阿滋海默症為常見的癡呆症之一，在病理學上有兩種特徵：（1）由Aβ（beta-amyloid）在神經元細胞外沈澱累積而形成的老化斑塊（senile plaque）（2）由高度磷酸化的Tau蛋白質在神經元細胞內沈澱累積而形成的神經纖維狀物質（neurofibrillary tangle，NFT）。Tau主要表現在大腦中，為一種微管結合蛋白。高度磷酸化的Tau會影響和微管之間的結合以及促進微管聚合的能力。已有一些能夠磷酸化PHF (paired helical filament) -Tau之proline-directed激酶被辨別出來，其中包括了Glycogen synthase kinase 3β (GSK3β)。在本實驗室先前之研究證明GSK3β可與PHF共同純化出來，而在本論文中，我們利用細胞轉殖證明了GSK3β能與Tau co-localize在老鼠神經融合瘤N18細胞中。也利用Tau、N-Tau、C-Tau、Tau T231A、Tau T231E、Tau 154~441、Tau S396A、Tau S400A、Tau S404A、Tau S413A及Tau S396A S400A來說明GSK3β磷酸化Tau的機制。我們認為Thr-231與Ser-404在GSK3β磷酸化Tau中扮演一重要角色。此外對於T231P motif，以合成之Tautide對GSK3β磷酸化Tau做一競爭性之抑制，說明了T231P motif對於GSK3β磷酸化及結合至Tau是必須的 (necessary) 也是絕對重要的 (critical)。
另外，我們想進一步探討磷酸化對於Tau功能上之調控，因此利用tubulin assembly assay來看Tau T231E、Tau S396E及Tau S400E單一胺基酸位置磷酸化之微管聚合情形。結果發現在上述Tau單一胺基酸位置磷酸化中，都具有促進微管聚合之能力。而利用細胞轉殖將這些質體送入老鼠神經融合瘤N18細胞中過度表現，發現其微管都有bundle情形產生。
而在本實驗室先前之研究中已發現Pin1可結合至被細胞週期中有絲分裂之重要激酶Cdc2磷酸化之Tau Thr-231胺基酸，並且可以回復高度磷酸化Tau失去促進微管聚合之能力。因此本論文中，我們也探討Pin1對於被GSK3β磷酸化之Tau是否也與被Cdc2磷酸化之Tau有相同之調控，結果顯示Pin1可結合至被GSK3β磷酸化之Tau Thr-231胺基酸，並且也可以回復Tau失去促進微管聚合之能力。

Alzheimer’s disease (AD), one of the most common dementia, is characterized by the formation two types of aggregation in the brain: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of hyperphosphorylated Tau. Tau protein mainly expressed in brain and was identified as one of the microtubule-associated proteins (MAPs). Hyperphophorylation on Tau affects its binding to tubulin and capacity to promote microtubule assembly. A number of proline-directed kinase capable of phosphorylating PHF-Tau have been identified, including Glycogen Synthase Kinase-3β (GSK-3β). Here we demonstrated that GSK3β can co-purify with PHFs and can co-localize with Tau in vitro in N18 cells. To examine the phosphorylation mechanism of Tau by GSK-3β, N-terminal, C-terminal, T231A, T231E, 154~441, S396A, S400A, S404A, S413A and S396A S400A mutants of Tau were used, respectively. We were able to demonstrate that phosphorylation on Thr231 and Ser404 in Tau may play important roles for GSK3β phosphorylation and its functional regulation. Most importantly, we have proved that T231P motif is necessary and critical for Tau phosphorylation by GSK3β.
Moreover, we used T231E, S396E and S400E mutants of Tau to understand the functional regulation of Tau by GSK3β phosphorylation by tubulin assembly assay. Surprisingly, we observed all of these Tau mutants can promote tubulin assembly and form tubulin bundles in N18 cells.
It has been proved that Pin1 WW domain can bind to Cdc2-phosphorylated Thr-231-Pro motif of Tau and restore the ability of Tau to promote tubulin assembly. In this study, we also studied whether Pin1 can regulate GSK3β- phosphorylated Tau. The results show that Pin1 WW domain can bind to phosphorylated Thr-231 of Tau by GSK3β and restore the ability of Tau to promote tubulin assembly.

目錄
ㄧ、中文摘要.............................i
二、英文摘要...........................iii
三、英文縮寫表...........................v
四、序言.................................1
五、材料與方法..........................13
六、結果................................35
七、討論................................52
八、參考文獻............................59
九、表..................................64
十、圖..................................67
十一、附錄..............................89

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