Developmental disturbance in model mice with abnormal pterin metabolism

四氢生物蝶呤（BH 4）是苯丙氨酸羟化酶、酪氨酸羟化酶（TH）、色氨酸羟化酶（TPH）和所有类型的一氧化氮合酶的必需辅因子。先前，我们通过破坏编码6-乙酰四氢蝶呤合酶（PTS）的基因建立了BH 4-缺陷小鼠（Pts^-/->），所述PTS催化BH 4生物合成的第二步。对于出生后不久死亡的Pts^<-/->小鼠，我们通过转基因导入在5.8kb人多巴胺β-羟化酶5'启动子（DPS）控制下的人PTS cDNA来拯救那些小鼠。我们使用2-7个月大的雄性DPS拯救的小鼠（Pts <-/->，DPS）进行生物化学分析。在DPS拯救的小鼠的肝脏中的PTS活性是野生型（Pts <+/+>）小鼠的2.1%。血浆苯丙氨酸浓度的DPS拯救的小鼠比野生型高37倍，并在腹腔注射20 g/kg BH 4后1小时降至15%。通过使用抗人PTS抗体的免疫荧光染色，以及使用抗人PTS抗体和抗牛TH抗体的双重免疫荧光染色，证实了人PTS在肾上腺髓质和去甲肾上腺素能细胞（包括蓝斑）中有效表达。而人PTS仅在黑质和腹侧被盖区的部分多巴胺能细胞表达。结果表明，经DPS治疗的小鼠中脑生物蝶呤和多巴胺含量分别为野生型的84%和52%。另一方面，DPS拯救的小鼠纹状体中的生物蝶呤和多巴胺的含量是野生型的21%。结果表明，DPS组小鼠的纹状体内侧部和丘脑外侧核对TH抗体的免疫反应与野生型小鼠一样强。相反，抗TH抗体对DPS拯救小鼠纹状体外侧区的染色非常弱。因此，我们认为，TH蛋白的多巴胺能神经末梢投射到纹状体的外侧区是最敏感的BH-4缺乏。

Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and all types of nitric oxide synthase. Previously, we established BH4-defficient mice (Pts^<-/->) by disruption of the gene encoding 6-pyruvoyltetrahydropterin synthase (PTS), which catalyzes the second step of BH4 biosynthesis. For Pts^<-/-> mice died shortly after birth, we rescued those by transgenic introduction of human PTS cDNA under the control of 5.8 kb human dopamine β-hydroxyklase 5' promoter (DPS). We performed biochemical analysis using 2-7 months old male DPS-rescued mice (Pts^<-/->,DPS). PTS activity in the liver of DPS-rescued mice was 2.1 % of that of wild type (Pts^<+/+>) ones. Plasma phenylalanine concentration of DPS-rescued mice was 37 times higher than that of wild type, and reduced to 15% 1 hour after intraperitoneal injection of 20g/kg BH4. It was confirmed that human PTS efficiently expressed in the adrenal medulla and noradrenergic cells including locus ceruleus by immunohistochemistory using anti-human PTS antibody, and also by double-immunofluorescence staining using anti-human PTS antibody with anti-bovine TH antibody. However, human PTS expressed in a part of the dopaminergic cells in the substantia nigra and ventral tegmental area. Content of biopterin and dopamine in the midbrain of DPS-rescued mice were 84% and 52% of those of wild type, respectively. On the other hand, content of biopterin and dopamine in the striatum of DPS-rescued mice were 21% of those of wild type. The nucleus accumbens and medial part of the striatum of DPS-rescued mice were immunoreactive to anti-TH antibody as strongly as those of wild type. On the contrary, lateral region of the striatum of DPS-rescued mice was far weakly stained by anti-TH antibody. Thus we concluded that TH protein in dopaminergic nerve terminals projecting to the lateral region of striatum is the most sensitive to BH4-insyfficiency.