Joint National Laboratory for Antibody Drug Engineering, The First Affiliated Hospital, School of Medicine, Henan University, Kaifeng, China.
Beijing Institute of Basic Medical Sciences, Beijing, China.
Invest Ophthalmol Vis Sci. 2023 Jun 1;64(7):5. doi: 10.1167/iovs.64.7.5.
HSF4 mutations are responsible for congenital cataract formation. Dysfunction of HSF4 leads to defects in lens terminal differentiation. We aimed to study the mechanism of how HSF4 promotes organelle degradation during lens differentiation.
HSF4del42 mutant mice that developed congenital cataracts were employed. The organelle degradation and autophagic function in lens fibers were detected by immunofluorescence and Immunoblotting. Transcriptome analysis was performed to investigate the differentially expressed genes in HSF4del42 lenses, whereas luciferase report assay and ChIP assay were used to confirm the directly transcriptional regulation of ATG9a by HSF4.
HSF4del42 mice displayed delayed organelle clearance and impaired autophagic degradation function in lens fibers. Activation of autophagy by rapamycin ameliorated the defects in organelle clearance in HSF4del42 lenses ex vivo and in vivo. Depletion of HSF4 attenuated autophagic flux by disrupting autophagosome biogenesis and maturation in lens epithelial cells. HSF4 directly transcriptionally activated the core autophagy protein ATG9a. Instead of the canonical ATG9a isoform, the ATG9a-X2 isoform was predominantly expressed in the lens and alleviated autophagic defects in HSF4 KO lens epithelial cells. The ATG9a-X2 protein displayed a short half-life, and rapamycin treatment restored its levels in HSF4 KO lens epithelial cells and HSF4del42 lenses.
Our findings demonstrate that HSF4 facilitates organelle degradation probably by transcriptionally activating autophagy during lens terminal differentiation. We first report the involvement of HSF4 in autophagy and the tissue specific splicing of ATG9a. Our study indicates that autophagy activation is a possible therapeutic strategy for HSF4-related congenital cataracts.
HSF4 突变是先天性白内障形成的原因。HSF4 功能障碍导致晶状体终末分化缺陷。我们旨在研究 HSF4 在晶状体分化过程中促进细胞器降解的机制。
使用发生先天性白内障的 HSF4del42 突变小鼠。通过免疫荧光和免疫印迹检测晶状体纤维中的细胞器降解和自噬功能。进行转录组分析以研究 HSF4del42 晶状体中差异表达的基因，而荧光素酶报告测定和 ChIP 测定用于确认 HSF4 对 ATG9a 的直接转录调控。
HSF4del42 小鼠显示晶状体纤维中细胞器清除延迟和自噬降解功能受损。雷帕霉素激活自噬可改善 HSF4del42 晶状体中细胞器清除的缺陷。在晶状体上皮细胞中，HSF4 的缺失减弱了自噬流，破坏了自噬体的生物发生和成熟。HSF4 直接转录激活核心自噬蛋白 ATG9a。在晶状体中主要表达的不是典型的 ATG9a 同工型，而是 ATG9a-X2 同工型，它减轻了 HSF4 KO 晶状体上皮细胞中的自噬缺陷。ATG9a-X2 蛋白的半衰期较短，雷帕霉素处理可恢复 HSF4 KO 晶状体上皮细胞和 HSF4del42 晶状体中的水平。
我们的研究结果表明，HSF4 在晶状体终末分化过程中通过转录激活自噬来促进细胞器降解。我们首次报道了 HSF4 参与自噬和 ATG9a 的组织特异性剪接。我们的研究表明，自噬激活可能是治疗 HSF4 相关先天性白内障的一种潜在策略。
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