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ABSTRACT: 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 of Xenopus 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.
Cell 02/2012; 148(4):752-64. · 32.40 Impact Factor
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ABSTRACT: During axon pathfinding, growth cones commonly show changes in sensitivity to guidance cues that follow a cell-intrinsic timetable. The cellular timer mechanisms that regulate such changes are, however, poorly understood. Here we have investigated microRNAs (miRNAs) in the timing control of sensitivity to the semaphorin Sema3A in Xenopus laevis retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant neuropilin-1 (NRP1) receptor expression and caused cell-autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was newly identified as a target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 is important in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that miRNAs may act broadly as linear timers in vertebrate neuronal development.
Nature Neuroscience 12/2011; 15(1):29-38. · 15.53 Impact Factor
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ABSTRACT: Precise navigation of axons to their targets is critical for establishing proper neuronal networks during development. Axon elongation, whereby axons extend far beyond the site of initiation to reach their target cells, is an essential step in this process, but the precise molecular pathways that regulate axon growth remain uncharacterized. Here we show that 14-3-3/14-3-3ς proteins-adaptor proteins that modulate diverse cellular processes including cytoskeletal dynamics-play a critical role in Xenopus retinal ganglion cell (RGC) axon elongation in vivo and in vitro. We have identified the expression of 14-3-3/14-3-3ς transcripts and proteins in retinal growth cones, with higher levels of expression occurring during the phase of rapid pathway extension. Competitive inhibition of 14-3-3/14-3-3ς by expression of a genetically encoded peptide, R18, in RGCs resulted in a marked decrease in the length of the initial retinotectal projection in vivo and a corresponding decrease in axon elongation rate in vitro (30-40%). Furthermore, 14-3-3/14-3-3ς (R1) co-localized with Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) in RGC growth cones. Inhibition of 14-3-3/14-3-3ς function with either R18 or morpholinos reduced the level of inactive pXAC and increased the sensitivity to collapse by the repulsive cue, Slit2. Collectively, these results demonstrate that14-3-3/14-3-3ς participates in the regulation of retinal axon elongation, in part by modulating XAC activity.
Developmental Neurobiology 07/2011; 72(4):600-14. · 3.55 Impact Factor
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ABSTRACT: 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.
RNA 01/2011; 17(1):85-98. · 5.09 Impact Factor
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ABSTRACT: 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 the mRNA 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.
Journal of Neuroscience 11/2010; 30(46):15464-78. · 7.11 Impact Factor
