Background: Tetrahydrobiopterin (BH4) and its oxidized derivative dihydrobiopterin (BH2) were found to be strongly elevated in ME/CFS patients with orthostatic intolerance (ME + OI).
Objective: However, the molecular mechanism of biopterin biogenesis is poorly understood in ME + OI subjects. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biogenesis of biopterins (BH4 and BH2) in ME + OI subjects.
Research design and results: Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the regulations of biopterins. To further investigate, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of biopterin metabolism via ELISA, immunoblot, and dual immunocytochemical analyses. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the bioavailability of phosphoribosyl pyrophosphate (PRPP), reduced the expressions of purine biosynthetic enzymes, attenuated GTP cyclohydrolase 1 (GTPCH1), and suppressed subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2 via the purine biosynthetic pathway. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients.
Conclusion: Taken together, our current research highlights that the induction of non-oxidative PPP regulates the biogenesis of biopterins contributing to ME/CFS pathogenesis.
Keywords: Inducible nitric oxide; ME/CFS; Orthostatic intolerance; Tetrahydrobiopterin; dihydrobiopterin.
Tetrahydrobiopterin (BH4) metabolism is tightly regulated in a healthy individual. Recently, our research showed that BH4 level is upregulated in the plasma samples of ME/CFS patients with orthostatic intolerance. While investigating the molecular mechanism, our current study identified that the pentose phosphate pathway (PPP) induction is critical for the upregulated expression of BH4. A novel hypoxia-based cell culture model is introduced to study PPP in human microglial cells. Subsequently, a comprehensive RNA array study, different immunoassay, and biochemical analyses of enzyme activities confirmed that the induction of non-oxidative PPP in microglial cells enhanced expressions of PPP-regulatory genes and enzymes, induced enzyme activities, activated purine biosynthesis, and finally upregulated biopterin biogenesis.
© The Author(s) 2024.