Mitochondrial Network Integrity Research Articles

Abstract A049: Epithelial to mesenchymal transition confers sensitivity to cytotoxic agent Ophiobolin A via alterations in mitochondrial function and metabolic pathways

Abstract Triple negative breast cancer (TNBC) is characterized by its high metastatic potential, resistance to therapeutics and elevated degree of disease reoccurrence. In patients with TNBC, metastasis occurs in approximately half of all cases and is tightly correlated with poor overall survival, made worse by limited therapeutic options. Metastatic events are hypothesized to be facilitated by the epithelial to mesenchymal transition (EMT), gaining enhanced self-renewal, migration, invasion and multidrug resistant capabilities. Thus, targeting these cells remains a primary therapeutic goal, despite significant difficulty. Previous attempts in pursuing these cells through cytotoxic chemotherapeutics or targeted agents has largely failed to significantly improve patient outcomes. However, the fungus-derived sesterterpenoid Ophiobolin A (OpA) has demonstrated nanomolar cytotoxic capabilities and a high therapeutic index, specific to EMT+cells in breast cancer. The properties conferring this sensitivity, and possible novel therapeutic target, though, remain unclear. As the drug-like small molecule presents a major therapeutic opportunity, the details of its mechanism of action must be determined. To this end, we generated mitochondrial cybrids and quantified activity in individual electron transport chain (ETC) complexes, and found OpA to act in a mitochondria-targeted manner. Expanding upon this, we designed an EMT based model of breast cancer and examined alterations in mitochondrial function and metabolic pathways. Targeted liquid chromatography-mass spectronomy of metabolites indicated significant variations in many of those involved in glycolysis, OXPHOS and glutaminolysis. Flow cytometry further revealed a significant decrease in membrane potential and increase in mitochondrial reactive oxygen species (ROS), indicating mitochondrial damage. Dynamic changes between OXPHOS and glycolysis were also detected via Seahorse Bioanalyzer. Transmission electron microscopy (TEM) images and fluorescent microscopy showed distinct alterations in mitochondrial morphology, spatial dynamics and network integrity. Finally, using targeted fluorescent staining, an increase in mitochondrial labile iron was uncovered. This work reveals novel metabolic variations in the context of EMT and exploitable therapeutic targets. Thus, based on these EMT-specific responses, we conclude that EMT imparts distinct alterations to mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu, Joseph Taube. Epithelial to mesenchymal transition confers sensitivity to cytotoxic agent Ophiobolin A via alterations in mitochondrial function and metabolic pathways [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A049.

Propionic acid induces alterations in mitochondrial morphology and dynamics in SH-SY5Y cells

Propionic acid (PPA) is used to study the role of mitochondrial dysfunction in neurodevelopmental conditions like autism spectrum disorders. PPA is known to disrupt mitochondrial biogenesis, metabolism, and turnover. However, the effect of PPA on mitochondrial dynamics, fission, and fusion remains challenging to study due to the complex temporal nature of these mechanisms. Here, we use complementary quantitative visualization techniques to examine how PPA influences mitochondrial ultrastructure, morphology, and dynamics in neuronal-like SH-SY5Y cells. PPA (5 mM) induced a significant decrease in mitochondrial area (p < 0.01), Feret's diameter and perimeter (p < 0.05), and in area2 (p < 0.01). Mitochondrial event localiser analysis demonstrated a significant increase in fission and fusion events (p < 0.05) that preserved mitochondrial network integrity under stress. Moreover, mRNA expression of cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001), and OPA1 (p < 0.01) was significantly decreased. This illustrates a remodeling of mitochondrial morphology, biogenesis, and dynamics to preserve function under stress. Our data provide new insights into the influence of PPA on mitochondrial dynamics and highlight the utility of visualization techniques to study the complex regulatory mechanisms involved in the mitochondrial stress response.

Twenty-four hour rhythmicity in mitochondrial network connectivity and mitochondrial respiration; a study in human skeletal muscle biopsies of young lean and older individuals with obesity

ObjectiveMitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day–night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h MethodsMuscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day ResultsYoung healthy lean individuals displayed a day–night rhythmicity in mitochondrial network morphology, which aligned with the day–night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity ConclusionsOur data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism

ObjectiveSGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. MethodsTwenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. ResultsMitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm2 (0.01–0.06), p < 0.05) and number (0.003 μm−2 (−0.001–0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4–OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue−1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (−40.0774 (−64.4766, −15.6782) pmoles∗mg tissue−1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. ConclusionDapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux.The trial is registered with ClinicalTrials.gov, number NCT03338855.

Mitoquinone mitigates paraquat-induced A549 lung epithelial cell injury by promoting MFN1/MFN2-mediated mitochondrial fusion.

Paraquat (PQ) poisoning often leads to severe lung injuries, in which the mitochondria damage plays a critical role. Mitoquinone (MitoQ), a newly designed mitochondria-targeted antioxidant, has been proved for its benefit in mitochondria protection. However, the role of MitoQ in PQ-induced lung injury remains unclear. Thus, this study was performed to investigate the effect of MitoQ on PQ-induced lung injury and its underlying mechanisms. Our work showed that PQ caused the inhibition of A549 lung epithelial cell viability in a dose-dependent manner, while MitoQ remarkably mitigated the PQ-induced cell viability suppression. Besides this, PQ-mediated apoptosis of A549 cells was significantly attenuated by MitoQ, as indicated by the TUNEL assay and mitochondria membrane potential assay. Moreover, the intracellular reactive oxygen species (ROS) production was also dramatically suppressed when cotreated MitoQ with PQ. This could be ascribed to enhanced mitochondrial fusion mediated by Mitofusin 1 (MFN1)/Mitofusin 2 (MFN2), because MitoQ preserved mitochondrial network integrity, as reflected by MitoTracker staining, and MitoQ also increased the expression of MFN1/MFN2 in A549 cells after PQ treatment. Our data suggested MitoQ mitigated PQ-induced lung epithelial cell injury by promoting MFN1/MFN2-mediated mitochondrial fusion, and MitoQ might be a potential candidate drug for the treatment of PQ-induced lung injury.

Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated β-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic β-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with β-cell–selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps–based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the β-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in β-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

Metabolic Reprogramming of the Retinal Pigment Epithelium Drives TGFβ2‐Induced Epithelial‐Mesenchymal Transition

Transforming growth factor-beta 2 (TGFβ2) is a key orchestrator of retinal wound healing through induction of epithelial-mesenchymal-transition (EMT) in retinal pigment epithelial cells (RPE). Here we describe a previously unrecognized function of TGFβ2 in modulating mitochondrial morphology and metabolic function in human RPE cells. Treating ARPE-19 (human RPE cell line) with TGFβ2 (10 ng/ml) induced defects in mitochondrial network integrity with increased sphericity and fragmentation. Correspondingly, TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial oxidative phosphorylation (OXPHOS) levels consistent with reduced expression of NDUFB5, a key gene of Complex I of the electron transport chain. The reduced mitochondrial respiration was associated with a compensatory increase in gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA) and glycolytic reserve. TGFβ2 induced a severe suppression of PGC-1α gene expression and treatment with the selective small molecule activator of PGC-1α, ZLN005, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a profound metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. The PGC-1α promoter, ZLN005, effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment for subretinal fibrosis.

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

When Friendship Turns Sour: Effective Communication Between Mitochondria and Intracellular Organelles in Parkinson's Disease.

Parkinson's disease (PD) is a complex neurodegenerative disease with pathological hallmarks including progressive neuronal loss from the substantia nigra pars compacta and α-synuclein intraneuronal inclusions, known as Lewy bodies. Although the etiology of PD remains elusive, mitochondrial damage has been established to take center stage in the pathogenesis of PD. Mitochondria are critical to cellular energy production, metabolism, homeostasis, and stress responses; the association with PD emphasizes the importance of maintenance of mitochondrial network integrity. To accomplish the pleiotropic functions, mitochondria are dynamic not only within their own network but also in orchestrated coordination with other organelles in the cellular community. Through physical contact sites, signal transduction, and vesicle transport, mitochondria and intracellular organelles achieve the goals of calcium homeostasis, redox homeostasis, protein homeostasis, autophagy, and apoptosis. Herein, we review the finely tuned interactions between mitochondria and surrounding intracellular organelles, with focus on the nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, and lysosomes. Participants that may contribute to the pathogenic mechanisms of PD will be highlighted in this review.

Microbial BMAA elicits mitochondrial dysfunction, innate immunity activation, and Alzheimer\u2019s disease features in cortical neurons

BackgroundAfter decades of research recognizing it as a complex multifactorial disorder, sporadic Alzheimer’s disease (sAD) still has no known etiology. Adding to the myriad of different pathways involved, bacterial neurotoxins are assuming greater importance in the etiology and/or progression of sAD. β-N-Methylamino-l-alanine (BMAA), a neurotoxin produced by some microorganisms namely cyanobacteria, was previously detected in the brains of AD patients. Indeed, the consumption of BMAA-enriched foods has been proposed to induce amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), which implicated this microbial metabolite in neurodegeneration mechanisms.MethodsFreshly isolated mitochondria from C57BL/6 mice were treated with BMAA and O2 consumption rates were determined. O2 consumption and glycolysis rates were also measured in mouse primary cortical neuronal cultures. Further, mitochondrial membrane potential and ROS production were evaluated by fluorimetry and the integrity of mitochondrial network was examined by immunofluorescence. Finally, the ability of BMAA to activate neuronal innate immunity was quantified by addressing TLRs (Toll-like receptors) expression, p65 NF-κB translocation into the nucleus, increased expression of NLRP3 (Nod-like receptor 3), and pro-IL-1β. Caspase-1 activity was evaluated using a colorimetric substrate and mature IL-1β levels were also determined by ELISA.ResultsTreatment with BMAA reduced O2 consumption rates in both isolated mitochondria and in primary cortical cultures, with additional reduced glycolytic rates, decrease mitochondrial potential and increased ROS production. The mitochondrial network was found to be fragmented, which resulted in cardiolipin exposure that stimulated inflammasome NLRP3, reinforced by decreased mitochondrial turnover, as indicated by increased p62 levels. BMAA treatment also activated neuronal extracellular TLR4 and intracellular TLR3, inducing p65 NF-κB translocation into the nucleus and activating the transcription of NLRP3 and pro-IL-1β. Increased caspase-1 activity resulted in elevated levels of mature IL-1β. These alterations in mitochondrial metabolism and inflammation increased Tau phosphorylation and Aβ peptides production, two hallmarks of AD.ConclusionsHere we propose a unifying mechanism for AD neurodegeneration in which a microbial toxin can induce mitochondrial dysfunction and activate neuronal innate immunity, which ultimately results in Tau and Aβ pathology. Our data show that neurons, alone, can mount inflammatory responses, a role previously attributed exclusively to glial cells.

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.

Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE)

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain healthy retinal function. RPE dysfunction is a key feature of age‐related macular degeneration (AMD), the leading cause of blindness in developed countries. Tumor necrosis factor‐alpha (TNFα), a potent pro‐inflammatory cytokine, has been implicated in the pathogenesis of AMD. Growing evidence supports metabolic dysfunction as another key mechanism driving AMD. To date, there is no literature on the metabolic effects of TNFα on RPE and thus, this study sheds light on the impact of TNFα on mitochondrial morphology and metabolic function in RPE. Matured ARPE‐19 (human RPE cell line) were treated with TNFα (10 ng/ml) for up to 72h. TNFα induced ARPE‐19 to elongate into spindle‐shaped cells, reminiscent of epithelial‐mesenchymal transition (EMT). However, qPCR showed that TNFα reduced the expression of EMT genes (α‐SMA, Col1A1, fibronectin, MMP2, CTGF) indicating that the elongated cells were not mesenchymal in nature. To explore the effects of TNFα on metabolism, two major energy‐generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis were assessed by Seahorse high‐resolution respirometry and qPCR. The Seahorse Mito Stress Test revealed an increase in basal respiration but reduced spare respiratory capacity following TNFα treatment. TNFα increased glycolytic reserve and capacity. Despite the increased OXPHOS and glycolysis, qPCR showed a significant reduction in expression of OXPHOS (ATP5O, COX4I1, COX5B, NDUFB5) and glycolysis (G6P, PFKL, PFKFB3, PGK1, LDHA) genes with TNFα treatment. ARPE‐19 were transfected with mitochondria‐tagged GFP and imaged using confocal microscopy. Parameters on mitochondrial morphology were extracted from the confocal images using automated image segmentation and revealed that TNFα disrupted mitochondrial network integrity. This was supported by qPCR data showing significantly reduced expression of genes regulating mitochondrial function (PGC‐1α, TFAM, POLG, MFN1, MFN2, FIS1, OPA1). TNFα significantly elevated SOD2, a mitochondrial antioxidant enzyme, but not SOD1. Taken together, we find that TNFα robustly disrupts mitochondrial function and morphology in RPE, although shifting the bioenergetic profile in a paradoxical manner, i.e. TNFα raised the levels of basal respiration and glycolysis despite the suppression of genes regulating OXPHOS and glycolysis. These findings highlight the potential of targeting metabolic pathways in RPE as a promising therapeutic avenue for AMD. Further research is required to elucidate the mechanisms underlying these intriguing TNFα‐driven metabolic changes.Support or Funding InformationGrimshaw‐Gudewicz Charitable Foundation; National Council of Scientific and Technological Development—Brazil (PDE 210474/2014‐9); The Iraty Award; NEI Core Grant P30EYE003790; Department of Defense, Spinal Vision Research Program under Award no. VR180132

MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day-1·mouse-1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment.NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons.

Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration.

Dynamic Proteomics Profiling of &lt;i&gt;Legionella pneumophila&lt;/i&gt; Infection Unveils Modulation of the Host Mitochondrial Stress Response Pathway

The human pathogen Legionella pneumophila (L.p.) secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of most L.p. effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome during L.p. infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show that L.p. substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmt by a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterized L.p. effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.

Inhibition of the proteasome preserves Mitofusin-2 and mitochondrial integrity, protecting cardiomyocytes during ischemia-reperfusion injury

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.

MitoQ Regulates Redox-related Non-coding RNAs to Improve Mitochondrial Network Integrity in Pressure Overload Heart Failure

We present high-throughput amplicon sequence (HTS) datasets of the microbial metacommunity DNA of the gut tissue and the gut digesta of naturally occurring (n = 3) and laboratory aquaculture (n = 2) green sea urchins, Lytechinus variegatus. The HTS datasets were generated on an Illumina MiSeq by targeting the amplicons of the V4 region of the 16S rRNA gene. After the raw sequences were quality checked and filtered, 88% of the sequence reads were subjected to bioinformatics analyses to generate operation taxonomic units (OTUs), which were then verified for saturation by using rarefaction analysis at a 3% sequence variation. Further, the OTUs were randomly subsampled to the minimum sequence count values. Then, the FASTA-formatted representative sequences of the microbiota were assigned taxonomic identities through multiple databases using the SILVA ACT: Alignment, Classification and Tree Service (www.arb-silva.de/aligner). The HTS datasets of this metagenome can be accessed from the BioSample Submission Portal (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject IDs PRJNA291441 and PRJNA326427.

The Role of Mitophagy in Innate Immunity.

Mitochondria are cellular organelles essential for multiple biological processes, including energy production, metabolites biosynthesis, cell death, and immunological responses among others. Recent advances in the field of immunology research reveal the pivotal role of energy metabolism in innate immune cells fate and function. Therefore, the maintenance of mitochondrial network integrity and activity is a prerequisite for immune system homeostasis. Mitochondrial selective autophagy, known as mitophagy, surveils mitochondrial population eliminating superfluous and/or impaired organelles and mediating cellular survival and viability in response to injury/trauma and infection. Defective removal of damaged mitochondria leads to hyperactivation of inflammatory signaling pathways and subsequently to chronic systemic inflammation and development of inflammatory diseases. Here, we review the molecular mechanisms of mitophagy and highlight its critical role in the innate immune system homeostasis.

Muscle strength deficiency and mitochondrial dysfunction in a muscular dystrophy model of Caenorhabditis elegans and its functional response to drugs.

ABSTRACTMuscle strength is a key clinical parameter used to monitor the progression of human muscular dystrophies, including Duchenne and Becker muscular dystrophies. Although Caenorhabditis elegans is an established genetic model for studying the mechanisms and treatments of muscular dystrophies, analogous strength-based measurements in this disease model are lacking. Here, we describe the first demonstration of the direct measurement of muscular strength in dystrophin-deficient C. elegans mutants using a micropillar-based force measurement system called NemaFlex. We show that dys-1(eg33) mutants, but not dys-1(cx18) mutants, are significantly weaker than their wild-type counterparts in early adulthood, cannot thrash in liquid at wild-type rates, display mitochondrial network fragmentation in the body wall muscles, and have an abnormally high baseline mitochondrial respiration. Furthermore, treatment with prednisone, the standard treatment for muscular dystrophy in humans, and melatonin both improve muscular strength, thrashing rate and mitochondrial network integrity in dys-1(eg33), and prednisone treatment also returns baseline respiration to normal levels. Thus, our results demonstrate that the dys-1(eg33) strain is more clinically relevant than dys-1(cx18) for muscular dystrophy studies in C. elegans. This finding, in combination with the novel NemaFlex platform, can be used as an efficient workflow for identifying candidate compounds that can improve strength in the C. elegans muscular dystrophy model. Our study also lays the foundation for further probing of the mechanism of muscle function loss in dystrophin-deficient C. elegans, leading to knowledge translatable to human muscular dystrophy.This article has an associated First Person interview with the first author of the paper.