Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia.

Upstream open reading frames (uORFs) have emerged as major post-transcriptional regulatory elements in eukaryotic species. In general, uORFs are initiated by a translation start codon within the 5' untranslated region of a gene (upstream ATG; uATG), and they are negatively correlated with translational efficiency. In addition to their translational regulatory role, some uORFs can code for biologically active short peptides. The importance of uATGs/uORFs is further underscored by human diseases associated with single nucleotide polymorphisms (SNPs), which disrupt existing uORFs or introduce novel uORFs. Although several functional proteins translated from naturally occurring uORFs have been described, the coding potential of uORFs created by SNPs has been ignored because of the a priori assumption that these proteins are short-lived with no likely impact on protein homeostasis. Thus, studies on SNP-created uORFs are limited to their translational effects, leaving unexplored the potential cellular consequences of a SNP/uORF-encoded protein. Here, we investigate functionality of a uATG/uORF introduced by a +142C>T SNP within the GCH1 gene and associated with a familial form of DOPA Responsive Dystonia. We report that the +142C>T SNP represses GCH1 translation, and introduces a short, frame shifted uORF that encodes a 73-amino acid peptide. This peptide is localized within the nucleus and compromises cell viability upon proteasome inhibition. Our work extends the list of uATG/uORF associated diseases and advances research on peptides translated from SNP-introduced uORFs, a neglected component of the proteome.

Isoflurane favorably modulates guanosine triphosphate cyclohydrolase-1 and endothelial nitric oxide synthase during myocardial ischemia and reperfusion injury in rats.

BACKGROUND: The authors investigated the hypothesis that isoflurane modulates nitric oxide (NO) synthesis and protection against myocardial infarction through time-dependent changes in expression of key NO regulatory proteins, guanosine triphosphate cyclohydrolase (GTPCH)-1, the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin and endothelial nitric oxide synthase (eNOS). METHODS: Myocardial infarct size, NO production (ozone-mediated chemiluminescence), GTPCH-1, and eNOS expression (real-time reverse transcriptase polymerase chain reaction and western blotting) were measured in male Wistar rats with or without anesthetic preconditioning (APC; 1.0 minimum alveolar concentration isoflurane for 30 min) and in the presence or absence of an inhibitor of GTPCH-1, 2,4-diamino-6-hydroxypyrimidine. RESULTS: NO2 production (158 ± 16 and 150 ± 13 pmol/mg protein at baseline in control and APC groups, respectively) was significantly (P < 0.05) increased 1.5 ± 0.1 and 1.4 ± 0.1 fold by APC (n = 4) at 60 and 90 min of reperfusion, respectively, concomitantly, with increased expression of GTPCH-1 (1.3 ± 0.3 fold; n = 5) and eNOS (1.3 ± 0.2 fold; n = 5). In contrast, total NO (NO2 and NO3) was decreased after reperfusion in control experiments. Myocardial infarct size was decreased (43 ± 2% of the area at risk for infarction; n = 6) by APC compared with control experiments (57 ± 1%; n = 6). 2, 4-Diamino-6-hydroxypyrimidine decreased total NO production at baseline (221 ± 25 and 175 ± 31 pmol/mg protein at baseline in control and APC groups, respectively), abolished isoflurane-induced increases in NO at reperfusion, and prevented reductions of myocardial infarct size by APC (60 ± 2%; n = 6). CONCLUSION: APC favorably modulated a NO biosynthetic pathway by up-regulating GTPCH-1 and eNOS, and this action contributed to protection of myocardium against ischemia and reperfusion injury.

Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension.

Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and pulmonary artery (PA) remodeling. Although comprising a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SOD(loxp/loxp) × Tg(cre/SMMHC) mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days, and PH was assessed by right ventricular systolic pressure measurements and right ventricle hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined cGMP content and soluble guanylate cyclase expression and activity in lung, lung phosphodiesterase 5 (PDE5) expression and activity, and expression of endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased PA EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung soluble guanylate cyclase or PDE5, yet it blunted the hypoxia-induced increase in cGMP. Although total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of PA EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function.

Design of a single AAV vector for coexpression of TH and GCH1 to establish continuous DOPA synthesis in a rat model of Parkinson's disease.

Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.

Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis.

BACKGROUND: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. METHODS AND RESULTS: In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. CONCLUSIONS: The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Differential regulation of dopaminergic gene expression by Er81.

A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase.

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.

2,4-Diamino-6-hydroxypyrimidine (DAHP) suppresses cytokine-induced VCAM-1 expression on the cell surface of human umbilical vein endothelial cells in a BH(4)-independent manner.

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH(4) biosynthesis and is widely used in order to elucidate the possible biological function of BH(4) in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH(4)) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH(4) might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH(4) and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH(4) level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH(4) level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH(4) production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH(4)-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappaB (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappaB level in the nucleus of HUVEC.

Regulation of tetrahydrobiopterin biosynthesis by shear stress.

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the alpha prime (alpha') subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2-dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.

Oral administration of tetrahydrobiopterin attenuates testicular damage by reducing nitric oxide synthase activity in a cryptorchid mouse model.

Experimental cryptorchidism has been shown to induce germ cell apoptosis. Nitric oxide (NO), a ubiquitous free radical produced by NO synthases (NOSs), has been associated with apoptosis in a number of cell types. However, the regulation of NOSs in experimental cryptorchid testes remains unknown. Tetrahydrobiopterin (BH4), an essential cofactor of NOS, plays an important role in the generation of NO. It has been reported that activation of the immune system stimulates an increase in endogenous BH4 rate-limiting enzyme GTP cyclohydrolase I (GTPCH I) activity, resulting in an increase in intracellular BH4 levels and BH4-dependent NO synthesis in various cells. We examined the effect of dietary treatment with BH4 on GTPCH I, BH4 synthesis, NO production, and testicular damage in cryptorchid model mice. Male mice were treated with oral BH4 starting from age 4 weeks or received standard diet only, and right cryptorchid testes were created surgically at age 10 weeks. The testes were evaluated 0, 3, 5, 7, and 10 days after surgery by assays of testicular weight, BH4 and dihydrobiopterin (oxidized BH4) levels, GTPCH I mRNA levels, NOS protein expression levels, NO concentration, and nitrotyrosine (product of ONOO(-); determinant of NO-dependent damage) levels. In untreated mice, GTPCH I mRNA and BH4 levels increased and eNOS protein expression, NO concentration, and nitrotyrosine levels increased gradually. BH4 treatment decreased GTPCH I mRNA and BH4 levels, with concomitant reduction of eNOS protein levels, nitrotyrosine levels, and NO concentration, resulting in reduced testicular damage. Our findings demonstrate that supplementation with BH4 could provide a new therapeutic intervention for heat stress-based testicular dysfunction.

Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-kappaB and Stat1/Stat3.

Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-gamma (IFN-gamma) induced endothelial cell GTPCH I protein and BH4 modestly, whereas high-level induction required combinations of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha). In the presence of IFN-gamma, TNF-alpha increased GTPCH I mRNA in a manner dependent on nuclear factor-kappaB (NF-kappaB), as this effect was abrogated by overexpression of a dominant-negative IkappaB construct. HUVEC IFN-gamma treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-gamma in supporting TNF-alpha-mediated GTPCH I induction. The role of IFN-gamma was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-alpha-mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-gamma in this system. These data implicate both NF-kappaB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-gamma and TNF-alpha exert distinct but cooperative roles for BH4 biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.

The Nrf2/GCH1/BH4 Axis Ameliorates Radiation-Induced Skin Injury by Modulating the ROS Cascade.

Radiation-induced skin injury is a common side effect of radiotherapy and can limit the duration and dose of radiotherapy. Most early work focused on elimination of reactive oxygen species (ROS) after radiation; however, less is known about the mechanisms underlying amplification of ROS and consequent skin injury by radiation. 5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for all nitric oxide synthases. Inadequate availability of BH4 leads to uncoupling of nitric oxide synthases and production of highly oxidative radicals. In this study, we demonstrated that radiation disrupted BH4, which resulted in nitric oxide synthases uncoupling and augmented radiation-induced ROS. Overexpression of GTP cyclohydrolase I (GCH1), the rate-limiting enzyme for BH4 synthesis, restored cellular BH4 levels and nitric oxide production and decreased radiation-induced ROS. GCH1 also protected skin cells and rat skins against radiation-induced damage. We found that GCH1 was regulated by NF-E2-related factor 2, a key mediator of the cellular antioxidant response. Importantly, we identified GCH1 as a key effector for NF-E2-related factor 2-mediated protection against radiation-induced skin injury by inhibiting ROS production. Taken together, the findings of this study illustrate the key role of the NF-E2-related factor 2/GCH1/BH4 axis during radiation-induced skin damage.

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice.

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.

Nucleotide Variants of the BH4 Biosynthesis Pathway Gene GCH1 and the Risk of Orofacial Clefts.

A deficiency of GTP cyclohydrolase, encoded by the GCH1 gene, results in two neurological diseases: hyperphenylalaninaemia type HPABH4B and DOPA-responsive dystonia. Genes involved in neurotransmitter metabolism and motor systems may contribute to palatogenesis. The purpose of the study was to analyse polymorphic variants of the GCH1 gene as risk factors for non-syndromic cleft lip with or without cleft palate (NSCL/P). Genotyping of nine polymorphisms was conducted in a group of 281 NSCL/P patients and 574 controls. The GCH1 variant rs17128077 was associated with a 1.7-fold higher risk for NSCL/P (95 %CI = 1.224-2.325; p = 0.001). We also found a significant correlation between the rs8004018 and rs17128050 variants and an increased risk of oral clefts (p trend = 0.003 and 0.004, respectively). The best evidence of the global haplotype association was observed for rs17128050 and rs8004018 (p corr = 0.0152). This study demonstrates that the risk of NSCL/P is associated with variants of the GCH1 gene related to BH4 metabolism and provides some evidence of the relationships between morphological/functional shifts in the central nervous system and orofacial clefts.

Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production.

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.

Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.

GTP cyclohydrolase I mRNA induction and tetrahydrobiopterin synthesis in human endothelial cells.

The key role of tetrahydrobiopterin (BH4) in the synthesis of nitric oxide by human umbilical vein endothelial cells (HUVEC) has been demonstrated. We characterized the induction of BH4 synthesis in a cell line (ECV) derived from HUVEC and primary HUVEC. A significant induction of guanosine triphosphate cyclohydrolase I (GTPCH) mRNA was observed in response to TNF, IL-1beta, and IFNgamma in ECV and HUVEC. The induction of GTPCH mRNA was abolished by actinomycin D. The cytokines led to an increased accumulation of BH4 in ECV. This effect was prevented by 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTPCH, as well as by actinomycin D and by cycloheximide. Results provide evidence for an increase in GTPCH activity and in BH4 levels in response to immunostimulants in human endothelial cells.

Tetrahydrobiopterin biosynthesis in white and brown adipose tissues is enhanced following intraperitoneal administration of bacterial lipopolysaccharide.

Tetrahydrobiopterin is an essential cofactor for nitric oxide synthase (NOS). This study was undertaken to examine the effects of intraperitoneally injected lipopolysaccharide on tetrahydrobiopterin biosynthesis in murine white and brown adipose tissues. Tetrahydrobiopterin content, catalytic activity and mRNA expression level of GTP cyclohydrolase I (GCH), rate-controlling enzyme in de novo biosynthesis of tetrahydrobiopterin, in both adipose tissues were up-regulated by 500-microg lipopolysaccharide at 6 h after the injection. On the contrary, treatment of 3T3-L1 adipocytes with lipopolysaccharide alone did not affect GCH mRNA expression level, whereas the combination of lipopolysaccharide, tumor necrosis factor (TNF)-alpha, and interferon gamma induced the increase in expression levels of GCH mRNA and CD14 mRNA. Collectively, our results showed that tetrahydrobiopterin biosynthesis can be augmented by increased GCH activity caused by a synergistic effect of lipopolysaccharide and cytokines in white and brown adipose tissues. These observations support the view that tetrahydrobiopterin biosynthesis in the adipose tissues is a target of inflammatory events triggered by peripheral LPS injection.

Functional tetrahydrobiopterin synthesis in human platelets.

BACKGROUND: Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. METHODS AND RESULTS: We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. CONCLUSIONS: Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice.

OBJECTIVE: Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). METHODS AND RESULTS: Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. CONCLUSIONS: These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosis and is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression.