Data_Sheet_1_EFhd2 co-aggregates with monomeric and filamentous tau in vitro.pdf

Tauopathies are characterized by the abnormal buildup of tau protein, with early oligomeric forms associated with neurodegeneration and the later neurofibrillary tangles possibly conferring neuroprotection. The molecular mechanisms governing the formation of these tau species are unclear. Lately, there has been an increased focus on examining the interactions between tau and other proteins, along with their influence on the aggregation of tau. Our previous work revealed EFhd2’s association with pathological tau in animal models and tauopathy brains. Herein, we examined the impact of EFhd2 on monomeric and filamentous tau in vitro. The results demonstrated that EFhd2 incubation with monomeric full length human tau (hTau40) formed amorphous aggregates, where both EFhd2 and hTau40 colocalized. Moreover, EFhd2 is entangled with arachidonic acid (ARA)-induced filamentous hTau40. Furthermore, EFhd2-induced aggregation with monomeric and filamentous hTau40 is EFhd2 concentration dependent. Using sandwich ELISA assays, we assessed the reactivity of TOC1 and Alz50—two conformation-specific tau antibodies—to EFhd2-hTau40 aggregates (in absence and presence of ARA). No TOC1 signal was detected in EFhd2 aggregates with monomeric hTau40 whereas EFhd2 aggregates with hTau in the presence of ARA showed a higher signal compared to hTau40 filaments. In contrast, EFhd2 aggregates with both monomeric and filamentous hTau40 reduced Alz50 reactivity. Taken together, our results illustrate for the first time that EFhd2, a tau-associated protein, interacts with monomeric and filamentous hTau40 to form large aggregates that are starkly different from tau oligomers and filaments. Given these findings and previous research, we hypothesize that EFhd2 may play a role in the formation of tau aggregates. Nevertheless, further in vivo studies are imperative to test this hypothesis.

tau蛋白病（tauopathies）以tau蛋白的异常聚集为特征，其早期寡聚体形式与神经退行性变相关，而晚期形成的神经原纤维缠结则可能发挥神经保护作用。目前，调控这类tau聚集体形式形成的分子机制仍不明确。近年来，研究焦点逐渐转向tau与其他蛋白的相互作用，以及二者对tau聚集过程的影响。我们前期研究发现，EFhd2与动物模型及tau蛋白病患者脑组织中的病理性tau存在关联。本研究中，我们体外探究了EFhd2对单体及丝状tau的影响。结果显示，将EFhd2与全长人类单体tau（hTau40）共孵育后，可形成无定形聚集体，且EFhd2与hTau40在此聚集体中共定位。此外，EFhd2可与花生四烯酸（arachidonic acid, ARA）诱导的丝状hTau40发生相互缠结。进一步研究表明，EFhd2诱导单体及丝状hTau40聚集的效应呈EFhd2浓度依赖性。我们采用夹心酶联免疫吸附试验（sandwich ELISA），检测了两种构象特异性tau抗体TOC1和Alz50对EFhd2-hTau40聚集体（在有、无ARA条件下）的反应性。结果显示，仅由单体hTau40与EFhd2形成的聚集体中未检测到TOC1信号；而在ARA存在时，EFhd2与hTau形成的聚集体的TOC1信号强度高于单纯hTau40丝状聚集体。与之相反，无论是单体还是丝状hTau40与EFhd2形成的聚集体，其Alz50反应性均有所降低。综上，本研究首次证实，tau相关蛋白EFhd2可与单体及丝状hTau40相互作用，形成与tau寡聚体及丝状tau结构截然不同的大型聚集体。结合本研究结果及既往研究，我们推测EFhd2可能参与tau聚集体的形成过程。不过，仍需开展进一步的体内实验以验证这一假说。