In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.

https://www.embopress.org/doi/pdf/10.15252/embj.2020104705

Molecular mechanisms and physiological functions of mitophagy.

Inflammasomes are cytoplasmic multiprotein complexes comprising a sensor protein, inflammatory caspases, and in some but not all cases an adapter protein connecting the two. They can be activated by a repertoire of endogenous and exogenous stimuli, leading to enzymatic activation of canonical caspase-1, noncanonical caspase-11 (or the equivalent caspase-4 and caspase-5 in humans) or caspase-8, resulting in secretion of IL-1β and IL-18, as well as apoptotic and pyroptotic cell death. Appropriate inflammasome activation is vital for the host to cope with foreign pathogens or tissue damage, while aberrant inflammasome activation can cause uncontrolled tissue responses that may contribute to various diseases, including autoinflammatory disorders, cardiometabolic diseases, cancer and neurodegenerative diseases. Therefore, it is imperative to maintain a fine balance between inflammasome activation and inhibition, which requires a fine-tuned regulation of inflammasome assembly and effector function. Recently, a growing body of studies have been focusing on delineating the structural and molecular mechanisms underlying the regulation of inflammasome signaling. In the present review, we summarize the most recent advances and remaining challenges in understanding the ordered inflammasome assembly and activation upon sensing of diverse stimuli, as well as the tight regulations of these processes. Furthermore, we review recent progress and challenges in translating inflammasome research into therapeutic tools, aimed at modifying inflammasome-regulated human diseases.

/pdf/inflammasome-activation-and-regulation-toward-a-better-21xmkghegc.pdf

Inflammasome activation and regulation: toward a better understanding of complex mechanisms.

Contrast-induced acute kidney injury (CI-AKI) occurs in more than 30% of patients after intravenous iodinated contrast media and causes serious complications, including renal failure and mortality. Recent research has demonstrated that routine antioxidant and alkaline therapy failed to show benefits in CI-AKI patients with high risk for renal complications. Mitophagy is a mechanism of selective autophagy, which controls mitochondrial quality and mitochondrial reactive oxygen species (ROS) through degradation of damaged mitochondria. The role of mitophagy and its regulation of apoptosis in CI-AKI are poorly understood. In this study, we demonstrated that mitophagy was induced in renal tubular epithelial cells (RTECs) during CI-AKI, both in vivo and in vitro. Meanwhile, contrast media-induced mitophagy was abolished when silencing PINK1 or PARK2 (Parkin), indicating a dominant role of the PINK1-Parkin pathway in mitophagy. Moreover, mitochondrial damage, mitochondrial ROS, RTEC apoptosis, and renal injury under contrast exposure were more severe in PINK1- or PARK2-deficient cells and mice than in wild-type groups. Functionally, PINK1-Parkin-mediated mitophagy prevented RTEC apoptosis and tissue damage in CI-AKI through reducing mitochondrial ROS and subsequent NLRP3 inflammasome activation. These results demonstrated that PINK1-Parkin-mediated mitophagy played a protective role in CI-AKI by reducing NLRP3 inflammasome activation.

PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation.

The NLRP3 inflammasome is cytosolic multi-protein complex that induces inflammation and pyroptotic cell death in response to both pathogen (PAMPs) and endogenous activators (DAMPs). Recognition of PAMPs or DAMPs leads to formation of the inflammasome complex, which results in activation of caspase-1, followed by cleavage and release of pro-inflammatory cytokines. Excessive activation of NLRP3 inflammasome can contribute to development of inflammatory diseases and cancer. Autophagy is vital intracellular process for recycling and removal of damaged proteins and organelles, as well as destruction of intracellular pathogens. Cytosolic components are sequestered in a double-membrane vesicle-autophagosome, which then fuses with lysosome resulting in degradation of the cargo. The autophagy dysfunction can lead to diseases with hyperinflammation and excessive activation of NLRP3 inflammasome and thus acts as a major regulator of inflammasomes. Autophagic removal of NLRP3 inflammasome activators, such as intracellular DAMPs, NLRP3 inflammasome components, and cytokines can reduce inflammasome activation and inflammatory response. Likewise, inflammasome signaling pathways can regulate autophagic process necessary for balance between required host defense inflammatory response and prevention of excessive and detrimental inflammation. Autophagy has a protective role in some inflammatory diseases associated with NLRP3 inflammasome, including gouty arthritis, familial Mediterranean fever (FMF), and sepsis. Understanding the interregulation between these two essential biological processes is necessary to comprehend the biological mechanisms and designing possible treatments for multiple inflammatory diseases.

/pdf/interplay-between-nlrp3-inflammasome-and-autophagy-19mnbeanr9.pdf

Interplay Between NLRP3 Inflammasome and Autophagy.

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory ...

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2016.1183081

Ji Hwan Oh

Papers

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages