TY - JOUR AU - Khalid, Melati AU - Mohd Moklas, Mohamad Aris PY - 2019/06/30 Y2 - 2024/03/29 TI - An Inhibited Dopamine Synthesizing Cell Model of AADC Deficiency JF - Life Sciences, Medicine and Biomedicine JA - LSMB VL - 3 IS - 6 SE - DO - 10.28916/lsmb.3.6.2019.24 UR - https://biomescientia.com/index.php/lsmb/article/view/24 SP - AB - <p><strong>Introduction:</strong> Aromatic L-amino acid decarboxylase deficiency (AADC) is a rare autosomal recessive pediatric neurotransmitter disease. To date it remains poorly understood mainly due to an absence of a disease model. The dopaminergic neuroblastoma cell SH-SY5Y was chosen to develop our AADC deficiency model. These cells are not native dopamine synthesizers. <strong>Objective:</strong> To develop a dopamine-producing cellular model of AADC deficiency using SH-SY5Y neuroblastoma cells. <strong>Methods:</strong>&nbsp;Dopamine pathway proteins were identified with Western Blotting. Dopaminergic differentiation was attempted using all-trans retinoic acid (ATRA) with dopamine detection via HPLC-ECD post alumina extraction. Treatment with <sub>L</sub>-DOPA provided SH-SY5Y with excess precursor. RT-PCR was used to determine the expression of markers of mature neurons.<strong> Results:</strong>&nbsp;Western Blot screening identified AADC, dopamine β-hydroxylase and tyrosine hyrdoxylase proteins, indicative of a dopaminergic pathway. ATRA was unsuccessful in producing dopamine from the cells. <sub>L</sub>-DOPA treatment however, generated dopamine first visible as a HPLC-ECD peak 30 minutes post-incubation. Prior to this, SH-SY5Y dopamine synthesis from <sub>L</sub>-DOPA has never been documented. This de novo synthesis is then inhibited using benserazide to form our AADC deficiency cell model. RT-PCR showed that SH-SY5Y cells express markers of mature neurons in its ‘native’ state and is not affected by <sub>L</sub>-DOPA and benserazide treatment. This cell model will potentially benefit many areas of AADC deficiency research. <strong>Conclusion:</strong>&nbsp;SH-SY5Y cells &nbsp;&nbsp;produced HPLC-ECD measureable amounts of dopamine with the addition of <sub>L</sub>-DOPA. &nbsp;&nbsp;&nbsp;Our model of AADC deficiency is generated by quelling the dopamine production with Benserazide.</p> ER -