TDRD7 participates in lens development and spermiogenesis by mediating autophagosome maturation

In humans, <i>TDRD7</i> (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7’s functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in <i>tdrd7-</i>deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to <i>Tbc1d20</i> mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that <i>tdrd7</i> deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human. <b>Abbreviations</b>: CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type.

在人类中，<i>TDRD7</i>（tudor domain containing 7）突变会引发一种兼具先天性白内障（congenital cataracts, CCs）与非梗阻性无精子症（non-obstructive azoospermia, NOA）的综合征，其特征为晶状体发育异常与精子发生缺陷。然而，TDRD7在眼部与睾丸发育中发挥功能的分子机制目前仍在很大程度上未被阐明。
本研究显示，在小鼠与人类细胞中该基因的缺失会导致自噬体（autophagosome）蓄积，并破坏巨自噬/自噬流（macroautophagic/autophagic flux）。在<i>tdrd7</i>缺陷的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中，自噬流受阻是由于自噬体无法与溶酶体融合所致。
此外，转录组分析与生化实验结果表明，TDRD7可直接结合<i>Tbc1d20</i> mRNA并下调其表达——而TBC1D20是调控自噬体成熟的关键因子，这最终导致自噬体成熟受阻。
另外，本研究提供证据表明，TDRD7介导的自噬体成熟可通过清除晶状体纤维细胞中的损伤蛋白与细胞器，并促进顶体生物发生，从而维持晶状体透明度。
综上，本研究结果表明，TDRD7在自噬体成熟过程中发挥关键作用；<i>tdrd7</i>基因敲除可导致小鼠出现眼部缺陷与睾丸异常，这提示自噬受阻可能是人类先天性白内障合并非梗阻性无精子症患者出现晶状体发育与精子发生缺陷的分子机制。

<b>缩略语</b>：CB：拟染色体（chromatoid bodies, CB）；CC：先天性白内障（congenital cataract, CC）；CTSD：组织蛋白酶D（cathepsin D, CTSD）；DMSO：二甲基亚砜（dimethyl sulfoxide, DMSO）；LAMP1：溶酶体相关膜蛋白1（lysosomal-associated membrane protein 1, LAMP1）；LECs：晶状体上皮细胞（lens epithelial cells, LECs）；MAP1LC3/LC3/Atg8：微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, MAP1LC3/LC3/Atg8）；MEFs：小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）；NOA：非梗阻性无精子症（non-obstructive azoospermia, NOA）；OFZ：无细胞器区（organelle-free zone, OFZ）；RG：RNA颗粒（RNA granules, RG）；SQSTM1/p62：自噬衔接蛋白1（sequestosome 1, SQSTM1/p62）；TBC1D20：TBC结构域家族成员20（TBC1 domain family member 20, TBC1D20）；TDRD7：含tudor结构域蛋白7（tudor domain containing 7, TDRD7）；TEM：透射电子显微镜（transmission electron microscopy, TEM）；WT：野生型（wild type, WT）。