Depletion of AADC activity in caudate nucleus and putamen of Parkinson's disease patients; implications for ongoing AAV2-AADC gene therapy trial

Abstract

In Parkinson's disease (PD), aromatic L-amino acid decarboxylase (AADC) is the rate-limiting enzyme in the conversion of L-DOPA (Sinemet) to dopamine (DA). Previous studies in PD animal models demonstrated that lesion of dopaminergic neurons is associated with profound loss of AADC activity in the striatum, blocking efficient conversion of L-DOPA to DA. Relatively few studies have directly analyzed AADC in PD brains. Thus, the aim of this study was to gain a better understanding of regional changes in AADC activity, DA, serotonin and their monoamine metabolites in the striatum of PD patients and experimentally lesioned animals (rat and MPTP-treated nonhuman primate, NHP). Striatal AADC activity was determined post mortem in neuropathologically confirmed PD subjects, animal models and controls. A regional analysis was performed for striatal AADC activity and monoamine levels in NHP tissue. Interestingly, analysis of putaminal AADC activity revealed that control human striatum contained much less AADC activity than rat and NHP striata. Moreover, a dramatic loss of AADC activity in PD striatum compared to controls was detected. In MPTP-treated NHP, caudate nucleus was almost as greatly affected as putamen, although mean DA turnover was higher in caudate nucleus. Similarly, DA and DA metabolites were dramatically reduced in different regions of PD brains, including caudate nucleus, whereas serotonin was relatively spared. After L-DOPA administration in MPTP-treated NHP, very poor conversion to DA was detected, suggesting that AADC in NHP nigrostriatal fibers is mainly responsible for L-DOPA to DA conversion. These data support further the rationale behind viral gene therapy with AAV2-hAADC to restore AADC levels in putamen and suggest further the advisability of expanding vector delivery to include coverage of anterior putamen and the caudate nucleus.

Fig 1. Dopamine metabolism after L-DOPA treatment in overlesioned MPTP monkeys.

Dopamine and its metabolites were evaluated in different regions of the striatum of asymmetrically parkinsonian NHP that had been administered different doses of Sinemet 45 min prior to necropsy. Each graph shows data obtained from an individual animal receiving a different Sinemet dose. HPLC analysis of the different brain regions revealed that the severely lesioned side (ICA + iv MPTP) was almost completely devoid of DA and its metabolites compared to the partially lesioned side (iv MPTP), in which reduction was also milder in most of the regions analyzed (A). Analysis of the same regions in MPTP animals euthanized 45 min after the last dose of Sinemet indicated a similarly reduced conversion of L-DOPA into DA to that seen in the control animal (A), regardless of the dose of Sinemet received [(B) Sinemet 100 mg, (C) Sinemet 250 mg or (D) Sinemet 350 mg]. Another animal was chronically treated for 2 months with 200 mg Sinemet daily and euthanized 45 min after the last Sinemet dose (E). This animal had higher L-DOPA levels on both sides, whereas DA levels remained as low as those in the acutely challenged animals. Abbreviations: acM: nucleus accumbens; dC: dorsal caudate nucleus dP: dorsal putamen; ICA: intracarotid artery; iv: intravenous; vC: ventral caudate nucleus; vP: ventral putamen; oC: temporal cortex; tC: temporal cortex.

Fig 2. Dopaminergic metabolites in Parkinson’s patients and age-matched controls.

AADC activity (A), dopamine levels (B), DOPAC levels (C) and HVA (D) levels in anterior portion of the caudate nucleus and different sub-regions of the putamen in control subjects (o, n = 8) and patients with PD (•, n = 8). Reduction of AADC activity was accompanied by greatly reduced levels of DA and its metabolites throughout the putamen. Caudate nucleus exhibited the same magnitude of reduction observed in putamen. Scatter plots depict mean ± SEM for each cohort and differences were analyzed between groups by unpaired, two-tailed t-test. *: p ≤ 0.05; **: p ≤ 0.01, ***: p ≤ 0.001.

Fig 3. Serotonergic metabolism in Parkinson’s disease patients and age-matched controls.

Levels of serotonin (A) and its metabolite, 5-HIAA (B) were measured by HPLC in the anterior caudate nucleus and different sub-regions of the putamen in control subjects (o, n = 8) and patients with PD (•, n = 8). All regions analyzed showed serotonin and 5-HIAA data overlap between control and PD groups and were only significantly reduced in the anterior dorsal putamen and the anterior caudate nucleus. Scattered plots depict mean ± SEM for each cohort and differences were analyzed between groups by unpaired, two-tailed t-test. *: p ≤ 0.05; **: p ≤ 0.01, ***: p ≤ 0.001.

Fig 4. Monoamine levels measurement in overlesioned parkinsonian monkeys.

MPTP administration resulted in an asymmetric parkinsonism in NHP presenting a more severe dopaminergic degeneration on the right striatum than that on the left. Accordingly, analysis of monoamine levels by HPLC revealed that there was a dramatic reduction of DA and its metabolites in the more severely lesioned putamen (right) compared to the mildly lesioned one (left) (a). In contrast, serotonin and its metabolite levels, measured in the same samples, were similarly depleted in both hemispheres (b).