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Parkinson’s disease (PD), like other neurodegenerative diseases, lacks a disease-modifying therapy. While we have solutions to counteract the dopaminergic depletion that ensues at the level of the striatum following the progressive loss of substantia nigra neurons in the midbrain, and therefore, are capable of reversing the motor symptoms experienced by patients, our understanding of the molecular mechanisms that trigger degeneration is minimal. Even following the discovery of single mutations in several genes that can cause familial forms of PD, like PINK1 and PRKN, we are still deprived of a deep appreciation of the basic biological mechanisms that drive disease initiation and progression. In Chapter 1 of this thesis, I describe the remarkable research progress that was made in the last two centuries following the original description of the disease. I detail how the mitochondrial hypothesis of PD emerged from brilliant discoveries, the advent of new biochemical and genomic tools, and even a few accidents, and how it may underlie the selective vulnerability of midbrain dopamine neurons that die in PD. Finally, I introduce some key unanswered questions in the field of PD research that I begin to address in this thesis. In Chapter 2, I describe the novel approach we developed to study a pathway of mitochondrial turnover that is critical in PD, which allowed us to track individual mitochondria at every step of the degradation process in neurons. We gained unprecedented insight into the fate of these mitochondria once they reach the lysosomal compartment which led to us to identify new steps of PINK1/PRKN-mediated mitochondrial degradation. These findings may have important implications for both basic biology and disease pathophysiology.In Chapter 3, I describe a mouse model we developed to assess the impact that chronic changes in neural activity have on the function and survival of midbrain dopamine neurons. We find that the substantia nigra dopamine neurons are preferentially vulnerable to chronic increases in neural activity, while other dopamine neurons are more resilient, suggesting a potential role for chronic hyperactivity as a driver of disease.Lastly, in Chapter 4, I elaborate and speculate on disease implications of the findings described in Chapter 2. I contextualize our contribution within the broader effort to understand why PINK1 and PRKN are so crucial to neurons under certain high-stress conditions, but are dispensable at baseline.

NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant.To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to COThese findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts pleiotropic effects on macrophages and is required for self-renewal but the mechanisms responsible are unknown. Using mouse models with disrupted GM-CSF signaling, we show GM-CSF is critical for mitochondrial turnover, functions, and integrity. GM-CSF signaling is essential for fatty acid β-oxidation and markedly increased tricarboxylic acid cycle activity, oxidative phosphorylation, and ATP production. GM-CSF also regulated cytosolic pathways including glycolysis, pentose phosphate pathway, and amino acid synthesis. We conclude that GM-CSF regulates macrophages in part through a critical role in maintaining mitochondria, which are necessary for cellular metabolism as well as proliferation and self-renewal.

Oocyte health is tightly tied to mitochondria given their role in energy production, metabolite supply, calcium (Ca(2+)) buffering, and cell death regulation, among others. In turn, mitochondrial function strongly relies on these organelle dynamics once cyclic events of fusion and fission (division) are required for mitochondrial turnover, positioning, content homogenization, metabolic flexibility, interaction with subcellular compartments, etc. Importantly, during oogenesis, mitochondria change their architecture from an “orthodox” elongated shape characterized by the presence of numerous transversely oriented cristae to a round-to-oval morphology containing arched and concentrically arranged cristae. This, along with evidence showing that mitochondrial function is kept quiescent during most part of oocyte development, suggests an important role of mitochondrial dynamics in oogenesis. To investigate this, recent works have downregulated/upregulated in oocytes the expression of key effectors of mitochondrial dynamics, including mitofusins 1 (MFN1) and 2 (MFN2) and the dynamin-related protein 1 (DRP1). As a result, both MFN1 and DRP1 were found to be essential to oogenesis and fertility, while MFN2 deletion led to offspring with increased weight gain and glucose intolerance. Curiously, neither MFN1/MFN2 deficiency nor DRP1 overexpression enhanced mitochondrial fragmentation, indicating that mitochondrial size is strictly regulated in oocytes. Therefore, the present work seeks to discuss the role of mitochondria in supporting oogenesis as well as recent findings connecting defective mitochondrial dynamics in oocytes with infertility and transmission of metabolic disorders.

The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24 h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. Key points: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.

Mitochondria are important organelles in eukaryotic cells. Normal mitochondrial homeostasis is subject to a strict mitochondrial quality control system, including the strict regulation of mitochondrial production, fission/fusion and mitophagy. The strict and accurate modulation of the mitochondrial quality control system, comprising the mitochondrial fission/fusion, mitophagy and other processes, can ameliorate the myocardial injury of myocardial ischaemia and ischaemia‐reperfusion after myocardial infarction, which plays an important role in myocardial protection after myocardial infarction. Further research into the mechanism will help identify new therapeutic targets and drugs for the treatment of myocardial infarction. This article aims to summarize the recent research regarding the mitochondrial quality control system and its molecular mechanism involved in myocardial infarction, as well as the potential therapeutic targets in the future.

Autism spectrum disorders (ASDs) are a group of complex neurodevelopmental disorders, including autistic disorder, Asperger's syndrome, pervasive developmental disorder and childhood disintegrative disorder. Mitochondria not only provide neurons with energy in the form of ATP to sustain neuron growth, proliferation and neurodevelopment, but also regulate neuron apoptosis, intracellular calcium ion (Ca2+) homeostasis, and reactive oxygen species (ROS) clearance. Due to their postmitotic state and high energy-demanded feature, neurons are particularly prone to mitophagy and mitochondrial disfunction. Mitophagy, a selective autophagy, is critical for sustaining mitochondrial turnover and quality control via eliminating unwanted and dysfunctional mitochondria in neurons. Dysfunctional mitochondria and dysregulated mitophagy have been closely associated with the onset of ASDs. In this review, we summarize the mechanism of mitophagy and its role in neurons, and the consequence of mitophagy dysfunction in ASDs. Deeper appreciation of the role of mitophagy in ASDs pathology is required for developing new therapeutic approaches.

ÖZET Mitokondri, hücresel fonksiyonlarda kullanılan ATP'yi üreten hücrenin güç merkezleri olarak tanımlanmaktadır. Mitokondriyal disfonksiyon yaşlanma sürecinin önemli bir parçasını oluşturmaktadır. Hücreler yaşlandıkça solunum zincirinin etkinliği azalmakta, elektron kaçağı artarak ve ATP oluşumunu azalmasına neden olmaktadır. Farklı hücre tipleri farklı sayıda mitokondriye sahiptir ve kendi DNA’larına sahip oldukları için, içinde bulundukları hücreden bağımsız olarak bölünebilmektedirler. Diğer bir ifadeyle, mitokondriyal replikasyon hücre bölünmesine bağlı değildir. Üreme sırasında, bir çocuğun DNA'sının yarısı babalarından, yarısı da annelerinden gelmektedir. Bununla birlikte, çocuk her zaman Mitokondriyal DNA’larını (mtDNA) annelerinden almaktadır. Mitokondriyal hastalıkların çoğunluğu, mitokondriye neden olan ürünleri etkileyen nükleer DNA'daki mutasyonlardan kaynaklanmaktadır. Bu mutasyonlar kalıtsal veya kendiliğinden olabilmektedir. Mitokondri çalışmayı bıraktığında, içinde bulundukları hücre enerjiden yoksun kalır. Bu nedenle, hücrenin tipine bağlı olarak semptomlar büyük ölçüde değişebilmektedir. Kalp kası hücreleri ve sinirler gibi en çok miktarda enerjiye ihtiyaç duyan hücreler, en çok hatalı mitokondrilerden etkilenmektedir. 62 yıl önce Harman, yaşlanmaya reaktif oksijen türleri (ROS) ile oluşan makromolekül hasarının aracılık ettiğini ileri sürmüştür. ROS mitokondride enerji üretiminin bir yan ürünü olarak üretilir ve bu yapılar DNA'ya, yağlara ve proteinlere zarar vermektedir. Artık yaşlanmada hedef mitokondriye odaklanmıştır. Yapılan çalışmalar, oksidatif stresin, mitokondriyal DNA mutasyonlarının, mitokondri turnover'ındaki azalmanın hücresel yaşlanmaya aracılık ettiğini ortaya koymaktadır. Bu derlemede, mitokondrinin insan yaşlanma sürecindeki rolü, elde edilen son bulgular temelinde gözden geçirilmesi hedeflenmiştir. Anahtar Kelimeler: Mitokondrial DNA; mitokondri; yaşlanma; serbest radikaller; oksidatif stres

Significance Mitochondria are organelles that make cellular energy. In the retina, mitochondrial health is important for photoreceptor cell function and normal vision. Photoreceptors need more energy at night, but it is unclear how their mitochondria adapt to changing energy demands. We investigated zebrafish cone photoreceptor mitochondria throughout the day. At night cone mitochondria are smaller, more numerous, and more metabolically active. Material from mitochondria is also released from photoreceptors during nighttime, forming stalks and networks outside of the cell. In daytime, photoreceptor mitochondria associate with ER and autophagosomes, organelles that mediate mitochondrial turnover. These daily changes to mitochondrial structure may help photoreceptors adapt to metabolic demands of daytime and nighttime., Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.

The maintenance of a healthy mitochondrial network and the ability to adjust organelle population in response to internal or external stimuli are essential for the function and the survival of eukaryotic cells. Over the last two decades several studies have demonstrated the paramount importance of mitophagy, a selective form of autophagy that removes damaged and/or superfluous organelles, in organismal physiology. Post-mitotic neuronal cells are particularly vulnerable to mitochondrial damage, and mitophagy impairment has emerged as a causative factor in multiple neurodegenerative pathologies, including Alzheimer's disease and Parkinson's disease among others. Although mitochondrial turnover is a multifaceted process, neurons have to tackle additional complications, arising from their pronounced bioenergetic demands and their unique architecture and cellular polarisation that render the degradation of distal organelles challenging. Mounting evidence indicates that despite the functional conservation of mitophagy pathways, the unique features of neuronal physiology have led to the adaptation of compartmentalised solutions, which serve to ensure seamless mitochondrial removal in every part of the cell. In this review, we summarise the current knowledge concerning the molecular mechanisms that mediate mitophagy compartmentalisation and discuss their implications in various human pathologies.

Background Cachexia presents in 80% of advanced cancer patients; however, cardiac atrophy in cachectic patients receives little attention. This cardiomyopathy contributes to increased occurrence of adverse cardiac events compared with age‐matched population norms. Research on cardiac atrophy has focused on remodelling; however, alterations in metabolic properties may be a primary contributor. The purpose of the study is to determine how cancer‐induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery, and in vitro oxidative characteristics. Methods Lewis lung carcinoma (LLC) tumours were implanted in C57BL6/J mice and grown for 28 days to induce cardiac atrophy. Endogenous metabolic species and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC‐conditioned media with (out) the antioxidant MitoTempo. Cells were analysed for reactive oxygen species (ROS), oxidative capacity, and hypoxic resistance. Results LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio [flavin adenine dinucleotide (FAD)/FAD + nicotinamide adenine dinucleotide hydrogen (NADH)] compared with phosphate‐buffered saline controls. When compared with phosphate‐buffered saline, LLC hearts showed ~50% greater Cytochrome‐C oxidase subunit 4 (COX‐IV) and Voltage‐dependent anion channel (VDAC), attributed to ~50% lower mitophagy markers. mt‐mRNA translation machinery was elevated similarly to markers of mitochondrial content. Mitochondrial DNA‐encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx‐3 and GPx‐7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC‐conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal and 75% at maximal), and greater susceptibility to hypoxia (~25%), which was reversed by MitoTempo. Conclusions These results substantiate metabolic cardiotoxic effects attributable to tumour‐associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity, and hypoxia resistance.

Sirtuin-3 (SirT3) and AMPK stimulate mitochondrial biogenesis, which increases mitochondrial turnover and cardiomyocyte regeneration. We studied the effects of SirT3, AMPK, and mitochondrial biogenesis on sepsis-induced myocardial injury. Our data showed that after treating cardiomyocytes with lipopolysaccharide, SirT3 and AMPK levels decreased, and this was followed by mitochondrial dysfunction and cardiomyocyte death. Overexpression of SirT3 activated the AMPK pathway and improved mitochondrial biogenesis, which is required to sustain mitochondrial redox balance, maintain mitochondrial respiration, and suppress mitochondrial apoptosis. Inhibition of mitochondrial biogenesis abolished SirT3/AMPK-induced cardioprotection by causing mitochondrial damage. These findings indicate that SirT3 reduces sepsis-induced myocardial injury by activating AMPK-related mitochondrial biogenesis.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, genetic and extrinsic factors influence senescent decline and ageing. Numerous gene mutations and treatments have been shown to extend the lifespan of organisms ranging from the unicellular Saccharomyces cerevisiae to primates. Most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to counter both intrinsic stress and extrinsic stress. Mitochondria, the main energy hub of the cell, are highly dynamic organelles, playing essential roles in cell physiology. Mitochondrial function impinges on several signalling pathways modulating cellular metabolism, survival and healthspan. Maintenance of mitochondrial function and energy homeostasis requires both generation of new healthy mitochondria and elimination of the dysfunctional ones. Here, we survey the mechanisms regulating mitochondrial number in cells, with particular emphasis on neurons. We, further, discuss recent findings implicating perturbation of mitochondrial homeostasis in cellular senescence and organismal ageing as well as in age-associated neurodegenerative diseases.

The only enzyme in the glycosphingolipid (GSL) metabolic pathway, which produces glucosylceramide (GlcCer) de novo is UDP-glucose ceramide glucosyltransferase (UGCG). UGCG is linked to pro-cancerous processes such as multidrug resistance development and increased proliferation in several cancer types. Previously, we showed an UGCG-dependent glutamine metabolism adaption to nutrient-poor environment of breast cancer cells. This adaption includes reinforced oxidative stress response and fueling the tricarboxylic acid (TCA) cycle by increased glutamine oxidation. In the current study, we investigated glycolytic and oxidative metabolic phenotypes following UGCG overexpression (OE). UGCG overexpressing MCF-7 cells underwent a metabolic shift from quiescent/aerobic to energetic metabolism by increasing both glycolysis and oxidative glucose metabolism. The energetic metabolic phenotype was not associated with increased mitochondrial mass, however, markers of mitochondrial turnover were increased. UGCG OE altered sphingolipid composition of the endoplasmic reticulum (ER)/mitochondria fractions that may contribute to increased mitochondrial turnover and increased cell metabolism. Our data indicate that GSL are closely connected to cell energy metabolism and this finding might contribute to development of novel therapeutic strategies for cancer treatment.

Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-β signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5−/− cells, respectively. Young but not aging Pgam5−/− mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases., Mitochondria are a hub that can direct cellular outcomes in response to stress. Here, the authors show that mitochondrial phosphatase PGAM5 has a role in mitochondrial turnover and regulation of cellular senescence by altering organellar dynamics via fission regulator Drp1.

Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson’s disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

Neurodegenerative disorders, including chemotherapy-induced cognitive impairment, are associated with neuronal mitochondrial dysfunction. Cisplatin, a commonly used chemotherapeutic, induces neuronal mitochondrial dysfunction in vivo and in vitro. Astrocytes are key players in supporting neuronal development, synaptogenesis, axonal growth, metabolism and, potentially mitochondrial health. We tested the hypothesis that astrocytes transfer healthy mitochondria to neurons after cisplatin treatment to restore neuronal health.We used an in vitro system in which astrocytes containing mito-mCherry-labeled mitochondria were co-cultured with primary cortical neurons damaged by cisplatin. Culture of primary cortical neurons with cisplatin reduced neuronal survival and depolarized neuronal mitochondrial membrane potential. Cisplatin induced abnormalities in neuronal calcium dynamics that were characterized by increased resting calcium levels, reduced calcium responses to stimulation with KCl, and slower calcium clearance. The same dose of cisplatin that caused neuronal damage did not affect astrocyte survival or astrocytic mitochondrial respiration. Co-culture of cisplatin-treated neurons with astrocytes increased neuronal survival, restored neuronal mitochondrial membrane potential, and normalized neuronal calcium dynamics especially in neurons that had received mitochondria from astrocytes which underlines the importance of mitochondrial transfer. These beneficial effects of astrocytes were associated with transfer of mitochondria from astrocytes to cisplatin-treated neurons. We show that siRNA-mediated knockdown of the Rho-GTPase Miro-1 in astrocytes reduced mitochondrial transfer from astrocytes to neurons and prevented the normalization of neuronal calcium dynamics.In conclusion, we showed that transfer of mitochondria from astrocytes to neurons rescues neurons from the damage induced by cisplatin treatment. Astrocytes are far more resistant to cisplatin than cortical neurons. We propose that transfer of functional mitochondria from astrocytes to neurons is an important repair mechanism to protect the vulnerable cortical neurons against the toxic effects of cisplatin.

Understanding the mechanisms regulating mitochondrial respiratory function and adaptations to metabolic challenges, such as exercise and high dietary fat, is necessary to promote skeletal muscle health and attenuate metabolic disease. Autophagy is a constitutively active degradation pathway that promotes mitochondrial turnover and transiently increases postexercise. Recent evidence indicates Bcl2 mediates exercise-induced autophagy and skeletal muscle adaptions to training during high-fat diet. We determined if improvements in mitochondrial respiration due to exercise training required Bcl2-mediated autophagy using a transgenic mouse model of impaired inducible autophagy (Bcl2(AAA)). Mitochondrial adaptations to a treadmill exercise training protocol, in either low-fat or high-fat diet fed mice, did not require Bcl2-mediated autophagy activation. Instead, training increased protein synthesis rates and basal autophagy in the Bcl2(AAA) mice, while acute exercise activated BNIP3 and Parkin autophagy. High-fat diet stimulated lipid-specific mitochondrial adaptations. These data demonstrate increases in basal mitochondrial turnover, not transient activation with exercise, mediate adaptations to exercise and high-fat diet.

Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a ‘basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.

To gain understanding on the mechanisms that drive immunosenescence in humans, we examined CD4+ T cells obtained from younger (20-39 years-old) and older (70+ years-old) healthy participants of the Baltimore Longitudinal Study on Aging (BLSA). We found that mitochondrial proteins involved in the electron transport chain were overrepresented in cells from older participants, with prevalent dysregulation of oxidative phosphorylation and energy metabolism molecular pathways. Surprisingly, gene transcripts coding for mitochondrial proteins pertaining to oxidative phosphorylation and electron transport chain pathways were underrepresented in older individuals. Paralleling the observed decrease in gene expression, mitochondrial respiration was impaired in CD4+ T cells from older subjects. Though mitochondrial number in both naive and memory cells visualized with electron microcopy was similar in older versus younger participants, there were a significantly higher number of autophagosomes, many of them containing undegraded mitochondria, in older individuals. The presence of mitochondria inside the accumulated autophagic compartments in CD4+ T cells from older individuals was confirmed by immunofluorescence. These findings suggest that older age is associated with persistence of dysfunctional mitochondria in CD4+ T lymphocytes caused by defective mitochondrial turnover by autophagy, which may trigger chronic inflammation and contribute to the impairment of immune defense in older persons.

Mitochondria are essential energy-providing organelles that are required in the maintenance of healthy skeletal muscle. As such, the removal of damaged mitochondria, through mitophagy, is necessary to maintain mitochondrial quality. In aging muscle, mitochondrial content and function are often found to be reduced compared to young individuals. This occurs despite the fact that measures of mitophagy are elevated, suggesting that mitophagy is insufficiently high to remove all of the dysfunctional organelles in aging muscle. Recent evidence has shown that acute exercise promotes mitophagic signaling, leading to organelle degradation. This exercise-induced signaling is attenuated in aging muscle, suggesting that aging muscle loses its capacity for mitochondrial turnover in response to exercise. This contributes to the reduction in muscle health in elderly individuals. Chronic exercise training improves mitochondrial content and function, even in aging muscle, leading to reduced mitophagy signaling. Thus, exercise training should be prescribed for both young and elderly populations to promote the maintenance of a healthy mitochondrial pool, through the stimulation of both organelle biogenesis and mitophagy.

Proper skeletal muscle development, maintenance, and function is necessary for movement. Decline in muscle function with age and disease is directly associated with a diminished quality of life. Radiation therapy is commonly used to treat certain forms of childhood cancer based on the cytotoxic effects of radiation on cancerous tissue. However, the adverse effects elicited by radiation are not always constrained to the diseased tissue and can accelerate muscle wasting and decline, which is particularly detrimental to juvenile cancer survivors. Exercise is effective at limiting muscle decline and improving muscle function in various diseases. Thus, we hypothesized 1 mo of voluntary endurance exercise following juvenile radiation treatment will reduce muscle damage and restore functional deficits that occur following radiation. Here, we show that following juvenile radiation, 1 mo of voluntary wheel running significantly improved muscle function in mice by promoting adaptations in intracellular calcium handling, improving mitochondrial turnover and reducing oxidative stress resulting from radiation-induced mitochondrial damage. These findings help guide caregivers in their approach to childhood cancer survivor recovery and have implications for other diseases where similar mechanisms of calcium handling and mitochondrial function are disrupted.

Background: Despite the development of radiation therapy (RT) techniques, concern regarding the serious and irreversible heart injury induced by RT has grown due to the lack of early intervention measures. Although exercise can act as an effective and economic nonpharmacologic strategy to combat fatigue and improve quality of life for cancer survivors, limited data on its application in radiation-induced heart disease (RIHD) and the underlying molecular mechanism are available.Methods: Fifteen young adult male mice were enrolled in this study and divided into 3 groups (including exercised RIHD group, sedentary RIHD group, and controls; n =5 samples/group). While the mice in the control group were kept in cages without irradiation, those in the exercised RIHD group underwent 3weeks of aerobic exercise on the treadmill after radiotherapy. At the end of the 3rd week following RT, FNDC5/irisin expression, cardiac function, aerobic fitness, cardiomyocyte apoptosis, mitochondrial function, and mitochondrial turnover in the myocardium were assessed to identify the protective role of exercise in RIHD and investigate the potential mechanism.Results: While sedentary RIHD group had impaired cardiac function and aerobic fitness than controls, the exercised RIHD mice had improved cardiac function and aerobic fitness, elevated ATP production and the mitochondrial protein content, decreased mitochondrial length, and increased formation of mitophagosomes compared with sedentary RIHD mice. These changes were accompanied by the elevated expression of FNDC5/irisin, a fission marker (DRP1) and mitophagy markers (PINK1 and LC3B) in exercised RIHD group than that of sedentary RIHD group, but the expression of biogenesis (TFAM) and fusion (MFN2) markers was not significantly changed.Conclusion: Exercise could enhance cardiac function and aerobic fitness in RIHD mice partly through an autocrine mechanism via FNDC5/irisin, in which autophagy was selectively activated, suggesting that FNDC5/irisin may act as an intervening target to prevent the development of RIHD.

Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and posttranslational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g., oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases, the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1, which upon activation recruits the ubiquitin ligase parkin. Here, we review mechanisms of mitophagy with an emphasis on posttranslational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on posttranslational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and antiapoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.

Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of PINK1 and Parkin, two key regulators of mitophagy, are amongst the most common causes of heritable parkinsonism. This has led to the hypothesis that pharmacological stimulation of mitophagy may be a feasible approach to combat neurodegeneration. Toward this end, we screened ~ 45,000 small molecules using a high-throughput, whole-organism, phenotypic screen that monitored accumulation of PINK-1 protein, a key event in mitophagic activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes, including delaying paralysis in a C. elegans β-amyloid aggregation model in a PINK-1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.