An inhibited dopamine synthesizing cell model of AADC deficiency
Introduction: 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 deficienc...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Biome Scientia
2019
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| Online Access: | http://psasir.upm.edu.my/id/eprint/80606/1/AADC.pdf |
| _version_ | 1796980790433677312 |
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| author | Khalid, Melati Mohd Moklas, Mohamad Aris |
| author_facet | Khalid, Melati Mohd Moklas, Mohamad Aris |
| author_sort | Khalid, Melati |
| collection | UPM |
| description | Introduction: 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. Objective: To develop a dopamine-producing cellular model of AADC deficiency using SH-SY5Y neuroblastoma cells. Methods: 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 L-DOPA provided SH-SY5Y with excess precursor. RT-PCR was used to determine the expression of markers of mature neurons. Results: 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. L-DOPA treatment however, generated dopamine first visible as a HPLC-ECD peak 30 minutes post-incubation. Prior to this, SH-SY5Y dopamine synthesis from L-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 L-DOPA and benserazide treatment. This cell model will potentially benefit many areas of AADC deficiency research. Conclusion: SH-SY5Y cells produced HPLC-ECD measureable amounts of dopamine with the addition of L-DOPA. Our model of AADC deficiency is generated by quelling the dopamine production with Benserazide. |
| first_indexed | 2024-03-06T10:28:14Z |
| format | Article |
| id | upm.eprints-80606 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T10:28:14Z |
| publishDate | 2019 |
| publisher | Biome Scientia |
| record_format | dspace |
| spelling | upm.eprints-806062020-11-05T18:49:02Z http://psasir.upm.edu.my/id/eprint/80606/ An inhibited dopamine synthesizing cell model of AADC deficiency Khalid, Melati Mohd Moklas, Mohamad Aris Introduction: 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. Objective: To develop a dopamine-producing cellular model of AADC deficiency using SH-SY5Y neuroblastoma cells. Methods: 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 L-DOPA provided SH-SY5Y with excess precursor. RT-PCR was used to determine the expression of markers of mature neurons. Results: 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. L-DOPA treatment however, generated dopamine first visible as a HPLC-ECD peak 30 minutes post-incubation. Prior to this, SH-SY5Y dopamine synthesis from L-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 L-DOPA and benserazide treatment. This cell model will potentially benefit many areas of AADC deficiency research. Conclusion: SH-SY5Y cells produced HPLC-ECD measureable amounts of dopamine with the addition of L-DOPA. Our model of AADC deficiency is generated by quelling the dopamine production with Benserazide. Biome Scientia 2019 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/80606/1/AADC.pdf Khalid, Melati and Mohd Moklas, Mohamad Aris (2019) An inhibited dopamine synthesizing cell model of AADC deficiency. Life Sciences, Medicine and Biomedicine, 3 (6). pp. 1-7. ISSN 2600-7207 https://biomescientia.com/index.php/lsmb/article/view/24 10.28916/lsmb.3.6.2019.24 |
| spellingShingle | Khalid, Melati Mohd Moklas, Mohamad Aris An inhibited dopamine synthesizing cell model of AADC deficiency |
| title | An inhibited dopamine synthesizing cell model of AADC deficiency |
| title_full | An inhibited dopamine synthesizing cell model of AADC deficiency |
| title_fullStr | An inhibited dopamine synthesizing cell model of AADC deficiency |
| title_full_unstemmed | An inhibited dopamine synthesizing cell model of AADC deficiency |
| title_short | An inhibited dopamine synthesizing cell model of AADC deficiency |
| title_sort | inhibited dopamine synthesizing cell model of aadc deficiency |
| url | http://psasir.upm.edu.my/id/eprint/80606/1/AADC.pdf |
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