The cellular mechanism of gene therapy in AADC deficiency
Date Issued
2014
Date
2014
Author(s)
Chen, Yi-Jye
Abstract
Aromatic L-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. AADC deficiency is an autosomal recessive disease. The metabolic abnormalities at birth, caused by mutations in the AADC gene, lead to severely reduced AADC activity. Hypotonia and oculogyric crises are the two most common symptoms of AADC deficiency. In addition, other common symptoms include developmental delay, hypokinesia, choreoathetosis, dystonia, limb hypertonia, insomnia, irritability, feeding and speech difficulties. AADC deficiency has an increased prevalence in the Taiwanese population due to the founder mutation IVS6+4A>T. Currently, treatment options are limited; only patients with relatively mild forms of the disease respond to drugs, and patients obtain relief from only a limited subset of symptoms. Drug therapy provides little or no benefit for many patients who often die during childhood. The gene therapy trial using the human AADC gene is currently undergo in Taiwan. The gene therapy is achieved by using an adeno-associated virus (AAV) type 2 vector to deliver the AADC gene into a brain area called the putamen in the patients with AADC deficiency. All patients showed improved motor function after treatment. However intracellular storage of dopamine requires expression of monoamine vesicular transporter, how could this gene therapy enhance the activity of the post-synaptic neuron in the putamen of striatum and persisted for long time is unclear. So we cultured neuroblastoma (N2a cells), primary medium spiny neurons and dopaminergic neurons in vitro, to investigate whether AADC produced by AADC-transfected cell could be secreted into medium (extracellular). 24hr post-transfection, culture medium and cell lysate was harvested for AADC activity analysis. HPLC analysis was used to detect ADDC activity by converting L-dopa to dopamine.
The results reveal that AADC can be detected both extracellular and intracellular in AADC-transfected N2a cell, and the AADC activity were 0.264±0.05 nmol/min/ml and 3.12±0.53 nmol/min/mg (n=8) respectively. AADC activity released into medium was expressed by the ratio of AADC activity obtaining from culture medium divides by the combined AADC activity from cell lysate and culture medium. Our result showed that AADC can be secreted into medium (ratio=25 ±5%, n=8) while the cytotoxicity index (LDH release) is 12±2% (n=8). To investigate the underlining mechanism of AADC secretion, we treated N2a cell with Brefeldin A for inhibition of protein secretion and synthesis or transfected dominat negative Rab11 for blocking the traffick of proteins or vesicles between the trans-Golgi network (TGN) and recycling endosome. Neither treated BFA nor transfected dominat negative Rab11 can reduce AADC secretion.
In conclusion, we transfected AADC gene into N2a cells and proved AADC can be secreted into extracellular space, although the cellular mechanism of secretion was still unclear. This study also established the primary culture experiment and provided valuable insight into cellular mechanism of gene therapy in AADC deficiency.
Subjects
芳香族L-胺基酸類脫羧基酵素
AADC缺乏症
基因治療
AADC活性分析
中型棘神經細胞
多巴胺能神經細胞
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-103-P01448009-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):c619698223c44de32dfb376897d04688