Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

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AMPK: guardian of metabolism and mitochondrial homeostasis.

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)

Although the development of mitochondrial therapies has largely focused on diseases caused by mutations in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins, it has been found that mitochondrial dysfunction also contributes to the pathology of many common disorders, including neurodegeneration, metabolic disease, heart failure, ischaemia-reperfusion injury and protozoal infections. Mitochondria therefore represent an important drug target for these highly prevalent diseases. Several strategies aimed at therapeutically restoring mitochondrial function are emerging, and a small number of agents have entered clinical trials. This Review discusses the opportunities and challenges faced for the further development of mitochondrial pharmacology for common pathologies.

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Mitochondria as a therapeutic target for common pathologies.

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.

How the Warburg effect supports aggressiveness and drug resistance of cancer cells

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.

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Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy

Our understanding of the molecular mechanisms underlying adaptations to resistance exercise remains elusive despite the significant biological and clinical relevance. We developed a novel voluntary mouse weightlifting model, which elicits squat-like activities against adjustable load during feeding, to investigate the resistance exercise-induced contractile and metabolic adaptations. RNAseq analysis revealed that a single bout of weightlifting induced significant transcriptome responses of genes that function in posttranslational modification, metabolism, and muscle differentiation in recruited skeletal muscles, which were confirmed by increased expression of fibroblast growth factor-inducible 14 (Fn14), Down syndrome critical region 1 (Dscr1) and Nuclear receptor subfamily 4, group A, member 3 (Nr4a3) genes. Long-term (8 weeks) voluntary weightlifting training resulted in significantly increases of muscle mass, protein synthesis (puromycin incorporation in SUnSET assay) and mTOR pathway protein expression (raptor, 4e-bp-1, and p70S6K proteins) along with enhanced muscle power (specific torque and contraction speed), but not endurance capacity, mitochondrial biogenesis, and fiber type transformation. Importantly, weightlifting training profound improved whole-body glucose clearance and skeletal muscle insulin sensitivity along with enhanced autophagy (increased LC3 and LC3-II/I ratio, and decreased p62/Sqstm1). These data suggest that resistance training in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fj.201903055R

A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle.

The common clinical symptoms of Friedreich’s ataxia (FRDA) include ataxia, muscle weakness, type 2 diabetes and heart failure, which are caused by impaired mitochondrial function due to the loss of frataxin (FXN) expression. Endurance exercise is the most powerful intervention for promoting mitochondrial function; however, its impact on FRDA has not been studied. Here we found that mice with genetic knockout and knock-in of the Fxn gene (KIKO mice) developed exercise intolerance, glucose intolerance and moderate cardiac dysfunction at 6 months of age. These abnormalities were associated with impaired mitochondrial respiratory function concurrent with reduced iron regulatory protein 1 (Irp1) expression as well as increased oxidative stress, which were not due to loss of mitochondrial content and antioxidant enzyme expression. Importantly, long-term (4 months) voluntary running in KIKO mice starting at a young age (2 months) completely prevented the functional abnormalities along with restored Irp1 expression, improved mitochondrial function and reduced oxidative stress in skeletal muscle without restoring Fxn expression. We conclude that endurance exercise training prevents symptomatic onset of FRDA in mice associated with improved mitochondrial function and reduced oxidative stress. These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients.

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Bevan M. Lewellen

Papers

Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy

A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle.

Long-term voluntary running prevents the onset of symptomatic Friedreich's ataxia in mice.