在神經病理相關的蛋白堆積當中有許多過磷酸化並截短的各種tau 蛋白，具有
這種病理特徵的疾病統稱為tauopathies。然而，是否這些修飾之間的相互作用會
影響tau 蛋白的毒性仍然未知。通過表達人屬tau 蛋白並將其中疾病相關的絲氨酸(Ser)/蘇氨酸(Thr)殘基做替換以模擬過磷酸化狀態，我們發現儘管全長tau 蛋白會導致嚴重的神經退化，但是在天冬氨酸421(Asp421)這個位點如果有截短的話tau蛋白的毒性會減輕。細胞學和生物化學分析顯示過磷酸化的全長tau 蛋白在細胞體，軸突，以及軸突末端都有著廣泛的分佈。然而天冬氨酸421 截短的tau 蛋白很少存在於軸突末梢。這個差異與果蠅表達過磷酸化全長tau 而非截短的tau 蛋白導致軸突損傷,包括軸突膨脹與異常的肌動蛋白堆積有關。過磷酸化的tau 蛋白在軸突末梢的量與果蠅的運動能力能力有關，因為天冬氨酸421 截短的tau 蛋白導致的運動能力缺陷相對輕微，說明突觸功能很好的被保存。天冬氨酸421 截短的tau 蛋白作為蛋白水解物常見於神經纖維纏結中。我們的發現說明不同的翻譯後修飾對tau 蛋白的亞細胞分佈及毒性有重要影響，這個信息對於我們考慮tau 作為治療選項時也許會有意義。

Hyperphosphorylated and truncated tau variants are enriched in neuropathological aggregates in diseases known as tauopathies. However, whether the interaction of these posttranslational modifications affects tau toxicity as a whole remains unresolved. By expressing human tau with disease-related Ser/Thr residues to simulate hyperphosphorylation, we show that despite severe neurodegeneration in full-length tau, with the truncation at Asp421, the toxicity is ameliorated. Cytological and biochemical analyses reveal that hyperphosphorylated full-length tau distributes in the soma, the axon,
and the axonal terminal without evident distinction, whereas the Asp421-truncated version is mostly restricted from the axonal terminal. This discrepancy is correlated with the fact that fly expressing hyperphosphorylated full-length tau, but not Asp421-cleaved one,
develops axonopathy lesions, including axonal spheroids and aberrant actin accumulations. The reduced presence of hyperphosphorylated tau in the axonal terminal is corroborated with the observation that flies expressing Asp421-truncated variants showed less motor deficit, suggesting synaptic function is preserved. The Asp421 cleavage of tau is a proteolytic product commonly found in the neurofibrillary tangles. Our finding suggests the coordination of different posttranslational modifications on tau may have an unexpected impact on the protein subcellular localization and cytotoxicity, which may be valuable
when considering tau for therapeutic purposes.

Table of Contents
CHAPTER 1 - Introduction
1.1 Tau biology ……………………………………………………………...1
1.2 Tau pathology …………………………………………………………...4
1.2.1 Neuropathological features in tauopathy …………………………4
1.2.2 Neuronal cell death in tauopathy …………………………………8
1.3 Posttranslational modifications of tau …………………………………10
1.3.1 Phosphorylation ………………………………………………….11
1.3.2 Truncation ………………………………………………………..14
CHAPTER 2 – Establishment of Drosophila tauopathy models
2.1 Introduction ……………………………………………………………16
2.2 Designed tau mutants ………………………………………………….18
2.3 Results ………………………………………………………………….19
2.3.1 Expression of the wild-type tau failed to induce rough eye
Phenotype ………………………………………………………..19
2.3.2 Expression of the hyper/hypophosphorylated tau induced rough eye
Phenotype ………………………………………………………..20
2.3.3 Neuronal driver screening identified specific driver suitable to
study tau brain pathology ………………………………………..21
2.4 Discussion ……………………………………………………………...23
CHAPTER 3 – Investigation of combinational impact of
hyper/hypophosphorylation and Asp421 truncation
on modulating tau toxicity
3.1 Introduction ……………………………………………………………23
3.2 Results ………………………………………………………………….24
3.2.1 Hyper/hypophosphorylated tau disrupted the internal structure
of Drosophila compound eye …………………………………….24
3.2.2 Hyper/hypophosphorylated tau disrupted the neuronal
transmission and induced severe motor dysfunction …………….25
3.2.3 Tau truncation at D421 does not affect the protein stability ……..26
3.2.4 Tau truncation at D421 alters the distribution of tau protein in
neurons ……………………………………………………………28
3.2.5 D421 truncation of hyperphosphorylated tau protects neurons
against the formation of axonal spheroids and aberrant actin
accumulation ….………………………………………………......29
3.2.6 Phosphorylated tau aggregates are not responsible for the
observed toxicity .…………………………………………………31
3.2.7 Hyperphosphorylation contributes to caspase activation and D421
truncation prevented the disruption of neuronal membrane integrity
……………………………………………………………………32
3.3 Discussion ……………………………………………………………...33
CHAPTER 4 – Investigation of the impact of modulating S433 on
tau toxicity
4.1 Introduction ……………………………………………………………40
4.2 Results ………………………………………………………………….40
4.2.1 The toxicity of WTS433A is moderate in drosophila brain
cholinergic neurons ……………………………………………...40
4.2.2 Modulating S433 under the hyperphosphorylation condition can
modulate the tau axonopathy …………………………………….42
4.2.3 The modulation of tau toxicity could be associated with the changes
of the tau protein structure ……………………………………….43
4.3 Discussion ……………………………………………………………...44
CHAPTER 5 – Conclusion Remarks …………………………………45
CHAPTER 6 – Materials and Methods ……………………………...47
6.1 DNA constructs ………………………………………………………...47
6.2 Fly genetics ………………………………………………………….....49
6.3 Subcellular fractions …………………………………………………...50
6.4 Sarkosyl extraction ………………………………………………..…..50
6.5 Western blot ……………………………………………………………51
6.6 Immunocytochemistry ………………………………………………….52
6.7 Fly behavior ……………………………………………………………53
6.8 PI live staining …………………………………………………………54
6.9 Thioflavin S staining …………………………………………………...54
6.10 Image analysis ………………………………………………………..55
Figures ……………………………………………………………………..57
References …………………………………………………………………81

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