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標題: | 遺傳性巴金森氏症致病基因,LRRK2,的分子功能性研究與基因轉殖果蠅動物模式之建立 Genetic epidemiology and functional study of LRRK2 gene in the pathogenesis of Parkinson’s Disease by using Drosophila as a model system |
作者: | Chin-Hsien Lin 林靜嫻 |
指導教授: | 吳瑞美,簡正鼎 |
關鍵字: | 巴金森氏症,LRRK2 (Leucine-rich repeat kinase),ATP13A2,G2019S,G2385R,果蠅,神經細胞, Parkinson's disease,LRRK2(Leucine-rich repeat kinase),G2019S(Glycine2019Serine),G2385R (Glycine2385Arginine),Drosophila melanogaster,animal model,neurons, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 研究背景及目的
巴金森氏症(巴病)為一重要的神經退化性疾病,截至西元2010年為止,已有11個基因的突變是引起少數遺傳性巴病的致病因。其中,LRRK2 (Leucine-rich repat kinase)是引起顯性遺傳以及老年型巴病病患最重要的ㄧ個基因。LRRK2主要分布在細胞質中,具有5個重要的功能區塊,包括ankyrin、LRR (Leucine-rich repeat)、ROC(Ras of complex protein)、COR(C-terminal of Ras)、kinase 以及WD40等區塊。過去在白種人的研究中發現,LRRK2基因上的G2019S為巴病病患的突變熱點,若是帶有此ㄧ突變,幾乎都會罹患此病症(penetrance >95%)。G2019S位點座落在LRRK2 kinase 區塊上,過去許多研究已經證實G2019S會導致LRRK2 蛋白的激脢活性增加,因此導致帶G2019S突變的神經細胞比帶正常LRRK2的神經細胞提早死亡。相對而言,在亞洲人的研究中卻都沒有發現這些突變﹔相反的,我們在exon48上發現了會顯著增加亞洲人罹病危險性的變異點- G2385R。雖然目前為止對於LRRK2基因在不同種族的變異分析的臨床研究甚多,但是LRRK2基因在神經細胞中究竟扮演何種功能以及其確切的致病機轉目前尚未被證實。因此,本研究欲探討LRRK2基因突變對神經細胞所產生的功能性變化,同時建立基因轉植果蠅動物模式,以了解LRRK2基因突變對於神經細胞的影響。 研究材料與方法 本研究分兩階段進行:第一階段為探討在台灣人遺傳性巴病患者中LRRK2 基因突變之基因流行病學研究,本研究由台大醫院巴金森氏症暨動作障礙中心中的病患基因檢體庫中,研究台灣漢人巴金森症患者LRRK2基因突變的流行病學;第二階段則以果蠅 (Drosophila melanogaster)為動物模式,以基因轉植(transgenic)的方式,分別建立野生型與突變型的基因轉植果蠅,以比較突變型的果蠅表現型(phenotype)的變化以及LRRK2蛋白其在果蠅神經細胞中的表現是否有改變,以及是否會影響神經細胞的存活,同時進一部探討LRRK2導致神經細胞死亡的分子機轉。 研究結果 在第一階段的臨床研究中,我們初步篩檢了27位遺傳性巴病病患,其中只有1位是R1441H基因突變(3.7%),而有6位是G2385R基因變異者(22.2%)。此外,為了進一步探討這些基因變異的功能性意義,我們利用EB virus transform LRRK2 變病患以及帶有G2385R病患的白血球細胞株進行進ㄧ步的in vitro實驗,我們發現,對於低濃度MG-132毒物處理,帶有R1441H的細胞株比起同年齡的對照組細胞株而言有顯著增加的細胞凋亡率以及細胞內氧化自由基的累積﹔但是帶有G2385R的細胞株則需接觸到高濃度的MG-132才會產生細胞毒性。我們因此在第二階段的基礎研究中建立in vivo的基因轉殖果蠅模式,我們發現LRRK2 G2019S 以及R1441C會顯著的導致果蠅周邊神經以及多巴胺神經細胞的樹突退化萎縮(特別是G2019S),而這種導致樹突萎縮的作用取決於LRRK2 kinase activity的大小,接下來我們以果蠅周邊神經樹突分支神經系(dendritic arborization neurons)為模式,我們發現LRRK2 G2019S之所以會導致神經樹突退化萎縮的其中之一的機轉為藉由調控Glycogen Synthase Kinase 3 beta (GSK3β)而增加 phospho-tau (T212 of tau)的產生,並將之由軸突不正常的運送到樹突,因而導致樹突中microtubule的不穩定及崩解。我們在LRRK2果蠅模式中觀察到的現象進一步驗證了在LRRK2病人死後腦組織切片中發現的大量tau蛋白沉積,並且重現了多巴胺神經細胞隨著老化而死亡的病理特徵。 結論 本研究延續過去台大醫院巴金森氏症暨動作障礙中心過去幾年來所進行的巴金森氏症之基因流行病學研究,使我們對於台灣漢人巴金森氏症之常見基因變異、基因型與表現型之關連性、以及其致病機制,有了基本的了解。本研究同時也證實了LRRK2果蠅模式可以重現許多重要的疾病病理特徵,此現象增加了運用LRRK2果蠅模式來治療巴金森氏症的可能性。 Background Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, with a prevalence close to 1% after age 65. Causal genes for Mendelian-inherited PD have been reported. The recent discovery of LRRK2 (Leucine-rich repeat kianse 2) as a causative PD gene has provided insights into the pathophysiology of the disease. Because the clinical phenotype of LRRK2 mutations resembles idiopathic PD, LRRK2 has emerged as the most relevant player in PD pathogenesis identified to date. Many LRRK2 gene mutations have been reported. The G2019S mutation is the hot spot mutation in Caucasians and the G2385R polymorphism is reported to be a genetic risk factor in Easten populations. Mutant Lrrk2 carrying human dominant mutations has enhanced kinase activity, resulting in cell toxicity and neurite shrinkage. Knock-down or ablation of LRRK2 in mammalian cultured neurons promotes neurite outgrowth through actin cytoskeletal rearrangment. However, the information regarding the LRRK2 mutation in the Asian population is rare and the functional relevance of these mutations (such as G2019S) or risk variant (G2385R) are unclear. In addition, very recently, aother gene for early onset-PD (PARK9/ATP13A2) was identified. The phenotype of affected individuals is juvenile onset of PD and may combine the phenotypes of dementia and pyramidal degeneration. The ATP13A2 protein is assumed to be the neuronal P-type ATPase and the intracellylar location is primarily in the lysosome. The mechanism by which loss of ATP13A2 causes parkinsonism and the possible function is unclear. To date there have been few studies examining the frequency of ATP13A2 mutations in parkinsonism, nor in different populations. The data in Asian populations are lack. Purpose We propose this research proposal with the following aims to evaluate the the frequency and functional significance of LRRK2 and ATP13A2 mutations in patients with PD in Taiwanese. 1. To determine the frequency of mutations of the ATP13A2 gene in PD patients of Taiwanese. 2. To determine the frequency of mutations of the LRRK2 gene in PD patients of Taiwanese. 3. To elucidate the functional relevance of LRRK2 genetic substitutions using lymphoblastoid cell lines derived from patients with LRRK2 substitutions. 4. Employing Drosophila da neurons as a model system, we aim to characterize the role of LRRK2 mutations in Drosophila dendrite morphogenesis by creating transgenic Drosophila models. Materials and methods We recruit more than 500 PD patients and control subjects to evaluate the frequency of ATP13A2 and LRRK2 mutations in Taiwanese populations using the method of direct sequencing in the first set of the study. In the second part, we use EBV transformed lymphoblastoid cell lines derived from patients carring LRRK2 mutations to elucidate the possible molecular functional changes. We also create transgenic Drosophila models carrying wild type LRRK2, G2019S, R1441C and G2385R to elucidate the molecular effects of these LRRK2 mutatuins in the in vivo model system. Results We identified one novel missense variant, Ala746Thr, in a single heterozygous state in three patients (1.7% in EOPD). The variant was not observed in 589 ethnicity matched controls. The frequency of this variant was significantly higher in PD cases than controls (p=0.01, relative risk 4.3, 95%CI 1.9-4.3). The clinical phenotype and 18F-dopa PET image of ATP13A2 Ala78Thr carriers are similar to that seen in idiopathic PD. The variant is located between the highly conserved phosphorylation region and the 5th transmembrane domain of the ATP13A2 protein. We also found the frequencies of R1441H and G2385R in familial PD patients were 3.7% and 22.2%, respectively. The clinical phenotypes and [18F]-dopa PET findings for subjects with R1441H or G2385R resembled those of patients with idiopathic PD; however, their lymphoblastoid cell lines showed increased apoptosis following exposure to a proteosome inhibitor. Thus, LRRK2 mutations are rare in Taiwanese with familial PD. By using Drosophila as a model system, we found that expression of G2019S mutant in Drosophila dendritic arborization neurons induces mislocalization of the axonal protein tau in dendrites and causes dendrite degeneration. G2019S-induced dendrite degeneration is suppressed by reducing the level of tau protein and aggravated by tau coexpression. Further genetic analyses suggest that G2019S and Tau function synergistically to cause microtubule fragmentation, inclusion formation and dendrite degeneration. Mechanistically, hyperactivated G2019S promotes tau phosphorylation at the T212 site by the Drosophila GSK3β homolog Shaggy (Sgg). G2019S increases the recruitment of autoactivated Sgg, thus inducing hyperphosphorylation and mislocalization of tau with resultant dendrite degeneration. Conclusions Our study not only provides a genetic epidemiology information regarding the muattaion frequency of ATP13A2 and LRRK2 in Taiwnaese PD patients but also provide a molecular and cellular mechanism in understanding the regulation of neurite degeneration by LRRK2. Our restuls will be stretched to understand the pathomechanism of LRRK2-linked PD and the transgenic LRRK2 Drosophila model could be a plateform for further drug screening. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48616 |
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顯示於系所單位: | 臨床醫學研究所 |
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