Stereotactic injection of shrna GSK-3β-AAV promotes axonal regeneration after spinal cord injury

Abstract

Evidence suggested that glycogen synthase kinase-3β (GSK-3β) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3β-adeno associated virus (GSK-3β-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3β-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.

Keywords

neurites / wound healing / protein-serine-threonine kinases / myelin proteins

References

[1] YorkEM, PetitA, RoskamsAJ. Epigenetics of neural repair following spinal cord injury. Neurotherapeutics, 2013, 10(4): 757-770 PMID: 24081781 PMCID: 3805854

[2] FilbinMT. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci, 2003, 4(9): 703-713 PMID: 12951563

[3] FinelliMJ, WongJK, ZouH. Epigenetic regulation of sensory axon regeneration after spinal cord injury. J Neurosci, 2013, 33(50): 19664-19676 PMID: 24336730 PMCID: 3858634

[4] ZhangC, TuF, ZhangJ Y, et al. . E-cadherin-transfected neural stem cells transplantation for spinal cord injury in rats. J Huazhong Univ Sci Technolog Med Sci, 2014, 34(4): 554-558 PMID: 25135726

[5] RuschelJ, HellalF, FlynnKC, et al. . Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science, 2015, 348(6232): 347-352 PMID: 25765066

[6] GaoW, YuLG, LiuYL, et al. . Mechanism of GABA receptors involved in spasticity inhibition induced by transcranial magnetic stimulation following spinal cord injury. J Huazhong Univ Sci Technolog Med Sci, 2015, 35(2): 241-247 PMID: 25877359

[7] KottisV, ThibaultP, MikolD, et al. . Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. J Neurochem, 2002, 82(6): 1566-1569 PMID: 12354307

[8] SchwabME. Functions of Nogo proteins and their receptors in the nervous system. Nat Rev Neurosci, 2010, 11(12): 799-811 PMID: 21045861

[9] GrandpreT, LiS, StrittmatterSM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 2002, 417(6888): 547-551 PMID: 12037567

[10] YiuG, HeZ. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci, 2006, 7(8): 617-627 PMID: 16858390 PMCID: 2693386

[11] LiuCM, HurEM, ZhouFQ. Coordinating gene expression and axon assembly to control axon growth: potential role of gsk3 signaling. Front Mol Neurosci, 2012, 5: 3 PMID: 22347166 PMCID: 3272657

[12] ZhouFQ, SniderWD. Cell biology. GSK-3beta and microtubule assembly in axons. Science, 2005, 308(5719): 211-214 PMID: 15825222

[13] LizMA, MarFM, SantosTE, et al. . Neuronal deletion of GSK3beta increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2. BMC Biol, 2014, 12: 47 PMID: 24923837 PMCID: 4229956

[14] AlabedYZ, PoolM, OngTS, et al. . GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci, 2010, 30(16): 5635-5643 PMID: 20410116

[15] ZhouFQ, ZhouJ, DedharS, et al. . NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron, 2004, 42(6): 897-912 PMID: 15207235

[16] AlabedYZ, PoolM, OngTS, et al. . GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci, 2010, 30(16): 5635-5643 PMID: 20410116

[17] ShenJY, YiXX, XiongNX, et al. . GSK-3beta activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells. Neurosci Lett, 2011, 505(2): 165-170 PMID: 22019984

[18] KawajiriH, YashiroM, ShintoO, et al. . A novel transforming growth factor beta receptor kinase inhibitor, A-77, prevents the peritoneal dissemination of scirrhous gastric carcinoma. Clin Cancer Res, 2008, 14(9): 2850-2860 PMID: 18451253

[19] WangH, ShenJ, XiongN, et al. . Protein kinase B is involved in Nogo-66 inhibiting neurite outgrowth in PC12 cells. Neuroreport, 2011, 22(15): 733-738 PMID: 21862940

[20] HuZ, TuJ. The roads to mitochondrial dysfunction in a rat model of posttraumatic syringomyelia. Biomed Res Int, 2015, 2015: 831490 PMID: 25685811 PMCID: 4309244

[21] BradburyEJ, McmahonSB. Spinal cord repair strategies: why do they work?. Nat Rev Neurosci, 2006, 7(8): 644-653 PMID: 16858392

[22] SchwabME, BartholdiD. Degeneration and regeneration of axons in the lesioned spinal cord. Physiol Rev, 1996, 76(2): 319-370 PMID: 8618960

[23] FitchMT, DollerC, CombsCK, et al. . Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J Neurosci, 1999, 19(19): 8182-8198 PMID: 10493720

[24] DusartI, SchwabME. Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci, 1994, 6(5): 712-724 PMID: 8075816

[25] PopovichPG, GuanZ, WeiP, et al. . Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury. Exp Neurol, 1999, 158(2): 351-365 PMID: 10415142

[26] WeiWJ, YuZY, YangHJ, et al. . Cellular expression profile of RhoA in rats with spinal cord injury. J Huazhong Univ Sci Technolog Med Sci, 2014, 34(5): 657-662 PMID: 25318874

[27] BradburyEJ, KhemaniS, VonR, et al. . NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur J Neurosci, 1999, 11(11): 3873-3883 PMID: 10583476

[28] QiuJ, CaiD, DaiH, et al. . Spinal axon regeneration induced by elevation of cyclic AMP. Neuron, 2002, 34(6): 895-903 PMID: 12086638

[29] BregmanBS, Kunkel-BagdenE, SchnellL, et al. . Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature, 1995, 378(6556): 498-501 PMID: 7477407

[30] BradburyEJ, MoonLD, PopatRJ, et al. . Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature, 2002, 416(6881): 636-640 PMID: 11948352

[31] SeiraO, DelRJ. Glycogen synthase kinase 3 beta (GSK3beta) at the tip of neuronal development and regeneration. Mol Neurobiol, 2014, 49(2): 931-944 PMID: 24158777

[32] YoshimuraT, KawanoY, ArimuraN, et al. . GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell, 2005, 120(1): 137-149 PMID: 15652488

[33] ZhouFQ, ZhouJ, DedharS, et al. . NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron, 2004, 42(6): 897-912 PMID: 15207235

[34] JiangH, GuoW, LiangX, et al. . Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators. Cell, 2005, 120(1): 123-135 PMID: 15652487

[35] WangT, WuX, YinC, et al. . CRMP-2 is involved in axon growth inhibition induced by RGMa in vitro and in vivo. Mol Neurobiol, 2013, 47(3): 903-913 PMID: 23275173

[36] DillJ, WangH, ZhouF, et al. . Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J Neurosci, 2008, 28(36): 8914-8928 PMID: 18768685