Tetramethylrhodamine Ethyl Ester Research Articles

TRANSCRIPTOME PROFILE OF NICOTINIC RECEPTOR–LINKED, BETA AMYLOID-INDUCED NEUROTOXICITY

Alzheimer's disease (AD) is a significant neurodegenerative disorder of the elderly. In order to develop potential therapeutic strategies for the management of this order, it is very critical to obtain a better understanding of the global gene changes underlying the progression of AD pathogenesis. In this study, we used RNA-seq technology to analyze global differential gene expression in the model neuroblastoma cell line NG108-15, transfected with α4β2 nAChRs, and treated or untreated with full-length beta amyloid (Aβ1-42), to gain insights into the underlying mechanisms of beta amyloid-induced neurotoxicity. Cells were treated with media or 100nM of Aβ1-42 for 3 days, followed by extraction and purification of their RNA. Multiplexed RNA-seq libraries were prepared from the cellular RNA and paired-end 100-bp sequencing was conducted using an Illumina HiSeq 2500 sequencer. Fold changes in the expression of transcripts (and hence, genes) between untreated and Aβ-treated samples were determined. Ingenuity pathway analysis (IPA) was employed to determine the cellular pathways. Treatment with Aβ1-42 in α4β2 nAChR-transfected cells differentially modulated the expression of 15 prominent genes as compared to that of untreated cells. Most notably, chrna4 and rcan3 (Regulator of Calcineurin) were upregulated 3-fold and 2-fold, respectively. The differential gene expression data identified by RNA-seq was validated by real time PCR, western blot and immunocytochemistry. Additionally, we used Cas9/CRISPR gene-editing system to knock out Rcan3. The sorted cells were further treated or not with Aβ1-42 for 3 days and the impact of loss of Rcan3 was examined in terms of oxidative stress, mitochondrial membrane potential and cell viability. The absence of Rcan3 was expected to reduce the extent of oxidative stress, as measured by counting reactive oxygen species (ROS)-positive cells and mitochondrial membrane potential, as determined by tetramethyl rhodamine ethyl ester (TMRE) staining, on exposure to beta amyloid. This is the first study to conduct an entire transcriptome profile of model nerve cells expressing a defined high-affinity target receptor, namely nAChR, for beta amyloid. Characterizing the genes associated with calcium signaling, specifically Rcan3 after beta amyloid treatment will have significant impact on developing neuroprotective agents to reduce or prevent neuronal death in AD patients.

Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.

Dehydroabietic acid isolated from Pinus elliottii exerts in vitro antileishmanial action by pro-oxidant effect, inducing ROS production in promastigote and downregulating Nrf2/ferritin expression in amastigote forms of Leishmania amazonensis

Dehydroabietic acid (DHA) is one of the main constituents of the resin that have antiprotozoal activity against Leishmania spp., but the leishmanicidal mechanism is unknown. The objective of the study was to investigate in vitro the leishmanicidal activity of the natural compound DHA against intracellular and extracellular forms of L. amazonensis and the mechanism of action involved. The antileishmanial activity of DHA was evaluated in vitro against promastigote forms of L. amazonensis by counting in Neubauer chamber. The morphological changes were observed by scanning electron microscopy and cell death mechanism by fluorescence assay using 2′,7′-dichlorofluorescein diacetate probe (H2DCFDA), tetramethylrhodamine ethyl ester (TMRE), annexin-V and propidium iodide (PI). The antiamastigote effect was observed by counting the number of amastigotes per macrophage and percentage of infected cells. In addition, reactive oxygen species (ROS) production, nitric oxide (NO), cytokines, free iron and total iron-binding capacity (TIBC), expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin were evaluated. DHA inhibited the proliferation of promastigotes at all times tested. The compound (IC50, 40 ± 0.1458 μg/mL) altered the morphology of the promastigote forms, caused mitochondrial depolarization, induced ROS production, increased phosphatidylserine exposure and caused loss of plasma membrane integrity. DHA also reduced the number of amastigotes and the percentage of infected macrophages by increasing ROS production, free iron and TIBC, and also caused downregulation of Nrf2 and ferritin expression. DHA was effective in the elimination of L. amazonensis both in its promastigote forms by apoptosis-like mechanisms and intracellular form by ROS production.

Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 (“Warburg effect”), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor’s adaptive behavior.

Mitofusin-Dependent ER Stress Triggers Glial Dysfunction and Nervous System Degeneration in a Drosophila Model of Friedreich's Ataxia.

Friedreich’s ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial protein frataxin. Despite its pivotal effect on biosynthesis of iron-sulfur clusters and mitochondrial energy production, little is known about the influence of frataxin depletion on homeostasis of the cellular mitochondrial network. We have carried out a forward genetic screen to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. Our screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). Thus, we have analyzed the effects of frataxin knockdown on mitochondrial morphology, mitophagy and ER function in our fly FRDA model using different histological and molecular markers such as tetramethylrhodamine, ethyl ester (TMRE), mitochondria-targeted GFP (mitoGFP), p62, ATG8a, LAMP1, Xbp1 and BiP/GRP78. Furthermore, we have generated the first Drosophila transgenic line containing the mtRosella construct under the UAS control to study the progression of the mitophagy process in vivo. Our results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, we demonstrate that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in our FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, our results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target.

Arginase II inhibition prevents interleukin-8 production through regulation of p38 MAPK phosphorylation activated by loss of mitochondrial membrane potential in nLDL-stimulated hAoSMCs

Arginase inhibition exhibits beneficial effects in vascular endothelial and smooth muscle cells. In human aortic smooth muscle cells (hAoSMCs), native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. Therefore, we examined the effect of arginase inhibition on IL-8 production and the underlying mechanism. In hAoSMCs, reverse transcription–PCR, western blotting and immunocytochemistry with MitoTracker confirmed that arginase II was confined predominantly to mitochondria. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine ethyl ester. The MMP decreased upon nLDL stimulation but was restored upon arginase inhibition. MMP loss caused by nLDL was prevented by treatment with the intracellular Ca2+ chelator BAPTA-AM. In mitochondrial Ca2+ measurements using Rhod-2 AM, increased mitochondrial Ca2+ levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca2+ movement. The nLDL-induced MMP change resulted in p38 mitogen-activated protein kinase (MAPK) phosphorylation and IL-8 production and was prevented by the arginase inhibitors BAPTA and ruthenium 360. In isolated AoSMCs from ApoE−/− mice fed a high-cholesterol diet, arginase activity, p38 MAPK phosphorylation, spermine and mitochondrial Ca2+ levels and keratinocyte-derived chemokine (KC) production were increased compared with wild-type (WT) mice. However, in AoSMCs isolated from arginase II-null mice, increases in MMP and decreases in mitochondrial Ca2+ levels were noted compared with WT and were associated with p38 MAPK activation and IL-8 production. These data suggest that arginase activity regulates the change in MMP through Ca2+ uptake that is essential for p38 MAPK phosphorylation and IL-8 production.

MiR-195 modulates oxidative stress-induced apoptosis and mitochondrial energy production in human trophoblasts via flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 and pyruvate dehydrogenase phosphatase regulatory subunit.

Preeclampsia is a severe pregnancy-specific syndrome defined as newly onset hypertension and proteinuria. Abnormal placental development has been generally accepted as the initial cause of the disorder. Recently, miR-195 was identified as one of the downregulated small RNAs in preeclamptic placentas. The potential targets of miR-195 in human trophoblast cells were screened by isobaric tags for relative and absolute quantification-based mass spectrum analysis. Localization of miR-195 and its targets was examined by in-situ hybridization and immunohistochemistry in human placenta. Real-time PCR, western blotting and luciferase assay were used for target validation. Apoptosis was accessed by Annexin V/PI costaining, whereas mitochondrial function by ATP measurement and tetramethylrhodamine ethyl ester fluorescence. Two mitochondria-associated proteins, flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 (FOXRED1) and pyruvate dehydrogenase phosphatase regulatory subunit (PDPR), were identified as targets of miR-195. Overexpression of miR-195 in HTR8/SVneo cells resulted in enhanced apoptosis, decreased mitochondrial membrane potential and cellular ATP content upon hydrogen peroxide stimulation. The effects could be partially rescued by FOXRED1 or PDPR. In preeclamptic patients, lowered circulating level of miR-195 were found at early-to-mid gestation and term pregnancy, and marked increase in FOXRED1 and PDPR expression were observed in the placenta when compared with gestational week-matched controls. In addition, chronic hydrogen peroxide stimuli suppressed miR-195 expression in trophoblast cells. MiR-195 could suppress mitochondrial energy production via targeting FOXRED1 and PDPR, and lead to trophoblast cell apoptosis under oxidative stress. In preeclamptic placenta, lowered level of miR-195 might be induced by chorionic oxidative stress and subsequently form a compensation mechanism to defend the disturbed energy production and cell apoptosis upon oxidative stress.

Abstract TP404: A Novel Adipokine DPPIV Induces Inflammation and Apoptosis of Carotid Artery Smooth Muscle Cells: Implications for Atherosclerotic Plaque Instability

Background: Carotid Atherosclerotic (CA) plaque rupture is associated with ischemic stroke. Smooth muscle cells (SMC) play key role in plaque development. SMC also produce collagen fibers and maintain the fibrous cap thickness and strength, and SMC apoptosis and inflammation can lead to plaque rupture. Molecular mechanisms leading to plaque instability and stroke are not completely understood. Our study has shown that an adipokine dipeptidyl peptidase (DPPIV) is significantly upregulated in post-stroke rat brain, and in the plasma of over 75% of CA patients, suggesting a role for DPPIV in CA and stroke. In this study, we examined the effects of DPPIV on SMC viability, apoptosis, and identified responsible signaling pathways. Methods: Mouse carotid artery SMC were exposed to oxygen glucose deprivation (OGD). Cells were treated with either physiological (200-500 ng/ml) or high (1000-2000 ng/ml) dose of DPPIV. Using transcriptome array, we examined the expression profile of apoptotic and inflammatory pathway specific genes in these cells. DPPIV levels were examined by PCR and immunofluorescence staining. MTT assay was used to assess cell viability. Live/dead cell assay detected apoptosis. Mitochondrial membrane potential was detected using tetra-methyl-rhodamine ethyl ester (TMRE). Results: Ischemia/hypoxia upregulates DPPIV mRNA and protein levels in association with increased pro-apoptotic genes including caspase 7, Nod1, and Gadd45a. Transcriptome array data demonstrated that higher dose of exogenous DPPIV results in significant increase in same pro-apoptotic pathway genes including caspase 3,7, Nod1, Trp63, and Gadd45a. On the other hand, DPPIV decreased the expression of anti-apoptotic genes including Atf5 and Birc3. Levels of pro-inflammatory cytokines including IL1-beta and Ccl26/Eotaxin were increased in both DPPIV and ischemic SMCs. Higher dose of DPPIV increased apoptosis, decreased viability and mitochondrial membrane potential in SMCs. Conclusion: Higher levels of DPP4 activates the inflammatory and apoptotic signaling cascade resulting in the apoptosis of SMC. Further research in this area will enhance our understanding of the mechanism of CA progression and may provide clues to efficient therapeutic intervention for CA.

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries.

Application of H2S (“sulfide”) elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 μM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 μM) and by 50 μM ryanodine; the Ca2+ channel blocker nifedipine (1 μM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 μM), the NADPH oxidase (NOX) blocker VAS2870 (10 μM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 μM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 μM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

Chronic exposure to a glyphosate-containing pesticide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans

Glyphosate-containing herbicides are among the most widely-used in the world. Although glyphosate itself is relatively non-toxic, growing evidence suggests that commercial herbicide formulations may lead to increased oxidative stress and mitochondrial inhibition. In order to assess these mechanisms in vivo, we chronically (24h) exposed Caenorhabditis elegans to various concentrations of the glyphosate-containing herbicide TouchDown (TD). Following TD exposure, we evaluated the function of specific mitochondrial electron transport chain complexes. Initial oxygen consumption studies demonstrated inhibition in mid- and high-TD concentration treatment groups compared to controls. Results from tetramethylrhodamine ethyl ester and ATP assays indicated reductions in the proton gradient and ATP levels, respectively. Additional studies were designed to determine whether TD exposure resulted in increased reactive oxygen species (ROS) production. Data from hydrogen peroxide, but not superoxide or hydroxyl radical, assays showed statistically significant increases in this specific ROS. Taken together, these data indicate that exposure of Caenorhabditis elegans to TD leads to mitochondrial inhibition and hydrogen peroxide production.

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

High-throughput BioSorter quantification of relative mitochondrial content and membrane potential in living Caenorhabditis elegans

Mitochondrial respiratory chain disease is caused by a wide range of individually rare genetic disorders that impair cellular energy metabolism. While fluorescence microscopy analysis of nematodes fed MitoTracker Green (MTG) and tetramethylrhodamine ethyl ester (TMRE) can reliably quantify relative mitochondrial density and membrane potential, respectively, in C. elegans models of mitochondrial dysfunction, it is a tedious process with limitations in the number and age of animals that can be studied. A novel, large particle, flow cytometry-based method reported here accelerates and automates the relative quantitation of mitochondrial physiology in nematode populations. Relative fluorescence profiles of nematode populations co-labeled with MTG and TMRE were obtained and analyzed by BioSorter (Union Biometrica). Variables tested included genetic mutation (wild-type N2 Bristol versus nuclear-encoded respiratory chain complex I mutant gas-1(fc21) worms), animal age (day 1 versus day 4 adults), classical respiratory chain inhibitor and uncoupler effects (oligomycin, FCCP), and pharmacologic therapy duration (24h versus 96h treatments with glucose or nicotinic acid). A custom MATLAB script, which can be run on any computer with MATLAB runtime, was written to automatically quantify and analyze results in large animal populations. BioSorter analysis independently validated relative MTG and TMRE changes that we had previously performed by fluorescence microscopy in a variety of experimental conditions, with notably greater animal population sizes and substantially reduced experimental time. Older, fragile animal populations that are difficult to study by microscopy approaches were readily amenable to analysis with the BioSorter method. Overall, this high-throughput method enables efficient relative quantitation of in vivo mitochondrial physiology over time in a living animal in response to gene mutations and candidate therapies, which can be used to accelerate the translation of basic research into optimization of clinical therapies for mitochondrial disease.

Maintenance of cellular respiration indicates drug resistance in acute myeloid leukemia

Primary resistance to induction therapy is an unsolved clinical problem in acute myeloid leukemia (AML). Here we investigated drug resistance in AML at the level of cellular metabolism in order to identify early predictors of therapeutic response. Using extracellular flux analysis, we compared metabolic drug responses in AML cell lines sensitive or resistant to cytarabine or sorafenib after 24h of drug treatment to a small cell lung cancer (SCLC) cell line exposed to etoposide. Only drug-resistant AML cells maintained oxidative metabolism upon drug exposure while SCLC cells displayed an overall metabolic shift towards glycolysis, i.e. a Warburg effect to escape drug toxicity. Moreover, primary AML blasts displayed very low glycolytic activity, while oxygen consumption was readily detectable, indicating an essential role of oxidative pathways in the bioenergetics of AML blasts. In line with these observations, analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester staining and flow cytometry allowed for clear discrimination between drug sensitive and resistant AML cell line clones and primary blasts after 24h of treatment with cytarabine or sorafenib. Our data reveal a distinct metabolic phenotype of resistant AML cells and suggest that disrupting oxidative metabolism rather than glycolysis may enhance the cytotoxic effects of chemotherapy in AML.

Amyloid β-42 induces neuronal apoptosis by targeting mitochondria.

Alzheimer's disease (AD), with a typical pathological hallmark of amyloid-beta (Aβ)-containing plaques and neurofibrillary tangles, is one of the most common types of chronic neurodegenerative diseases. Aβ oligomers serve a crucial role in the pathogenesis of AD, and lead to neuronal loss. However, the precise mechanism of Aβ oligomers in AD remains to be elucidated. The present study demonstrated that 10 µM Aβ-42 activated the caspase signaling pathway, and induced significant apoptosis in primary cultured mouse cerebral cortical neurons. The results of reverse transcription-quantitative polymerase chain reaction and western blotting demonstrated that Aβ-42 (10 µM) also significantly upregulated the transcription and expression of the mitochondrial fission protein dynamin-related protein 1 (Drp1), and downregulated the transcription and expression of mitochondrial fusion proteins, including mitofusin 1/2 (Mfn1/2) and mitochondrial dynamin like GTPase (OPA-1). Neurons were transfected with pDsRed2-Mito for mitochondrial imaging, which revealed that 10 µM Aβ-42 induced mitochondrial fission in cortical neurons. In addition, 2′,7′-dichlorodihydrofluorescein diacetate and tetramethylrhodamine ethyl ester staining indicated that Aβ-42 increased the reactive oxygen species (ROS) level and reduced mitochondrial membrane potential in neurons. Inhibition of Drp1 activity by Mdivi-1 efficiently prevented Aβ-42-induced ROS production and disruption of mitochondrial membrane potential. Loss of mitochondrial membrane potential may activate PTEN-induced putative kinase 1 (Pink1), the prominent sensor for mitochondrial damage, and trigger the process of mitophagy to remove the damaged mitochondria. In the present study, western blotting revealed that the levels of autophagy marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) and Pink1 were upregulated after Aβ-42 stimulation. In conclusion, these data indicated that Aβ-42 induces neuronal apoptosis by targeting mitochondria, including promotion of mitochondrial fission, disruption of mitochondrial membrane potential, increasing intracellular ROS level and activation of the process of mitophagy. Therefore, mitochondria may represent a potential therapeutic target for AD in the future.

Apoptin Gene Delivery by the Functionalized Polyamidoamine (PAMAM) Dendrimer Modified with Ornithine Induces Cell Death of HepG2 Cells.

The use of tumor-specific therapeutic agents is a promising option for efficient and safe nonviral gene transfer in gene therapy. In this study, we describe the efficacy of polyamidoamine (PAMAM)-based nonviral gene delivery carriers, namely, an ornithine conjugated PAMAM (PAMAM-O) dendrimer in delivering apoptin, a tumor-specific killer gene, into human hepatocellular carcinoma (HepG2 cells) and dermal fibroblasts. We analyzed the transfection efficiency by the luciferase assay and assessed cell viability in both cell types. The transfection efficiency of the PAMAM-O dendrimer was found to be higher than that of the PAMAM dendrimer. Moreover, the cytotoxicity of the PAMAM-O dendrimer was very low. We treated both cell types with a polyplex of PAMAM-O dendrimer with apoptin, and analyzed its cellular uptake and localization by confocal microscopy. Cell cycle distribution, tetramethylrhodamine, ethyl ester (TMRE) analysis, and transmission electron microscopy imaging showed that apoptin induced cell death in HepG2 cells. We therefore demonstrated that a PAMAM-O/apoptin polyplex can be used as an effective therapeutic strategy in cancer owing to its effectiveness as a suitable nonviral gene vector for gene therapy.

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 μm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria’s response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

Label-free detection of myocardial ischaemia in the perfused rat heart by spontaneous Raman spectroscopy

Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.

Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer's disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1-5mM) in primary culture for 48h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.

Tumor suppressor BLU promotes TRAIL-induced apoptosis by downregulating NF-κB signaling in nasopharyngeal carcinoma

A putative tumor suppressor BLU mapped on the chromosomal 3p21 region, is frequently lost in human tumors including nasopharyngeal carcinoma (NPC). To explore the underlying mechanism of tumor suppression by BLU, its potential to promote apoptosis induced by TRAIL, an effector molecule elaborated by natural killer-T (NKT) cells was investigated. BLU was re-expressed in NPC-derived HNE1 cells by recombinant adenoviral infection and the cells were challenged with recombinant TRAIL. The growth inhibition of BLU was assayed and apoptosis was examined by flow cytometry-based tetramethylrhodamine ethyl ester (TMRE) and annexin V staining, cleavage of pro-caspase-8 and poly ADP ribose polymerase (PARP). The modulation of NF-κB pathway by BLU was evaluated by the reporter activity and estimation of the level of the molecules involved such as IKKalpha, p65 NF-κB, as well as NF-κB induced anti-apoptotic factors cFLIPL and cIAP2. The expression of BLU exerted in vitro and in vivo growth inhibitory effect and promoted TRAIL-induced apoptosis. This phenomenon was validated by FACS-based assays of mitochondrial membrane potential (BLU vs. Vector 87.8% ± 7.7% and 72.1%±6.7% at 6h exposure to TRAIL) and phosphatidylserine turnover (BLU vs. vector: 28.7%±2.9% and 22.6%±2.5%), as well as, enhanced caspapse-8 cleavage. Similar with the findings that BLU promotes chemotherapeutic agent-induced apoptosis, it also augmented death receptor-induced pathway through NF-κB pathway inhibition. In conclusion, BLU suppressed tumor formation by strengthening the antitumor immunity.

Measuring Mitochondrial Transmembrane Potential by TMRE Staining.

Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide the majority of this ATP by a process known as oxidative phosphorylation. This process involves active transfer of positively charged protons across the mitochondrial inner membrane resulting in a net internal negative charge, known as the mitochondrial transmembrane potential (ΔΨm). The proton gradient is then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free phosphate. The net negative charge across a healthy mitochondrion is maintained at approximately -180 mV, which can be detected by staining cells with positively charged dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low ΔΨm. Cytochrome c is essential for producing ΔΨm because it promotes the pumping the protons into the mitochondrial intermembrane space as it shuttles electrons from Complex III to Complex IV along the electron transport chain. Cytochrome c is released from the mitochondrial intermembrane space into the cytosol during apoptosis. This impairs its ability to shuttle electrons between Complex III and Complex IV and results in rapid dissipation of ΔΨm. Loss of ΔΨm is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.