Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Journal qf Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic L-Amino Acid Decarboxylase: Neurochemical Effects Farida G. Kaddis, Edward D. Clarkson, *Mjche~J. Weber, tDavid J. Vandenbergh, tDavid M. Donovan, ~Jacques Mallet, ~Phi1ippe Horellou, 1~GeorgeR. Uhi, and Curt R. Freed Departments of Medicine and Pharmacology, University of Colorado School of Medicine, Denver, Colorado; lMolecular 5Laboratoire de Biologic Neurobiology Branch, NIDA Addiction Research Center, Baltimore, Maryland, U.S.A.; Moleculaire Eucaryote, CNRS, Toulouse; and ~LGN, CNRS, Hdpital de Ia Pitié Salpbtrière, Paris, France

Abstract: To study the possibility that increasing striatal activity of aromatic [-amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to [-3,4-dihydroxyphenylalanine (L-DOPA) administration, we grafted Cos cells stably expressing the human MDC gene (Coshaadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14002 from L[140]DOPA.The Km value for [-DOPA in intact and disrupted cells was 0.60 and 0.56 mM, respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 mM. The pH optimum for enzyme activity was 6.8. Grafting Cos-haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of L-DOPA. When measured 2 h after L-DOPA administration, the mean dopamine level in the striata of Cos-haadc-grafted animals was 2 ptg/g of tissue, representing 31 % of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 ~sg/g.At 6—8 h after LDOPA administration, striatal dopamine content in the Cos-haadc-grafted animals was 0.67 ~eg/gof tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal MDC activity can improve dopamine bioformation in response to systemically administered L-DOPA. Key Words: Aromatic Lamino acid decarboxylase—Stable expression—Transfected cells—Cos cells—L-DOPA— Parkinson’s disease—Transplantation. J. Neurochem. 68, 1520—1526 (1997).

L-3,4-Dihydroxyphenylalanine (L-DOPA) administration improves the motor disabilities associated with parkinsonism (Cotzias et al., 1967). The drug is be-

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lieved to exert its beneficial effects after being converted to dopamine by decarboxylation by aromatic Lamino acid decarboxylase (AADC; EC 4.1.1.28) (Lloyd et al., 1975; Rinne, 1978). Long-term treatment of Parkinson’s disease with L-DOPA is associated with a progressive decline of L-DOPA efficacy characterized by fluctuating motor responses and dyskinesias (Marsden and Parkes, 1977; Yahr, 1977; Lesser et al., 1979; Nutt, 1990). Variation in the qua!ity of movement in response to L-DOPA in advanced parkinsonism may result from (a) the loss of capacity of striatal dopamine nerve terminals to store and release dopamine (Marsden and Parkes, 1977; Marsden and Jenner, 1981; Mouradian and Chase, 1988; Freed et a!., 1990, 1992a,h), (b) postsynaptic changes presumably at the dopamine receptor level (Mouradian and Chase, 1988; Mouradian et al., 1988), and/or (c) diminished conversion of L-DOPA to dopamine caused by reduced striatal AADC activity (Hefti and Melamed, 1980; Hefti et al., 1981; Melamed et al., 1983; Takashima et a]., 1987). In the striatum, AADC activity is present in dopaminergic and serotonergic nerve terminals (Hökfelt et al., 1973; Sims et a!., 1973; Cavilli-Sforza et a!., 1974), capillary endothelium (Bertler et al., 1966). and striatal interneurons (Duvoisin and Mytilineou. Resubmitted manuscript received October 3, 996; revised manuscript received November 26, 1996; accepted November 28, 1996. Address correspondence and reprint requests to Dr. C. R. Freed at Departments of Medicine and Pharmacology, Division of Clinical Pharmacology, University of Colorado Health Sciences Center, 4201) East 9th Avenue, C237. Denver, CO 80262, U.S.A. Abbreviations used: AADC, aromatic L-amino acid decarboxylase; Cos-ut cells, untransfected Cos cells; L-DOPA, L-3,4-dihydrOXyphenylalanine; haodc, human aromatic L-amino acid decarhoxylase eDNA; NSD- 1015, 3-hydroxybenzylhydrazine; 6-OHDA, 6-hydroxydopamine: PLP, pyridoxal 5-phosphate.

HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE 1978; Hefti et al., 1981; Mura et al., 1995). These structures are postulated to be the striatal sites for decarboxylation of L-DOPA. In Parkinson’s disease and in 6-hydroxydopamine (6-OHDA)-denervated rat striatum, severe loss of dopaminergic nerve terminals is associated with an 80—95% depletion of striatal AADC activity (Lloyd and Hornykiewicz, 1970; Duvoisin and Mytilineou, 1978; Hefti and Melamed, 1980; Hefti et a!., 1981; Zhong eta!., 1995). Genes encoding aadc have been cloned from neuronal and nonneuronal tissues of the rat (Krieger et al., 1991), cow (Albert et a!., 1987; Kang and Joh, 1990), and human (Le Van Thai et a!., 1993). With the hypothesis that enhanced striatal AADC activity could improve dopamine bioformation in response to L-DOPA administration, we have developed a line of Cos cells that stably express human AADC cDNA (haadc) and have grafted these cells into 6-OHDAdenervated rat striatum. We have then measured the effects of the grafted haadc-expressing Cos cells on striatal dopamine concentrations after systemic administration of i~-D0PA.

MATERIALS AND METHODS Expression of haadc in mammalian cells The eDNA encoding haadc was inserted between the Xba! and EcoRV sites of the eukaryotic expression vector pcDNAI/Amp (Invitrogen Corp., San Diego, CA, U.S.A.). The plasmid containing the haadc insert was characterized by restriction enzyme digestion and by plasmid/insert junction sequencing. Cos cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mI penicillin, and 100 pg/ml streptomycin. Cells (10~)were then cotransfected by electroporation with 20 pg of haadc/pcDNAl and 2 pg of neomycin resistance plasmid pSV2neo (Clontech Laboratories, Palo Alto, CA, U.S.A.). Stably transformed cells were selected by culture in medium containing 400 pg/mI geneticin (G-418 sulfate; GibcoBRL, Grand Island, NY, U.S.A.).

Assay for AADC activity in Cos cells Confluent cultures of geneticin-resistant cells (!0~)were harvested with a cell scraper or by trypsin treatment, centrifuged at 800 rpm for 5 mm, and either resuspended in icecold 150 mM potassium phosphate buffer and kept intact or disrupted by sonication in 100 mM potassium phosphate buffer containing 0.1 mM EDTA and 1 mM dithiothreitol. The activity of human AADC was measured by the forma4C0 4C I DOPA according to tion of ‘ 2 from the by the method developed substrate Waymire L- [ ‘et a!. (1971) with some modifications. In brief, 30 p1 of cell suspension (140 ± 5 pg of protein) was mixed with 5 p1 of 6 mM pyridoxal 5-phosphate (PLP; Sigma Chemical Co., St. Louis, MO, U.S.A.), and the reaction was initiated by a S-p! mixture of varying concentrations of L-DOPA (Sigma) and 80 pM DL[‘4C]DOPA (DL- 3,4 - [alanine- 1 - ‘4C]dihydroxyphenyl alanine; 54.6 mCi/mmol; New England Nuclear Corp., Boston, MA, U.S.A.). Then, a small p!astic vial containing a 4- X 0.4-cm strip of Whatman filter, soaked with 30 pl of

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M benzthonium hydroxide, was placed in the reaction mixture tube, which was then capped and incubated at 37°Cfor 15 mm. The reaction was stopped by adding 200 pl of I M sulfuric acid followed by incubation for 20 mm at 37°C. Filter paper strips were collected, and 14C content was assessed by liquid scintillation spectroscopy (model LS 6000TA; Beckman). Total AADC activity in Cos-haadc cells was compared with that in untransfected Cos cells (Cos-ut cells) in intact cells using 1 mM L-DOPA and 0.6 mM PLP at pH 7.2. The specificity of the reaction was determined by including the AADC inhibitor 3-hydroxybenzylhydrazine (NSD-10l5) at 5.0 mM. The kinetics of human AADC were assayed at different concentrations of L-DOPA with the PLP cofactor concentration he!d constant at 0.6 mM. Then, the effect of PLP cofactor concentration on AADC activity was determined at PLP concentrations ranging from 1.0 pM to 1.0 mM at a constant L-DOPA concentration of 1.0 mM. Finally, the effect of pH on the activity of human AADC was determined for the pH range 6.0—8.0 in the presence of 1.0 mM L-DOPA and 0.6 mM PLP.

Animal lesion and transplantation procedures Female Sprague—Dawley rats (weighing 250—300 g; Sasco, Wilmington, MA, U.S.A.) were anesthetized with equithesin (3.0 mI/kg) and injected stereotaxically into the left medial forebrain bundle with 5.0 pl of 2.5 pg/pl of 6OHDA dissolved in norma! saline solution containing 0.25% ascorbic acid (RBI, Natick, MA, U.S.A.) at a rate of 1.0 pl/ mm for 5 mm (AP —4.5 mm from bregma, L !.3 mm and V —7.8 mm from dura). The needle (28 gauge) was left in place for 2 mm after injection. Successful lesion of the substantia nigra was determined by testing the animals for circling behavior induced by apomorphine (0.05 mg/kg, s.c.; Sigma). Complete lesions were confirmed postmortem by immunohistochemical staining for tyrosine hydroxylase immunoreactivity in the midbrain (data not shown). Intrastriatal transplantation was performed stereotaxically with the animal under equithesin anesthesia (3.0 mI/kg, i.p.). Animals were transplanted with 250,000 Cos-haadc cells or the same number of Cos-ut cells suspended in Hanks’ buffered salt solution containing 0.6% glucose at a concentration of 250,000 viable cells/4.0 p1. The cells were deposited into the 6-OHDA-lesioned striatum at AP 0 mm from bregma, L 3 mm and V starting at —7.5 mm and ending at —3.5 mm from dura at a rate of 1.0 p1/mm. To prevent rejection of the grafted primate cells, the animals were treated with cyclosporine (10 mg/kg/day, s.c.). Cyclosporine treatment was started 48 h before cell grafting and was continued until the conclusion of the experiment.

HPLC assessment for striatal dopamine and L-DOPA content Two weeks after grafting, animals were killed at 2 or 6— 8 h after L-DOPA injection by an overdose of chloral hydrate and then decapitated. Basal dopamine levels were determined in animals that were not treated with L-DOPA. Brains were immediately dissected and placed on ice, and the striaturn was isolated from both sides of the brain and placed into a weighed vial containing 500 p1 of ice-cold 0.1 M perchloric acid. The solution contained 20 pg/pl of 3,4dihydroxybenzylamine, which was used as an internal standard for subsequent HPLC measurement of levels of dopa-

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FIG. 1. Total activity of AADC in Cos-haadc cells in comparison with endogenous AADC activity in Cos-ut cells. AADC activity 14C0 14C]DOPA, in

was measuredandby presence the generation the absence of theofspecific AADCL-[inhibitor 2 from

NSD1015 at 5.0 mM. Cos-haadc cells without NSD-1015 had activity significantly different from all other groups at p < 0.0001 by Student’s Newman—Keuls test. Data are mean ± SEM (bars) values (n = 6).

mine and DOPA. The wet tissue weight was determined, and the tissue was homogenized and centrifuged at 13,000 rpm for 20 mm at 4°C.The supernatant was filtered (pore size, 0.2 pm) and stored at —20°Cuntil analyzed. To determine the dopamine and L-DOPA content, a S-pl sample containing 100 pg of internal standard was injected onto a reversed-phase C18 column (1 x 100 mm; Spherisorb, ODS 002; Keystone Scientific, Bellefonte, PA, U.S.A.). The mobile phase (0.07 M phosphoric acid contamnmng 0.04 M trichloroacetic acid, 1.34 mM EDTA, and 1.8 mM octy! sodium sulfate, pH 3.0) was pumped at a rate of 110 p1/mm. The potential at the glassy carbon working electrode was maintained at 0.7 V.

Statistical analysis Statistical analysis of data from two groups was carried out using unpaired one-tailed Student’s t test. For comparing data from several groups, one-way ANOVA followed by Student’s Newman—Keuls test was performed using the Instat computer program (GraphPad Biostatistics, San Diego, CA, U.S.A.).

FIG. 2. Lineweaver—Burk plot of activity of human AADC stably expressed in Cos cells measured in intact (A) and disrupted (0) cells. The activity of AADC was determined at different L-DOPA concentrations and 0.6mM PLP. The Km value for L-DOPA determined from the Lineweaver—Burk plot was 0.60 and 0.56 mM, respectively. Data are mean ± SEM (bars) values of four measures.

disrupted cells, human AADC activity was maximal at a PLP concentration of 0.1 mM (Fig. 3). In intact cells, the optimal PLP concentration was 0.6 mM (data not shown). Finally, the effect of pH on decarboxylase activity was measured for the pH range 6.0—8.0. The pH optimum for human AADC activity in disrupted cells was 6.8 (Fig. 4).

Dopamine and i~-DOPAstriatal content following systemic L-DOPA administration The effect of L-DOPA on dopamine bioformation in the striatum was determined after systemic L-DOPA administration. Carbidopa was coadministered along with L-DOPA to block the metabolism of L-DOPA to dopamine in peripheral tissues and to maximize the

RESULTS Characteristics of stable expression of human AADC in Cos cells The activity of human AADC stably expressed in Cos-haadc cells was 10.6-fold higher than that in CosUt cells (p < 0.0001). The decarboxylase activity in both cell types was almost completely blocked by 5.0 mMNSD-l015, an AADC inhibitor (Fig. 1). The kinetics of the enzyme were measured at various L-DOPA concentrations. As shown in Fig. 2, the K,,, value for L-DOPA calculated from a Lineweaver—Burk plot was 5.6 x iO~ M when determined in disrupted cells and 6.0 x iO~M when determined in intact cells. The cofactor PLP produced a concentration-dependent effect on the activity of human AADC. Measured in

j.

Neurochem., Vol. 68, No. 4, 1997

FIG. 3. Effect of the cofactor PLP concentration on activity of human AADC stably expressed in Cos cells. Activity of AADC was assayed at the indicated PLP concentrations with the LDOPA concentration held at 1.0 mM and at pH 6.8. Data are mean ± SEM (bars) values of three measures.

HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE

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grafted animals, respectively (Fig. 5C). The corresponding values for the intact striata were 0.29 ±0.08 and 0.33 ±0.14 pg/g of wet tissue weight (Fig. SD). Thus, L-DOPA was equally available in all the striata

assayed. Dopamine and L-DOPA striatal content in the absence of i.-DOPA administration In the absence of L-DOPA administration, dopamine tissue content was below detection limits in the lesioned striata of both the Cos-ut and Cos-haadc groups.

Therefore, without a source of L-DOPA, the grafted FIG. 4. Activity of human AADC stably expressed in Cos cells

Cos-haadc cells did not alter the dopamine levels in

as a function of pH. Activity of AADC was determined at 1.0 mM L-DOPA and 0.6 mM PLP. Data are mean ±SEM (bars) values of six measures.

dose of L-DOPA reaching the CNS. Striatal tissue dopamine content measured 2 h after L-DOPA administration was 2.00 ±0.37 and 1.06 ±0.11 pg/g of wet tissue weight of the lesioned striatum of Cos-haadc and Cos-ut groups, respectively (p < 0.02; Fig. 5A).

These dopamine levels represented 31 and 15% of the basal dopamine content in normal striatum, respectively (6.5 fig/mg; see below). L-DOPA administration increased dopamine levels in the intact striatal side to 26.2 ±2.8 and 30.2 ±4.2 pg/g of wet tissue weight in the Cos-haadc and Cos-ut cells, respectively (Fig.

SB). The L-DOPA levels in the lesioned striata were 4.8 ±0.4 ,uglg for Cos-haadc-grafted group and 4.4 ±0.5 pg/g of wet tissue weight for the Cos-ut-grafted

group (Fig. SA). The corresponding L-DOPA tissue levels in the intact striata were 4.3 ±0.6 and 4.6 ±0.8 pg/g (statistically not different, ANOVA; Fig. SB). When measured 6—8 h after L-DOPA administration, the dopamine concentration in the lesioned stria-

turn of the Cos-haadc-grafted group was 0.67 ±0.27 ftg/g, which represented 9% of the dopamine tissue content in the normal striatum. By contrast, the dopamine levels in the lesioned striaturn of the Cos-utgrafted group was below detection limits (3 ng/g of tissue) in four of five animals. The detection limit was

FIG. 5. Dopamine and L-DOPA tissue contents of 6-OHDA-le-

used to calculate the average dopamine concentration

sioned (A and C) and intact (B and D) striatum of Cos-ut- and Cos-haadc-grafted animals following L-DOPA administration. Whole striatal dopamine and L-DOPA contents were measured by HPLC 2 weeks postgrafting at 2 h (A and B) or 6—8 h (C and D) after intraperitoneal administration of 50 mg/kg L-DOPA with 5 mg/kg carbidopa. The mean dopamine concentration in the Cos-haadc-grafted group was significantly higher than that of the Cos-ut-grafted group at 2 h (°p < 0.02). At 6—8 h after LDOPA administration, the mean ±SEM dopamine concentration in the Cos-haadc-grafted striata was 0.67 ±0.27 pg/mg of tissue weight, whereas that of the Cos-ut-grafted group was not different from 0. The mean value of dopamine concentration in the intact striatum of the Cos-haadc-grafted group was not significantly different from that in the Cos-ut control. The mean values of DOPA content in the lesioned and intact sides of both Cosut and Cos-haadc striatum were not statistically different (n =5_7).

for this group, which was 0.04 ± 0.04 pg/g of wet

tissue weight, which was not different from a concentration of dopamine of 0 ftg/g. Thus, striata of Cosut-grafted animals had no detectable doparnine at this later time point (Fig. SC). The dopamine concentrations of the intact side of the striatum were 7.4 ± I I ~ig/g for Cos-haadc and 9.9 ±1.8 pg/g for Cos-ut .

(Fig. SD). At 6—8 h after L-DOPA administration,

dopamine content in the intact striata approached the normal baseline concentration (6.5 ~.tg/mg; see below). The striatal L-DOPA levels, measured at 6—8 h following L-DOPA administration, were 0.31 ±0.56 and 0.28 ± 0.12 ,uglg for Cos-haadc- and Cos-ut-

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F. G. KADDIS ET AL.

the 6-OHDA-lesioned striatum. The dopamine content in the intact side of the striatum was 6.4 ±0.6 (n = 5) and 6.5 ±0.7 ftglg (n = 5) for Cos-ut and Cos-haadc cells, respectively. The L-DOPA levels were below detection limits in both intact and lesioned sides of the striatum in both groups. DISCUSSION In the present study, we have demonstrated stable expression of recombinant human AADC in Cos cells in vitro. We have also shown that Cos cells expressing haadc that have been grafted into the 6-OHDA-denervated rat striatum have led to an enhanced and prolonged rise of striatal dopamine concentration after LDOPA administration. In our in vitro studies, the recombinant human AADC protein had a K,,, value for L-DOPA of 5.6 < 10 ~ and 6.0 x l0—~M in disrupted and intact cells, respectively. These values are comparable to the value reported by Sims et a!. (1973) for AADC from rat brain homogenate but higher than the K,,, value reported for human AADC purified from pheochromocytoma cells (Ichinose et al., 1985), recombinant human AADC transiently expressed in Cos cells (Sumi et a!., 1990), or recombinant bovine AADC expressed in the C 127 mouse cell line (Park et al., 1992). The cofactor concentration and pH optimum for enzyme activity obtained in our study are in agreement with the reported values for AADC purified from pheochromocytoma (Ichinose et al., 1985) and recombinant AADC (Sumi et al., 1990). Our finding that human AADC kinetics are similar in both intact and disrupted cells

reveals that Cos cells are capable of taking up L-DOPA. Kang et a!. (1993) demonstrated that L-DOPA and dopamine move freely across the membrane of fibroblasts expressing bovine AADC. Transplantation of human AADC-producing cells (Cos-haadc) into 6-OHDA-denervated rat striatum improved the striatal response to systemic administration of L-DOPA. The 6-OHDA-denervated rat striatum model was selected (a) to minimize the role of endogenous AADC and dopamine and (b) because the 6OHDA-denervated rat striatum is a useful model for Parkinson’s disease, a condition in which L-DOPA is used therapeutically. The neurochemical response was measured as striatal dopamine concentration early (2

h) and late (6—8 h) following L-DOPA administration. The selection of these time points was based on the observation that the rise in dopamine levels in both intact and 6-OHDA-lesioned striata peaks at ~2 h following L-DOPA administration. At 6—8 h after L-

DOPA administration, L-DOPA-induced increases in dopamine levels decline to baseline (Zetterstrom et a!., 1986; Karoum et al., 1988). The L-DOPA-induced dopamine production measured at 2 and 6—8 h after L-DOPA administration in

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the striata of Cos-haadc-grafted animals was higher

than in Cos-ut-grafted controls. The conversion of

L-

DOPA to dopamine is believed to be the primary explanation for the therapeutic benefit of L-DOPA for treatment of Parkinson’s disease (Lloyd et al., 1975; Rinne,

1978). The sites of conversion of L-DOPA to dopamine in the 6-OHDA-denervated striatum have been previously postulated to be capillary endothelium, serotonergic nerve terminals, or striatal interneurons (Bertler et a!., 1966; Ng et a!., 1972; Duvoisin and Mytilineou, 1978; Hefti et al., 1981; Mura et al.,

1995). In the current study, AADC in endothelium is unlikely to be the site of L-DOPA decarboxylation because carbidopa was administered along with LDOPA. Carbidopa is an inhibitor of peripheral AADC activity, which includes the brain endothelium (Duvoisin and Mytilineou, 1978). The role of serotonergic neurons in L-DOPA decarboxylation has been reexamined by Melamed et al. (1980) and was found to be limited. Thus, in our study, conversion of L-DOPA to dopamine is most likely have taken place in striatal interneurons in addition to in Cos-haadc cells. The higher dopamine levels observed in the Cos-haadcgrafted animals is due to the AADC activity in the Cos cells. Gene therapy approaches for treatment of Parkinson’s disease have focused on restoration of striatal dopamine levels by improving tyrosine hydroxylase activity in the striatum. Grafts of cells transduced with the tyrosine hydroxylase gene (Wolff et al., 1989; forellou et al., 1990a,b; Fisher et al., 1991), direct injection of tyrosine hydroxylase-expressing virus (During et al., 1994; Horellou et al., 1994), or liposomes that carry tyrosine hydroxylase plasmid DNA (Cao et al., I 995) are examples of the explored strategies. Our study demonstrates an alternative to tyrosine hydroxylase replacement. This study was not designed to be a model for behavioral improvement of parkinsonism because the Cos cell line continues to divide in vivo and will form a tumor mass that limits the postgrafting period. Nonetheless, these experiments demonstrate that genetically modified cells expressing AADC activity can provide substantial increases in striatal dopamine concentration from a single dose of L-DOPA and thus is an appropriate alternative to replacing the tyrosine hydroxylase activity approach. Stable expression of AADC in primary cells, which do not divide in vivo, could be a useful strategy for treatment of advanced parkinsonism. Acknowledgment: This work was supported by grants from the National Parkinson Foundation, the Seaver Institute, NIH NS 18639 and NS 23918, the lntraagency Personnel Agreement from the Intramural Research Program, NIDA, NIH, Association Française contre les Myopathies (AFM), and Institute pour Ia Recherche sur la Moelle Epinière (IRME). The University of Colorado is a National Parkinson Foundation Center of Excellence.

HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE

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