scholarly journals Loss of vacuolar type H+-ATPase induces caspase-independent necrosis-like death of hair cells in zebrafish neuromast

2021 ◽  
Author(s):  
Peu Santra ◽  
Jeffrey D. Amack
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Hair Cells ◽  
Mitochondrial Membrane ◽  
Sensorineural Deafness ◽  
Specific Cell ◽  
Cell Type ◽  
Specific Cell Type

The vacuolar type H+-ATPase (V-ATPase) is a ubiquitous membrane-bound, multi-subunit proton pump that regulates pH of cellular compartments. V-ATPase activity is known to modulate several cellular processes, but cell type-specific V-ATPase functions remain poorly understood. Patients with mutations in specific V-ATPase subunits can develop sensorineural deafness, but underlying mechanisms are unclear. Here, we show that V-ATPase mutations disrupt formation of zebrafish neuromasts, which serve as a model system to investigate the underpinnings of hearing loss. Neuromasts consist of support cells surrounding mechanosensory hair cells that function similarly to hair cells in the mammalian inner ear. In V-ATPase mutant zebrafish embryos, neuromasts are small, malformed, and contain pyknotic nuclei that denote dying cells. Using molecular markers and live imaging, we find that loss of V-ATPase induces hair cells, but not neighboring support cells, to undergo caspase-independent necrosis-like cell death. This is the first demonstration that loss of V-ATPase can lead to necrosis-like cell death in a specific cell type in vivo. Mechanistically, loss of V-ATPase reduces mitochondrial membrane potential in hair cells, which has previously been associated with necrotic cell death. Modulating the mitochondrial permeability transition pore, which regulates mitochondrial membrane potential, improves hair cell survival. These results have implications for understanding causes of sensorineural deafness, and more broadly, reveal functions for V-ATPase in regulating mitochondrial function and promoting survival of a specific cell type in vivo.

scholarly journals Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

2021 ◽  
Vol 7 (2) ◽  
pp. 130
Author(s):  
Nathan P. Wiederhold
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Candida Species ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Fungal Infections ◽  
Published Data ◽  
Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

scholarly journals Mitochondrial membrane potential in 2-cell stage embryos correlates with the success of preimplantation development

Reproduction ◽  
2014 ◽  
Vol 147 (5) ◽  
pp. 627-638 ◽  
Author(s):  
Kouji Komatsu ◽  
Akira Iwase ◽  
Miki Mawatari ◽  
Jingwen Wang ◽  
Mamoru Yamashita ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Process ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Hormonal Stimulation ◽  
Maternal Mrna

Hormonal stimulation in superovulation induces female mice to ovulate more oocytes than spontaneous ovulation. Because the superovulated oocytes contain a number of oocytes that normally regress before spontaneous ovulation or immature oocytes, the development of some embryos that derive from these oocytes by IVF is prevented. Therefore, the quality of superovulated oocytes should differ from that of spontaneously ovulated oocytes. In this study, we evaluated the quality of superovulated oocytes, by examining 1- and 2-cell stage embryos, in which the development mainly depends on the maternal mRNA, proteins, and mitochondria that are contained in the oocytes, and we then measured the mitochondrial membrane potential (ΔΨm) of the 1- and 2-cell stage,in vivo-fertilized, and IVF embryos. The ΔΨmof 1-cell stage IVF embryos was lower than that ofin vivo-fertilized embryos; however, there was no difference between IVF embryos. During the developmental process from 1- to 2-cell stage, the ΔΨmofin vivo-fertilized embryos was highly upregulated, whereas a number of IVF embryos remained unchanged. As a result, 2-cell stage embryos were divided into two groups: high- and low- ΔΨm2-cell stage IVF embryos. The development of low-ΔΨm2-cell stage IVF embryos tended to be arrested after the 2-cell stage. These results indicated that the upregulation of ΔΨmduring the 1- to 2-cell stage was important in the development of early preimplantation embryos; there were some defects in the mitochondria of superovulated oocytes, which prevented their development.

Abstract 371: Mitochondrial Antiviral Signaling (MAVS) Protects Cardiomyocytes Under Oxidative Stress by Interfering with Bax-Mediated Cell Death

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Toshitaka Yajima ◽  
Stanley Park ◽  
Hanbing Zhou ◽  
Michinari Nakamura ◽  
Mitsuyo Machida ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Antiviral Signaling ◽  
Cell Death Pathway ◽  
The Impact

MAVS is a mitochondrial outer membrane protein that activates innate antiviral signaling by recognizing cytosolic viral RNAs and DNAs. While the discovery of MAVS is the first molecular evidence that links mitochondria to innate immune mechanisms, it is still unclear whether MAVS affects mitochondrial cell death as a member of caspase activation and recruitment domain (CARD)-containing proteins. We found that MAVS interacts with Bax through CARD by Yeast two-hybrid and a series of immunoprecipitation (IP) assay, which led us to hypothesize that MAVS functions not only in the innate antiviral mechanisms but also in the mitochondrial cell death pathway. Methods: 1) We examined molecular interaction between MAVS and Bax under oxidative stress by IP using isolated myocytes with H2O2 stimulation and the heart post ischemia-reperfusion (I/R). 2) We evaluated the effect of MAVS on mitochondrial membrane potential and apoptosis under H2O2 stimulation using isolated myocytes with adenoviral MAVS knockdown. 3) We investigated the impact of MAVS on %myocardial infarction (%MI) post I/R using cardiac-specific MAVS knockout (cKO) and transgenic (cTg) mice which we have originally generated. 4) We examined the effect of MAVS on recombinant Bax (rBax)-mediated cytochrome c release using isolated mitochondria from wild type (WT) and MAVS KO mice. Results: 1) The amount of Bax pulled down with MAVS was significantly increased in isolated myocytes with 0.2 mM H2O2 compared to those without stimulation (mean±SD; 1.808±0.14, n=5, p<0.001) and in the heart post I/R compared to sham (2.2±1.19, n=3, p=0.0081). 2) Myocytes with MAVS knockdown showed clear abnormalities in mitochondrial membrane potential and caspace-3 cleavage with 0.2 mM H2O2 compared to control cardiomyocytes. 3) MAVS cKO had significantly larger %MI than WT (81.9 ± 5.8% vs. 42.6 ± 13.6%, n=8, p=0.0008). In contrast, MAVS cTg had significantly smaller %MI that WT (30.0 ± 4.8% vs. 49.2 ± 4.8%, n=10, p=0.0113). 4) Mitochondria from MAVS KO exhibited cytochrome c release after incubation with 2.5 μ g of rBax while those from WT required 10 μ g of rBax. Conclusion: These results demonstrate that MAVS protects cardiomyocyte under oxidative stress by interfering with Bax-mediated cytochrome c release from mitochondria.

scholarly journals In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer

Nature ◽  
2019 ◽  
Vol 575 (7782) ◽  
pp. 380-384 ◽  
Author(s):  
Milica Momcilovic ◽  
Anthony Jones ◽  
Sean T. Bailey ◽  
Christopher M. Waldmann ◽  
Rui Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Membrane Potential ◽  
Small Cell Lung Cancer ◽  
Mitochondrial Membrane Potential ◽  
Cell Lung Cancer ◽  
In Vivo Imaging ◽  
Mitochondrial Membrane ◽  
Small Cell ◽  
Small Cell Lung

scholarly journals Meloxicam, a Selective COX-2 Inhibitor, Mediates Hypoxia-Inducible Factor- (HIF-) 1α Signaling in Hepatocellular Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Yinghong Zhou ◽  
Xiaofeng Dong ◽  
Peng Xiu ◽  
Xin Wang ◽  
Jianrong Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Transcriptional Activation ◽  
Mitochondrial Membrane ◽  
Hypoxia Inducible Factor ◽  
Luciferase Assay ◽  
Cox 2

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

scholarly journals Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

2020 ◽  
Vol 117 (37) ◽  
pp. 23113-23124
Author(s):  
Helena M. Viola ◽  
Ashay A. Shah ◽  
Victoria P. A. Johnstone ◽  
Henrietta Cserne Szappanos ◽  
Mark P. Hodson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Hypertrophic Cardiomyopathy ◽  
Mitochondrial Membrane Potential ◽  
Metabolic Activity ◽  
Murine Model ◽  
Mitochondrial Membrane ◽  
Cardiac Myocyte ◽  
Human Condition ◽  
Preventative Therapy

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in “at risk” patients with identified Gly203Ser gene mutations.

scholarly journals Effects of inflammatory and anti-inflammatory environments on the macrophage mitochondrial function

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dong Ji ◽  
Jian-yun Yin ◽  
Dan-feng Li ◽  
Chang-tai Zhu ◽  
Jian-ping Ye ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Raw 264.7 Cells ◽  
Metabolic Adaptation ◽  
Isocitrate Dehydrogenase 2 ◽  
Anti Inflammatory

AbstractMitochondrial response to inflammation is crucial in the metabolic adaptation to infection. This study aimed to explore the mitochondrial response under inflammatory and anti-inflammatory environments, with a focus on the tricarboxylic acid (TCA) cycle. Expression levels of key TCA cycle enzymes and the autophagy-related protein light chain 3b (LC3b) were determined in raw 264.7 cells treated with lipopolysaccharide (LPS) and metformin (Met). Additionally, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed using flow cytometry. Moreover, 8-week-old C57BL/6J mice were intraperitoneally injected with LPS and Met to assess the mitochondrial response in vivo. Upon LPS stimulation, the expression of key TCA enzymes, including citrate synthase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase 2, and the mitochondrial membrane potential decreased, whereas the levels of LC3b and ROS increased. However, treatment with Met inhibited the reduction of LPS-induced enzyme levels as well as the elevation of LC3b and ROS levels. In conclusion, the mitochondrial TCA cycle is affected by the inflammatory environment, and the LPS-induced effects can be reversed by Met treatment.

scholarly journals Protein Kinase Cδ Targets Mitochondria, Alters Mitochondrial Membrane Potential, and Induces Apoptosis in Normal and Neoplastic Keratinocytes When Overexpressed by an Adenoviral Vector

1999 ◽  
Vol 19 (12) ◽  
pp. 8547-8558 ◽  
Author(s):  
Luowei Li ◽  
Patricia S. Lorenzo ◽  
Krisztina Bogi ◽  
Peter M. Blumberg ◽  
Stuart H. Yuspa
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Protein Kinase ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Protein Fusion ◽  
Cell Death Pathway ◽  
Morphological Criteria ◽  
Protein Kinase Cδ

ABSTRACT Inactivation of protein kinase Cδ (PKCδ) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCδ in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCδ under a cytomegalovirus promoter to overexpress PKCδ in normal and neoplastic keratinocytes. While PKCδ overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCα, PKCɛ, PKCζ, and PKCη, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCδ adenovirus. Activation of PKCδ by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCδ. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCδ translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCδ-green fluorescent protein fusion protein. Furthermore, activation of PKCδ in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCδ-overexpressing keratinocytes. These results indicate that PKCδ can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

scholarly journals The U95 Protein of Human Herpesvirus 6B Interacts with Human GRIM-19: Silencing of U95 Expression Reduces Viral Load and Abrogates Loss of Mitochondrial Membrane Potential

2007 ◽  
Vol 82 (2) ◽  
pp. 1011-1020 ◽  
Author(s):  
W. M. Yeo ◽  
Yuji Isegawa ◽  
Vincent T. K. Chow
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Human Herpesvirus ◽  
Ultrastructural Changes ◽  
Gene Encoding ◽  
Interacting Protein ◽  
Functional Aspects ◽  
Infected Cells

ABSTRACT To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.

scholarly journals Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes

1992 ◽  
Vol 288 (1) ◽  
pp. 207-213 ◽  
Author(s):  
J P Zoeteweij ◽  
B van de Water ◽  
H J de Bont ◽  
G J Mulder ◽  
J F Nagelkerke
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Rat Hepatocytes ◽  
Extracellular Atp ◽  
Complete Loss ◽  
Isolated Rat Hepatocytes ◽  
Isolated Mitochondria ◽  
Isolated Rat

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

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