OBJECTIVE Pleckstrin homologylike domain family A member 1(PHLDA1),also known as TDAG51,was first identified as a potential transcription factor required for Fas expression and activation-induced apoptosis in mouse T ...OBJECTIVE Pleckstrin homologylike domain family A member 1(PHLDA1),also known as TDAG51,was first identified as a potential transcription factor required for Fas expression and activation-induced apoptosis in mouse T cell hybridomas.Subsequent research showed that PHILDA1 plays crucial roles in cell proliferation and apoptosis,PHLDA1 deficiency caused several phenotypic changes in macrophages that increased cytoprotection against oxidative and endoplasmic reticulum(ER) stress.However,little is known about the critical role of PHLDA1 in the neuroinflammation and neuronal apoptosis.METHODS Mouse Parkinson disease(PD) model was induced by intraperitoneal injection(ip) of MPTP for 7 d.Immunohistofluorescence staining was used to observe TH,Iba-1 and PHLDA1 in brain slides.Primary microglia and BV2 microglial cell lines were exposed to lipopolysacchrides(LPS) at different time points to determine PHLDA1 mRNA and protein level in vitro.BV2 cells with PHLDA1 knockdown using lentivirus experessing shRNA were exposed to LPS for the detection of proinflammatory genes expression by q-PCR or Western blotting.The NF-κB and MAPK pathways were detected to reveal the underlying mechanisms of PHLDA1 regulate microglia activation.Furthermore,to determine the effect of PHLDA1 deletion in the substantia nigra(SN) on motor dysfunction,the mice were previously infected with AAV-GFP(control) or AAV-shP HLDA1 in the SN in PD model,then were administered with behavior assessment.RESULTS PHLDA1 was expressed in microglia.PHLDA1 was increased in primary microglia or BV2 microglia cell lines by LPS treatment,acted as a positive regulator of TLR4 signaling.PHLDA1 deficiency suppressed LPS-induced microglia activation.The underlying mechanisms of PHLDA1 might be through directly interacting with TRAF6 to promote its auto-ubiquitination consequently enhanced neuroinflammatory.In PD model,PHLDA1 deletion in the SN limits microglial activation and protects dopaminergic neurons.PHLDA1 deficiency mice have a good behavioral score.CONCLUSION PHLDA1 deficiency in SN limits microglial inflammatory responses and protects dopaminergic neurons through attenuating auto-ubiquitination of TRAF6 in PD model.展开更多
目的研究人Plecstrin同源域家族A成员1(Pleckstrin homology like domain family A member 1,PHLDA1)与胶质瘤恶性表型关系。方法收集马鞍山市中心医院神经外科和上海新华医院神经外科2015年1月至2020年4月65例原发脑胶质瘤的速冻标本...目的研究人Plecstrin同源域家族A成员1(Pleckstrin homology like domain family A member 1,PHLDA1)与胶质瘤恶性表型关系。方法收集马鞍山市中心医院神经外科和上海新华医院神经外科2015年1月至2020年4月65例原发脑胶质瘤的速冻标本。从接受尸检的非胶质瘤患者中收集了5份脑组织样本作为对照。利用免疫组化方法检测样品中的PHLDA1表达。采用免疫荧光、流式细胞术、集落形成法、体外细胞迁移侵袭法对成人胶质瘤U87细胞系和U251细胞系进行胶质瘤表型分析。结果免疫组化结果显示,随着胶质瘤WHO分级的增加,PHLDA1染色强度增加(x^(2)趋势=24.145,P<0.01)。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法(MTT试验)结果显示,与空白对照组和阴性干扰组相比,当SiRNA干扰敲低PHLDA1后,U87和U251培养48 h后的增殖率显著降低(P<0.05)。流式细胞仪检测显示,PHLDA1干扰后,U87和U251细胞凋亡率高于对照组(P<0.01),G0-G1期细胞比例低于对照组(P<0.05)而S期比例高于对照组(P<0.01)。transwell试验显示,PHLDA1干扰组的U87和U251迁移率明显低于对照组(P<0.01)。结论PHLDA1在胶质瘤中过表达。功能分析显示PHLDA1在胶质瘤的迁移和侵袭中起重要作用。PHLDA1可能成为胶质瘤新的潜在治疗靶点。展开更多
基金National Natural Science Foundation of China(81373382,81773702,81130023)PriorityAcademic Program Development of JiangsuHigher Education Institutes (PAPD).
文摘OBJECTIVE Pleckstrin homologylike domain family A member 1(PHLDA1),also known as TDAG51,was first identified as a potential transcription factor required for Fas expression and activation-induced apoptosis in mouse T cell hybridomas.Subsequent research showed that PHILDA1 plays crucial roles in cell proliferation and apoptosis,PHLDA1 deficiency caused several phenotypic changes in macrophages that increased cytoprotection against oxidative and endoplasmic reticulum(ER) stress.However,little is known about the critical role of PHLDA1 in the neuroinflammation and neuronal apoptosis.METHODS Mouse Parkinson disease(PD) model was induced by intraperitoneal injection(ip) of MPTP for 7 d.Immunohistofluorescence staining was used to observe TH,Iba-1 and PHLDA1 in brain slides.Primary microglia and BV2 microglial cell lines were exposed to lipopolysacchrides(LPS) at different time points to determine PHLDA1 mRNA and protein level in vitro.BV2 cells with PHLDA1 knockdown using lentivirus experessing shRNA were exposed to LPS for the detection of proinflammatory genes expression by q-PCR or Western blotting.The NF-κB and MAPK pathways were detected to reveal the underlying mechanisms of PHLDA1 regulate microglia activation.Furthermore,to determine the effect of PHLDA1 deletion in the substantia nigra(SN) on motor dysfunction,the mice were previously infected with AAV-GFP(control) or AAV-shP HLDA1 in the SN in PD model,then were administered with behavior assessment.RESULTS PHLDA1 was expressed in microglia.PHLDA1 was increased in primary microglia or BV2 microglia cell lines by LPS treatment,acted as a positive regulator of TLR4 signaling.PHLDA1 deficiency suppressed LPS-induced microglia activation.The underlying mechanisms of PHLDA1 might be through directly interacting with TRAF6 to promote its auto-ubiquitination consequently enhanced neuroinflammatory.In PD model,PHLDA1 deletion in the SN limits microglial activation and protects dopaminergic neurons.PHLDA1 deficiency mice have a good behavioral score.CONCLUSION PHLDA1 deficiency in SN limits microglial inflammatory responses and protects dopaminergic neurons through attenuating auto-ubiquitination of TRAF6 in PD model.
文摘目的研究人Plecstrin同源域家族A成员1(Pleckstrin homology like domain family A member 1,PHLDA1)与胶质瘤恶性表型关系。方法收集马鞍山市中心医院神经外科和上海新华医院神经外科2015年1月至2020年4月65例原发脑胶质瘤的速冻标本。从接受尸检的非胶质瘤患者中收集了5份脑组织样本作为对照。利用免疫组化方法检测样品中的PHLDA1表达。采用免疫荧光、流式细胞术、集落形成法、体外细胞迁移侵袭法对成人胶质瘤U87细胞系和U251细胞系进行胶质瘤表型分析。结果免疫组化结果显示,随着胶质瘤WHO分级的增加,PHLDA1染色强度增加(x^(2)趋势=24.145,P<0.01)。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法(MTT试验)结果显示,与空白对照组和阴性干扰组相比,当SiRNA干扰敲低PHLDA1后,U87和U251培养48 h后的增殖率显著降低(P<0.05)。流式细胞仪检测显示,PHLDA1干扰后,U87和U251细胞凋亡率高于对照组(P<0.01),G0-G1期细胞比例低于对照组(P<0.05)而S期比例高于对照组(P<0.01)。transwell试验显示,PHLDA1干扰组的U87和U251迁移率明显低于对照组(P<0.01)。结论PHLDA1在胶质瘤中过表达。功能分析显示PHLDA1在胶质瘤的迁移和侵袭中起重要作用。PHLDA1可能成为胶质瘤新的潜在治疗靶点。
