- Email: [email protected]
A bacterial model to study Leigh syndrome
Abstracts
Thus, metabolic strategies may constitute promising therapeutic strategies against mitochondrial disorders. References 1. V. Desquiret-Dumas, N. Gueguen, M. Barth, A. Chevrollier, S.Hancock, D.C. Wallace, P. A. Bonneau, D. Henrion, D.Bonneau, P. Reynier, V. Procaccio, Metabolically induced heteroplasmy shifting and L-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS, Biochim Biophys Acta 1822(6) (2012) 1019–1029. doi:10.1016/j.bbabio.2016.04.217
10.03 A bacterial model to study Leigh syndrome Emmanuel Gnandta, Karoline Aierstocka,b, Klaudia Fricka, Lothar Kussmaulb, Thorsten Friedricha a Albert-Ludwigs Universität Freiburg, Albertstraße 21, 79104 Freiburg, Germany b Department of CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach an der Riß, Germany E-mail address: [email protected] (E. Gnandt) Dysfunction of the mitochondrial NADH ubiquinone oxidoreductase (respiratory complex I) is known to be related to several neurodegenerative diseases [1]. Amino acids Tyr 204 and Cys206 close to the NADH binding site on subunit NDUFV1 of complex I have been shown to be heterozygously mutated in a patient suffering from Leigh syndrome [2]. These mutations have been partially described in a yeast model system for Leigh syndrome [3]. We developed a bacterial model for mutational studies in Escherichia coli. We introduced the mutations Y178C and C180G at orthologous positions by site-directed and λ-Red mediated mutagenesis and measured the impact of these mutations on NADH oxidase activity. We purified the variants and characterized their NADH:ferricyanide and NADH:decyl-ubiquinone oxidoreductase activities. The production of ROS was quantified by means of the AmplexRed assay. The FMN binding to the variants was determined by the ThermoFAD assay. We discuss the impact of these mutations as molecular causes for Leigh syndrome. References 1. A.H.V Schapira, Human complex I defects in neurodegenerative diseases, Biochim. Biophys. Acta 1364 (1998) 261-270. 2. P. Bénit, D. Chretien, N. Kadhom, P. de Lonlay-Debeney, V. Cormier-Daire, A. Cabral, et al., Large-Scale Deletion and Point Mutations of the Nuclear NDUFV1 and NDUFS1 Genes in Mitochondrial Complex I Deficiency, Am. J. Hum. Genet. 68 (2001) 1344-1352. 3. F. Varghese, E. Atcheson, H. R. Bridges, J. Hirst, Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system, Hum. Mol. Genet. 24 (2015) 6350-6360. doi:10.1016/j.bbabio.2016.04.218
10.04 MicroRNAs as modulators of mitochondrial pathogenesis Francesca Grespia,b, Laura Reffoa,b, Stefano Cagninb,c, Gerolamo Lanfranchib,c, Luca Scorranoa,b
e103
a
Venetian Institute of Molecular Medicine, Padova, Italy Dept. of Biology, University of Padova, Italy c CRIBI Biotechnology Centre, University of Padova, Italy E-mail address: [email protected] (F. Grespi) b
Opa1 is a dynamin-related protein of the inner mitochondrial membrane mediating fusion and cristae remodeling, a key mechanism in apoptosis. Indeed, Opa1 is mutated in Autosomal Dominant Optic Atrophy (ADOA) a genetic disorder characterized by a high degree of phenotypic variability. Nevertheless, triggers determining the severity of the disease are still unknown. Intriguingly, emerging evidence highlight a link between mitochondrial fusion/fission and microRNAs, well-known epigenetic regulators. This suggests that miRNAs might participate in modulation of penetrance of diseases affecting mitochondrial shape. Accordingly, we performed an in silico screening to identify miRNAs putatively targeting the 3’UTR of Opa1. Our preliminary data demonstrate that specific miRNAs can directly modulate Opa1 levels, thus influencing mitochondrial shape and apoptosis. We identified specific cell death triggers inducing miRNAs expression, indicating that candidates are important at the beginning of the apoptotic cascade. For this reason, we hypothesize that the differential penetrance of ADOA is mediated by miRNAs and this feature can be exploited therapeutically. Indeed, our project is proposing an innovative mechanism of modulating ADOA penetrance that has never been investigated before. We believe that increasing levels of Opa1, even if mutated, by modulation of miRNAs by AntagomiRs, could ameliorate the phenotype of ADOA patients. In conclusion, our study could provide new potential therapeutic targets for a currently untreatable disease. doi:10.1016/j.bbabio.2016.04.219
10.05 Human VDAC isoform effects on viability of Saccharomyces cerevisiae model of Huntington disease Daria Grobys, Andonis Karachitos, Hanna Kmita Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland E-mail address: [email protected] (D. Grobys) Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by a loss of neurons in striatum and cerebral cortex. HD is caused by CAG trinucleotide repeat expansion in exon 1 of IT15 gene encoding huntingtin (Htt). The repeats number higher than 35 results in an abnormally long polyglutamine tract in Htt N terminus that gives rise to its mutated form (mHtt). It is now obvious that mitochondria play a vital role in HD pathogenesis while Voltage-Dependent Anion selective Channel (VDAC), a major protein of the mitochondrial outer membrane, is crucial for mitochondria functioning. It has been shown that VDAC can regulate activity, import and expression of mitochondrial proteins as well as proteins crucial to communications between nucleus and mitochondria or interact with proteins involved in these processes. To investigate the role of human VDAC isoforms for cell viability in HD pathogenesis, we applied Saccharomyces cerevisiae HD model based on induced expression of IT15 gene exon 1 containing 25 or 103 repeats of glutamine codon that results in expression of Htt and mHtt, respectively. The expression of the proteins is induced by galactose, and monitored due to GFP labeling. We also investigate the role of temperature stress (37 °C)
Thus, metabolic strategies may constitute promising therapeutic strategies against mitochondrial disorders. References 1. V. Desquiret-Dumas, N. Gueguen, M. Barth, A. Chevrollier, S.Hancock, D.C. Wallace, P. A. Bonneau, D. Henrion, D.Bonneau, P. Reynier, V. Procaccio, Metabolically induced heteroplasmy shifting and L-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS, Biochim Biophys Acta 1822(6) (2012) 1019–1029. doi:10.1016/j.bbabio.2016.04.217
10.03 A bacterial model to study Leigh syndrome Emmanuel Gnandta, Karoline Aierstocka,b, Klaudia Fricka, Lothar Kussmaulb, Thorsten Friedricha a Albert-Ludwigs Universität Freiburg, Albertstraße 21, 79104 Freiburg, Germany b Department of CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach an der Riß, Germany E-mail address: [email protected] (E. Gnandt) Dysfunction of the mitochondrial NADH ubiquinone oxidoreductase (respiratory complex I) is known to be related to several neurodegenerative diseases [1]. Amino acids Tyr 204 and Cys206 close to the NADH binding site on subunit NDUFV1 of complex I have been shown to be heterozygously mutated in a patient suffering from Leigh syndrome [2]. These mutations have been partially described in a yeast model system for Leigh syndrome [3]. We developed a bacterial model for mutational studies in Escherichia coli. We introduced the mutations Y178C and C180G at orthologous positions by site-directed and λ-Red mediated mutagenesis and measured the impact of these mutations on NADH oxidase activity. We purified the variants and characterized their NADH:ferricyanide and NADH:decyl-ubiquinone oxidoreductase activities. The production of ROS was quantified by means of the AmplexRed assay. The FMN binding to the variants was determined by the ThermoFAD assay. We discuss the impact of these mutations as molecular causes for Leigh syndrome. References 1. A.H.V Schapira, Human complex I defects in neurodegenerative diseases, Biochim. Biophys. Acta 1364 (1998) 261-270. 2. P. Bénit, D. Chretien, N. Kadhom, P. de Lonlay-Debeney, V. Cormier-Daire, A. Cabral, et al., Large-Scale Deletion and Point Mutations of the Nuclear NDUFV1 and NDUFS1 Genes in Mitochondrial Complex I Deficiency, Am. J. Hum. Genet. 68 (2001) 1344-1352. 3. F. Varghese, E. Atcheson, H. R. Bridges, J. Hirst, Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system, Hum. Mol. Genet. 24 (2015) 6350-6360. doi:10.1016/j.bbabio.2016.04.218
10.04 MicroRNAs as modulators of mitochondrial pathogenesis Francesca Grespia,b, Laura Reffoa,b, Stefano Cagninb,c, Gerolamo Lanfranchib,c, Luca Scorranoa,b
e103
a
Venetian Institute of Molecular Medicine, Padova, Italy Dept. of Biology, University of Padova, Italy c CRIBI Biotechnology Centre, University of Padova, Italy E-mail address: [email protected] (F. Grespi) b
Opa1 is a dynamin-related protein of the inner mitochondrial membrane mediating fusion and cristae remodeling, a key mechanism in apoptosis. Indeed, Opa1 is mutated in Autosomal Dominant Optic Atrophy (ADOA) a genetic disorder characterized by a high degree of phenotypic variability. Nevertheless, triggers determining the severity of the disease are still unknown. Intriguingly, emerging evidence highlight a link between mitochondrial fusion/fission and microRNAs, well-known epigenetic regulators. This suggests that miRNAs might participate in modulation of penetrance of diseases affecting mitochondrial shape. Accordingly, we performed an in silico screening to identify miRNAs putatively targeting the 3’UTR of Opa1. Our preliminary data demonstrate that specific miRNAs can directly modulate Opa1 levels, thus influencing mitochondrial shape and apoptosis. We identified specific cell death triggers inducing miRNAs expression, indicating that candidates are important at the beginning of the apoptotic cascade. For this reason, we hypothesize that the differential penetrance of ADOA is mediated by miRNAs and this feature can be exploited therapeutically. Indeed, our project is proposing an innovative mechanism of modulating ADOA penetrance that has never been investigated before. We believe that increasing levels of Opa1, even if mutated, by modulation of miRNAs by AntagomiRs, could ameliorate the phenotype of ADOA patients. In conclusion, our study could provide new potential therapeutic targets for a currently untreatable disease. doi:10.1016/j.bbabio.2016.04.219
10.05 Human VDAC isoform effects on viability of Saccharomyces cerevisiae model of Huntington disease Daria Grobys, Andonis Karachitos, Hanna Kmita Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland E-mail address: [email protected] (D. Grobys) Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by a loss of neurons in striatum and cerebral cortex. HD is caused by CAG trinucleotide repeat expansion in exon 1 of IT15 gene encoding huntingtin (Htt). The repeats number higher than 35 results in an abnormally long polyglutamine tract in Htt N terminus that gives rise to its mutated form (mHtt). It is now obvious that mitochondria play a vital role in HD pathogenesis while Voltage-Dependent Anion selective Channel (VDAC), a major protein of the mitochondrial outer membrane, is crucial for mitochondria functioning. It has been shown that VDAC can regulate activity, import and expression of mitochondrial proteins as well as proteins crucial to communications between nucleus and mitochondria or interact with proteins involved in these processes. To investigate the role of human VDAC isoforms for cell viability in HD pathogenesis, we applied Saccharomyces cerevisiae HD model based on induced expression of IT15 gene exon 1 containing 25 or 103 repeats of glutamine codon that results in expression of Htt and mHtt, respectively. The expression of the proteins is induced by galactose, and monitored due to GFP labeling. We also investigate the role of temperature stress (37 °C)