Netrin-G2在耐药性癫痫患者及大鼠模型脑组织中的表达及意义
摘要
目的:Netrin-G2是一种主要在中枢神经系统表达的神经轴突导向因子,在轴突的形成及神经细胞的迁移中发挥作用。本实验通过拟通过检测Netrin-G2在耐药性癫痫患者及氯化锂-匹罗卡品大鼠癫痫模型脑组织中的表达,探讨其在耐药性癫痫发生发展中的作用。
方法:按随机化原则从课题组前期建立的耐药性癫痫患者脑组织库中抽取35例颞叶组织作为实验组,15例因外伤导致颅内高压行减压术患者的颞叶组织作为对照组。56只SD成年大鼠腹腔注射氯化锂-匹罗卡品诱导慢性颞叶癫痫大鼠模型,并于成功建立模型后分别于6小时、1天、2天、7天、14天、30天和60天各时间点随机抽取8只大鼠处死,取其海马及邻近皮质作为实验组;8只腹腔注射生理盐水的SD成年大鼠作为对照组。分别采用免疫组化、免疫荧光双标和Western-blot方法检测上述样本脑组织中Netrin-G2的表达。
结果:Netrin-G2在正常人及大鼠脑组织中主要分布于神经细胞胞浆及胞膜。耐药性癫痫患者脑组织中Netrin-G2含量较对照组显著增高。动物模型中Netrin-G2主要集中表达在齿状回、CA1和CA3区,实验组中的表达高于对照组:动物点燃后6小时开始增高,约2天达到高峰,并维持较高表达量,持续到1-2月时下降至对照组水平。
结论:在耐药性癫痫患者和动物模型中,神经轴突导向因子Netrin-G2在实验组的表达量比对照组升高,提示其可能参与了耐药性癫痫形成的病理过程,可能在异常的轴突发育及神经细胞迁移中起着重要作用,参与了异常兴奋性突触重构和病理性神经网络重组,是耐药性癫痫耐药性形成的可能原因。
The membrane-bound axon guidance molecule Netrin-G2 is preferentially expressed in the central nervous system and plays a role in synapse formation and maintenance. Using immunohistochemistry, immunofluorescence, and western blotting, we investigated the possible correlation between Netrin-G2 expression and intractable epilepsy (IE) using surgical samples from epilepsy patients. We used 35 samples of temporal neocortex from patients undergoing surgery for drug-refractory epilepsy and 15 autopsy samples from individuals who died in traffic accidents (i.e., samples of normal human brain). We also examined Netrin-G2 expression in the hippocampus and adjacent cortex of rats with temporal lobe epilepsy (lithium chloride-pilocarpine model). Netrin-G2 was expressed in the membrane and cytoplasm of neurons from control specimens, and expression was higher in tissue from patients with intractable epilepsy. Western blotting of rat brain tissue showed that Netrin-G2 was upregulated starting at 6 h after kindling. Maximal expression was seen around 2 days, and relatively high expression was maintained until 30 days. Expression then returned to normal levels at 60 days, which was consistent with the immunohistochemical and immunofluorescence results. These data implicate Netrin-G2 in the pathophysiology of epilepsy and are consistent with the hypothesis that this protein may participate in the abnormal development of synapses and in neuron migration.
方法:按随机化原则从课题组前期建立的耐药性癫痫患者脑组织库中抽取35例颞叶组织作为实验组,15例因外伤导致颅内高压行减压术患者的颞叶组织作为对照组。56只SD成年大鼠腹腔注射氯化锂-匹罗卡品诱导慢性颞叶癫痫大鼠模型,并于成功建立模型后分别于6小时、1天、2天、7天、14天、30天和60天各时间点随机抽取8只大鼠处死,取其海马及邻近皮质作为实验组;8只腹腔注射生理盐水的SD成年大鼠作为对照组。分别采用免疫组化、免疫荧光双标和Western-blot方法检测上述样本脑组织中Netrin-G2的表达。
结果:Netrin-G2在正常人及大鼠脑组织中主要分布于神经细胞胞浆及胞膜。耐药性癫痫患者脑组织中Netrin-G2含量较对照组显著增高。动物模型中Netrin-G2主要集中表达在齿状回、CA1和CA3区,实验组中的表达高于对照组:动物点燃后6小时开始增高,约2天达到高峰,并维持较高表达量,持续到1-2月时下降至对照组水平。
结论:在耐药性癫痫患者和动物模型中,神经轴突导向因子Netrin-G2在实验组的表达量比对照组升高,提示其可能参与了耐药性癫痫形成的病理过程,可能在异常的轴突发育及神经细胞迁移中起着重要作用,参与了异常兴奋性突触重构和病理性神经网络重组,是耐药性癫痫耐药性形成的可能原因。
The membrane-bound axon guidance molecule Netrin-G2 is preferentially expressed in the central nervous system and plays a role in synapse formation and maintenance. Using immunohistochemistry, immunofluorescence, and western blotting, we investigated the possible correlation between Netrin-G2 expression and intractable epilepsy (IE) using surgical samples from epilepsy patients. We used 35 samples of temporal neocortex from patients undergoing surgery for drug-refractory epilepsy and 15 autopsy samples from individuals who died in traffic accidents (i.e., samples of normal human brain). We also examined Netrin-G2 expression in the hippocampus and adjacent cortex of rats with temporal lobe epilepsy (lithium chloride-pilocarpine model). Netrin-G2 was expressed in the membrane and cytoplasm of neurons from control specimens, and expression was higher in tissue from patients with intractable epilepsy. Western blotting of rat brain tissue showed that Netrin-G2 was upregulated starting at 6 h after kindling. Maximal expression was seen around 2 days, and relatively high expression was maintained until 30 days. Expression then returned to normal levels at 60 days, which was consistent with the immunohistochemical and immunofluorescence results. These data implicate Netrin-G2 in the pathophysiology of epilepsy and are consistent with the hypothesis that this protein may participate in the abnormal development of synapses and in neuron migration.
引文
[1] Lin J.C., Ho W.H., Gurney A., et al. The netrin-G1 ligand NGL-1 promotes the outgrowth of thalamocortical axons[J]. Nat Neurosci. 2003, 6(12):1270–1276.
[2] Kim, S., Burette, A., Chung, H.S., et al. NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation[J]. Nat Neurosci.2006, 9(10): 1294-1301.
[3] Nakashiba T., Nishimura S., Ikeda T., et al. Complementary expression and neurite outgrowth activity of netrin-G subfamily members[J]. Mech Dev.2002,111(1-2):47-60.
[4]Nakashiba T., Ikeda T., Nishimura S., et al. Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrin that is functionally divergent from classical netrins[J]. Neurosci. 2000, 20(17): 6540-6550.
[5] Yin Y, Miner JH, Sanes JR. Laminets: laminin- and netrin-related genes expressed in distinct neuronal subsets[J]. Mol Cell Neurosci.2002,19(3):344-358.
[6] Zhang, W., Rajan, I., Savelieva, K. V., et al. Netrin-G2 and Netrin-G2 ligand are both required for normal auditory responsiveness[J]. Genes Brain Behav. 2008, 7(4):385-392.
[7] Eastwood, SL, and Harrison, PJ. Decreased mRNA expression of netrin-G1 and Netrin-G2 in the temporal lobe in schizophrenia and bipolar disorder[J]. Neuropsychopharmacology. 2008,33(4), 933-945.
[8] W.Xun, S.Dinglie, Intractable epilepsy[J]. Chinese Journal of neurology. 1998,31:4-5.
[9]International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures. From the Commission on Classification and Terminology of the International League Against Epilepsy. Epilepsia.1981,2:489-501.
[10] Sato M, Racine RJ, Mc Intyre DC. Kindling: basic mechanisms and clinical validity[J].Electroencephalography Clin Neurophysiology.1990,76(5):459-472.
[11] Buckmaster PS, Zhang GF, and Yamawaki R. Axon sprouting in a model of temporal lobe epilepsy creates a predominantly excitatory feedback circuit[J]. Neurosci. 2002,22(1):6650-6658.
[12] Woo J, Kwon S K, Kim E. The NGL family of leucine-rich repeat-containing synaptic adhesion molecules[J]. Mol Cell Neurosci. 2009,42(1):1-10.
[13] Nadler JV, Perry BW, Gentry C, et al. Loss and reacquisition of hippocampal synapses after selective destruction of CA3-CA4 afferents with kainic acid[J]. BrainRes. 1980,191(2): 387-403.
[14]Barth PG. Disorders of neuronal migration[J]. Can J Neurol Sci, 1987,14(1):1-16.
[15] Gordon N. Epilepsy and disorders of neuronal migration. II: Epilepsy as a symptom of neuronal migration defects[J]. Dev Med Child Neurol. 1996, 38(12): 1131-1134.
[16] Nishimura-Akiyoshi S., Niimi K., Nakashiba T., et al. Axonal netrin-Gs transneuronally determine lamina-specific subdendritic segments[J]. Proc Natl Acad Sci U S A. 2007,104(37): 14801-14806.
[17] Tanaka DH, Yamauchi K, and Murakami F. Guidance mechanisms in neuronal and axonal migration[J]. Brain Nerve. 2008,60(4):405-413.
[18] Davy A., Gale N.W., Murray E.W., et al. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion[J]. Genes Dev.1999,13(23): 3125-3135.
[19] Ikezawa H. Glycosylphosphatidylinositol (GPI)-anchored proteins[J]. Biol Pharm Bull. 2002,25(4): 409-417.
[20] Biederer T. Hooking up new synapses[J]. Nat Neurosci. 2006, 9(10), 1203-1204.
[21] Garner CC, Nash J, Huganir RL. PDZ domains in synapse assembly and signaling[J]. Trends Cell Biol. 2000, 10(7), 274-280.
[22] Kim E, Sheng M. PDZ domain proteins of synapses[J]. Nat Rev Neurosci.2004, 5(10), 771-781.
[1] Hardesty I. On the development and nature of the neuroglia [J]. Am J Anat.1904, 3:229-268
[2] Cajal S, Rsmon Y. Histology of the nervous system,translated by Swanson, N., and L. W. Swanson, Oxford University Press, New York. 1995.
[3] Rakic P.Neuron-glia relationship during granule cell migration in developing cerebellar cortex [J]. J Comp Neurol. 1971,141(3):283-312.
[4] Barth PG. Disorders of nruronal migration [J].Can J Neurol Sci.1987, 14(1):1-16.
[5] Gordon N.Epilepsy and disorders of neuronal migration.Ⅱ:Epilepsy as a symptom of neuronal migration defects [J].Dev Med Child Neurol.1996,38(12):1131-1134.
[6] Tessier-Lavigne M., Goodman, C.S. The molecular biology of axon Guidance[J]. Science.1996,274(5290):l123-l133.
[7] Koch M,Murrell JR,Hunter DD,et al.A novel member of the Netrin family,β-Netrin,shares homology withβchain of laminin:identification,expression,and functional characterization [J].Journal of Cell Biology. 2000, 151(2):221-234.
[8] Dai KS, Liew CC. Chromosomal, in silico and in vitro expression analysis of cardiovascular-based genes encoding zinc finger proteins [J].J Mol Cell Cardiol. 1999,31(9):1749-1769.
[9]Miyashita T, Nishimura-Akiyoshi S, Itohara S, et al.. Strong expression of NETRIN-G2 in the monkey claustrum [J]. Neuroscience. 2005, 136(2): 487-496.
[10] Lise M.F., El-Husseini A.. The neuroligin and neurexin families: from structure to function at the synapse [J].Cell Mol Life Sci. 2006, 63(16): 1833–1849.
[11] Serafini T.,Kennedy T.E.,Galko M.J.,et al.The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6 [J]. Cell. 1994, 78(3): 409-424.
[12] Shewan D., Dwivedy A.,Anderson R.,et a1.Age-related changes underlie switch in Netrin-1 responsiveness as growth cones advance along visual pathway [J].Nature Neuroscience. 2002, 5(10): 953-962.
[13] Deiner M.S.,Sretavan D.W..Altered midline axon pathways and ectopic neurons in the developing hypothalamus of Netrin·l and DCC-deficient mice [J].J Neuroeci. 1999, 19(22): 9900-99l2.
[14] Torre J.R.,Hopker V.H.,Ming G.L.,et al.Turning of retinal growth cones in a Netrin-1 gradient mediated by the Netrin receptor DCC [J].Neuron. 1997, 19(6): 1211-1224.
[15] Stein E., Zou Y.,Poo M.M., et a1.Binding of DCC by Netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation [J].Science. 2001, 291(5510): 1976-1982.
[16] Dickson B.J..Molecular mechanisms of axon guidance [J].Science. 2002, 298(5600):1959-1964.
[17] Keleman K.,Dickson B.J. Short and long-range repulsion by the Drosophils Unc-5 Netrin receptor [J].Neuron. 2001, 32(4): 605-617.
[18] Yee K.T.,Simon H.H.,Tessier-Lavigne M.,et al.Extension of long leading processes and neuronal migration in the mammalian brain directed by the chemoattractant Netrin-l [J].Neuron. 1999, 24(3): 607-622.
[19]Winberg M.L.,Mitchell K.J.,Goodman C.S..Genetic analysis of the mechanisms controlling target selection:complementary and combinatorial functions of Netrlns,semaphorins,and IgCAMs [J].Cell. l998, 93(4):581-591.
[20]Kennedy T.E..Cellular mechanisms of Netrin function:long-range and short-range actions [J].Biochem Cell Biol. 2000,78(5):569-575.
[21] Manitt C,,Colicos M,A.,Thompson K.M. et a1.Widespread expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian spinal cord [J].J Neurosci. 2001, 21(11): 3911-3922.
[22] Tsai H.H.,Tessier-La vigne M.,Miller R.H..Netrin 1 mediates spinal cord oligodendrocyte precursor dispersal [J].Development. 2003, 130(10): 2095.
[23] Von Hilchen C M, Hein I, Technau GM, et al. Netrins guide migration of distinct glial cells in the Drosophila embryo[J].Development. 2010, 137(8):1251-1262.
[24] Alcantara S.,Ruiz M.,Castro F.D.,et a1.Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system [J].Development. 2000, 127(7): 1359-1372.
[25] Forcet C.,Stein E.,Pays L..Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation [J].Nature. 2002,417(6887):443-447.
[26] Nishiyama M,Hoshino A,Tsai L. Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning [J].Nature. 2003, 424(6943): 990-995.
[2] Kim, S., Burette, A., Chung, H.S., et al. NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation[J]. Nat Neurosci.2006, 9(10): 1294-1301.
[3] Nakashiba T., Nishimura S., Ikeda T., et al. Complementary expression and neurite outgrowth activity of netrin-G subfamily members[J]. Mech Dev.2002,111(1-2):47-60.
[4]Nakashiba T., Ikeda T., Nishimura S., et al. Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrin that is functionally divergent from classical netrins[J]. Neurosci. 2000, 20(17): 6540-6550.
[5] Yin Y, Miner JH, Sanes JR. Laminets: laminin- and netrin-related genes expressed in distinct neuronal subsets[J]. Mol Cell Neurosci.2002,19(3):344-358.
[6] Zhang, W., Rajan, I., Savelieva, K. V., et al. Netrin-G2 and Netrin-G2 ligand are both required for normal auditory responsiveness[J]. Genes Brain Behav. 2008, 7(4):385-392.
[7] Eastwood, SL, and Harrison, PJ. Decreased mRNA expression of netrin-G1 and Netrin-G2 in the temporal lobe in schizophrenia and bipolar disorder[J]. Neuropsychopharmacology. 2008,33(4), 933-945.
[8] W.Xun, S.Dinglie, Intractable epilepsy[J]. Chinese Journal of neurology. 1998,31:4-5.
[9]International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures. From the Commission on Classification and Terminology of the International League Against Epilepsy. Epilepsia.1981,2:489-501.
[10] Sato M, Racine RJ, Mc Intyre DC. Kindling: basic mechanisms and clinical validity[J].Electroencephalography Clin Neurophysiology.1990,76(5):459-472.
[11] Buckmaster PS, Zhang GF, and Yamawaki R. Axon sprouting in a model of temporal lobe epilepsy creates a predominantly excitatory feedback circuit[J]. Neurosci. 2002,22(1):6650-6658.
[12] Woo J, Kwon S K, Kim E. The NGL family of leucine-rich repeat-containing synaptic adhesion molecules[J]. Mol Cell Neurosci. 2009,42(1):1-10.
[13] Nadler JV, Perry BW, Gentry C, et al. Loss and reacquisition of hippocampal synapses after selective destruction of CA3-CA4 afferents with kainic acid[J]. BrainRes. 1980,191(2): 387-403.
[14]Barth PG. Disorders of neuronal migration[J]. Can J Neurol Sci, 1987,14(1):1-16.
[15] Gordon N. Epilepsy and disorders of neuronal migration. II: Epilepsy as a symptom of neuronal migration defects[J]. Dev Med Child Neurol. 1996, 38(12): 1131-1134.
[16] Nishimura-Akiyoshi S., Niimi K., Nakashiba T., et al. Axonal netrin-Gs transneuronally determine lamina-specific subdendritic segments[J]. Proc Natl Acad Sci U S A. 2007,104(37): 14801-14806.
[17] Tanaka DH, Yamauchi K, and Murakami F. Guidance mechanisms in neuronal and axonal migration[J]. Brain Nerve. 2008,60(4):405-413.
[18] Davy A., Gale N.W., Murray E.W., et al. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion[J]. Genes Dev.1999,13(23): 3125-3135.
[19] Ikezawa H. Glycosylphosphatidylinositol (GPI)-anchored proteins[J]. Biol Pharm Bull. 2002,25(4): 409-417.
[20] Biederer T. Hooking up new synapses[J]. Nat Neurosci. 2006, 9(10), 1203-1204.
[21] Garner CC, Nash J, Huganir RL. PDZ domains in synapse assembly and signaling[J]. Trends Cell Biol. 2000, 10(7), 274-280.
[22] Kim E, Sheng M. PDZ domain proteins of synapses[J]. Nat Rev Neurosci.2004, 5(10), 771-781.
[1] Hardesty I. On the development and nature of the neuroglia [J]. Am J Anat.1904, 3:229-268
[2] Cajal S, Rsmon Y. Histology of the nervous system,translated by Swanson, N., and L. W. Swanson, Oxford University Press, New York. 1995.
[3] Rakic P.Neuron-glia relationship during granule cell migration in developing cerebellar cortex [J]. J Comp Neurol. 1971,141(3):283-312.
[4] Barth PG. Disorders of nruronal migration [J].Can J Neurol Sci.1987, 14(1):1-16.
[5] Gordon N.Epilepsy and disorders of neuronal migration.Ⅱ:Epilepsy as a symptom of neuronal migration defects [J].Dev Med Child Neurol.1996,38(12):1131-1134.
[6] Tessier-Lavigne M., Goodman, C.S. The molecular biology of axon Guidance[J]. Science.1996,274(5290):l123-l133.
[7] Koch M,Murrell JR,Hunter DD,et al.A novel member of the Netrin family,β-Netrin,shares homology withβchain of laminin:identification,expression,and functional characterization [J].Journal of Cell Biology. 2000, 151(2):221-234.
[8] Dai KS, Liew CC. Chromosomal, in silico and in vitro expression analysis of cardiovascular-based genes encoding zinc finger proteins [J].J Mol Cell Cardiol. 1999,31(9):1749-1769.
[9]Miyashita T, Nishimura-Akiyoshi S, Itohara S, et al.. Strong expression of NETRIN-G2 in the monkey claustrum [J]. Neuroscience. 2005, 136(2): 487-496.
[10] Lise M.F., El-Husseini A.. The neuroligin and neurexin families: from structure to function at the synapse [J].Cell Mol Life Sci. 2006, 63(16): 1833–1849.
[11] Serafini T.,Kennedy T.E.,Galko M.J.,et al.The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6 [J]. Cell. 1994, 78(3): 409-424.
[12] Shewan D., Dwivedy A.,Anderson R.,et a1.Age-related changes underlie switch in Netrin-1 responsiveness as growth cones advance along visual pathway [J].Nature Neuroscience. 2002, 5(10): 953-962.
[13] Deiner M.S.,Sretavan D.W..Altered midline axon pathways and ectopic neurons in the developing hypothalamus of Netrin·l and DCC-deficient mice [J].J Neuroeci. 1999, 19(22): 9900-99l2.
[14] Torre J.R.,Hopker V.H.,Ming G.L.,et al.Turning of retinal growth cones in a Netrin-1 gradient mediated by the Netrin receptor DCC [J].Neuron. 1997, 19(6): 1211-1224.
[15] Stein E., Zou Y.,Poo M.M., et a1.Binding of DCC by Netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation [J].Science. 2001, 291(5510): 1976-1982.
[16] Dickson B.J..Molecular mechanisms of axon guidance [J].Science. 2002, 298(5600):1959-1964.
[17] Keleman K.,Dickson B.J. Short and long-range repulsion by the Drosophils Unc-5 Netrin receptor [J].Neuron. 2001, 32(4): 605-617.
[18] Yee K.T.,Simon H.H.,Tessier-Lavigne M.,et al.Extension of long leading processes and neuronal migration in the mammalian brain directed by the chemoattractant Netrin-l [J].Neuron. 1999, 24(3): 607-622.
[19]Winberg M.L.,Mitchell K.J.,Goodman C.S..Genetic analysis of the mechanisms controlling target selection:complementary and combinatorial functions of Netrlns,semaphorins,and IgCAMs [J].Cell. l998, 93(4):581-591.
[20]Kennedy T.E..Cellular mechanisms of Netrin function:long-range and short-range actions [J].Biochem Cell Biol. 2000,78(5):569-575.
[21] Manitt C,,Colicos M,A.,Thompson K.M. et a1.Widespread expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian spinal cord [J].J Neurosci. 2001, 21(11): 3911-3922.
[22] Tsai H.H.,Tessier-La vigne M.,Miller R.H..Netrin 1 mediates spinal cord oligodendrocyte precursor dispersal [J].Development. 2003, 130(10): 2095.
[23] Von Hilchen C M, Hein I, Technau GM, et al. Netrins guide migration of distinct glial cells in the Drosophila embryo[J].Development. 2010, 137(8):1251-1262.
[24] Alcantara S.,Ruiz M.,Castro F.D.,et a1.Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system [J].Development. 2000, 127(7): 1359-1372.
[25] Forcet C.,Stein E.,Pays L..Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation [J].Nature. 2002,417(6887):443-447.
[26] Nishiyama M,Hoshino A,Tsai L. Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning [J].Nature. 2003, 424(6943): 990-995.