单纯疱疹病毒Ⅰ型诱导人成神经细胞瘤细胞SH-SY5Y表达β-淀粉样蛋白
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摘要
目的探讨单纯疱疹病毒Ⅰ型(HSV-1)感染对人成神经细胞瘤细胞SH-SY5Yβ-淀粉样蛋白(Aβ)表达的影响。方法通过非洲绿猴肾细胞Vero扩增HSV-1,测定病毒滴度。取感染复数(MOI)为10的3.2×108PFU/mL HSV-1分别感染SH-SY5Y细胞12、24 h,显微镜下观察SH-SY5Y细胞的形态变化,使用PCR检测细胞中HSV-1 DNA的表达;双色免疫荧光检测SH-SY5Y细胞中Aβ和载脂蛋白E(ApoE)的表达;蛋白质印迹法检测淀粉样蛋白前体(APP)、黑素代谢酶(MME)、ApoE、糖原合成酶激酶3β(GSK-3β)、磷酸化糖原合成酶激酶3β(p-GSK-3β)蛋白的表达。结果 HSV-1感染SH-SY5Y细胞12 h后,镜下可见细胞突触减少,突起回缩;感染24 h后细胞开始聚集,呈圆形,并有细胞脱落。与磷酸盐缓冲液(PBS)对照组相比,HSV-1感染12 h后细胞中Aβ表达增加(P<0.01),ApoE蛋白表达无明显改变;感染24 h后,Aβ表达仍增加(P<0.05),但弱于12 h时(P<0.01),APOE蛋白表达增加(P<0.01)。蛋白质印迹分析结果显示,与PBS对照组比较,HSV-1感染12 h后细胞中APP蛋白的表达水平降低(P<0.05),GSK-3β总蛋白的表达水平增加(P<0.05)且磷酸化活性增强(P<0.05),MME蛋白的表达水平增加(P<0.05);感染24 h时MME蛋白的表达水平降低(P<0.05),ApoE蛋白的表达水平增加(P<0.05)。结论 HSV-1感染后可诱导SH-SY5Y细胞Aβ表达增加,在感染12 h时可能通过促进APP代谢、GSK-3βSer9磷酸化诱导Aβ产生,而在24 h时主要是通过抑制Aβ降解促使Aβ表达增加。
Objective To explore the effect of herpes simplex virus type Ⅰ(HSV-1) infection on expression of β-amyloid(Aβ) in human neuroblastoma cell lines SH-SY5 Y. Methods African green monkey kidney cell lines Vero cells were used to amplified HSV-1, and the virus titers were measured. SH-SY5 Y cells were infected with HSV-1 3.2×108 plaque forming unit(PFU)/mL at a multiplicity of infection(MOI) of 10 for 12 h or 24 h, and the morphological changes of the cells were observed under microscope. PCR was used to detect the expression of HSV-1 DNA. Double-color immunofluorescence assay was performed to show the expression of Aβ and apolipoprotein E(ApoE). Western blotting was used to detect the expression levels of amyloid precursor protein(APP), melanin metabolic enzyme(MME), ApoE, glycogen synthase kinase-3β(GSK-3β) and phosphorylated glycogen synthase kinase-3β(p-GSK-3β). Results After infection with HSV-1 for 12 h, the SH-SY5 Y cells had synapse reduction and neurite retraction and few neurites. And after 24 h of infection, the cells began to aggregate, and were round and shed. Compared with phosphate buffer saline(PBS) control group, the expression of Aβwas significantly increased after infection with HSV-1 for 12 h(P<0.01), while the expression of ApoE protein was not significantly changed. After 24 h of infection, the expressions of Aβ and ApoE were significantly increased(P<0.05), but the expression of Aβ was significantly lower than that on 12 h of post-infection(P<0.01). Western blotting analysis showed that, compared with PBS control group, the expression of APP was significantly decreased on 12 h of post-infection(P<0.05), and the expressions of GSK-3β, p-GSK-3β and MME were significantly increased(P<0.05). However, the expression of MME was significantly decreased(P<0.05) and the expression of ApoE was significantly increased(P<0.05) 24 h post-infection. Conclusion HSV-1 infection induces the expression of Aβ in SH-SY5 Y cells through promoting APP metabolism and Ser9 phosphorylation of GSK-3β on 12 h of post-infection, and inhibiting the degradation of Aβ on 24 h of post-infection.
Objective To explore the effect of herpes simplex virus type Ⅰ(HSV-1) infection on expression of β-amyloid(Aβ) in human neuroblastoma cell lines SH-SY5 Y. Methods African green monkey kidney cell lines Vero cells were used to amplified HSV-1, and the virus titers were measured. SH-SY5 Y cells were infected with HSV-1 3.2×108 plaque forming unit(PFU)/mL at a multiplicity of infection(MOI) of 10 for 12 h or 24 h, and the morphological changes of the cells were observed under microscope. PCR was used to detect the expression of HSV-1 DNA. Double-color immunofluorescence assay was performed to show the expression of Aβ and apolipoprotein E(ApoE). Western blotting was used to detect the expression levels of amyloid precursor protein(APP), melanin metabolic enzyme(MME), ApoE, glycogen synthase kinase-3β(GSK-3β) and phosphorylated glycogen synthase kinase-3β(p-GSK-3β). Results After infection with HSV-1 for 12 h, the SH-SY5 Y cells had synapse reduction and neurite retraction and few neurites. And after 24 h of infection, the cells began to aggregate, and were round and shed. Compared with phosphate buffer saline(PBS) control group, the expression of Aβwas significantly increased after infection with HSV-1 for 12 h(P<0.01), while the expression of ApoE protein was not significantly changed. After 24 h of infection, the expressions of Aβ and ApoE were significantly increased(P<0.05), but the expression of Aβ was significantly lower than that on 12 h of post-infection(P<0.01). Western blotting analysis showed that, compared with PBS control group, the expression of APP was significantly decreased on 12 h of post-infection(P<0.05), and the expressions of GSK-3β, p-GSK-3β and MME were significantly increased(P<0.05). However, the expression of MME was significantly decreased(P<0.05) and the expression of ApoE was significantly increased(P<0.05) 24 h post-infection. Conclusion HSV-1 infection induces the expression of Aβ in SH-SY5 Y cells through promoting APP metabolism and Ser9 phosphorylation of GSK-3β on 12 h of post-infection, and inhibiting the degradation of Aβ on 24 h of post-infection.
引文
[1]MIKLOSSY J.Emerging roles of pathogens in Alzheimer disease[J/OL].Expert Rev Mol Med,2011,13:e30.doi:10.1017/s1462399411002006.
[2]JAMIESON G A,MAITLAND N J,WILCOCK G K,CRASKE J,ITZHAKI R F.Latent herpes simplex virus type 1 in normal and Alzheimer’s disease brains[J].JMed Virol,1991,33:224-227.
[3]WOZNIAK M A,ITZHAKI R F,SHIPLEY S J,DOBSON C B.Herpes simplex virus infection causes cellularβ-amyloid accumulation and secretase upregulation[J].Neurosci Lett,2007,429(2/3):95-100.
[4]SANTANA S,RECUERO M,BULLIDO M J,VALDIVIESO F,ALDUDO J.Herpes simplex virus typeⅠinduces the accumulation of intracellular beta-amyloid in autophagic compartments and the inhibition of the non-amyloidogenic pathway in human neuroblastoma cells[J].Neurobiol Aging,2012,33:430.
[5]CRIBBS D H,AZIZEH B Y,COTMAN C W,LAFERLAF M.Fibril formation and neurotoxicity by a herpes simplex virus glycoprotein B fragment with homology to the Alzheimer’s Aβpeptide[J].Biochemistry,2000,39:5988-5994.
[6]BOURGADE K,GARNEAU H,GIROUX G,LEPAGE A Y,BOCTI C,DUPUIS G,et al.β-Amyloid peptides display protective activity against the human Alzheimer’s disease-associated herpes simplex virus-1[J].Biogerontology,2014,16:85-98.
[7]ITZHAKI R F,WOZNIAK M A.Herpes simplex virus type 1 in Alzheimer’s disease:the enemy within[J].JAlzheimers Dis,2008,13:393-405.
[8]MCNAMARA J,ANN MURRAY T A.Connections between herpes simplex virus type 1 and Alzheimer’s disease pathogenesis[J].Curr Alzheimer Res,2016,13:996-1005.
[9]SHIPLEY M M,MANGOLD C A,KUNY C V,SZPARA M L.Differentiated human SH-SY5Y cells provide a reductionist model of herpes simplex virus1 neurotropism[J/OL].J Virol,2017,91.doi:10.1128/jvi.00958-17.
[10]SPEAR P G.Herpes simplex virus:receptors and ligands for cell entry[J].Cell Microbiol,2004,6:401-410.
[11]WOZNIAK M A,MEE A P,ITZHAKI R F.Herpes simplex virus type 1 DNA is located within Alzheimer’s disease amyloid plaques[J].J Pathol,2009,217:131-138.
[12]YOUMANS K L,TAI L M,KANEKIYO T,STINEW B,MICHON S C,NWABUISI-HEATH E,et al.Intraneuronal Aβdetection in 5xFAD mice by a new Aβ-specific antibody[J].Mol Neurodegener,2012,7:8.
[13]DE CHIARA G,MARCOCCI M E,CIVITELLI L,ARGNANI R,PIACENTINI R,RIPOLI C,et al.APPprocessing induced by herpes simplex virus type 1(HSV-1)yields several APP fragments in human and rat neuronal cells[J/OL].PLoS One,2010,5:e13989.doi:10.1371/journal.pone.0013989.
[14]HARRIS S A,HARRIS E A.Molecular mechanisms for herpes simplex virus type 1 pathogenesis in Alzheimer’s disease[J].Front Aging Neurosci,2018,10:48.
[15]PIACENTINI R,LI PUMA D D,RIPOLI C,MARCOCCI M E,DE CHIARA G,GARACI E,et al.Herpes simplex virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-beta protein accumulation[J/OL].Sci Rep,2015,5:15444.doi:10.1038/srep15444.
[16]CIVITELLI L,MARCOCCI M E,CELESTINO I,PIACENTINI R,GARACI E,GRASSI C,et al.Herpes simplex virus type 1 infection in neurons leads to production and nuclear localization of APP intracellular domain(AICD):implications for Alzheimer’s disease pathogenesis[J].J Neurovirol,2015,21:480-490.
[17]NAGHAVI M H,GUNDERSEN G G,WALSH D.Plus-end tracking proteins,CLASPs,and a viral Akt mimic regulate herpesvirus-induced stable microtubule formation and virus spread[J].Proc Natl Acad Sci USA,2013,110:18268-18273.
[2]JAMIESON G A,MAITLAND N J,WILCOCK G K,CRASKE J,ITZHAKI R F.Latent herpes simplex virus type 1 in normal and Alzheimer’s disease brains[J].JMed Virol,1991,33:224-227.
[3]WOZNIAK M A,ITZHAKI R F,SHIPLEY S J,DOBSON C B.Herpes simplex virus infection causes cellularβ-amyloid accumulation and secretase upregulation[J].Neurosci Lett,2007,429(2/3):95-100.
[4]SANTANA S,RECUERO M,BULLIDO M J,VALDIVIESO F,ALDUDO J.Herpes simplex virus typeⅠinduces the accumulation of intracellular beta-amyloid in autophagic compartments and the inhibition of the non-amyloidogenic pathway in human neuroblastoma cells[J].Neurobiol Aging,2012,33:430.
[5]CRIBBS D H,AZIZEH B Y,COTMAN C W,LAFERLAF M.Fibril formation and neurotoxicity by a herpes simplex virus glycoprotein B fragment with homology to the Alzheimer’s Aβpeptide[J].Biochemistry,2000,39:5988-5994.
[6]BOURGADE K,GARNEAU H,GIROUX G,LEPAGE A Y,BOCTI C,DUPUIS G,et al.β-Amyloid peptides display protective activity against the human Alzheimer’s disease-associated herpes simplex virus-1[J].Biogerontology,2014,16:85-98.
[7]ITZHAKI R F,WOZNIAK M A.Herpes simplex virus type 1 in Alzheimer’s disease:the enemy within[J].JAlzheimers Dis,2008,13:393-405.
[8]MCNAMARA J,ANN MURRAY T A.Connections between herpes simplex virus type 1 and Alzheimer’s disease pathogenesis[J].Curr Alzheimer Res,2016,13:996-1005.
[9]SHIPLEY M M,MANGOLD C A,KUNY C V,SZPARA M L.Differentiated human SH-SY5Y cells provide a reductionist model of herpes simplex virus1 neurotropism[J/OL].J Virol,2017,91.doi:10.1128/jvi.00958-17.
[10]SPEAR P G.Herpes simplex virus:receptors and ligands for cell entry[J].Cell Microbiol,2004,6:401-410.
[11]WOZNIAK M A,MEE A P,ITZHAKI R F.Herpes simplex virus type 1 DNA is located within Alzheimer’s disease amyloid plaques[J].J Pathol,2009,217:131-138.
[12]YOUMANS K L,TAI L M,KANEKIYO T,STINEW B,MICHON S C,NWABUISI-HEATH E,et al.Intraneuronal Aβdetection in 5xFAD mice by a new Aβ-specific antibody[J].Mol Neurodegener,2012,7:8.
[13]DE CHIARA G,MARCOCCI M E,CIVITELLI L,ARGNANI R,PIACENTINI R,RIPOLI C,et al.APPprocessing induced by herpes simplex virus type 1(HSV-1)yields several APP fragments in human and rat neuronal cells[J/OL].PLoS One,2010,5:e13989.doi:10.1371/journal.pone.0013989.
[14]HARRIS S A,HARRIS E A.Molecular mechanisms for herpes simplex virus type 1 pathogenesis in Alzheimer’s disease[J].Front Aging Neurosci,2018,10:48.
[15]PIACENTINI R,LI PUMA D D,RIPOLI C,MARCOCCI M E,DE CHIARA G,GARACI E,et al.Herpes simplex virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-beta protein accumulation[J/OL].Sci Rep,2015,5:15444.doi:10.1038/srep15444.
[16]CIVITELLI L,MARCOCCI M E,CELESTINO I,PIACENTINI R,GARACI E,GRASSI C,et al.Herpes simplex virus type 1 infection in neurons leads to production and nuclear localization of APP intracellular domain(AICD):implications for Alzheimer’s disease pathogenesis[J].J Neurovirol,2015,21:480-490.
[17]NAGHAVI M H,GUNDERSEN G G,WALSH D.Plus-end tracking proteins,CLASPs,and a viral Akt mimic regulate herpesvirus-induced stable microtubule formation and virus spread[J].Proc Natl Acad Sci USA,2013,110:18268-18273.