The cellular mechanism of gene therapy in AADC deficiency

AADC缺乏症為一種罕見的體染色體隱性疾病，其致病原因是由於負責產生L-3,4-dihydroxyphenylalanine (L-DOPA) 與5- hydroxytryptophan (5-HTP) 的AADC酵素缺乏，因而造成身體多巴胺 (dopamine) 與血清素 (serotonin) 的不足，症狀有嚴重的發展遲緩、肌肉張力低下、轉眼危象以及自律神經系統功能失調。在臺灣的AADC缺乏症常常是由一種創造者變異(founder mutation) 引起，所以患者特別多。2007年首次AADC缺乏症的基因治療人道救援在臺灣進行，基因治療的方法是以腺相關病毒第二型（AAV2）作為病毒載體，將AADC基因利用立體定位手術送入患者的紋狀體殼核 (putamen) ，治療後所有的患者皆有很大的改善。但是基因治療如何能改善此突觸後神經元的活性，並維持長時間的效應， 及AADC在細胞中的作用為何目前還不清楚。因此我們分別培養神經母細胞瘤細胞 (N2a) 和體外培養中型棘神經細胞 (medium spiny neurons) 及多巴胺能細胞 (dopaminergic neurons) 進行三個階段的實驗，以探討AADC在細胞中生成後是否會釋放到細胞外。我們將細胞轉染AADC基因後，透過免疫染色法觀察AADC的表現，再以高效液相層析儀HPLC （偵測L-DOPA轉換成多巴胺的量）分析 AADC酵素的活性。 在N2a細胞的實驗中，N2a 細胞經轉染AADC後可以被表現，而在其細胞培養液及細胞中皆可測得AADC活性，其值分別為0.264±0.05 nmol/min/ml及3.12±0.53 nmol/min/mg，顯示細胞內外皆有AADC酵素的存在；我們想進一步確認在培養液中測得的AADC活性是由細胞所釋放，而非細胞死亡造成的，因此以LDH的釋放百分比來估算細胞受損率，結論是釋出細胞外的AADC活性25%（培養液測得之AADC活性/培養液及細胞測得之AADC活性的總和）遠大於細胞死亡率12%；且我們另外針對轉染AADC的細胞進行細胞內外測得之AADC活性與其細胞受損率關係的比較，結果顯示隨著細胞受損率上昇，其細胞內測得之AADC活性減少，但其對應培養液所測得的AADC活性並沒有跟隨細胞受損率的增加而改變，因此我們認為培養液中測得的AADC活性應是由細胞所釋放出去的。而在探討AADC釋放機制的實驗中，我們分別加上BFA (Brefeldin A) 阻斷阻斷蛋白質從內質網傳送到高基氏體的路徑，或使用顯性失活Rab11阻斷蛋白質及囊泡運輸及核內體回收，結果兩種阻斷蛋白質運送的方式皆無法使釋放到AADC的活性減少，顯示AADC的釋放路徑可能非一般的分泌路徑。在中型棘神經細胞實驗與多巴胺能神經細胞實驗中，皆僅得到小於1% 的中型棘神經細胞或多巴胺能神經細胞。我們轉染AADC至中型棘神經細胞，但其細胞內外的AADC活性因太低皆無法被HPLC 測得。 本研究成功轉染AADC至N2a細胞內表現，並證實AADC可以被細胞釋放出來，但其釋放機制尚未釐清。體外培養中型棘神經細胞及多巴胺能細胞雖然未能得到足夠的細胞數目，但本研究建立培養技術與實驗流程提供了未來研究的基礎，並經由了解AADC的釋放機制以幫助基因治療的研究與執行。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.口試委員會審定書 i 誌謝 ii 中文摘要 iii ABSTRACT v 目錄 vii 圖目錄 x Chapter 1 緒論 1 1.1 單胺類代謝路徑 1 1.2 單胺類神經傳導物質 2 1.3 芳香族L-胺基酸類脫羧基酵素 (Aromatic L-amino acid decarboxylase, AADC) 3 1.4 AADC酵素存在位置 4 1.5 AADC缺乏症 4 1.6 AADC 缺乏症的診斷 5 1.7 AADC缺乏症的藥物治療 5 1.8 巴金森氏症的基因治療 6 1.9 AADC缺乏症的基因治療 8 1.10 研究假說 9 Chapter 2 材料與方法 11 2.1 實驗設計 11 2.1.1 N2a細胞實驗 11 2.1.2 中型棘神經細胞實驗 12 2.1.3 多巴胺能神經細胞實驗 13 2.2 細胞培養 13 2.2.1 N2a細胞培養 13 2.2.2 神經膠質細胞培養 14 2.2.3 中型棘神經細胞培養 (medium spiny neurons) 14 2.2.4 多巴胺能神經細胞培養 15 2.3 DNA 16 2.4 細胞轉染技術 (transfection) 16 2.5 探討AADC釋放機制的細胞處理 16 2.5.1 BFA (brefeldin A) 16 2.5.2 DN-Rab11（顯性失活Rab11） 17 2.6 免疫螢光染色 17 2.7 AADC酵素活性分析 18 2.7.1 培養液 (extracellular) 之樣品處理 18 2.7.2 細胞 (intracellular) 之樣品處理 18 2.7.3 HPLC分析 18 2.7.4 AADC酵素活性計算方式 19 2.8 乳酸脫氫酶 (LDH) 活性分析 20 2.9 統計分析 20 2.10 蛋白質定量 21 Chapter 3 結果 22 3.1 N2a細胞實驗 22 3.1.1 轉染AADC基因後可以表現AADC 22 3.1.2 表現AADC之N2a細胞培養液中AADC酵素活性 22 3.1.3 LDH活性分析評估細胞受損率 23 3.1.4 AADC酵素活性與細胞受損率的關係 23 3.1.5 以BFA處理細胞探討AADC釋放機制 24 3.1.6 轉染顯性失活的Rab11探討AADC釋放機制 25 3.2 中型棘神經細胞之實驗 26 3.2.1 初代培養中型棘神經細胞的免疫染色 26 3.2.2 轉染AADC及AADC活性分析的結果 27 3.3 多巴胺能神經細胞之實驗 27 Chapter 4 討論 28 4.1 基因治療與細胞實驗 28 4.2 AADC可被釋放到細胞外 29 4.3 影響AADC活性分析的因素 30 4.4 探討AADC釋放機制 31 4.5 初代神經細胞培養實驗 31 REFERENCE 3