背景:Lesch-Nyhan病(LND)是由次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HGprt)的遗传缺陷引起的严重神经系统疾病,参与嘌呤合成的一种酶。为了弥补这种不足,嘌呤从头生物合成途径加速。大多数研究未能在从患者获得的培养细胞中发现嘌呤核苷酸的任何一致异常。最近,已显示5-氨基咪唑-4-甲酰胺核苷5'-单磷酸酯(ZMP),从头途径的中间部分,在含有生理水平(25nM)叶酸(FA)的RPMI维持的LND成纤维细胞中积累,这与常规细胞培养基的FA水平(2200nM)有很大不同。然而,RPMI和其他标准培养基含有非生理水平的许多营养素,对细胞代谢有很大影响,不能准确概括细胞的体内行为。
方法:我们准备了一种新的培养基,其中含有所有营养物质的生理水平,包括维生素(Plasmax-PV),研究可能被非生理介质的使用所掩盖的LND成纤维细胞的潜在变化。我们定量了不同培养条件下的ZMP积累,并评估了两种已知的ZMP靶蛋白(AMPK和ADSL)的活性,叶酸载体SLC19A1的mRNA表达,LND成纤维细胞中可能的线粒体改变和功能后果。
结果:用Plasmax-PV维持的LND成纤维细胞表现出代谢适应,例如更高的糖酵解能力,与对照相比,叶酸载体SCL19A1的表达增加,以及诸如线粒体电位降低和细胞迀移减少的功能改变。这些改变可以用高水平的叶酸逆转,这表明叶酸补充剂可能是LND的潜在治疗方法。
结论:完整的生理细胞培养基揭示了Lesch-Nyhan病的新变化。这项工作强调了在研究代谢紊乱时使用生理细胞培养条件的重要性。
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 5\'-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.