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Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies.
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2019-08-19 , DOI: 10.1021/acschemneuro.9b00217 Elian Dupré 1 , Clément Danis 1, 2 , Alexis Arrial 3 , Xavier Hanoulle 1 , Mégane Homa 2 , François-Xavier Cantrelle 1 , Hamida Merzougui 1 , Morvane Colin 2 , Jean-Christophe Rain 3 , Luc Buée 2 , Isabelle Landrieu 1
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2019-08-19 , DOI: 10.1021/acschemneuro.9b00217 Elian Dupré 1 , Clément Danis 1, 2 , Alexis Arrial 3 , Xavier Hanoulle 1 , Mégane Homa 2 , François-Xavier Cantrelle 1 , Hamida Merzougui 1 , Morvane Colin 2 , Jean-Christophe Rain 3 , Luc Buée 2 , Isabelle Landrieu 1
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Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood. New tools are thus needed to decipher tau pathways involved in the diseases. In this context, a family of novel single domain antibody fragments, or VHHs, directed against tau were generated and characterized. Among the selected VHHs obtained from screening of a synthetic library, a family of six VHHs shared the same CDR3 recognition loop and recognized the same epitope, located in the C-terminal domain of tau. Affinity parameters characterizing the tau/VHHs interaction were next evaluated using surface plasmon resonance spectroscopy. The equilibrium constants KD were in the micromolar range, but despite conservation of the CDR3 loop sequence, a range of affinities was observed for this VHH family. One of these VHHs, named F8-2, was additionally shown to bind tau upon expression in a neuronal cell line model. Optimization of VHH F8-2 by yeast two-hybrid allowed the generation of an optimized VHH family characterized by lower KD than that of the F8-2 wild-type counterpart, and recognizing the same epitope. The optimized VHHs can also be used as antibodies for detecting tau in transgenic mice brain tissues. These results validate the use of these VHHs for in vitro studies, but also their potential for in-cell expression and assays in mouse models, to explore the mechanisms underlying tau physiopathology.
中文翻译：
单域抗体片段作为检测细胞和小鼠研究中神经元Tau蛋白的新工具。
Tau是一种神经元蛋白，与称为Tauopathies（包括阿尔茨海默氏病）的病理相关。在阿尔茨海默氏病中，tau聚集成细丝，导致观察到神经内神经原纤维缠结。导致tau聚集和tau病理学扩散通过大脑区域的分子机制仍不完全清楚。因此，需要新的工具来破译与疾病有关的tau途径。在这种情况下，产生并表征了针对tau的新的单结构域抗体片段或VHHs家族。在通过筛选合成文库获得的选定VHH中，六个家族的VHH共享相同的CDR3识别环，并识别位于tau C端结构域的相同表位。接下来，使用表面等离振子共振光谱法评估表征tau / VHHs相互作用的亲和力参数。平衡常数KD在微摩尔范围内，但是尽管CDR3环序列保守，但仍观察到该VHH家族的亲和力范围。还显示了这些VHH之一，名为F8-2，在神经元细胞系模型中表达后可与tau结合。通过酵母双杂交对VHH F8-2的优化允许生成一个优化的VHH家族，其特征是其KD比F8-2野生型对应物的KD低，并识别相同的表位。优化的VHHs也可以用作在转基因小鼠脑组织中检测tau的抗体。这些结果验证了这些VHH在体外研究中的用途，也证明了它们在小鼠模型中进行细胞内表达和测定的潜力，
更新日期：2019-08-05
中文翻译：
单域抗体片段作为检测细胞和小鼠研究中神经元Tau蛋白的新工具。
Tau是一种神经元蛋白，与称为Tauopathies（包括阿尔茨海默氏病）的病理相关。在阿尔茨海默氏病中，tau聚集成细丝，导致观察到神经内神经原纤维缠结。导致tau聚集和tau病理学扩散通过大脑区域的分子机制仍不完全清楚。因此，需要新的工具来破译与疾病有关的tau途径。在这种情况下，产生并表征了针对tau的新的单结构域抗体片段或VHHs家族。在通过筛选合成文库获得的选定VHH中，六个家族的VHH共享相同的CDR3识别环，并识别位于tau C端结构域的相同表位。接下来，使用表面等离振子共振光谱法评估表征tau / VHHs相互作用的亲和力参数。平衡常数KD在微摩尔范围内，但是尽管CDR3环序列保守，但仍观察到该VHH家族的亲和力范围。还显示了这些VHH之一，名为F8-2，在神经元细胞系模型中表达后可与tau结合。通过酵母双杂交对VHH F8-2的优化允许生成一个优化的VHH家族，其特征是其KD比F8-2野生型对应物的KD低，并识别相同的表位。优化的VHHs也可以用作在转基因小鼠脑组织中检测tau的抗体。这些结果验证了这些VHH在体外研究中的用途，也证明了它们在小鼠模型中进行细胞内表达和测定的潜力，
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