Functional consequences of modulated expression of SLC25A51 on cellular NAD metabolism
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- Master theses 
Nicotinamide adenine dinucleotide is a small molecule involved in the regulation of signaling pathways and energy transduction. In the compartmentalized human cell, mitochondria are considered to constitute the major pool of this dinucleotide, where it is involved in key processes, such as the tricaboxylic acid cycle and NAD+-dependent deacylation by Sirtuins. Since biological membranes are considered impermeable to NAD+, the mechanisms underlying the generation and maintenance of the mitochondrial NAD+ pool in human cells were largely unknown. The recent characterization of solute carrier family 25 member 25 (SLC25A51) as a mitochondrial NAD+ transporter challenged the role of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) in contributing to NAD+ biosynthesis within the mitochondrial matrix. In this study, the impact of modulated expression of SLC25A51 on cellular NAD+ metabolism was studied. To that end, a cell line harboring a deficient gene encoding SLC25A51 as well a cell line constitutively expressing the NAD+ transporter from recombinant DNA were used as experimental systems. The knockout of SLC25A51 promoted a switch from oxidative phosphorylation to aerobic glycolysis in those cells and a consequent reduction in cell proliferation, which could be connected to the depleted mitochondrial NAD+ pool. Interestingly, the depletion of the mitochondrial NAD+ pool did not affect the levels of whole cell NAD+ content. The overexpression of this transporter, though, did not affect the availability of mitochondrial NAD+, but affected whole cell NAD+ content. The influence of the depleted mitochondrial NAD+ pool in SLC25A51-ko cells in the generation and maintenance of the peroxisomal NAD+ pool was assessed using an established NAD+ biosensor that relies on the targeted expression of the catalytic domain of poly(ADP)ribose polymerase 1 (PARP1cd) and immunodetection of poly(ADP)ribose (PAR) as readout. Stable expression of PARP1cd in the peroxisomes in the context of an SLC25A51-knockout revealed that the availability of NAD+ in the peroxisomes was unaffected by the absence of a functional mitochondrial NAD+ transporter. Furthermore, none of the cell lines used showed a compensatory regulation of neither NMNAT2 nor NamPT under the experimental conditions used. VI Stable expression of PARP1cd in the mitochondria in the context of an SLC25A51-knockout followed by PAR immunodetection revealed a vast reduction of available NAD+ in that compartment. This finding substantiated the function of SLC25A51 as a mitochondrial NAD+ transporter and provided the possibility to investigate alternative pathways for the generation and maintenance of the mitochondrial NAD+ pool in the absence of a functional mitochondrial NAD+ transporter. Strikingly, treatment of these engineered cells with the reduced form of nicotinamide riboside (NRH) suggested this metabolite supports NAD+ availability in the mitochondria in the absence of a functional mitochondrial NAD+ transporter. This finding may contribute to the identification of a novel pathway for the generation and maintenance of the mitochondrial NAD+ pool, which may require the catalytic activity of NMNAT3 in these organelles.