Mutations in the Sepiapterin Reductase Gene Cause a Novel Tetrahydrobiopterin-Dependent Monoamine-Neurotransmitter Deficiency without Hyperphenylalaninemia

Classic tetrahydrobiopterin (BH(4)) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH(4). Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC-->CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397-1401delAGAAC) resulting in abolished SPR-gene expression and an A-->G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH(4) biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH(4) in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH(4) and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.     

A murine model for human sepiapterin-reductase deficiency

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.     

Genetically rescued tetrahydrobiopterin-depleted mice survive with hyperphenylalaninemia and region-specific monoaminergic abnormalities

One of the possibly mutated genes in DOPA-responsive dystonia (DRD, Segawa's disease) is the gene encoding GTP cyclohydrolase I, which is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. Based on our findings on 6-pyruvoyltetrahydropterin synthase (PTS) gene-disrupted (Pts(-/-)) mice, we suggested that the amount of tyrosine hydroxylase (TH) protein in dopaminergic nerve terminals is regulated by the intracellular concentration of BH4. In this present work, we rescued Pts(-/-) mice by transgenic introduction of human PTS cDNA under the control of the dopamine beta-hydroxylase promoter to examine regional differences in the sensitivity of dopaminergic neurons to BH4-insufficiency. The DPS-rescued (Pts(-/-), DPS) mice showed severe hyperphenylalaninemia. Human PTS was efficiently expressed in noradrenergic regions but only in a small number of dopaminergic neurons. Biopterin and dopamine contents, and TH activity in the striatum were poorly restored compared with those in the midbrain. TH-immunoreactivity in the lateral region of the striatum was far weaker than that in the medial region or in the nucleus accumbens. We concluded that dopaminergic nerve terminals projecting to the lateral region of the striatum are the most sensitive to BH4-insufficiency. Biochemical and pathological changes in DPS-rescued mice were similar to those in human malignant hyperphenylalaninemia and DRD.     

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain

Tyrosine hydroxylase (TH) is a rate‐limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4‐deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4‐deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post‐natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post‐natal period is critical for the developmental regulation of TH protein level.     

Tetrahydrobiopterin Availability in Parkinson’s and Alzheimer’s Disease; Potential Pathogenic Mechanisms

Within the central nervous system, tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis. In addition, BH4 is now established to be an essential cofactor for all isoforms of nitric oxide synthase (NOS). Inborn errors of metabolism affecting BH4 availability are well documented and the clinical presentation can be attributed to a paucity of dopamine, serotonin, and nitric oxide (NO) generation. In this article, we have focussed upon the sensitivity of BH4 to oxidative catabolism and the observation that when BH4 is limiting some cellular sources of NOS may generate superoxide whilst other BH4 saturated NOS enzymes may be generating NO. Such a scenario could favor peroxynitrite generation. If peroxynitrite is not scavenged, e.g., by antioxidants such as reduced glutathione, irreversible damage to critical cellular enzymes could ensue. Such targets include components of the mitochondrial electron transport chain, alpha ketoglutarate dehydrogenase and possibly pyruvate dehydrogenase. Such a cascade of events is hypothesized, in this article, to occur in neurodegerative conditions such as Parkinson’s and Alzheimer’s disease.     

Tetrahydrobiopterin deficiency exaggerates intimal hyperplasia after vascular injury

Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 (n = 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 +/- 0.3 vs. 4.5 +/- 0.4 pmol/min/mg protein, P < 0.01), and higher aortic superoxide content (5.2 +/- 2.0 x 10(5) cpm vs. 1.6 +/- 0.7 x 10(5) cpm, P < 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 +/- 0.3 to 4.1 +/- 0.4 pmol.min(-1).mg protein(-1), P < 0.05), and attenuated superoxide production (from 5.2 +/- 2.0 x 10(5) cpm to 0.8 +/- 0.7 x 10(5) cpm, P < 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 +/- 0.5 vs. wild-type 0.7 +/- 0.1, P < 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 +/- 0.1 vs. hph-1, P < 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis.     

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.     

Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system

Insulin-like growth factor-1 (IGF-1) is an essential growth factor for normal intrauterine development and postnatal growth. Mice with a complete deficiency of IGF-1 (IGF-1-null mice), created by homologous recombination, were found to exhibit postnatal lethality, growth retardation, infertility, and profound defects in the development of major organ systems. Furthermore, IGF-1-null mice were resistant to growth hormone (GH) treatment in peri-pubertal somatic growth. Using the Cre/loxP-induced conditional knockout system, we generated a mouse that lacks IGF-1 specifically in the liver, the primary site of IGF-1 production. Interestingly, although circulating and serum levels of IGF-1 were decreased by approximately 75% in these mice, they exhibited no defect in growth or development. When administered exogenously, GH stimulated IGF-1 production in several extra-hepatic tissues as well as body growth. The "Somatomedin hypothesis" originally proposed that circulating IGF-1 acting in various tissues mediate the effects of GH. These striking in vivo results, obtained using homologous recombination technology, call for a major modification of the Somatomedin hypothesis. These gene targeting studies confirm that IGF-1 is essential for GH-stimulated postnatal body growth. However, liver-derived (endocrine) IGF-1 is not essential for normal postnatal growth, though it does exert a negative feedback on GH secretion. Instead, local production of IGF-1, acting in a paracrine/autocrine fashion, appears to mediate GH-induced somatic growth. This review will discuss the effects of tissue-specific IGF-1 gene deficiency created by the Cre/loxP system versus the conventional IGF-1 knockout.     

Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH(4)). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH(4) becomes oxidized in organs of apolipoprotein E-deficient (ApoE(-/-)) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH(4) were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE(-/-) mice or ApoE(-/-) mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE(-/-) mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH(4) oxidation in heart, lung, and kidney of atherogenic ApoE(-/-) mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE(-/-) mice and that these species differentially contribute to protein 3-NT accumulation and BH(4) oxidation in a tissue-selective manner. Since BH(4) oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.     

Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

Neurochemical Effects Following Peripheral Administration of Tetrahydropterin Derivatives to the hph-1 Mouse

Abstract: The hph-1 mouse, which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 µmol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N -benzyl-1,4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1–4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 µmol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.     

Effect of experimental hyperphenylalaninemia on biogenic amine synthesis at later stages of brain development

The effects of experimental hyperphenylalaninemia on catecholamine and serotonin synthesis in brain at a later stage of brain development were investigated. A group of 35-day-old rats treated with normal chow supplemented with 5% Phe + 0.4% alpha-methylphenylalanine, alpha MP, for the previous 10 days showed decreases in dopa, norepinephrine, and epinephrine versus controls. A group treated with a normal diet supplemented with 0.4% alpha MP showed similar decreases and these differences could be attributed to the presence of the phenylalanine hydroxylase and tyrosine hydroxylase inhibitor, alpha MP, rather than the hyperphenylalaninemia condition. No differences in dopamine were observed. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) were decreased 50% in the HyPhe condition and were unaffected in the presence of alpha MP alone, indicating that the decreases in serotonin and 5HIAA were due to the increases in phenylalanine rather than the presence of the inhibitor. These abnormalities in serotonin metabolism at later stages of brain development may be relevant to early discontinuation of dietary therapy in the PKU patient and implies a role in tryptophan supplementation to increase intracerebral serotonin values.     

Molecular Biology of Catecholamine-Related Enzymes in Relation to Parkinson's Disease

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels.2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4.3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)].4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.     

An experimental model of partial insulin-like growth factor-1 deficiency in mice

Insulin-like growth factor-1 (IGF-1) is responsible for many systemic growth hormone (GH) functions although it has an extensive number of inherent activities (anabolic, cytoprotective, and anti-inflammatory). The potential options for IGF-1 therapy arise as a promising strategy in a wide list of human diseases. However, deeper studies are needed from a suitable animal model. All human conditions of IGF-1 deficiency consist in partially decreased IGF-1 levels since total absence of this hormone is hardly compatible with life. The aim of this work was to confirm that heterozygous Igf-1 ^+/? mice (Hz) may be considered as an appropriate animal model to study conditions of IGF-1 deficiency, focusing on early ages. Heterozygous Igf-1 ^+/? mice were compared to homozygous Igf-1 ^+/+ by assessing gene expression by quantitative PCR, serum circulating levels by ELISA, and tissue staining. Compared to controls, Hz mice (25 days old) showed a partial but significant reduction of IGF-1 circulating levels, correlating with a reduced body weight and diminished serum IGFBP-3 levels. Hz mice presented a significant decrease of IGF-1 gene expression in related organs (liver, bone, testicles, and brain) while IGF-1 receptor showed a normal expression. However, gene expression of growth hormone receptor (GHR) was increased in the liver but reduced in the bone, testicles, and brain. In addition, a significant reduction of cortical bone thickness and histopathological alterations in the testicles were found in Hz mice when compared to controls. Finally, the lifelong evolution of IGF-1 serum levels showed significant differences throughout life until aging in mice. Results in this paper provide evidence for considering heterozygous mice as a suitable experimental model, from early stages, to get more insight into the mechanisms of the beneficial actions induced by IGF-1 replacement therapy.     

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.