Mitochondrial Cluster Research Articles

Glutathione deficiency decreases lipid droplet stores and increases reactive oxygen species in mouse oocytes†.

Glutathione (GSH) is a tripeptide thiol antioxidant that has been shown to be important to overall reproductive health. Glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis consists of a catalytic and a modifier (GCLM) subunit. We previously showed that oxidative stress in the ovary and oocytes of Gclm-/- mice is associated with accelerated age-related decline in ovarian follicles and decreased female fertility due to preimplantation embryonic mortality. Mammalian preimplantation development is a highly regulated and energy-intensive process that primarily relies on coordination between lipid droplets (LDs) and mitochondria to maintain cellular homeostasis. In this study, we hypothesized that GSH deficiency in oocytes increases oxidative stress, leading to increased mitochondrial dysfunction and decreased LD consumption, thereby decreasing oocyte developmental competence. We observed that Gclm-/- oocytes have increased oxidative stress, primarily in the form of mitochondrial superoxide and decreased subcortical mitochondrial clusters. Further, Gclm-/- oocytes have decreased mitochondrial membrane potential (ΔΨm) compared with Gclm+/+. We surmise this is likely due to the decreased availability of LDs, as we observed a significant decrease in LD content in Gclm-/- oocytes compared with Gclm+/+. The decreased oocyte LD content is likely related to an altered serum lipidome, with Gclm-/- serum having relatively lower unsaturated fatty acids and triglycerides than that of Gclm+/+ and Gclm+/- females. Altogether these data support that decreased LDs and increased oxidative stress are primary drivers of decreased oocyte developmental competence in GSH-deficient oocytes.

Localized glucose import, glycolytic processing, and mitochondria generate a focused ATP burst to power basement-membrane invasion.

Invasive cells use transient, energy-consuming protrusions to breach basement membrane (BM) barriers. Using the ATP sensor PercevalHR during anchor cell (AC) invasion in Caenorhabditis elegans, we show that BM invasion is accompanied by an ATP burst from mitochondria at the invasive front. RNAi screening and visualization of a glucose biosensor identified two glucose transporters, FGT-1 and FGT-2, which bathe invasive front mitochondria with glucose and facilitate the ATP burst to form protrusions. FGT-1 localizes at high levels along the invasive membrane, while FGT-2 is adaptive, enriching most strongly during BM breaching and when FGT-1 is absent. Cytosolic glycolytic enzymes that process glucose for mitochondrial ATP production cluster with invasive front mitochondria and promote higher mitochondrial membrane potential and ATP levels. Finally, we show that UNC-6 (netrin), which polarizes invasive protrusions, also orients FGT-1. These studies reveal a robust and integrated energy acquisition, processing, and delivery network that powers BM breaching.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Electron Microscopy Reveals Evidence of Perinuclear Clustering of Mitochondria in Cardiac Biopsy-Proven Allograft Rejection.

Acute cellular rejection is a major complication in heart transplantation. We focus on the analysis of new ultrastructural findings in cardiac biopsy rejection based on mitochondrial intracellular organization. This study includes heart transplanted patients from a single center who were referred for endomyocardial biopsies as a scheduled routine screening. Participants were divided into two groups: patients transplanted without allograft rejection (Grade 0R), and patients with biopsy-proven allograft rejection (Grade ≥ 2R). Using electronic microscopy, we detected a significant increase in the volume density of mitochondria (p < 0.0001) and dense bodies (p < 0.01) in the rejection group. The most relevant finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and molding the morphology of this membrane in all rejection samples (100%). We identified this perinuclear clustering of mitochondria phenomenon in a 68 ± 27% of the total cardiac nucleus observed from rejection samples. We did not observe this phenomenon in any non-rejection samples, reflecting excellent sensitivity and specificity. We have identified a specific phenomenon affecting the architecture of the nuclear membrane—perinuclear clustering of mitochondria—in endomyocardial biopsies from patients with cardiac rejection. This ultrastructural approach might complement and improve the diagnosis of rejection.

Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila.

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.

Expression signature of the Leigh syndrome French-Canadian type

As a result of a founder effect, a Leigh syndrome variant called Leigh syndrome, French-Canadian type (LSFC, MIM / 220,111) is more frequent in Saguenay–Lac-Saint-Jean (SLSJ), a geographically isolated region on northeastern Quebec, Canada. LSFC is a rare autosomal recessive mitochondrial neurodegenerative disorder due to damage in mitochondrial energy production. LSFC is caused by pathogenic variants in the nuclear gene leucine-rich pentatricopeptide repeat-containing (LRPPRC). Despite progress understanding the molecular mode of action of LRPPRC gene, there is no treatment for this disease.The present study aims to identify the biological pathways altered in the LSFC disorder through microarray-based transcriptomic profile analysis of twelve LSFC cell lines compared to twelve healthy ones, followed by gene ontology (GO) and pathway analyses.A set of 84 significantly differentially expressed genes were obtained (p ≥ 0.05; Fold change (Flc) ≥ 1.5). 45 genes were more expressed (53.57%) in LSFC cell lines compared to controls and 39 (46.43%) had lower expression levels. Gene ontology analysis highlighted altered expression of genes involved in the mitochondrial respiratory chain and energy production, glucose and lipids metabolism, oncogenesis, inflammation and immune response, cell growth and apoptosis, transcription, and signal transduction. Considering the metabolic nature of LSFC disease, genes included in the mitochondrial respiratory chain and energy production cluster stood out as the most important ones to be involved in LSFC mitochondrial disorder. In addition, the protein-protein interaction network indicated a strong interaction between the genes included in this cluster. The mitochondrial gene NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2), with higher expression in LSFC cells, represents a target for functional studies to explain the role of this gene in LSFC disease.This work provides, for the first time, the LSFC gene expression profile in fibroblasts isolated from affected individuals. This represents a valuable resource to understand the pathogenic basis and consequences of LRPPRC dysfunction.

A mitochondria cluster at the proximal axon initial segment controls axodendritic TAU trafficking in rodent primary and human iPSC-derived neurons

Loss of neuronal polarity and missorting of the axonal microtubule-associated-protein TAU are hallmarks of Alzheimer’s disease (AD) and related tauopathies. Impairment of mitochondrial function is causative for various mitochondriopathies, but the role of mitochondria in tauopathies and in axonal TAU-sorting is unclear. The axon-initial-segment (AIS) is vital for maintaining neuronal polarity, action potential generation, and—here important—TAU-sorting. Here, we investigate the role of mitochondria in the AIS for maintenance of TAU cellular polarity. Using not only global and local mitochondria impairment via inhibitors of the respiratory chain and a locally activatable protonophore/uncoupler, but also live-cell-imaging and photoconversion methods, we specifically tracked and selectively impaired mitochondria in the AIS in primary mouse and human iPSC-derived forebrain/cortical neurons, and assessed somatic presence of TAU. Global application of mitochondrial toxins efficiently induced tauopathy-like TAU-missorting, indicating involvement of mitochondria in TAU-polarity. Mitochondria show a biased distribution within the AIS, with a proximal cluster and relative absence in the central AIS. The mitochondria of this cluster are largely immobile and only sparsely participate in axonal mitochondria-trafficking. Locally constricted impairment of the AIS-mitochondria-cluster leads to detectable increases of somatic TAU, reminiscent of AD-like TAU-missorting. Mechanistically, mitochondrial impairment sufficient to induce TAU-missorting results in decreases of calcium oscillation but increases in baseline calcium, yet chelating intracellular calcium did not prevent mitochondrial impairment-induced TAU-missorting. Stabilizing microtubules via taxol prevented TAU-missorting, hinting towards a role for impaired microtubule dynamics in mitochondrial-dysfunction-induced TAU-missorting. We provide evidence that the mitochondrial distribution within the proximal axon is biased towards the proximal AIS and that proper function of this newly described mitochondrial cluster may be essential for the maintenance of TAU polarity. Mitochondrial impairment may be an upstream event in and therapeutic target for AD/tauopathy.

The dynamin-related protein 1 is decreased and the mitochondrial network is altered in Friedreich’s ataxia cardiomyopathy

Friedreich ataxia is an autosomal recessive congenital neurodegenerative disease caused by a deficiency in the frataxin protein and is often diagnosed in young adulthood. An expansion of guanine-adenine-adenine repeats in the first intron of the FXN gene leads to decreased frataxin expression. Frataxin plays an essential role in mitochondrial metabolism. Most Friedreich ataxia patients are diagnosed with left ventricular hypertrophic cardiomyopathy, and 60% of patients die with hypertrophic cardiomyopathy. However, the mitochondrial anatomy in Friedreich ataxia hypertrophic cardiomyopathy is still poorly understood. We investigated mitochondrial fission, fusion, and function using biochemical, microscopy, and computational stochastic analysis in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy and a healthy individual. We found a significantly higher mitochondrial footprint, decreased mitochondrial fission protein dynamin-related protein, and mitochondrial fission rate over fusion with more giant mitochondrial clusters in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy, compared to an unaffected individual. We also found significantly depolarized mitochondrial membrane potential and higher reactive oxygen species levels in Friedreich ataxia human induced pluripotent stem cell cardiomyocytes. Our results show that frataxin's depletion may dampen the mitochondrial fission machinery by reducing dynamin-related protein1. The loss of mitochondrial fission might lead to elevated reactive oxygen species and depolarized mitochondrial membrane potential, which may cause oxidative damage in Friedreich ataxia hypertrophic cardiomyopathy. Further investigations are needed to identify the mechanism of downregulating dynamin-related protein1 due to the frataxin deficiency in Friedreich ataxia hypertrophic cardiomyopathy.

Salidroside inhibits doxorubicin-induced cardiomyopathy by modulating a ferroptosis-dependent pathway

BackgroundDoxorubicin-induced cardiotoxicity (DIC) limits the clinical application of the drug in treatment of cancers and imposes a severe health burden on the patients. Therefore, there is an urgent need to develop alternative therapeutic strategies or drugs to minimize DIC. Salidroside is a phenylpropanoid glycoside extracted from Rhodiola rosea with multiple biological effects such as anti-inflammation and antioxidant properties. However, its mechanism of action in DIC is still poorly understood. PurposeThe present study was aimed to investigate the role of salidroside in DIC and associated mechanism of action for the described effects. MethodsCardiac dysfunction was induced through treatment of mice with doxorubicin in vivo and in vitro. The mechanism of action of salidroside was investigated using western blot assay, qPCR, immunofluorescence, histochemistry, echocardiography, and high-content imaging system. ResultsResults of the current study found that treatment of mice with salidroside significantly improved doxorubicin-induced cardiac dysfunction, ferroptosis-like cell damage, and fibrosis in vivo. Further, it was noted that salidroside inhibited ferroptosis in vivo and in vitro by limiting iron accumulation, restoring GPX4-dependent antioxidant capacity, and preventing lipid peroxidation at the cellular or mitochondrial levels. Mechanistically, salidroside inhibited DOX-induced mitochondrial ROS, Fe2+, and lipid peroxidation as well as restored mitochondrial membrane potential by promoting mitochondrial biogenesis, improving mitochondrial iron-sulfur clusters, and restoring mitochondrial OXPHOS complexes, thereby improving mitochondrial function. In addition, AMPK is a key protein that coordinates mitochondria, metabolism, and ferroptosis. Therefore, it was found that compound C (CC), an AMPK inhibitor, disrupted the regulation of cellular lipid metabolism and mitochondrial function of salidroside as well as led to failure of the protective effect of salidroside against ferroptotic cell death. ConclusionsThe present study evidently demonstrated the cardioprotective effects of salidroside against doxorubicin-induced cardiomyopathy. Further, salidroside markedly down-regulated ferroptotic cell death by activating AMPK-dependent signaling pathways including regulating abnormal fatty acid metabolism and maintaining mitochondrial function. Therefore, salidroside is can be exploited to develop a novel medication for clinical DIC and salidroside may represent a novel treatment that improves recovery from DIC by targeting ferroptosis.

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering.

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity.

During FeS cluster biogenesis, ferredoxin and frataxin use overlapping binding sites on yeast cysteine desulfurase Nfs1

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron–sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1–Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.

Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation.

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

Aberrant Mitochondrial Dynamics and Exacerbated Response to Neuroinflammation in a Novel Mouse Model of CMT2A.

Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common hereditary axonal neuropathy caused by mutations in MFN2 encoding Mitofusin-2, a multifunctional protein located in the outer mitochondrial membrane. In order to study the effects of a novel MFN2K357T mutation associated with early onset, autosomal dominant severe CMT2A, we generated a knock-in mouse model. While Mfn2K357T/K357T mouse pups were postnatally lethal, Mfn2+/K357T heterozygous mice were asymptomatic and had no histopathological changes in their sciatic nerves up to 10 months of age. However, immunofluorescence analysis of Mfn2+/K357T mice revealed aberrant mitochondrial clustering in the sciatic nerves from 6 months of age, in optic nerves from 8 months, and in lumbar spinal cord white matter at 10 months, along with microglia activation. Ultrastructural analyses confirmed dysmorphic mitochondrial aggregates in sciatic and optic nerves. After exposure of 6-month-old mice to lipopolysaccharide, Mfn2+/K357T mice displayed a higher immune response, a more severe motor impairment, and increased CNS inflammation, microglia activation, and macrophage infiltrates. Overall, ubiquitous Mfn2K357T expression renders the CNS and peripheral nerves of Mfn2+/K357T mice more susceptible to mitochondrial clustering, and augments their response to inflammation, modeling some cellular mechanisms that may be relevant for the development of neuropathy in patients with CMT2A.

Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites.

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.

MitoWave: Spatiotemporal analysis of mitochondrial membrane potential fluctuations during I/R

Mitochondria exhibit unstable inner membrane potentials (ΔΨm) when subjected to stress, such as during ischemia/reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called “MitoWave” that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨm changes in the cardiac mitochondrial network during I/R. In vitro ischemia was affected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes for 1 h and removing the coverslip to allow for reperfusion, revealing complex oscillatory ΔΨm. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors were quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time point of ΔΨm depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from neonatal mouse ventricular myocytes subjected to I/R oscillate in the frequency range of 8.6–45 mHz, with a mean of 8.73 ± 4.35 mHz. Oscillating clusters had smaller areas ranging from 49.8 ± 1.2 μm2, whereas nonoscillating clusters had larger areas 66 ± 1.5 μm2. A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semiautomated method to quantify complex time-resolved mitochondrial behavior in an easy-to-follow workflow, enabling unbiased, reproducible quantitation of complex nonstationary cellular phenomena.

Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands

Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. Dysfunction of mitochondrial reprogramming can occur during bacterial and viral infections compromising hosts’ immune signaling. Comparative evaluation of these alterations in response to bacterial and viral ligands can provide insights into a cell’s ability to mount pathogen-specific responses. In this study, we used two-photon excitation fluorescence (TPEF) imaging to quantify reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels in the cell and to calculate the optical redox ratio (ORR), an indicator of mitochondrial dysfunction. Analyses were performed on RAW264.7 cells and murine bone marrow derived macrophages (BMM) stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. Responses were cell type dependent, with primary cells having significantly higher levels of FAD and higher oxygen consumption rates suggesting BMM may be more dependent on mitochondrial metabolism. Our findings also suggest that FAD-TPEF intensity may be a better predictor of mitochondrial activity and localization since it demonstrates unique mitochondrial clustering patterns in LPS vs. Poly(I:C) stimulated macrophages. Collectively, we demonstrate that TPEF imaging is a powerful label-free approach for quantifying changes in mitochondrial function and organization in macrophages following bacterial and viral stimuli.

Thiophene-2-carboxamide derivatives of anthraquinone: A new potent antitumor chemotype.

The anthraquinone scaffold has long been known as a source of efficacious antitumor drugs. In particular, the various chemical modifications of the side chains in this scaffold have yielded the compounds potent for the wild type tumor cells, their counterparts with molecular determinants of altered drug response, as well as invivo settings. Further exploring the chemotype of anticancer heteroarene-fused anthraquinones, we herein demonstrate that derivative of anthra[2,3-b]thiophene-2-carboxamide, (compound 8) is highly potent against a panel of human tumor cell lines and their drug resistant variants. Treatment with submicromolar or low micromolar concentrations of 8 for only 30min was sufficient to trigger lethal damage of K562 chronic myelogenous leukemia cells. Compound 8 (2.5μM, 3-6h) induced an apoptotic cell death as determined by concomitant activation of caspases 3 and 9, cleavage of poly(ADP-ribose) polymerase, increase of Annexin V/propidium iodide double stained cells, DNA fragmentation (subG1 fraction) and a decrease of mitochondrial membrane potential. Neither a significant interaction with double stranded DNA nor strong inhibition of the DNA dependent enzyme topoisomerase 1 by 8 were detectable in cell free systems. Laser scanning confocal microscopy revealed that some amount of 8 was detectable in mitochondria as early as 5min after the addition to the cells; exposure for 1h caused significant morphological changes and clustering of mitochondria. The bioisosteric analog 2 in which the thiophene ring was replaced with furan was less active although the patterns of cytotoxicity of both derivatives were similar. These results point at the specific role of the sulfur atom in the antitumor properties of carboxamide derivatives of heteroarene-fused anthraquinone.

In Vivo/Ex Vivo EPR Investigation of the Brain Redox Status and Blood-Brain Barrier Integrity in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive decline and total brain atrophy. Despite the substantial scientific effort, the pathological mechanisms underlying neurodegeneration in AD are currently unknown. In most studies, amyloid β peptide has been considered the key pathological change in AD. However, numerous Aβ-targeting treatments have failed in clinical trials. This implies the need to shift the research focus from Aβ to other pathological features of the disease. The aim of this study was to examine the interplay between mitochondrial dysfunction, oxidative stress and blood-brain barrier (BBB) disruption in AD pathology, using a novel approach that involves the application of electron paramagnetic resonance (EPR) spectroscopy. In vivo and ex vivo EPR spectroscopy using two spin probes (aminoxyl radicals) exhibiting different cell-membrane and BBB permeability were employed to assess BBB integrity and brain tissue redox status in the 5xFAD mouse model of AD. In vivo spin probe reduction decay was analyzed using a two-compartment pharmacokinetic model. Furthermore, 15 K EPR spectroscopy was employed to investigate the brain metal content. This study has revealed an altered brain redox state, BBB breakdown, as well as ROS-mediated damage to mitochondrial iron-sulfur clusters, and up-regulation of MnSOD in the 5xFAD model. The EPR spin probes were shown to be excellent in vivo reporters of the 5xFAD neuronal tissue redox state, as well as the BBB integrity, indicating the importance of in vivo EPR spectroscopy application in preclinical studies of neurodegenerative diseases.

An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development.

Hepatitis B virus X protein C-terminal 127 amino acid truncation is often found expressed in hepatocellular carcinoma (HCC) tissue samples. The present in vitro study tried to determine the role of this truncation mutant in the hepatitis B–related liver diseases such as fibrosis, cirrhosis, HCC, and metastasis. HBx gene and its 127 amino acid truncation mutant were cloned in mammalian expression vectors and transfected in human hepatoma cell line. Changes in cell growth/proliferation, cell cycle phase distribution, expression of cell cycle regulatory genes, mitochondrial depolarization, and intracellular reactive oxygen species (ROS) level were analyzed. Green fluorescent protein (GFP)–tagged version of HBx and the truncation mutant were also created and the effects of truncation on HBx intracellular expression pattern and localization were studied. Effect of time lapse on protein expression pattern was also analyzed. The truncation mutant of HBx is more efficient in inducing cell proliferation, and causes more ROS production and less mitochondrial depolarization as compared with wild type (wt) HBx. In addition, gene expression is altered in favor of carcinogenesis in the presence of the truncation mutant. Furthermore, mitochondrial perinuclear aggregation is achieved earlier in the presence of the truncation mutant. Therefore, HBx C-terminal 127 amino acid truncation might be playing important roles in the development of hepatitis B–related liver diseases by inducing cell proliferation, altering gene expression, altering mitochondrial potential, inducing mitochondrial clustering and oxidative stress, and changing HBx expression pattern.