A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

• Type of Material: Article
• Authors: Paula Escudero-Ferruz, Universitat Autònoma de Barcelona; Neus Ontiveros Ontiveros, Universitat Autònoma de Barcelona; Claudia Cano-Estrada Cano-Estrada, Universitat Autònoma de Barcelona; Diane J Sutcliffe, Emory University; Hyder A Jinnah, Emory University; Rosa J Torres, Instituto de Salud Carlos III (ISCIII); José M López, Universitat Autònoma de Barcelona
• Language: English
• Date: 2024-01-03
• Publisher: Springer Nature
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2023
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): https://molmed.biomedcentral.com/articles/10.1186/s10020-023-00774-8
• Title of Journal or Parent Work: Molecular Medicine
• Volume: 30
• Grant/Funding Information: Obtaining cells was supported in part by NIH grants NS109242 and NS119758.; This research was supported by Fundation La Marató de TV3, grant 776/C/2020, and grant PID2020-113124RB-C21 funded by MCIN/AEI/10.13039/501100011033.; Obtaining urine samples was supported by grant PID2020-113124RB-C22 funded by 10.13039/501100011033.
• Keywords: Folic acid; ZMP; Purine nucleotides; Plasmax; Lesch-Nyhan disease; AICAR
• Research Categories: Health Sciences, General; Biology, Cell

Abstract

Background Lesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells. Methods We prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts. Results LND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND. Conclusions A complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.