Tryptophan Metabolism in Obesity: The Indoleamine 2,3-Dioxygenase-1 Activity and Therapeutic Options.

Obesity activates both innate and adaptive immune responses in adipose tissue. Adipose tissue macrophages are functional antigen-presenting cells that promote the proliferation of interferon-gamma (IFN-γ)-producing cluster of differentiation (CD)4+ T cells in adipose tissue of obese subjects. The increased formation of neopterin and degradation of tryptophan may result in decreased T-cell responsiveness and lead to immunodeficiency. The activity of inducible indoleamine 2,3-dioxygenase-1 (IDO1) plays a major role in pro-inflammatory, IFN-γ-dominated settings. The expression of several kynurenine pathway enzyme genes is significantly increased in obesity. IDO1 in obesity shifts tryptophan metabolism from serotonin and melatonin synthesis to the formation of kynurenines and increases the ratio of kynurenine to tryptophan as well as with neopterin production. Reduction in serotonin (5-hydroxytryptamine; 5-HT) production provokes satiety dysregulation that leads to increased caloric uptake and obesity. According to the monoamine-deficiency hypothesis, a deficiency of cerebral serotonin is involved in neuropsychiatric symptomatology of depression, mania, and psychosis. Indeed, bipolar disorder (BD) and related cognitive deficits are accompanied by a higher prevalence of overweight and obesity. Furthermore, the accumulation of amyloid-ß in Alzheimer's disease brains has several toxic effects as well as IDO induction. Hence, abdominal obesity is associated with vascular endothelial dysfunction. kynurenines and their ratios are prognostic parameters in coronary artery disease. Increased kynurenine/tryptophan ratio correlates with increased intima-media thickness and represents advanced atherosclerosis. However, after bariatric surgery, weight reduction does not lead to the normalization of IDO1 activity and atherosclerosis. IDO1 is involved in the mechanisms of immune tolerance and in the concept of tumor immuno-editing process in cancer development. Serum IDO1 activity is still used as a parameter in cancer development and growth. IDO-producing tumors show a high total IDO immunostaining score, and thus, using IDO inhibitors, such as Epacadostat, Navoximod, and L isomer of 1-methyl-tryptophan, seems an important modality for cancer treatment. There is an inverse correlation between serum folate concentration and body mass index, thus folate deficiency leads to hyperhomocysteinemia-induced oxidative stress. Immune checkpoint blockade targeting cytotoxic T-lymphocyte-associated protein-4 synergizes with imatinib, which is an inhibitor of mitochondrial folate-mediated one-carbon (1C) metabolism. Antitumor effects of imatinib are enhanced by increasing T-cell effector function in the presence of IDO inhibition. Combining IDO targeting with chemotherapy, radiotherapy and/or immunotherapy, may be an effective tool against a wide range of malignancies. However, there are some controversial results regarding the efficacy of IDO1 inhibitors in cancer treatment.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Acute Hypobaric Hypoxia Exposure Causes Neurobehavioral Impairments in Rats: Role of Brain Catecholamines and Tetrahydrobiopterin Alterations.

Hypoxia is a state in which the body or a specific part of the body is deprived of adequate oxygen supply at the tissue level. Sojourners involved in different activities at high altitudes (> 2500 m) face hypobaric hypoxia (HH) due to low oxygen in the atmosphere. HH is an example of generalized hypoxia, where the homeostasis of the entire body of an organism is affected and results in neurochemical changes. It is known that lower O2 levels affect catecholamines (CA), severely impairing cognitive and locomotor behavior. However, there is less evidence on the effect of HH-mediated alteration in brain Tetrahydrobiopterin (BH4) levels and its role in neurobehavioral impairments. Hence, this study aimed to shed light on the effect of acute HH on CA and BH4 levels with its neurobehavioral impact on Wistar rat models. After HH exposure, significant alteration of the CA levels in the discrete brain regions, viz., frontal cortex, hippocampus, midbrain, and cerebellum was observed. HH exposure significantly reduced spontaneous motor activity, motor coordination, and spatial memory. The present study suggests that the HH-induced behavioral changes might be related to the alteration of the expression pattern of CA and BH4-related genes and proteins in different rat brain regions. Overall, this study provides novel insights into the role of BH4 and CA in HH-induced neurobehavioral impairments.

Catecholamines and Parkinson's disease: tyrosine hydroxylase (TH) over tetrahydrobiopterin (BH4) and GTP cyclohydrolase I (GCH1) to cytokines, neuromelanin, and gene therapy: a historical overview.

The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine ß-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR.

Pathophysiology associated with Huntington's disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant.

Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).

Endothelium-Specific GTP Cyclohydrolase I Overexpression Restores Endothelial Function in Aged Mice.

This study tested the hypothesis that endothelium-specific GTP cyclohydrolase I (GTPCH I) overexpression (Tg-GCH) restores age-associated endothelial dysfunction in vivo. Aortic GTPCH I expression and serum nitric oxide (NO) release were measured in young and aged mice. Aortic rings from young and aged wild-type (WT) mice and aged Tg-GCH mice were suspended for isometric tension recording. A hind limb ischemia model was used to measure blood flow recovery. Aged mice showed reduced GTPCH I expression in the aorta and decreased NO levels in serum. Compared with aged WT mice, Tg-GCH significantly elevated NO levels in serum in aged Tg-GCH mice, restored the impaired aortic relaxation in response to acetylcholine, and significantly elevated aortic constriction in response to L-NAME. Importantly, aged Tg-GCH mice displayed a significant increase in blood flow recovery compared with aged WT mice. GTPCH I reduction contributes to aging-associated endothelial dysfunction, which can be retarded by Tg-GCH.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age.

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity. In GTP-CH deficient mice, the aortic BH4 levels were decreased, by -77% in 12 week-middle-aged mice (young) and by -83% in 35-45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (-46%), in flow-mediated vasodilation of mesenteric artery (-31%), in coronary hyperemia response measured in isolated heart following transient ischemia (-27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (-69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age. Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

Comprehensive analysis of GTP cyclohydrolase I activity in Mycobacterium tuberculosis H37 Rv via in silico studies.

GTP cyclohydrolase I enzyme (GTPCH-I) is a rate limiting enzyme in the biosynthesis pathway of tetrahydrobiopterin (BH4) and tetrahydrofolate (THF) compounds; latter being are an essential compounds involved in many biological functions. This enzyme has been evaluated structurally and functionally in many organisms to understand its putative role in cell processes, kinetics, regulations, drug targeting in infectious diseases, pain sensitivity in humans, and so on. In Mycobacterium tuberculosis (a human pathogen causing tuberculosis), this GTPCH-I activity has been predicted to be present in Rv3609c gene (folE) of H37 Rv strain, which till date has not been studied in detail. In order to understand in depth, the structure and function of folE protein in M. tuberculosis H37 Rv, in silico study was designed by using many different bioinformatics tools. Comparative and structural analysis predicts that Rv3609c gene is similar to folE protein ortholog of Listeria monocytogenes (cause food born disease), and uses zinc ion as a cofactor for its catalysis. Result shows that mutation of folE protein at 52th residue from tyrosine to glycine or variation in pH and temperature can lead to high destability in protein structure. Studies here have also predicted about the functional regions and interacting partners involved with folE protein. This study has provided clues to carry out experimentally the analysis of folE protein in mycobacteria and if found suitable will be used for drug targeting.

Zinc supplementation protects against diabetic endothelial dysfunction via GTP cyclohydrolase 1 restoration.

This study explored whether zinc supplementation alleviates diabetic endothelial dysfunction and the possible mechanisms underlying. We found that high glucose exposure significantly increased reactive oxygen species (ROS) and decreased guanosine 5'-triphosphate cyclohydrolase 1 (GTPCH1) and tetrahydrobiopterin (BH4) levels in bovine aortic endothelial cells (BAECs) in a time-dependent manner. High glucose increased zinc release from GTPCH1 in a similar trend. Zinc supplementation restored GTPCH1 and BH4 levels and blocked ROS accumulation in both BACEs and wild type GTPCH1 transfected HEK293 cells, but not in the zinc-free C141R mutant of GTPCH1 transfected ones. In vivo experiments showed that exogenous supplementation of zinc to streptozotocin (STZ)-induced diabetic mice partially improved the impaired maximal endothelium-dependent vasorelaxation, reversed the aberrant reduction of GTPCH1 and BH4, and suppressed the elevation of ROS in the aortas. In conclusion, our study demonstrated a novel mechanism that via GTPCH1 restoration zinc supplementation exerts a protective benefit on diabetic endothelial dysfunction.

Improved folate accumulation in genetically modified maize and wheat.

Folates are indispensable co-factors for one-carbon metabolism in all organisms. In humans, suboptimal folate intake results in serious disorders. One promising strategy for improving human folate status is to enhance folate levels in food crops by metabolic engineering. In this study, we cloned two GmGCHI (GTP cyclohydrolase I) genes (Gm8gGCHI and Gm3gGCHI) and one GmADCS (aminodeoxychorismate synthase) gene from soybean, which are responsible for synthesizing the folate precursors pterin and p-aminobenzoate, respectively. We initially confirmed their functions in transgenic Arabidopsis plants and found that Gm8gGCHI increased pterin and folate production more than Gm3gGCHI did. We then co-expressed Gm8gGCHI and GmADCS driven by endosperm-specific promoters in maize and wheat, two major staple crops, to boost their folate metabolic flux. A 4.2-fold and 2.3-fold increase in folate levels were observed in transgenic maize and wheat grains, respectively. To optimize wheat folate enhancement, codon-optimized Gm8gGCHI and tomato LeADCS genes under the control of a wheat endosperm-specific glutenin promoter (1Dx5) were co-transformed. This yielded a 5.6-fold increase in folate in transgenic wheat grains (Gm8gGCHI+/LeADCS+). This two-gene co-expression strategy therefore has the potential to greatly enhance folate levels in maize and wheat, thus improving their nutritional value.

Human tyrosine hydroxylase in Parkinson's disease and in related disorders.

Parkinson's disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD; (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene; and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.

Exercise training induces eNOS coupling and restores relaxation in coronary arteries of heart failure rats.

Exercise training (ET) has emerged as a nonpharmacological therapy for cardiovascular diseases because of its helpful milieu for improving vascular function. The aim of the present study was to assess whether ET reverses the alterations in vascular reactivity observed in heart failure (HF)-related coronary arteries and to elucidate the molecular mechanisms involved in these adjustments. Male Wistar rats were subjected to either coronary artery ligation or sham operation. Four weeks after the surgery, rats were divided into two groups: untrained HF (UHF) and exercise-trained HF (THF). ET was conducted on a treadmill for 8 wk. An untrained SO group was included in the study as a normal control. ET restored the impaired acetylcholine (ACh)- and sodium nitroprusside-induced relaxation in coronary arteries to levels of the control. Oxidative stress and reduced nitric oxide (NO) production were observed in UHF, whereas ET restored both parameters to the levels of the control. Expression levels of endothelial NO synthase (eNOS) and soluble guanylyl cyclase subunits were increased in coronary arteries of UHF rats but reduced in THF rats. Tetrahydrobiopterin restored ACh-induced NO production in the UHF group, indicating that eNOS was uncoupled. ET increased the eNOS dimer-to-monomer ratio and expression of GTP cyclohydrolase 1, thus increasing NO bioavailability. Taken together, these findings demonstrate that ET reverses the dysfunction of the NO/soluble guanylyl cyclase pathway present in coronary arteries of HF rats. These effects of ET are associated with increased GTP cyclohydrolase 1 expression, restoration of NO bioavailability, and reduced oxidative stress through eNOS coupling. NEW & NOTEWORTHY The present study provides a molecular basis for the exercise-induced improvement in coronary arteries function in heart failure. Increasing the expression of GTP cyclohydrolase 1, the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin, exercise training couples endothelial nitric oxide synthase, reduces oxidative stress, and increases nitric oxide bioavailability and sensitivity in coronary arteries of heart failure rats.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

De novo tetrahydrobiopterin biosynthesis is impaired in the inflammed striatum of parkin(-/-) mice.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disease, is primarily characterized by neurodegeneration in the substantia nigra pars compacta, resulting in motor impairment. Loss-of-function mutations in parkin are the major cause of the early onset familial form of the disease. Although rodents deficient in parkin (parkin(-/-) ) have some dopaminergic system dysfunction associated with central oxidative stress and energy metabolism deficiencies, these animals only display nigrostriatal pathway degeneration under inflammatory conditions. This study investigated the impact of the inflammatory stimulus induced by lypopolisaccharide (LPS) on tetrahydrobiopterin (BH4) synthesizing enzymes (de novo and salvage pathways), since this cofactor is essential for dopamine synthesis. The mitochondrial content and architecture was investigated in the striatum of LPS-exposed parkin(-/-) mice. As expected, the LPS (0.33 mg/kg; i.p.) challenge compromised spontaneous locomotion and social interaction with juvenile parkin(-/-) and WT mice. Moreover, the genotype impacted the kinetics of the investigation of the juvenile. The inflammatory scenario did not induce apparent changes in mitochondrial ultrastructure; however, it increased the quantity of mitochondria, which were of smaller size, and provoked the perinuclear distribution of the organelle. Furthermore, the BH4 de novo biosynthetic pathway failed to be up-regulated in the LPS challenge, a well-known stimulus for its activation. The LPS treatment increased sepiapterin reductase (SPR) expression, suggesting compensation by the salvage pathway. This might indicate that dopamine synthesis is compromised in parkin(-/-) mice under inflammatory conditions. Finally, this scenario impaired the striatal expression of the transcription factor BDNF, possibly favoring cell death.

Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo.

BACKGROUND: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation. METHODS: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization. RESULTS: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin. CONCLUSIONS: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

The Interactions Between Kynurenine, Folate, Methionine and Pteridine Pathways in Obesity.

Obesity activates both innate and adaptive immune responses in adipose tissue. Elevated levels of eosinophils with depression of monocyte and neutrophil indicate the deficiencies in the immune system of morbidly obese individuals. Actually, adipose tissue macrophages are functional antigen-presenting cells that promote the proliferation of interferon-gamma (IFN-gamma)-producing CD4+ T cells in adipose tissue of obese subjects. Eventually, diet-induced obesity is associated with the loss of tissue homeostasis and development of type 1 inflammatory responses in visceral adipose tissue. Activity of inducible indoleamine 2,3-dioxygenase-1 (IDO-1) plays a major role under pro-inflammatory, IFN-gamma dominated settings. One of the two rate-limiting enzymes which can metabolize tryptophan to kynurenine is IDO-1. Tumor necrosis factor-alpha (TNF-alpha) correlates with IDO-1 in adipose compartments. Actually, IDO-1-mediated tryptophan catabolism due to chronic immune activation is the cause of reduced tryptophan plasma levels and be considered as the driving force for food intake in morbidly obese patients. Thus, decrease in plasma tryptophan levels and subsequent reduction in serotonin (5-HT) production provokes satiety dysregulation that leads to increased caloric uptake and obesity. However, after bariatric surgery, weight reduction does not lead to normalization of IDO-1 activity. Furthermore, there is a connection between arginine and tryptophan metabolic pathways in the generation of reactive nitrogen intermediates. Hence, abdominal obesity is associated with vascular endothelial dysfunction and reduced nitric oxide (NO) availability. IFN-gamma-induced activation of the inducible nitric oxide synthase (iNOS) and dissociation of endothelial adenosine monophosphate activated protein kinase (AMPK)- phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt)- endothelial NO synthase (eNOS) pathway enhances oxidative stress production secondary to high-fat diet. Thus, reduced endothelial NO availability correlates with the increase in plasma non-esterified fatty acids and triglycerides levels. Additionally, in obese patients, folate-deficiency leads to hyperhomocysteinemia. Folic acid confers protection against hyperhomocysteinemia-induced oxidative stress.

Redox Signaling and Persistent Pulmonary Hypertension of the Newborn.

Reactive oxygen species (ROS) are redox-signaling molecules that are critically involved in regulating endothelial cell functions, host defense, aging, and cellular adaptation. Mitochondria are the major sources of ROS and important sources of redox signaling in pulmonary circulation. It is becoming increasingly evident that increased mitochondrial oxidative stress and aberrant signaling through redox-sensitive pathways play a direct causative role in the pathogenesis of many cardiopulmonary disorders including persistent pulmonary hypertension of the newborn (PPHN). This chapter highlights redox signaling in endothelial cells, antioxidant defense mechanism, cell responses to oxidative stress, and their contributions to disease pathogenesis.

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.).

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed-specific overexpression of AtGCHI caused significant increases of up to 150-fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 µg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.

Coffee extracts suppress tryptophan breakdown in mitogen-stimulated peripheral blood mononuclear cells.

OBJECTIVES: Coffee consumption is considered to exert an influence on mood, the immune system, cardiovascular disease, and cancer development, but the mechanisms of action of coffee and its compounds are only partly known and understood. METHODS: Immunomodulatory effects of filtered extracts of coffee and decaffeinated coffee as well as coffee compounds were investigated in human peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA). The activation of PBMCs was monitored by the breakdown of tryptophan to kynurenine via enzyme indoleamine 2,3-dioxygenase (IDO) and the production of the immune activation marker neopterin by GTP-cyclohydrolase I (GCH1). Both of these biochemical pathways are induced during cellular immune activation in response to the Th1-type cytokine interferon-γ (IFN-γ). RESULTS: Filtered extracts of coffee and decaffeinated coffee both suppressed tryptophan breakdown and neopterin formation in mitogen-stimulated PBMCs efficiently and in a dose-dependent manner. Of 4 coffee compounds tested individually, only gallic acid and less strong also caffeic acid had a consistent suppressive influence but also affected cell viability, whereas pure caffeine and chlorogenic acid exerted no relevant effect in the PBMC assay. CONCLUSION: The parallel influence of extracts on tryptophan breakdown and neopterin production shows an anti-inflammatory and immunosuppressive property of coffee extracts and some of its compounds. When extrapolating the in vitro results to in vivo, IFN-γ-mediated breakdown of tryptophan could be counteracted by the consumption of coffee or decaffeinated coffee. This may increase tryptophan availability for the biosynthesis of the neurotransmitter 5-hydroxytryptamine (serotonin) and thereby improve mood and quality of life.