Expression Of Voltage-dependent Anion Channel Research Articles

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltage-dependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.

MAVS Regulates Apoptotic Cell Death by Decreasing K48-Linked Ubiquitination of Voltage-Dependent Anion Channel 1

The mitochondrial antiviral signaling protein MAVS (IPS-1, VISA, or Cardif) plays an important role in the host defense against viral infection by inducing type I interferon. Recent reports have shown that MAVS is also critical for virus-induced apoptosis. However, the mechanism of MAVS-mediated apoptosis induction remains unclear. Here, we show that MAVS binds to voltage-dependent anion channel 1 (VDAC1) and induces apoptosis by caspase-3 activation, which is independent of its role in innate immunity. MAVS modulates VDAC1 protein stability by decreasing its degradative K48-linked ubiquitination. In addition, MAVS knockout mouse embryonic fibroblasts (MEFs) display reduced VDAC1 expression with a consequent reduction of the vesicular stomatitis virus (VSV)-induced apoptosis response. Notably, the upregulation of VDAC1 triggered by VSV infection is completely abolished in MAVS knockout MEFs. We thus identify VDAC1 as a target of MAVS and describe a novel mechanism of MAVS control of virus-induced apoptotic cell death.

Benzo[a]pyrene-mediated toxicity in primary pig bladder epithelial cells: A proteomic approach

The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 μM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis. Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen.

Novel mitochondrial mechanisms mediate enhanced vasodilation of rat middle cerebral arteries to mitochondrial depolarization following ischemia‐reperfusion injury

Mitochondrial depolarization leads to vasodilation via activation of SR/ER Ca2+ release. Our objective was to determine the role of mitochondria in vasodilation of middle cerebral arteries (MCA) following ischemia‐reperfusion injury (I‐RI). Male Sprague Dawley rats were subjected to 90 minutes MCA occlusion followed by 48 hours reperfusion. Vasodilation in response to bradykinin (BK), nitroprusside (NTP) and mitochondrial depolarization by diazoxide (DZ, a mitochondrial KATP channel activator) was determined in isolated MCAs by videomicroscopy. Mitochondrial membrane potential (Ψm)was determined by fluorescence microscopy with specific fluoroprobe, TMRE. Western blots determined the expression of markers of mitochondrial biogenesis [Fusion: mitofusins 1 & 2 (MFNs) and Fission: dynamin‐related protein 1 (DRP1)], voltage‐dependent anion channels (VDAC), ER stress (phosphorylated eukaryotic initiation factor 2 α subunit (p‐eIF2α) and neuronal nitric oxide synthase (nNOS). DZ induced vasodilation was enhanced on ipsilateral MCA (iMCA) compared to contralateral (cMCA) whereas BK and NTP responses were similar. Mitochondrial in iMCA displayed increased baseline Ψm and enhanced depolarization to DZ compared to cMCA. Expression of MFNs and VDAC were unchanged, DRP1 was decreased and nNOS and p‐eIF2α were increased in iMCA. Thus, MCAs respond to I‐RI by reduced mitochondrial fission and increasing Ψm leading to increased ROS response to depolarization, ER Ca2+ release and nNOS activation.

Tim50 in Trypanosoma brucei Possesses a Dual Specificity Phosphatase Activity and Is Critical for Mitochondrial Protein Import

In eukaryotes, proteins are imported into mitochondria via multiprotein translocases of the mitochondrial outer and inner membranes, TOM and TIM, respectively. Trypanosoma brucei, a hemoflagellated parasitic protozoan and the causative agent of African trypanosomiasis, imports about a thousand proteins into the mitochondrion; however, the mitochondrial protein import machinery in this organism is largely unidentified. Here, we characterized a homolog of Tim50 that is localized in the mitochondrial membrane in T. brucei. Similar to Tim50 proteins from fungi and mammals, Tim50 in T. brucei (TbTim50) possesses a mitochondrial targeting signal at its N terminus and a C-terminal domain phosphatase motif at its C terminus. Knockdown of TbTim50 reduced cell growth and inhibited import of proteins that contain N-terminal targeting signals. Co-immunoprecipitation analysis revealed that TbTim50 interacts with TbTim17. Unlike its fungal counterpart but similar to the human homolog of Tim50, recombinant TbTim50 possesses a dual specificity phosphatase activity with a greater affinity for protein tyrosine phosphate than for protein serine/threonine phosphate. Mutation of the aspartic acid residues to alanine in the C-terminal domain phosphatase motif (242)DXDX(V/T)(246) abolished activity for both type of substrates. TbTim50 knockdown increased and its overexpression decreased the level of voltage-dependent anion channel (VDAC). However, the VDAC level was unaltered when the phosphatase-inactive mutant of TbTim50 was overexpressed, suggesting that the phosphatase activity of TbTim50 plays a role in regulation of VDAC expression. In contrast, phosphatase activity of the TbTim50 is required neither for mitochondrial protein import nor for its interaction with TbTim17. Overall, our results show that TbTim50 plays additional roles in mitochondrial activities besides preprotein translocation.

Peripheral benzodiazepine receptor regulates vascular endothelial activations via suppression of the voltage-dependent anion channel-1

Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor-α (TNF-α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM-1, and ICAM-1 expression in TNF-α-activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF-α-induced VCAM-1 expression. Overexpression of PBR inhibits voltage-dependent anion channel-1 (VDAC-1) expression and the silencing of PBR increases VDAC-1 expression in endothelial cells. Moreover, TNF-α-induced VCAM-1 expression is suppressed by VDAC-1 gene silencing. PBR overexpression significantly decreases TNF-α-induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC-1 expression in endothelial cells.

Abstract LB-229: Arsenic trioxide, ascorbic acid and disulfiram (AAA) arrests pancreatic cancer development in a transgenic mouse model with p16 knockout and activated Kras

Abstract Previously, we found that four human pancreatic cancer (PC) cell lines underwent robust cell death associated with dramatic ATP decline and ROS accumulation when treated with clinically achievable concentrations of arsenic trioxide, ascorbic acid and disulfiram (AAA). Early apoptosis was shifted to aponecrosis, and dependent on the voltage-dependent anion channel (VDAC), since shRNA directed against VDAC-1 abrogated the ROS accumulation and blocked cell death. In this study, assessment of AAA was performed in Pdx1-Cre transgenic mice with a conditional p16 knockout (p16flox/flox), and oncogenic KrasG12D. The KrasG12D, p16flox/flox; LSL-KrasG12D; Pdx1-Cre mice develop the full spectrum of pancreatic intraepithelial neoplasia (mPanIN) lesions, pancreatic ductal adenocarcinoma, and hepatic and peritoneal metastases. Over 95% of human PC have p16 deficiency and Kras mutation. Eight week old mice were treated for 8 weeks, 3 times/week with AAA, sacrificed, and the development of PC assessed. Little or no toxicity of the mice to the AAA treatment was observed. Frank PC was zero in AAA-treated mice and 31% in controls. The mean number of PanIN-3 lesions/mouse declined from 14.1 in controls to 1.58 in AAA-treated mice (p=0.008). The mean number of PanIN-2 lesions declined from 5.18 in controls to 1.67 in AAA treated mice (p=0.06). The mean number of PanIN-1 lesions in control mice were 14.2 and in AAA-treated mice were 16.0, p=0.51 indicating no difference in PanIN-1 occurrence. Preliminary results from immunohistochemical staining of the PanIN lesions for VDAC, indicated that PanIN-1 lesions have low, and PanIN-2 and 3 lesions have high, VDAC expression. This result parallels the in vitro human cell line data showing VDAC expression is critical to AAA sensitivity, and suggests a similar mechanism of action in vivo as in vitro. Although collection of data is ongoing, this is the first treatment that has been shown to arrest PanIN and tumor development in any a genetically-engineered mouse model for PC. The data suggests AAA either arrests the development of pre-cancerous lesions after the PanIN-1 stage, or the PanIN-2 and PanIN-3 lesions are destroyed by AAA in a VDAC-dependent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2011-LB-229

Effects of intracellular zinc depletion on the expression of VDAC in cultured hippocampal neurons

An experiment was conducted to investigate whether intracellular zinc depletion can actually change expression of voltage-dependent anion channel 1 (VDAC1) and VPAC2 in cultured hippocampal neurons as well as their significance. Hippocampal neurons were obtained by primary culture from hippocampus of newborn Wistar rats. Cultured hippocampal neurons were exposed to a cell membrane-permeable zinc chelator N,N,N′,N′-tetrakis (2-pyridyl methyl) ethylenediamine (TPEN) (2 µM), and to TPEN plus zinc sulfate (5 µM) for 1 or 24 hours. Cultures were then processed to detect neuronal injury by lactate dehydrogenase (LDH) assay, intracellular Ca2+ with the fluorescent probe fluo-3/AM, reactive oxygen species (ROS) generation using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay, nuclear morphology by Hoechst 33342, VDAC1, and VDAC2 protein levels by western blot, and VDAC1 and VDAC2 mRNA levels by RT–PCR. The results demonstrated that exposure of hippocampal neurons to TPEN (2 µM) for 24 hours induced notably neuronal injury, significantly increased the number of apoptotic nuclei, up-regulated the expression of VDAC1 protein level and down-regulated the expression of VDAC2 protein level. Significant down-regulation of mRNA levels for VDAC1 and VDAC2 were observed in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced neuronal injury and above alterations in VDAC1 and VDAC2 protein levels and mRNA levels. Present results implicate a possibility that up-regulation of VDAC1 and down-regulation of VDAC2 may participate in hippocampal neuron injury induced by zinc deficiency.

Enhanced Expression of the Voltage-Dependent Anion Channel 1 (VDAC1) in Alzheimer's Disease Transgenic Mice: An Insight into the Pathogenic Effects of Amyloid-β

The mitochondrial voltage-dependent anion channel 1 (VDAC1) is involved in the release of apoptotic proteins with possible relevance in Alzheimer's disease (AD) neuropathology. Through proteomic analysis followed by Western blotting and immunohistochemical techniques, we have found that VDAC1 is overexpressed in the hippocampus from amyloidogenic AD transgenic mice models. VDAC1 was also overexpressed in postmortem brain tissue from AD patients at an advanced stage of the disease. Interestingly, amyloid-β (Aβ) soluble oligomers were able to induce upregulation of VDAC1 in a human neuroblastoma cell line, further supporting a correlation between Aβ levels and VDAC1 expression. In hippocampal extracts from transgenic mice, a significant increase was observed in the levels of VDAC1 phosphorylated at an epitope that is susceptible to phosphorylation by glycogen synthase kinase-3β, whose activity was also increased. The levels of hexokinase I (HXKI), which interacts with VDAC1 and affects its function, were decreased in mitochondrial samples from AD models. Since phospho-VDAC and reduced HXKI levels favors a VDAC1 conformational state more prone to the release proapoptotic factors, regulation of the function of this channel may be a promising therapeutic approach to combat AD.

Mitochondrial ROS fuel the inflammasome

Mitochondrial ROS fuel the inflammasome

Hepatoprotection through regulation of voltage dependent anion channel expression by Amomum subulatum Roxb seeds extract

Background and Purpose:Voltage dependent anion channel (VDAC) plays an important role in triggering the opening of the mitochondrial permeability transition pore (PTP) that leads to mitochondrial damage and induce apoptic or necrotic cell death. In the present study, the methanolic extract of Amomum subulatum Roxb. seeds (MEAS) was used to examine its effect on VDAC. Aminotransferase activity, mitochondrial membrane potential, calcium-induced liver MPT, and VDAC expression were used to evaluate the hepato protective effect of MEAS.Results:Pretreatment of mice with MEAS (100 and 300 mg/kg) significantly blocked the CCl4-induced increase in AST and ALT activities. Pretreatment with MEAS showed significant preservation of mitochondrial membrane potential as compared to CCl4 control demonstrating the mitochondrial protection. In addition, pretreatment with MEAS at various concentrations exerted a dose-dependent effect against sensitivity to mitochondrial swelling induced by calcium. In addition, MEAS (300 mg/kg) significantly increased the transcription and translation of VDAC.Conclusion:Our data suggest that MEAS significantly prevents the damage to liver mitochondria through regulation of VDAC expression.

Salinity-related variation in gene expression in wild populations of the black-chinned tilapia from various West African coastal marine, estuarine and freshwater habitats

This study measured the relative expression of the genes coding for Na +, K +-ATPase 1α(NAKA), voltage-dependent anion channel (VDAC), cytochrome c oxidase-1 (COX), and NADH dehydrogenase (NDH), in gills of six wild populations of a West African tilapia species, acclimatised to a range of seasonal (rainy or dry) salinities in coastal, estuarine and freshwater sites. Previous laboratory experiments have demonstrated that these genes, involved in active ion transport, oxidative phosphorylation, and intra-cellular ATP transport, are relatively over-expressed in gill tissues of this species acclimated to high salinity. Positive correlations between relative expression and ambient salinity were found for all genes in the wild populations (Spearman rank correlation, p < 0.05), although for some genes these were only significant in either the rainy season or dry season. Most significantly, however, relative expression was positively correlated amongst the four genes, indicating that they are functionally interrelated in adaptation of Sarotherodon melanotheron to salinity variations in its natural environment. In the rainy season, when salinity was unstable and ranged between zero and 37 psu across the sites, overall mean expression of the genes was higher than in the dry season, which may have reflected more variable particularly sudden fluctuations in salinity and poorer overall water quality. In the dry season, when the salinity is more stable but ranged between zero and 100 psu across the sites, NAKA, NDH and VDAC expression revealed U-shaped relationships with lowest relative expression at salinities approaching seawater, between 25 and 45 psu. Although it is not simple to establish direct relationship between gene expression levels and energy requirement for osmoregulation, these results may indicate that costs of adaptation to salinity are lowest in seawater, the natural environment of this species. While S. melanotheron can colonise environments with extremely high salinities, up to 100 psu, this was related to high relative expression for all genes studied, indicating that this imposes increased energy demand for osmotic homeostasis in gill tissues. This study is the first to demonstrate, in fish and in wild populations, that expression of NAKA, VDAC, NDH and COX are interrelated in gill tissues, and are involved in long-term acclimatisation to a salinity range between 0 and 100 psu.

A study of association between expression of hOGG1, VDAC1, HK-2 and cervical carcinoma

BackgroundHuman 8-oguanine glycosylase 1(hOGG1), voltage-dependent anion channel 1(VDAC1), hexokinase 2(HK-2), represented the process of oxidative DNA damage, cell apoptosis and glycolysis, respectively. This study aims to explore the association between expression of hOGG1, VDAC1, HK-2 and cervical carcinoma.MethodsA case-control study was conducted. 65 cervical biopsy samples consist of 20 control and 45 cases. The expression of hOGG1, VDAC1 and HK-2 were examined with immunohistochemistry(IHC), immunolabeling was evaluated with stereological cell counts.ResultsThe data showed that the positive proportion of hOGG1 and HK-2 in the case group was higher than that of the control group (P < 0.05). Further, there was an increasing trend for the positive proportion and expression degree of hOGG1 and HK-2 from Control, Mild cervical carcinoma (MCC), Intermediate cervical carcinoma(ICC) to Severe cervical carcinoma(SCC) in order (P < 0.05). To VDAC1, the significant result was not obtained.ConclusionsThe results suggested that there was a close association between expression of hOGG1, HK-2 and cervical cancer. hOGG1 and HK-2 might play a key role at the early stage of cervical cancer, and the findings of hOGG1 and HK-2 should be considered as a significant biomarker at the early stage of cervical cancer.

Analysis and difference of voltage-dependent anion channel mRNA in ejaculated spermatozoa from normozoospermic fertile donors and infertile patients with idiopathic asthenozoospermia

to analyze the abundance and difference of voltage-dependent anion channel (VDAC) mRNA in ejaculated spermatozoa from normozoospermic fertile donors and infertile patients with idiopathic asthenozoospermia. high motile and low motile spermatozoa were separated respectively from ejaculates of 36 donors and 40 patients using a discontinuous Percoll gradient centrifugation. Real-Time PCR was performed to detect mRNA abundance and difference of three VDAC subtypes between two groups with different sperm motility. real-Time PCR demonstrated that three VDAC mRNAs were present in mature spermatozoa. The VDAC2 mRNA level in ejaculated spermatozoa of patients was significantly higher than that of donors. No significant differences of VDAC1 and VDAC3 mRNA levels were found between two groups. the high abundance of VDAC2 mRNA seemed to have a positive correlation with low sperm motility. The abnormal expression of VDAC might be related to male infertility with idiopathic asthenozoospermia.

Chronic low-level Pb exposure during development decreases the expression of the voltage-dependent anion channel in auditory neurons of the brainstem

Lead (Pb) exposure is a risk factor for neurological dysfunction. How Pb produces these behavioral deficits is unknown, but Pb exposure during development is associated with auditory temporal processing deficits in both humans and animals. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain high rates of synaptic activity. The voltage-dependent anion channel (VDAC) is involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. We have previously demonstrated that VDAC is an in vitro target for Pb, therefore, VDAC may represent a potential target for Pb in the auditory system. In order to determine whether Pb alters VDAC expression in central auditory neurons, CBA/CaJ mice ( n = 3–5/group) were exposed to 0.01 mM, or 0.1 mM Pb acetate during development via drinking water. At P21, immunohistochemistry reveals a significant decrease for VDAC in neurons of the Medial Nucleus of the Trapezoid Body. Western blot analysis confirms that Pb results in a significant decrease for VDAC. Decreases in VDAC expression could lead to an upregulation of other cellular energy producing systems as a compensatory mechanism, and a Pb-induced increase in brain type creatine kinase is observed in auditory regions of the brainstem. In addition, comparative proteomic analysis shows that several proteins of the glycolytic pathway, the phosphocreatine circuit, and oxidative phosphorylation are also upregulated in response to developmental Pb exposure. Thus, Pb-induced decreases in VDAC could have a significant effect on the function of auditory neurons.

Підвищена експресія потенціалзалежного аніонного каналу й аденіннуклеотидтранслокази та чутливість кальційіндукованої мітохондріальної пори в серці старих щурів

We investigated mRNA and protein expression of voltage-dependent anion channel (VDAC), mRNA adenine nucleotide translocase (ANT) as well as the sensitivity of the mitochondrial permeability transition pore opening (MPTP) to Ca2+ in the adult and old rat heart. It was shown that in the old rats hearts VDAC mRNA expression increased by 1.7 (p < 0.05) times and mRNA ANT expression increased by 1.8 (p < 0.05) times in comparison with adult animals. The Western Blot analysis showed that the level of VDAC protein expression in the old rat hearts also significantly increased compared with adult animals. In the hearts of old rats, the sensitivity of MPTP opening to calcium (10(-7)-10(-4) mol/l) determined by mitochondria swelling, increased two-fold (p < 0.05). Therefore, an increased VDAC and ANT expression, as the main structural functional components of the MPTP, and an increased sensitivity of MPTP opening to Ca2+ caused an increase in the permeability of mitochondrial membranes in aging. Each of these factors may contribute to alterations in mitochondrial barrier properties and lead to mitochondrial dysfunction.

Downregulation of voltage-dependent anion channel-1 expression by RNA interference prevents cancer cell growth in vivo

The voltage-dependent anion channel 1 (VDAC1), located at the outer mitochondrial membrane (OMM), is a key protein in regulating the exchange of ions, nucleotides and variety of metabolites in and out of the mitochondria. As such, VDAC serves a crucial role in cellular energy maintenance. We have previously shown that silencing VDAC1 expression in T-Rex-293cells by means of RNA interference using shRNA directed to human (h)VDAC1 resulted in reduced ATP production and a decrease in cell growth. Since cancer cells highly express VDAC, we examined here the effects of shRNA-hVDAC1-mediated silencing of VDAC1 expression on cancer cell growth in an animal model. To this end, HeLa cervical cancer cells were stably transfected with an inducible shRNA plasmid encoding for a sequence directed to hVDAC1. VDAC1 expression in these cells was decreased almost completely. Stably transfected shRNA-hVDAC1 HeLa cells proliferated much slower than did control cells, pointing to VDAC1 expression as being essential for normal growth of HeLa cancer cells. We further studied the ability of hVDAC1 silencing to block cancer cell proliferation in vivo. Nude mice subcutaneously inoculated with stably transfected shRNA-hVDAC1 HeLa cells developed about 40-fold smaller tumors than did mice inoculated with control HeLa cells. Taken together, the results of this study demonstrate, for the first time, the anti-cancer therapeutic potential of VDAC1 down-regulation by means of shRNA.

Activation of the nitric oxide-mitochondrial hyperpolarization-mTOR-Rab4 signaling pathway precedes disease development in lupus T cells (93.36)

Abstract Nitric oxide (NO) dependent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation in Systemic Lupus Erythematosus. Activity of mTOR, which senses the mitochondrial membrane potential, is increased in lupus T cells, which promotes HRES-1/Rab4 expression. Rab4 overexpression enhances recycling and lysosomal degradation of immune synapse components, including CD4 and TCRζ. We investigated activity of the NO-MHP-mTOR-Rab4 signaling pathway in lupus-prone mice. Overexpression of Rab4 and loss of TCRζ were detected prior to disease onset of NZB/NZW (F1) mice at 5 months. Increased NO production, mitochondrial transmembrane potential, and mitochondrial mass were present after disease onset at 11 months. MRL/lpr mice exhibited increased expression of the mitochondrial voltage-dependent anion channel 1(VDAC1) protein, transaldolase (TAL), Rab4, mTOR activation, and enhanced CD3 and CD4 recycling at 2-3 months, prior to disease development. In conclusion, lupus-prone mice exhibit mitochondrial dysfunction, with increased NO production, MHP, elevated expression of VDAC1 and TAL, and mTOR activation. Increased Rab4 expression was associated with enhanced endocytic recycling of CD4 and CD3. The results suggest that activation of the NO-MHP-mTOR-Rab4 signaling pathway is detectable and precedes disease onset in lupus-prone mice.

Age-dependent posttranslational modifications of voltage-dependent anion channel 1

The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems.In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, we previously found a set of conserved biomarkers across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, we found age-related redundant isoforms of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms.Building on these results, we examined the expression of VDAC-1 in an in vitro model of excitotoxicity. We show that glutamate-induced calcium toxicity in neurons induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation.

Increased Expression of Voltage-Dependent Anion Channel and Adenine Nucleotide Translocase and the Sensitivity of Ca2+-Induced Mitochondrial Permeability Transition Opening Pore in Old Rat Heart

Increased Expression of Voltage-Dependent Anion Channel and Adenine Nucleotide Translocase and the Sensitivity of Ca2+-Induced Mitochondrial Permeability Transition Opening Pore in Old Rat Heart