阿茲海默氏症(Alzheimer's Disease,AD)是一種神經退化性疾病。臨床症狀的特徵是大腦認知功能逐漸的衰退，而病理上的特徵則是大腦神經細胞內出現神經纖維糾結(neurofibrillary tangle,NFT)及神經細胞外堆積的老年斑塊(senile plaque, SP)。其中NFT的主要成分是一種與細胞內微管相連結但被過度磷酸化的Tau蛋白質,而SP主要是由貝它類澱粉(Aβ)堆積而成，其中大部分是Aβ1-42胜肽(Aβ42)。由於臨床上需要更好的工具來輔助AD早期及精確診斷，預測AD疾病的進展，及監測AD治療藥物的療效，因此腦脊髓液(CSF)生化標記蛋白的運用發展，便成為快速成長的研究領域。本研究計劃，我們總共納入28位AD病人，16位非AD(non-AD)的失智症病人，14位其他神經科疾病(OND)的病人，及21位智能正常者(HC)成為研究對象。我們的結果顯示AD病人相較於非失智症的控制組，腦脊髓液的tau蛋白有明顯上升而Aβ42則明顯下降。而AD及非AD二組失智病人，CSF tau蛋白及Aβ42的量則沒有明顯的差異。這些結果建議CSF的tau蛋白及Aβ42二者都是很好的生化標記蛋白可幫助區別AD及非失智症，但卻無法有效鑑別AD及non-AD二組失智症病人。進一步分析CSF生化標記蛋白與臨床症狀相關性，我們結果發現，只有CSF tau蛋白而非Aβ42，與AD病人的短期記憶測驗分數呈現有意義的負相關(spearman: r= -0.444; p=0.018)這些結果顯示AD病人中，較高的CSF tau蛋白會合併有較多的NFT病灶及較嚴重的短期記憶力障礙。在研究tau蛋白被肝醣合成酶激酶3β(GSK3β)過度磷酸化的分子機轉方面，我們的結果顯示Thr231位置是GSK3β原始磷酸化tau蛋白的位置，而且tau胜肽，tau227-237 (AVVRTPPKSPS)包含了T231P232 motif被辨識為GSK3β的結合處而且結合係數(kd)高達0.82±0.16μM。我們的結果發現GSK3β直接結合並磷酸化T231P232 motif後，導致tau蛋白結構的改變，並進而使N端tau失去了抑制C端tau蛋白被磷酸化的能力，最後導致C端tau蛋白被GSK3β順序性磷酸化。再者，實驗結果顯示tau蛋白被過度磷酸化後會減弱它促進微小管聚合的能力並導致N18細胞膜周圍無法形成微管束(bundle)，而T231A的突變細胞內，tau可以完全不被GSK3β磷酸化而保有它促進微小管聚合能力並形成微管束。總之，我們的結果顯示GSK3β在T231位置的磷酸化對於tau蛋白的過度磷酸化及功能調控扮演重要角色。

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions and the presence of intracellular neurofibrillary tangles (NFT) and extraneuronal senile plaques (SP). The major component of NFT is the hyperphosphorylated microtubules-associated protein tau. SP is consistent of extracellular deposition of β-amyloid (Aβ), mainly Aβ1-42 peptide (Aβ42). Given the need of tools for early and accurate diagnosis and prediction of disease progression and monitoring the efficacy of therapeutic agents for AD, development of cerebrospinal fluid (CSF) biomarkers have become a rapidly growing research field. In our study, patients with AD (n=28), non-AD dementia (n=16), other neurological disorder (OND, n=14) and healthy controls (HC, n=21) were included. Our results revealed that AD patients have significant higher CSF total tau (t-tau) and lower Aβ42 levels than HC and OND groups. There is no significant difference of both CSF t-tau and Aβ42 levels between AD and non-AD dementia groups. These results suggest that both CSF t-tau and Aβ42 are good biomarkers for distinguishing AD from non-dementia control subjects but demonstrate less discriminating power in differentiating AD from non-AD dementia. Moreover, our results show only CSF t-tau level but not Aβ42 has an inverse correlation with the score of short-term memory patients with AD (spearman: r = -0.444; p=0.018). These data indicate the higher CSF t-tau level is associated with much NFT pathology and more severe impairment of short-term memory in AD patients. In the study of the moleacular mechanism of tau’s hyperphosphorylation by glycogen synthase kinase 3b (GSK3b), we show that the T231 is the primary phosphorylation site for GSK3b and the tau227-237 (AVVRTPPKSPS) derived from tau containing T231P232 motif is identified as the GSK3b binding site with high affinity of a Kd value 0.82 ± 0.16 mM. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3b induces conformational change of tau and consequentially alters the inhibitory activity of its N-terminus that allows the sequential phosphorylation of C-terminus of tau by GSK3b. Furthermore, hyperphosphorylation reduces tau’s ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes tau phosphorylation by GSK3b and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3b may play an important role in tau’s hyperphosphorylation and functional regulation.

Contents
Chapter 1. General introduction………………………………………...……1
1.1 Alzheimer’s disease…………………………………………………..2
1.2 Cerebrospinal fluid biomarkers and Alzheimer’s disease……………3
1.3 Tau protein and Alzheimer’s disease…………………………………4
Chapter 2. Analysis of cerebrospinal fluid biomarkers in Alzheimer’s disease………………………………………………………………..8
2.1 Introduction…………………………………………………………….9
2.2 Meterials and methods………………………………….…………..…13
2.3 Results……………………………………………….………………..18
2.4 Discussion…………………………………………….……….………23
Chapter 3. Molecular mechanism of tau’s hyperphosphorylation by
glycogen synthase kinase 3β……………………………..……….36
3.1 Introduction…………………………………………….….….………37
3.2 Experimental procedures…………………….……………….……….40
3.3 Results………………………………………….……………………..46
3.4 Discussion………………………………………….…………………56
Chapter 4. Conclusions and future perspective…………….….……………76
References……………………………………………………….…………….79
Figures contents

Figure 2.1 Scattergraphs and measures of CSF levels of t-tau and Aβ42 in
demented patients and controls subjects…………………………30
Figure 2.2 Receiver operating characteristic curve analysis of CSF t-tau and
Aβ42 level………………………………………………………31
Figure 2.3 Western blot assays of total tau protein from CSF samples of four groups…………………………………………………………….32
Figure 3.1 Preparation charactarization and cleavage of Tau and Tau’s
mutants…………………………………………………………...65
Figure 3.2 Phosphorylation of T231 induced the conformation change is critical for Tau hyperphosphorylation….………………………….……..67
Figure 3.3 Priming phosphorylation at S235 is not an absolutely required event for T231 prephosphorylation……….……………………………...68
Figure 3.4 Biochemical characterization of the interaction between GSK3

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