Complementary approaches to investigate dopamine alteration: from Aromatic L-amino acid decarboxylase deficiency to Parkinson’s disease

Dopamine is an essential monoamine neurotransmitter that plays a crucial role in motor and behavioral functions. Impairment of dopamine can lead to a wide range of diseases, including monoamine neurotransmitter disorders such as Aromatic L-Amino Acid Decarboxylase (AADC) deficiency and neurological diseases like Parkinson’s disease (PD). AADC deficiency is an autosomal recessive inborn disorder caused by mutations of DDC gene, which may result in altered AADC protein. AADC is the fundamental enzyme that converts L-Dopa and L-5-hydroxytryptophan into dopamine and serotonin, respectively. Impairment of AADC leads to a systemic lack of dopamine. In contrast, PD is the second most common neurodegenerative disease characterized by dopaminergic neuronal loss in the substantia nigra pars compacta. Despite significant efforts, the underlying causes and mechanisms of PD pathology remain only partially understood. Although the differences between AADC deficiency and PD, patients have similar symptoms due to the lack of dopamine. In this study, AADC deficiency has been investigated with two different approaches: i. in-depth biochemical characterization of 18 pathogenic recombinant protein AADC variants, including 11 that have never been characterized before, and ii. investigation of 2 severe catalytic variants in DDC-KO SH-SY5Y neuroblastoma cell line. Characterization of AADC pathogenic variants, spread across the entire protein, provides a molecular basis for the AADC deficiency and further insight into AADC molecular mechanism function. The most affected variants map to the N-terminal domain and to the active site. Notably, eight of the characterized pathogenic variants exhibit a different kinetic behavior compared to the previous reported, showing positive or negative cooperativity interaction between the two active sites. Furthermore, a DDC knockout (KO) model in SH-SY5Y neuroblastoma has been obtained. Combination of morphology features and characterization of dopaminergic metabolic pathway confirmed its reliability as AADC deficiency model. Transient transfection in DDC-KO SH-SY5Y cells of two pathogenic variants, known to impair catalysis without altering protein structure, confirms their AADC loss-of-function and exhibits dopamine pathway metabolite patterns that mirror those observed clinically. Additionally, different subcultures of the SH-SY5Y cell line have been selected and subsequently treated with a new differentiation protocol to obtain dopaminergic-like neurons in the neuroblastoma cell line. Indeed, the selection of a subculture is required to obtain a reliable model for studying dopamine-related disorders. Dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) have been used to investigate the potential therapeutic mechanism of N-acetyl-L-Leucine (NALL) in PD. iPSC-derived dopaminergic neurons carrying LRRK2 mutations exhibit a markedly reduced phosphorylated Ser129-α-synuclein upon NALL treatment, likely driven by an increase in HTRA1 levels following treatment with NALL. Furthermore, NALL treatment increases parkin levels, resulting in increased synaptic membrane-associated synaptojanin-1, and accelerated synaptic vesicle endocytosis, also corroborated by an increase of vesicular monoamine transporter 2. These findings highlight the therapeutic potential of NALL in PD. Overall, this work underscores the importance of studying dopamine-related disorders with different approaches. Biochemical characterization of recombinant protein variants of AADC provides a deeper insight into AADC molecular function, which is further supported by DDC-KO neuroblastoma cells model. Cell models increase in biological complexity provides a valuable platform for studying complex dopamine-related disorders, including PD. The developed differentiation protocol for neuroblastoma dopaminergic-like neurons and iPSC-derived dopaminergic neurons are helpful for evaluating potential therapeutic candidates.