永津 俊治

Catecholamine neurotransmitters-dopamine, noradrenaline (norepinephrine), adrenaline (epinephrine) -are synthesized in catecholaminergic neurons from tyrosine, via dopa, dopamine and noradrenaline, to adrenaline. Four enzymes are involved in the biosynthesis of adrenaline: (1) tyrosine 3-mono-oxygenase (tyrosine hydroxylase, TH); (2) aromatic L-amino acid decarboxylase (AADC, or DOPA decarboxylase, DDC); (3) dopamine beta-mono-oxygenase (dopamine beta-hydroxylase, DBH); and (4) noradrenaline N-methyltransferase (phenylethanolamine N-methyltransferase, PNMT). We cloned full-length complementary DNAs (cDNAs) and genomic DNAs of human catecholamine-synthesizing enzymes (TH, AADC, DBH, PNMT) and determined the nucleotide sequences and the deduced amino acid sequences. We discovered multiple messenger RNAs (mRNAs) of human TH, human DBH, and human PNMT. Four types (types 1, 2, 3, and 4) of human TH mRNAs are produced by alternative mRNA splicing mechanism from a single gene. We found the multiple forms of TH in two species of monkeys, but only a single mRNA corresponding to human TH type 1 in Sunkus murinus and rat, suggesting that the multiplicity of TH mRNA is primate-specific. Total TH mRNA, especially the most abundant type 2 and type 1 mRNAs in the human brain, were found to be reduced during the process of aging. The multiple forms of human TH may give additional regulation to the human enzyme, probably through altered phosphorylation and activation. We have succeeded in producing transgenic mice carrying multiple copies of the human TH gene in brain and adrenal medulla. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, Western blot analysis, and TH activity measurements proved definitely increased TH in transgenic mice, though not comparable to the increment of the mRNA. However, catecholamine levels in transgenics were not significantly different from those in non-transgenics. The results suggest complex regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice. Kohsaka and Uchida in collaboration with us applied genetically engineered (human TH cDNA-transfected) non-neuronal cells to brain tissue transplantation in parkinsonian rat models. We isolated and sequenced a full-length cDNA encoding human AADC. We expressed a recombinant human AADC in COS cells and proved that the expressed enzyme decarboxylated both L-DOPA and L-5-hydroxytryptophan. We isolated two different cDNAs (types A and B) for human DBH and the genomic DNA, and showed that the two mRNAs are generated through alternative polyadenylation from a single gene. Type A mRNA (2.7 kilobase pairs, kb) and type B mRNA (2.4 kb) encoded the same amino acid sequence and were different only in the 3'-untranslated region. Type A mRNA contained a 3'-extension of 300 base pairs (bp) at the end of the type B mRNA sequence. We assigned the human PNMT gene to chromosome 17. We observed the presence of a minor human PNMT mRNA (type B, 1.7 kb) besides the major mRNA (type A, 1.0 kb). Type B mRNA of human PNMT carries an approximately 700-bp-long untranslated region in the 5'-terminus, suggesting that the two types of human PNMT mRNA are produced from a single gene through the use of two alternative promoters. The 5'-flanking regions of the genes of human TH, DBH and PNMT contain possible transcription regulatory elements such as cyclic AMP response element (CRE) (TH, DBH, and PNMT), glucocorticoid response element (GRE) (DBH and PNMT), and Sp1 binding site (TH and PNMT).

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH)gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

We raised antibodies against 3 oligopeptide segments of human dopamine-beta-hydroxylase (hDBH) corresponding to the N-terminal (hDBH-N), the intermediate (hDBH-I, and the C-terminal (hDBH-C) amino acid sequences (residues 26-43, 452-468, and 582-598), respectively. We characterized the antibodies in terms of specificity by means of Western blotting and immunohistochemistry. Anti-hDBH-N antiserum recognized DBH in the brain (noradrenergic neurons in the pons and medulla oblongata) and adrenal medulla, not only of human but also of mouse, rat and house shrew. In contrast, anti-hDBH-C antiserum recognized only human DBH. These observations suggest that the antibody raised against the hDBH-C terminal peptide may specifically recognize only human DBH.