中文摘要
由於醫療的進步與死亡率的大幅降低，現代社會已經趨向老齡化發展，神經退化性疾病的難題已經無法避免。在基因組分析的研究中，指出不同的突變蛋白皆與神經退化性疾病有關，包含了阿茲海默症(Alzheimer's disease, AD)、帕金森氏症(Parkinson’s disease, PD)、亨丁頓舞蹈症(Huntington's disease)與肌萎縮性脊髓側索硬化症(Amyotrophic lateral sclerosis, ALS)等疾病(如：SOD1突變與ALS的致病有關、Tau突變與AD的致病有關、LRRK2與PD的致病高度相關)。過去的研究指出帕金森氏症病人的組織中可以發現LRRK2的突變，而在Wanli W.Smith等人藉由果蠅模式生物的研究中，也指出LRRK2 WT(wild-type)與G2019S突變和PD的致病高度相關。過去研究多是調查LRRK2突變與PD的關係，但是對於LRRK2 WT的功能依然所知甚少。另外一些研究也指出，阿茲海默症相關的Tau蛋白也被發現與PD高度相關，而LRRK2的蛋白功能分析中也顯示與Tau蛋白有關聯。然而對於這兩個蛋白的交互作用，或各別對於帕金森氏症的病理機制依然不甚清楚。因此我們利用果蠅的動物模式，來表現帕金森氏症相關的人類TauWT, LRRK2WT與LRRK2G2019S突變，並觀察LRRK2與Tau在細胞內的分子機轉。結果發現神經細胞中表現LRRK2WT相較於沒有表現 的野生型果蠅，果蠅的生命週期有延長的現象。西方墨點法分析結果發現，LRRK2WT表現時會提高Akt473S磷酸化。我們也對於Akt上游PI3K蛋白做研究，發現LRRK2WT並不會影響PI3K的磷酸化或是蛋白總量上的表現。接著，我們針對Akt下游FoxO1(細胞存活相關蛋白)的研究中發現，LRRK2表現會提高FoxO1256S磷酸化且蛋白總量顯著下降。這現象指出當LRRK2表現時可以提高細胞存活。我們也針對與細胞存活相關的蛋白進行研究，發現LRRK2表現也會增加細胞存活相關蛋白GSK3 beta(GSK3β)9S的磷酸化，且提高神經細胞的存活。我們利用果蠅的動物模式，表現帕金森氏症相關的人類TauWT(Wild type)，發現神經細胞型態會隨著時間逐漸改變成紡錘狀的結構。我們還發現Tau蛋白會隨著時間逐漸堆積，並形成線條狀的結構纏繞在一起，這些結構的分子量大約是90-230 kDa左右。過去的研究指出，Tau蛋白在過度磷酸化的情況下容易形成高分子量的聚集，但是我們也發現當 Tau蛋白處於Hypophosphorylation 的情況下，仍然會形成高分子量聚集，且依然具有細胞毒性來引發細胞結構的改變與凋亡。我們也分析細胞凋亡的相關的分子Casepase3與Cytochrome C，發現Tau蛋白的表現確實會引發細胞凋亡的分子訊號進而凋亡。由於先前的研究結果指出，LRRK2WT可以通過影響細胞存活機轉來提高細胞存活，因此我們想了解在Tau蛋白與LRRK2的共同表現下，是否LRRK2仍然具有保護細胞的功能。我們利用果蠅的動物模式共同表現 TauWT、LRRK2WT與LRRK2G2019S，結果驚訝發現LRRK2WT確實可以抑制高分子量Tau蛋白的產生。我們也發現單分子的Tau蛋白明顯表現量低於Tau單獨表現的組別，但是在LRRK2G2019S突變的情況下，卻發現會提升高分子量Tau蛋白的累積。綜合以上結果，我們提出當LRRK2WT表現時可以通過提高Akt-FoxO1與GSK3β細胞傳遞路徑來增加細胞存活率，且LRRK2 WT可以通過降低Tau蛋白的表現量來抑制高分子的Tau生成提高細胞存活，但是這個功能會因為LRRK2的突變而失去。

關鍵字：帕金森氏症(Parkinson’s disease, PD) 、LRRK2 WT (wild-type) LRRK2 G2019S、Tau 蛋白、過度磷酸化表現、Hypophosphorylate、紡錘狀細胞、neurofibrillary tangles、Phosphoinositide 3-kinase (PI3K)、Akt、Forkhead box protein O1(FoxO1)、Glycogen synthase kinase 3 beta (GSK3β)

Abstract
Numerous genome-wide association studies have revealed that many abnormal proteins are involved in diverse neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis. Related pathological studies detected abnormal protein aggregation or accumulation in patient’s brain tissue (such as superoxide dismutase 1 mutations associated with amyotrophic lateral sclerosis; Tau mutations associated with Alzheimer’s disease and PD, and leucine-rich repeat kinase 2 (LRRK2) mutations associated with PD). Recent studies also revealed differential mutations in LRRK2 and Tau in PD patient’s brain tissues. A previous study employed a Drosophila model to express wild type LRRK2 or LRRK2G2019S mutation, and found neurodegeneration in Drosophila eye tissues, also indicating that wild type LRRK2 can induce neurodegeneration, which is associated with PD. LRRK2 mutations are commonly known in both autosomal dominant and sporadic cases, with age as the largest risk factor. Another study shows that Tau H1 haplotype is associated with an increased risk of PD, and also shows that the elevated expression of Tau may increase the risk of PD via a genetically gain-of-function mechanism. However, the mechanism of Tau and wild-type LRRK2 in PD remains unclear. In this study, we investigated the effect of Tau and wild-type LRRK2 in Drosophila PD model. First, we used Drosophila model system to stably express wild-type LRRK2 in neurons and performed a survival assay. The survival assay showed that wild-type LRRK2 enhanced survival compared to the control group in the Drosophila model system. The western blot analysis showed that LRRK2 expression increases the phosphorylation of Akt in Serine 473(473S), but found no difference in Akt upstream kinase protein PI3K. The phosphorylation level of FoxO1 protein, an Akt downstream protein kinase involved in cell apoptosis, was increased by LRRK2 expression. We also conducted a western blot assay to screen other survival-associated proteins and found that GSK3 beta (GSK3β) increased phosphorylation levels by LRRK2 expression. Second, we expressed Tau in Drosophila DA neurons and found that this will trigger the age-dependent formation of Tau containing neurofibrillary tangle-like structures with progressive losses of DA neuron. Surprisingly, contrary to common belief that hyperphosphorylated Tau could aggravate toxicity, the aggregation of Tau is found in DA neurons expressing the modified hypophosphorylated TauAP. Third, we co-expressed wild-type LRRK2 and Tau together in the Drosophila model system and found that the wild-type LRRK2 will decrease the Tau aggregation with protective effects in DA neuron. These data suggest that Tau alone will induce the aggregation and formation of neurofibrillary tangle-like structures in neurons and wild-type LRRK2 expression increases the Akt-FoxO1 and GSK3β pathways to promote cell survival. However, co-expression of Tau and LRRK2 suggests that LRRK2 have a powerful protective function, which can decrease neurofibrillary tangle-like structure formations against Tau protein induced neuron death. These results provide the first in vivo evidence supporting the pro-survival function of LRRK2 via activation of Akt and GSK3β anti-apoptotic mechanisms and protecting cells against Tau induced neuron death.

Keywords: Parkinson’s disease, PD, Tau, LRRK2 WT (wild-type), LRRK2 G2019S, Tau, hyperphosphorylate, hypophosphorylated, Tangle like structure, neurofibrillary tangles, Phosphoinositide 3-kinase (PI3K), Akt, Forkhead box protein O1 (FoxO1, Glycogen synthase kinase 3 beta (GSK3β)

Table of Contents
Table of Contents………………………………………...………………………………………………………….…….……..I
致謝………………………………………..…………………………………………………………………………………….………V
中文摘要……………………………………………………………………………………………………………….…….………..1
Abstract…………………………………………………………………………………………………………………..…..……...3
Introduction……………………………………..……………………………………………………………………………………5
Parkinson’s disease (PD) ………………………………………………………………………………………….………..…5
Biochemical and Physiological roles of Tau protein……………………………………………………...……..…6
Biochemical and Physiological roles of leucine-rich repeat kinase 2 (LRRK2)………..………………7
Tau and mutation of LRRK2 is associated with PD ……..………………………………………….....…...……8
PD and MAPK signaling pathway……………………………………………………………………………....………….9
PI3K/Akt signaling pathway…………..……………………..………………………………………………......…………10
Experimental Procedures……………………………………………………………………………………………………..13
Drosophila Strains and Assays…………………………………….………………………………………………..……..13
Survival rate…………………………………………………………………………………………………………..….....……..13
Western blotting …………………………………………………………………………………………………...…..………..13
Sarkosyl insolubility assay…………………………………………………………………………………….…....……….14
Scanning electron microscopy (SEM) …………………………………………………………………….……....…..15
Statistical Analysis………………………………………………………………………………………………………..……..15
Results……………………………………………………………………………………………………………………….…...…..16
LRRK2 can increase Drosophila survival rate………………………………………………………………..……...16
LRRK2 increase survival, but not through the ERK pathway…………………………………………….…...17
LRRK2 increase survival rate by increasing Akt phosphorylation and inhibiting FoxO1 activity…………………………………………………………………………………………………………………………………19
PD-linked lrrk2 G2019S and R1441C mutations compromise anti-apoptosis function because of a failure in activating AKT …………………..…………………………………………………………………………..22
LRRK2 increase survival rate through GSK3β signaling pathway……....…………………………………23
Formation of abnormal oligomeric tau and tangle-like pathology in DA neurons………………....24
The high molecular weight of tau come from not only hyperphosphorylation but also hypophosphorylation tau accumulation..........................………………………….............................26
Overexpression of tau will induce apoptosis in neuron cell......…………………….........................28
LRRK2 can inhibit Tau protein accumulation into high molecular weight in neuron cell..........28
Discussion………………………………..…………………………………………………………………………………..……30
References………………………………..………………………………………………………………………………….……37
Figure…………………………………………………………………………………………………………………………...……46
Figure 1. Western blotting analysis of humam wild typr LRRK2 express in Drosophila model system. ………………………………………………………………………………………………………………..……………46
Figure 2. The expression of human LRRK2 does not affect ERK phosphorylation levels. ….…47
Figure 3. The expression of human wild type LRRK2 can specifically promote Akt phosphorylation at Ser473. ……………………………………………………………….………….……………..……48
Figure 4. The expression of human wild type LRRK2 cannot promote Akt phosphorylation at Ser308, which controls Akt activation loop……….…………………………………………………………….…49
Figure 5. Human wild type LRRK2 does not function through PI3K to promote Akt phosphorylation at 473S. ……………………….…………………………………………………………………...……50
Figure 6. Human wild type LRRK2 decreases the protein expression level of FoxO1.....…….…51
Figure 7. Human wild type LRRK2 can increase the phosphorylation level of FoxO1 at 256S to promote cell survival. ……………………………………………………………………………………..…………….……52
Figure 8. Human wild type LRRK2 inhibits phosphorylation of GSK3β at 9S to promote cell survival. …………………………………..………………………………………………………………………..………...……53
Figure 9. Human wild-type LRRK2 promotes cell survival, but not through the Akt-mTOR pathway. ………………..………………...…………………………………………………………………………………….…54
Figure 10. PD-linked lrrk2 mutants G2019S and R1441C inhibit the survival promotion function because of failure in Akt phosphorylation…....……………………………………………………....…….....…55
Figure 11. Western blotting analysis of human tauWT, tauAP and tauE14 in Drosophila model system..………...…………………………………………………………………………………….......….…………..…...…56
Figure 12. Expression of htau will induce DA neuron to change cell body into neurofibrillary tangles like structure..………………...……….….....….….…………………………………………………………..…57
Figure 13. Expression of htau can promote the Tau protein age dependent formation into high molecular structure in DA neuron..………...……….....………………………………………………………………59
Figure 14. Expression of htau can lead to Tau protein filamentous aggregates structures in the Drosophila brains..…………………...……………………………………………………………………...…………………61
Figure 15. The high molecular weight Tau proteins come from the hypophosphorylation hTau....………...……………...…………………..……………………………………………………………………………...…62
Figure 16. Overexpression of Tau will promote the high molecular weight formation and induce apoptosis in neuron cell..………...…………………………………………………………………......................…64
Figure 17. LRRK2WT can protect cell regarding total level and high molecular weight of tau but LRRK2G2019S cannot..………...……...…………………………………………………………………...…………...…65
Supplemental Information………………………………………………………………............………………….…….66
Figure S1. Protein domains of human LRRK2. …………………………………………………...……….….……66
Figure S2. The Human wild type lrrk2 expression promotes the Drosophila lifespan……....……67
Figure S3. The model of wild type of LRRK2 protective functions……………………………………..…69
Figure S4. The model of co-expression lrrk2G2019S mutant and human wild type tau….….…70
Table S1. The Antibodies used in this study…..……………..….………………………………………....………71

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