High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes (original) (raw)
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Scientific Reports
Nandrolone is a testosterone analogue with anabolic properties commonly abused worldwide, recently utilized also as therapeutic agent in chronic diseases, cancer included. Here we investigated the impact of nandrolone on the metabolic phenotype in HepG2 cell line. The results attained show that pharmacological dosage of nandrolone, slowing cell growth, repressed mitochondrial respiration, inhibited the respiratory chain complexes I and III and enhanced mitochondrial reactive oxygen species (ROS) production. Intriguingly, nandrolone caused a significant increase of stemness-markers in both 2D and 3D cultures, which resulted to be CxIII-ROS dependent. Notably, nandrolone negatively affected differentiation both in healthy hematopoietic and mesenchymal stem cells. Finally, nandrolone administration in mice confirmed the up-regulation of stemness-markers in liver, spleen and kidney. Our observations show, for the first time, that chronic administration of nandrolone, favoring maintenanc...
Cellular and molecular life sciences : CMLS, 2015
Alzheimer's disease (AD) is an age-related neurodegenerative disease marked by a progressive cognitive decline. Metabolic impairments are common hallmarks of AD, and amyloid-β (Aβ) peptide and hyperphosphorylated tau protein-the two foremost histopathological signs of AD-have been implicated in mitochondrial dysfunction. Neurosteroids have recently shown promise in alleviating cognitive and neuronal sequelae of AD. The present study evaluates the impact of neurosteroids belonging to the sex hormone family (progesterone, estradiol, estrone, testosterone, 3α-androstanediol) on mitochondrial dysfunction in cellular models of AD: human neuroblastoma cells (SH-SY5Y) stably transfected with constructs encoding (1) the human amyloid precursor protein (APP) resulting in overexpression of APP and Aβ, (2) wild-type tau (wtTau), and (3) mutant tau (P301L), that induces abnormal tau hyperphosphorylation. We show that while APP and P301L cells both display a drop in ATP levels, they present ...
Journal of Experimental & Clinical Cancer Research
Background Neuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR). Methods Cell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lip...
2015
Aims Results 2.1 Mediterrean CLN1 mutations are associated with mitochondrial dysfunction 2.1.1 Analysis of mitochondrial compartment in CLN1 fibroblasts 2.1.2 Differentiation of CLN1 neuroblastoma stable clones 2.1.3 Analysis of mitochondrial compartment in CLN1 neuroblastoma stable clones 2.2 CLN5 is a partner of ceramide synthase enzymes 2.2.2 Intracellular localisation of CLN5 and ceramide synthases proteins 2.2.3 Analyses of intracellular trafficking of CLN5 together with the CLN8 interacting protein 2.2.4 Lysosomal enzyme activities in CLN5 fibroblasts 2.2.5 Mitochondrial respiration 2.3 Impairment of cell autophagy in primary CTSF/CLN13 cells Discussion Conclusions Materials and Methods 4.1 Cell cultures and differentiation media 4.2 Transfection 4.3 Molecular analysis 4.4 Immunofuorescence assay 4.5 Aggresome detection 4.6 SDS-PAGE and Western Blotting 4.7 Mitochondrial Protein Preparation 4.8 Blue Native Analysis 4.9 IGA assay 4.10 Total and mitochondrial ATP assay 4.11 Transmission electron microscopy 4.12 Cloning 4.13 Bimolecular fluorescence complementation (BiFC) analyses 4.14 Co-immunoprecipitation 4.15 Enzyme activity assays 4.16 OCR and ECAR measurements References Appendix Human neuronal ceroid lipofuscinoses variants Disease Eponym OMIM Clinical phenotype Gene Protein CLN1 Haltia-Santavuori 256730 Classic infantile, late infantile, juvenile, adult* CLN1 (PPT1) PPT-1 CLN2 Janský-Bielschowsky 204500 Classic late infantile, juvenile* CLN2 (TPP1) TPP-1 CLN3 Spielmeyer-Sjögren 204200 Juvenile* CLN3 CLN3 protein (battenin) CLN4 Parry 162350 Adult autosomal dominant* CLN4 (DNAJC5) DnaJ homologue subfamily C member CLN5 Finnish variant late infantile, variant juvenile (previously CLN9) 256731 Late infantile variant, juvenile, adult* CLN5 Protein CLN5 CLN6 Early juvenile (Lake Cavanaugh), late infantile Costa Rican-Indian variant, adult Kuf type A 601780 Late infantile variant, adult* (Kuf, type A)* CLN6 Protein CLN6 CLN7 Turkish variant late infantile 610951 Late infantile variant*, juvenile*, adult* CLN7 (MFSD8) Major facilitator superfamily domain-containing protein CLN8 Northern epilepsy, progressive EPMR 610003 Late infantile variant EPMR* CLN8 Protein CLN8 CLN10 Congenital 610127 Congenital classic*, late infantile*, adult* CLN10 (CTSD) Cathepsin D CLN11 Adult variant-Adult* CLN11 (GRN) Progranulin CLN12 Juvenile variant-Juvenile, Kufor-Rakeb syndrome* CLN12 (ATP13A2)-CLN13 Adult Kuf type B-Adult Kuf type* CLN13 (CTSF) Cathepsin F CLN14 Infantile-Infantile, progressive myoclonus epilepsy 3* CLN14 (KCTD7) Potassium channel tetramerization domain-containing protein *These diseases have neurological involvement. Abbreviation: EPMR, epilepsy with mental retardation. Table1. Human ceroid lipofuscinoses variants (Boustany et al., 2013). They are considered lysosomal storage disorders (LSD), because of their characteristic accumulation of autofluorescent ceroid lipopigments, subunit
Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells
PLOS ONE, 2015
Chronic exposure to polychlorinated biphenyls (PCBs), ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254) in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 μg/ml) reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase), measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.
Scientific Reports
We previously reported that kenpaullone, which inhibits GSK-3a/b and CDKs inhibited CCCP mediated mitochondrial depolarisation and augments the mitochondrial network. To investigate the actions of this class of drug further, we compared the ability of kenpaullone, alsterpaullone, 1-azakenapaullone, AZD5438, AT7519 (CDK and GSK-3a/b inhibitors) and dexpramipexole and olesoxime (mitochondrial permeability transition pore inhibitors) to prevent CCCP mediated mitochondrial depolarisation and found that AZD5438 and AT7519, were the most effective. Furthermore, treatment with AZD5438 alone increased the complexity of the mitochondrial network. We also found that AZD5438 prevented the rotenone induced decrease in PGC-1alpha and TOM20 levels and that it mediated powerful anti-apoptotic effects and promoted glycolytic respiration. Importantly, experiments in human iPSC derived cortical and midbrain neurons showed AZD5438 mediated significant protective effects, preventing the neuronal cell d...