Abstract
The importance of mRNA localization and localized protein synthesis to spatially modulate protein levels in distinct subcellular domains has increasingly been recognized in recent years. Axonal and dendritic processes of neurons represent separate functional domains of the cell that have shown the capacity to autonomously respond to extracellular stimuli through localized protein synthesis. With the vast distance often separating distal axons and dendrites from the neuronal cell body, these processes have provided an appealing and useful model system to study the mechanisms that drive mRNA localization and regulate localized mRNA translation. Here, we discuss the methodologies that have been used to isolate neuronal processes to purity, and provide an in-depth method for using a modified Boyden chamber to isolate axons from adult dorsal root ganglion neurons for analyses of axonal mRNA content. We further show how this method can be utilized to identify specific mRNAs whose transport into axons is altered in response to extracellular stimuli, providing a means to begin to understand how axonal protein synthesis contributes to the proper function of the neuron.
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Martin, K.C. and A. Ephrussi. (2009). mRNA localization: gene expression in the spatial dimension. Cell 136: 719 –30.
Steward, O. and W.B. Levy. (1982). Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus. J Neurosci 2: 284 –91.
Bramham, C.R. and D.G. Wells. (2007). Dendritic mRNA: transport, translation and function. Nat Rev Neurosci 8: 776 –89.
Lin, A.C. and C.E. Holt. (2008). Function and regulation of local axonal translation. Curr Opin Neurobiol 18: 60– 68.
Yoo, S., E.A. van Niekerk, T.T. Merianda, and J.L. Twiss. (2009). Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration. Exp Neurol 223: 19–27.
Holt, C.E. and S.L. Bullock. (2009). Subcellular mRNA localization in animal cells and why it matters. Science 326: 1212– 6.
Willis, D.E. and J.L. Twiss. (2010). Regulation of protein levels in subcellular domains through mRNA transport and localized translation. Mol Cell Proteomics in press 9: 952–62.
Ellis, L., F. Katz, and K.H. Pfenninger. (1985). Nerve growth cones isolated from fetal rat brain. J. Neurosci. 5: 1393 –1401.
Witzmann, F.A., R.J. Arnold, F. Bai, P. Hrncirova, M.W. Kimpel, Y.S. Mechref, W.J. McBride, M.V. Novotny, N.M. Pedrick, H.N. Ringham, and J.R. Simon. (2005). A proteomic survey of rat cerebral cortical synaptosomes. Proteomics 5: 2177 – 201.
Jimenez, C.R., M. Eyman, Z.S. Lavina, A. Gioio, K.W. Li, R.C. van der Schors, W.P. Geraerts, A. Giuditta, B.B. Kaplan, and J. van Minnen. (2002). Protein synthesis in synaptosomes: a proteomics analysis. J Neurochem 81: 735 – 44.
Matsumoto, M., M. Setou, and K. Inokuchi. (2007). Transcriptome analysis reveals the population of dendritic RNAs and their redistribution by neural activity. Neurosci Res 57: 411–23.
Williams, C., R. Mehrian Shai, Y. Wu, Y.H. Hsu, T. Sitzer, B. Spann, C. McCleary, Y. Mo, and C.A. Miller. (2009). Transcriptome analysis of synaptoneurosomes identifies neuroplasticity genes overexpressed in incipient Alzheimer’s disease. PLoS One 4: e4936.
Weiler, I.J. and W.T. Greenough. (1993). Metabotropic glutamate receptors trigger postsynaptic protein synthesis. Proc. Natl. Acad. Sci., USA 90: 7168–7171.
Klemmer, P., A.B. Smit, and K.W. Li. (2009). Proteomics analysis of immuno-precipitated synaptic protein complexes. J Proteomics 72: 82–90.
Schrimpf, S.P., V. Meskenaite, E. Brunner, D. Rutishauser, P. Walther, J. Eng, R. Aebersold, and P. Sonderegger. (2005). Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry. Proteomics 5: 2531– 41.
Cotrina, M.L., J.H. Lin, J.C. Lopez-Garcia, C.C. Naus, and M. Nedergaard. (2000). ATP-mediated glia signaling. J Neurosci 20: 2835– 44.
Slezak, M. and F.W. Pfrieger. (2003). New roles for astrocytes: regulation of CNS synaptogenesis. Trends Neurosci 26: 531–5.
Shea, T.B. (1994). Toxic and trophic effects of glial-derived factors on neuronal cultures. Neuroreport 5: 797–800.
Olink-Coux, M. and P.J. Hollenbeck. (1996). Localization and Active Transport of mRNA in Axons of Sympathetic Neurons in Culture. Journal of Neuroscience 16: 1346 –1358.
Moccia, R., D. Chen, V. Lyles, E. Kapuya, Y. E, S. Kalachikov, C.M. Spahn, J. Frank, E.R. Kandel, M. Barad, and K.C. Martin. (2003). An unbiased cDNA library prepared from isolated Aplysia sensory neuron processes is enriched for cytoskeletal and translational mRNAs. J Neurosci 23: 9409 –17.
Eng, H., K. Lund, and R.B. Campenot. (1999). Synthesis of beta-tubulin, actin, and other proteins in axons of sympathetic neurons in compartmented cultures. J Neurosci 19: 1–9.
Vogelaar, C.F., N.M. Gervasi, L.F. Gumy, D.J. Story, R. Raha-Chowdhury, K.M. Leung, C.E. Holt, and J.W. Fawcett. (2009). Axonal mRNAs: characterisation and role in the growth and regeneration of dorsal root ganglion axons and growth cones. Mol Cell Neurosci 42: 102–15.
Hillefors, M., A. Gioio, M. Mameza, and B. Kaplan. (2007). Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons. Cell Mol Neurobiol 27: 701–16.
Torre, E.R. and O. Steward. (1996). Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture. J Neurosci 16: 5967–78.
Zheng, J.-Q., T. Kelly, B. Chang, S. Ryazantsev, A. Rajasekaran, K. Martin, and J. Twiss. (2001). A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons. J Neurosci 21: 9291–9303.
Wu, K.Y., U. Hengst, L.J. Cox, E.Z. Macosko, A. Jeromin, E.R. Urquhart, and S.R. Jaffrey. (2005). Local translation of RhoA regulates growth cone collapse. Nature 436: 1020– 4.
Poon, M.M., S.H. Choi, C.A. Jamieson, D.H. Geschwind, and K.C. Martin. (2006). Identification of process-localized mRNAs from cultured rodent hippocampal neurons. J Neurosci 26: 13390 –9.
Mili, S., K. Moissoglu, and I.G. Macara. (2008). Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions. Nature 453: 115 – 9.
Taylor, A.M., N.C. Berchtold, V.M. Perreau, C.H. Tu, N. Li Jeon, and C.W. Cotman. (2009). Axonal mRNA in uninjured and regenerating cortical mammalian axons. J Neurosci 29: 4697–707.
Taylor, A.M., M. Blurton-Jones, S.W. Rhee, D.H. Cribbs, C.W. Cotman, and N.L. Jeon. (2005). A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2: 599– 605.
Cox, L.J., U. Hengst, N.G. Gurskaya, K.A. Lukyanov, and S.R. Jaffrey. (2008). Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival. Nat Cell Biol 10: 149 –59.
Wu, H.I., G.H. Cheng, Y.Y. Wong, C.M. Lin, W. Fang, W.Y. Chow, and Y.C. Chang. A lab-on-a-chip platform for studying the subcellular functional proteome of neuronal axons. Lab Chip 10: 647– 53.
Rishal, I., I. Michaelevski, M. Rozenbaum, V. Shinder, K.F. Medzihradszky, A.L. Burlingame, and M. Fainzilber. (2009). Axoplasm isolation from peripheral nerve. Dev Neurobiol 70: 126–33.
Knowles, R.B. and K.S. Kosik. (1997). Neurotrophin-3 signals redistribute RNA in neurons. Proceedings of the National Academy of Sciences of the United States of America 94: 14804 –8.
Zhang, H.L., R.H. Singer, and G.J. Bassell. (1999). Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones. J Cell Biol 147: 59–70.
Willis, D., K.W. Li, J.Q. Zheng, J.H. Chang, A. Smit, T. Kelly, T.T. Merianda, J. Sylvester, J. van Minnen, and J.L. Twiss. (2005). Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons. J Neurosci 25: 778–91.
Willis, D.E., E.A. van Niekerk, Y. Sasaki, M. Mesngon, T.T. Merianda, G.G. Williams, M. Kendall, D.S. Smith, G.J. Bassell, and J.L. Twiss. (2007). Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs. J Cell Biol 178: 965–80.
Aakalu, G., W.B. Smith, N. Nguyen, C. Jiang, and E.M. Schuman. (2001). Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 30: 489 – 502.
Yudin, D., S. Hanz, S. Yoo, E. Iavnilovitch, D. Willis, Y. Segal-Ruder, D. Vuppalanchi, K. Ben-Yaakov, M. Hieda, Y. Yoneda, J. Twiss, and M. Fainzilber. (2008). Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve. Neuron 59: 241–52.
Hanz, S., E. Perlson, D. Willis, J.Q. Zheng, R. Massarwa, J.J. Huerta, M. Koltzenburg, M. Kohler, J. van-Minnen, J.L. Twiss, and M. Fainzilber. (2003). Axoplasmic importins enable retrograde injury signaling in lesioned nerve. Neuron 40: 1095–104.
van Niekerk, E.A., D.E. Willis, J.H. Chang, K. Reumann, T. Heise, and J.L. Twiss. (2007). Sumoylation in axons triggers retrograde transport of the RNA-binding protein La. Proc Natl Acad Sci U S A 104: 12913–8.
Wang, W., E. van Niekerk, D.E. Willis, and J.L. Twiss. (2007). RNA transport and localized protein synthesis in neurological disorders and neural repair. Develop Neurobiol 67: 1166 –82.
Acknowledgements
The methods presented here were developed using funds from the National Institutes of Health (R01-NS041596 and R01-NS049041 to JLT and K99-NR010797 to DEW).
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Willis, D.E., Twiss, J.L. (2011). Profiling Axonal mRNA Transport. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_21
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DOI: https://doi.org/10.1007/978-1-61779-005-8_21
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