The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients.

Abbreviation: atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1

先兆子痫（PE）是一种严重的妊娠并发症，其病因仍然是一个谜。我们已经证明，蛋白质病是神经退行性疾病的一种病理特征，是 PE 患者胎盘和血清中的一个关键观察结果。我们假设介导聚集蛋白降解和受损细胞器的巨自噬/自噬机制在 PE 中受损。在这里，我们发现，溶酶体生物发生的主要转录调节因子 TFEB（转录因子 EB）及其调节蛋白 LAMP1、LAMP2 和 CTSD（组织蛋白酶 D）在早期和晚期 PE 分娩的胎盘中失调。原代人滋养层细胞和永生化绒毛外滋养层细胞 (EVT) 在缺氧时表现出 TFEB 表达和核易位以及溶酶体蛋白含量降低。缺氧暴露的滋养层细胞还表现出 PPP3/钙调神经磷酸酶活性降低和 XPO1/CRM1（输出蛋白 1）增加，这些事件抑制 TFEB 核转位。这些蛋白质在 PE 胎盘中也失调。这些结果得到了在缺氧处理的原代人滋养层中观察到的溶酶体超微结构缺陷以及自溶酶体数量减少的支持。自噬缺陷的人类 EVT 表现出较差的 TFEB 核转位、溶酶体蛋白表达和功能降低以及 MTORC1 活性增加。 PE 患者的血清诱导了 EVT 中的这些特征和蛋白质聚集。重要的是，滋养层特异性条件性atg7敲除小鼠表现出 TFEB 表达减少，胎盘中蛋白质聚集体沉积增加。 这些结果为 PE 患者胎盘中蛋白质聚集体的积累与 TFEB 介导的溶酶体生物发生和自噬受损之间的调节联系提供了令人信服的证据。

缩写： atg7 ：自噬相关7； CTSD：组织蛋白酶D； ER：内质网； EVT：绒毛外滋养层细胞； KRT7：角蛋白7； LAMP1：溶酶体相关膜蛋白1； LAMP2：溶酶体相关膜蛋白2； mSt：m草莓； MTORC1：雷帕霉素复合物1的机制靶点； NP：正常妊娠； NPS：正常妊娠血清； PE：先兆子痫； PES：子痫前期血清； p-RPS6KB：磷酸化核糖体蛋白S6激酶B1； SQSTM1/p62: 隔离体 1; TEM：透射电子显微镜； TFEB：转录因子EB； XPO1/CRM1：导出 1