oligomycins and Disease-Models--Animal

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line, Tumor; Cyclic N-Oxides; Disease Models, Animal; Drug Evaluation, Preclinical; Free Radical Scavengers; Glucose; Infarction, Middle Cerebral Artery; Lipid Peroxidation; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Molecular Structure; Neuroblastoma; Neurons; Neuroprotection; Neuroprotective Agents; Nitrogen Oxides; Oligomycins; Oxygen; Rotenone

Huntington's disease (HD) is a genetic disease caused by a CAG trinucleotide repeat expansion encoding a polyglutamine tract in the huntingtin (HTT) protein, ultimately leading to neuronal loss and consequent cognitive decline and death. As no treatments for HD currently exist, several chemical screens have been performed using cell-based models of mutant HTT toxicity. These screens measured single disease-related endpoints, such as cell death, but had low 'hit rates' and limited dimensionality for therapeutic detection. Here, we have employed gene expression microarray analysis of HD samples--a snapshot of the expression of 25,000 genes--to define a gene expression signature for HD from publically available data. We used this information to mine a database for chemicals positively and negatively correlated to the HD gene expression signature using the Connectivity Map, a tool for comparing large sets of gene expression patterns. Chemicals with negatively correlated expression profiles were highly enriched for protective characteristics against mutant HTT fragment toxicity in in vitro and in vivo models. This study demonstrates the potential of using gene expression to mine chemical activity, guide chemical screening, and detect potential novel therapeutic compounds.. Single-endpoint chemical screens have low therapeutic discovery hit-rates. In the context of HD, we guided a chemical screen using gene expression data. The resulting chemicals were highly enriched for suppressors of mutant HTT fragment toxicity. This study provides a proof of concept for wider usage in all chemical screening.

Topics: Animals; Caspases; Cell Line; Cluster Analysis; Connectome; Deferoxamine; Disease Models, Animal; Drosophila; Drug Discovery; Gene Expression Regulation; Humans; Huntington Disease; Inclusion Bodies; Mice; Models, Biological; Mutation; Nerve Tissue Proteins; Oligomycins; Phenotype

Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various in vivo fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. In vivo imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction in vivo.

Topics: Animals; Anti-Bacterial Agents; Anticonvulsants; Benzophenones; Carbocyanines; Chromans; Disease Models, Animal; Fluorescent Dyes; Hypoglycemic Agents; Membrane Potential, Mitochondrial; Mitochondria; Nitrophenols; Oligomycins; Optical Imaging; Pioglitazone; Thiazolidinediones; Tolcapone; Toxicity Tests; Troglitazone; Zebrafish; Zebrafish Proteins

Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL(+) leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph(+) leukemia and AML upon TK inhibition.. Ph(+) cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined.. We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL(+) leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo.. TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL(+) and FLT3(ITD) leukemias.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Dihydrolipoyllysine-Residue Acetyltransferase; Disease Models, Animal; Female; fms-Like Tyrosine Kinase 3; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Ketone Oxidoreductases; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Oligomycins; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; RNA Interference; RNA, Small Interfering; Superoxides

ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimer's disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca(2+) overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimer's disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Calcium Signaling; Cell Line; Cerebral Infarction; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Drug Evaluation, Preclinical; Hippocampus; Memory Disorders; Mice; Molecular Structure; Molecular Targeted Therapy; Naphthyridines; Nerve Tissue Proteins; Neuroprotective Agents; Oligomycins; Oxidative Stress; Phosphorylation; Protein Phosphatase 2; Protein Processing, Post-Translational; Rats; Rotenone; Scopolamine; tau Proteins

Mitochondria are present at high concentration at the site of sensory transduction in the peripheral terminals of nociceptors. Because nerve growth factor (NGF), which induces nociceptor sensitization by acting on the high-affinity tropomyosin receptor kinase A (TrkA) receptor, also produces local recruitment of mitochondria in DRG neurons, we evaluated the role of mitochondria in NGF-induced mechanical hyperalgesia. Inhibition of 3 major mitochondrial functions-oxidation of nutrients, adenosine triphosphate (ATP) production, and generation of reactive oxygen species--markedly attenuated NGF-induced mechanical hyperalgesia in the rat. Disruption of microtubules, which are required for the trafficking and subcellular localization of mitochondria, also attenuated NGF-induced hyperalgesia. Our results suggest a contribution of mitochondrial localization and function to NGF-dependent pain syndromes.

Topics: Acetylcysteine; Analysis of Variance; Animals; Antiparasitic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Hyperalgesia; Male; Mitochondria; Nerve Growth Factor; Oligomycins; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley

Adenosine is an endogenous nucleoside that signals through G-protein coupled receptors. Extracellular adenosine is required for receptor activation and two pathways have been identified for formation and cellular release of adenosine. The CLASSICAL pathway relies on intracellular formation of adenosine from adenine nucleotides and cellular efflux of adenosine via equilibrative nucleoside transporters (ENTs). The ALTERNATE pathway involves cellular release of adenine nucleotides, hydrolysis via ecto-5'-nucleotidases and extracellular formation of adenosine.. A rat model of cerebral ischemia and primary cultures of rat forebrain astrocytes and neurons were used.. Using a rat model of cerebral ischemia, the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) significantly increased post-ischemic forebrain adenosine levels and significantly decreased hippocampal neuron injury relative to saline-treatment. NBMPR-induced increases in adenosine receptor activation were not detected, suggesting that altering the intracellular:extracellular distribution of adenosine can affect ischemic outcome. Using primary cultures of rat forebrain astrocytes and neurons, adenosine release was evoked by ischemic-like conditions. Dipyridamole, an inhibitor of ENTs, was more effective at inhibiting adenosine release from neurons than from astrocytes. In contrast, alpha , beta-methylene ADP, an inhibitor of ecto-5'-nucleotidase, was effective at inhibiting adenosine release from astrocytes, but not from neurons. Thus, during ischemic-like conditions, neurons released adenosine via the CLASSICAL pathway, while astrocytes released adenosine via the ALTERNATE pathway.. These cell type differences in pathways for adenosine formation during ischemia may allow transport inhibitors to block simultaneously adenosine release from neurons and adenosine uptake into astrocytes. In principle, this could improve neuronal ATP levels without decreasing adenosine receptor activation.

Topics: Adenosine; Affinity Labels; Animals; Astrocytes; Brain Ischemia; Cells, Cultured; Deoxyglucose; Dipyridamole; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Glucose; Hypoxia; Inosine; Models, Biological; Neurons; Oligomycins; Phosphodiesterase Inhibitors; Prosencephalon; Purines; Rats; Thioinosine; Tritium

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

Topics: Animals; Apoptosis; Calcium; Collagen Type VI; Cyclosporine; Disease Models, Animal; Enzyme Inhibitors; Female; Fibroblasts; Homozygote; Immunosuppressive Agents; In Situ Nick-End Labeling; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Muscle; Mitochondrial Diseases; Muscle, Skeletal; Muscular Diseases; Oligomycins; Sarcoplasmic Reticulum