Dysregulation of tetrahydrobiopterin metabolism in ME/CFS by pentose phosphate pathway, 2024, Bulbule, Roy et al

This is the preprint. See post #4 for the published paper

Augmentation of Anaerobic Pentose Phosphate Pathway Dysregulates Tetrahydrobiopterin Metabolism in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) Patients with Orthostatic Intolerance: A Pilot Study

Sarojini Bulbule, Carl Gunnar Gottschalk, Molly E Drosen, Daniel Peterson, Leggy A Arnold, Avik Roy

Tetrahydrobiopterin (BH4), an essential cofactor of amino acid metabolism, was found to be strongly elevated in ME/CFS patients with Orthostatic intolerance (ME + OI). However, the molecular mechanism of BH4 upregulation is poorly understood in ME + OI patients.

Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biosynthesis of BH4 in ME + OI patients. 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 pathogenesis of ME + OI.

Along with the upregulated anaerobic PPP enzymes, we observed that biopterin metabolites such as BH4 and dihydrobiopterin (BH2) are strongly upregulated suggesting the disruption of biopterin homeostasis in ME + OI patients. To explore the molecular role of anaerobic PPP in biopterin metabolism, 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 BH4 and BH2 upregulation via ELISA, immunoblot and dual immunocytochemical analyses. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2.

Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the expression of GTP cyclohydrolase 1 (GTPCH1) and subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. 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.

https://www.researchsquare.com/article/rs-3716093/v1

 

Years ago I read that BH4 is involved in an alternative loop for forming methionine if there are issues with folate and B12 . I may be misremembering , but it's part of a very complex interdependent system for many compounds , some compromised by genetics and epigenetic expression .

We need to look at all of these as interdependent.

 

Another impact re BH4 availability - reduced recycling of BH2 to BH4 if folate availability is reduced.

uncoupling of NOS by either too little BH4 or too little arginine produces not only superoxide and but also BH2 (a partially oxidized pterin). Both BH4 and BH2 bind to iNOS with equal affinity but only BH4 provides the desired enzymatic effect of producing NO. Therefore, BH2 can block BH4 from binding to iNOS. unbound BH4 is exposed to superoxide which converts the BH4 to peroxynitrite. So BH2 promotes more oxidative stress. To maintain BH4 homeostasis, BH2 is normally recycled back into BH4 by quinoid dihydropteridin reductase (QDPR) using dihydrofolate (DHF). This recycling pathway is also important because the use of BH4 as a co-factors in the production of neurotransmitters (serotonin, dopamine, norepinephrine, and epinephrine) always produces BH2 as an output. This recycling pathway is therefore a major source of BH4 and must remain reliably functional. However, efficiency is reduced by two types of genetic mutations -
mutations of QDPR have been shown to impair this recycling pathway.
folate mutations (chiefly MTHFR C677T and MTHFR A1298C) and/or a diet deficient in fresh green vegetables can reduce the availability of dihydrofolate for the recycling pathway .

If BH2 is upregulated as suggested in the paper, then having the above genetic mutations may underlie this or exacerbate it ?

 

Now published:

Bulbule S, Gottschalk CG, Drosen ME, Peterson D, Arnold LA, Roy A. Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway. Journal of Central Nervous System Disease. 2024;16. doi:10.1177/11795735241271675

Abstract

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.

Plain Language Summary

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.

 

Opposite the Ron Davis group finding re BH4, correct?

 

Has the Ron Davis group study been concluded yet?

Andrew Miller found that inflammation reduces the levels of a cofactor called BH4 – which helps to produce tyrosine – the precursor to dopamine. Miller believes inflammation or oxidative stress may be whacking the BH4 co-enzyme in ME/CFS, thus reducing tyrosine and ultimately dopamine levels. Interestingly, Ron Davis is exploring the role BH4 plays in ME/CFS right now.

 

Quotes from this study.

"we first performed a comprehensive microarray screening of 46 genes from the PAXgene total RNA samples of ME + OI (n = 4) subjects"

"Based on the microarray data, we further validated the expressions of 3 critical enzymes of PPP including G6PDH (Figure 1(D)), TALDO (transaldolase) (Figure 1(E)), and 6PGL (6-phosphogluconolactonase) (Figure 1(F)) by realtime-PCR analyses in n = 10 ME + OI subjects"

"Acquisition of blood samples and questionnaire data were previously collected32 under the supervision of Dr Daniel Peterson"

Ref 32 is to this paper, Elevated ATG13 in serum of pwME stimulates oxidative stress response in microglial cells , 2022, Gottschalk et al, which is paywalled, so I'm unable to determine what criteria, if any, the participants met.

 

It does seem different although I think technically his finding is bh4 as a % of total biopterin rather than absolute levels. His work is also n=1 and the model patient is who you think.

Ron says he gave Whitney some synthetic BH2 which made this ratio even lower and Whitney felt much worse.

 

https://my.theopenscholar.com/files/simmaronlab/files/1-s2.0-s1044743122000379-main_1.pdf

 

So if I was just working based on logic with those two statements if Whitney had a lower BH4 /biopterin vs HC/norm and this says BH4 is strongly elevated compared to HCs
the condition for both to be true would involve high biopterin. Given Whitney's % is half of norms then far more than double normal biopterin (for BH4 to also be 'strongly elevated')

Is that possible? surely that would be what was reported?

 

Regarding the new (2024) paper:

I'm confused. I don't understand how the causal direction was determined. Red to highlight the conclusions confusing me.

Intro:
Interestingly, our siRNA-mediated silencing of the transaldolase (taldo1) gene followed by the evaluation of BH4 expression, iNOS activation, and nitric oxide production demonstrated that the augmentation of non-oxidative (non-ox) PPP followed by the upregulation of BH4 is critical in the pathogenesis of OI.

Discussion:
BH4 Upregulation: Is it a Cause or Result of Metabolic Impairment in ME + OI Subjects?
BH4 is an important cofactor in many essential metabolic pathways. Upregulation of BH4 is beneficial in reducing inflammation and stress in metabolic diseases ranging from diabetes to cancer, neuroinflammatory diseases to rare genetic disorders. Therefore, it is puzzling to decode the role of upregulated BH4 in the pathogenesis of ME + OI. At first, we asked if the upregulation of BH4 was the central and independent mechanism that contributed to the pathogenesis of ME + OI or if it was a secondary response to some other upstream metabolic impairments. Impaired glucose metabolism and the resultant deficit of mitochondrial energy are shown to be involved in ME/CFS pathogenesis. As an alternative strategy, the pentose phosphate pathway could be operative for glucose utilization. A recent study [32] showed that PPP was truly activated in ME/CFS subjects. However, our study showed that the augmented PPP did not shunt the glycolytic energy production mechanism but continued to proceed through the anaerobic stage, which might facilitate R5P to follow the purine nucleotide biosynthetic pathway for the synthesis of GTP, induced GTPCH1 enzyme activity that further caused the upregulated synthesis of BH4 (graphical summary at Figure 8). Therefore, BH4 upregulation possibly happened as a secondary event due to the augmentation of non-oxidative PPP.

How did BH4 Contribute to the Pathogenesis of ME + OI?
Our study showed that the augmentation of anaerobic PPP and the resultant upregulation of BH4 could stimulate iNOS in microglial cells. First, a fluorimetric NO measurement assay revealed that the activation of anaerobic PPP by treating microglial cells with 10 μM R5P under hypoxic conditions stimulated NO. Interestingly, under similar conditions, we observed a strong upregulation of BH4. Second, A dual immunofluorescence study also revealed that the induction of anaerobic PPP as indicated by enhanced TALDO1 expression also upregulated iNOS expression, and the knocking down of TALDO1 expression by siRNA also ameliorated iNOS expression. Interestingly, under similar conditions, we observed that taldo1 siRNA also inhibited BH4 expression in C20 microglial cells. Third, the exogenous addition of ME + OI serum samples with high BH4 (>15 pmol/mg) induced iNOS expression and NO production, whereas administration of HC serum samples with low BH4 (<5 pmol/mg protein) was unable to induce the same. Finally, A parametric correlation statistical analysis between the levels of BH4 and NO production capacity in a total of 20 serum samples (n = 10 ME + OI and n = 10 HC) demonstrated that the elevated BH4 could be directly linked to the induced NO production under hypoxic conditions. Is that NO pathologically relevant? Interestingly, we also observed a strong upregulation of BH2 in ME + OI serum samples. Implementing non-oxidative PPP also upregulated BH2 in C20 microglia. Studies suggest37 that the accelerated conversion of BH4 to BH2 could uncouple physiological NO production and induce cellular stress. Therefore, based on our analyses, a significant proportion of the elevated BH4 may be converted to BH2 in ME + OI subjects and directly correlated with the increased iNOS activation and NO production in microglial cells.



PS: G6PD was up in my ME/CFS cell lines here: https://www.mdpi.com/1422-0067/22/4/2046 (concordant)

 

Somewhat related. In mice.

Mast cell–derived BH4 and serotonin are critical mediators of postoperative pain
https://www.science.org/doi/10.1126/sciimmunol.adh0545

Postoperative pain affects most patients after major surgery and can transition to chronic pain. The considerable side effects and limited efficacy of current treatments underline the need for new therapeutic options. We observed increased amounts of the metabolites BH4 and serotonin after skin injury. Mast cells were primary postoperative sources of Gch1, the rate-limiting enzyme in BH4 synthesis, itself an obligate cofactor in serotonin production by tryptophan hydroxylase (Tph1). Mice deficient in mast cells or in mast cell–specific Gch1 or Tph1 showed drastically decreased postoperative pain. We found that injury induced the nociceptive neuropeptide substance P, mast cell degranulation, and granule nerve colocalization. Substance P triggered serotonin release in mouse and human mast cells, and substance P receptor blockade substantially ameliorated pain hypersensitivity. Our findings highlight the importance of mast cells at the neuroimmune interface and substance P–driven mast cell BH4 and serotonin production as a therapeutic target for postoperative pain treatment.​

 

Solve ME/CFS Initiative

In a new publication of a Solve-funded study, Ramsay Research Grant winner Dr. Avik Roy and his co-authors examine orthostatic imbalance in people with ME/CFS using an interesting new experimental model to dissect the metabolic missteps responsible for this effect.

Read our summary of the study here: https://solvecfs.org/tetrahydrobiopterin-a-cause-of-orthostatic-imbalance-in-people-with-me-cfs/

 

Twitter thread from the group

https://twitter.com/user/status/1835386394308665528

 

Simmaron Research:

Our 2nd publication on BH4 seeks to answer questions like: why do #ME patients with Orthostatic Intolerance have high BH4? How does BH4 become elevated in this subset? What is the mechanism that governs this process?

#OI #MECFS #longcovid

https://simmaronresearch.salsalabs.org/bh4inme