摘要:巴金森氏症(巴病)為最常見的神經退化性疾病之ㄧ,平均發生率佔60歲以上老年人口1-2%。可惜的是,目前此病症並無根治之治療方法,臨床上的治療方針仍侷限在左多巴的補充,病情仍會隨病患年齡增加而益趨惡化。研究顯示,帶有巴病易感基因的變異加上環境危險因子的暴露是導致此病症發病的機制,目前已知有11個基因的突變是引起少數遺傳性巴病的致病基因,在這些基因中,Leucine-rich repeat kinase(LRRK2)是引起顯性遺傳以及老年 型巴病最常見的基因。過去的研究中發現,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。臨床表現型上,帶有LRRK2 G2019S突變的病患,其症狀和偶發型的巴病病患幾乎無異。因此,釐清LRRK2 G2019S導致神經細胞死亡的機轉將對於探討巴病中多巴胺神經細胞死亡的機轉提供一道曙光。過去的研究指出G2019S此突變會導致LRRK2蛋白的激脢活性增加,導致神經細胞提早死亡,但其機轉未明。我們過去已成功建立LRRK2 G2019S基因轉殖果蠅,發現LRRK2 G2019S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,而這樣的變化與LRRK2的激脢活性強弱成顯著相關性,進一步我們證實帶有G2019S突變點的LRRK2蛋白會招募活性化的GSK3b激酶,提升下游的tau蛋白磷酸化程度,因此使得以軸突分布為主的磷酸化tau蛋白不正常的出現在樹突中,這樣的結果導致樹突分支的微管結構(microtubules)破壞進而導致神經細胞樹突退化萎縮以及神經細胞提早死亡。除此之外,我們也成功在此基因轉殖果蠅身上發現如同人類病患一般的動作遲緩以及多巴胺神經細胞隨老化死亡的病徵,顯示此果蠅模式不僅可以用來釐清分子致病機轉,另可以做為快速藥物篩選的動物模式平台。最近研究亦發現,以LRRK2 G2019S基因轉殖果蠅以及線蟲為動物模式篩選化學小分子化合物,發現LRRK2激酶活性抑制劑,GW5074 以及sorafenib,均顯示出可以顯著抑制LRRK2蛋白自身磷酸化的活性。這兩種化合物後來被證明可以在基因轉殖的線蟲和果蠅中有效地挽救多巴胺細胞死亡,以及減少局部運動障礙的功能。然而,儘管GW5074和sorafenib代表可能是將來可以根治巴病的抑制劑,但是這兩種化合物距離上市藥物的階段仍有一段距離,它們在人體的生物利用度和可能毒性都未明。同時,新研發藥物距離人體試驗的過程漫長,以及幾乎所有的藥物都有眾多的脫靶效應(off-target effect),因此目前一些藥物的研發以"老藥新用"的潛在新治療作用為目標,以期縮短臨床藥物試驗的時間與金錢,同時也可以更加釐清多巴胺神經細胞死亡的機轉。基於上述背景以及過去吾人的研究結果,我們有以下四個子目的:1. 驗證LRRK2 G2019S果蠅周邊樹突分支神經細胞(dendritic arborization neurons, da neurons) 為合適的巴病藥物篩選模式。2. 以上述da神經細胞樹突退化萎縮為篩選表現型,篩選不同濃度的640個FDA已經核准的藥物資料庫,以找出可回復神經樹突萎縮的可能標的藥物。3. 檢視不同濃度的標的藥物對於LRRK2 G2019S果蠅的生命,老化程度以及多巴胺神經細胞的影響。4. 以LRRK2 G2019S基因轉殖鼠的多巴胺神經細胞為模式,探究此藥物對於挽救神經細胞退化的分子機轉,以更釐清多巴胺神經細胞死亡的機轉。目前初步研究結果: 我們目前已初步篩選100種不同濃度的藥物,發現有6種藥物(Bexarotene, Clobetasol Propionate, Flufenamic acid, Gentamycin, Pronoprofen, Vidarabine)可以顯著或是部分的挽回LRRK2 G2019S 樹突退化的情形。我們將進一步完成所有藥物的篩選,並進行可能機轉的分析,以及在大鼠多巴胺神經細胞細胞模式中做進一步的療效驗證。
Abstract: Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, with anestimated incidence of 1% in persons over age 65. It is currently thought that PD results from a combination of genetic and environmental susceptibility factors. During the past decade, identification of causative genes linked to familial forms of PD has shed light on the pathogenesis of PD. Among these causative genes, mutations in Leucine-rich repeat kinase (LRRK2) are the most prevalent in both familial and sporadic PD. Among LRRK2 mutations, G2019S is the mutation hot spot in both familial and sporadic PD patients. As opposed to other genetic familial PDs, the majority of reported LRRK2 G2019S mutation carriers are late-onset, over age 50 years, and responsive to levodopa treatment, features typically found in sporadic PD cases. These clinical and pathological phenotypes of LRRK2 PD patients are similar to classic late-onset PD, further emphasizing the potential importance of LRRK2.In our previous work, we have demonstrated that Lrrk2 G2019S causes dendritedegeneration through phosphorylation and mis-localization of tau into dendrites by recruitingautoactivated GSK3b in Drosophila in vivo model system. Our transgenic LRRK2 G2019Sfly model also showed loss of dopaminergic neurons, decreased longevity and behavioraldeficits, which recapitulates several key features of human parkinsonism. Currently there isno therapeutic treatment to slow or ameliorate dopaminergic neuron degeneration in PD. Withthe well-known arduous process of drug development, it might be several years potentialLRRK2 kinase inhibitors are approved for use in humans. The rigors of drug developmenthave led some investigative teams to examine drugs already in use for potential beneficialeffects in ‘off-label’ situations. In addition, the molecular mechanisms that these medicationswork could further clarify the pathogenetic mechanism of PD.Based on the above background, we proposed to use our previously developed LRRK2 G2019S fly model to screen for 640 FDA approved medications and identify potential drugs that rescue the dendrite degeneration phenotypes of either dendritic arborization (da) neurosn or dopaminergic neurons. Furthermore, we will examine the therapeutic effects of these potential drugs on the aspects of motor function, life span and loss of dopaminergic neurons of LRRK2 G2019S flies. The results of this study will be extended to primary dopaminergic mammalian cells of LRRK2 G2019S transgenic mouse to confirm the therapeutic effects seen in the fly model and further dissecting the possible mechanism of its therapeutic effects. Our findings could be further extended to human clinical trials and will have strong implications in designing therapeutic targets for halting the devastating process in PD patients.