Brain Research 954 (2002) 237–246www.elsevier.com/ locate/bres

Research report

L ocalization of sepiapterin reductase in the human braina,b,c , d d d*Keiko Ikemoto , Takahiro Suzuki , Hiroshi Ichinose , Tamae Ohye ,

b b a dAkiyoshi Nishimura , Katsuji Nishi , Ikuko Nagatsu , Toshiharu NagatsuaDepartment of Anatomy, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan

bDepartment of Legal Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, JapancClinical Research Institute, National Minami Hanamaki Hospital, Hanamaki, Iwate 025-0033, Japan

dInstitute for Comprehensive Medical Science, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan

Accepted 17 June 2002

Abstract

Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor forphenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR isessential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purifiedrecombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein andSPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebralcortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPRantibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPRprotein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I(GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistryrevealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of thehypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPRwere colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOSneuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4biosynthesis. 2002 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Other neurotransmitters

Keywords: Sepiapterin reductase; Human; Immunohistochemistry; Tyrosine hydroxylase; Tetrahydrobiopterin; Monoamine

1 . Introduction [15,50]. The genomic organization and chromosomallocalization of SPR in rats [47], mice [29], and humans

Sepiapterin reductase (SPR), a polypeptide of 30 kDa [45,56] have already been determined. It has been estab-generally existing in a homodimeric form [53], is the lished that the first-step and rate-limiting BH4-synthesizingterminal-step enzyme for the synthesis of tetrahydrobio- enzyme is guanosine triphosphate (GTP) cyclohydrolase Ipterin (BH4) [27,28,34,52], which is the reduced pteridine (GCH), and the second-step one, pyruvoyltetrahydropterincofactor for pteridine-requiring enzymes such as phenylal- synthase (PTPS) [1,16,36].anine hydroxylase, tyrosine hydroxylase (TH), tryptophan Catecholamines (dopamine, noradrenaline, and ad-hydroxylase (TPH) [37], and nitric oxide synthase (NOS) renaline) are synthesized from tyrosine starting with TH:

tyrosine→dopa→dopamine→noradrenaline→adrenaline:and indoleamines (serotonin and melatonin) from*Corresponding author. Tel.:181-198-24-0511; fax:181-198-24-tryptophan, starting with TPH: tryptophan→5-hydroxytry-1721.

E-mail address: ikemoto@shanamaki.hosp.go.jp(K. Ikemoto). ptophan→ serotonin→ N-acetylserotonin→ melatonin.

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03341-3

238 K. Ikemoto et al. / Brain Research 954 (2002) 237–246

Catecholamines and serotonin are known as monoamines. (pH 8.0), 500 mM NaCl, and 100 mM EDTA). The eluateNitric oxide (NO) is synthesized by NOS from arginine: was dialyzed against PBS and stored at280 8C until used.arginine→NO1citrulline.

BH4 also plays important roles in immune or cytokine 2 .2. Production of an anti-hSPR antibodysystems [26,39,51,58,59,62] and in cell proliferation [55].In humans, the lack of BH4 due to mutation of GCH gene A rabbit polyclonal antibody against the purified recom-is a cause of dopa-responsive dystonia (Segawa’s disease) binant SPR was raised. For immunization, a solution[20,24]. containing the purified recombinant protein (1 mg/ml) was

Although previous studies examining human and rodent emulsified with Freud’s complete adjuvant having twobrain tissues indicated that GCH immunoreactivity or GCH times the volume of the antigen solution. A rabbit receivedmRNA expression was localized in monoamine neurons, a dose of 0.5 mg of the protein intradermally at multipleneither could be detected in pteridine-requiring NOS sites on its back. Doses of 0.5 mg of the protein inneuronal fields [33,40–42]. The significance of such a Freund’s incomplete adjuvant were then given as boosterdiscrepancy is unclear, and the question arises as to injections at intervals of 2 weeks by subcutaneous in-whether a BH4 biosynthetic pathway where GCH is not jections. More than 10 booster injections were necessary toinvolved might exist. obtain a satisfactory antibody titer.

The complementary DNAs (cDNAs) of SPR have beencloned from rat [8], mouse [31,46], and human [19] 2 .3. Immunoblot analysissources: and the primary structures of their proteins wereelucidated. Recently we produced for the first time a highly Western blot analysis of human and mouse liver lysatesspecific antibody against human SPR (hSPR) protein. and of human brain homogenates from the substantia nigraUsing it, we conducted the present study to determine the (SN), caudate nucleus (CN), gray and white matters of thelocalization of SPR enzyme activity and SPR immuno- cerebral cortex (CTX) and dorsal and ventral parts of thereactivity in the human brain by Western blotting and medulla oblongata (MO) was performed. With the consentimmunohistochemistry. The coexistence of SPR and TH of the bereaved family, the human brain tissue waswas also examined by a double-staining immunohisto- obtained from a pathological autopsy case (78-year-oldchemical method. female, with tissues taken 5 h postmortem) with no clinical

or pathological signs of neurological or psychiatric dis-eases at Nagoya University Hospital. The tissues were

2 . Materials and methods homogenized in 20 mM Tris–HCl buffer (pH 7.5) con-taining 100 mM KCl, 1 mM EDTA, 1 mM DTT, 10

2 .1. Purification of recombinant hSPR for use as an mg/ml aprotinin, 10mg/ml leupeptin, and 1 mM PMSF.immunogen The homogenate was then centrifuged at 15003g for 10

min, and the supernatant was stored at280 8C until used.Human SPR cDNA was introduced into theNdeI and The extract or the purified protein was subjected to SDS–

BamHI sites of a pET16b vector (Novagen), which expres- 12% polyacrylamide gel electrophoresis (SDS–PAGE),ses a foreign gene as the fusion protein with a stretch of 10 and the proteins transferred to a PVDF membrane (Bio-histidine residues.Escherichia coli (E. coli) strain BL21 Rad) at a constant voltage of 15 V for 1 h. The membrane,(DE3, pLysS) carrying the vector were cultured at 378C to after having been blocked with 3% skim milk, wasa late-log phase, and the expression of the fusion protein incubated with the antibodies (1:1000 dilution), and thenwas induced by the addition of isopropylb-D-thiogalac- incubated with anti-rabbit IgG conjugated to horseradishtopyranoside (IPTG). Protein concentrations were deter- peroxidase. Following repeated washing of the membrane,mined by the method of Bradford [7], with bovineg- the signals were visualized with ECL Plus (Amersham–globulin used as a standard. Bacterial cells containing the Pharmacia Biotech).fusion proteins were sonicated in ice-cold phosphate-buf-fered saline (PBS), and the lysate was centrifuged at 2 .4. Measurement of SPR and GCH activities100 0003g for 30 min. The supernatant was filteredthrough a 0.22-mm filter (Millipore) and applied to a The enzyme activities were measured by using 10mg of

21Ni -chelating column (Amersham–Pharmacia Biotech) the brain homogenate, which was prepared as describedequilibrated with binding buffer (20 mM Tris–HCl (pH above. SPR activity was assayed as described previously8.0), 500 mM NaCl, and 5 mM imidazol) at 48C. The with a slight modification [11]. The incubation mixturecolumn was washed with 10 ml of binding buffer, then (total volume of 50ml) contained 100 mM potassiumwith 6 ml of washing buffer (20 mM Tris–HCl (pH 8.0), phosphate buffer (pH 6.4), 0.1 mM NADPH, and 0.15 mM500 mM NaCl, and 50 mM imidazol) and then again with sepiapterin as substrate. The reaction was carried out at10 ml of binding buffer. Subsequently elution was 378C for 1 h and then terminated by the addition of 25mlachieved with 20 ml of stripping buffer (20 mM Tris–HCl of iodine solution (1% iodine and 2% KI) and kept at room

K. Ikemoto et al. / Brain Research 954 (2002) 237–246 239

temperature in the dark for oxidation of the product, al. [35], and Kitahama et al. [30] were used to determine7,8-dihydrobiopterin, to biopterin. After centrifugation to the anatomical territories.remove precipitated proteins, the excess of iodine wasreduced by adding 25ml of 2% ascorbic acid. Biopterin 2 .5.2.2. Double staining. For SPR/TH double-stainingwas separated by high-performance liquid chromatography immunohistochemistry, sections were incubated in a mix-(HPLC) on a reverse-phase HPLC column and detected ture of mouse anti-TH antibody (LNCI; Incstar, Stillwater,fluorometrically. GCH activity was assayed as described MN; diluted 1:1000) and the rabbit anti-hSPR antibodypreviously with a slight modification [54]. The incubation (diluted 1:10 000) for 1 week at 48C. They were thenmixture (total volume of 100ml) contained 100 mM rinsed in PBST (10 min33), and were incubated with aTris–HCl (pH 8.0), 300 mM KCl, 2.5 mM EDTA, 10% mixture of secondary antibodies, i.e., Cy3-conjugatedglycerol, and 1 mM GTP as substrate. The reaction was donkey anti-mouse IgG (AP192C; Chemicon, Temecula,carried out at 378C for 1 h. The product, 7,8-dihydroneop- CA; 1:200) for TH and fluorescein-conjugated donkeyterin triphosphate, was oxidized with iodine and dephos- anti-rabbit IgG (AP182F; Chemicon, 1:200) for hSPR.phorylated with alkaline phosphatase to neopterin. Neo- Sections were rinsed with PBST (10 min33) and sub-pterin was also separated and detected by the same method sequently mounted on glass slides and coverslipped withas used for biopterin. medium containing 70% glycerol and 0.1%p-phenyl-

enediamine in PBS (Vector, Burlingame, CA). Finally thespecimens were examined under a confocal laser-scanning2 .5. Immunohistochemistrymicroscope (LSM410; Zeiss, Oberkochen, Germany) withappropriate excitation laser beams and emission filters (for

2 .5.1. Tissue processingCy3, excitation at 543 nm, emission at4570 nm; for

Human brains were obtained from five autopsy casesfluorescein, excitation at 488 nm, emission at 510–525 nm)

(27–72 years old, both sexes, postmortem interval: 4–5 h)[22].

at the Department of Legal Medicine, Shiga University ofMedical Science, Japan, in compliance with the ethicalcode of the Ethical Committee of the Japanese Society of

3 . ResultsLegal Medicine. Each case had died from natural causes,and no signs of neurological or psychiatric diseases were

3 .1. SPR is widely distributed in the human braindetected clinically or pathologically. The brains wereimmediately sliced into 1-cm slabs and immersed in fresh

The Western blot analysis of human and mouse liverfixative at 48C for 48–72 h. The slices were then

lysates showed that the anti-hSPR antibody recognized atransferred to phosphate buffer containing 15% sucrose

single major band of approximately 30 kDa and did notand 0.1% sodium azide for storage at 48C. The brain

cross-react with other proteins (lanes 1 and 2 of Fig. 1).sections were cut in coronal planes to a thickness of 50mm

The hSPR migrated more slowly than the mouse one,and thereafter treated with 40% methanol and 1% H O2 2 probably due to differences in the amino acid compositionfor 20 min to inhibit endogenous peroxidase [21].

of SPR proteins between the two species. We also elec-

2 .5.2. Immunocytochemical methods

2 .5.2.1. Single staining. The sections were incubated withantibodies against hSPR (diluted 1:1000–20 000) or TH(diluted 1:10 000) [38] in PBS containing 0.3% TritonX-100 (PBST) at 48C for 1 week. They were then rinsedin PBST (10 min33), incubated in biotinylated rabbit IgG(Vector Laboratory, 1:1000) for 12 h at room temperature,washed again in PBST (10 min33), and incubated withavidin–biotin–peroxidase complex (Vector Laboratory,1:1000) for 1 h at room temperature. After final washes inPBST (10 min33), the peroxidase activity was visualizedwith 50 mM Tris–HCl buffer (pH 7.6) containing 0.0003%H O , 0.01% 3,3 diaminobenzidine–4HCl (DAB), and 1%2 2

nickel ammonium sulfate. The sections were then rinsed inFig. 1. Western blot analysis of human and mouse liver extract and ofPBS, mounted on gelatin-coated glass slides, dehydrated,purified recombinant hSPR. Western blotting was performed with anti-

cleared in xylene, and coverslipped. Some sections werehSPR antibody. One hundred micrograms of lysate protein of humancounterstained with neutral red. Atlases of Pearson et al. (lane 1) and of mouse (lane 2) liver and 1 ng (lane 3) and 10 ng (lane 4)

of His-hSPR were used.[49], Hokfelt et al. [17], Paxinos and Huang [48], Mai et

240 K. Ikemoto et al. / Brain Research 954 (2002) 237–246

Table 1trophoresed 1 and 10 ng of the purified His-hSPR andActivities of SPR and GCH in the human brain regionssubjected these minute amounts to Western blot analysis.

Sepiapterin reductase GTP cyclohydrolase IWe could detect even 1 ng of the protein with the anti-activity (nmol /h per activity (pmol /h perhSPR antibody (lanes 3 and 4 of Fig. 1).mg protein) mg protein)Next we examined the distribution of SPR in the human

Substantia nigra 44.562.8 3.8260.25brain and compared it with that of TH. Human brainCaudate nucleus 22.562.3 1.8260.26homogenates from the SN, CN, gray and white matters ofCerebral cortex:the cerebral CTX, and dorsal and ventral parts of the MOGray matter 23.760.2 N. D.were subjected to the Western blot analysis with anti-hSPRWhite matter 26.861.6 N. D.antibody (Fig. 2A) or with anti-TH antibody (Fig. 2B).Medulla oblongata:Whereas TH protein showed a restricted localizationDorsal part 26.560.6 4.1560.64mainly to the SN and CN, SPR protein was detected in allVentral part 42.061.1 2.5460.28

brain regions examined (Fig. 2). Further, we measuredResults represent the mean (S.D. from three independent experiments.SPR activity in the brain homogenates (Table 1). The SPRN.D., not detectable (,0.1 pmol /h per mg protein).activity was relatively high compared with the activity of

GCH, the rate-limiting enzyme in the biosynthesis of BH4. (Fig. 3D). In the globus pallidus, SPR-immunopositiveThe difference between the SPR and GCH activities in neurons were sparse, with the staining intensity rangingeach brain region was less than 2-fold, while the relatively from low to moderate. The neurons of the paraterminalhigher SPR activity was found in the SN and ventral part gyrus and anterior olfactory nucleus also showed SPRof the MO. The SPR activity was more widely distributed immunoreactivity.than the GCH activity (Table 1).

3 .2.3. Hypothalamus3 .2. Single-staining immunohistochemistry SPR-ir neurons were found in the dopaminergic fields of

the posterior (A11) and caudal dorsal hypothalamic area3 .2.1. Cerebral cortex (A13), as well as in the arcuate (A12) and periventricular

The pyramidal neurons in layers III and V of the (A14) zones [17,30] (not shown).cerebral CTX showed low to moderate SPR immuno-reactivity (Fig. 3A,B), as did the granular cells. 3 .2.4. Midbrain

Moderately to intensely stained SPR-ir neuronal cell3 .2.2. Basal ganglia bodies were found in the retrorubral region, SN (Fig. 4A),

The CN, putamen, and nucleus accumbens contained and ventral tegmental area, corresponding to the A8–10SPR-immunoreactive (-ir) cells and fibers (Fig. 3C). At dopaminergic fields [49]. A number of positive neuronshigh magnification, SPR-ir cell bodies and fibers were were also found in the dorsal raphe nucleus (DR: Fig.found around the fiber bundle of the internal capsule (ic) 3E,F), corresponding to the B7 serotonergic field [3].

Fig. 2. Western blot analysis of human brain extract. Western blotting was performed by using anti-hSPR antibody (A) or anti-TH antibody (B). Twentymicrograms of lysate protein of the substantia nigra (SN) (lane 1), caudate nucleus (CN) (lane 2), cerebral gray matter (lane 3), cerebral white matter (lane4), and dorsal (lane 5) and ventral (lane 6) parts of the medulla oblongata (MO) were used. The positions of SPR and TH are indicated by the arrows.

K. Ikemoto et al. / Brain Research 954 (2002) 237–246 241

Fig. 3. Immunostained sections of SPR through the cerebral CTX (A,B), striatum (C,D), dorsal raphe nucleus (DR: E,F) and cerebral peduncle (cp: G).SPR-ir neurons indicated by an arrow in ‘E’ are magnified in ‘F’. ic, internal capsule; Aq, cerebral aqueduct. Bar: 100mm.

242 K. Ikemoto et al. / Brain Research 954 (2002) 237–246

Fig. 4. (A) Immunostained sections of SPR through the SN. (B) Absorption test in the SN. No staining is shown in sections incubated with anti-humanSPR serum preabsorbed with the human SPR antigen. Only neuromelanin pigments are seen. Bar: 100mm.

Fig. 5. SPR-immunostained sections through (A) the A1 of the ventrolateral medulla, (B) nucleus ambiguus (Amb), (C) olivary nucleus, and (D) rapheobscurus (ROb). IO, inferior olivary nucleus. Bar: 100mm.

K. Ikemoto et al. / Brain Research 954 (2002) 237–246 243

There were many SPR-ir neurons packed within the fiberbundle of the cerebral peduncle (cp: Fig. 3G). No stainingcould be obtained in sections incubated in anti-hSPRantiserum preabsorbed with the protein antigen (Fig. 4B).

3 .2.5. PonsIn the locus coeruleus (LC) neurons, corresponding to

the A6 noradrenergic field [49], intense SPR immuno-reactivity was found (not shown).

3 .2.6. Medulla oblongata (MO)SPR-ir neurons were seen in the A1 or C1 of the

ventrolateral medulla (Fig. 5A), and in the A2 or C2 of thesolitary tract nucleus [49]. The neuronal cell bodies in thenucleus ambiguus (Amb: Fig. 5B), raphe obscurus (ROb:Fig. 5D), and the olivary nucleus (Fig. 5C) showed intenseSPR immunoreactivity.

3 .3. Double immunostaining

Double staining for TH and SPR in the neurons of theSN showed many neurons doubly labeled by anti-TH andanti-SPR (Fig. 6A–C).

4 . Discussion

The present study showed that SPR protein and SPRactivity were widely distributed in the human brain. Asexpected from its role as the enzyme in the final step ofBH4 synthesis. SPR could be detected in the neuronalfields where pteridine-requiring enzymes such as TPH [2],TH [49,57] and NOS [10,12,13,43,44,61] are known to belocated. The present study also showed that the distributionof GCH activity was rather restricted mainly to themonoaminergic neurons. This finding coincides with ourprevious observations using human materials [42].

4 .1. Localization of SPR in relation to monoamineneurons

The present study demonstrated SPR protein and SPRactivity by biochemical methods in the human brain areaswhere monoaminergic neurons are distributed. The SPRimmunohistochemical results confirmed SPR immuno-reactivity in the hypothalamic and midbrain dopaminergicfields, locus coeruleus noradrenergic region, and medullaryC1–2 adrenergic and A1–2 noradrenergic regions[17,30,49]. In these areas of the human brain, immuno-reactivity of GCH, the first-step and rate-limiting BH4-synthesizing enzyme was earlier described [42]. Some ofthe serotonin neurons in the dorsal raphe nucleus werepreviously shown to contain NOS [25,32,60]. In ourobservation, the number of SPR-ir neurons was greaterFig. 6. Double-immunostaining: (A,B) SPR (red)- and TH (green)than that of GCH-ir neurons [42], suggesting the existenceimmunoreactivities in the SN. (C) SPR (1) /TH (1) neurons are shown

in yellow. Bar: 25mm. of SPR-positive, GCH-negative neurons.

244 K. Ikemoto et al. / Brain Research 954 (2002) 237–246

review of human and animal studies and a report onMacaca4 .2. Relationship between the distribution of NOS andfascicularis, Adv. Neurol. 43 (1986) 407–468.SPR

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diaphorase/nitric oxide synthase containing neurons in normal andcerebral CTX, as well as in the striatum, nucleus am-Parkinson’s disease putamen, J. Neural Transm. Parkinson’s Dis.biguus, nucleus tractus solitarii, raphe nucleus, and inferiorDement. Sect. 7 (1994) 115–121.olivary nucleus [10,12,13,43,44,61]. The present study

[6] L. Bonafe, B. Thony, J.M. Penzien, B. Czarnecki, N. Blau,showed SPR-ir neurons in these areas, though the previous Mutations in the sepiapterin reductase gene cause a novel tetrahy-studies did not describe GCH immunoreactivity or GCH drobiopterin-dependent monoamine neurotransmitter deficiency

without hyperphenylalaninemia, Am. J. Hum. Genet. 69 (2001)mRNA expression in them [33,40,41]. This indicates that269–277.there might be an unknown biosynthetic pathway of BH4

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