Neuroscience Letters, 151 (1993) 55-58 0 1993 Elsevier Scientitic Publishers Ireland Ltd. All rights reserved 0304_394O/93/$06OO

55

NSL 093 11

Regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying human tyrosine hydroxylase gene

Kiyomi Kiuchi”, Kazutoshi Kiuchib, Norio Kanedac, Toshikuni Sasaokad,*, Hiroyoshi Hidaka” and Toshiharu Nagatsu”

“Department of Pharmacology bRadioisotope Center Medical Division, ‘Department of Biochemistry, Nagoya University School of Medicine, Nagoya (Japan), dDivision of Developmental Biotechnology, Central Institute for Experimental Animals, Kawasaki (Japan) and 'Institute for Comprehensive

Medical Science, Fujita Health University School of Medicine, Toyoake, Aichi (Japan)

(Received 27 October 1992; Revised version received 20 November 1992; Accepted 20 November 1992)

Key words: Tyrosine hydroxylase; Transgenic mouse; Striatum; Phosphorylation; (6R)-Tetrahydrobiopterin; DOPA formation

We investigated the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human tyrosine hydroxylase (TH). The in vitro TH activity of transgenic striatum at pH 7.0 was approximately 2.8-fold higher than that of non-transgenic striatum. This augmentation is similar to that of the in vitro TH activity at pH 6.0, indicating that the expression of human TH in transgenic striatum induced little change in the phosphorylation level of TH. L-3,4_Dihydroxyphenylalanine (DOPA) formation in striatal slices of transgenic mice was approxi- mately 2.7-fold higher than that of non-transgenic mice. The addition of 0.5 mM (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-SK) to the incuba- tion medium brought a negligible increase in DOPA formation in both cases. These results suggest that 6R-BH, is not the limiting factor of TH in situ both in the transgenic and non-transgenic mice.

In our previous report [4], we have obtained three in- dependent transgenic mice carrying the human tyrosine hydroxylase (TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine: oxygen oxidoreductase [3-hydroxyl- ating]; EC 1.14.16.2.) gene and reported the detailed characterization of one strain. We observed a discrep- ancy in the transgenic striatum between the in vitro TH activity and the dopamine content. The in vitro TH ac- tivity in homogenates of transgenic striatum at pH 6.0 was approximately 3.4-fold higher than that of non- transgenic striatum, but the dopamine content in trans- genie striatum was 1.05fold higher than that in non- transgenic striatum [4]. One possible explanation for this discrepancy is that the decrease in phosphorylation level of TH in transgenic mice may minimize the effect of the transgene. TH catalyzes the first and rate-limiting step of catecholamine biosynthesis [7, 121, and phosphorylation by cyclic AMP dependent protein kinase is known to enhance the in vitro TH activity at neutral pH by increas-

Correspondence: T. Nagatsu, Institute for Comprehensive Medical Sci- ence, Fujita Health University School of Medicine, Toyoake, Aichi 470-l 1, Japan. *Present address: Medical Institute of Bioregulation, Kyushu Univer- sity, Fukuoka, 812, Japan.

ing V,,,, as well as reducing the K,,, for the cofactor [6,9, 15-171, but to induce little change in V,,, at pH 6.0 [6, 151. Mogi et al. [l l] reported the increase in ho- mospecific activity (V,,,Jenzyme protein) of TH in the Parkinsonian brains where the TH protein content was reduced, which is a case opposite to transgenic striatum where TH protein is overproduced. If the phosphoryla- tion level of TH in transgenic striatum is curtailed to maintain the dopamine content as normal, TH activities at neutral pH could recede. Another possibility is that TH requires (6R)-L-eryrhro-5,6,7,8_tetrahydrobiopterin (6R-BH,) as cofactor and 6R-BH, may serve as a limiting factor of tyrosine hydroxylation in situ if its content in striatum of transgenic mice is insufficient. In rat stria- tum, TH is reported to be finely regulated through the availability of 6R-BH, [3, 5, lo]. If the 6R-BH, concen- tration in transgenic striatum is not enough to draw the full activity of TH, the enzymatic activity could recede and as a result the dopamine content may settle down to normal. In this paper, we measured the in vitro striatal TH activity at both pH 6.0 and pH 7.0, and the in situ TH activity in striatal slices to clarify the regulatory mechanism of dopamine biosynthesis in the transgenic striatum according to the above hypotheses.

The production of transgenic mice carrying human

56

I 1 I 0.0 0.1 0.2 0.3 0.4 0.5 0.6

TH activity (nmollminlmg protein)

Fig. 1. In vitro TH activity in the striatum of transgenic and non- transgenic mice. TH activity in tissue homogenate was measured by the HPLC-ECD method. The blank columns show the in vitro TH activity at pH 6.0; the hatched columns at pH 7.0. Each column represents the mean f S.E.M. of duplicate determinations from 6 independent ani-

mals.

TH gene was described in the previous paper [4]. The DNA 11 kb fragment of the human TH gene consisting of 2.5 kb of the 5’ upstream region, the entire exon-intron sequence and 0.5 kb of the 3’ flanking region, was micro- injected into fertilized mouse eggs (C57BW 65 x C57BL/ 65). The in vitro TH activity was measured by high-per- formance liquid chromatography with electrochemical detection (HPLC-ECD) as described previously [ 131 using an Irika amperometric detector E-502. Mouse stri- atum was homogenized with 100 ~1 of 0.32 M sucrose containing 20 mM Tris-HCl buffer (pH 7.3), 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 pg/ml leupeptin, 1 &ml pepstatin, 0.2 mM phenylmethylsul- fonylfluoride (PMSF) and 1 PM okadaic acid. The standard assay mixture (200 ~1) contained 40 nmol of L-tyrosine, 200 nmol of 2-mercaptoethanol, 40 pg (2,600 U) of catalase, and enzyme in either 0.2 M sodium ace- tate buffer (pH 6.0) or 0.1 M 2-(N-morpholino)-ethane- sulfonic acid (MES) buffer (pH 7.0) containing 10 PM NSD-1055. The in situ TH activity of striatal slices was measured according to Hirata et al. [3]. Under an atmos- phere of 95% 0,/s% CO,, striatal slices (0.22 mm in thickness) were incubated at 37°C for 1 h in 500 ,ul of Krebs-Ringer bicarbonate medium containing 10 PM NSD-1055, an inhibitor of aromatic L-amino acid decar- boxylase, and 25 mM 2-mercaptoethanol with or with- out 0.5 mM 6R-BH4. Protein concentrations were deter- mined by a micro-assay using a Bio-Rad protein assay kit with bovine y-globulin as a standard [l].

To compare the phosphorylation level of striatal TH in transgenic and non-transgenic mice, the in vitro TH activities both at pH 6.0 and at pH 7.0 were measured. Decreased phosphorylation of TH reduces the ratio of the in vitro activity at pH 7.0 to that at pH 6.0 [6, 151. Since protein phosphatase 2A was the major TH phos-

phatase in adrenal medulla and corpus striatum [2], mouse striatum was homogenized in the presence of 1 PM okadaic acid. As shown in Fig. 1, the in vitro TH activity of transgenic striatum at pH 7.0 was approxi- mately 2.8-fold higher than that of non-transgenic mice, while the augmentation of in vitro TH activity at pH 6.0 was approximately 3.1 -fold. This result indicates that the phosphorylation level of TH is parallel between trans- genie and non-transgenic striatum, and suggests that the rate of dopamine biosynthesis in transgenic striatum is not reduced to normal by accelerating dephosphoryla- tion of TH. The next possible mechanism is that 6R-BH, may be the limiting factor of tyrosine hydroxylation in situ in transgenic striatum. In order to test this hypothe- sis, we measured in situ TH activity in striatal slices to elucidate whether or not the 6R-BH, content is sufficient for TH activity. As shown in Fig. 2, DOPA formation in transgenic striatum was approximately 2.7-fold higher than that in non-transgenic striatum, indicating that human TH overproduced in mouse striatum really ex- hibits the enzymatic activity as well as original mouse TH. When 6R-BH, was added to the Krebs-Ringer bi- carbonate medium at a final concentration of 0.5 mM, a 1.15-fold increase in DOPA formation was observed in striatal slices of both non-transgenic and transgenic mice (Fig. 2). This augmentation is negligible compared with that in rat striatal slices (a 2.5-fold increase [3]). These results suggest that the 6R-BH4 content in the striatum of transgenic mice is sufficient for in situ TH activity despite the 2.7-fold increase of DOPA formation, and that 6R- BH, is not the limiting factor of tyrosine hydroxylation in situ both in the transgenic and non-transgenic mice. Therefore, neither the phosphorylation level of TH nor the 6R-BH, content reduces the dopamine content in transgenic striatum to normal. If the in vivo TH activity

nontransgmk

0.0 0.1 0.2 0.3 0.4

DOPA formed (nmollhlmg protein)

0.6

Fig. 2. In situ TH activity in striatal slices of transgenic and non-trans- genie mice. DOPA formed in striatal slices was measured by the HPLC-ECD method. The blank columns show the in situ TH activity in the absence of 6R-BH,; the hatched columns in the presence of 6R- BH,. Each column represents the mean f S.E.M. of 7 independent

animals.

TABLE I

DOPAMINE METABOLITE LEVELS IN STRIATUM OF TRANSGENIC AND NON-TRANSGENIC MICE

Dopamine and its metabolites in transgenic and non-transgenic striatum were measured by the method of Oka et al. [14] with some modification. 3,4-Dihydroxyhydrocinnamic acid was used as an internal standard instead of 3,Cdihydroxycinnamic acid. Each value represents the mean f S.E.M., expressed in nmol per mg of protein, from 3 independent animals.

Non-transgenic mice (control)

Transgenic mice

DA DOPAC HVA

0.503 f 0.028 0.0708 f 0.0017 0.0819 f 0.0011

0.524 f 0.036 0.0701 * 0.0034 0.0718 f 0.0049

in transgenic striatum increases comparable to the in situ TH activity in the striatal slices, dopamine metabolites might be augmented due to enhancement in the rate of dopamine turnover. According to Oka et al. [14], with some modification, we measured the contents of dopam- ine metabolites in mouse striatum. Mouse striatum was homogenized with 500 ,ul of 0.32 M sucrose containing 20 mM Tris-HCl buffer (pH 7.3), 1 mM EDTA, 1 mM DTT, 1 ,ug/ml leupeptin, 1 @ml pepstatin, 0.2 mM PMSF and 0.25 mM pargyline. The amounts of 3,4-dihy- droxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in transgenic and non-transgenic striatum were shown in Table I. There was no detectable amount of 3-methoxytyramine in both cases. The contents of dopamine metabolites in transgenic striatum were identi- cal with those of non-transgenic striatum, indicating that dopamine turnover in transgenic striatum does not accel- erate and the rate of dopamine biosynthesis may be par- allel between the two. Therefore, the in vivo TH activity of transgenic striatum could be reduced to the normal level as that of non-transgenic striatum. One possible mechanism is the feedback inhibition of dopamine to- ward the in vivo TH activity. Since the amount of DOPA formed in striatal slices was found mainly in Krebs- Ringer medium, 73.8 + 0.5% (n = 7) in case of transgenic mice and 72.1 f 1.5% (n = 7) in case of non-transgenic mice, DOPA did not interfere with the in situ TH activity as a feedback inhibitor. Recently, Liu et al. [8] have re- ported the cDNA sequences of chromaffin granule and synaptic vesicle amine transporters. The latter incorpo- rates dopamine into synaptic vesicles as a final gate of dopamine biosynthesis. If the synaptic vesicle amine transporter trims the dopamine content in synaptic vesi- cles, overproduced dopamine might remain in the cyto- plasm to inhibit the TH activity. Based on the findings presented here, further study such as in vivo microdialy- sis should make it possible to illustrate the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human TH.

1 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding, Anal. Biochem., 72 (1976) 248-254.

This work was supported by a Grant-in-aid for Spe- cially Promoted Research from the Ministry of Educa- tion, Science and Culture of Japan.

2

3

4

5

6

I

8

Haavik, J., Schelling, D.L., Campbell, D.G., Andersson, K.K., Flatmark, T. and Cohen, P., Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal me- dulla and corpus striatum: evidence from the effects of okadaic acid, FEBS Lett., 251 (1989) 3642. Hirata, Y., Togari, A. and Nagatsu, T., Studies on tyrosine hydrox- ylase system in rat brain slices using high-performance liquid chromatography with electrochemical detection, J. Neurochem., 40 (1983) 1585-1589. Kaneda, N., Sasaoka, T., Kobayashi, K., Kiuchi, K., Nagatsu, I., Kurosawa, Y., Fujita, K., Yokoyama, M., Nomura, T., Katsuki, M. and Nagatsu, T., Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice, Neuron, 6 (1991) 583-594. Kettler, R., Bartholini, G. and Pletscher, A., In vivo enhancement of tyrosine hydroxylation in rat striatum by tetrahydrobiopterin, Nature, 249 (1974) 476478. Lazar, M.A., Lockfeld, A.J., Truscott, R.J. and Barchas, J.D., Ty- rosine hydroxylase from bovine striatum: catalytic properties of the phosphorylated and non-phosphorylated forms of the purified en- zyme, J. Neurochem., 39 (1982) 409422. Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucida- tion of the rate-limiting step in norepinephrine biosynthesis in the perfusedguinea-pig heart, J. Pharmacol. Exp. Ther., 148 (1965) 1-8. Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G.G., Eisen- berg, D., Brecha, N. and Edwards, R.H., A cDNA that suppresses MPP’ toxicity encodes a vesicular amine transporter, Cell, 70 (1992) 539-551. Meligeni, J.A., Haycock, J.W., Bennett, W.F. and Waymire, J.C., Phosphorylation and activation of tyrosine hydroxylase mediate the CAMP-induced increase in catecholamine biosynthesis in adrenal chromaflin cells, J. Biol. Chem., 257 (1982) 12632-12640. Miwa, S., Watanabe, Y. and Hayaishi, O., 6R-L-q&o-5,6,7,8-

tetrahydrobiopterin as a regulator of dopamine and serotonin bio- synthesis in the rat brain, Arch. Biochem. Biophys., 239 (1985) 234 241. Magi, M., Harada, M., Kiuchi, K., Kojima, K., Kondo, T., Nara- bayashi, H., Rausch, D., Riederer, P., Jellinger, K. and Nagatsu, T., Homospecific activity (activity per enzyme protein) of tyrosine hy-

58

droxylase increases in parkinsonian brain, J. Neural Transm., 72 (1988) 77-82.

12 Nagatsu, T., Levitt, M. and Udenfriend, S., Tyrosine hydroxylase: the initial step in norepineprhine biosynthesis, J. Biol. Chem., 239 (1964) 291CL2917.

13 Nagatsu, T., Oka, K. and Kato, T., Highly sensitive assay for tyro- sine hydroxylase activity by high-performance liquid chromatogra- phy, J. Chromatogr., 163 (1979) 247-252.

14 Oka, K., Kojima, K., Togari, A., Nagatsu, T. and Kiss, B., An integrated scheme for the simultaneous determination of biogenic amines, precursor amino acids, and related metabolites by liquid chromatography with electrochemical detection, J. Chromatogr., 308 (1984) 43-53.

15 Pollock, R.J., Kapatos, G. and Kaufman, S., Effect of cyclic AMP- dependent protein phosphorylating conditions on the pa-depend- ent activity of tyrosine hydroxylase from beef and rat striata, J. Neurochem., 37 (1981) 855-860.

16 Vigny, A. and Henry, J.P., Mechanism of tyrosine hydroxylase acti- vation by phosphorylation, Biochem. Biophys. Res. Commun., 106 (1982) 1-7.

17 Yamauchi, T. and Fujisawa, H., In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3’:5’-monophosphate- dependent protein kinase, J. Biol. Chem., 254 (1979) 503507.