Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/ß2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

AMPK-mediated potentiation of GABAergic signalling drives hypoglycaemia-provoked spike-wave seizures.

Metabolism regulates neuronal activity and modulates the occurrence of epileptic seizures. Here, using two rodent models of absence epilepsy, we show that hypoglycaemia increases the occurrence of spike-wave seizures. We then show that selectively disrupting glycolysis in the thalamus, a structure implicated in absence epilepsy, is sufficient to increase spike-wave seizures. We propose that activation of thalamic AMP-activated protein kinase, a sensor of cellular energetic stress and potentiator of metabotropic GABAB-receptor function, is a significant driver of hypoglycaemia-induced spike-wave seizures. We show that AMP-activated protein kinase augments postsynaptic GABAB-receptor-mediated currents in thalamocortical neurons and strengthens epileptiform network activity evoked in thalamic brain slices. Selective thalamic AMP-activated protein kinase activation also increases spike-wave seizures. Finally, systemic administration of metformin, an AMP-activated protein kinase agonist and common diabetes treatment, profoundly increased spike-wave seizures. These results advance the decades-old observation that glucose metabolism regulates thalamocortical circuit excitability by demonstrating that AMP-activated protein kinase and GABAB-receptor cooperativity is sufficient to provoke spike-wave seizures.

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

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.

Exercise-Induced Mitophagy in Skeletal Muscle and Heart.

Regular exercise enhances mitochondrial function by promoting healthy mitochondrial remodeling, but the underlying mechanisms are not thoroughly understood. An emerging hypothesis suggests that, in addition to anabolic events such as mitochondria biogenesis, the selective degradation of dysfunctional mitochondria (i.e., mitophagy) also is a key component of exercise-mediated adaptations in striated muscle, which eventually leads to better mitochondrial functions.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

Mitophagy in maintaining skeletal muscle mitochondrial proteostasis and metabolic health with ageing.

Skeletal muscle is important for overall functionality and health. Ageing is associated with an accumulation of damage to mitochondrial DNA and proteins. In particular, damage to mitochondrial proteins in skeletal muscle, which is a loss of mitochondrial proteostasis, contributes to tissue dysfunction and negatively impacts systemic health. Therefore, understanding the mechanisms underlying the regulation of mitochondrial proteostasis and how those mechanisms change with age is important for the development of interventions to promote healthy ageing. Herein, we examine how impairment in the selective degradation of damaged/dysfunctional mitochondria through mitophagy may play a central role in the loss of mitochondrial proteostasis in skeletal muscle ageing, as well as its broader implications for systemic health. Further, we explore how stimulating mitophagy through exercise may promote healthy ageing.

Molecular mechanisms for mitochondrial adaptation to exercise training in skeletal muscle.

Exercise training enhances physical performance and confers health benefits, largely through adaptations in skeletal muscle. Mitochondrial adaptation, encompassing coordinated improvements in quantity (content) and quality (structure and function), is increasingly recognized as a key factor in the beneficial outcomes of exercise training. Exercise training has long been known to promote mitochondrial biogenesis, but recent work has demonstrated that it has a profound impact on mitochondrial dynamics (fusion and fission) and clearance (mitophagy), as well. In this review, we discuss the various mechanisms through which exercise training promotes mitochondrial quantity and quality in skeletal muscle.

Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation.

Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1.

Ulk1 Phosphorylation at S555 is Not Required in Exercise Training-mediated Improvements in Endurance and Metabolic Capacity in Healthy Mice.

Endurance exercise training improves exercise capacity as well as skeletal muscle and whole-body metabolism, which are hallmarks of high quality-of-life and healthy aging. However, its mechanisms are not yet fully understood. Exercise-induced mitophagy has merged as an important step in mitochondrial remodeling. ULK1, specifically its activation by phosphorylation at serine 555, was discovered as an autophagy driver and to be important for energetic stress-induced mitophagy in skeletal muscle, making it a potential mediator the benefit of exercise on mitochondrial remodeling. Here, we employed CRISPR/Cas9-mediated gene editing and generated knock-in mice with a serine-to-alanine mutation of Ulk1. We now report that these mice displayed normal endurance capacity and cardiac function at baseline with a mild impairment of energy metabolism as indicated by accelerated increase of respiratory exchange ratio (RER) during acute exercise stress; however, this was completely corrected by 8 weeks of voluntary running. Ulk1-S555A mice also completely retained the exercise-mediated improvements of endurance capacity. We conclude that Ulk1 phosphorylation at S555 is not required for exercise-mediated improvements of endurance and metabolic capacity in healthy mice.

Neuromuscular Dysfunction Precedes Cognitive Impairment in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) develops along a continuum that spans years prior to diagnosis. Decreased muscle function and mitochondrial respiration occur years earlier in those that develop AD; however, it is unknown what causes these peripheral phenotypes in a disease of the brain. Exercise promotes muscle, mitochondria, and cognitive health and is proposed to be a potential therapeutic for AD, but no study has investigated how skeletal muscle adapts to exercise training in an AD-like context. Utilizing 5xFAD mice, an AD model that develops ad-like pathology and cognitive impairments around 6 mo of age, we examined in vivo neuromuscular function and exercise adapations (mitochondrial respiration and RNA sequencing) before the manifestation of overt cognitive impairment. We found 5xFAD mice develop neuromuscular dysfunction beginning as early as 4 mo of age, characterized by impaired nerve-stimulated muscle torque production and compound nerve action potential of the sciatic nerve. Furthermore, skeletal muscle in 5xFAD mice had altered, sex-dependent, adaptive responses (mitochondrial respiration and gene expression) to exercise training in the absence of overt cognitive impairment. Changes in peripheral systems, specifically neural communication to skeletal muscle, may be harbingers for AD and have implications for lifestyle interventions, like exercise, in AD.

Early-stage Alzheimer's disease: are skeletal muscle and exercise the key?

Alzheimer's disease (AD) is the most common form of dementia affecting approximately 6.5 million people in the United States alone. The development of AD progresses over a span of years to possibly decades before resulting in cognitive impairment and clinically diagnosed AD. The time leading up to a clinical diagnosis is known as the preclinical phase, a time in which recent literature has noted a more severe loss of body mass and more specifically lean muscle mass and strength prior to diagnosis. Mitochondria dysfunction in neurons is also closely associated with AD, and mitochondrial dysfunction has been seen to occur in skeletal muscle with mild cognitive impairment prior to AD manifestation. Evidence from animal models of AD suggests a close link among skeletal muscle mass, mitochondria function, and cognition. Exercise is a powerful stimulus for improving mitochondria function and muscle health, and its benefits to cognition have been suggested as a possible therapeutic strategy for AD. However, evidence for beneficial effects of exercise in AD-afflicted populations and animal models has produced conflicting results. In this mini-review, we discuss these findings and highlight potential avenues for further investigation that may lead to the implementation of exercise as a therapeutic intervention to delay or prevent the development of AD.

Sensing local energetics to acutely regulate mitophagy in skeletal muscle.

The energetic requirements of skeletal muscle to sustain movement, as during exercise, is met largely by mitochondria, which form an intricate, interconnected reticulum. Maintenance of a healthy mitochondrial reticulum is essential for skeletal muscle function, suggesting quality control pathways are spatially governed. Mitophagy, the process by which damaged and/or dysfunctional regions of the mitochondrial reticulum are removed and degraded, has emerged as an integral part of the molecular response to exercise. Upregulation of mitophagy in response to acute exercise is directly connected to energetic sensing mechanisms through AMPK. In this review, we discuss the connection of mitophagy to muscle energetics and how AMPK may spatially control mitophagy through multiple potential means.

Mitochondrial plasticity supports proliferative outgrowth and invasion of ovarian cancer spheroids during adhesion.

Background: Ovarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion. Methods: Using our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities. Results: Independent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids. Discussion: Our studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.

Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog.

BACKGROUND: Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. RESULTS: Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. CONCLUSIONS: Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.

Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.

Ulk1, Not Ulk2, Is Required for Exercise Training-Induced Improvement of Insulin Response in Skeletal Muscle.

Unc51 like autophagy activating kinase 1 (Ulk1), the primary autophagy regulator, has been linked to metabolic adaptation in skeletal muscle to exercise training. Here we compared the roles of Ulk1 and homologous Ulk2 in skeletal muscle insulin action following exercise training to gain more mechanistic insights. Inducible, skeletal muscle-specific Ulk1 knock-out (Ulk1-iMKO) mice and global Ulk2 knock-out (Ulk2-/-) mice were subjected to voluntary wheel running for 6 weeks followed by assessment of exercise capacity, glucose tolerance, and insulin signaling in skeletal muscle after a bolus injection of insulin. Both Ulk1-iMKO and Ulk2-/- mice had improved endurance exercise capacity post-exercise. Ulk1-iMKO did not improve glucose clearance during glucose tolerance test, while Ulk2-/- had only marginal improvement. However, exercise training-induced improvement of insulin action in skeletal muscle, indicated by Akt-S473 phosphorylation, was only impaired in Ulk1-iMKO. These data suggest that Ulk1, but not Ulk2, is required for exercise training-induced improvement of insulin action in skeletal muscle, implicating crosstalk between catabolic and anabolic signaling as integral to metabolic adaptation to energetic stress.

AICAR treatment for 14 days normalizes obesity-induced dysregulation of TORC1 signaling and translational capacity in fasted skeletal muscle.

The aim of this study was to determine the effect of 14 days of 5-aminoimidazole-4-carboxamide-1ß-4-ribofuranoside (AICAR) treatment on mammalian target of rapamycin (mTOR) signaling and mTOR-regulated processes (i.e., translation initiation) in obese mouse skeletal muscle. Our hypothesis was that daily treatment (14 days) with AICAR would normalize obesity-induced alterations in skeletal muscle mTOR signaling and mTOR-regulated processes to lean levels and positively affect muscle mass. Fourteen-week-old male, lean (L; 31.3 g body wt) wild-type and ob/ob (O; 59.6 g body wt) mice were injected with the AMP-activated kinase (AMPK) activator AICAR (A) at 0.5 mg·g body wt(-1)·day(-1) or saline control (C) for 14 days. At 24 h after the last injection (including a 12-h fast), all mice were killed, and the plantar flexor complex muscle (gastrocnemius, soleus, and plantaris) was excised for analysis. Muscle mass was lower in OC (159 ± 12 mg) than LC, LA, and OA (176 ± 10, 178 ± 9, and 166 ± 16 mg, respectively) mice, independent of a body weight change. A decrease in obese muscle mass corresponded with higher muscle cross section staining intensity for lipid and glycogen, higher blood glucose and insulin levels, and lower nuclear-enriched fractions for peroxisome proliferator-activated receptor-γ coactivator-1α protein expression in OC skeletal muscle, which was normalized with AICAR treatment. AMPK and acetyl-cocarboxylase phosphorylation was reduced in OC mice and augmented by AICAR treatment in OA mice. Conversely, OC mice displayed higher activation of downstream targets (S6 kinase-1 and ribosomal protein S6) of mTOR and lower raptor-associated mTOR than LC mice, which were reciprocally altered after 14 days of AICAR treatment. Dysregulation of translational capacity was improved in OA mice, as assessed by sucrose density gradient fractionation of ribosomes, total and ribosome-associated RNA content, eukaryotic initiation factor 4F complex formation, and eukaryotic initiation factor 4G phosphorylation. These data show that short-term (14 days) AMPK agonist treatment augments regulatory processes in atrophic obese mouse skeletal muscle through the normalization of mTOR signaling and mRNA translation closer to lean levels.

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

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.

Targeting healthspan to optimally combat non-communicable disease in an aging world.

Human lifespan and life expectancy have increased worldwide, but the number of years that we spend free of chronic or debilitating disorders, known as healthspan, has not shifted along with increased lifespan. This unfavourable trend presents a tremendous global social-economical problem. We propose a model of promoting optimal human health with proactive, holistic interventions across the lifespan, which require multi-disciplinary, innovative approaches to research and care. We contend that this is the only hope that we have to face the challenges of population growth and aging, as well as the upward trend in non-communicable disease prevalence.