A degradative to secretory autophagy switch
mediates mitochondria clearance in the absence
of the mATG8-conjugation machinery
核心内容：
PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. PINK1-Parkin介导的线粒体自噬是一种选择性的自噬形式，是通过清除受损线粒体来控制线粒体质量的最重要机制之一。 Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. 尽管众人皆知mATG8（mammalian ATG8s）的脂质化是自噬过程的关键步骤，然而其在线粒体自噬过程中的作用仍有待研究。 ：这里的脂质化具体来说是mATG8结合了磷脂酰乙醇胺。脂质是种类繁多、结构复杂的一类大分子物质，是脂肪和类脂的总称。脂肪即甘油三酯(triglyceride,TG),也称三脂酰甘油(triacylglyc-erol)。类脂包括固醇及其酯、磷脂和糖脂等。磷脂又分为甘油磷脂和鞘磷脂。磷脂酰乙醇胺（phosphatidylethanolamine ，PE)属于甘油磷脂，其基本骨架是甘油（丙三醇、三羟基丙烷）。conjugation共轭（conjugate：记忆技巧：con 共同 + jug 牛轭 + ate 表动词 → 共同在轭下 → 成对，to give the different forms of a verb, as they vary according to number , person , tense, etc.） In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. 在本研究中，为了阐明mATG8脂质化在线粒体自噬中的功能，他们利用CRISPR技术敲除了多个介导mATG8脂质化的几个关键基因，包括ATG3、ATG5和ATG7等。意想不到的是，mATG8脂质化系统的缺陷并不影响受损线粒体的清除。 Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). 然而，（mATG8脂质化缺陷时）线粒体是通过自噬性分泌的形式被排出胞外的，这个过程我们定义为线粒体自噬分泌（ASM）。 Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. 在功能上，ASM的增多促进了受体细胞中天然免疫cGAS-STING途径的激活。 Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses. 总的来说，本研究揭示了当细胞mATG8脂质化功能失调时，ASM是线粒体质量控制中的一种机制，并强调了mATG8脂质化在抑制炎症反应中的关键作用。
【背景】
在酵母细胞中，ATG8脂质化是其自噬体形成所必须的。
Kirisako T，et al. Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol. 1999 Oct 18; https:///jcb/article-pdf/147/2/435/1288464/9907028.pdf
Nguyen TN，et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.J Cell Biol.2016 Dec 19;Tsuboyama K, et al. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane.Science. 2016 Nov 25;Kishi-Itakura C, et al. Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J Cell Sci. 2014 Sep 15; 自噬体可从细胞内降解途径转换为细胞外分泌途径。 Ponpuak M, et al. Secretory autophagy. Curr Opin Cell Biol.2015 Aug;Kimura T,et al. Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy.EMBO J. 2017 Jan 4;36(1):42-60. 【猜想&提问1】mATG8脂质化对自噬的活性很重要，自噬能清除受损线粒体，但哺乳动物细胞自噬体不依赖mATG8脂质化。那么mATG8脂质化缺陷时哺乳动物细胞的自噬能否清除损伤线粒体？
Fig. 1 The mATG8-conjugation system is not required for PINK1–Parkin-mediated mitochondria clearance.
a: WT or ATG7-KO HeLa stably expressing mCherry-Parkin were treated with antimycin A and oligomycin (A/O, 1 µM each) for the indicated duration. Mitochondria content was assessed by immunoblotting of proteins from different mitochondria compartments.
b:Quantification 定量 of mitochondrial protein changes from (a).
cd和ab类似,ef是DNA含量变化，和蛋白含量相呼应。
：线粒体各关键组分蛋白含量可表征线粒体含量，加 antimycin A and oligomycin (A/O, 1 µM )可诱导线粒体损伤。敲除了多个介导mATG8脂质化的几个关键基因，包括ATG7、ATG3和ATG5后各组分蛋白和WT细胞一样随线粒体损伤的积累而减少，DNA含量变化模式也类似，也就是说损伤的线粒体被细胞清除了。所以mATG8脂质化缺失后动物细胞能清除损伤线粒体。
Fig. 2 Clearance of damaged mitochondria requires key ATG components upstream of the mATG8-conjugation system.
a WT, ATG7-KO, and PINK1- KO, b TBK1-KO, c ATG9A-KO, d FIP200-KO HeLa cells stably expressing mCherry-Parkin were treated with A/O for the indicated duration.
Mitochondria content was assessed by immunoblotting of proteins from different mitochondria compartments.
e WT HeLa stably expressing mCherry-Parkin was treated with A/O, with or without SBI- 0206965 (25 µM).
f WT and ATG7-KO cells stably expressing mCherry-Parkin were treated with A/O for 24 h, with or without Bafilomycin A1 (BafA1, 200 nM) or MG132 (10 µM) to inhibit lysosomal or proteasome degradation, respectively.
g Representative immunofluorescence images of WT and ATG7-KO cells treated with A/O for 6 h and stained for HSP60 (red) and LAMP2 (green). Scale bar = 10 µm. h Quantification of A/O-treated groups in g showing percentage of LAMP2 puncta colocalised with HSP60.
Fig. 3 Global proteomics profiling of extracellular vesicles.
a Schematic diagram of SILAC labelling and extracellular vesicles (EV) isolation.
b log2[H/L] dotplot of EV proteins that had SILAC ratios in forward (WT heavy; ATG7-KO light) and reverse (ATG7-KO heavy; WT light) labelling experiments. Grey lines denote a 1.5× fold enrichment cutoff.
c Heatmap of proteins identified to have a >1.5 fold enrichment when comparing ATG7-KO to WT EVs in both forward and reverse labelling experiments.
d Gene ontology analysis (cellular compartment) of proteins that were enriched >1.5 fold in EVs of ATG7-KO vs. WT, plotted according to −log10 FDR.
Supplementary Fig. 3 Characterization of secreted mitochondria in other mATG8-conjugation defective cell lines.
a Conditioned media from WT, ATG5-KO, ATG3-KO HeLa stably expressing mCherry-Parkin treated with A/O for 24 hr were collected. Extracellular vesicles (EVs) were isolated by differential ultracentrifugation and immunoblotted for mitochondria markers.
b LDH release assay from WT, ATG7-KO, ATG5-KO and ATG3-KO cells treated with or without A/O for 24 hrs. Mean of n = 4 independent replicates ± SEM is shown. P values are calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ns denotes not significant. See source data for exact P values.
c Representative TEM images of extracellular regions from WT or ATG7-KO HeLa stably expressing mCherry-Parkin untreated or treated with A/O + BafA1. Scale bar represents 500 nm.
d Nanoparticle tracking analysis of EVs from WT and ATG5-KO cells untreated or treated with A/O for 24 hrs.
e nPLEX analysis of mitochondria cargo from immunocaptured CD63+ EVs isolated from WT and ATG5-KO cells stably expressing YFP-Parkin untreated or treated with A/O for 24 hrs. Mean of n = 3 technical replicates ± SD is shown.
Fig. 4 Damaged mitochondria are secreted via a pathway distinct from small EVs.
a Conditioned media from WT and ATG7-KO HeLa stably expressing mCherry-Parkin treated with A/O for 24 h were collected. Extracellular vesicles (EVs) were isolated by differential ultracentrifugation and immunoblotted for mitochondria and small EV markers.
b Control or muscle specific ATG7-KO (Atg7f/f;Ckmm-cre) mice were subjected to 3 consecutive days of exhaustive exercise. Serum EVs or TA muscle tissue were then harvested. Representative immunoblots and quantification of the mitochondria markers (ACO2 and SDHA) from serum EVs are shown. Mean of n = 4 mice per group ±SEM.
c Nanoparticle tracking analysis of EVs from WT and ATG7-KO cells untreated or treated with A/O for 24 h.
d Conditioned media from 24 h A/O treated ATG7-KO HeLa stably expressing mCherry-Parkin were filtered (0.22 µm cutoff) before isolation of EVs by differential ultracentrifugation and immunoblotting.
e Proteinase K protection assay of EVs isolated by differential ultracentrifugation from 24 h A/O treated ATG7-KO HeLa stably expressing mCherry-Parkin.
f EVs from 24 h A/O treated ATG7-KO cells were separated by a 5–40% bottom-up iodixanol density flotation gradient and subjected to immunoblotting. *Non-specific bands.
：免疫印迹证明ATG7 KO的细胞外囊泡组分有较高水平的磷酸化泛素。
Fig. 5 ATG7-independent mitochondria secretion occurs via secretory autophagy.
a Proteinase K protection assay of homogenates from WT or ATG7- KO cells stably expressing mCherry-Parkin treated with A/O and BafA1 (200 nM) for 12 h in the presence or absence of SAR405 (5 µM).
b Quantification of the percentage of NDP52 protected from proteinase K digestion for each treatment condition. Mean of n = 3 independent replicates ±SEM is shown. P values were calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test against the A/O + BafA1 group. *P < 0.05, **P < 0.01, ***P < 0.001, and ns denotes not significant. See source data for exact P values.
c Representative TEM images of mature autophagosomes containing mitochondria from WT or ATG7-KO cells stably expressing mCherry-Parkin treated with A/O + BafA1 for 16 h. Scale bar represents 500 nm.
d ATG7-KO and ATG14/ATG7 DKO;
e ATG7-KO and FIP200/ATG7 DKO;
f ATG7-KO and ATG9A/ATG7 DKO;
g ATG7-KO and SNAP23/ATG7 DKO;
h or ATG7-KO and RAB7A/ATG7 DKO HeLa stably-expressing mCherry-Parkin were treated with A/O for 24 h. Extracellular vesicles (EVs) were isolated by differential ultracentrifugation and immunoblotted for the indicated proteins.
Fig. 6 ATG7-independent mitochondria clearance is impaired when autophagic secretion of mitochondria is inhibited. 当线粒体的自噬分泌被抑制时，ATG7非依赖的线粒体清除功能受损。
这四张结果图说明mATG8脂质化缺陷的细胞（ATG3/5/7 KO）依赖自噬体的合成（ATG14和FIP200）将受损线粒体分泌到细胞外。 【猜想&提问5】 线粒体自噬缺陷时，mtDNA的清除受限，导致cGAS-STING信号通路的激活及炎症反应，与神经退行性疾病有密切关系。--Sliter DA, et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature. 2018 Sep; cGAS-STING信号通路在mATG8脂质化缺陷的细胞被激活？
Fig. 7 cGAS–STING is potently activated by secreted mitochondria.cGAS-STING信号通路被分泌的受损线粒体有效激活。
cGAS-STING信号通路被分泌的受损线粒体有效激活。这可能就是mATG8脂质化缺陷导致的神经性炎性的部分原因。 PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses. --------------------------------------------------------------
A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery Hayden Weng Siong Tan 1,2, Guang Lu 1,3, Han Dong1 , Yik-Lam Cho 1 , Auginia Natalia 4, Liming Wang1,5, Charlene Chan6, Dennis Kappei 6,7,8, Reshma Taneja 1,2, Shuo-Chien Ling 1 , Huilin Shao 4, Shih-Yin Tsai 1 , Wen-Xing Ding 9 & Han-Ming Shen 1,10✉
Kirisako T，et al. Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol. 1999 Oct 18; https:///jcb/article-pdf/147/2/435/1288464/9907028.pdf
Nguyen TN，et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.J Cell Biol.2016 Dec 19;Tsuboyama K, et al. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane.Science. 2016 Nov 25;Kishi-Itakura C, et al. Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J Cell Sci. 2014 Sep 15;Ponpuak M, et al. Secretory autophagy. Curr Opin Cell Biol.2015 Aug;Kimura T,et al. Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy.EMBO J. 2017 Jan 4;36(1):42-60.Sliter DA, et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature. 2018 Sep;
：这里的脂质化具体来说是mATG8结合了磷脂酰乙醇胺。脂质是种类繁多、结构复杂的一类大分子物质，是脂肪和类脂的总称。脂肪即甘油三酯(triglyceride,TG),也称三脂酰甘油(triacylglyc-erol)。类脂包括固醇及其酯、磷脂和糖脂等。磷脂又分为甘油磷脂和鞘磷脂。磷脂酰乙醇胺（phosphatidylethanolamine ，PE)属于甘油磷脂，其基本骨架是甘油（丙三醇、三羟基丙烷）。conjugation共轭（conjugate：记忆技巧：con 共同 + jug 牛轭 + ate 表动词 → 共同在轭下 → 成对，to give the different forms of a verb, as they vary according to number , person , tense, etc.）
：哺乳动物细胞中，线粒体清除不依赖mATG8脂质化，自噬体也不依赖mATG8脂质化，可能是自噬体合成通路跳过了mATG8脂质化这一步。作者敲除自噬体形成通路上游的基因，如FIP200和ATG9A，发现损伤线粒体的清除被有效阻断。ULK1作为自噬的起始，是自噬的关键蛋白之一，其抑制剂SBI-0206965可有效抑制损伤线粒体的清除。这些结果提示，mATG8脂质化缺陷时，线粒体清除仍需自噬体的合成。
