Aromatic l-amino acid decarboxylase turnover in vivo in rhesus macaque striatum: A microPET study

www.elsevier.com/locate/brainres

Brain Research 1054

Research Report

Aromatic l-amino acid decarboxylase turnover in vivo in

rhesus macaque striatum: A microPET study

O.T. DeJesusa,*, L.G. Floresa, D. Muralia, A.K. Converseb, R.M. Bartletta,

T.E. Barnharta, T.R. Oakesb, R.J. Nicklesa

aMedical Physics, University of Wisconsin Medical School, 1530 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USAbKeck Imaging Laboratory, Waisman Center, University of Wisconsin, Madison, WI 53706, USA

Accepted 25 June 2005

Available online 1 August 2005

Abstract

The aromatic l-amino acid decarboxylase (AAAD) is involved in the de novo synthesis of dopamine, a neurotransmitter crucial in

cognitive, neurobehavioral and motor functions. The goal of this study was to assess the in vivo turnover rate of AAAD enzyme protein in the

rhesus macaque striatum by monitoring, using microPET imaging with the tracer [18F]fluoro-m-tyrosine (FMT), the recovery of enzyme

activity after suicide inhibition. Results showed the AAAD turnover half-life to be about 86 h while total recovery was estimated to be 16

days after complete inhibition. Despite this relatively slow AAAD recovery, the animals displayed normal movement and behavior within 24

h. Based on the PET results, at 24 h, the animals have recovered about 20% of normal AAAD function. These findings show that normal

movement and behavior do not depend on complete recovery of AAAD function but likely on pre-synaptic and post-synaptic compensatory

mechanisms.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Monoamine synthesis

Keywords: Aromatic l-amino acid decarboxylase; Protein turnover; MicroPET imaging; Parkinson’s disease

1. Introduction

The aromatic l-amino acid decarboxylase (AAAD) (EC

4.1.1.28) is the second enzyme in the biosynthetic pathway

to important monoamine neurotransmitters—dopamine

(DA), norepinephrine (NE) and serotonin (5-hydroxytrypt-

amine or 5 HT). AAAD converts l-DOPA to DA which in

turn is converted in NE neurons by dopamine h-hydrox-ylase (DhH) to NE while 5-hydroxytryptophan is converted

by AAAD to 5 HT. Because the AAAD enzymic Km is

higher than the normal levels of cerebral l-DOPA,

conversion of l-DOPA to dopamine is rapid and has led

many to believe that AAAD is not involved in dopamine

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.06.086

* Corresponding author. Fax: +1 608 262 2413.

E-mail address: [email protected] (O.T. DeJesus).

regulation. However, there is now accumulating evidence

that pre- and post-synaptic mechanisms alter AAAD activity

via regulatory processes [6,29].

Parkinson’s disease (PD) is a movement disorder

characterized by a dramatic reduction in dopamine levels

in the terminals due to degeneration of nigrostriatal

dopamine neurons. Since its introduction in 1967 [7],

dopamine replacement using l-DOPA has remained to be

the most effective therapy to ameliorate PD symptoms.

Thus, AAAD is thought to play the important role as rate-

limiting step in l-DOPA therapy in PD. Because changes in

AAAD activity have clinical consequences [21], better

understanding of AAAD function is needed. One important

aspect of AAAD function which needs elucidation is its

turnover rate in vivo. In this study, AAAD turnover rate in

normal non-human primate striatum was assessed in vivo by

monitoring recovery of AAAD function after irreversible

(2005) 55 – 60

O.T. DeJesus et al. / Brain Research 1054 (2005) 55–6056

AAAD inhibition. This study involved the administration of

a single dose of an AAAD suicide inhibitor, a-monofluor-

omethyl-dl-3,4-dihydroxyphenylalanine (FMDOPA) [14]

followed by longitudinal assessment of striatal AAAD

recovery by non-invasive microPET imaging using 6-

[18F]fluoro-meta-tyrosine (FMT), a selective imaging agent

for estimating AAAD activity [8,9].

2. Materials and methods

2.1. Inhibitor synthesis and characterization

FMDOPA was synthesized de novo for this study

following the method of Bey et al. [3] as we previously

described in the synthesis of a close analog, a-monofluor-

omethyl-m-hydroxyphenylalanine (FM-m-tyrosine, FMFmT)

[19]. After purification, FMDOPA was authenticated by 13C-

and 1H NMR spectroscopy and validated by in vitro studies

using rat kidney and striatal homogenates. Consistent with

the results of Bey at al. [4], our in vitro results showed that

FMDOPA has IC50 value comparable to those of other

AAAD inhibitors–carbidopa, benserazide and NSD 1015–in

inhibiting AAAD activity in these rat tissue homogenates

(unpublished results).

2.2. Radiotracer synthesis

The PET tracer [18F]FMTwas prepared by the method of

Namavari et al. [20]. Briefly, to achieve regioselective

electrophilic radiofluorination, [18F]F2, produced as previ-

ously described [24], was bubbled into a fluorodichloro-

methane solution of a protected stannylated m-tyrosine

derivative purchased from ABX Biochemicals (Radeberg,

Germany). After removal of SnF2 and other by-products

using silica minicolumn, protective groups in the [18F]-

intermediate were removed by refluxing in 48% hydro-

bromic acid. The product [18F]FMT was purified using a

semi-preparative HPLC (Alltech C18 column, 10 A, 250 �10 mm) with a mobile phase which consisted of 0.02 M

NaOAc pH 3.5 and made up with sufficient NaCl to be

isotonic. The HPLC fraction containing the product was

collected and sterilized by filtration through a 0.22 A filter

into a vented sterile vial prior to administration into the

subjects.

2.3. Animal imaging: PET and MRI studies

Two male rhesus monkeys (designated as AT 97 and AU

18), both 7 years old and weighing 8 kg, were used in this

study. The animals were anesthetized with ketamine (15 mg/

kg IM) to allow administration dl-FMDOPA (25 mg/kg)

into the peritoneum. Both animals were scanned serially at

different times ranging from 4 h up to 8 days after

FMDOPA injection. The animals were fasted overnight

before each PET study. On the day of the PET study,

anesthesia was initially induced with ketamine (15 mg/kg

IM) to allow the transport of the animals to the Keck

MicroPET Laboratory at the University of Wisconsin

Waisman Center. Anesthesia during the scan was main-

tained with isoflurane (1%–2% in oxygen). The protocol

used in these studies was approved by the UWAnimal Care

and Use Committee in compliance with NIH regulations on

the use of non-human primates in research.

The microPET P4 scanner used in this study is a

commercial dedicated small animal research scanner (22 cm

bore) from Concorde Microsystems (Knoxville, TN). The

calculated image resolution, using filtered back projection

reconstruction, in 8 cm axial � 19 cm transaxial field of view

(FOV), is 2 � 2 � 2 mm3 with 2% sensitivity at center [27].

The anesthesized animal was positioned prone in the scanner

bed with its head comfortably immobilized in a stereotactic

holder (with ear, mouth and eye orbit bars) orienting the brain

such that the PET slices paralleled the orbitomeatal (OM)

line. Heart rate, SPO2, respiratory rate and temperature were

monitored throughout the scan and body temperature was

controlled using a loose-fitting wrap blowing warm air

around the animal (Bair Hugger, Arizant Healthcare, Eden

Prairie, MN). A 15-min transmission scan using a 68Ge point

source was performed to verify proper positioning and for use

in post-acquisition correction for photon attenuation. Data are

recorded in list mode, permitting maximum post-analysis

flexibility in time averaging. A 90-min dynamic 3D acqui-

sition was begun simultaneously with the intravenous

administration of 3.5–5.4 mCi [18F]FMT. Each animal had

previously undergone magnetic resonance imaging (MRI) on

an Advantx 1.5 T instrument (General Electric Medical

Systems, Waukesha, WI) under ketamine sedation. MR

imaging included a spoiled gradient-echo (SPGR) sequence,

a 3D acquisition reconstructed into contiguous 1.3-mm

coronal slices providing good differentiation between gray

matter and white matter.

2.4. PET image and region of interest analysis

After the scan, the list mode data were binned into

sinograms with 20 frames (5 � 1 min, 5 � 2 min, 5 � 5 min

and 5 � 10 min) and corrected for dead time and random

coincidences. Images were then reconstructed with Con-

corde Microsystems proprietary software using OSEM,

correcting for detector sensitivity and attenuation. The

images were converted to Analyze format using an in-house

imaging software program called SPAMALIZE [22].

Images were then smoothed with a 3 mm Gaussian filter

in Spm99. The PET image was manually co-registered to

each animal’s anatomical and stereotaxic MRI T1 weighted

image using SPAMALIZE and a region of interest (ROI)

approach was used. Striatal and cerebellar regions were

identified and drawn on the MRI T1 weighed images. The

ROIs were then applied to the co-registered PET images for

generating time activity curves (TACs) for the subsequent

Patlak analysis [23]. The Patlak Ki values obtained were

Fig. 1. Time course of FMT PET images of monkey 1 (AU18) Showing recovery of AAAD Activity. Pairs of transaxial (top) and coronal (bottom) views of the

striatum at different times after FMDOPA injection are shown. The head of the caudate separate from the putamen is seen at 0, 97 and 192 h images in the

coronal slices. The normalized images are displayed in a hot metal scale wherein black, red, orange, yellow and white correspond to increasing levels of

radioactivity.

O.T. DeJesus et al. / Brain Research 1054 (2005) 55–60 57

taken from a linear least squares fit of the time frames 15–

20 corresponding to 41–90 min of the scan. Patlak Ki

values are the model outcome measures related to AAAD

activity [9].

3. Results

Both animals were scanned twice before FMDOPA

treatment. Baseline Ki values of 0.01025 min�1 and

0.01030 min�1 for one animal (AU97) and 0.01112 min�1

and 0.00959 min�1 for the other (AU18) were obtained and

the mean test–retest reproducibility is about 5.4%. Four

hours after dl-FMDOPA treatment, microPET results

Fig. 2. Patlak plots calculated from PET data showing AAAD recovery for

monkey 1 (AU18). The slope, Ki, is the striatal uptake constant (using

cerebellum as reference tissue) for the irreversible tracer [18F]FMT and is a

model outcome measure related to AAAD activity.

showed complete inhibition of [18F]FMT uptake in subject

AU18. The PET images for subject AU18 (Fig. 1) show the

time course of [18F]FMT uptake in the caudate-putamen

regions in two views, transaxial (parallel to the OM line)

and coronal. Patlak plots based on these images for monkey

AU18 are shown in Fig. 2. The fractional recovery of

AAAD activity in both animals is plotted in Fig. 3, where

recovery in each animal is expressed as fraction of the

measured Ki relative to the mean baseline Ki for that

subject. Fitting the data in Fig. 3 to a simple exponential

equation showed good correlation (r = 0.94) and the half-

life of AAAD recovery is estimated to be 86 h after

treatment. Further, in Fig. 3, about 80% recovery was

observed within 192 h while complete AAAD recovery was

estimated to take about 16 days after FMDOPA treatment.

After recovering from anesthesia after dl-FMDOPA

treatment, both animals were bradykinetic and assumed

Fig. 3. Fractional AAAD recovery, calculated as ratios of Ki(t) at time = t to

baseline Ki(t = 0) for each animal, as a function of time after FMDOPA

administration for the 2 animals (circles for AU-18 and squares for AT-97).

Fitting the data to a simple exponential show a half-life of recovery of 86.4

h (r = 0.94).

O.T. DeJesus et al. / Brain Research 1054 (2005) 55–6058

crouched postures but were able to feed. However, both

animals appeared normal in their behavior and movement

within 24 h.

4. Discussion

The use of PET imaging to assess enzyme turnover in

vivo in monkeys was first reported by Arnett et al. [2] in the

study of MAO B turnover by irreversible inhibition with l-

deprenyl, a selective MAO B suicide inhibitor. This method

was later extended to assessing MAO A and MAO B

turnover in humans [11]. In the present study, a similar

approach was used to assess the turnover rate of striatal

AAAD enzyme in vivo in the primate brain after suicide

inhibition. While the study of Arnett et al. [2] used the same

compound, [11C]-l-deprenyl, both as PET imaging agent

and as suicide inhibitor, in the present study, an AAAD

substrate, [18F]FMT, was used as imaging agent but a

different compound, FMDOPA, was used as suicide

inhibitor. A subtle difference between these two approaches

arises from the tracer kinetic properties of the imaging

agents utilized, with each assessing a different aspect of

enzyme function. The tracer kinetics measured with

covalently bound [11C]-l-deprenyl reflects enzyme protein

levels while the tracer kinetics of a substrate such as

[18F]FMT, wherein the [18F]-labeled product formed is

essentially trapped, measures enzyme activity.

The mechanism for the suicide inhibition of AAAD by

FMDOPA was proposed by Maycock et al. [18] to involve

adduct-formation between the FMDOPA-derived inhibitor

and the AAAD enzyme. Recent crystal structural analysis of

AAAD by Burkhard et al. has provided additional insights

into the mechanism of FMDOPA suicide inhibition [5].

AAAD is a tightly associated a2-dimer belonging to the a-

family of pyridoxal 5V-phosphate (PLP)-dependent enzymes

[1]. In the first step in enzyme action, the co-factor PLP is

anchored in the active site of AAAD by an extended

hydrogen bond network through its phosphate group while

the protonated pyridine nitrogen stabilizes the carbanionic

Schiff base intermediate formed with the substrate [5].

FMDOPA, like l-DOPA, is thought to form this Schiff base

intermediate with the enzyme-bound PLP co-factor, with the

carboxylate group forming hydrogen bonds with His 192 in

the enzyme active site [5]. Elimination of carbon dioxide

yields a decarboxylated FMDOPA quinoid intermediate

which, instead of being released as FMdopamine analogous

to dopamine release after l-DOPA decarboxylation, under-

goes fluoride elimination leaving a reactive alkylating

electrophile which then forms a covalent bond at the

enzyme’s active site. This alkylation reaction essentially

blockades the AAAD active site thereby incapacitating the

enzyme. In order to recover decarboxylation function,

synthesis of new AAAD protein in the dopamine cell

bodies becomes necessary. New protein synthesis requires

gene transcription of new AAAD mRNA followed by

translation into new AAAD enzyme proteins as demon-

strated by Li et al. in vitro in PC12 cells using the dopamine

inhibitor NSD 1015 [17]. New AAAD enzyme protein

synthesized in the nigral cell bodies is then delivered by

axoplasmic transport to the striatal terminals [12]. Monitor-

ing recovery of striatal enzyme activity using a substrate as

tracer allows the estimation of the combined rates of AAAD

protein synthesis and axonal transport.

The inhibitor dose used in this study was selected based

on the report by Jung et al. evaluating the effects of dl-

FMDOPA in mice [14]. In mice, 4 h after the administration

of 25 mg/kg FMDOPA, AAAD activity in the kidney and

heart was almost completely inhibited while whole brain

AAAD was inhibited about 28% of control [14]. In contrast,

after administration of 25 mg/kg FMDOPA, both macaques

in this microPET study showed complete striatal AAAD

inhibition at 4 and 26 h, respectively. Although peripheral

AAAD activity was not assessed in this study, evidence for

significant peripheral AAAD inhibition, which would slow

[18F]FMT plasma clearance and allow more unmetabolized

[18F]FMT to enter the brain, can be inferred from the

normalized PET images shown in Fig. 1 wherein images

obtained at 4 h and 49 h after FMDOPA treatment showed

higher background radioactivity compared to the images at

the three other time points.

Consistent with the notion that AAAD protein synthesis

occurs in the nigral dopamine cell bodies, Gardner and

Richards found that AAAD recovery after FMDOPA

inhibition in synaptosomal fractions (P2 fraction) in the

mouse brain lagged the recovery in sub-cellular fractions

rich with cell bodies (S2 fraction) by about 2 days [12].

Based on axonal length consistent with the mouse brain,

axonal transport rate of 1–6 mm/day was estimated [12].

Although, we can identify the substantia nigra in the PET

images and calculate nigral Ki values, the sample of two

animals did not provide sufficient power to obtain statisti-

cally significant measure of AAAD recovery which would

have been useful in estimating axoplasmic transport rate

under these conditions.

Although complete AAAD recovery was not observed

until an estimated 16 days after the total enzyme blockade

by suicide inhibition (Fig. 3), both animals displayed normal

movement and behavior within a day. This is not

unexpected because of known plasticity of the dopamine

system and suggests that compensatory mechanisms are

involved [30]. In mice, 6 h after the highest single

FMDOPA dose used (500 mg/kg) was given wherein

complete AAAD inhibition is assumed, dopamine levels

on a per gram whole brain basis dropped to less than 40% of

control. Because of higher dopamine concentration in the

striatal terminals, the drop in synaptic dopamine levels in

the striatum is likely larger. In this study, based on the PET-

estimated half-life of AAAD recovery of 86 h, 24 h after

treatment, one can estimate the recovered AAAD activity to

be about 20% of control. The observed behavioral and

motor recovery at this level of AAAD activity suggests that

O.T. DeJesus et al. / Brain Research 1054 (2005) 55–60 59

the dopamine levels under this condition are sufficient to

allow normal function. It is likely that compensatory

mechanisms similar to those reported in dopamine deficient

mice wherein normal locomotor activity can be restored

with striatal dopamine levels amounting to 9% of normal

values [26] are involved. Reduced dopamine levels can

trigger upregulation of the rate-limiting conversion of p-

tyrosine to l-DOPA by tyrosine hydroxylase (TH) by post-

translational changes, e.g., phosphorylation [10]. However,

because of much reduced AAAD activity after FMDOPA

inhibition, the rate-limiting step may have shifted from the

TH step to the AAAD step. Another pre-synaptic compen-

satory response may be the downregulation of metabolism

involving re-uptake by the dopamine transporter (DAT)

similar to that seen in pre-clinical PD [16]. On the post-

synaptic side, dopamine receptors may become super-

sensitive because of reduced dopamine levels as first

reported in 6-OHDA-lesioned animals [28] and recently

found in the TH-deficient dopamine depleted mice [15].

Such supersensitivity is likely related to increased levels of

the high affinity states of D2 receptors [25].

It is interesting to note that the symptomatic threshold for

PD is associated with declines in striatal dopamine levels in

the order of about 20–40% of normal levels [13]. A recent

PET study with [18F]FDOPA estimated the PD symptomatic

threshold to be at 47–62% of normal putaminal uptake

values [16]. The recovery of both animals within 24 h after

complete AAAD inhibition may therefore be similar to the

pre-clinical stage of PD but since normal brains are involved

in this study the observed faster motor and behavioral

recovery is a reasonable outcome. A third animal given the

same dose of FMDOPA in the afternoon and whose PET

scan was planned for the following day was found to be

surprisingly alert and displayed normal movement and

behavior the next morning. The seeming lack of FMDOPA

effect on this animal prompted the misguided decision to

forego its PET study.

In summary, the in vivo turnover rate of AAAD enzyme

protein in the rhesus macaque striatum was assessed by

monitoring recovery of enzyme activity after suicide inhib-

ition by microPET imaging using the tracer [18F]FMT.

Results showed that the AAAD turnover half-life is about

86 h and total recovery is estimated to be within 16 days after

complete inhibition. However, normal movement and behav-

ior were observed within 24 h at which time AAAD function

was shown by the PET data to be about 20% of normal

levels. This suggests that pre- and post-synaptic compensa-

tory mechanisms allow recovery of normal movement and

behavior earlier than recovery of AAAD function. FMDOPA

inhibition and microPET imaging, using other pre- and post-

synaptic dopamine imaging agents, in non-human primates

may be useful in elucidating these compensatory mecha-

nisms. Furthermore, a possible use of AAAD suicide

inhibition is in generating a reversible non-human primate

model of pre-symptomatic Parkinson’s disease wherein the

consequences of dopamine depletion on dopamine receptors

and transporters and other dopamine-interacting neurotrans-

mitter systems such as serotonin and GABA, may be

investigated in vivo longitudinally using the appropriate

PET imaging agents.

Acknowledgment

The support of NIH Grant NS 26621 is gratefully

acknowledged.

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