2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/661267摘要：研究背景：巴金森氏症是僅次於阿茲海默氏症第二常見的神經退化性疾病，約佔60歲以上人口的1~2%。其主要病理變化是黑質（substantia nigra）多巴胺神經元退化，並伴隨著-synuclein堆積。巴金森氏症病患中只有少於10%的病患是具有家族史。目前已確定有七個基因是巴金森氏症的致病基因，分別為α-Synuclein、Parkin、UCHL-1（ubiquitinC-terminal hydrolase-1）、PINK1（PTEN-induced kinase 1）、DJ-1、LRRK2（leucine rich repeatkinase 2）及ATP13A2。其中，LRRK2基因突變會造成體染色體顯性巴金森氏症（Autosomaldominant Parkinson disease）。LRRK2蛋白屬於ROCO蛋白家族，具有5個重要的功能區塊，包括ANK（ankryn）、LRR （Leucine-rich repeat）ROC（Ras of complex protein）、COR（carboxy-terminal of Ras）、MAPKKK（mitogen activated protein kinase kinase kinase）以及WD40等domains。目前GTPase domain（ROC、COR）上已發現數個突變點，其中R1441H、R1628P突變點分別只在台灣及漢人的巴金森氏症患者上發現。許多研究試圖解釋ROC domain的突變調控kinase activity引發疾病的機轉，但其機制依舊不明。研究目的：探討LRRK2 GTPase domain基因突變R1441H及R1628P risk variant與細胞內Ca2+平衡、GTP水解及細胞死亡之相關性，並建立帶有疾病型LRRK2基因轉殖鼠以提供病程之研究與觀察，進一步評估發展多巴胺神經保護藥物之可能性。研究方法：將rat primary striatal neuron轉殖入人類野生型以及突變型（R1441H、R1628P）LRRK2基因，經環境荷爾蒙的處理後，進ㄧ步分析細胞凋亡、粒線體功能變化、細胞內Ca2+濃度以及細胞內氧化自由基的累積程度，觀察LRRK2基因的功能以及其對於多巴胺神經細胞死亡的影響。同時建立野生型以及突變型（R1441H、R1628P）之基因轉殖鼠，觀察LRRK2蛋白其在小鼠神經細胞中的表現量、蛋白質結構、表現位置、磷酸化能力等等是否有改變，以及是否會影響其行為模式。<br> Abstract: Background: Parkinson’s disease(PD) is second to Alzheimer’s disease as the most commonprogressive neurodegenerative disease affecting 1–2% of individuals > 60 years of age.Epidemiology of PD indicates that <10% of PD has a strict familial etiology while the bulk ofcases are sporadic. Seven clearly defined genetic involving α-Synuclein, Parkin, UCHL-1(ubiquitin C-terminal hydrolase-1), PINK1 (PTEN-induced kinase 1), DJ-1, LRRK2 (leucinerich repeat kinase 2) and ATP13A2 cause PD. Mutations in LRRK2 gene have been shown tobe the leading cause of autosomal dominant PD. LRRK2 is belonging to the ROCO proteinsuperfamily. LRRK2, multidomain protein, contains ankryn (ANK), leucine-rich repeats(LRRs), a Ras GTPase-like domain termed ROC (Ras of complex protein), amitogen-activated protein kinase kinase kinase (MAPKKK) domain and a C-terminal WD40repeats. An additional domain C-terminal to the GTPase domain is COR (forcarboxy-terminal of Ras). The GTPase domain ROCO (ROC and COR) have several aminoacid substitutions. R1441H was discovered in Taiwanese PD family and R1628P is a riskfactor in ethnic Chinese populations. Although many studies attempt to explore themechanism of ROC mutation domain regulates kinase activity leads to disease, it is alsounclear until now in vivo and in vitro.Purpose: The aims of this research are (1) to understand the correlation of homeostasis ofcellular calcium, GTP hydrolysis and cell death in striatal neurons after constructs of LRRK2wide-type, R1441H or R1628P variant transfection; (2) to establish mouse model carryingwith pathological LRRK2 gene mutation for functional study and (3) to estimate thefeasibility of developing the protective agents for dopaminergic neuron in mice modelcarrying LRRK2 gene.Methods: Wide-type, R1441H and R1628 LRRK2 are transfected into rat primary striatalneuron. The transfected cells are analyzed the apoptosis levels, intracellular ROS and calciumconcentrations and mitochondria functions by using FACS flow cytometry afterenvironmental hormones treatment to understand the function of LRRK2 gene and the effectof doapmine neurons death. In addation, generation of wide-type, R1441H and R1628LRRK2 transgenic mice are for investigating the expression, structure, location andphosphoration of LRRK2 protein in neuron system and the behavior effect.巴金森氏症leucine rich repeat kinase 2（LRRK2）GTPase domainR1628P基因轉殖鼠Parkinson’s diseaseleucine rich repeat kinase 2GTPase domainR1628Ptransgenic mice