Alzheimer’s disease (AD) is a complex neurodegenerative disease, pathologically characterized by the accumulation of β-amyloid peptide (Aβ) and intraneuronal neurofibrillary tangles. Production of the Aβpeptide by...Alzheimer’s disease (AD) is a complex neurodegenerative disease, pathologically characterized by the accumulation of β-amyloid peptide (Aβ) and intraneuronal neurofibrillary tangles. Production of the Aβpeptide by the regulated intra-membrane proteolysis of the β-amyloid precursor protein (APP) is a central event in AD.Cognitive decline observed in AD models is dependent on Aβgeneration. One of the downstream effects mediated by soluble Aβoligomersincludes the hyperactivation of calcineurin (CaN), a Ca2+and calmodulin (CaM)-dependent, serinethreonine protein phosphatase. CaN is one of the major phosphatase associated with cognitive and neurodegenerative effects of Aβ. A specific isoform of CaN, CaNAβ, is one of the most up-regulated mRNAs in brains of early stage AD patients. While Aβ’s ability to activate CaN has been well studied, not much is known about how increased levels of CaN in AD brain can contribute towards Aβgeneration. In this study we specifically investigate if increased levels of CaNAβ regulate APP metabolism. Our results demonstrate that increased CaNAβ expressionleads to increased APP levels and increased proteolytic products of APP, including Aβ. The data suggest that upregulation of CaN levels could contribute to neurodegeneration observed in AD by increasing levels ofAβ, potentially forming a pathogenic feed forward loop.展开更多
目的从原代培养的大鼠心肌细胞中扩增钙调磷酸酶(Calcineurin,CaN)AβcD N A序列,构建重组真核表达质粒pcDN A3.1-CaN,并检测其表达情况,为进一步研究C aN及其在心肌损伤的信号转导途径中的作用奠定基础。方法用RT-PCR方法从原代培养的...目的从原代培养的大鼠心肌细胞中扩增钙调磷酸酶(Calcineurin,CaN)AβcD N A序列,构建重组真核表达质粒pcDN A3.1-CaN,并检测其表达情况,为进一步研究C aN及其在心肌损伤的信号转导途径中的作用奠定基础。方法用RT-PCR方法从原代培养的大鼠心肌细胞R N A中扩增出C aN AβcD N A序列,测序后将其用限制性内切酶双酶切,然后与真核表达质粒pcD N A3.1(+)进行连接,再转化D H 5α,将鉴定为阳性的真核表达质粒转染人胚肾293T细胞,用免疫荧光细胞化学技术检测其表达情况。结果①将鉴定为阳性的质粒进行测序后,所测序列结果与G eneBank的C aN Aβ基因序列同源性达到91.7%;②真核表达载体pcDN A3.1-CaN转染293T细胞后经免疫荧光细胞化学染色检测到钙调磷酸酶基因在人胚肾293T细胞中表达。结论正确克隆出了大鼠心肌细胞C aN Aβ基因并建立了CaNAβ基因的真核表达载体pcDN A3.1-CaN。展开更多
文摘Alzheimer’s disease (AD) is a complex neurodegenerative disease, pathologically characterized by the accumulation of β-amyloid peptide (Aβ) and intraneuronal neurofibrillary tangles. Production of the Aβpeptide by the regulated intra-membrane proteolysis of the β-amyloid precursor protein (APP) is a central event in AD.Cognitive decline observed in AD models is dependent on Aβgeneration. One of the downstream effects mediated by soluble Aβoligomersincludes the hyperactivation of calcineurin (CaN), a Ca2+and calmodulin (CaM)-dependent, serinethreonine protein phosphatase. CaN is one of the major phosphatase associated with cognitive and neurodegenerative effects of Aβ. A specific isoform of CaN, CaNAβ, is one of the most up-regulated mRNAs in brains of early stage AD patients. While Aβ’s ability to activate CaN has been well studied, not much is known about how increased levels of CaN in AD brain can contribute towards Aβgeneration. In this study we specifically investigate if increased levels of CaNAβ regulate APP metabolism. Our results demonstrate that increased CaNAβ expressionleads to increased APP levels and increased proteolytic products of APP, including Aβ. The data suggest that upregulation of CaN levels could contribute to neurodegeneration observed in AD by increasing levels ofAβ, potentially forming a pathogenic feed forward loop.
文摘目的从原代培养的大鼠心肌细胞中扩增钙调磷酸酶(Calcineurin,CaN)AβcD N A序列,构建重组真核表达质粒pcDN A3.1-CaN,并检测其表达情况,为进一步研究C aN及其在心肌损伤的信号转导途径中的作用奠定基础。方法用RT-PCR方法从原代培养的大鼠心肌细胞R N A中扩增出C aN AβcD N A序列,测序后将其用限制性内切酶双酶切,然后与真核表达质粒pcD N A3.1(+)进行连接,再转化D H 5α,将鉴定为阳性的真核表达质粒转染人胚肾293T细胞,用免疫荧光细胞化学技术检测其表达情况。结果①将鉴定为阳性的质粒进行测序后,所测序列结果与G eneBank的C aN Aβ基因序列同源性达到91.7%;②真核表达载体pcDN A3.1-CaN转染293T细胞后经免疫荧光细胞化学染色检测到钙调磷酸酶基因在人胚肾293T细胞中表达。结论正确克隆出了大鼠心肌细胞C aN Aβ基因并建立了CaNAβ基因的真核表达载体pcDN A3.1-CaN。
