Fragmentation Of Mitochondrial Network Research Articles

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

DIPG-62. REPURPOSING OF ANTIPSYCHOTIC TRIFLUOPERAZINE FOR INHIBITING MITOCHONDRIAL FUNCTION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse Midline Gliomas (DMGs) present formidable challenges in neuro-oncology, prompting the exploration of innovative therapeutic strategies. Repurposing approved drugs, particularly those exhibiting multifaceted effects, holds promise for addressing these challenges efficiently. In this study, we investigate the potential of trifluoperazine (TFP), an antipsychotic medication, with a specific focus on its mitochondrial-targeted actions in DMGs. METHODS TFP, a phenothiazine derivative, displayed robust in vitro anticancer activity against DMGs. Our investigation explored the molecular mechanisms underlying TFP’s interaction with mitochondria, emphasizing its influence on mitochondrial function, membrane potential, and reactive oxygen species (ROS) production. TFP is well known to induce irreversible Ca2+ release from intracellular stores by directly interacting with calmodulin 2 (CaM2) at the inositol triphosphate receptor (IP3R), leading to IP3R opening. In DMGs, intracellular Ca2+ plays a pivotal role in gene expression, cell motility, differentiation, and survival. TFP disrupts calcium homeostasis by elevating intracellular Ca2+ concentration, thereby inhibiting DMG invasion and growth. DMG cell lines, exhibiting heightened CaM2 expression compared to control healthy astrocyte cells, display increased sensitivity to TFP. RESULTS Remarkably, mitochondrial homeostasis was seriously affected by TFP, and a loss of mitochondrial membrane potential, ROS overproduction and fragmentation of mitochondrial network was observed. This mitochondrial stress, coupled with ER stress, activated the integrated stress response. We demonstrated that TFP induces cell death triggered by a poor ATP content, observed in TFP-treated cells as a consequence of a dramatic decrease in OXPHOS metabolism due to mitochondrial stress. CONCLUSIONS Our findings underscore TFP’s potential as a targeted therapeutic agent against DMGs, offering a better understanding of its impact on mitochondrial and intracellular signaling pathways. As the mitochondrial landscape remains critical in DMG biology, the observed mitochondrial damage-induced oxidative stress provides valuable insights for future investigations into the clinical application of TFP in neuro-oncology.

Mechanical overload-induced release of extracellular mitochondrial particles from tendon cells leads to inflammation in tendinopathy

Tendinopathy is one of the most common musculoskeletal diseases, and mechanical overload is considered its primary cause. However, the underlying mechanism through which mechanical overload induces tendinopathy has not been determined. In this study, we identified for the first time that tendon cells can release extracellular mitochondria (ExtraMito) particles, a subtype of medium extracellular particles (mEPs), into the environment through a process regulated by mechanical loading. RNA sequencing systematically revealed that oxygen-related reactions, extracellular particles, and inflammation were present in diseased human tendons, suggesting that these factors play a role in the pathogenesis of tendinopathy. We simulated the disease condition by imposing a 9% strain overload on three-dimensional mouse tendon constructs in our cyclic uniaxial stretching bioreactor. The three-dimensional mouse tendon constructs under normal loading with 6% strain exhibited an extended mitochondrial network, as observed through live-cell confocal laser scanning microscopy. In contrast, mechanical overload led to a fragmented mitochondrial network. Our microscopic and immunoblot results demonstrated that mechanical loading induced tendon cells to release ExtraMito particles. Furthermore, we showed that mEPs released from tendon cells overloaded with a 9% strain (mEP9%) induced macrophage chemotaxis and increased the production of proinflammatory cytokines, including IL-6, CXCL1, and IL-18, from macrophages compared to mEP0%, mEP3%, and mEP6%. Partial depletion of the ExtraMito particles from mEP9% by magnetic-activated cell sorting significantly reduced macrophage chemotaxis. N-acetyl-L-cysteine treatment preserved the mitochondrial network in overloaded tendon cells, diminishing overload-induced macrophage chemotaxis toward mEP9%. These findings revealed a novel mechanism of tendinopathy; in an overloaded environment, ExtraMito particles convey mechanical response signals from tendon cells to the immune microenvironment, culminating in tendinopathy.

Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

Plastic response of macrophages to metal ions and nanoparticles in time mimicking metal implant body environment.

The paradigm of using metal biomaterials could be viewed from two sides - treatment of wide spectrum of degenerative diseases, and debris release from materials. After implant insertion, metal nanoparticles (NPs) and ions are released not only upon the first contact with cells/tissues, but in continual manner, which is immediately recognized by immune cells. In this work, the effects of metal nanoparticles (TiO2, Ni) and ions (Ni2+, Co2+, Cr3+, Mo6+) on primary human M0 macrophages from the blood samples of osteoarthritic patients undergoing total arthroplasty were studied in order to monitor immunomodulatory effects on the cells in a real-time format. Thehighest NiNPs concentration of 10µg/ml had no effect on any of macrophage parameters, while the Ni2+ ions cytotoxicity limit for the cells is 0.5mM. The cytotoxic effects of higher Ni2+ concentration revealed mitochondrial network fragmentation leading to mitochondrial dysfunction, accompanied by increased lysosomal activity and changes in pro-apoptotic markers. The suppression of M2 cell formation ability was connected to presence of Ni2+ ions (0.5mM) and TiO2NPs (10µg/ml). The immunomodulatory effect of Mo6+ ions, controversially, inhibit the formation of the cells with M1 phenotype and potentiate the thread-like shape M2s with increased chaotic cell movement. To summarize, metal toxicity depends on the debris form. Both, metal ions and nanoparticles affect macrophage size, morphological and functional parameters, but the effect of ions is more complex and likely more harmful, which has potential impact on healing and determines post-implantation reactions.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

ABSTRACT Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn’s disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1 α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate’s effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

Mitochondrial permeability transition regulator, cyclophilin D, is transcriptionally activated by C/EBP during adipogenesis

Age-related bone loss is associated with decreased bone formation, increased bone resorption and accumulation of bone marrow fat. During aging, differentiation potential of bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) is shifted towards an adipogenic lineage and away from an osteogenic lineage. In aged bone tissue, we previously observed pathological opening of the mitochondrial permeability transition pore (MPTP) which leads to mitochondrial dysfunction, oxidative phosphorylation uncoupling, and cell death. Cyclophilin D (CypD) is a mitochondrial protein that facilitates opening of the MPTP. We found earlier that CypD is downregulated during osteogenesis of BMSCs leading to lower MPTP activity and, thus, protecting mitochondria from dysfunction. However, during adipogenesis, a fate alternative to osteogenesis, the regulation of mitochondrial function and CypD expression is still unclear. In this study, we observed that BMSCs have increased CypD expression and MPTP activity, activated glycolysis, and fragmented mitochondrial network during adipogenesis. Adipogenic C/EBPα acts as a transcriptional activator of expression of the CypD gene, Ppif, during this process. Inflammation-associated transcription factor NF-κB shows a synergistic effect with C/EBPα inducing Ppif expression. Overall, we demonstrated changes in mitochondrial morphology and function during adipogenesis. We also identified C/EBPα as a transcriptional activator of CypD. The synergistic activation of CypD by C/EBPα and the NF-κB p65 subunit during this process suggests a potential link between adipogenic signaling, inflammation, and MPTP gain-of-function, thus altering BMSC fate during aging.

The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling.

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

APOE ε4 allele, along with G206D-PSEN1 mutation, alters mitochondrial networks and their degradation in Alzheimer's disease.

Alzheimer's disease remains the most common neurodegenerative disorder, depicted mainly by memory loss and the presence in the brain of senile plaques and neurofibrillary tangles. This disease is related to several cellular alterations like the loss of synapses, neuronal death, disruption of lipid homeostasis, mitochondrial fragmentation, or raised oxidative stress. Notably, changes in the autophagic pathway have turned out to be a key factor in the early development of the disease. The aim of this research is to determine the impact of the APOE allele ε4 and G206D-PSEN1 on the underlying mechanisms of Alzheimer's disease. Fibroblasts from Alzheimer's patients with APOE 3/4 + G206D-PSEN1 mutation and homozygous APOE ε4 were used to study the effects of APOE polymorphism and PSEN1 mutation on the autophagy pathway, mitochondrial network fragmentation, superoxide anion levels, lysosome clustering, and p62/SQSTM1 levels. We observed that the APOE allele ε4 in homozygosis induces mitochondrial network fragmentation that correlates with an increased colocalization with p62/SQSTM1, probably due to an inefficient autophagy. Moreover, G206D-PSEN1 mutation causes an impairment of the integrity of mitochondrial networks, triggering high superoxide anion levels and thus making APOE 3/4 + PSEN1 fibroblasts more vulnerable to cell death induced by oxidative stress. Of note, PSEN1 mutation induces accumulation and clustering of lysosomes that, along with an increase of global p62/SQSTM1, could compromise lysosomal function and, ultimately, its degradation. The findings suggest that all these modifications could eventually contribute to the neuronal degeneration that underlies the pathogenesis of Alzheimer's disease. Further research in this area may help to develop targeted therapies for the treatment of Alzheimer's disease.

Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy.

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFβ-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

Puerarin prevents cadmium-induced mitochondrial fission in AML-12 cells via Sirt1-dependent pathway

Recent investigations have revealed that puerarin (PU) alleviates cadmium (Cd)-caused hepatic damage via inhibiting oxidative stress. Mitochondria are dynamic organelles and play a critical part in regulating the occurrence of oxidative stress, but the role of mitochondria in the protection of PU against hepatocellular damage caused by Cd exposure remains unknown. Thus, this study was aimed to clarify this issue using mouse hepatocyte AML-12 cell line. Transmission electron microscopy analysis firstly showed that PU prevents Cd-induced mitochondrial ultrastructure damage. Mitochondrial network image analysis by confocal microscopy revealed that PU exerts the protection against Cd-induced cytotoxicity via restoring mitochondrial network fragmentation. Also, mitochondrial dynamic protein expression profiles showed that enhanced fission protein levels and inhibited fusion protein levels in Cd-treated cells were significantly reversed by PU, suggesting the protective effect of PU against Cd-induced mitochondrial fission. Moreover, changes of intracellular ATP level and protein levels of key regulators involving in mitochondrial biogenesis indicated that Sirtuin-1(Sirt1) pathway may be involved in the protection of Cd-impaired mitochondrial function by PU. Next, Sirt1 protein levels in treated cells were effectively regulated by genetic knockdown or chemical agonist SRT1720. Accordingly, alleviation of Cd-induced mitochondrial fission assays and cell viability by PU was markedly regulated by SRT1720 or Sirt1 knockdown, suggesting the indispensable role of Sirt1 in this process. Collectively, these findings highlight that PU prevents Cd-induced mitochondrial fission to alleviate cytotoxicity via Sirt1-dependent pathway, which provide novel evidences to fully understand the hepatoprotective action of PU against heavy metal toxicity.

Tissue imaging reveals disruption of epithelial mitochondrial networks and loss of mitochondria-associated cytochrome-C in inflamed human and murine colon

Mitochondrial dysfunction as defined by transcriptomic and proteomic analysis of biopsies or ultra-structure in transmission electron microscopy occurs in inflammatory bowel disease (IBD); however, mitochondrial dynamics in IBD have received minimal attention, with most investigations relying on cell-based in vitro models. We build on these studies by adapting the epithelial cell immunofluorescence workflow to imaging mitochondrial networks in normal and inflamed colonic tissue (i.e., murine di-nitrobenzene sulphonic acid (DNBS)-induced colitis, human ulcerative colitis). Using antibodies directed to TOMM20 (translocase of outer mitochondrial membrane 20) and cytochrome-C, we have translated the cell-based protocol for high-fidelity imaging to examine epithelial mitochondria networks in intact intestine. In epithelia of non-inflamed small or large intestinal tissue, the mitochondrial networks were dense and compact. This pattern was more pronounced in the basal region of the cell compared to that between the nucleus and apical surface facing the gut lumen. In comparison, mitochondrial networks in inflamed tissue displayed substantial loss of TOMM20+ staining. The remaining networks were less dense and fragmented, and contained isolated spherical mitochondrial fragments. The degree of mitochondrial network fragmentation mirrored the severity of inflammation, as assessed by blinded semi-quantitative scoring. As an indication of poor cell ‘health’ or viability, cytosolic cytochrome-C was observed in enterocytes with highly fragmented mitochondria. Thus, high-resolution and detailed visualization of mitochondrial networks in tissue is a feasible and valuable approach to assess disease, suited to characterizing mitochondrial abnormalities in tissue. We speculate that drugs that maintain a functional remodelling mitochondrial network and limit excess fragmentation could be a valuable addition to current therapies for IBD.

Glyphosate-induced mitochondrial reactive oxygen species overproduction activates parkin-dependent mitophagy to inhibit testosterone synthesis in mouse leydig cells

Glyphosate (GLY), one of the most extensively used herbicides in the world, has been shown to inhibit testosterone synthesis in male animals. Mitochondria are crucial organelles for testosterone synthesis and its dysfunction has been demonstrated to induce the inhibition of testosterone biosynthesis. However, whether low-dose GLY exposure targets mitochondria to inhibit testosterone synthesis and its underlying mechanism remains unclear. Here, an in vitro model of 10 μM GLY-exposed mouse Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that mitochondrial malfunction, mainly manifested by ultrastructure damage, disturbance of mitochondrial dynamics and mitochondrial reactive oxygen species (mtROS) overproduction, was responsible for GLY-decreased protein levels of steroidogenic enzymes, which leads to the inhibition of testosterone synthesis. Enhancement of autophagic flux and activation of mitophagy were shown in GLY-treated TM3 cells, and further studies have revealed that GLY-activated mitophagy is parkin-dependent. Notably, GLY-inhibited testosterone production was significantly improved by parkin knockdown. Finally, data showed that treatment with mitochondria-targeted antioxidant Mito-TEMPO (M-T) markedly reversed GLY-induced mitochondrial network fragmentation, activation of parkin-dependent mitophagy and consultant testosterone reduction. Overall, these findings demonstrate that GLY induces mtROS overproduction to activate parkin-dependent mitophagy, which contributes to the inhibition of testosterone synthesis. This study provides a potential mechanistic explanation for how GLY inhibits testosterone synthesis in mouse Leydig cells.

Mitochonic Acid 5 Improves Duchenne Muscular Dystrophy and Parkinson's Disease Model of Caenorhabditis elegans.

Mitochonic Acid 5 (MA-5) enhances mitochondrial ATP production, restores fibroblasts from mitochondrial disease patients and extends the lifespan of the disease model “Mitomouse”. Additionally, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) in mammalian cultured cells. Here, we used the nematode Caenorhabditis elegans to investigate whether MA-5 improves the Duchenne muscular dystrophy (DMD) model. Firstly, we confirmed the efficient penetration of MA-5 in the mitochondria of C. elegans. MA-5 also alleviated symptoms such as movement decline, muscular tone, mitochondrial fragmentation and Ca2+ accumulation of the DMD model. To assess the effect of MA-5 on mitochondria perturbation, we employed a low concentration of rotenone with or without MA-5. MA-5 significantly suppressed rotenone-induced mitochondria reactive oxygen species (ROS) increase, mitochondrial network fragmentation and nuclear destruction in body wall muscles as well as endogenous ATP levels decline. In addition, MA-5 suppressed rotenone-induced degeneration of dopaminergic cephalic (CEP) neurons seen in the Parkinson’s disease (PD) model. Furthermore, the application of MA-5 reduced mitochondrial swelling due to the immt-1 null mutation. These results indicate that MA-5 has broad mitochondrial homing and MINOS stabilizing activity in metazoans and may be a therapeutic agent for these by ameliorating mitochondrial dysfunction in DMD and PD.

Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.

Aging is associated with metabolic decline in skeletal muscle, which can be delayed by physical activity. Moreover, both lifelong and short-term exercise training have been shown to prevent age-associated fragmentation of the mitochondrial network in mouse skeletal muscle. However, whether lifelong endurance exercise training exerts the same effects in human skeletal muscle is still not clear. Therefore, the aim of the present study was to examine the effect of volume-dependent lifelong endurance exercise training on mitochondrial function and network connectivity in older human skeletal muscle. Skeletal muscle complex I+II-linked mitochondrial respiration per tissue mass was higher, but intrinsic complex I+II-linked mitochondrial respiration was lower in highly trained older subjects than in young untrained, older untrained, and older moderately trained subjects. Mitochondrial volume and connectivity were higher in highly trained older subjects than in untrained and moderately trained older subjects. Furthermore, the protein content of the ADP/ATP exchangers ANT1 + 2 and VDAC was higher and of the mitophagic marker parkin lower in skeletal muscle from the highly trained older subjects than from untrained and moderately trained older subjects. In contrast, H2O2 emission in skeletal muscle was not affected by either age or exercise training, but SOD2 protein content was higher in highly trained older subjects than in untrained and moderately trained older subjects. This suggests that healthy aging does not induce oxidative stress or mitochondrial network fragmentation in human skeletal muscle, but high-volume exercise training increases mitochondrial volume and network connectivity, thereby increasing oxidative capacity in older human skeletal muscle.

228-LB: Mitochondrial Architecture Controls Fatty Acid Utilization by Regulating CPT1 Sensitivity to Malonyl-CoA

Mitochondrial network fragmentation has been associated with metabolic dysregulation, although the underlying mechanisms remain unknown. Herein, using multiple cellular models where the physiological relevance of fatty acid is well-established, we show that mitochondrial fragmentation is highly associated with increased fatty acid oxidation (FAO) rates. Through confocal imaging and stable isotope tracing analysis, forced mitochondrial elongation using Mfn2 over-expression or Drp1 depletion significantly decreased FAO rates, while Mfn2 knockout leads to enhanced FAO rates (P-value =0.0003) , suggesting that mitochondrial morphology regulates fatty acid utilization. To determine the mechanism by which mitochondrial architecture controls fatty acid utilization we studied the effects of mitochondrial fragmentation and elongation on CPT1 activity. Remarkably, we find that CPT1 sensitivity to its main regulator, malonyl-CoA is strongly affected by molecular interventions that inhibit mitochondrial fusion and fission. Notably, the reduction in hepatic FAO rates upon mitochondrial elongation is attributed to a 14 fold increase in CPT1 sensitivity to malonyl-CoA. Consistent with this mechanism we show that the inhibition of FAO imposed by mitochondrial elongation can be overcome using CPT1 pharmacological activator that is malonyl-CoA-independent, but not by the malonyl-CoA-sensitive activator. Furthermore, we tested the functional implications of this mechanism in hepatocytes and beta cells. In hepatocytes we show that forced elongation by the inhibition of fission reduced gluconeogenesis by 30-70%. Whereas in islets, forced mitochondrial fragmentation through molecular inhibition of fusion, resulted in a 2.5 fold increase in insulin secretion at basal glucose concentrations, a phenomenon associated with prediabetes and obesity. Taken together, our study provides a biochemical and mechanistic explanation by which changes to mitochondrial architecture affects fuel utilization and lipid metabolism. Disclosure J. Ngo: None. D. Choi: None. L. Stiles: None. A. S. Divakaruni: None. M. Liesa: None. N. N. Danial: None. O. Shirihai: None. Funding American Diabetes Association (1-19-IBS-049)