[en] [en] BACKGROUND: Metabolism is error prone. For instance, the reduced forms of the central metabolic cofactors nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), can be converted into redox-inactive products, NADHX and NADPHX, through enzymatically catalyzed or spontaneous hydration. The metabolite repair enzymes NAXD and NAXE convert these damaged compounds back to the functional NAD(P)H cofactors. Pathogenic loss-of-function variants in NAXE and NAXD lead to development of the neurometabolic disorders progressive, early-onset encephalopathy with brain edema and/or leukoencephalopathy (PEBEL)1 and PEBEL2, respectively.
METHODS: To gain insights into the molecular disease mechanisms, we investigated the metabolic impact of NAXD deficiency in human cell models. Control and NAXD-deficient cells were cultivated under different conditions, followed by cell viability and mitochondrial function assays as well as metabolomic analyses without or with stable isotope labeling. Enzymatic assays with purified recombinant proteins were performed to confirm molecular mechanisms suggested by the cell culture experiments.
RESULTS: HAP1 NAXD knockout (NAXDko) cells showed growth impairment specifically in a basal medium containing galactose instead of glucose. Surprisingly, the galactose-grown NAXDko cells displayed only subtle signs of mitochondrial impairment, whereas metabolomic analyses revealed a strong inhibition of the cytosolic, de novo serine synthesis pathway in those cells as well as in NAXD patient-derived fibroblasts. We identified inhibition of 3-phosphoglycerate dehydrogenase as the root cause for this metabolic perturbation. The NAD precursor nicotinamide riboside (NR) and inosine exerted beneficial effects on HAP1 cell viability under galactose stress, with more pronounced effects in NAXDko cells. Metabolomic profiling in supplemented cells indicated that NR and inosine act via different mechanisms that at least partially involve the serine synthesis pathway.
CONCLUSIONS: Taken together, our study identifies a metabolic vulnerability in NAXD-deficient cells that can be targeted by small molecules such as NR or inosine, opening perspectives in the search for mechanism-based therapeutic interventions in PEBEL disorders.
Disciplines :
Biochemistry, biophysics & molecular biology Human health sciences: Multidisciplinary, general & others
Author, co-author :
WALVEKAR, Adhish ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine > Enzymology and Metabolism > Team Carole LINSTER
WARMOES, Marc Omer ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine > Scientific Central Services > Metabolomics Platform
CHEUNG, Dean ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Sikora, Tim ; Brain and Mitochondrial Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, 3002, Australia
SEYEDKATOULI, Najmesadat ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Enzymology and Metabolism
Gomez-Giro, Gemma ; Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367, Belvaux, Luxembourg
PERRONE, Sebastian ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine > Enzymology and Metabolism > Team Carole LINSTER
Dengler, Lisa ; Enzymology and Metabolism Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367, Belvaux, Luxembourg
Unger, François ; Enzymology and Metabolism Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367, Belvaux, Luxembourg
Santos, Bruno F R ; Disease Modeling and Screening Platform, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367, Belvaux and Luxembourg Institute of Health, L-1445, Strassen, Luxembourg
GAVOTTO, Floriane ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Scientific Central Services > Metabolomics Platform
SCHWAMBORN, Jens Christian ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Developmental and Cellular Biology
Van Bergen, Nicole J ; Brain and Mitochondrial Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, 3002, Australia ; Department of Paediatrics, University of Melbourne, Melbourne, VIC, 3002, Australia
Christodoulou, John ; Brain and Mitochondrial Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, 3002, Australia ; Department of Paediatrics, University of Melbourne, Melbourne, VIC, 3002, Australia ; Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, VIC, 3002, Australia
Fonds National de la Recherche Luxembourg Juniclair Foundation Mito Foundation
Funding text :
We thank J\u00E9r\u00F4me Oswald (LCSB) for support with generating the NAXD rescue plasmids and Alison Graham and Sara Howden from the Gene Editing Core facility at MCRI for generating the NAXD knockout iPSC lines. The author/s acknowledge the facilities, and the scientific and technical assistance of Murdoch Children\u2019s Research Institute iPSC and GE Core Facility. MCRI iPSC and GE Core Facility was established using a generous donation from the Stafford Fox Medical Research Foundation. It is currently supported by Phenomics Australia (PA), and the Novo Nordisk Foundation reNEW Center for Stem Cell Medicine (NNF21CC0073729). PA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We acknowledge Gezime Seferi (LCSB) for help with the NAXD gene expression analysis during the revision of the manuscript. We also acknowledge Siddhesh Kamat (Indian Institute of Science Education and Research Pune, India) for providing infrastructural support to A.S.W.This research was supported by a CORE grant from the Luxembourg National Research Fund (FNR) under project code C18/BM/12661133 and a donation from the Juniclair Foundation to C.L.L. The FNR also provided a PhD fellowship to N.K. within the doctoral training unit ACTIVE (PRIDE19/14063202) under the supervision of C.L.L. The research conducted at the Murdoch Children\u2019s Research Institute (MCRI) was supported by the State Government of Victoria\u2019s Operational Infrastructure Support Program. The work was supported by funding from the Mito Foundation to N.V.B. and J.C., the MCRI Near Miss grant to N.V.B., and the MCRI Strategic Pilot Project in Stem Cell and Genomics Medicine grant to N.V.B. and J.C. The Chair in Genomic Medicine awarded to J.C. is generously supported by The Royal Children\u2019s Hospital Foundation. We are grateful to the Crane and Perkins families for their generous financial support.
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