The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
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DOI：
10.3389/fnins.2015.00184
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发表时间：
2015-05-29
影响因子：
4.3
通讯作者：
Tolkovsky, Aviva M.
中科院分区：
文献类型：
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作者：
Brelstaff, Jack;Ossola, Bernardino;Tolkovsky, Aviva M.
在活细胞培养系统中鉴定标记神经退行性疾病特征的丝状蛋白质聚集体（例如阿尔茨海默病中的β-淀粉样蛋白和tau蛋白）的荧光染料以前是一个主要障碍。在这里，我们表明，五聚体甲酰噻吩乙酸（pFTAA）在成年P301 S tau转基因小鼠培养的活神经元中实现了这一功能。将pFTAA注射到5个月大的P301 S tau小鼠中，检测到皮质和DRG神经元对AT 100或MC 1具有免疫反应性，AT 100是一种仅识别丝状tau的抗体，MC 1是一种识别tau构象变化的抗体，其与阿尔茨海默病大脑中的神经元缠结形成相称。在背根神经节（DRG）神经元的固定培养物中，pFTAA结合，也确定了AT 100或MC 1 +ve神经元，遵循一个单一的，饱和的结合曲线，半饱和常数为0.14 μ M，第一次报告的β-折叠活性染料的结合亲和力的测量，以原代神经元窝藏丝状tau。用溶解丝状tau的甲酸处理，提取pFTAA，并防止pFTAA和MC 1的再结合，而不干扰可溶性tau的表达，使用抗人tau（HT 7）抗体检测。在活的培养物中，pFTAA仅鉴定了固定后为AT 100/MC 1 +ve的DRG神经元，证实了这些形式的tau预先存在于活的神经元中。pFTAA区分含有丝状tau的活神经元与其他神经元的效用通过显示在25天内比pFTAA-ve神经元更多的pFTAA+ve神经元死亡来证明。由于pFTAA鉴定了培养的活神经元和几种神经退行性疾病的动物模型以及人脑中的纤维状tau和其他错误折叠蛋白，因此它将在整理疾病机制和鉴定疾病调节药物方面具有相当大的应用，这些药物最终将有助于建立人类神经退行性疾病中的神经退行性机制。
Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as beta-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 mu M, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying diseasemodifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.