PhD Scientific Days 2022

Budapest, 6-7 July 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

The DLST+/- and DLD +/- Knockout α-ketoglutarate Dehydrogenase Complex Affects the Oxygen Consumption and the Reactive Oxygen Species Generation in Mouse Brain Mitochondria

Márton Kokas, Department of Biochemistry, Budapest

Text of the abstract

Introduction: The mitochondrial tricarboxylic acid cycle (TCA) is a key complex to energy alteration in the human body. The rate limiting step of the cycle catalized by the α-ketoglutarate dehydrogenase complex (αKGDH), which creates succinyl-coenzime A and CO2 from α-ketoglutarate, while an NAD+ is reduced to NADH. The αKGDH contains three subunits: oxoglutarate dehydrogenase (OGDH); dihydrolipoamide succinyltransferase (DLST); dihydrolipoamide dehydrogenase (DLD). it has been proven by many researchers that malfunctioned αKGDH is associated with neurodegenerative diseases. In the presence of elevated NADH/NAD+ ratio mitochondrial matrix dehydrogenases, like αKGDH can produce large amount of reactive oxygen sepcies (ROS), which also leads to patological condition in the brain.
Aims: The purpose of the present study is to examine how DLST+/- and DLD +/- heterozygous knockout in αKGDH affects oxygen consumption and ROS generation in mouse brain mitochondria in the presence of various mitochondrial substrates.
Methods: The mitochondria were isolated from adult mouse brain. In our study four genotypes of αKGDH were examined: DLST+/-; DLD +/-; DLST+/- and DLD +/- heterozygous mice; WT was used as control. Oxygen consumptionwas measured using high-resolution respirometry . H2O2 production was detected using Amplex UltraRed fluorescense assay.
Results: Mitochondrial oxygen consumption in the presence of ADP was significantly decreased in all of the KO groups compared to control WT mice, when the substrate was αKG. However, no difference was observed among the different groups using succinate or α-glycerophosphate as respiratory substrate. In the αKG-supported mitochondria ROS production was decreased in the transgenic groups after adding ADP and rotenone, which is a Complex I inhibitor. Nevertheless using succinate in the abscence of ADP there was a high rate of ROS production attributed to reverse electron transport, whichwas reduced in the KO groups compared to WT.
Conclusion: Our results show that αKGDH subunits have a vital role in mitochondrial ROS formation.
Funding: Supported by the ÚNKP-21-2-I-SE 30 new national excellence program of the ministry for innovation and technology from the source of national research, development and innovation fund.
Supported by Kiegészítő Kutatási Kiválósági Ösztöndíj (EFOP-3.6.3-VEKOP-16-2017-00009).