Mitochondrial Turnover Research Articles

The Acute and Chronic influence of Exercise on Mitochondrial Dynamics in Skeletal Muscle.

Exercise and nutritional modulation are potent stimuli for eliciting increases in mitochondrial mass and function. Collectively, these beneficial adaptations are increasingly recognized to coincide with improvements to skeletal muscle health. Mitochondrial dynamics of fission and fusion are increasingly implicated as having a central role in mediating aspects of key organelle adaptions that are seen with exercise. Exercise-induced mitochondrial adaptations that dynamics have been implicated in are: 1) Increases to mitochondrial turnover, resulting from elevated rates of mitochondrial synthesis (biogenesis) and degradative (mitophagy) processes. 2) Morphological changes to the 3D tubular network, known as the mitochondrial reticulum, that mitochondria form in skeletal muscle. Notably, mitochondrial fission has also been implicated in coordinating increases in mitophagy, following acute exercise. Further, increased fusion following exercise training promotes increased connectivity of the mitochondrial reticulum and is associated with improved metabolism and mitochondrial function. However, the molecular basis and fashion in which exercise infers beneficial mitochondrial adaptations through mitochondrial dynamics remains poorly understood. This review attempts to highlight recent developments investigating the effects of exercise on mitochondrial dynamics, while attempting to offer a perspective of the methodological refinements and potential variables, such as substrate/glycogen availability, which should be considered going forward.

Effects of intermittent exposure to hypobaric hypoxia and cold on skeletal muscle regeneration: Mitochondrial dynamics, protein oxidation and turnover

Muscle injuries and the subsequent regeneration events compromise muscle homeostasis at morphological, functional and molecular levels. Among the molecular alterations, those derived from the mitochondrial function are especially relevant. We analysed the mitochondrial dynamics, the redox balance, the protein oxidation and the main protein repairing mechanisms after 9 days of injury in the rat gastrocnemius muscle. During the recovery rats were exposed to intermittent cold exposure (ICE), intermittent hypobaric hypoxia (IHH), and both simultaneous combined stimuli. Non-injured contralateral legs were also analysed to evaluate the specific effects of the three environmental exposures. Our results showed that ICE enhanced mitochondrial adaptation by improving the electron transport chain efficiency during muscle recovery, decreased the expression of regulatory subunit of proteasome and accumulated oxidized proteins. Exposure to IHH did not show mitochondrial compensation or increased protein turnover mechanisms; however, no accumulation of oxidized proteins was observed. Both ICE and IHH, when applied separately, elicited an increased expression of eNOS, which could have played an important role in accelerating muscle recovery. The combined effect of ICE and IHH led to a complex response that could potentially impede optimal mitochondrial function and enhanced the accumulation of protein oxidation. These findings underscore the nuanced role of environmental stressors in the muscle healing process and their implications for optimizing recovery strategies.

Overexpression of SIMK in menadione-treated alfalfa enhances antioxidant machinery and leads to oxidative stress resistance

Mitogen-activated protein kinases (MAPKs) transduce stress and developmental signals related to the production of reactive oxygen species (ROS). Alfalfa (Medicago sativa L.) is a valuable forage and human nutrition crop, however, the involvement of MAPKs in plant resistance to oxidative stress is poorly understood in this species. Therefore, we elucidated the role of STRESS-INDUCED MAPK (SIMK) in alfalfa response to menadione, a compound inducing ROS generation, exploiting transgenic alfalfa lines with contrasting SIMK abundance. SIMK was activated by short-term menadione treatment and relocated from the nucleus to the cytoplasm. Proteomic analysis revealed that menadione caused changes in the abundance of proteins involved in metabolism, oxidative stress, biotic stress response, detoxification of carbonyl species, glutathione homeostasis, chloroplast protein turnover, photosynthesis, and membrane trafficking. Genetic manipulations of SIMK altered the abundance of proteins involved in mitochondrial and chloroplast protein import and processing, as well as GLUTATHIONE S-TRANSFERASES (GSTs). Increased GST abundance and activity in roots, and modifications in mitochondrial and chloroplast protein turnover might be responsible for the elevated oxidative stress resistance of alfalfa line overexpressing SIMK. This was supported by the reduced ROS levels in this line. These results reveal a complex nature of plant stress response and suggest a new role of SIMK in the alfalfa resistance to menadione-induced oxidative stress.

Activation of the Keap1/Nrf2 pathway suppresses mitochondrial dysfunction, oxidative stress, and motor phenotypes in C9orf72 ALS/FTD models.

Mitochondrial dysfunction is a common feature of C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD); however, it remains unclear whether this is a cause or consequence of the pathogenic process. Analysing multiple aspects of mitochondrial biology across several Drosophila models of C9orf72-ALS/FTD, we found morphology, oxidative stress, and mitophagy are commonly affected, which correlated with progressive loss of locomotor performance. Notably, only genetic manipulations that reversed the oxidative stress levels were also able to rescue C9orf72 locomotor deficits, supporting a causative link between mitochondrial dysfunction, oxidative stress, and behavioural phenotypes. Targeting the key antioxidant Keap1/Nrf2 pathway, we found that genetic reduction of Keap1 or pharmacological inhibition by dimethyl fumarate significantly rescued the C9orf72-related oxidative stress and motor deficits. Finally, mitochondrial ROS levels were also elevated in C9orf72 patient-derived iNeurons and were effectively suppressed by dimethyl fumarate treatment. These results indicate that mitochondrial oxidative stress is an important mechanistic contributor to C9orf72 pathogenesis, affecting multiple aspects of mitochondrial function and turnover. Targeting the Keap1/Nrf2 signalling pathway to combat oxidative stress represents a therapeutic strategy for C9orf72-related ALS/FTD.

ALS-Linked VapB P56S Mutation Alters Neuronal Mitochondrial Turnover at the Synapse.

Mitochondrial population maintenance in neurons is essential for neuron function and survival. Contact sites between mitochondria and the endoplasmic reticulum (ER) are poised to regulate mitochondrial homeostasis in neurons. These contact sites can facilitate transfer of calcium and lipids between the organelles and have been shown to regulate aspects of mitochondrial dynamics. Vesicle-associated membrane protein-associated protein B (VapB) is an ER membrane protein present at a subset of ER-mitochondrial contact sites. A proline-to-serine mutation in VapB at amino acid 56 (P56S) correlates with susceptibility to amyotrophic lateral sclerosis (ALS) type 8. Given the relationship between failed mitochondrial health and neurodegenerative disease, we investigated the function of VapB in mitochondrial population maintenance. We demonstrated that transgenic expression of VapBP56S in zebrafish larvae (sex undetermined) increased mitochondrial biogenesis, causing increased mitochondrial population size in the axon terminal. Expression of wild-type VapB did not alter biogenesis but, instead, increased mitophagy in the axon terminal. Using genetic manipulations to independently increase mitochondrial biogenesis, we show that biogenesis is normally balanced by mitophagy to maintain a constant mitochondrial population size. VapBP56S transgenics fail to increase mitophagy to compensate for the increase in mitochondrial biogenesis, suggesting an impaired mitophagic response. Finally, using a synthetic ER-mitochondrial tether, we show that VapB's function in mitochondrial turnover is likely independent of ER-mitochondrial tethering by contact sites. Our findings demonstrate that VapB can control mitochondrial turnover in the axon terminal, and this function is altered by the P56S ALS-linked mutation.

Involvement of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitochondrial turnover in according protection to hyperglycemic NRK-52E cells by Berberine

Aberrant accumulation of dysfunctional mitochondria in renal cells during hyperglycemia signifies perturbed autophagy and mitochondrial turnover. This study aims to focus on the underlying mechanism involved in autophagy and mitophagy inducing efficacy of Berberine (isoquinoline alkaloid) in hyperglycemic NRK-52E cells. Berberine mediated protection to hyperglycemic cells prevented alteration in mitochondrial structure and function. Treatment with SRT-1720 (Sirt1 activator) enhanced autophagy, decreased apoptosis, upregulated expression of downstream moieties (FoxO3a and Bnip3) and ameliorated mitochondria related anomalies while nicotinamide (Sirt1 inhibitor) treatment exhibited reversal of the same. GFP reporter assay ascertained enhanced transcriptional activity of FoxO in Berberine-treated hyperglycemic cells, which was found to be correlated to increased expression of downstream protein Bnip3. Knocking down FoxO3a disrupted autophagy and stimulated apoptosis. N-acetyl-L-cysteine pre-treatment confirmed that generation of ROS intervened high glucose induced toxicity in NRK-52E cells. Berberine co-treatment resulted in differential expressions of key proteins involved in autophagy and mitophagy like LC3B, ATGs, Beclin1, Sirt1, Bnip3, FoxO3a and Parkin. Further, enhanced mitophagy in Berberine-treated cells was confirmed by transmission electron microscopy. Thus, our findings give evidence that the protection accorded by Berberine against hyperglycemia in renal proximal tubular cells (NRK-52E) involves instigation of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitophagy induction.

Excitotoxicity, Oxytosis/Ferroptosis, and Neurodegeneration: Emerging Insights into Mitochondrial Mechanisms.

Mitochondrial dysfunction plays a pivotal role in the development of age-related diseases, particularly neurodegenerative disorders. The etiology of mitochondrial dysfunction involves a multitude of factors that remain elusive. This review centers on elucidating the role(s) of excitotoxicity, oxytosis/ferroptosis and neurodegeneration within the context of mitochondrial bioenergetics, biogenesis, mitophagy and oxidative stress and explores their intricate interplay in the pathogenesis of neurodegenerative diseases. The effective coordination of mitochondrial turnover processes, notably mitophagy and biogenesis, is assumed to be critically important for cellular resilience and longevity. However, the age-associated decrease in mitophagy impedes the elimination of dysfunctional mitochondria, consequently impairing mitochondrial biogenesis. This deleterious cascade results in the accumulation of damaged mitochondria and deterioration of cellular functions. Both excitotoxicity and oxytosis/ferroptosis have been demonstrated to contribute significantly to the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS). Excitotoxicity, characterized by excessive glutamate signaling, initiates a cascade of events involving calcium dysregulation, energy depletion, and oxidative stress and is intricately linked to mitochondrial dysfunction. Furthermore, emerging concepts surrounding oxytosis/ferroptosis underscore the importance of iron-dependent lipid peroxidation and mitochondrial engagement in the pathogenesis of neurodegeneration. This review not only discusses the individual contributions of excitotoxicity and ferroptosis but also emphasizes their convergence with mitochondrial dysfunction, a key driver of neurodegenerative diseases. Understanding the intricate crosstalk between excitotoxicity, oxytosis/ferroptosis, and mitochondrial dysfunction holds potential to pave the way for mitochondrion-targeted therapeutic strategies. Such strategies, with a focus on bioenergetics, biogenesis, mitophagy, and oxidative stress, emerge as promising avenues for therapeutic intervention.

Abstract Mo107: Age-Related Impairment of Mitochondrial Protein Turnover Exacerbates Pathogenesis of Heart Failure with Preserved Ejection Fraction in Old Mice

Introduction: Age is recognized as a major risk factor for heart failure with preserved ejection fraction (HFpEF), yet the precise role of aging in HFpEF development remains unknown. Disruption of mitochondrial protein turnover affects the quality and quantity of proteins crucial for energy metabolism. Although mitochondrial dysfunction is a key feature of both aging and HFpEF heart, the influence of age-related disturbances in mitochondrial protein turnover on HFpEF pathogenesis remains unclear. Hypothesis: Age-related impairment of mitochondrial protein turnover intensifies proteostatic perturbations induced by hypertensive and metabolic stress (2-hit stress) to exacerbate pathogenesis of cardiometabolic HFpEF. Methods: Young (3 mo.) and old (19 mo.) C57BL/6J male mice were exposed to high-fat diet and L-NAME to induce HFpEF development. Deuterium oxide (D 2 O) labeling coupled with mass spectrometry was used to investigate bulk and individual mitochondrial protein synthesis. The changes in individual mitochondrial protein abundance and protein degradation pathways were also studied. Results: Old mice presented exacerbated HFpEF characteristics compared to young mice, with higher mortality and worsened cardiac and extra-cardiac phenotypes. Regardless of age, mice subjected to 2-hit stress had 24% higher bulk mitochondrial protein synthesis compared to controls. Old control and HFpEF mice showed 17% lower mitochondrial protein synthesis compared to younger counterparts, potentially hindering the replacement of dysfunctional mitochondrial proteins. Aging reduced the synthesis rates of 30 out of 115 proteins synthesized more rapidly with HFpEF, including those involved in mitochondrial metabolism. Despite slower synthesis of numerous mitochondrial proteins with age, the overall abundance of these proteins remained unchanged or elevated compared to young counterparts, indicating impaired protein degradation. We found that aging and 2-hit stress differentially inhibited degradation pathways, including autophagy and the ubiquitin-proteasome system, further impairing protein turnover. Conclusion: Aging impairs mitochondrial protein turnover in cardiometabolic HFpEF and contributes to poorer disease outcomes.

Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

Inhibition of NLRP3 inflammasome ameliorates LPS-induced neuroinflammatory injury in mice via PINK1/Parkin pathway

Parkinson's disease (PD) is characterized by the severe loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor dysfunction. The onset of PD is often accompanied by neuroinflammation and α-Synuclein aggregation, and extensive research has focused on the activation of microglial NLRP3 inflammasomes in PD, which promotes the death of dopaminergic neurons. In this study, a model of cerebral inflammatory response was constructed in wild-type and Parkin＋/－ mice through bilateral intraventricular injection of LPS. LPS-induced activation of the NLRP3 inflammasome in wild-type mice promotes the progression of PD. The use of MCC950 in wild mice injected with LPS induces activation of Parkin/PINK and improves autophagy, which in turn improves mitochondrial turnover. It also inhibits LPS-induced inflammatory responses, improves motor function, protects dopaminergic neurons, and inhibits microglia activation. Furthermore, Parkin＋/－ mice exhibited motor dysfunction, loss of dopaminergic neurons, activation of the NLRP3 inflammasome, and α-Synuclein aggregation beginning at an early age. Parkin ± mice exhibited more pronounced microglia activation, greater NLRP3 inflammasome activation, more severe autophagy dysfunction, and more pronounced motor dysfunction after LPS injection compared to wild-type mice. Notably, the use of MCC950 in Parkin ± mice did not ameliorate NLRP3 inflammasome activation, autophagy dysfunction, or α-synuclein aggregation. Thus, MCC950 can only exert its effects in the presence of Parkin/PINK1, and targeting Parkin-mediated NLRP3 inflammasome activation is expected to be a potential therapeutic strategy for Parkinson's disease.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

Bnip3 expression is strongly associated with reelin-positive entorhinal cortex layer II neurons

In layer II of the entorhinal cortex, the principal neurons that project to the dentate gyrus and the CA3/2 hippocampal fields markedly express the large glycoprotein reelin (Re + ECLII neurons). In rodents, neurons located at the dorsal extreme of the EC, which border the rhinal fissure, express the highest levels, and the expression gradually decreases at levels successively further away from the rhinal fissure. Here, we test two predictions deducible from the hypothesis that reelin expression is strongly correlated with neuronal metabolic rate. Since the mitochondrial turnover rate serves as a proxy for energy expenditure, the mitophagy rate arguably also qualifies as such. Because messenger RNA of the canonical promitophagic BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) is known to be highly expressed in the EC, we predicted that Bnip3 would be upregulated in Re + ECLII neurons, and that the degree of upregulation would strongly correlate with the expression level of reelin in these neurons. We confirm both predictions, supporting that the energy requirement of Re + ECLII neurons is generally high and that there is a systematic increase in metabolic rate as one moves successively closer to the rhinal fissure. Intriguingly, the systematic variation in energy requirement of the neurons that manifest the observed reelin gradient appears to be consonant with the level of spatial and temporal detail by which they encode information about the external environment.

Mitochondrial DNA Instability Supersedes Parkin Mutations in Driving Mitochondrial Proteomic Alterations and Functional Deficits in Polg Mutator Mice.

Mitochondrial quality control is essential in mitochondrial function. To examine the importance of Parkin-dependent mechanisms in mitochondrial quality control, we assessed the impact of modulating Parkin on proteome flux and mitochondrial function in a context of reduced mtDNA fidelity. To accomplish this, we crossed either the Parkin knockout mouse or ParkinW402A knock-in mouse lines to the Polg mitochondrial mutator line to generate homozygous double mutants. In vivo longitudinal isotopic metabolic labeling was followed by isolation of liver mitochondria and synaptic terminals from the brain, which are rich in mitochondria. Mass spectrometry and bioenergetics analysis were assessed. We demonstrate that slower mitochondrial protein turnover is associated with loss of mtDNA fidelity in liver mitochondria but not synaptic terminals, and bioenergetic function in both tissues is impaired. Pathway analysis revealed loss of mtDNA fidelity is associated with disturbances of key metabolic pathways, consistent with its association with metabolic disorders and neurodegeneration. Furthermore, we find that loss of Parkin leads to exacerbation of Polg-driven proteomic consequences, though it may be bioenergetically protective in tissues exhibiting rapid mitochondrial turnover. Finally, we provide evidence that, surprisingly, dis-autoinhibition of Parkin (ParkinW402A) functionally resembles Parkin knockout and fails to rescue deleterious Polg-driven effects. Our study accomplishes three main outcomes: (1) it supports recent studies suggesting that Parkin dependence is low in response to an increased mtDNA mutational load, (2) it provides evidence of a potential protective role of Parkin insufficiency, and (3) it draws into question the therapeutic attractiveness of enhancing Parkin function.

Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic β cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.

Lower Mitochondrial Genome Turnover and Greater Mutation Frequency in Skeletal Muscle of Aged Compared to Adult OKC-HET Rats

Mitochondrial genomic integrity, which is supported by processes such as replication, repair, and turnover, is critical in supporting metabolism and organismal health. Accumulation of mitochondrial DNA (mtDNA) mutations contributes to mitochondrial and whole-tissue dysfunction, which lead to chronic diseases including sarcopenia. The underlying mechanisms that promote generation and age-related accumulation of mtDNA mutations are unclear. mtDNA mutants may have a replicative advantage over wild-type mtDNA that propagates the accumulation of mtDNA mutations with age. However, there is limited in vivo data regarding effects of age on mtDNA replication, particularly in skeletal muscle. We hypothesized mtDNA synthesis rates would increase with age as mutation burden increased in skeletal muscle in both male and female rats. Methods: We measured mtDNA synthesis over 14 days using the stable isotope tracer deuterium oxide (D2O) and assessed mtDNA copy number and mutation deletion frequency using digital PCR in quadriceps muscle of 9- and 26 month-old (mo) male and female OKC-HET rats, which have heterogenous mtDNA backgrounds. Results: There were no differences in mtDNA copy number between sexes or ages. However, 26 month-od rats had lower rates of mtDNA synthesis compared to 9 month-old rats (9 mo: 0.509 ± 0.009 %/day, 26 month: 0.371 ± 0.048 %/day; p=0.0024) and greater mtDNA half-lives (9 mo: 132 ± 1.79 days, 216 ± 25.63 days; p=0.0009). Concomitant with lower mtDNA synthesis, 26 month-old rats had greater (p=0.003) mtDNA deletion mutation frequency (2.023e-004 ± 7.18e-005) than 9 month-old rats (9.310e-005 ± 2.404e-005). 26 month-old female rats (1.309e-004 ± 2.386e-005) had a lower (p=0.008) mutation burden than male rats (2.74e-004 ± 4.484e-005). Conclusion: Contrary to our hypothesis, mtDNA synthesis declined with age as mutation burden increased in the quadriceps. Because mtDNA copy number was not different between ages, while synthesis decreased, mtDNA turnover declined with age. Altogether, these results suggest lower mtDNA turnover rates may contribute to age-related mtDNA mutation burden. Future studies should test how modulating mtDNA turnover affects mutation frequency and mitochondrial function. This study was supported by funding from NIH grants R21AG072137 and 5T32AG052363-04. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanical regulation of mitochondrial morphodynamics in cancer cells by extracellular microenvironment

Recently, it has been recognized that physical abnormalities (e.g. elevated solid stress, elevated interstitial fluid pressure, increased stiffness) are associated with tumor progression and development. Additionally, these mechanical forces originating from tumor cell environment through mechanotransduction pathways can affect metabolism. On the other hand, mitochondria are well-known as bioenergetic, biosynthetic, and signaling organelles crucial for sensing stress and facilitating cellular adaptation to the environment and physical stimuli. Disruptions in mitochondrial dynamics and function have been found to play a role in the initiation and advancement of cancer. Consequently, it is logical to hypothesize that mitochondria dynamics subjected to physical cues may play a pivotal role in mediating tumorigenesis. Recently mitochondrial biogenesis and turnover, fission and fusion dynamics was linked to mechanotransduction in cancer. However, how cancer cell mechanics and mitochondria functions are connected, still remain poorly understood. Here, we discuss recent studies that link mechanical stimuli exerted by the tumor cell environment and mitochondria dynamics and functions. This interplay between mechanics and mitochondria functions may shed light on how mitochondria regulate tumorigenesis.

Loss of FoxO1 activates an alternate mechanism of mitochondrial quality control for healthy adipose browning.

Browning of white adipose tissue is hallmarked by increased mitochondrial density and metabolic improvements. However, it remains largely unknown how mitochondrial turnover and quality control are regulated during adipose browning. In the present study, we found that mice lacking adipocyte FoxO1, a transcription factor that regulates autophagy, adopted an alternate mechanism of mitophagy to maintain mitochondrial turnover and quality control during adipose browning. Post-developmental deletion of adipocyte FoxO1 (adO1KO) suppressed Bnip3 but activated Fundc1/Drp1/OPA1 cascade, concurrent with up-regulation of Atg7 and CTSL. In addition, mitochondrial biogenesis was stimulated via the Pgc1α/Tfam pathway in adO1KO mice. These changes were associated with enhanced mitochondrial homeostasis and metabolic health (e.g., improved glucose tolerance and insulin sensitivity). By contrast, silencing Fundc1 or Pgc1α reversed the changes induced by silencing FoxO1, which impaired mitochondrial quality control and function. Ablation of Atg7 suppressed mitochondrial turnover and function, causing metabolic disorder (e.g., impaired glucose tolerance and insulin sensitivity), regardless of elevated markers of adipose browning. Consistently, suppression of autophagy via CTSL by high-fat diet was associated with a reversal of adO1KO-induced benefits. Our data reveal a unique role of FoxO1 in coordinating mitophagy receptors (Bnip3 and Fundc1) for a fine-tuned mitochondrial turnover and quality control, underscoring autophagic clearance of mitochondria as a prerequisite for healthy browning of adipose tissue.

Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

BackgroundAmong the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion.MethodsWestern blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively.ResultsWe observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity.ConclusionsDysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

NOX4 alleviates breast cancer cell aggressiveness by co-ordinating mitochondrial turnover through PGC1α/Drp1 axis

Triple Negative Breast Cancer (TNBC) is a highly aggressive form of breast cancer, with few treatment options. This study investigates the complex molecular mechanism by which NADPH oxidase 4 (NOX4), a major ROS producer in mitochondria, affects the aggressiveness of luminal and triple-negative breast cancer cells (TNBCs). We found that NOX4 expression was differentially regulated in luminal and TNBC cells, with a positive correlation to their epithelial characteristics. Time dependent analysis revealed that TNBCs exhibits higher steady-state ROS levels than luminal cells, but NOX4 silencing increased ROS levels in luminal breast cancer cells and enhanced their ability to migrate and invade. In contrast, NOX4 over expression in TNBCs had the opposite effect. The mouse tail-vein experiment showed that the group injected with NOX4 silenced luminal cells had a higher number of lung metastases compared to the control group. Mechanistically, NOX4 enhanced PGC1α dependent mitochondrial biogenesis and attenuated Drp1-mediated mitochondrial fission in luminal breast cancer cells, leading to an increased mitochondrial mass and elongated mitochondrial morphology. Interestingly, NOX4 silencing increased mitochondrial ROS (mtROS) levels without affecting mitochondrial (Δψm) and cellular integrity. Inhibition of Drp1-dependent fission with Mdivi1 reversed the effect of NOX4-dependent mitochondrial biogenesis, dynamics, and migration of breast cancer cells. Our findings suggest that NOX4 expression diminishes from luminal to a triple negative state, accompanied by elevated ROS levels, which may modulate mitochondrial turnover to attain an aggressive phenotype. The study provides potential insights for targeted therapies for TNBCs.

Role of Autophagy and Mitophagy in Neurodegenerative Disorders.

Autophagy is a self-destructive cellular process that removes essential metabolites and waste from inside the cell to maintain cellular health. Mitophagy is the process by which autophagy causes disruption inside mitochondria and the total removal of damaged or stressed mitochondria, hence enhancing cellular health. The mitochondria are the powerhouses of the cell, performing essential functions such as ATP (adenosine triphosphate) generation, metabolism, Ca2+ buffering, and signal transduction. Many different mechanisms, including endosomal and autophagosomal transport, bring these substrates to lysosomes for processing. Autophagy and endocytic processes each have distinct compartments, and they interact dynamically with one another to complete digestion. Since mitophagy is essential for maintaining cellular health and using genetics, cell biology, and proteomics techniques, it is necessary to understand its beginning, particularly in ubiquitin and receptor-dependent signalling in injured mitochondria. Despite their similar symptoms and emerging genetic foundations, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) have all been linked to abnormalities in autophagy and endolysosomal pathways associated with neuronal dysfunction. Mitophagy is responsible for normal mitochondrial turnover and, under certain physiological or pathological situations, may drive the elimination of faulty mitochondria. Due to their high energy requirements and post-mitotic origin, neurons are especially susceptible to autophagic and mitochondrial malfunction. This article focused on the importance of autophagy and mitophagy in neurodegenerative illnesses and how they might be used to create novel therapeutic approaches for treating a wide range of neurological disorders.