Voltage-dependent Anion Channel Research Articles

Silencing VDAC1 to Treat Mesothelioma Cancer: Tumor Reprograming and Altering Tumor Hallmarks.

Mesothelioma, an aggressive cancer with a poor prognosis, is linked to asbestos exposure. However, carbon nanotubes found in materials we are exposed to daily can cause mesothelioma cancer. Cancer cells reprogram their metabolism to support increased biosynthetic and energy demands required for their growth and motility. Here, we examined the effects of silencing the expression of the voltage-dependent anion channel 1 (VDAC1), controlling the metabolic and energetic crosstalk between mitochondria and the rest of the cell. We demonstrate that VDAC1 is overexpressed in mesothelioma patients; its levels increase with disease stage and are associated with low survival rates. Silencing VDAC1 expression using a specific siRNA identifying both mouse and human VDAC1 (si-m/hVDAC1-B) inhibits cell proliferation of mesothelioma cancer cells. Treatment of xenografts of human-derived H226 cells or mouse-derived AB1 cells with si-m/hVDAC1-B inhibited tumor growth and caused metabolism reprogramming, as reflected in the decreased expression of metabolism-related proteins, including glycolytic and tricarboxylic acid (-)cycle enzymes and the ATP-synthesizing enzyme. In addition, tumors depleted of VDAC1 showed altered microenvironments and inflammation, both associated with cancer progression. Finally, tumor VDAC1 silencing also eliminated cancer stem cells and induced cell differentiation to normal-like cells. The results show that silencing VDAC1 expression leads to reprogrammed metabolism and to multiple effects from tumor growth inhibition to modulation of the tumor microenvironment and inflammation, inducing differentiation of malignant cells. Thus, silencing VDAC1 is a potential therapeutic approach to treating mesothelioma.

Overexpressed VDAC1 in breast cancer as a novel prognostic biomarker and correlates with immune infiltrates

BackgroundMore and more evidence suggests that cancer is a mitochondrial metabolic disease recently and mitochondria dysfunction is critical to tumorigenesis. As a gatekeeper of mitochondria, the voltage-dependent anion channel 1 (VDAC1) is associated with the development of breast cancer (BC). However, its potential mechanism and clinical significance remain unclear; thus, in this research, we aimed to explore it.MethodsVDAC1 expression in BC tissues and normal tissues was obtained from The Cancer Genome Atlas (TCGA) and validated by datasets from the gene expression omnibus (GEO) database. Then, the relationships between VDAC1 expression and clinicopathological features were analyzed. Receiver operating characteristics (ROC) curves were used to identify the diagnostic value of VDAC1. The prognostic value was evaluated by Kaplan-Meier survival curves and Cox regression analysis. VDAC1 with its co-expression genes were subjected to enrichment analysis to explore potential mechanisms in BC and the protein-protein interaction (PPI) network was constructed. At last, the association between VDAC1 expression and infiltration levels of immune cell infiltration by various methods, as well as their corresponding markers, was analyzed. We also analyzed the correction between VDAC1 expression and eight immune checkpoint genes and the tumor immune dysfunction and exclusion (TIDE) scores of each BC sample in TCGA were calculated and the differences between high and low VDAC1 expression groups were analyzed.ResultsVDAC1 expression was remarkably elevated in BC (p < 0.001), and high expression of VDAC1 was associated with the positive expression of ER (p = 0.004), PR (p = 0.033), and HER2 (p = 0.001). ROC analysis suggested that VDAC1 had diagnosed value in BC. The Kaplan-Meier analysis suggested that higher expression of VDAC1 was associated with shorter overall survival (OS), and further Cox regression analysis revealed that VDAC1 was an independent factor of unfavorable prognosis in BC patients. Enrichment analysis of VDAC1 and its co-expression suggested that VDAC1 was related to the regulation of mitochondrial energy metabolism and protein modification, and the HIF-1 singing pathway might be the potential mechanism in BC. Notably, we found that VDAC1 expression was infiltration levels of most types of immune cells, as well as the expression of marker genes of immune cells. The ICGs PDCD1, CTLA4, LAG3, SIGLEC15, and TIGIT were negatively corrected with VDAC1 expression in BC. TIDE scores between the low and high expression groups showed no difference.ConclusionOverexpressed VDAC1 in BC could be severed as a novel biomarker for diagnosis and VDAC1 was an independent factor for adverse prognosis prediction. Our study revealed that VDAC1 might inhibit tumor immunity and might be a novel therapeutic target in BC.

Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outermembrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Traumatic and Diabetic Schwann Cell Demyelination Is Triggered by a Transient Mitochondrial Calcium Release through Voltage Dependent Anion Channel 1.

A large number of peripheral neuropathies, among which are traumatic and diabetic peripheral neuropathies, result from the degeneration of the myelin sheath, a process called demyelination. Demyelination does not result from Schwann cell death but from Schwann cell dedifferentiation, which includes reprograming and several catabolic and anabolic events. Starting around 4 h after nerve injury, activation of MAPK/cJun pathways is the earliest characterized step of this dedifferentiation program. Here we show, using real-time in vivo imaging, that Schwann cell mitochondrial pH, motility and calcium content are altered as soon as one hour after nerve injury. Mitochondrial calcium release occurred through the VDAC outer membrane channel and mPTP inner membrane channel. This calcium influx in the cytoplasm induced Schwann-cell demyelination via MAPK/c-Jun activation. Blocking calcium release through VDAC silencing or VDAC inhibitor TRO19622 prevented demyelination. We found that the kinetics of mitochondrial calcium release upon nerve injury were altered in the Schwann cells of diabetic mice suggesting a permanent leak of mitochondrial calcium in the cytoplasm. TRO19622 treatment alleviated peripheral nerve defects and motor deficit in diabetic mice. Together, these data indicate that mitochondrial calcium homeostasis is instrumental in the Schwann cell demyelination program and that blocking VDAC constitutes a molecular basis for developing anti-demyelinating drugs for diabetic peripheral neuropathy.

Cloning and expression analysis of voltage dependent anion channel (&amp;lt;italic&amp;gt;AhVDAC&amp;lt;/italic&amp;gt;) gene in the geotropism response of the peanut gynophores

<p id="C3">Peanut is a plant that blooms above ground and bears fruit underground. In our earlier studies, the voltage dependent anion channel (<italic>AhVDAC</italic>) gene was detected to be involved in gravitropism of peanut gynophores. In this study, the full-length cDNA of peanut <italic>AhVDAC</italic> gene was cloned, and the prokaryotic expression, subcellular localization, and the relative expression level of <italic>AhVDAC</italic> gene in gravitropism were analyzed. The results showed that the open reading frame of <italic>AhVDAC</italic> gene was 831 bp encoding a protein containing 276 amino acids, with a molecular weight of 29.7 kD and pI 6.38. Subcellular localization showed that <italic>AhVDAC</italic> gene was mainly located in cytoplasm. The prokaryotic expression vector pPROEXHTa-<italic>AhVDAC</italic> was constructed, and the 37 kD AhVDAC protein was induced, isolated, and purified. The relative expression level of <italic>AhVDAC</italic> in gynophores at different developmental days was analyzed by RT-PCR, and the results showed that the relative expression level of <italic>AhVDAC</italic> gene was the highest at the 2nd day, and then decreased gradually and maintained at a lower level. The application of exogenous CaCl₂ and LaCl₃ to peanut gynophores <italic>in vitro</italic> revealed that CaCl₂treatment could significantly promote the relative expression of <italic>AhVDAC</italic> gene and the geotropic bending of gynophores, whereas LaCl₃ treatment could decelerate the relative expression of <italic>AhVDAC</italic> gene and geotropic bending of gynophores. In conclusion, we speculated that Ca²⁺ accumulation may promote the expression of<italic> AhVDAC</italic> and Ca²⁺was asymmetrically distributed through the transport of AhVDAC on biological membrane, so that the growth direction of peanut gynophore was changed and geotropic bending occurs.

Tubulin Isotypes: Emerging Roles in Defining Cancer Stem Cell Niche.

Although the role of microtubule dynamics in cancer progression is well-established, the roles of tubulin isotypes, their cargos and their specific function in the induction and sustenance of cancer stem cells (CSCs) were poorly explored. But emerging reports urge to focus on the transport function of tubulin isotypes in defining orchestrated expression of functionally critical molecules in establishing a stem cell niche, which is the key for CSC regulation. In this review, we summarize the role of specific tubulin isotypes in the transport of functional molecules that regulate metabolic reprogramming, which leads to the induction of CSCs and immune evasion. Recently, the surface expression of GLUT1 and GRP78 as well as voltage-dependent anion channel (VDAC) permeability, regulated by specific isotypes of β-tubulins have been shown to impart CSC properties to cancer cells, by implementing a metabolic reprogramming. Moreover, βIVb tubulin is shown to be critical in modulating EphrinB1signaling to sustain CSCs in oral carcinoma. These tubulin-interacting molecules, Ephrins, GLUT1 and GRP78, are also important regulators of immune evasion, by evoking PD-L1 mediated T-cell suppression. Thus, the recent advances in the field implicate that tubulins play a role in the controlled transport of molecules involved in CSC niche. The indication of tubulin isotypes in the regulation of CSCs offers a strategy to specifically target those tubulin isotypes to eliminate CSCs, rather than the general inhibition of microtubules, which usually leads to therapy resistance.

Mitochondrial Dysfunction in Spinocerebellar Ataxia Type 3 Is Linked to VDAC1 Deubiquitination.

Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.

Neuronal ER-Signalosome Proteins as Early Biomarkers in Prodromal Alzheimer's Disease Independent of Amyloid-β Production and Tau Phosphorylation.

There exists considerable interest to unveil preclinical period and prodromal stages of Alzheimer's disease (AD). The mild cognitive impairment (MCI) is characterized by significant memory and/or other cognitive domains impairments, and is often considered the prodromal phase of AD. The cerebrospinal fluid (CSF) levels of β-amyloid (βA), total tau (t-tau), and phosphorylated tau (p-tau) have been used as biomarkers of AD albeit their significance as indicators during early stages of AD remains far from accurate. The new biomarkers are being intensively sought as to allow identification of pathological processes underlying early stages of AD. Fifty-three participants (75.4 ± 8.3 years) were classified in three groups as cognitively normal healthy controls (HC), MCI, and subjective memory complaints (SMC). The subjects were subjected to a battery of neurocognitive tests and underwent lumbar puncture for CSF extraction. The CSF levels of estrogen-receptor (ER)-signalosome proteins, βA, t-tau and p-tau, were submitted to univariate, bivariate, and multivariate statistical analyses. We have found that the components of the ER-signalosome, namely, caveolin-1, flotilin-1, and estrogen receptor alpha (ERα), insulin growth factor-1 receptor β (IGF1Rβ), prion protein (PrP), and plasmalemmal voltage dependent anion channel 1 (VDAC) could be detected in the CSF from all subjects of the HC, MCI, and SMC groups. The six proteins appeared elevated in MCI and slightly increased in SMC subjects compared to HC, suggesting that signalosome proteins undergo very early modifications in nerve cells. Using a multivariate approach, we have found that the combination of ERα, IGF-1Rβ, and VDAC are the main determinants of group segregation with resolution enough to predict the MCI stage. The analyses of bivariate relationships indicated that collinearity of ER-signalosome proteins vary depending on the stage, with some pairs displaying opposed relationships between HC and MCI groups, and the SMC stage showing either no relationships or behaviors similar to either HC or MCI stages. The multinomial logistic regression models of changes in ER-signalosome proteins provide reliable predictive criteria, particularly for the MCI. Notably, most of the statistical analyses revealed no significant relationships or interactions with classical AD biomarkers at either disease stage. Finally, the multivariate functions were highly correlated with outcomes from neurocognitive tests for episodic memory. These results demonstrate that alterations in ER-signalosome might provide useful diagnostic information on preclinical stages of AD, independently from classical biomarkers.

Mitochondria-specific peptide amphiphiles induce mitochondrial dysfunction and peripheral T-cell lymphomas (PTCL) damage.

BackgroundPeripheral T-cell lymphomas (PTCL) are aggressive lymphomas with poor prognosis, and therefore, there is a pressing need to explore new targets or compounds. Mitochondria may serve as a potential therapeutic target for PTCL. A designed positively-charged segment (pKV) is anchored to the specific 15 amino acid sequence (MIASHLLAYFFTELN) to yield a cell-penetrating peptide (pHK-pKV) and a lipid chain (Pal) is conjugated to the N-terminus of pHK-pKV (Pal-pHK-pKV) are bioactive amphiphilic peptide assemblies targeting the interaction between mitochondrial voltage dependent anion channel 1 (VDAC1) and hexokinase II (HKII).MethodsPTCL cell line H9 was treated with Pal-pHK-pKV and pHK-pKV, respectively. Cell proliferation in each group was measured by detecting cell viability and the corresponding marker Ki-67. Apoptosis was detected by immunofluorescence, flow cytometry and western blot. We also measured mitochondrial membrane potential, adenosine triphosphate (ATP) production, the cytochrome c distribution and the expression levels of B cell lymphoma 2 (BCL-2) and BCL-2 associated X protein (BAX). Western blot was used to detect the activation of the extracellular regulated protein kinases (ERK) signaling pathway.ResultsPal-pHK-pKV and pHK-pKV with 20 µM blocked the interaction between VDAC1 and HKII, and detached HKII from mitochondria, which depolarized the mitochondrial membrane potential, induced mitochondria dysfunction, and decreased ATP production. The decreased ATP subsequently inhibited the activation of the ERK/BCL-2 pathway and increased the BAX/BCL-2 ratio. Cytochrome c was then released from the mitochondria and induced capase-3 activation and subsequently apoptosis. Additionally, decreased ATP induced the expression of FAS and then apoptosis.ConclusionsMitochondria specific peptide amphiphiles induce mitochondrial dysfunction and provide a new approach for the treatment of PTCL.

Reticulon-1A mediates diabetic kidney disease progression through endoplasmic reticulum-mitochondrial contacts in tubular epithelial cells

Recent epidemiological studies suggest that some patients with diabetes progress to kidney failure without significant albuminuria and glomerular injury, suggesting a critical role of kidney tubular epithelial cell (TEC) injury in diabetic kidney disease (DKD) progression. However, the major risk factors contributing to TEC injury and progression in DKD remain unclear. We previously showed that expression of endoplasmic reticulum-resident protein Reticulon-1A (RTN1A) increased in human DKD, and the increased RTN1A expression promoted TEC injury through endoplasmic reticulum (ER) stress response. Here, we show that TEC-specific RTN1A overexpression worsened DKD in mice, evidenced by enhanced tubular injury, tubulointerstitial fibrosis, and kidney function decline. But RTN1A overexpression did not exacerbate diabetes-induced glomerular injury or albuminuria. Notably, RTN1A overexpression worsened both ER stress and mitochondrial dysfunction in TECs under diabetic conditions by regulation of ER-mitochondria contacts. Mechanistically, ER-bound RTN1A interacted with mitochondrial hexokinase-1 and the voltage-dependent anion channel-1 (VDAC1), interfering with their association. This disengagement of VDAC1 from hexokinase-1 resulted in activation of apoptotic and inflammasome pathways, leading to TEC injury and loss. Thus, our observations highlight the importance of ER-mitochondrial crosstalk in TEC injury and the salient role of RTN1A-mediated ER-mitochondrial contact regulation in DKD progression.

Small molecules targeting the NADH-binding pocket of VDAC modulate mitochondrial metabolism in hepatocarcinoma cells.

Voltage dependent anion channels (VDAC) control the flux of most anionic respiratory substrates, ATP, ADP, and small cations, crossing the outer mitochondrial membrane. VDAC closure contributes to the partial suppression of mitochondrial metabolism that favors the Warburg phenotype of cancer cells. Recently, it has been shown that NADH binds to a specific pocket in the inner surface of VDAC1, also conserved in VDAC2 and 3, closing the channel. We hypothesized that binding of small molecules to the NADH pocket, maintain VDAC in an open configuration by preventing closure induced by NADH and possible other endogenous regulators. We screened in silico, the South Carolina Compound Collection SC3 (~ 100,000 proprietary molecules), using shape-based queries of the NADH binding region of VDAC. After molecular docking of selected compounds, we physically screened candidates using mitochondrial membrane potential (ΔΨm), as an overall readout of mitochondrial metabolism. We identified SC18, as the most potent compound. SC18 bound to VDAC1, as assessed by a thermal shift assay. Short-term treatment with SC18 decreased ΔΨm in SNU-449 and HepG2 human hepatocarcinoma cells. Mitochondrial depolarization was similar in wild type, VDAC1/2, 1/3, and 2/3 double KO HepG2 cells indicating that the effect of SC18 was not VDAC isoform-dependent. In addition, SC18 decreased mitochondrial NADH and cellular ATP production; and increased basal respiration. Long-term exposure to SC18, decreased cell proliferation as determined by wound-healing and cell viability assays. In summary, SC18 is a novel VDAC-targeting small molecule that induces mitochondrial dysfunction and inhibits cell proliferation.

Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells

ABSTRACT Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

MORTALIN-Ca2+ axis drives innate rituximab resistance in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma, with the combination of rituximab and chemotherapy being the standard treatment for it. Although rituximab monotherapy has a remarkable response rate, drug resistance with unclear mechanisms and lack of effective second-line therapy limit the survival benefits of patients with lymphoma. Here, we report that MORTALIN is highly expressed and correlates with resistance to rituximab-based therapy and poor survival in patients with DLBCL. Mechanistically, gain- and loss-of-function experiments revealed that the voltage-dependent anion channel 1-binding protein, MORTALIN, regulated Ca2+ release from the endoplasmic reticulum through mitochondria-associated membrane, facilitating AP1-mediated cell proliferation and YY-1-mediated downregulation of FAS in DLBCL cells. These dual mechanisms contribute to rituximab resistance. In mouse models, genetic depletion of MORTALIN markedly increased the antitumor activity of rituximab. We shed mechanistic light on MORTALIN-Ca2+-CaMKII-AP1-mediated proliferation and MORTALIN-Ca2+-CaMKII-inhibited death receptor in DLBCL, leading to rituximab resistance, and propose MORTALIN as a novel target for the treatment of DLBCL.

Unbiased proteomics, histochemistry, and mitochondrial DNA copy number reveal better mitochondrial health in muscle of high-functioning octogenarians.

Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear. We performed muscle biopsies in 15 octogenarian world-class track and field MAs and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mitochondrial DNA (mtDNA) copy number, and proteomics by liquid-chromatography mass spectrometry. MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage-dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and that are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA. We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with underrepresentation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging. Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, USA.

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury.

Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases.

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside.

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

PcVDAC promotes WSSV infection by regulating the apoptotic activity of haemocytes in Procambarus clarkii.

Apoptosis is programmed cell death that is strictly regulated by a series of related genes and is of great importance in resisting pathogen invasion and maintaining cell environment homeostasis. Among apoptotic proteins, the voltage-dependent anion channel protein (VDAC) plays a key role in the mitochondrial apoptosis pathway because of its close connection with changes in mitochondrial membrane potential. However, the role of VDAC in apoptosis and immune regulation in Procambarus clarkii is poorly understood. In this study, the VDAC gene in P. clarkii (PcVDAC) was cloned by rapid amplification of cDNA ends (RACE) technology. The gene was found to have a total length of 2277bp, including a 194-bp 5'-UTR, 1234-bp 3'-UTR and 849-bp open reading frame (ORF), and to encode 282 amino acids. PcVDAC was expressed in all tissues tested, and its expression was upregulated after white spot syndrome virus (WSSV) infection (P<0.05). The RNA interference (RNAi) method was used to explore the role of PcVDAC in WSSV infection. The results showed that the number of WSSV copies in haemocytes was significantly reduced after RNAi (P<0.05), and the survival rate was significantly increased. In addition, after RNAi, the apoptosis rate was significantly increased (P<0.05), the mitochondrial membrane potential was reduced (P<0.01), and the expression of caspase-3 and other genes was upregulated (P<0.05). These results indicate that PcVDAC promotes the replication of WSSV in P. clarkii by inhibiting haemocytes apoptosis. Therefore, the results presented in this paper provide new insights into the immune response of P. clarkii infected with WSSV.

Calcium-Calmodulin-Involved Heat Shock Response of Neoporphyra haitanensis

Increasing global temperatures have seriously affected the sustainable development of Neoporphyra haitanensis cultivation. Although several pathways are reported to be involved in the response of N. haitanensis to heat stress, it is unknown which ones are activated by signal transduction. Previously, we detected a large influx of calcium ions (Ca2+) in N. haitanensis under heat stress. In this study, we further investigated the specificity of Ca2+ signaling and how is it transduced. Transmission electron microscopy and Fv/Fm analyses showed that the Ca2+ signal derived from extracellular Ca2+ formed at the early stage of the response to heat stress, and the signal was recognized and decoded by N. haitanensis calmodulin (NhCaM). In yeast two-hybrid assays, DnaJ, a voltage-dependent anion channel, and a bromodomain-containing protein interacted with PhCaM1 in vivo. The transcript levels of the genes encoding these proteins increased significantly in response to heat stress, but decreased upon inhibition of NhCaM1, indicating that these interacting factors were positively related to NhCaM1. Additionally, a comparative transcriptome analysis indicated that Ca2+ signal transduction is involved in phosphatidylinositol, photosystem processes, and energy metabolism in N. haitanensis under heat stress. Our results suggest that Ca2+-CaM plays important roles in signal transduction in response to heat stress in N. haitanensis.

Late embryogenesis abundant gene LEA3 (Gh_A08G0694) enhances drought and salt stress tolerance in cotton

Plants have evolved a complex and organized response to abiotic stress that involves physiological and metabolic reprogramming, transcription control, epigenetic regulation, and expressions of thousand interacting genes for instance the late embryogenesis abundant (LEA) proteins are expressed in multiple environmental variables during the plant developmental period, and thus play critical role in enhancing drought and salt stress tolerance. A comprehensive molecular and functional characterization of the LEA3 gene was carried out in cotton under abiotic stress conditions in order to elucidate their functions. Seventy eight genes were identified in cotton, and were clustered into six clades moreover; the LEA genes were more upregulated in the tissues of the tetraploid cotton compared to the diploid type. A key gene, Gh_A08G0694 was the most upregulated, and was knocked in tetraploid cotton, the knocked out significantly increased the susceptibility of cotton plants to salinity and drought stresses, moreover, several ABA/stress-associated genes were down regulated. Similarly, overexpression of the key gene, significantly increased tolerance of the overexpressed plants to drought and salinity stress. The key gene is homologous to GhLEA3 protein, found to have strong interaction to key abiotic stress tolerance genes, voltage-dependent anion channel 1 (VDAC1) and glyceraldehyde-3-phosphate dehydrogenase A (gapA).