Aromatic L-amino Acid Decarboxylase Activity Research Articles

P.037 Refractory status dystonicus and hypotension after cardiac arrest in a child with AADC deficiency post gene therapy: a case report

Background: Aromatic l-amino acid decarboxylase (AADC) deficiency is a metabolic disorder that causes deficient serotonin, dopamine, and catecholamine synthesis. How children respond to neurological insult post intracranial gene therapy remains underreported. We present a 10 year old girl with profound neurological injury after a brief in-hospital cardiac arrest, secondary to viral infection-induced respiratory failure, 4 years after gene therapy. Methods: Patient’s chart review included brain imaging, clinical notes, laboratory results, and treatment. Results: MRI showed symmetric abnormalities in the basal ganglia, thalami, cortex, and cerebellar hemispheres. CSF analysis showed homovanillic acid 27 nmol/L (reference range 167-563) and 5-hydroxyindoleacetic acid 7 nmol/L (reference range 67-189). She developed generalized dystonia and oculogyric crises which were not seen since before gene therapy. There was poor catecholamine production causing refractory hypotension. She required a one-month stay in ICU for hypotension and status dystonicus. Dystonia was controlled with high doses of 6 agents. Conclusions: We describe a patient with AADC deficiency post gene therapy who experienced disproportionately severe neurological injury and decreased AADC activity after hypoxic neurological insult. There may be unique considerations of dopaminergic neuron integrity, AADC gene promoter sensitivity, and cerebrovascular autoregulation in children with AADC deficiency post gene therapy.

A novel mechanism of idiopathic orthostatic hypotension and hypocatecholaminemia due to autoimmunity against aromatic l-Amino acid decarboxylase

Orthostatic hypotension (OH) is a common condition. Many potential etiologies of OH have been identified, but in clinical practice the underlying cause of OH is often unknown. In the present study, we identified a novel and extraordinary etiology of OH. We describe a first case of acquired severe OH with syncope, and the female patient had extremely low levels of catecholamines and serotonin in plasma, urine and cerebrospinal fluid (CSF). Her clinical and biochemical evidence showed a deficiency of the enzyme aromatic l-amino acid decarboxylase (AADC), which converts l-DOPA to dopamine, and 5-hydroxytryptophan to serotonin, respectively. The consequence of pharmacologic stimulation of catecholaminergic nerves and radionuclide examination revealed her catecholaminergic nerves denervation. Moreover, we found that the patient's serum showed presence of autoantibodies against AADC, and that isolated peripheral blood mononuclear cells (PBMCs) from the patient showed cytokine-induced toxicity against AADC. These observations suggest that her autoimmunity against AADC is highly likely to cause toxicity to adrenal medulla and catecholaminergic nerves which contain AADC, resulting in hypocatecholaminemia and severe OH. Administration of vitamin B6, an essential cofactor of AADC, enhanced her residual AADC activity and drastically improved her symptoms. Our data thus provide a new insight into pathogenesis and pathophysiology of OH.

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n=143) and genotype (n=151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency=32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency=21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

Gene therapy for aromatic L-amino acid decarboxylase deficiency by MR-guided direct delivery of AAV2-AADC to midbrain dopaminergic neurons

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4–9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

Peripheral decarboxylase inhibitors paradoxically induce aromatic L-amino acid decarboxylase

Peripheral decarboxylase inhibitors (PDIs) prevent conversion of levodopa to dopamine in the blood by the enzyme aromatic L-amino acid decarboxylase (AADC). Alterations in enzyme activity may contribute to the required higher dosages of levodopa observed in many patients with Parkinson’s disease. We evaluated the effect of levodopa/PDI use on serum AADC enzyme activity. Serum AADC enzyme activity was evaluated in three independent cohorts of patients with Parkinson’s disease or parkinsonism (n = 301) and compared between patients on levodopa/PDI vs. patients not on this medication. AADC enzyme activity was elevated in 62% of patients on levodopa/PDI treatment, compared to 19% of patients not on levodopa/PDI (median 90 mU/L vs. 50 mU/L, p < 0.001). Patients with elevated AADC activity had longer disease duration and higher doses of levodopa/PDI. These findings may implicate that peripheral AADC induction could underlie a waning effect of levodopa, necessitating dose increases to maintain a sustained therapeutic effect.

Dnmt3a2/Dnmt3L Overexpression in the Dopaminergic System of Mice Increases Exercise Behavior through Signaling Changes in the Hypothalamus.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

Anterior pituitary gland synthesises dopamine from l-3,4-dihydroxyphenylalanine (l-dopa).

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l-3,4-dihydroxyphenylalanine (l-dopa). Accordingly, we investigated the expression of aromatic l-amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l-dopa and its release in vitro. Then, we explored the effects of l-dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l-dopa. Also, the presence of l-dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l-dopa. In addition, l-dopa reduced corticotrophin-releasing hormone-stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD-1015. Moreover, DA formation from l-dopa increased apoptosis and decreased proliferation. However, in the presence of NSD-1015, l-dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l-dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l-dopa exerts direct actions independently from its metabolisation to DA.

Dnmt3a2/Dnmt3L Overexpression in the Dopaminergic System of Mice Increases Exercise Behavior Through Signaling Changes in the Hypothalamus

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt and controls. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, regulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis. Keywords: DNA methylation, transgenic mouse model, behavioral paradigms, positron emission tomography, dopaminergic system.

Longitudinal study of striatal aromatic l-amino acid decarboxylase activity in patients with idiopathic rapid eye movement sleep behavior disorder

Study objectivesTo determine if nigrostriatal dopaminergic system function, evaluated by aromatic l-amino acid decarboxylase (AADC) activity using 6-[18F]fluoro-meta-tyrosine brain positron emission tomography (FMT-PET) can accurately and efficiently identify idiopathic rapid-eye-movement behavior disorder (IRBD) individuals at risk for conversion to a clinical diagnosis of Parkinson's disease (PD) or dementia with Lewy bodies (DLB). MethodsWe assessed prospectively striatal aromatic l-amino acid decarboxylase activity using FMT brain PET imaging in IRBD patients who were followed systematically every 1–3 months for 1–10 years. IRBD patients (n = 27) were enrolled in this prospective cohort study starting in 2009. Those who underwent follow-up scans between January 2011 and September 2014 (n = 24) were analyzed in the present study. ResultsOf the 24 IRBD patients with baseline and follow-up FMT-PET scans, 11 (45.8%) developed PD (n = 6) or DLB (n = 5). Compared to IRBD patients who were still disease-free, those who developed PD (n = 5) or DLB with parkinsonism (n = 1) had significantly reduced bilateral putaminal FMT uptake during the follow-up. Furthermore, the rate of FMT decline between baseline and follow-up scans was higher in all converted patients, even for those with DLB without parkinsonism, than in IRBD patients who remained disease-free. ConclusionsFMT-PET, which represents a dynamic change in AADC activity over time, may also be a useful predictor for the risk of conversion to PD or DLB over short-term clinical follow-up periods, or when testing neuroprotective and restorative strategies in the prodromal phases of PD or DLB.

11 C-PE2I and 18 F-Dopa PET for assessing progression rate in Parkinson's: A longitudinal study.

18 F-dopa PET measuring aromatic l-amino acid decarboxylase activity is regarded as the gold standard for evaluating dopaminergic function in Parkinson's disease. Radioligands for dopamine transporters are also used in clinical trials and for confirming PD diagnosis. Currently, it is not clear which imaging marker is more reliable for assessing clinical severity and rate of progression. The objective of this study was to directly compare 18 F-dopa with the highly selective dopamine transporter radioligand 11 C-PE2I for the assessment of motor severity and rate of progression in PD. Thirty-three mild-moderate PD patients underwent 18 F-dopa and 11 C-PE2I PET at baseline. Twenty-three were followed up for 18.8 ± 3.4 months. Standard multiple regression at baseline indicated that 11 C-PE2I BPND predicted UPDRS-III and bradykinesia-rigidity scores (P < 0.05), whereas 18 F-dopa Ki did not make significant unique explanatory contributions. Voxel-wise analysis showed negative correlations between 11 C-PE2I BPND and motor severity across the whole striatum bilaterally. 18 F-Dopa Ki clusters were restricted to the most affected putamen and caudate. Longitudinally, negative correlations were found between striatal Δ11 C-PE2I BPND , ΔUPDRS-III, and Δbradykinesia-rigidity, whereas no significant associations were found for Δ18 F-dopa Ki . One cluster in the most affected putamen was identified in the longitudinal voxel-wise analysis showing a negative relationship between Δ11 C-PE2I BPND and Δbradykinesia-rigidity. Striatal 11 C-PE2I appears to show greater sensitivity for detecting differences in motor severity than 18 F-dopa. Furthermore, dopamine transporter decline is closely associated with motor progression over time, whereas no such relationship was found with aromatic l-amino acid decarboxylase. 11 C-PE2I may be more effective for evaluating the efficacy of neuroprotective treatments in PD. © 2017 International Parkinson and Movement Disorder Society.

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

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.

201. Neuron-Specific Systemic Gene Therapy for Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that causes defective synthesis of dopamine and serotonin, and children with AADC deficiency exhibit severe motor, behavioral and autonomic dysfunctions. We have created an IVS6+4A>T knock-in mouse model of AADC (DdcKI mice) and shown that gene therapy at the neonatal stage can rescue the phenotype. In this study, we extended the treatment to systemic therapy on young mice. After intraperitoneal injection of 7-day-old mice with either AAV9-CMV-hAADC (AAV9-AADC) or yfAAV9/3-Syn-I-mAADC (AAVN-AADC), the treated DdcKI mice showed improvements in weight gain, survival, motor function, autonomic function, and behavior, but the effects of AAVN-AADC were superior. The survival of AAVN-AADC treated DdcKI mice (95%) was slightly better than that of the AAV9-AADC treated mice (78%), but this difference was not significant. Brain AADC activity of both AAV9-AADC and AAVN-AADC treated mice was slightly elevated (2.3% and 2.7% of wild-type, respectively). Untreated DdcKI mice had difficulties maintaining body temperature during cold exposure, and both AAV9-AADC- and AAVN-AADC-treated DdcKI mice exhibited body temperature control similar to that of WT mice in cold environments. Moreover, the AAV9-AADC-treated DdcKI mice exhibited slight hyperactivity. Under low magnification, AADC-positive cells were found in the cortex of AAV9-AADC-treated mice. Therefore, mice with a neurotransmitter deficiency can be rescued at a young age using systemic gene therapy, but a neuron-specific vector may be necessary.

Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

Progressively blunted response to L-DOPA in Parkinson’s disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of function of AAV2-hAADC transgene product in PD gene therapy.

Spinal Cord Hemisection Facilitates Aromatic L-Amino Acid Decarboxylase Cells to Produce Serotonin in the Subchronic but Not the Chronic Phase.

Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC) cells to produce 5-HT from its precursor (5-hydroxytryptophan, 5-HTP) is dramatically increased following complete spinal cord transection. In this study, we investigated whether a partial loss of 5-HT innervation could similarly increase AADC activity. Adult rats with spinal cord hemisected at thoracic level (T11/T12) were used with a postoperation interval at 5 days or 60 days. Using immunohistochemistry, first, we observed a significant reduction in the density of 5-HT-immunoreactive fibers in the spinal cord below the lesion on the injured side for both groups. Second, we found that the AADC cells were similarly expressed on both injured and uninjured sides in both groups. Third, increased production of 5-HT in AADC cells following 5-HTP was seen in 5-day but not in 60-day postinjury group. These results suggest that plastic changes of the 5-HT system might happen primarily in the subchronic phase and for longer period its function could be compensated by plastic changes of other intrinsic and/or supraspinal modulation systems.

Renalase regulates peripheral and central dopaminergic activities.

Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

Aromatic L-Amino acid decarboxylase deficiency: A new case from Turkey with a novel mutation

Aromatic L-amino acid decarboxylase (AADC), a vitamin B6-requiring enzyme that converts L-dopa to dopamine and 5-hydroxytryptophan to serotonin. Deficiency of this enzyme results in developmental delay, muscular hypotonia, dystonia, involuntary movements, autonomic dysfunction, and oculogyric crises. We now report a 2-year-old Turkish boy with AADC deficiency confirmed by greatly reduced AADC activity in the plasma and by genetic studies. Mutation analysis revealed a homozygous mutation c.208C > T (p. His70Tyr) in exon 3 of the AADC gene which has not been described to date.

Regulation of the dopaminergic system in a murine model of aromatic l-amino acid decarboxylase deficiency

Aromatic l-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. Children with AADC deficiency exhibit compromised development, particularly with regard to their motor functions. Currently, no animal model of AADC deficiency exists. We inserted an AADC gene mutation (IVS6+4A>T) and a neomycin-resistance gene into intron 6 of the mouse AADC (Ddc) gene. In the brains of homozygous knock-in (KI) mice (DdcIVS6/IVS6), AADC mRNA lacked exon 6, and AADC activity was <0.3% of that in wild-type mice. Half of the KI mice were born alive but grew poorly and exhibited severe dyskinesia and hindlimb clasping after birth. Two-thirds of the live-born KI mice survived the weaning period, with subsequent improvements in their growth and motor functions; however, these mice still displayed cardiovascular dysfunction and behavioral problems due to serotonin deficiencies. The brain dopamine levels in the KI mice increased from 9.39% of the levels in wild-type mice at 2weeks of age to 37.86% of the levels in wild-type mice at 8weeks of age. Adult KI mice also exhibited an exaggerated response to apomorphine and an elevation of striatal c-Fos expression, suggesting post-synaptic adaptations. Therefore, we generated an AADC deficient mouse model, in which compensatory regulation allowed the mice to survive to adulthood. This mouse model will be useful both for developing gene therapies for AADC deficiency and for designing treatments for diseases associated with neurotransmitter deficiency.

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.

Blunted renal dopaminergic system in a mouse model of diet-induced obesity

Obesity has reached epidemic proportions in the Western world and is implicated in the pathophysiology of essential hypertension. The aim of the present study was to evaluate sodium handling, blood pressure and renal dopaminergic system activity in a mouse model of obesity induced by exposure to a hypercaloric diet. From six to 18 weeks of age, animals were fed with a control diet or a high-fat high-simple-carbohydrate (HFHSC) diet. Renal function, blood pressure and urinary and plasmatic catecholamines and biochemical parameters were evaluated in both groups. In parallel, the effects of high sodium intake (HS, 1.0% NaCl, 3 days) on natriuresis, urinary catecholamine excretion and aromatic l-amino acid decarboxylase (AADC) activity were evaluated in control and obese mice. Mice exposed to the HFHSC diet presented obesity, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia and increased blood pressure. This was accompanied, in obese mice, by decreases in urinary excretion of dopamine and metabolites as well as reduced AADC activity in renal tissues. During HS intake, absolute urinary dopamine excretion increased in control, but not in obese mice. This was accompanied in obese mice by a natriuretic resistance on day 1 of the HS diet. In addition, obese mice presented increased urinary and plasmatic noradrenaline levels, as well as an increased heart rate when compared with control mice. In conclusion, in this model of diet-induced obesity hyperinsulinemia, insulin resistance and increased sympathetic tone are associated with blunted renal dopaminergic activity. It is suggested that this may contribute to compromised sodium excretion and increased blood pressure in obesity.

Safety and Tolerability of Magnetic Resonance Imaging-Guided Convection-Enhanced Delivery of AAV2-hAADC with a Novel Delivery Platform in Nonhuman Primate Striatum

Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.