Our results thus suggest that axonally synthesized lamin B plays a crucial role in axon maintenance by promoting mitochondrial function