Abstract
Mammalian sterile 20-like kinase-3b (Mst3b, encoded by Stk24), regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using short hairpin RNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.
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References
Ramon y Cajal, S. Degeneration and Regeneration of the Nervous System (Oxford University Press, New York, 1991).
Berry, M., Carlile, J. & Hunter, A. Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J. Neurocytol. 25, 147–170 (1996).
Leon, S., Yin, Y., Nguyen, J., Irwin, N. & Benowitz, L.I. Lens injury stimulates axon regeneration in the mature rat optic nerve. J. Neurosci. 20, 4615–4626 (2000).
Fischer, D., Heiduschka, P. & Thanos, S. Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats. Exp. Neurol. 172, 257–272 (2001).
Pernet, V. & Di Polo, A. Synergistic action of brain-derived neurotrophic factor and lens injury promotes retinal ganglion cell survival, but leads to optic nerve dystrophy in vivo. Brain 129, 1014–1026 (2006).
Yin, Y. et al. Macrophage-derived factors stimulate optic nerve regeneration. J. Neurosci. 23, 2284–2293 (2003).
Lorber, B., Berry, M. & Logan, A. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors. Eur. J. Neurosci. 21, 2029–2034 (2005).
Cen, L.P. et al. Chemotactic effect of ciliary neurotrophic factor on macrophages in retinal ganglion cell survival and axonal regeneration. Invest. Ophthalmol. Vis. Sci. 48, 4257–4266 (2007).
Yin, Y. et al. Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat. Neurosci. 9, 843–852 (2006).
Yin, Y. et al. Oncomodulin links inflammation to optic nerve regeneration. Proc. Natl. Acad. Sci. USA (in the press).
Makwana, M. & Raivich, G. Molecular mechanisms in successful peripheral regeneration. FEBS J. 272, 2628–2638 (2005).
Barrette, B. et al. Requirement of myeloid cells for axon regeneration. J. Neurosci. 28, 9363–9376 (2008).
Yudin, D. et al. Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve. Neuron 59, 241–252 (2008).
Greene, L.A., Volonte, C. & Chalazonitis, A. Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons. J. Neurosci. 10, 1479–1485 (1990).
Benowitz, L.I. et al. Axon outgrowth is regulated by an intracellular purine-sensitive mechanism in retinal ganglion cells. J. Biol. Chem. 273, 29626–29634 (1998).
Irwin, N., Li, Y.M., O'Toole, J.E. & Benowitz, L.I. Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth. Proc. Natl. Acad. Sci. USA 103, 18320–18325 (2006).
Volonte, C. & Greene, L.A. Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography. J. Biol. Chem. 267, 21663–21670 (1992).
Zurn, A.D. & Do, K.Q. Purine metabolite inosine is an adrenergic neurotrophic substance for cultured chicken sympathetic neurons. Proc. Natl. Acad. Sci. USA 85, 8301–8305 (1988).
Chen, P., Goldberg, D.E., Kolb, B., Lanser, M. & Benowitz, L.I. Inosine induces axonal rewiring and improves behavioral outcome after stroke. Proc. Natl. Acad. Sci. USA 99, 9031–9036 (2002).
Smith, J.M. et al. Inosine promotes recovery of skilled motor function in a model of focal brain injury. Brain 130, 915–925 (2007).
Zai, L. et al. Inosine alters gene expression and axonal projections in neurons contralateral to a cortical infarct and improves skilled use of the impaired limb. J. Neurosci. 29, 8187–8197 (2009).
Bontioti, E.N., Kanje, M. & Dahlin, L.B. Regeneration and functional recovery in the upper extremity of rats after various types of nerve injuries. J. Peripher. Nerv. Syst. 8, 159–168 (2003).
Lu, T.J. et al. Inhibition of cell migration by autophosphorylated mammalian sterile 20-like kinase 3 (MST3) involves paxillin and protein-tyrosine phosphatase-PEST. J. Biol. Chem. 281, 38405–38417 (2006).
Martin, K.R., Klein, R.L. & Quigley, H.A. Gene delivery to the eye using adeno-associated viral vectors. Methods 28, 267–275 (2002).
Sapieha, P.S., Peltier, M., Rendahl, K.G., Manning, W.C. & Di Polo, A. Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury. Mol. Cell. Neurosci. 24, 656–672 (2003).
Fischer, D., He, Z. & Benowitz, L.I. Counteracting the Nogo receptor enhances optic nerve regeneration if retinal ganglion cells are in an active growth state. J. Neurosci. 24, 1646–1651 (2004).
Li, Y., Irwin, N., Yin, Y., Lanser, M. & Benowitz, L.I. Axon regeneration in goldfish and rat retinal ganglion cells: differential responsiveness to carbohydrates and cAMP. J. Neurosci. 23, 7830–7838 (2003).
Schaden, H., Stuermer, C.A. & Bahr, M. GAP-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat. J. Neurobiol. 25, 1570–1578 (1994).
Kermer, P. et al. Caspase-9: involvement in secondary death of axotomized rat retinal ganglion cells in vivo. Brain Res. Mol. Brain Res. 85, 144–150 (2000).
Berkelaar, M., Clarke, D.B., Wang, Y.C., Bray, G.M. & Aguayo, A.J. Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats. J. Neurosci. 14, 4368–4374 (1994).
Ygge, J. Central projections of the rat radial nerve investigated with transganglionic degeneration and transganglionic transport of horseradish peroxidase. J. Comp. Neurol. 279, 199–211 (1989).
Markus, A., Zhong, J. & Snider, W.D. Raf and akt mediate distinct aspects of sensory axon growth. Neuron 35, 65–76 (2002).
Zhong, J. et al. Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo. Nat. Neurosci. 10, 598–607 (2007).
Cowley, S., Paterson, H., Kemp, P. & Marshall, C.J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841–852 (1994).
Kaplan, D.R. & Miller, F.D. Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10, 381–391 (2000).
Tucker, B.A., Rahimtula, M. & Mearow, K.M. A procedure for selecting and culturing subpopulations of neurons from rat dorsal root ganglia using magnetic beads. Brain Res. Brain Res. Protoc. 16, 50–57 (2005).
Schwab, M.E. Repairing the injured spinal cord. Science 295, 1029–1031 (2002).
Aguayo, A.J. et al. Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals. Phil. Trans. R. Soc. Lond. B 331, 337–343 (1991).
Dan, I., Watanabe, N.M. & Kusumi, A. The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol. 11, 220–230 (2001).
Herskowitz, I. MAP kinase pathways in yeast: for mating and more. Cell 80, 187–197 (1995).
Fischer, D., Petkova, V., Thanos, S. & Benowitz, L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J. Neurosci. 24, 8726–8740 (2004).
Costigan, M. et al. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3, 16 (2002).
Bonilla, I.E., Tanabe, K. & Strittmatter, S.M. Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth. J. Neurosci. 22, 1303–1315 (2002).
Fan, M., Mi, R., Yew, D.T. & Chan, W.Y. Analysis of gene expression following sciatic nerve crush and spinal cord hemisection in the mouse by microarray expression profiling. Cell. Mol. Neurobiol. 21, 497–508 (2001).
Benowitz, L.I., Apostolides, P.J., Perrone-Bizzozero, N., Finklestein, S.P. & Zwiers, H. Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain. J. Neurosci. 8, 339–352 (1988).
Cui, Q., Yip, H.K., Zhao, R.C., So, K.F. & Harvey, A.R. Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons. Mol. Cell. Neurosci. 22, 49–61 (2003).
Moskowitz, P.F. & Oblinger, M.M. Sensory neurons selectively upregulate synthesis and transport of the beta III-tubulin protein during axonal regeneration. J. Neurosci. 15, 1545–1555 (1995).
Schwalb, J.M. et al. Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture. J. Neurosci. 15, 5514–5525 (1995).
Leclere, P.G. et al. Effective gene delivery to adult neurons by a modified form of electroporation. J. Neurosci. Methods 142, 137–143 (2005).
Gavazzi, I., Kumar, R.D., McMahon, S.B. & Cohen, J. Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro. Eur. J. Neurosci. 11, 3405–3414 (1999).
Acknowledgements
We thank A. Logan and M. Berry for advice on DRG injections and use of facilities, Y. Yin for surgical assistance in the preliminary optic nerve studies, D. Kim and L. Zai for surgical assistance in the Mst3b activation studies, M.T. Henzl (University of Missouri) for oncomodulin, and the Developmental Disabilities Research Center of Children's Hospital (US National Institutes of Health (NIH) P30 HD018655) for use of core facilities and expertise. We are grateful for the support of the NIH (EY05690 to L.I.B.), the European Union (Marie Curie Outgoing International Fellowship MOIF-CT-2004-008424 to B.L.), Alseres, Inc. and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.
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B.L. helped design some of the experiments, carried out most in vivo and in vitro studies, drafted much of paper and did most of the data analysis. M.L.H. assisted with many cell culture studies and in vivo work. L.I.B. helped conceive the overall structure of the study, supervised parts of it, carried out some of the data analysis and wrote parts of the manuscript. N.I. generated the core idea of the study, helped conceive the overall structure of the study, performed preliminary optic nerve studies, constructed the viral vectors, made the mutations in Mstb3, supervised parts of the study, did some of the data analysis and wrote parts of the manuscript.
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Lorber, B., Howe, M., Benowitz, L. et al. Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways. Nat Neurosci 12, 1407–1414 (2009). https://doi.org/10.1038/nn.2414
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DOI: https://doi.org/10.1038/nn.2414
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