A pathogenic S250F missense mutation results in a mouse model of mild aromatic l-amino acid decarboxylase (AADC) deficiency

Aromatic l-amino acid decarboxylase (AADC) is a pyridoxal phosphate-dependent enzyme responsible for catalyzing the final step in the production of the monoamine neurotransmitters, dopamine and serotonin (1,2). Dopamine underlies the control of voluntary movement, the modulation of behavior, reward-based learning and the regulated release of various hormones. It is also a precursor of the catecholamines, noradrenaline and adrenaline. Serotonin serves as the substrate for melatonin, and is an important neuromodulator, regulating gastrointestinal function, the respiratory and cardiovascular systems, circadian rhythm, body temperature and the sensation of pain. Paucity of AADC therefore impacts many of the myriad physiological functions associated with dopamine, serotonin and their metabolites, and results in the rare but potentially life-threatening autosomal recessive disorder, AADC deficiency (3–8). AADC deficient patients become symptomatic as infants and characteristically exhibit hypotonia, hypokinesia, choreoathetosis and oculogyric crises. Autonomic dysfunction in these patients encompasses nasal congestion, diaphoresis and temperature instability (8).

AADC deficiency is customarily treated with monoamine oxidase (MAO) inhibitors, vitamin B6, usually in the form of pyridoxine, dopamine agonists or some combination thereof, but patients generally respond poorly, and those on the more severe end of the disease spectrum often succumb to the disorder during the first decade of life (3,8,9). A gene replacement strategy in four patients reportedly provided therapeutic benefit, but appeared to restore protein to only localized regions of the putamen, failed to greatly alter disease biomarkers and triggered dyskinesias or severe apnea in a proportion of the treated individuals (10). Accordingly, there is still an unmet medical need for a safe, reliable and effective treatment for human AADC deficiency.

AADC deficiency was initially described in 1990 and involved an instance of relatively mildly affected monozygotic twins who were found to harbor a homozygous mutation in the AADC gene, have markedly low levels of AADC activity and correspondingly little homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA)—catabolic products respectively of dopamine and serotonin (11). Consistent with the signature features of the disease, cerebrospinal fluid (CSF) levels of 3,4-dihydroxyphenylalanine (l-Dopa) and 5-hydroxytryptophan (5-HTP), precursors, respectively, of dopamine and serotonin, were grossly—12–15 times normal values—elevated and blood serotonin reduced by ∼95% in the patients (11). Following the initial description of the disease, ∼100 additional cases have been reported involving ∼28 different missense mutations (12). Although a definitive genotype–phenotype correlation has not emerged from the literature, patients harboring an intron 6 splice-site mutation, reportedly more frequent in the Chinese population, are more severely affected. In contrast, individuals, including the index cases, bearing a relatively frequent homozygous S250F mutation are milder. In an attempt to model AADC deficiency and better understand the human disease, Lee et al. knocked in the IVS 6 + 4A>T splice site mutation into the mouse genome (13). However, the mutation, devoid of an accompanying neo selection cassette, failed to mimic the splicing error observed in human patients, resulting in mice that were reportedly disease-free. Retention of the neo cassette within the engineered allele disrupted protein expression and triggered disease, but the resulting mutants do not owe their phenotype to a bona fide human mutation. In contrast, ablating murine AADC by excising exon 8 fails to produce viable mice, rendering it impossible to study the disease postnatally in constitutive knockouts (14). In this study, we report a novel mouse model of AADC deficiency that harbors the S250F mutation identified in the index patients. We show that although model mice are viable they express profoundly low levels of active AADC. Yet, dopamine levels in the basal ganglia of the mice were only modestly altered and neurons in the substantia nigra remained intact. In contrast, we found that serotonin levels in the mutants were markedly reduced and this perturbed behavior as well as autonomic function. We conclude that even minimal amounts of active AADC are sufficient to produce significant concentrations of dopamine and that low amounts of the enzyme per se do not impact the dopaminergic system. The AADC S250F mutant mouse we report here is expected to serve as a useful tool in the pre-clinical development of effective therapies for human AADC deficiency.


Generation of AADC S250F mutant mice

To create our murine model of AADC deficiency, we exploited an 835C>T missense mutation in exon 7 of the human AADC gene. The C>T change induces a S250F amino acid alteration in the AADC protein sequence and constitutes both the initial as well as second most commonly reported genetic lesion in AADC deficiency (4,11). Importantly, the serine residue that is replaced at this position by the mutation constitutes part of a highly conserved five amino acid domain, a suggestion that it is of structural and/or functional importance to the AADC protein (Fig. 1A). To mutate the corresponding amino acid in the murine genome to a phenylalanine, a knock-in strategy was employed. This involved a C>T change at position 890 of the murine Aadc gene; the targeting strategy is depicted in Figure 1B. Following electroporation of the targeting construct into embryonic stem (ES) cells, correctly targeted clones were identified by PCR and then confirmed by Southern blot analysis (Fig. 1C). They were then introduced into mouse blastocysts to generate knock-in mice. This resulted in the derivation of a single founder whose heterozygous state was detected by PCR and then confirmed by sequence analysis (Fig. 1D). The founder was further documented to transmit the mutant Aadc allele through the germline to his progeny.