Submitted Abstract
Over the past years it has become clear that the notions of damage and repair, in biology, do not only apply to DNA and proteins, but also to the low molecular weight components of the cell, i.e. metabolites. The latter can be damaged by spontaneous chemical as well as enzymatic side reactions and enzymes dedicated to remove the resulting ‘abnormal’ metabolites are being identified at an increasingly rapid pace. In that context, we have discovered in 2011 a widely conserved metabolite repair system that acts on NADHX and NADPHX (hydrated, redox-inactive forms of the central metabolic cofactors NADH and NADPH). This repair system consists of two enzymes, a dehydratase and an epimerase, that are encoded in humans by the NAXD and NAXE genes, respectively. The vital role of these enzymes was demonstrated recently, through the identification of paediatric patients with mutations in the NAXD or NAXE genes and suffering from a fatal febrile-induced neurodegenerative disorder. In this project, we will establish and analyze cellular and animal models of this disorder, with the aim of elucidating the disease mechanism and testing therapeutic interventions. In addition, we will develop an enzyme activity based method to non-invasively diagnose both the NAXE and NAXD diseases in patient cell extracts. Our main hypothesis is that NAD(P) depletion and/or NAD(P)HX accumulation play major roles in disease development by causing metabolic blockages, similar to what we previously demonstrated in a yeast model of NAD(P)HX repair deficiency. To test this, we will, in addition to NAXE and NAXD knockout immortalized human cell lines that we use already, establish more advanced disease models, by deriving neuronal and glial cells from NAD(P)HX repair deficient human induced pluripotent stem cells and by generating NAXD and NAXE mutant zebrafish lines. In the cellular models, our focus will be to identify metabolic perturbations using metabolomics based approaches, but other phenotypes, such as cell morphology, viability, metabolic stress resistance, and mitochondrial function will also be investigated. Morphological and various functional analyses will be carried out in the mutant zebrafish larvae. In addition to helping us progress in the understanding of the disease mechanism, some of the identified phenotypes will then be used as readouts to evaluate the therapeutic effect of small molecule treatments. Our experiments build on our extensive previous fundamental research experience with and findings on the NAD(P)HX repair system, but aim to translate into results that should directly benefit patients and their families by providing diagnostic tests as well as efficient treatment approaches. Beyond the specific disorder under investigation, this project will also elucidate new molecular mechanisms likely to be involved in neurodegeneration more generally.