From the
Division of Clinical Chemistry and
Biochemistry, Department of Pediatrics, ¶Division
of Animal Facility, University of Zürich, Steinwiesstrasse 75, CH-8032
Zurich, Switzerland
Received for publication, April 16, 2003 , and in revised form, May 5, 2003.
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ABSTRACT |
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INTRODUCTION |
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The BH4 cofactor is synthesized from guanosine triphosphate (GTP) by a set of reactions involving three enzymes. GTP cyclohydrolase I (GTPCH), the first enzyme in BH4 biosynthesis, catalyzes the formation of dihydroneopterin triphosphate from GTP (3). GTPCH activity is regulated by its substrate GTP, BH4, and phenylalanine. A physiological mechanism for post-translational control of GTPCH activity involves feedback inhibition by BH4. Notably, feedback inhibition results from BH4-induced complex formation of GTPCH with a regulatory protein known as GTPCH feedback regulatory protein (GFRP) (46). In the second step, the 6-pyruvoyltetrahydropterin synthase (PTPS) catalyzes the conversion of dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin. PTPS must be phosphorylated to be fully active (7, 8). Sepiapterin reductase (SR) is required for the final step reductions of the diketo intermediate, 6-pyruvoyl-tetrahydropterin to BH4.
BH4 is required as cofactor for phenylalanine hydroxylase (PAH),
tyrosine hydroxylase, and tryptophan hydroxylase. The latter two are key
enzymes in the biosynthesis of the neurotransmitters dopamine and serotonin
(9). The complete hydroxylating
system of aromatic amino acids consists of the two additional
BH4-regenerating enzymes: pterin 4-carbinolamine dehydratase
and dihydropteridine reductase (DHPR)
(10). BH4 is also
required for the nitric oxide synthase enzymes
(11).
A deficiency of phenylalanine catabolism, leading to hyperphenylalaninemia
(HPA), comprises a heterogeneous group of disorders caused by a partial or
complete deficiency of the hepatic apoenzyme PAH, or by one of the enzymes
involved in cofactor biosynthesis (GTPCH or PTPS)
(12,
13), or regeneration (DHPR and
pterin 4-carbinolamine dehydratase)
(1416).
Whereas severe HPA, leading to classic phenylketonuria, can only be treated
with a low phenylalanine diet, patients with BH4-responsive PAH
deficiency can be treated with BH4 alone
(17). Two disorders of
BH4 metabolism may present without HPA. These are dopa-responsive
dystonia (Segawa disease) (18)
and sepiapterin reductase deficiency
(19,
20). Although dopa-responsive
dystonia is caused by a mutation in the GTPCH gene and is inherited in an
autosomal dominant manner, SR deficiency is an autosomal recessive trait. Both
diseases manifest severe biogenic amine deficiencies.
To diagnose BH4 deficiencies and follow-up of the resulting pathologies, only limited possibilities are available, including measurements of metabolites in body fluids, and follow-up of the disease development almost exclusively under treated conditions. Diagnosis starts in most cases with screening of HPA with the Guthrie card and determination of plasma phenylalanine levels as an indirect measurement of hepatic phenylalanine hydroxylase (PAH) activity. Analysis of phenylalanine and tyrosine in serum or plasma before and after a BH4 challenge are often applied as an additional diagnostic tool for differentiation between classic phenylketonuria and biopterin variants. Furthermore, urinary pterin analysis and enzymatic measurements in erythrocytes or skin fibroblasts are carried out to gather information on biopterin-metabolizing enzymes. These data are then combined with a neurotransmitter status. For neurotransmitters, the dopamine and serotonin neurotransmitter degradation products homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), respectively, are measured in cerebrospinal fluid (CSF), thus following the activities of tyrosine and tryptophan hydroxylases. Furthermore, NO metabolites at least for brain nitric-oxide synthase (NOS) isoenzyme activity are determined in CSF.
Symptoms of BH4 deficiency include a vast range of abnormalities of the central nervous system, including microcephaly, seizures, hypertonia, hypersalivation, temperature instability, feeding difficulties, and mental retardation. The goals of treatment are to control HPA by dietary restriction of phenylalanine (in PAH deficiency) or BH4 administration (in GTPCH and PTPS deficiency) and to restore neurotransmitter homeostasis by oral administration of the dopamine and serotonin precursors L-dopa and 5-hydroxytryptophan, respectively, in BH4 deficiencies. Late detection and introduction of treatment leads to irreversible brain damage. For patients with BH4 deficiency, HPA is controllable with oral doses of 210 mg of synthetic BH4/kg/day. However, such relatively low doses of BH4 do not allow the cofactor to penetrate the blood-brain barrier efficiently (21, 22). To some extent, this problem can be overcome by administering higher doses of BH4, up to 20 mg/kg/day, together with corresponding neurotransmitter precursors (23). The combined therapy is mandatory to avoid neurological damage; however, this treatment is not sufficient in every case (24).
To analyze in more detail the consequences of BH4 deficiency and its treatment, and to study pathologies in the organism, the use of animal models is required. Here we report on the generation of an animal model for PTPS deficiency by knocking out the Pts gene in the mouse. This led to perinatal lethality of otherwise normal born animals. Treatment studies with daily oral administration of different concentrations of BH4, L-dopa, and 5-hydroxytryptophan for BH4 led to the observation that mice can be rescued but exhibit severe growth deficiency leading to dwarfism due to low serum insulin-like growth factor-1 (IGF-1).
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EXPERIMENTAL PROCEDURES |
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Replacement Therapy
For an overview of concentrations of drugs used for treatments, see
Table I. For the
"low" treatment, tablets from Schircks Laboratories (Jona,
Switzerland) containing 50 mg of BH4, 50 mg of ascorbic acid, and
25 mg of N-acetyl-L-cysteine were dissolved in 10 ml of
H2O. For L-dopa, tablets containing 100 mg of levodopa
and 25 mg of carbidopa (Sinemet MSD, Glattbrugg, Switzerland), and for
5-hydroxytryptophan, tablets containing 100 mg of oxitriptanum (Sigma-tau)
were each dissolved in 20 ml of H2O. For the medium treatment, the
following stock solution was prepared: 65 mg of BH4-2HCl, 50 mg of
ascorbic acid, 10 mg of L-dopa, 6 mg of 5-hydroxytryptophan, 2.5 mg
of carbidopa and 25 mg of N-acetyl-L-cysteine dissolved
in 1 ml of H2O. For the high treatment, the following stock
solution was prepared: 130 mg of BH4-2HCl, 100 mg of ascorbic acid,
10 mg of L-dopa, 6 mg of 5-hydroxytryptophan, 2.5 mg of carbidopa,
and 50 mg of N-acetyl-L-cysteine dissolved in 1 ml of
H2O. To determine the actual concentrations of BH4,
L-dopa and 5-hydroxytryptophan, the dissolved compounds were
analyzed by standard HPLC (see below). Aliquots were kept frozen, thawed
before used, and buffered with sodium citrate to pH 5. The solutions were
diluted with water and 1020 µl were orally administered using yellow
tips and a Gilson pipette. For the medium and high treatment protocols, the
daily aliquots were divided into two daily doses.
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Preparation of Mouse Tissues
After scarifying the animals, brains and livers were withdrawn immediately
for preparing tissue homogenates. Homogenizing buffer containing 50
mM Tris-HCl, pH 7.5, 0.1 M KCl, 1 mM EDTA, 1
mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride,
1 µM leupeptin, and 1 µM pepstatin was added to
whole brain (4 µl/mg tissue), or liver (5 µl/mg tissue). Tissues were
blended at 4 °C with an electric homogenizer (Kinematic GmbH,
Littau-Luzern, Switzerland) and centrifuged at 15,000 x g for
20 min at 4 °C. Supernatants were kept frozen at 80 °C.
Neopterin, Biopterin, and Neurotransmitter Measurements
A volume of 50 µl of liver or 50 µl of brain tissue homogenates were
adjusted to 100 µl and oxidized with 10 µl of oxidizing solution (5
g/liter iodine and 10 g/liter potassium iodide in 1 M HCl). After
oxidation in the dark for 60 min, the reaction was stopped by adding 10 µl
of freshly prepared ascorbic acid (20 g/liter). A total of 14 µl of 1
M NaOH was added to adjust the mixture to pH 8.5, followed by
incubating with 20 µl of an alkaline phosphatase solution at 37 °C for
1 h (300 units/ml calf intestine alkaline phosphatase from Roche Applied
Science in 0.1 M Tris-HCl, pH 8.0, 1 mM
MgCl2, 0.1 mM ZnCl2). The mixture was
adjusted to pH 2 by adding 5 µl of 2 M HCl and deproteinized
through an Ultrafree-MC filter (Millipore). Neopterin and biopterin are
measured from the filtrate by HPLC
(28). The concentrations are
expressed as picomoles per mg of protein. Monoamine neurotransmitters were
determined according to a published method
(46).
Enzymatic Assays
A volume of 100-µl tissue homogenates was desalted on a spin column
(MicroSpinTM G-25 columns, Amersham Biosciences), and 100 µg of protein
from the liver filtrate or 200 µg of protein from the brain filtrate was
used for GTPCH and PTPS assays, respectively.
GTPCH AssayA final volume of filtrate was adjusted to 50 µl and added to 148 µl of homogenizing buffer and 2 µl of 100 mM GTP (Roche Applied Science). This mixture was divided into two 100-µl portions. One portion was immediately oxidized as blank with cell extract, and the second portion was incubated for 60 min at 37 °C. The reaction was stopped by cooling the sample on ice and adding 10 µl of oxidizing solution (5 g/liter iodine and 10 g/liter potassium iodide in 1 M HCl). After oxidation in the dark for 60 min, the reaction was stopped by adding 10 µl of 20 g/liter ascorbic acid (freshly prepared). The mixture was adjusted to pH 8.5 by adding 14 µl of 1 M NaOH, and the sample was incubated with 20 µl of alkaline phosphatase solution at 37 °C for 1 h (300 units/ml calf intestine alkaline phosphatase (Roche Applied Science); see above). The mixture was adjusted to pH 2 by adding 5 µl of 2 M HCl and deproteinized through an Ultrafree-MC filter (Millipore). Neopterin was measured from the filtrate by HPLC. One unit of GTPCH produces 1 µmol of neopterin per minute at 37 °C.
PTPS AssayA final volume of filtrate was adjusted to 50 µl and was added to 60 µl of reaction mixture (100 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM NADPH, 1 mM NADH, 3 milliunits of SR, 140 milliunit of DHPR from Roche Applied Science, and 60 µmol/liter dihydroneopterin triphosphate) in a final volume of 110 µl. This mixture was divided into two portions, one 50-µl aliquot was incubated for 2 h at 37 °C, and another 50 µl was used as a blank. A blank without cell extract was incubated at the same time; it contained 50 µl of reaction buffer and 60 µl of reaction mixture. The reaction was stopped by adding 15 µl of 300 g/liter trichloroacetic acid for protein precipitation, and cooling on ice for at least 10 min, followed by oxidation with 10 µl of oxidizing solution (10 g/liter iodine and 10 g/liter potassium iodide in H2O) for 60 min in the dark. Excess iodine is destroyed by adding 15 µl of ascorbic acid (10 g/liter ascorbic acid in H2O). For the blanks, the same procedure was used. After 2 min of centrifugation at 15,000 x g, the supernatant was deproteinized through an Ultrafree-MC filter (Millipore) and analyzed by HPLC. One unit of PTPS produces 1 µmol of biopterin per minute at 37 °C.
NO AssayThe NO, which is the product of NOS, is extremely
reactive and undergoes a series of reaction. Nitrite
() and nitrate
(
) are the final products. The sum
of these two products (nitrite plus nitrate) was measured using a commercial
Colorimetric assay kit (Cayman Chemical, Ann Arbor, MI). During this assay,
nitrate was converted to nitrite utilizing nitrate reductase and measured by
using the Griess reagent. Absorbance was read at 570/620 nm in a Micro-ELISA
autoreader MR 530 (Dynatech, Chantilly, VA).
Phenylalanine Hydroxylase AssayThis assay was adapted from Ledley et al. (29). Liver homogenate containing 50100 µg of total protein was used for PAH assay. For the blank, the appropriate amount of liver homogenate was adjusted to a final volume of 104 µl with water and incubated for 5 min in a 96 °C heating block. For the sample, the appropriate amount of liver homogenate was adjusted to a final volume of 77.5 µl with water. A volume of 22 µl of master mixture was added to each sample. The master mixture contained 0.6 mM phenylalanine, 3.6 units of catalase (Sigma), 0.15 M KCl in a 0.2 M potassium phosphate buffer, pH 6.8. After preincubation at room temperature, the reaction was started by adding 2 µl of 0.1 M dithiothreitol and 2 µl of 4.5 mM 6-methyltetrahydropterin (Schircks Laboratories) to the samples and incubated for 60 min at 25 °C. The reaction was stopped by incubation for 5 min in a 96 °C heating block and centrifuged for 5 min at 13,000 rpm. The supernatant was filtrated in an Ultrafree-MC filter device and centrifuged again at 5000 x g for 15 min. Phenylalanine and tyrosine were quantified with a standard amino acid analyzer (Biochrom 20 Plus, Amersham Biosciences).
Phenylalanine Concentration in the Blood
The blood from the mice was collected on filter paper cards (Guthrie card).
Phenylalanine (and tyrosine) concentrations were measured using electrospray
ionization tandem mass spectrometry.
Protein Measurement
Protein concentrations in homogenized tissues were determined by the
spectrophotometric method described by Bradford, using -globulin as a
calibrator (30). The
activities of the various enzymes are expressed as units per milligram of
protein.
Immunoassays
Blood was collected from 35- and 44-day-old mice at the time animals were
sacrificed.
Insulin-like Growth Factor-1Serum IGF-1 was separated from clotted blood by centrifugation. It was measured after extraction with acid-ethanol (40 µl of serum and 160 µl of acid-ethanol). The mixture was incubated for 30 min at room temperature and centrifuged, and 100 µl of supernatant was diluted 1:6 before analysis. Serum IGF-1 was determined by radioimmunoassay (RIA) using a rat IGF-1 RIA kit (DSL-2900, Bühlmann Laboratories AG, Switzerland).
Growth HormoneSerum GH was measured by RIA using a specific rabbit anti-rat antiserum and rat GH as standard. The rat GH RIA kit (AH R012) was obtained from Bühlmann Laboratories AG.
Blood ThyroxinT4 was measured by fluoroimmunoassay using the mouse anti-thyroxine IgG as first antibody and the anti-mouse IgG as second antibody. The blood was dried on filter paper cards (Guthrie card). The total amount of T4 was determined in the test. The AutoDELFIATM neonatal thyroxine (T4) kit was obtained from PerkinElmer Life Sciences, Wallac-ADL AG (Switzerland).
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RESULTS |
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Upon PCR and Southern blot analyses
(Fig. 1, B and
C), a few
Pts+/ ES cell clones were
used for blastocyst injection and subsequent generation of PTPS-null mice.
Homozygous mice developed with normal morphology in utero and were
born at the expected Mendelian ratio (25% wild-type, 48% heterozygotes, 27%
Pts-null mice; n = 159). However, most of the
Pts/ mice died within
the first hours after birth; at maximum we found 4 mice out of 26 surviving
for 7 days. A more detailed analysis of brain development and fine structure
of Pts/ mice is now in
preparation.2 Southern
blot analyses with genomic mouse tail DNA and a 5'-external probe
(Fig. 1C), or an
internal neo-probe (Probe B in
Fig. 1A; results not
shown) revealed correct homologous recombination at the single mouse
Pts gene locus. As compiled in
Table II, newborn knockout mice
at day 1 had no PTPS (<0.05 microunit/mg) and normal GTPCH activity
(0.10.3 microunit/mg). Heterozygous animals showed intermediate PTPS
activity (1.5 microunit/mg) compared with normal activity in wild-type mice
(8.0 microunits/mg). Liver neopterin was almost 200-fold higher than normal
(59.0 pmol/mg in
Pts/), and biopterin
was only 4% of wild-type (0.9 pmol/mg). Furthermore, the
Pts/ animals had
hyperphenylalaninemia with blood values of 1352 µmol/liter phenylalanine
(normal control levels were between 34 and 85 µmol/liter), and no
detectable or very low brain dopamine and serotonin levels. Expression of
-galactosidase was observed in heterozygous and homozygous Pts
mutants (not shown). A more detailed study on developmental expression of the
PTPS-LacZ fusion is now in preparation.2
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Treatment of Pts/ Mice Resulted in Rescue from Lethality but Severe DwarfismIn the next step, we sought to rescue the knockout mice by applying a replacement therapy protocol based on the recommended standard concentrations for treatment of human PTPS patients (1). This included daily oral administration of BH4 to control blood phenylalanine and the neurotransmitter precursors L-dopa and 5-hydroxyptryptophan. Unexpectedly, we learned that, with this standard treatment protocol, Pts/ mice survived not longer than for about 3 weeks. We thus extended our treatment studies with three types of application levels (Table I; see also "Experimental Procedures"): standard or low treatment, a medium treatment with 2- to 5-fold higher concentrations of BH4 and neurotransmitter precursors, and a high treatment with roughly 3- to 10-fold higher concentrations. Each treatment group contained 714 control animals, i.e. wild-type or heterozygotes, and 36 Pts/ mice. The body weight of each animal was monitored daily and is depicted for each treatment group in Fig. 2.
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As mentioned before, the Pts/ animals with the low treatment did not survive for more than 3 weeks. A similar situation was encountered with the medium treatment, where survival of the Pts/ animals was prolonged but they eventually died between day 31 and 40 after birth (at a certain point knockout animals had to be sacrificed due to progressively poor conditions and in agreement with the Rules and Guidelines for the Care and Use of Laboratory Animals of the State of Zurich). In contrast, all Pts/ animals with the high treatment survived and were in relatively good health conditions. The experiment was stopped after 6 weeks of treatment, where all animals were sacrificed for biochemical analysis (see below). As shown in Fig. 2(AC), newborn mice regardless of the treatment mode gained weight without significant differences between genotypes for about 2 weeks and underwent pronounced growth retardation during week 3 (days 1521). Only the Pts/ mice with low treatment seemed to be slightly different from their normal littermates, as they had reduced growth rate almost from birth (see low at day 3 in Fig. 3). This developmental difference in the low treatment group was even more pronounced later, as illustrated also in Fig. 2D, which shows a 7-day-old Pts/ animal in comparison with an age-matched heterozygous littermate. After the period of growth stagnation, the Pts/ animals stopped gaining weight independently of the treatment level, whereas all wild-type and heterozygous mice grew normally. The diminutive body size of Pts/ mice was best visible for those that survived due to high treatment, where the body weight was 34% of control at the age of 7 weeks (7.8 ± 1.5 g for Pts/ mice compared with 23.1 ± 2.4 g for combined controls; Fig. 3 medium and high at days 23, 31, and 44). Further qualitative characteristics of these otherwise healthy dwarf mice included reduced activity, hypersalivation, difficulty in swallowing, dystonia, tremor, and no signs of sexual maturation. Additional but less pronounced or steady characteristics were fair hair due to light pigmentation, hair loss, and hypothermia. In summary, treatment of Pts/ mice with BH4 and neurotransmitter precursors resulted in rescue from lethality but severe dwarfism.
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Biochemical Analysis of Sacrificed Mice following Different Treatment ProtocolsFor enzymatic and metabolite analyses in liver, blood, and brain, all groups of treated mice were sacrificed at day 23 for low, day 31 for medium, and day 44 for high treatments. Liver analysis was also carried out with untreated mice at day 1 after birth (see above), whereas the limited brain material from newborns allowed us to measure only nitrite plus nitrate (compare with Tables II and III).
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As expected, PTPS activity in liver and brain was completely abolished in the Pts-knockout mice and reduced to roughly 45% in heterozygous animals compared with wild-type. Furthermore, in liver and brain of phenotypically normal mice, i.e. wild-type and heterozygotes, PTPS activity increased with age. Independent of the treatment level, blood phenylalanine, and liver BH4 and GTPCH activity decreased in 23, 31, and 44-day-old knockout mice compared with controls. Low GTPCH activity in Pts/ mice was surprising, because it was expected that hyperphenylalaninemia and low BH4 levels, as observed in these mice, result in an increase of GTPCH activity via the stimulatory action of GFRP (6) (see also "Discussion").
Brain serotonin was severely lowered only in 23-day-old knockout mice with
the low treatment, but 50% of normal in knockouts with medium or high
treatment. Instead, brain dopamine was not detectable in knockouts with the
low treatment and also very low, between 311% of control, in the medium
and high treatment. The brain metabolites for dopamine and serotonin, HVA and
5-HIAA, were only slightly reduced in
Pts/ mice even under
the high treatment conditions, with 52% of normal for HVA and 60% of normal
for 5-HIAA (day 44 of treatment). Brain dihydroxyphenylacetic acid (DOPAC),
which is the first degradation product of dopamine following the action of
dopamine hydroxylase, was indistinguishable among knockout and wild-type or
heterozygous animals from the medium and high treatment groups (between 30.2
and 67.9 pmol/mg; not shown).
The NOS activity in brain, as determined by measuring the sum of nitrate plus nitrite, revealed no difference among knockout and control animals and was independent of treatment levels. NOS activity in the brain of untreated Pts/ or normal mice at day 1 was severalfold higher compared with older animals but was also indistinguishable between the phenotypes (100.0117.3 nmol/g of tissue at day 1).
The Pts/ knockout animals, independent of medium or high treatment conditions, exhibited extremely low dopamine and sub-optimal levels of BH4 and serotonin. Furthermore, the only metabolic difference we observed among these two treatment conditions, which eventually lead to the death of animals with only the medium treatment, was the intermediate HPA.
IGF-1 Is Severely Lowered in Rescued Pts/ Mice with DwarfismBecause the phenotypic characteristics of the dwarf mice might be a consequence of abnormal feeding behavior due to low dopamine and/or of hormonal deregulation, we wondered whether the pituitary growth hormone (GH), the thyroid hormone thyroxin (T4), and the insulin-like growth factor-1 (IGF-1) in serum of knockout animals were reduced. As shown in Fig. 4, the serum IGF-1 levels in the knockout mice were reduced by a factor of 7 in comparison with age-matched controls (knockouts 79 ± 36 ng/ml; controls 541 ± 155 ng/ml). The expected sexual dimorphism between females (482 ± 170 ng/ml) and males (613 ± 113 ng/ml) is also clearly visible as published before (31). Furthermore, we also tested whether the pituitary-derived growth hormone (GH) and the thyroxin-stimulating hormone (TSH)-dependent thyroxin (T4) were also reduced in these animals. However, we found no change in GH and T4 (not shown), indicating that the pituitary gland is normally developed and thus not the primary reason for dwarfism in these treated Pts/ mice. Low IGF-1 might thus be the biochemical reason for the dwarfism, probably caused by the limiting brain dopamine, because abnormal feeding behavior was reported in dopamine-deficient mice (see "Discussion") (32).
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DISCUSSION |
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We found only a few knockout mice surviving for up to 7 days after birth,
probably due to the BH4 present in mother's milk
(35), while the amount of milk
available is in turn dependent on the litter size and/or the mother's
behavior. Data from 250 PTPS patients, as compiled in the data base
www.bh4.org,
does not reveal perinatal lethality, and, although symptoms may be noted
during the neonatal period, abnormalities develop typically during the first
weeks of life (1). Furthermore,
low birth weight and microcephaly, which are typical for PTPS deficiency in
human newborns (36), were not
observed in our mice. On the other hand, symptoms like hypersalivation and
temperature instability are found in PTPS mice and human patients. Analysis of
metabolites showed HPA and neopterin accumulation due to complete absence of
PTPS activity, as expected (day 1, Table
II). Brain metabolites at this age were only determined for NOS
due to the limited material. However, monoamine neurotransmitters must be low
as inferred from measurements of 23-day-old knockouts under low treatment that
died despite initial treatment and had very low or not detectable brain
serotonin and dopamine levels. Although BH4 has a central role for
the function of the three NOS isoenzymes, i.e. vasorelaxation, immune
response, and neurotransmission, no direct association with such pathologies
in BH4-deficient patients has been made. Only recently, however, we
found that patients revealed reduced NO metabolites in cerebrospinal fluids
independent of treatment. Implications from this study are that, under
BH4-deficient conditions NOS is uncoupled and produces by-products
that are neurotoxic and thus responsible for neuronal cell pathology through
peroxynitrite generation (2,
37,
38). Regarding the NOS
activity in our mutant mouse presented here, we found no alterations in brain
nitrate and nitrite levels, independently of treatment level or age
(Table III). Also the mouse
brain NOS activity was not reduced in untreated newborn knockouts, a
phenomenon that may be explained by the fact that mother's milk contains high
concentrations of BH4 and that all NOS have a low
KD for BH4 binding compared with the
aromatic amino acid hydroxylases (KD of
100600 µM compared with KD
of 0.2 µM for nNOS)
(39). Despite lethality during
the first days of life, which is not typical for BH4 deficiency in
humans, we think that this Pts knockout is a suitable animal model
for studying the pathophysiology and treatment of BH4
deficiency.
During the initial treatment study where the recommended concentration of compounds and precursors for the treatment of human patients was administered orally (low treatment), we learned that this therapy did not rescue the mice. Accordingly, we found no normalization of the metabolites that are followed today to control treatment in human patients, i.e. plasma phenylalanine, brain biopterin, HVA, and 5-HIAA (Tables II and III). Furthermore, brain dopamine and serotonin were extremely low in the knockout mice with low treatment. These neurotransmitters are below detection levels in human CSF and thus can not be determined in patients. A simple explanation for the requirement of higher doses of precursors and compounds for treatment may be the fact that mice have a much higher metabolic rate than humans. For instance, the enzymatic efficiency of PTPS is roughly ten times higher for the mouse compared with the human enzyme (kcat/Km of recombinant PTPS from mouse is 2.5 x 104 and from human 2.8 x 103) (25). This hypothesis was corroborated by the fact that we could eventually rescue the animals by increasing the treatment doses.
The biochemical parameters under high treatment conditions revealed that
plasma phenylalanine was normalized, and biopterin was in the same range as in
controls (Tables II and
III). Serotonin and the
neurotransmitter metabolites HVA and 5-HIAA were in the subnormal range
(50% of normal), whereas brain dopamine was unexpectedly low at 3% of
normal (see below). Furthermore, plasma and brain neopterin remained elevated,
and GTPCH was below normal activity, although hyperphenylalaninemia is
expected to result in an increase of GTPCH activity. Moreover, under
conditions of low BH4 levels, as in
Pts/ mice with low
treatment, stimulation of GTPCH by the GTPCH·GFRP complex was expected
to be even more pronounced, because the inhibitory action of BH4
should also be diminished (4,
6). From the data presented
here, we conclude that PTPS may have a direct or indirect effect on GTPCH
expression or GTPCH·GFRP activity. Regarding hyperphenylalaninemia,
increasing levels of oral BH4 lead to a gradually decrease of blood
phenylalanine. Whereas the medium treatment exhibited an intermediate
phenylalanine level, the high treatment conditions were required to completely
normalize blood phenylalanine levels. A further observation that cannot be
explained sufficiently at this point is that knockout animals under medium
treatment consistently did not survive for more than 4 weeks, whereas under
high treatment none of the mutants died. The only metabolic difference we
observed between these two treatment procedures was the slightly elevated
plasma phenylalanine levels in the animals treated with the medium dose (see
Tables II and
III). It is unlikely that a
mild HPA has such a dramatic effect on growth and development, and additional
treatment studies must be conducted to learn more about these differences.
The most remarkable observation made while treating the Pts/ mice was the consistently reduced growth starting almost from the first days of life, leading to dwarfism. Normal growth and development are largely programmed during the first weeks of postnatal life by the pituitary growth hormone (GH) and thyroxin-stimulating hormone (TSH) (40). Furthermore, somatic growth is mediated mainly by circulating IGF-I, an insulin-like hormone produced mainly in the liver but also in many other tissues. At least two examples of dwarf mice are well described: the so-called Ames and Snell dwarf mice with recessive mutations in the homeotic genes Pit-1 or Prop-1, respectively, with developmental arrest in pituitary ontogeny (40). Phenotypic characteristics are decreased growth rate post-weaning and reduced body size of adults, having approximately one-third of the weight of their normal siblings, similar to what we found in our Pts knockout mice. Further characteristics reminiscent of our dwarf mice include delayed puberty, reduction of body temperature, tendency to experience hair loss, and reduction in plasma IGF-1. This prompted us to determine these hormones in our treated mice. In contrast to the Ames and Snell dwarf mice, we found normal levels for the pituitary GH- and TSH-dependent T4, indicating that the pituitary gland developed normally and can thus be excluded as the primary reason for dwarfism of treated Pts/ mice. The IGF-1 level is influenced by GH but also by the nutritional status and food intake, which in turn is regulated by the dopamine and norepinephrine levels (4143). For instance, it was reported that a knockout mouse that does not express tyrosine hydroxylase was unable to initiate feeding, an ability that can be restored by gene delivery of tyrosine hydroxylase into the striatum (32, 33, 44, 45). As also mentioned before, we found that the brain serotonin, and the neurotransmitter metabolites HVA, DOPAC, and 5-HIAA, although slightly below normal, were not much different from control levels. This is in sharp contrast with the actual dopamine levels of 3% of normal in the brain of mice under high treatment. Furthermore, the DOPAC/dopamine ratio (and the HVA/dopamine ratio), which is an index of dopamine turnover rate, was 1.8 in the wild-type and heterozygous mice group under medium and high treatment, but was 48 in both knockout mice groups, presumably reflecting the high turnover of the small dopamine pool (not shown). The extremely low brain dopamine together with the wealth of literature on the feeding behavior in dopamine-deficient mice mentioned above supports the assumption that abnormal (hypothalamic) neurotransmission is associated in our mice with disturbance of eating behavior. Although we did not measure daily food or water intake, we consistently observed difficulties in swallowing in our knockout mice groups and conclude that control of appetite is compromised in the treated Pts/ mice, and thus chronic undernutrition is responsible for low IGF-1 and dwarfism. Such feeding difficulties have been described for BH4-deficient patients, but so far there was no indication of growth retardation or dwarfism. Nevertheless, we tested for IGF-1 in a first study with a very small group of BH4-deficient patients and found specifically reduced plasma IGF-1 levels in newborns with PTPS deficiency. Although these results are only preliminary, we believe it will be important to collect more data on IGF-1 levels and to follow growth and development in human patients with BH4 deficiency.
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FOOTNOTES |
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Present address: European Patent Office, 80339 München, Germany.
|| To whom correspondence should be addressed. Tel.: 41-1-266-7622; Fax: 41-1-266-7169; E-mail: beat.thony{at}kispi.unizh.ch.
1 The abbreviations used are: BH4, tetrahydrobiopterin; NOS,
nitricoxide synthase; GTPCH, GTP cyclohydrolase I; GFRP, GTPCH feedback
regulatory protein; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin
reductase; PAH, phenylalanine hydroxylase; DHPR, dihydropteridine reductase;
HPA, hyperphenylalaninemia; HVA, homovanillic acid; 5-HIAA,
5-hydroxyindoleacetic acid; CSF, cerebrospinal fluid; IGF-1, insulin-like
growth factor-1; ES, embryonic stem; HPLC, high-performance liquid
chromatography; RIA, radioimmuno-assay; GH, growth hormone; T4,
thyroxine; DOPAC, dihydroxyphenylacetic acid; TSH, thyroxin-stimulating
hormone; dopa, L-dihydroxyphenylalanine.
2 L. Elzaouk, W. Leimbacher, M. Turri, B. Ledermann, K. Bürki, N. Blau,
and B. Thöny, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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