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A Dose-Ranging Study of AAV-hAADC Therapy in Parkinsonian Monkeys
Abstract
The main medication for idiopathic Parkinson disease is l-Dopa. Drug efficacy declines steadily in part because the converting enzyme, aromatic l-amino acid decarboxylase (AADC), is lost concomitant with substantia nigra atrophy. Over the past decade, we have developed a gene therapy approach in which AADC activity is restored to the brain by infusion into the striatum of a recombinant adeno-associated virus carrying human AADC cDNA. We report here the results of an investigation of the relationship between vector dose and a series of efficacy markers, such as PET, l-Dopa response, and AADC enzymatic activity. At low doses of vector, no effect of vector was seen on PET or behavioral response. At higher doses, a sharp improvement in both parameters was observed, resulting in an approximate 50% improvement in l-Dopa responsiveness. The relationship between vector dose and AADC enzymatic activity in tissue extracts was linear. We conclude that little behavioral improvement can be seen until AADC activity reaches a level that is no longer rate limiting for conversion of clinical doses of l-Dopa into dopamine or for trapping of the PET tracer FMT. These findings have implications for the design and interpretation of clinical studies of AAV-hAADC gene therapy.
INTRODUCTION
In Parkinson disease (PD), neurons that originate in the substantia nigra and project to the striatum (caudate and putamen) atrophy, thereby causing a dopamine deficiency in the striatum that leads in turn to the principal neuropathology of the disorder [1,2,4]. The mesolimbic pathway is also involved in idiopathic PD [7], but degeneration occurs at a much slower rate. Although some degeneration usually occurs also in the peripheral sympathetic nervous system precipitating orthostatic hypotension and intestinal symptoms [20], the most serious and debilitating symptoms arise from imbalances in dopaminergic activity within the nigrostriatal pathway and between the mesolimbic and nigrostriatal systems.
Dopamine synthesis requires the conversion of tyrosine to l-3,4-hydroxyphenylalanine (l-Dopa) by tyrosine hydroxylase (TH) and then decarboxylation of l-Dopa by aromatic l-amino acid decarboxylase (AADC). Because dopamine cannot cross the blood–brain barrier, l-Dopa has become the standard pharmacological therapy for PD and is highly effective early in the disease, presumably because there is still enough AADC left in the diminishing nigral terminals to exceed a rate-limiting level [11,12]. With disease progression, however, AADC becomes more severely rate limiting, and both increased and more frequent doses of substrate (l-Dopa) are required for clinical response [15]. Increased l-Dopa (and dopamine) results in excessive stimulation of the relatively intact mesolimbic system, which may be responsible for some of the side effects such as hallucinations. Nonuniform degeneration within the nigrostriatal system may be responsible for some complications of treatment such as involuntary movements termed l-Dopa-induced dyskinesias (LID) [12]. Thus, dose escalations of l-Dopa are invariably associated with the development of LID and other side effects, the management of which represents one of the greatest challenges in the long-term treatment of PD [14,16]. We have developed a strategy to overcome this loss of efficiency in conversion of l-Dopa to dopamine. Overexpression of AADC in the striatum of MPTP-lesioned monkeys leads to substantial restoration of dopaminergic activity [3].
In the present study, we dosed pairs of parkinsonian monkeys with one of six doses of AAV-hAADC over about a 2-log range. Low doses of vector had little effect either upon positron emission tomography (PET) signal or upon behavioral response to l-Dopa. Higher doses of vector effected a sharp transition to complete restoration of PET signal equivalent to that seen in normal monkey striatum. This was accompanied by an equally dramatic increase in sensitivity to l-Dopa. In contrast, AADC enzyme activity in tissue extracts increased linearly with vector dose. We argue that increases both in PET signal and in behavioral response to l-Dopa depend critically on the achievement of non-rate-limiting concentrations of AADC in the striatum with respect to relevant concentrations of FMT radiotracer or behaviorally effective l-Dopa levels. This insight has significant implications for the design and interpretation of clinical studies with AAV-hAADC now under way.
RESULTS
Experimental Time Line
In this study, we infused pairs of hemiparkinsonian monkeys with AAV-hAADC over a broad range of doses. We euthanized all animals 6 months after AAV infusion, at which time we dissected the brains to perform enzymatic assays. We subjected all animals to a schedule of treatments that commenced with MPTP lesioning on one side of the brain (ipsilateral). This involves a single intracarotid infusion of MPTP as described under Materials and Methods, followed by a combination of repeated behavioral testing and intravenous MPTP until a stable lesion is obtained, i.e., no change in behavioral rating over a 4-month period. Next, we gave the animals a PET scan to ensure a complete lesion on the ipsilateral striatum that included caudate nucleus, putamen, and globus pallidus. We then infused the animals with AAV as described above. We allowed 6 months for them to recover from surgery and to express transgene fully. They then underwent several months of acute l-Dopa testing in which we scored them on a scale similar to the Unified Parkinson’s Disease Rating Scale after they received intramuscular injections of l-Dopa. When testing was complete, we euthanized the animals, and necropsy yielded an ipsilateral hemisphere used for assay of AADC activity in striatum and a contralateral hemisphere fixed in formalin for histochemical analysis (manuscript in preparation).
FMT-PET Scanning
We used PET to monitor AADC activity both before and approximately 6 weeks after gene transfer. Before MPTP-lesioning, the mean FMT-PET ratio in the ipsilateral striatum was 2.72 ± 0.63 (n = 8). After lesioning, animals displayed ipsilateral ratios of 1.51 ± 0.23 (n = 12). As shown in Fig. 1, dosing monkeys with 6–55 Units AAV-hAADC per striatum or less resulted in little apparent effect on FMT ratio. At the 55-Unit dose, one animal had a ratio of approximately 2.3 and the other a ratio of 1.7. At doses greater than that, however, animals exceeded a ratio of 2.0, close to what is seen in normal animals. These data argue for the presence of a distinct transition at about 55 Units. Even so, FMT-PET afforded us the ability to pool animals into an unresponsive group (A) and a responsive group (B), irrespective of the dose of vector they had received. The vertical dimensions of the two boxes in Fig. 1 indicate the range of PET ratios in lesioned (A) and unlesioned (B) animals. All but one of the animals that showed restoration of AADC activity by PET imaging had received a dose of AAV-hAADC of 100 Units or greater per striatum.
Effect of dose of AAV-hAADC on FMT-PET. Pairs of rhesus monkeys were infused in each striatum with the indicated dose of vector. Note that 1 unit AAV = 1 × 109 vg. Animals shown as receiving a dose of 0 received 500 units AAV-GFP per striatum. FMT-PET was performed on all animals 4–6 weeks after vector infusion. Individual PET data points from the lesioned (ipsilateral) side of the brain are presented as a ratio of striatal signal to background signal derived from cerebellum. PET ratios fell into two groups: those in box A have values that approximate values obtained from a historical mean of 12 MPTP-lesioned animals of 1.51 ± 0.23. The vertical dimensions of boxes A and B indicate the range of values. After lesioning, animals displayed ipsilateral ratios of 1.51 ± 0.23 (n = 12). In contrast, data in box B are within the range of ratios found in normal brain (2.72 ± 0.63; n = 8). This segregation of data was used to evaluate corresponding clinical response to l-Dopa in the two groups, and the results from this analysis is shown in Fig. 3. Two animals in group A are from the control group that received no AAV-hAADC but received 500 units AAV-GFP per striatum instead. Group A contains data from seven animals, and group B five animals.
We suspected that this transition derived from the fact that we were measuring expression of an enzyme (AADC) and that the PET signal showed a dramatic increase only when AADC levels exceeded a rate-limiting tissue concentration. If so, the correlation between PET and dose in group A should have been slightly positive as the increasing enzyme content in the target tissue permitted better conversion of FMT [10]. When AADC levels reached a non-rate-limiting level, the correlation between dose and PET signal should change. As shown in Fig. 2A the correlation between AAV-hAADC dose and PET signal in group A has a shallow positive slope indicating an appropriate relationship between AADC expression and PET signal. In group B (Fig. 2B), there is no positive slope, in fact a slightly negative correlation, indicating that increases in dose beyond 55 Units are no longer rate limiting. It should be noted, however, that the effect of dose on PET signal is muted by the fact that the concentration of FMT in the brain during scans is very low relative to the Km of AADC for l-Dopa of approxi mately 0.1 mM (J. R. Forsayeth and Z. Zhen, unpublished data), assuming that the Km for FMT is similar to that of l-Dopa. Also, the scans represent accumulation of FMT metabolites over 30 min and do not, therefore, measure a true linear enzymatic rate. It is all the more remarkable then that this difference in effect between low and high doses of vector emerged in the FMT-PET experiment.
Behavioral Response
We compared acute responses to l-Dopa administration in both group A and group B, by determining the dose of l-Dopa that most effectively minimized the clinical rating scale (CRS) before and after gene therapy (Fig. 3). Testing monkeys for l-Dopa response is complicated by the necessity to limit stress and harm to the animals. Dose escalation is not continued beyond those at which side effects begin to appear. Thus, under some conditions, a dose of 20 mg/kg is acceptable, but not under other conditions under which some hyperactivity, dis-tractibility, and apparent hallucinations have been observed at 10 mg/kg. Similarly, if little response was observed at 3 mg/kg l-Dopa, animals were not subjected to the stress of being tested at lower doses. Because each animal displays a different baseline CRS, response to l-Dopa is reported as “% behavioral improvement,” and the decrease in CRS in response to drug relative to baseline is recorded. It is also common to see statistically anomalous differences between randomized cohorts of lesioned animals in terms of absolute response to l-Dopa. Thus, in the preinfusion testing, group B happened to be somewhat more responsive to high doses of l-Dopa than was group A. Thus, the valid point of comparison is between the post- and the pre-infusion responses, rather than between groups A and B. Infusion of low doses of AAV-hAADC (0–55 units AAV) had no significant effect on acute l-Dopa response (Fig. 3A). In contrast, higher doses of vector (55–500 Units AAV) gave a dramatically improved sensitivity to l-Dopa (Fig. 3B), widening the therapeutic window considerably (global P < 0.05). Animals in group B improved their behavioral rating by approximately 50% at doses of l-Dopa as low as 1 mg/kg. The nonresponsive, low-vector-dose group barely responded to l-Dopa at 3 mg/kg. These data indicate that, at doses of AAV-hAADC that normalize the FMT-PET, the therapeutic window for l-Dopa can be dramatically improved.
l-Dopa dose response in groups A and B from Fig. 1. Animals were sorted into two groups, A and B, based upon whether their PET scan gave a ratio of (A) less than or (B) greater than 2.0. Note that all animals with a score of greater than 2.0 had received a dose of AAV-hAADC greater than 55 Units AAV per striatum. In this experiment, animals were exposed acutely (see Materials and Methods) to various doses of l-Dopa 1 month before (blue) or 6 months after (red) infusion of AAV-hAADC vector. One of the AAV-CFP animals was excluded from this analysis because it developed postsurgical behavioral abnormalities that interfered with testing. Thus, (A) represents data from six animals, and (B) represents data from five animals.
Postmortem Tissue Analysis
Subsequent to behavioral evaluation, we euthanized the animals to perform biochemical analyses to investigate dose-dependent effects of vector infusion. Convection-enhanced delivery (CED) of AAV-hAADC distributes into three regions: putamen, globus pallidus, and caudate. We have previously described a perivascular transport mechanism that actively distributes macromolecules, viruses, and nanoparticles effectively into these regions from a single infusion site [8]. We have found that the relative distribution into these three areas varies somewhat from animal to animal. However, all three areas are detected by the PET camera. Hence, it makes sense to assay enzymatic activity in a composite region to relate it back to the FMT-PET data. Accordingly, we measured AADC content in the ipsilateral hemisphere by dissecting those regions of the basal ganglia that contributed to the PET signal, namely putamen, caudate nucleus, and globus pallidus. These regions are sized relatively as 1:0.5:0.3, respectively. Measures of AADC per milligram of protein were similarly weighted to give a fairly accurate measure of the contribution of each region in terms of specific activity to the overall specific activity in the basal ganglia. As shown in Fig. 4, vector dose produced a linear increase in AADC activity at doses of vector <55 Units and an apparent plateau above 55 Units. Thus, in contrast to the PET data and the behavioral data, there is no evidence of a sudden transition in AADC activity with respect to vector dose. We concluded from these data that vector dosing produces a progressive increase in enzymatic activity until, at high doses, increased vector dose produces little further increase in expression of AADC transgene. This result demonstrates that transition from rate-limiting levels of AADC to non-rate-limiting levels is key in generating optimal improvement in response to l-Dopa and suggests that the therapeutic dose of AAV-hAADC in MPTP-lesioned monkeys needs only to exceed 55 Units AAV per striatum to achieve a plateau. The broad dose-ranging strategy employed here, however, does not permit fine-scale mapping of the dose range between 55 and 170 Units wherein the transition occurs.
Effects of dose of AAV-hAADC on AADC activity in tissue extracts. Brain tissue was isolated from animals euthanized 6 months after AAV-hAADC infusion. Punches from several brain regions (putamen, globus pallidus, and caudate) were taken and flash-frozen. Thawed samples were homogenized and assayed for AADC activity as described under Materials and Methods. The data represent a pool of the three sampled tissues to correlate as closely as possible with the region of interest for the PET scanning. However, since the three regions in question are not of equal size, their overall contribution to specific activity also differs. From the MRI scans made of all 12 animals, we were able to weight the relative contribution as follows: putamen:caudate:-globus pallidus 1.0:0.5:0.3. The activity obtained represents the sum of the adjusted activities from the three regions.
DISCUSSION
Individuals with idiopathic PD progress inexorably toward dependence on l-Dopa (Sinemet) therapy, which then begins to fail despite the use of adjunct therapies such as MAO-B and COMT inhibitors [2,18,19]. Because the enzyme that converts l-Dopa to dopamine, aromatic l-amino acid decarboxylase, is rate limiting in primates [22], loss of substantia nigra neurons results in progressive decline in l-Dopa responsiveness as the concentration of AADC enzyme declines. A potential therapeutic strategy, therefore, is to use gene therapy to restore adequate levels of AADC in the striatum, and thereby re-establish satisfactory l-Dopa responsiveness. To achieve this, we developed an AAV vector that drives constitutive expression of human AADC in neurons, along with a highly efficient means of infusing the virus into striatum (CED). This therapy is now being investigated in a clinical trial at the University of California San Francisco.
To understand the relationship between vector dose and behavioral effect, we infused six pairs of animals with a broad (2-log) range of vector doses. In this type of experiment, the emphasis is on overall correlations rather than on individual values. Even though vector was also infused on the slightly lesioned contralateral side (data not shown), we did not correlate vector dose and PET signal because of the high basal levels of endogenous activity on that side of the brain. Nevertheless, vector infusion correlated somewhat with PET signal, despite the confounding effect of the high background signal (data not shown). There is clearly no overall linear relationship between dose of AAV-hAADC and resulting PET ratios (Fig. 1). There is, however, a striking grouping of the data into low PET ratios below a ratio of 1.6 (group A) and another above 2.0 (group B). Within the high- and low-dose groups, however, weak linear correlations could be detected between vector dose. As expected, low doses of vector had a positive correlation with PET, but high doses appeared to have a slight negative correlation (Fig. 2). Our conclusion was that only non-rate-limiting levels of AADC can drive an improvement in PET signal. The slope of the curve is quite modest, most probably because the concentration of FMT in the PET procedure is very low, and the time scale over which scans are performed (30 min) means that we probably did not measure a true linear rate of accumulation of FMT metabolite in striatal neurons.
We asked whether such a grouping was associated with altered behavioral response to l-Dopa. As shown in Fig. 3, pooling of acute l-Dopa response data before, and 6 months after, vector infusion yielded dramatically different results. In the group of animals with low PET ratios, there was no significant change in response to l-Dopa over a range of doses. In contrast, in those animals with a PET ratio greater than 2.0, indicating high levels of AADC expression, a remarkable widening of the therapeutic window for l-Dopa was observed. On average, animals in this range improved their response to l-Dopa by 10- to 20-fold. If such an effect were recapitulated in humans, a substantial lowering of l-Dopa requirement would be expected with a concomitant decrease in l-Dopa-dependent side effects.
The most direct readout of vector dose versus AADC expression came from assaying AADC activity in homogenates of brain tissue (Fig. 4). We observed a very tight, linear relationship between vector dose and AADC expression at lower vector doses. However, at the highest two doses, there appeared to be a saturation phenomenon in which increases in dose beyond 170 Units AAV per striatum produced no increase in AADC activity. Because the data are much more scattered at the 170 and 500 Unit doses, further experiments will be required to determine unequivocally whether there is indeed a plateau above 170 Units. A potential deleterious consequence of AADC overexpression might be observed in terms of increased side effects in animals that received high doses of vector (Group B, Fig. 3) and high doses of l-Dopa. We observed side effects in high-dose animals at l-Dopa in excess of 10 mg/kg, consistent with the enhanced efficiency of l-Dopa conversion. However, it is equally clear that the remarkable shift leftward in dose response experienced in the high-dose group B would permit, if replicated in human studies, l-Dopa dosing to be reduced to a level only 10% of the level at which side effects would be encountered. It should be emphasized that AAV-hAADC is not a therapeutic agent per se. l-Dopa remains the therapeutic agent.
This study was designed to obtain a broad overview of how AAV-hAADC dose variation affects outcome measures in a primate model of PD. The study of this therapy is complicated by the fact that AAV-hAADC drives expression of an enzyme and thus confers merely a therapeutic capacity on the striatum. This capacity drives improved l-Dopa response in experimental animals, and clearly l-Dopa remains the therapeutic agent in AAV-hAADC therapy. Dose ranging with AAV-hAADC, therefore, requires an understanding of how enzyme kinetics play into the generation of behavioral responses. Our data indicate that the key driver of clinical efficacy may be whether enough AADC transgene is expressed to reach levels of catalysis that are no longer rate limiting. That level in monkeys appears to be reached at doses beyond about 55 Units AAV per striatum. At doses higher than this the amount of enzyme expression drives a dramatic improvement in behavioral responsiveness in hemipar-kinsonian animals. Future studies will focus on further defining the relationship between vector dose and behavioral effect in the range from 50 to 200 Units AAV per striatum.
MATERIALS AND METHODS
Induction of parkinsonism in rhesus monkeys
The 12 young adult (3–5 kg) rhesus monkeys used in this study were housed individually with food and water freely available, as well as a variety of novelty foods and environmental enrichment. Experiments were performed according to the NIH guidelines and to protocols approved by the UCSF Institutional Animal Care and Use Committee. All monkeys were lesioned by infusion of 2.5–3 mg of MPTP-HCl through the right internal carotid artery (referred to as the ipsilateral side) followed by four intravenous doses of 0.3 mg/kg MPTP-HCl to ensure a very stable, hemiparkinsonian syndrome [17]. Animals were evaluated weekly by a behavioral rating scale and monitoring of activity for 5 months prior to gene transfer. This model mimics, in one hemisphere, the biochemical and histopathological changes seen in advanced stages of idiopathic Parkinson disease. After MPTP administration, animals developed signs of Parkinson disease manifested by general slowness, bradykinesia, rigidity, balance disturbances, and flexed posture. The left arm was less frequently used than the right in all monkeys; all showed signs of tremor. All monkeys had moderate-to-severe, stable parkinsonian scores during the 5-month period after MPTP lesion.
AAV vectors
The human AADC cDNA was cloned into an AAV2 shuttle plasmid, and a recombinant AAV2 containing hAADC under the control of the cytomegalovirus promoter was generated by a triple-transfection technique and subsequent purification by CsCl gradient centrifugation [13,21]. AAV-hAADC was concentrated to approximately 4 × 1012 vector genomes (vg/ml) as determined by quantitative PCR. In the text, we define 1 × 109 vg as 1.0 Unit AAV to facilitate dose comparisons. Thus interconversion of vector genomes and Units AAV is easily accomplished by inserting or removing the term “× 109” after the quoted number of Units AAV. Our use of this term refers only to the quantitative measure of numbers of vector genomes in a given sample determined by quantitative PCR and is not per se a measure of biological activity.
CED of AAV vector
A day or so prior to surgery, animals were given an MRI scan while wearing a MRI-compatible stereotactic frame to obtain images for registration of precise stereotactic cannula insertion. Animals were sedated, prepared for surgery, and placed in the stereotactic frame. Vital signs were monitored, and a bone flap was made in the skull with a dental drill to expose areas of the dura over target sites. Fused silica cannulae, prepared as previously described [3], were stereotactically guided into the brain with coordinates generated by MRI. This unique type of cannula has a stepped design that prevents reflux of infused material. Care was taken to avoid any possibility of leakage into internal capsule or nontarget tissue.
AAV-hAADC or control AAV-GFP was infused bilaterally at four sites (two per hemisphere) into postcommissural putamen at the following rates: 0.1 µl/min (10 min), 0.2 µl/min (10 min), 0.5 µl/min (10 min), 0.8 µl/min (10 min), and 1.0 µl/min (36 min). The doses of AAV-hAADC vector used for each pair of animals were 0, 6, 18, 55, 170, or 500 Units AAV-hAADC per hemisphere and were diluted from concentrate (4000 Units AAV/ml) such that each indicated dose was delivered in a volume of approximately 50 µl at each infusion site. The pair of animals that received no AAV-hAADC received instead AAV-GFP at 500 Units per hemisphere. Although animals were lesioned on only one hemisphere, vector was delivered to both hemispheres to mimic insofar as was possible the intended human clinical study and also to provide tissue in the contralateral hemisphere for histological analysis (manuscript in preparation).
Approximately 10 min after infusion, the cannulae were raised at a rate of 1 mm/min until clear of both the striatum and the overlying cortex. Animals were monitored for full recovery and observed for behavior twice per day over the next 7 days.
Behavioral rating scale
The modified Parkinson CRS employed approxi mates those reported in the literature [9]. The scale evaluates 14 parkinsonian features, each of which receives a score from 0 to 3 in order of increasing severity. Individual scores are summed to arrive at a final score. Features evaluated include tremor (right and left sides), locomotion, “freezing,” fine motor skills (right and left sides), bradykinesia (right and left sides), hypokinesia, balance, posture, startle response, and gross motor skills (right and left sides). Normal animals score in the range 0–4, and severely parkinsonian monkeys score in the range 30–42. In these studies, we obtained animals with a CRS from 15 to 25. A single, highly experienced rater, blinded to the assignment of animals to dose cohorts, was used throughout. It should be noted that each animal achieves a stable score unique to that animal. As a result, absolute responses must be scaled relative to the baseline score and are quoted as “percentage improvement” at a given l-Dopa dose.
Behavioral response to L-Dopa
Each dosing session consisted of an intramuscular injection of l-DOPA and benserazide (to inhibit peripheral dopa decarboxylase) followed by a clinical evaluation that was performed approximately 45–60 min after administration of drug. Ascending and descending doses of l-DOPA were administered with a 3-day washout period between each dose. In most cases, animals were challenged with the same dose several times on different days. l-Dopa dosing commenced at a normally moderate dose from 5 to 10 mg/kg. Depending on the response observed, the following session either increased the l-Dopa dose or decreased it. Doses were not tested below a dose at which no behavioral improvement was observed nor above a dose at which side effects began to appear.
PET
PET was performed on a Siemens-CTI ECAT EXACT (Model 951) 31-slice scanner in 2D acquisition mode. The scanner has a resolution of approximately 5.8 mm FWHM at center and 7.7 mm radially (at 20 cm), with axial resolution 5 mm at the center and 7.1 mm FWHM at R = 20. The axial field of view of the scanner is 10.2 cm. Sensitivity is 110K cps/µCi/ml for a 20-cm cylinder phantom in 2D. [18F]FMT was produced with an on-site cyclotron, located directly adjacent to the scanner facility by a modification of the procedure described previously [5]. Immediately prior to PET imaging, the monkeys were anesthetized with an intramuscular injection of ketamine (15 mg/kg), intubated, and placed on isoflurane anesthesia. Normothermia was maintained with a heating blanket, and animals were kept hydrated via a saphenous catheter. An oximeter was used to monitor pO2, and arterial blood pressure was monitored with an intra-arterial catheter/transducer combination. The animals were placed in a stereotactic frame identical to that used for MRI. Images were acquired in the coronal plane. All animals were pretreated with an intravenous injection of benserazide (2 mg/kg), a peripheral decarboxylase inhibitor, 30 min before injection of the tracer. A 20-min transmission scan was obtained prior to the emission scan to correct for photon attenuation using a rotating 68Ge source consisting of three rods of about 2 mCi/rod. Emission data were collected for 30 min beginning 45 min after the injection of approximately 5 mCi of FMT.
The PET and MRI data sets were co-registered and regions of interest (ROIs) were drawn for the striatum and the cerebellum. ROIs were drawn directly on the MRI and subsequently mapped onto the PET images. Radioactivity counts were determined for each ROI. Radioactivity count ratios were created with the cerebellum as a reference tissue. The cerebellum was selected as a reference region because FMT uptake is negligible and should not change between baseline and post-treatment studies. A detailed discussion of this method may be found in Eberling et al. [6].
Measurement of AADC activity
AADC activity in tissue homogenates (10–30 mg) was determined by measuring the linear rate of dopamine production (µmol/min/mg protein). Where necessary, homogenates were diluted with assay buffer (50 mM phosphate buffer, pH 7.3, 200 µM pyridoxal phosphate, 200 µM pargyline phosphate, and 0.05 mM EDTA) to maintain a linear rate over the assay period (5–10 min). The assay was commenced by the addition of l-dopa (330 mM) to homogenate and terminated by the addition of 0.1 M perchloric acid containing 1% ethanol and 5 mM EDTA, and the reaction mix was then filtered through a 0.2-µm filter. The dopamine content of the filtrate was determined by HPLC with electrochemical detection by an ESA Coulochem II detector. The assay components were optimized to ensure that the assay measured AADC activity at Vmax. Homogenates (baseline) without substrate were also included as controls. Standard preparations of l-dopa and dopamine (Sigma Aldrich, St. Louis, MO, USA) were spiked into some control homogenates to calibrate the detection system.
ACKNOWLEDGMENTS
The authors thank Avigen for providing the AAV vector. We thank Dr. Fraser Wright for vector development and Dr. Jurg Sommer for advice on the enzyme assay. Funding for this work was provided by the NINDS Intramural Research Program, a U54 grant from NINDS, and Avigen, Inc. (Alameda, CA, USA) to K.S.B.