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標題: | 巴金森氏症BST1、HTRA2及LRRK2基因變異的分子遺傳及功能研究 Molecular Genetic and Functional Studies of BST1, HTRA2 and LRRK2 Gene Variations in Parkinson's Disease |
作者: | Meng-Ling Chen 陳孟伶 |
指導教授: | 吳瑞美(Ruey-Meei Wu) |
關鍵字: | 巴金森病,BST1,HTRA2,LRRK2,環境因子,粒腺體,自噬體, Parkinson’s disease,BST1,HTRA2,LRRK2,environmental factor,mitochondria,autophagosome, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 巴金森病是一個複雜性神經退化疾病,目前認為其病因可能由於基因與環境產生交互作用所引起的。最近相關研究顯示bone marrow stromal cell antigen 1 (BST1)、High temperature requirement A2(HTRA2)與Leucine-rich repeat kinase(LRRK2)基因突變或變異與巴金森病的發生有不同強度的相關性。
我們分析台灣巴金森病患者HTRA2的基因變異,結果共發現二個變異點(Pro143Ala與c.906 +3 G>A)。證明台灣巴金森病與HTRA2的基因變異有關。進一步建構GFP標記的cDNA質體,表現於初代多巴胺神經細胞,觀察發現Pro143Ala引起神經軸退化。人類神經瘤細胞株SH-SY5Y轉殖GFP標記的cDNA質體,經rotenone處理後,結果顯示相對於野生型SH-SY5Y細胞,Pro143Ala細胞可顯著觀察到粒腺體超顯微結構異常、粒腺體功能失調與細胞凋亡。此外,Pro143Ala增加HTRA2蛋白磷酸化表現,進而導致粒腺體功能失調。 根據最近全基因組關聯研究顯示BST1的單一核苷酸多型性rs11724635會提高罹患巴金森病的危險。因此我們探討在台灣巴金森病患者中BST1 rs11724635之基因流行病學研究研究中收集了468位巴金森病患者與487位健康對照組。結果顯示BST1 rs11724635表現頻率在病患組與健康組之間沒有顯者的差異,井水飲用在病患組與健康組之間有顯者的差異;然而國人若同時帶有BST1 rs11724635基因且曾飲用過井水則會明顯提高罹患巴金森病的危險。 LRRK2是最近被發現會導致偶發性與家族性巴金森病,LRRK2蛋白質包括armadillo (ARM)、ankyrin repeat (ANK)、leucine-rich repeat (LRR)、Ras of complex proteins: GTPase (ROC)、C-terminal of ROC (COR)、WD-40 (WD40)區塊。雖然目前對突變的LRRK2引起的致病機轉仍不明瞭,但已有許多研究發現LRRK2參與神經細胞的極性化、神經傳導物質、細胞膜與細胞骨架的動態平衡及蛋白質降解的調控。另外,亦有許多研究顯示細胞自噬可能為巴金森病的病理機轉之一。因此我們假設細胞自噬的機制可能參與LRRK2引起的巴金森病致病機轉。因此本實驗利用數種LRRK2基因轉殖鼠來探討此機制。結果發現hLRRK2 R1441G HET與HOM相對於同年齡的野生型老鼠皆有明顯地體重減輕、探究行為次數減少、步態異常與姿勢不穩。發現hLRRK2 R1441G HOM小鼠的GTase活性有增加之趨勢。另一方面,進一步穿透式電子顯微鏡與西方墨點法分析後發現12個月大的hLRRK2 R1441G HOM小鼠的黑質區發現溶體及粒腺體型態的改變且有自噬體的形成。因此,本實驗更加確認細胞自噬與GTase活性異常可能參與LRRK2 ROC區塊突變引起的巴金森病致病機轉。 Parkinson‘s disease is a complex neurodegenerative disease, caused by a combination of various genetic and environmental factors. Recently, several studies indicates the bone marrow stromal cell antigen 1 (BST1), high temperature requirement A2 (HTRA2) and leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson’s disease (PD). Mutations in HTRA2 re inconsistently associated with a risk of PD. We identified two novel heterozygous variants, Pro143Ala and c.906 +3 G>A. Expressing Pro143Ala variant of HTRA2 in primary dopaminergic neurons causes neurite degeneration. Following exposure to rotenone, the ultrastructural mitochondrial abnormality, the percentage of mitochondrial dysfunction and apoptosis in SH-SY5Y cells carrying the HTRA2 Pro143Ala a variant was significantly higher than wild-type cells. Mechanistically, protein level of phosphorylated HTRA2 was increased in cells carrying the Pro143Ala variant, suggesting Pro143Ala variant promotes HTRA2 phosphorylation with resultant mitochondrial dysfunction. Our results support a biologically relevant role of HTRA2 in PD susceptibility in Taiwanese. Further studies are warranted to confirm the role of HTRA2 Pro143Ala variant in the risk of PD. A recently published genome-wide association study in Caucasian and Asian populations showed a significant association between the BST1 SNP rs11724635 and increased risk for PD. We conduct a case control study to investigate whether BST1 rs11724635 increases the risk of PD, either by itself or in combination with environmental factors. 468 PD patients and 487 controls were evaluated. Compared with the AA genotype, the frequency of both CA and CC genotypes was not significantly different between the patient and control groups. The adjust odds ratios for CAand CC were 0.962 (95% CI = 0.643-1.439, p = 0.850) and 0.992 (95% CI = 0.654-1.503, p = 0.969) respectively. Of note, ever use of well water before the onset of PD symptoms had an impact on the occurrence of PD through interactions with BST1 rs11724635 AC and CC (OR = 1.623, p = 0.008). Our results show that the BST1 rs11724635 SNP alone is not associated with the development of PD, but it can interact with well water drinking to increase the risk of PD in this Taiwanese population. Mutations in LRRK2 are found in a significant proportion of sporadic and familial Parkinson’s disease (PD) patients. The LRRK2 protein contains multi-domains, including a GTPase and kinase domains. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, several in vitro and in vivo studies suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. In addition, recent studies in the brains of PD patients and in animal models of PD indicate the emerging role of autophagy during PD pathogenesis. We hypothesized that autophagy potentiates associated LRRK2 pathophysiology. We used the transgenic mouse model overexpressing the human LRRK2 gene and R1441G heterozygous (R1441G HET) and homozygous (R1441G HOM) mutation in the human LRRK2 gene. We have performed a comprehensive analysis of these mice up to 12 months of age, including evaluation of body weight, behavioral testing, GTPase activity, TEM image and protein expression. Our results show that both of hLRRK2 R1441G HET and HOM mice reduce weight loss. Moreover, both of R1441G HET and HOM mice exhibit less exploratory rearing behavior compared with hLRRK2 mice at 9 and 12 months old. hLRRK2 R1441G HOM mice decreased the swing speed, stride length, and front paws of base of support comparing to hLRRK2 mice. In contrast, the stand, step cycle, duty cycle, and hind paws of base of support increased in hLRRK2 R1441G HOM mice. And hLRRK2R1441G HOM resulted in an increase in GTPase activity. As expected, lysosome and mitochondria defects and autophagosome were investigated in SN region of hLRRK2 R1441G HOM brain by transmission electron microscopy (TEM) at 12 months old. In addition, our also observed the impaired autophagy- related makers and mitochondria dynamic proteins by western blot. Finally, we confirm that mutation of LRRK2 ROC domain accompanied by autophagic and GTPase activity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77456 |
DOI: | 10.6342/NTU201803986 |
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顯示於系所單位: | 生命科學系 |
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