Association of poly(A) mRNA with microtubules in cultured neurons.
ABSTRACT The structural basis for the synthesis of specific proteins within distinct intraneuronal compartments is unknown. We studied the distribution of poly(A) mRNA within cultured cerebrocortical neurons using high resolution in situ hybridization to identify cytoskeletal components that may anchor mRNA. After 1 day in culture, poly(A) mRNA was distributed throughout all of the initial neurites, including the axon-like process. At 4 days in culture, poly(A) mRNA was distributed throughout the cell body and dendritic processes, but confined to the proximal segment of the axon. Poly(A) mRNA was bound to the cytoskeleton as demonstrated by resistance to detergent extraction. Perturbation of microtubules with colchicine resulted in a major reduction of dendritic poly(A) mRNA; however, this distribution was unaffected by cytochalasin. Ultrastructural in situ hybridization revealed that poly(A) mRNA and associated ribosomes were excluded from tightly bundled microtubules.
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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. DOI:10.1261/rna.2386111 · 4.62 Impact Factor
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ABSTRACT: Protein synthesis-dependent late-long term potentiation (L-LTP) is an enduring form of synaptic plasticity that has been shown to rely on, at least partly, protein synthesis at synaptic and/or dendritic sites. Evidence suggests that somatic transcription of new mRNAs may provide a significant contribution to the availability of mRNAs at synaptic sites where they are made available for dendritic translation. Transport of mRNAs from somatic to dendritic sites might be expected to involve movement along a microtubule network. In this study we examined whether it was possible to maintain L-LTP in hippocampal slices with destabilized microtubule networks. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from rat hippocampal slices and following a period of baseline recording, stimuli were given that induced LTP. LTP was monitored for 5 h in both control slices and slices treated with vincristine to depolymerize tubulin. L-LTP was induced and maintained in vincristine-treated slices. Four hours after tetanic stimulation fEPSPs were 196+/-19% of baseline values. The magnitude of potentiation was similar to that seen in untreated slices (175+/-15%). L-LTP in vincristine-treated slices was, however, not maintained in the presence of the protein synthesis inhibitor, rapamycin. Immunohistochemistry and confocal microscopy of vincristine-treated slices verified that the microtubule network had been destabilized. Communication between somatic and synaptic sites through protein and/or mRNA trafficking via an intact microtubule network is not required for protein synthesis dependent L-LTP.British Journal of Pharmacology 09/2007; 151(7):1071-7. DOI:10.1038/sj.bjp.0707314 · 4.99 Impact Factor