肌萎缩侧索硬化症相关基因VAPB对皮质神经元存活及突触蛋白表达的影响
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摘要
目的探讨肌萎缩侧索硬化症(ALS)致病基因VAPB P56S突变对原代培养皮质神经元的细胞活性及突触蛋白表达的影响。方法以非转基因(nTg)和VAPB P56S转基因(P56S Tg)新生小鼠为细胞来源,体外培养皮质神经元10d和20d后,分别利用MTT法和Western blot法检测皮质神经元存活率以及突触蛋白表达水平。结果与nTg小鼠的皮质神经元比较,VAPB P56S转基因小鼠的皮质神经元在体外培养20d时细胞活力显著降低(P<0.0001),而在体外培养10d时无明显差异(P=0.5140),但突触囊泡膜蛋白SV2C(P<0.0001)、突触囊泡结合蛋白VAMP2(P=0.0047)、突触小体相关蛋白SNAP25(P<0.0001)和突触后致密蛋白PSD95(P<0.0001)表达水平均显著降低。结论 VAPB P56S降低原代皮质神经元活性,并在神经元变性死亡前即损害突触结构和功能相关蛋白的表达。
Objective To investigate the effects of amyotrophic lateral sclerosis(ALS) related VAPB P56 S mutation on cell viability and expression of synaptic proteins of cultured mouse cortical neurons. Method The cortical neurons from non-transgenic(nTg) and VAPB P56 S transgenic(P56 S Tg) newborn mice were cultured in vitro for 10 to 20 days. The cell viability and the expressions of synaptic proteins were detected with MTT assay and western blot, respectively. Results Compared to n Tg mice, cortical neurons from P56 S Tg mice showed reduced cell viabilities on 20 days in vitro(P<0.0001) and comparable cell viability on 10 days in vitro. On 10 days in vitro, cortical neurons from P56 S Tg mice showed decreased level of synaptic vesicle glycoprotein 2 C(SV2C)(P<0.0001), vesicle associated membrane protein 2(VAMP2)(P=0.0047), synaptosome associated protein 25(SNAP25)(P<0.0001) and postsynaptic density protein 95(PSD95)(P<0.0001). Conclusion VAPB P56 S can impair the cell viability, as well as reduce the expression of synaptic structure and function related proteins of cultured cortical neurons before neuron degeneration.
Objective To investigate the effects of amyotrophic lateral sclerosis(ALS) related VAPB P56 S mutation on cell viability and expression of synaptic proteins of cultured mouse cortical neurons. Method The cortical neurons from non-transgenic(nTg) and VAPB P56 S transgenic(P56 S Tg) newborn mice were cultured in vitro for 10 to 20 days. The cell viability and the expressions of synaptic proteins were detected with MTT assay and western blot, respectively. Results Compared to n Tg mice, cortical neurons from P56 S Tg mice showed reduced cell viabilities on 20 days in vitro(P<0.0001) and comparable cell viability on 10 days in vitro. On 10 days in vitro, cortical neurons from P56 S Tg mice showed decreased level of synaptic vesicle glycoprotein 2 C(SV2C)(P<0.0001), vesicle associated membrane protein 2(VAMP2)(P=0.0047), synaptosome associated protein 25(SNAP25)(P<0.0001) and postsynaptic density protein 95(PSD95)(P<0.0001). Conclusion VAPB P56 S can impair the cell viability, as well as reduce the expression of synaptic structure and function related proteins of cultured cortical neurons before neuron degeneration.
引文
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[18]Tran D,Chalhoub A,Schooley A,et al.A mutation in VAPB that causes amyotrophic lateral sclerosis also causes a nuclear envelope defect[J].J Cell Sci,2012,125(Pt 12):2831-2836.
[19]De Vos KJ,Morotz GM,Stoica R,et al.VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis[J].Hum Mol Genet,2012,21(6):1299-1311.
[20]Rocha N,Kuijl C,van der Kant R,et al.Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning[J].J Cell Biol,2009,185(7):1209-12025.
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[23]Martin LJ,Chang Q.Inhibitory synaptic regulation of motoneurons:a new target of disease mechanisms in amyotrophic lateral sclerosis[J].Mol Neurobiol,2012,45(1):30-42.
[24]Jiang M,Schuster JE,Fu R,et al.Progressive changes in synaptic inputs to motoneurons in adult sacral spinal cord of a mouse model of amyotrophic lateral sclerosis[J].J Neurosci,2009,29(48):15031-15038.
[25]Chai A,Withers J,Koh YH,et al.h VAPB,the causative gene of a heterogeneous group of motor neuron diseases in humans,is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction[J].Hum Mol Genet,2008,17(2):266-280.
[26]Pennetta G,Hiesinger PR,Fabian-Fine R,et al.Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner[J].Neuron,2002,35(2):291-306.
[27]Yang Z,Huh SU,Drennan JM,et al.Drosophila Vap-33 is required for axonal localization of Dscam isoforms[J].J Neurosci,2012,32(48):17241-17250.
[28]Kuijpers M,Yu KL,Teuling E,et al.The ALS8 protein VAPB interacts with the ER-Golgi recycling protein YIF1A and regulates membrane delivery into dendrites[J].EMBO J,2013,32(14):2056-2072.
[29]Ratnaparkhi A,Lawless GM,Schweizer FE,et al.A Drosophila model of ALS:human ALS-associated mutation in VAP33A suggests a dominant negative mechanism[J].PLo S One,2008,3(6):e2334.
[30]Larroquette F,Seto L,Gaub PL,et al.Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response[J].Hum Mol Genet,2015,24(22):6515-6529.
[2]Murphy SE,Levine TP.VAP,a versatile access point for the endoplasmic reticulum:review and analysis of FFAT-like motifs in the VAPome[J].Biochim Biophys Acta,2016,1861(8 Pt B):952-961.
[3]Lev S,Ben Halevy D,Peretti D,et al.The VAP protein family:from cellular functions to motor neuron disease[J].Trends Cell Biol,2008,18(6):282-290.
[4]Jackson CL,Walch L,Verbavatz JM.Lipids and their trafficking:an integral part of cellular organization[J].Dev Cell,2016,39(2):139-153.
[5]Aliaga L,Lai C,Yu J,et al.Amyotrophic lateral sclerosis-related VAPB P56S mutation differentially affects the function and survival of corticospinal and spinal motor neurons[J].Hum Mol Genet,2013,22(21):4293-4305.
[6]Parisiadou L,Yu J,Sgobio C,et al.LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity[J].Nat Neurosci,2014,17(3):367-376.
[7]Yu J,Sun M,Chen Z,et al.Magnesium modulates amyloid-beta protein precursor trafficking and processing[J].J Alzheimers Dis,2010,20(4):1091-1106.
[8]Cho HJ,Yu J,Xie C,et al.Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER-Golgi export[J].EMBO J,2014,33(20):2314-2331.
[9]Shahrizaila N,Sobue G,Kuwabara S,et al.Amyotrophic lateral sclerosis and motor neuron syndromes in Asia[J].J Neurol Neurosurg Psychiatry,2016,87(8):821-830.
[10]Taylor JP,Brown RH Jr,Cleveland DW.Decoding ALS:from genes to mechanism[J].Nature,2016,539(7628):197-206.
[11]Chen HJ,Anagnostou G,Chai A,et al.Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis[J].J Biol Chem,2010,285(51):40266-40281.
[12]Di L,Chen H,Da Y,et al.Atypical familial amyotrophic lateral sclerosis with initial symptoms of pain or tremor in a Chinese family harboring VAPB-P56S mutation[J].J Neurol,2016,263(2):263-268.
[13]Nishimura AL,Mitne-Neto M,Silva HC,et al.A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis[J].Am J Hum Genet,2004,75(5):822-831.
[14]van Blitterswijk M,van Es MA,Koppers M,et al.VAPB and C9orf72mutations in 1 familial amyotrophic lateral sclerosis patient[J].Neurobiol Aging,2012,33(12):2950 e1-4.
[15]Anagnostou G,Akbar MT,Paul P,et al.Vesicle associated membrane protein B(VAPB)is decreased in ALS spinal cord[J].Neurobiol Aging,2010,31(6):969-985.
[16]Gkogkas C,Middleton S,Kremer AM,et al.VAPB interacts with and modulates the activity of ATF6[J].Hum Mol Genet,2008,17(11):1517-15126.
[17]Peretti D,Dahan N,Shimoni E,et al.Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport[J].Mol Biol Cell,2008,19(9):3871-3884.
[18]Tran D,Chalhoub A,Schooley A,et al.A mutation in VAPB that causes amyotrophic lateral sclerosis also causes a nuclear envelope defect[J].J Cell Sci,2012,125(Pt 12):2831-2836.
[19]De Vos KJ,Morotz GM,Stoica R,et al.VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis[J].Hum Mol Genet,2012,21(6):1299-1311.
[20]Rocha N,Kuijl C,van der Kant R,et al.Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning[J].J Cell Biol,2009,185(7):1209-12025.
[21]Forrest S,Chai A,Sanhueza M,et al.Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis[J].Hum Mol Genet,2013,22(13):2689-2704.
[22]van Zundert B,Izaurieta P,Fritz E,et al.Early pathogenesis in the adult-onset neurodegenerative disease amyotrophic lateral sclerosis[J].J Cell Biochem,2012,113(11):3301-3312.
[23]Martin LJ,Chang Q.Inhibitory synaptic regulation of motoneurons:a new target of disease mechanisms in amyotrophic lateral sclerosis[J].Mol Neurobiol,2012,45(1):30-42.
[24]Jiang M,Schuster JE,Fu R,et al.Progressive changes in synaptic inputs to motoneurons in adult sacral spinal cord of a mouse model of amyotrophic lateral sclerosis[J].J Neurosci,2009,29(48):15031-15038.
[25]Chai A,Withers J,Koh YH,et al.h VAPB,the causative gene of a heterogeneous group of motor neuron diseases in humans,is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction[J].Hum Mol Genet,2008,17(2):266-280.
[26]Pennetta G,Hiesinger PR,Fabian-Fine R,et al.Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner[J].Neuron,2002,35(2):291-306.
[27]Yang Z,Huh SU,Drennan JM,et al.Drosophila Vap-33 is required for axonal localization of Dscam isoforms[J].J Neurosci,2012,32(48):17241-17250.
[28]Kuijpers M,Yu KL,Teuling E,et al.The ALS8 protein VAPB interacts with the ER-Golgi recycling protein YIF1A and regulates membrane delivery into dendrites[J].EMBO J,2013,32(14):2056-2072.
[29]Ratnaparkhi A,Lawless GM,Schweizer FE,et al.A Drosophila model of ALS:human ALS-associated mutation in VAP33A suggests a dominant negative mechanism[J].PLo S One,2008,3(6):e2334.
[30]Larroquette F,Seto L,Gaub PL,et al.Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response[J].Hum Mol Genet,2015,24(22):6515-6529.