CASP9 (caspase 9) is a well-known initiator caspase which triggers intrinsic apoptosis. Recent studies also suggest various non-apoptotic roles of CASP9, including macroautophagy/autophagy regulation. However, the involvement of CASP9 in autophagy and its molecular mechanisms are not well understood. Here we report the non-apoptotic function of CASP9 in positive regulation of autophagy through maintenance of mitochondrial homeostasis. Growth factor or amino acid deprivation-induced autophagy activated CASP9, but without apoptotic features. Pharmacological inhibition or genetic ablation of CASP9 decreased autophagy flux, while ectopic expression of CASP9 rescued autophagy defects. In CASP9 knockout (KO) cells, initiation and elongation of phagophore membranes were normal, but sealing of the membranes and autophagosome maturation were impaired, and the lifetime of autophagosomes was prolonged. Ablation of CASP9 caused an accumulation of inactive ATG3 and decreased lipidation of the Atg8-family members, most severely that of GABARAPL1. Moreover, it resulted in abnormal mitochondrial morphology with depolarization of the membrane potential, reduced reactive oxygen species production, and aberrant accumulation of mitochondrial fusion-fission proteins. CASP9 expression or exogenously added H₂O₂ in the CASP9 KO cells corrected the ATG3 level and lipidation status of Atg8-family members, and restored autophagy flux. Of note, only CASP9 expression but not H₂O₂ rescued mitochondrial defects, revealing regulation of mitochondrial homeostasis by CASP9. Our findings suggest a new regulatory link between mitochondria and autophagy through CASP9 activity, especially for the proper operation of the Atg8-family conjugation system and autophagosome closure and maturation.

Abbreviations

AA: amino acid; ACD: autophagic cell death; ACTB: actin beta; ANXA5: annexin A5; APAF1: apoptotic peptidase activating factor 1; Atg: autophagy related; ATG16L1: autophagy related 16 like 1; BafA₁: bafilomycin A₁; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; CARD: caspase recruitment domain containing; CASP: caspase; CM-H₂DCFDA: chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; Δψm: mitochondrial membrane potential; DN: dominant-negative; DNM1L/DRP1: dynamin 1 like; EBSS: Earle’s balanced salt solution; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; HCN: hippocampal neural stem cells; IAM: inner autophagosome membrane; INS: insulin; KO: knockout; LEHD: Z-LEHD-fmk; MAP1LC3: microtubule associated protein 1 light chain 3; MFN1: mitofusin 1; MFN2: mitofusin 2; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP1: poly(ADP-ribose) polymerase 1; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; ROS: reactive oxygen species; sgRNA: single guide RNA; SR-SIM: super-resolution structured illumination microscopy; SQSTM1: sequestosome 1; STS: staurosporine; STX17: syntaxin 17; TMRE: tetramethylrhodamine ethyl ester; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1

中文翻译：

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CASP9 (caspase 9) 通过调节线粒体稳态对自噬体成熟至关重要。

CASP9 (caspase 9) 是众所周知的引发内在细胞凋亡的启动子 caspase。最近的研究还表明 CASP9 具有多种非凋亡作用，包括巨自噬/自噬调节。然而，CASP9 参与自噬及其分子机制尚不清楚。在这里，我们报告了 CASP9 通过维持线粒体稳态在自噬的正向调节中的非凋亡功能。生长因子或氨基酸剥夺诱导的自噬激活 CASP9，但没有凋亡特征。CASP9 的药理抑制或基因消融降低了自噬通量，而 CASP9 的异位表达挽救了自噬缺陷。在CASP9 中敲除（KO）细胞，吞噬细胞膜的起始和伸长正常，但膜的封闭和自噬体成熟受损，自噬体的寿命延长。CASP9 的消融导致无活性 ATG3 的积累并降低 Atg8 家族成员的脂化，最严重的是 GABARAPL1。此外，它导致线粒体形态异常，膜电位去极化，活性氧产生减少，线粒体融合裂变蛋白异常积累。CASP9 表达或在CASP9 中外源添加 H ₂ O ₂KO 细胞纠正了 Atg8 家族成员的 ATG3 水平和脂化状态，并恢复了自噬通量。值得注意的是，只有 CASP9 表达而不是 H ₂ O ₂拯救了线粒体缺陷，揭示了 CASP9 对线粒体稳态的调节。我们的研究结果表明，线粒体和自噬之间通过 CASP9 活性建立了新的调节联系，特别是对于 Atg8 家族结合系统的正常运行以及自噬体的关闭和成熟。

缩写

AA：氨基酸；ACD：自噬细胞死亡；ACTB：肌动蛋白β；ANXA5：膜联蛋白A5；APAF1：凋亡肽酶激活因子1；Atg：自噬相关；ATG16L1：自噬相关 16 like 1；BafA ₁：巴弗洛霉素 A ₁；BCL2：BCL2 凋亡调节剂；BECN1: beclin 1; CARD：包含caspase招募域；CASP：半胱天冬酶；CM-H ₂DCFDA：氯甲基-2ʹ,7ʹ-二氯二氢荧光素二乙酸酯；Δψm：线粒体膜电位；DN：显性阴性；DNM1L/DRP1：dynamin 1 like；EBSS：厄尔平衡盐溶液；GABARAP：GABA A 型受体相关蛋白；GABARAPL1：GABA A 型受体相关蛋白像 1；GABARAPL2：GABA A 型受体相关蛋白像 2；HCN：海马神经干细胞；IAM：内自噬体膜；INS：胰岛素；KO：淘汰赛；LEHD：Z-LEHD-fmk；MAP1LC3：微管相关蛋白1轻链3；MFN1：线粒体融合素 1；MFN2：线粒体融合素 2；MTORC1：雷帕霉素激酶复合物 1 的机制靶点；PARP1：聚（ADP-核糖）聚合酶 1；PBS：磷酸盐缓冲盐水；PE：磷脂酰乙醇胺；ROS：活性氧；sgRNA：单向导RNA；SR-SIM：超分辨率结构照明显微镜；SQSTM1：螯合体 1; STS：星形孢菌素；STX17：syntaxin 17；TMRE：四甲基若丹明乙酯；TUBB：微管蛋白β I类；ULK1：unc-51 类似自噬激活激酶 1；WT：野生型；ZFYVE1/DFCP1：锌指FYVE型含1