International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Linc00673-V3 positively regulates autophagy by promoting Smad3-mediated LC3B transcription in NSCLC.

Since its first discovery, long noncoding RNA Linc00673 has been linked to carcinogenesis and metastasis of various human cancers. Linc00673 had five transcriptional isoforms and their biological functions remained to be explored. Here we have reported that Linc00673-V3, one of the isoforms of Linc00673, promoted non-small cell lung cancer chemoresistance, and increased Linc00673-V3 expression level was associated with enhanced autophagy. Mechanistically, we discerned the existence of a stem-loop configuration engendered by the 1-100-nt and 2200-2275-nt fragments within Linc00673-V3. This structure inherently interacted with Smad3, thereby impeding its ubiquitination and subsequent degradation orchestrated by E3 ligase STUB1. The accumulation of Smad3 contributed to autophagy via up-regulation of LC3B transcription and ultimately conferred chemoresistance in NSCLC. Our results revealed a novel transcriptional regulation network between Linc00673-V3, Smad3, and LC3B, which provided an important insight into the interplay between autophagy regulation and non-canonical function of Smad3. Furthermore, the results from in vivo experiments suggested Linc00673-V3 targeted antisense oligonucleotide as a promising therapeutic strategy to overcome chemotherapy resistance in NSCLC.

FBP1 inhibits NSCLC stemness by promoting ubiquitination of Notch1 intracellular domain and accelerating degradation.

The existence of cancer stem cells is widely acknowledged as the underlying cause for the challenging curability and high relapse rates observed in various tumor types, including non-small cell lung cancer (NSCLC). Despite extensive research on numerous therapeutic targets for NSCLC treatment, the strategies to effectively combat NSCLC stemness and achieve a definitive cure are still not well defined. The primary objective of this study was to examine the underlying mechanism through which Fructose-1,6-bisphosphatase 1 (FBP1), a gluconeogenic enzyme, functions as a tumor suppressor to regulate the stemness of NSCLC. Herein, we showed that overexpression of FBP1 led to a decrease in the proportion of CD133-positive cells, weakened tumorigenicity, and decreased expression of stemness factors. FBP1 inhibited the activation of Notch signaling, while it had no impact on the transcription level of Notch 1 intracellular domain (NICD1). Instead, FBP1 interacted with NICD1 and the E3 ubiquitin ligase FBXW7 to facilitate the degradation of NICD1 through the ubiquitin-proteasome pathway, which is independent of the metabolic enzymatic activity of FBP1. The aforementioned studies suggest that targeting the FBP1-FBXW7-NICD1 axis holds promise as a therapeutic approach for addressing the challenges of NSCLC recurrence and drug resistance.

Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation.

Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage-mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3-associated phagocytosis (LAP), a non-canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G-protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL-induced efferocytosis, anti-colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3-associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.

Low dose PFDA induces DNA damage and DNA repair inhibition by promoting nuclear cGAS accumulation in ovarian epithelial cells.

Per- and polyfluoroalkyl substances (PFAS), the versatile anthropogenic chemicals, are popular with the markets and manufactured in large quantities yearly. Accumulation of PFAS has various adverse health effects on human. Albeit certain members of PFAS were found to have genotoxicity in previous studies, the mechanisms underlying their effects on DNA damage repair remain unclear. Here, we investigated the effects of Perfluorodecanoic acid (PFDA) on DNA damage and DNA damage repair in ovarian epithelial cells through a series of in vivo and in vitro experiments. At environmentally relevant concentration, we firstly found that PFDA can cause DNA damage in primary mouse ovarian epithelial cells and IOSE-80 cells. Moreover, nuclear cGAS increased in PFDA-treated cells, which leaded to the efficiency of DNA homologous recombination (HR) decreased and DNA double-strand breaks perpetuated. In vivo experiments also verified that PFDA can induce more DNA double-strand breaks lesions and nuclear cGAS in ovarian tissue. Taken together, our results unveiled that low dose PFDA can cause deleterious effects on DNA and DNA damage repair (DDR) in ovarian epithelial cells and induce genomic instability.

Chemical mitophagy modulators: Drug development strategies and novel regulatory mechanisms.

Maintaining mitochondrial homeostasis is a potential therapeutic strategy for various diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic disorders, and cancer. Selective degradation of mitochondria by autophagy (mitophagy) is a fundamental mitochondrial quality control mechanism conserved from yeast to humans. Indeed, small-molecule modulators of mitophagy are valuable pharmaceutical tools that can be used to dissect complex biological processes and turn them into potential drugs. In the past few years, pharmacological regulation of mitophagy has shown promising therapeutic efficacy in various disease models. However, with the increasing number of chemical mitophagy modulator studies, frequent methodological flaws can be observed, leading some studies to draw unreliable or misleading conclusions. This review attempts (a) to summarize the molecular mechanisms of mitophagy; (b) to propose a Mitophagy Modulator Characterization System (MMCS); (c) to perform a comprehensive analysis of methods used to characterize mitophagy modulators, covering publications over the past 20 years; (d) to provide novel targets for pharmacological intervention of mitophagy. We believe this review will provide a panorama of current research on chemical mitophagy modulators and promote the development of safe and robust mitophagy modulators with therapeutic potential by introducing high methodological standards.

STING directly recruits WIPI2 for autophagosome formation during STING-induced autophagy.

The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation.

Autophagic secretion of mitochondria (ASM): an alternative way for getting rid of damaged mitochondria.

PINK1-PRKN/Parkin-mediated mitophagy represents an important mitochondrial quality control (MQC) pathway that clears damaged/dysfunctional mitochondria. Although the conjugation of mammalian Atg8-family proteins (mATG8s) to phosphatidylethanolamine (PE) is a defining step in autophagy, its role in mitophagy remains unclear. In our recent study, we found that the mATG8 conjugation system is not required for PINK1-PRKN-mediated mitochondria clearance. Instead, mATG8 conjugation system-independent mitochondria clearance relies on secretory autophagy, a process we term as the autophagic secretion of mitochondria (ASM). As ASM results in the spurious activation of the CGAS-STING1 pathway, we propose that defects in mATG8 lipidation may promote inflammation through ASM.

The emerging mechanisms and functions of microautophagy.

'Autophagy' refers to an evolutionarily conserved process through which cellular contents, such as damaged organelles and protein aggregates, are delivered to lysosomes for degradation. Different forms of autophagy have been described on the basis of the nature of the cargoes and the means used to deliver them to lysosomes. At present, the prevailing categories of autophagy in mammalian cells are macroautophagy, microautophagy and chaperone-mediated autophagy. The molecular mechanisms and biological functions of macroautophagy and chaperone-mediated autophagy have been extensively studied, but microautophagy has received much less attention. In recent years, there has been a growth in research on microautophagy, first in yeast and then in mammalian cells. Here we review this form of autophagy, focusing on selective forms of microautophagy. We also discuss the upstream regulatory mechanisms, the crosstalk between macroautophagy and microautophagy, and the functional implications of microautophagy in diseases such as cancer and neurodegenerative disorders in humans. Future research into microautophagy will provide opportunities to develop novel interventional strategies for autophagy- and lysosome-related diseases.

Modulation of Atg genes expression in aged rat liver, brain, and kidney by caloric restriction analyzed via single-nucleus/cell RNA sequencing.

Dysregulation of macroautophagy/autophagy has been closely implicated in aging. Caloric restriction (CR) is an effective intervention of aging partially via activation of autophagy. Recently, a high-throughput single-cell RNA-seq technique has been employed to detect the comprehensive transcriptomes of individual cells. However, the transcriptional networks of ATG (autophagy related) genes in the aging process and the modulation of ATG genes expression by CR at the single-cell level have not been elucidated. Here, by performing data analysis of single nucleus/cells RNA sequencing in rats undergoing aging and the modulation by CR, we demonstrate that the transcription patterns of Atg genes in different cell types of rat liver, brain, and kidney are highly heterogeneous. Importantly, CR reversed aging-induced changes of multiple Atg genes across different cell types in the brain, liver, and kidney. In summary, our results, for the first time, provide comprehensive information on Atg gene expression in specific cell types of different organs in a mammal during aging and give novel insight into the protective role of autophagy and CR in aging at the single-cell resolution.Abbreviations: ATG genes: autophagy-related genes; Atg5: autophagy related 5; Atg7: autophagy related 7; CR: caloric restriction; DEATG: differentially expressed autophagy-related; NAFLD: nonalcoholic fatty liver disease; ScRNA-seq: single-cell RNA sequencing.

Targeting mitophagy to promote apoptosis is a potential therapeutic strategy for cancer.

Many anticancer agents exert cytotoxicity and trigger apoptosis through the induction of mitochondrial dysfunction. Mitophagy, as a key mitochondrial quality control mechanism, can remove damaged mitochondria in an effective and timely manner, which may result in drug resistance. Although the implication of mitophagy in neurodegenerative diseases has been extensively studied, the role and mechanism of mitophagy in tumorigenesis and cancer therapy are largely unknown. In a recent study, we found that the inhibition of PINK1-PRKN-mediated mitophagy can significantly enhance the anticancer efficacy of magnolol, a natural product with potential anticancer properties. On the one hand, magnolol can induce severe mitochondrial dysfunction, including mitochondrial depolarization, excessive mitochondrial fragmentation and the generation of mitochondrial ROS, leading to apoptosis. On the other hand, magnolol induces PINK1-PRKN-dependent mitophagy via activation of two rounds of feedforward amplification loops. The blockage of mitophagy through genetic or pharmacological approaches promotes rather than attenuates magnolol-induced cell death. Furthermore, inhibition of mitophagy by using distinct inhibitors targeting different mitophagic stages effectively enhances magnolol's anticancer efficacy in vivo. Taken together, our findings strongly indicate that manipulation of mitophagy in cancer treatment will be a promising therapeutic strategy for overcoming cancer drug resistance and improving the therapeutic efficacy of anticancer agents.

Targeting mitophagy as a novel therapeutic approach in liver cancer.

Ischemia may be the most common pathological occurrence to restrict nutrient availability and induce macroautophagy/autophagy. As a self-digestive process, autophagy helps sustain nutrient/energy and restrict damages in short-term scenarios, but it switches to a self-destructive process leading to cell death in long-term scenarios. Notably, ischemia has been used as one clinical application to treat cancer, particularly transarterial embolization (TAE) and chemoembolization (TACE) as the first-line treatments of intermediate-stage hepatocellular carcinoma (HCC, the predominant type of liver cancer). Partly due to the induced autophagy together with hypoxia-induced angiogenesis, TAE/TACE is not successful to treat HCC in many cases. Our recent work demonstrated that simultaneous treatments with sorafenib (a first-line therapeutic agent for advanced HCC) can sensitize HCC cells to cell death induced by glucose starvation via impairing mitophagy, a mitochondria-specific form of autophagy. Moreover, we identified SIAH1 as an important E3 ubiquitin ligase for mitophagic induction in HCC cells.

Morusin and mulberrin extend the lifespans of yeast and C. elegans via suppressing nutrient-sensing pathways.

Compounds with lifespan extension activity are rare, although increasing research efforts have been invested in this field to find ways to extend healthy lifespan. By applying a yeast-based high-throughput assay to identify the chronological lifespan extension activity of mulberry extracts rapidly, we demonstrated that a group of prenylated flavones, particularly morusin and mulberrin, could extend the chronological lifespan of budding yeast via a nutrient-dependent regime by at least partially targeting SCH9. Their antiaging activity could be extended to C. elegans by promoting its longevity, dependent on the full functions of genes akt-1 or akt-2. Moreover, additional benefits were observed from morusin- and mulberrin-treated worms, including increased reproduction without the influence of worm health (pumping rate, pumping decline, and reproduction span). In the human HeLa cell model, morusin and mulberrin inhibited the phosphorylation of p70S6K1, promoted autophagy, and slowed cell senescence. The molecular docking study showed that mulberrin and morusin bind to the same pocket of p70S6K1. Collectively, our findings open up a potential class of prenylated flavones performing their antiaging activity via nutrient-sensing pathways.

Simultaneous treatment with sorafenib and glucose restriction inhibits hepatocellular carcinoma in vitro and in vivo by impairing SIAH1-mediated mitophagy.

Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical significance to explore the synergistic effect of TACE with antiangiogenic inhibitors and the molecular mechanisms involved. This study determined that glucose, but not other analyzed nutrients, offered significant protection against cell death induced by sorafenib, as indicated by glucose deprivation sensitizing cells to sorafenib-induced cell death. Next, this synergistic effect was found to be specific to sorafenib, not to lenvatinib or the chemotherapeutic drugs cisplatin and doxorubicin. Mechanistically, sorafenib-induced mitophagy, as indicated by PINK1 accumulation, increased the phospho-poly-ubiquitination modification, accelerated mitochondrial membrane protein and mitochondrial DNA degradation, and increased the amount of mitochondrion-localized mKeima-Red engulfed by lysosomes. Among several E3 ubiquitin ligases tested, SIAH1 was found to be essential for inducing mitophagy; that is, SIAH1 silencing markedly repressed mitophagy and sensitized cells to sorafenib-induced death. Notably, the combined treatment of glucose restriction and sorafenib abolished ATP generation and mitophagy, which led to a high cell death rate. Oligomycin and antimycin, inhibitors of electron transport chain complexes, mimicked the synergistic effect of sorafenib with glucose restriction to promote cell death mediated via mitophagy inhibition. Finally, inhibition of the glucose transporter by canagliflozin (a clinically available drug used for type-II diabetes) effectively synergized with sorafenib to induce HCC cell death in vitro and to inhibit xenograft tumor growth in vivo. This study demonstrates that simultaneous treatment with sorafenib and glucose restriction is an effective approach to treat HCC, suggesting a promising combination strategy such as transarterial sorafenib-embolization (TASE) for the treatment of unresectable HCC.

Photocatalytic Pt(IV)-Coordinated Carbon Dots for Precision Tumor Therapy.

Rapid, efficient, and precise cancer therapy is highly desired. Here, this work reports solvothermally synthesized photoactivatable Pt(IV)-coordinated carbon dots (Pt-CDs) and their bovine serum albumin (BSA) complex (Pt-CDs@BSA) as a novel orange light-triggered anti-tumor therapeutic agent. The homogeneously distributed Pt(IV) in the Pt-CDs (Pt: 17.2 wt%) and their carbon cores with significant visible absorption exhibit excellent photocatalytic properties, which not only efficiently releases cytotoxic Pt(II) species but also promotes hydroxy radical generation from water under orange light. When triggered with a 589 nm laser, Pt-CDs@BSA possesses the ultrastrong cancer cell killing capacities of intracellular Pt(II) species release, hydroxyl radical generation, and acidification, which induce powerful immunogenic cell death. Activation of Pt-CDs@BSA by a single treatment with a 589 nm laser effectively eliminated the primary tumor and inhibited distant tumor growth and lung metastasis. This study thus presents a new concept for building photoactivatable Pt(IV)-enriched nanodrug-based CDs for precision cancer therapy.

The resurrection of RIP kinase 1 as an early cell death checkpoint regulator-a potential target for therapy in the necroptosis era.

Receptor-interacting serine threonine protein kinase 1 (RIPK1) has emerged as a central molecular switch in controlling the balance between cell survival and cell death. The pro-survival role of RIPK1 in maintaining cell survival is achieved via its ability to induce NF-κB-dependent expression of anti-apoptotic genes. However, recent advances have identified the pro-death function of RIPK1: posttranslational modifications of RIPK1 in the tumor necrosis factor receptor 1 (TNFR1)-associated complex-I, in the cytosolic complex-IIb or in necrosomes regulate the cytotoxic potential of RIPK1, forming an early cell death checkpoint. Since the kinase activity of RIPK1 is indispensable in RIPK3- and MLKL-mediated necroptosis induction, while it is dispensable in apoptosis, a better understanding of this early cell death checkpoint via RIPK1 might lead to new insights into the molecular mechanisms controlling both apoptotic and necroptotic modes of cell death and help develop novel therapeutic approaches for cancer. Here, we present an emerging view of the regulatory mechanisms for RIPK1 activity, especially with respect to the early cell death checkpoint. We also discuss the impact of dysregulated RIPK1 activity in pathophysiological settings and highlight its therapeutic potential in treating human diseases.

Friend or Foe? Implication of the autophagy-lysosome pathway in SARS-CoV-2 infection and COVID-19.

There is increasing amount of evidence indicating the close interplays between the replication cycle of SARS-CoV-2 and the autophagy-lysosome pathway in the host cells. While autophagy machinery is known to either assist or inhibit the viral replication process, the reciprocal effects of the SARS-CoV-2 on the autophagy-lysosome pathway have also been increasingly appreciated. More importantly, despite the disappointing results from the clinical trials of chloroquine and hydroxychloroquine in treatment of COVID-19, there is still ongoing effort in discovering new therapeutics targeting the autophagy-lysosome pathway. In this review, we provide an update-to-date summary of the interplays between the autophagy-lysosome pathway in the host cells and the pathogen SARS-CoV-2 at the molecular level, to highlight the prognostic value of autophagy markers in COVID-19 patients and to discuss the potential of developing novel therapeutic strategies for COVID-19 by targeting the autophagy-lysosome pathway. Thus, understanding the nature of such interactions between SARS-CoV-2 and the autophagy-lysosome pathway in the host cells is expected to provide novel strategies in battling against this global pandemic.

Autophagy in health and disease: From molecular mechanisms to therapeutic target.

Macroautophagy/autophagy is an evolutionally conserved catabolic process in which cytosolic contents, such as aggregated proteins, dysfunctional organelle, or invading pathogens, are sequestered by the double-membrane structure termed autophagosome and delivered to lysosome for degradation. Over the past two decades, autophagy has been extensively studied, from the molecular mechanisms, biological functions, implications in various human diseases, to development of autophagy-related therapeutics. This review will focus on the latest development of autophagy research, covering molecular mechanisms in control of autophagosome biogenesis and autophagosome-lysosome fusion, and the upstream regulatory pathways including the AMPK and MTORC1 pathways. We will also provide a systematic discussion on the implication of autophagy in various human diseases, including cancer, neurodegenerative disorders (Alzheimer disease, Parkinson disease, Huntington's disease, and Amyotrophic lateral sclerosis), metabolic diseases (obesity and diabetes), viral infection especially SARS-Cov-2 and COVID-19, cardiovascular diseases (cardiac ischemia/reperfusion and cardiomyopathy), and aging. Finally, we will also summarize the development of pharmacological agents that have therapeutic potential for clinical applications via targeting the autophagy pathway. It is believed that decades of hard work on autophagy research is eventually to bring real and tangible benefits for improvement of human health and control of human diseases.

A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.

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.