TDP-43 seeding induces cytoplasmic aggregation heterogeneity and nuclear loss-of-function of TDP-43

Cytoplasmic aggregation and nuclear depletion of TDP-43 are hallmarks of several age-related neurodegenerative disorders. Yet, recapitulating both features in cellular systems has been a major challenge. Here, we produced amyloid-like fibrils from the recombinant low-complexity-domain of TDP-43, and demonstrate that sonicated fibrils trigger TDP-43 pathology in human cell lines and iPSC-derived neurons. Fibril-induced cytoplasmic TDP-43 inclusions acquire distinct biophysical properties, recapitulate pathological hallmarks such as phosphorylation, ubiquitin- and p62-accumulation, and recruit nuclear endogenous TDP-43 leading to nuclear-loss-of-function-driven disease-specific cryptic splicing defects. Cytoplasmic TDP-43 aggregates exhibit, with time, distinct heterogeneous morphologies as in patients including compacted, filamentous or fragmented, through the activation of cellular protein clearance pathways. Cell-specific progressive toxicity is provoked by seeded TDP-43 pathology in human neurons. These findings identify templated aggregation of TDP-43 as a key mechanism driving both cytoplasmic gain- and nuclear-loss-of-function, offering a valuable approach to identify modifiers of sporadic TDP-43 proteinopathies. TDP-43-mNLS-Clover expressing U2OS cells (doxycycline induced) were transfected with fibril seeds produced from recombinant TDP-43 LCD (50nM) to induce TDP-43 aggregation. In control conditions, TDP-43-mNLS-Clover expressing U2OS cells were mock transfected with protein storage buffer. Each transfection reaction was performed in triplicates, serving as 3 independent biological replicates. 48 hours after fibril (or control) transfection, cells were collected and image-based FACS sorting was performed using FACSDiscover S8 to isolate cells with TDP-43-Clover aggregates (from fibril transfected condition) or cells with diffuse TDP-43-Clover (from mock transfected condition). FACS-purified cell populations (aggregates vs no_aggregates) were subsequently processed for RNA-Seq.

TDP-43的细胞质聚集与核耗竭是多种年龄相关性神经退行性疾病的标志性病理特征。然而，在细胞体系中同时重现这两种表型一直是一项重大挑战。本研究从重组表达的TDP-43低复杂度结构域（low-complexity-domain, LCD）中制备了淀粉样蛋白样纤维，并证实经超声处理的纤维可在人类细胞系及诱导多能干细胞（induced pluripotent stem cell, iPSC）衍生神经元中诱发TDP-43病理改变。
纤维诱导产生的细胞质TDP-43包涵体具备独特的生物物理特性，可重现磷酸化、泛素积累及p62蛋白聚集等典型病理特征，并会募集内源性核TDP-43，进而引发由核功能缺失介导的疾病特异性隐蔽剪接缺陷。
随着时间推移，细胞质TDP-43聚集体会呈现出与患者体内一致的异质性形态谱，包括紧密聚集型、丝状及碎片化结构，该过程伴随细胞蛋白质清除通路的激活。
人类神经元中的种子诱导型TDP-43病理可引发细胞类型特异性的进行性毒性损伤。
本研究结果证实，TDP-43的模板化聚集是同时驱动细胞质功能获得与核功能缺失的关键机制，为识别散发性TDP-43蛋白病的修饰因子提供了极具价值的研究策略。
将表达TDP-43-mNLS-Clover的U2OS细胞（经多西环素诱导）用重组TDP-43 LCD制备的纤维种子（50nM）进行转染，以诱导TDP-43聚集。
对照组中，表达TDP-43-mNLS-Clover的U2OS细胞采用蛋白质储存缓冲液进行空白转染。
每组转染实验均设置三次重复，作为3个独立的生物学重复样本。
在纤维（或对照）转染48小时后，收集细胞并使用FACSDiscover S8设备开展基于成像的荧光激活细胞分选（fluorescence-activated cell sorting, FACS），以分离出带有TDP-43-Clover聚集体的细胞（来自纤维转染组）或呈现弥散性TDP-43-Clover信号的细胞（来自空白转染组）。
随后将经FACS分选得到的细胞群（聚集体组与无聚集体组）进行RNA测序（RNA-Seq）分析。