臺灣博碩士論文加值系統

經突觸（synapse）位於兩個神經細胞（neuron）的連接處，對於神經傳導扮演重要的角色。N-cadherin為一個位於突觸的細胞黏著因子（cell adhesion molecule），藉由同型親合交互作用（homophilic interaction）來連接突觸前後的神經細胞。N-cadherin 的細胞質部份（cytoplasmic domain）藉由與 β-catenin、α-catenin 的交互作用可與肌動蛋白骨架（Actin cytoskeleton）作連結。在先前的研究指出，當甲基天門冬胺酸受體（N-methyl D-aspartate receptor, NMDAR）被活化時，可能經由一條去駱氨酸磷酸化（tyrosine dephosphorylation）的途徑使得 β-catenin 從樹突分支（dendritic shaft）移往樹突小刺（dendritic spine），並持續的累積。此一累積會調控位於樹突小刺上突觸的大小與受體的總量。已知位於 β-catenin 上第 654 胺基酸的去磷酸化可調控 β-catenin 往突觸小刺的轉位（translocation），然而 NMDAR 活化是否可調控 β-catenin 上其他駱氨酸的磷酸化，進而影響 β-catenin 的轉位還不清楚。
本論文發現 NMDAR 的活化可降低 β-catenin 上第 142 胺基酸的磷酸化。我們將 β-catenin 接上綠螢光蛋白（green fluorescent protein, GFP）並將第 142 胺基酸突變為無法被磷酸化的苯丙胺酸（phenolalanine），大量表現在神經細胞中觀察 β-catenin 的轉位。我們發現，將第 142 胺基酸突變為苯丙胺酸並不會影響 NMDAR 活化所造成的 β-catenin 轉位。然而，當利用 genistein 這個駱胺酸去磷酸酶抑制劑 (tyrosine phosphatase inhibitor) 誘導 β-catenin 轉位時，此突變卻能使 β-catenin 不再繼續累積在樹突小刺，證明 β-catenin 第 142 胺基酸的磷酸化與否對於調控 β-catenin 在神經細胞中的位置扮演重要的角色。
最後，我們藉由免疫螢光染色（immunostaining）觀察內生的（endogenous） β-catenin 在 NMDAR 活化時是否會表現在神經細胞的細胞核。我們發現無論 NMDAR 活化與否，β-catenin 都不會表現在細胞核中，因此我們認為NMDAR 活化造成的 β-catenin 轉位並非由於 β-catenin 進核活化下游基因所導致。

Synapse is a junction that mediating neural transmission between two neurons. N-cadherin is a cell adhesion molecule at synapses. It mediates actin cytoskeleton organization and transsynaptic communication through the interaction with β-catenin and α-catenin. Beta-catenin responds to NMDAR activation and stabilizes N-cadherin at the surface. Previous studies have suggested that NMDAR-dependent neural activity drives β-catenin accumulated in spines possibly through the dephosphorylation of multiple tyrosine residues. Beta-catenin Y654 residue has been found to be one of these sites, however, a few other sites remain to be investigated.
Here, we found that phosphorylation on β-catenin Y142 residue was reduced upon NMDA treatment. By infecting neurons with GFP fusioned β-catenin Y142F, a phosphorylation-prevented mutant, we observed that NMDA-dependent β-catenin spine accumulation was not inhibited by this construct. However, no accumulation of this mutant in the spine was observed in genistein-treated neurons comparing to theβ-catenin WT control group. This result suggests that regulation of phosphorylation on β-catenin Y142 residue is important for β-catenin spine accumulation.
Since β-catenin is known as a coactivator in neuceli, we also examined the subcellular localization of β-catenin by immunostaining to observe whether NMDA can drive β-catenin not only into the spines but also into the nuclei. No endogenous β-catenin was observed in the nuclei upon NMDA treatment, suggesting that NMDA-dependent β-catenin synaptic translocation was not result from activation of its target genes.

誌謝……………………………………….………………………………………..…. I
摘要………………………………………………………..………………………….III
Abstract…...…….…………………….…………………......................………….…IV
Table of contents……………………………………...…………………………….. V
Introduction……………………………………………………………………….…...1
1.1 CNS glutamatergic synapses…………………………………………..……..1
1.2 N-cadherin-catenin adhesion complex……………………………...…...……3
1.3 Structure of beta-catenin ………….………………......................……...……3
1.4 Tyrosine phosphorylation of beta-catenin regulates structure and function of the synapse……………………………………………………………...…….5
Materials and Methods…………………………………………………………...……8
2.1 Cultured neuron………………………………………………………………8
2.2 Mammalian cell cultures……………………………………..………………9
2.3 Plasmids and recombinant DNA techniques……………………..……..……9
2.4 Sindbis virus production……………………………………………………..9
2.5 Viral infection……………………………………………………………....10
2.6 Antibodies………………………………………….……………………….10
2.7 Live-cell imaging………………………………………………………...…11
2.8 Immunoprecipitation………………………………………………………..11
2.9 2D Electrophoresis…………………………………………………...……..12
2.10 Electrophoresis and immunoblotting……………………………………..13
2.11 Synaptosome preparation…………………………………………….……13
2.12 Coomassie blue staining………………………………………………...…14
2.13 Surface biotinylation…………………………………………………...….14
2.14 Immunostaining………………………………………………………..…..15
2.15 Reverse transcription PCR………………………………………………..15
2.16 Image analysis and statistics…………………………………………….16
Results……………………………………………………………………………….17
3.1 NMDA treatment reduced phosphorylation of β-catenin on Y142 residue…17
3.2 Overexpression of β-catenin Y142F does not alter the density of dendritic spines, but increase of spine head width upon NMDA treatment…………...18
3.3 β-catenin Y142F infected neurons exhibited NMDA-dependent β-catenin spine translocation…………………………….……………………………..19
3.4 β-catenin Y142F mutant blocked genistein induced β-catenin spine translocation………………………………………………………………….21
3.5 The amount of surface N-cadherin and membrane-bound β-catenin were not increased by the NMDA and genistein treatment……………………………21
3.6 NMDA treatment does not cause nuclear translocation of β-catenin…….....22
Discussion……………………………………………………………………………24
4.1 The mechanisms involved in the dephosphorylation of β-catenin Y142 by NMDAR activity…………………………………………………………….24
4.2 Dephosphorylation of β-catenin Y142 residue regulates β-catenin spine translocation……………………………………………………………...….25
4.3 NMDAR activity caused spine shape change in β-catenin Y142F overexpressed neurons……………………………………………………….26
4.4 Multiple tyrosine phosphorylation sites of β-catenin………………………..27
4.5 Functions of synaptic β-catenin…………………………………………...…28
4.6 The amount of surface N-cadherin and membrane-bound β-catenin were not increased by the NMDA and genistein treatment……………………………28
4.7 NMDA-dependent β-catenin synaptic translocation was an independent pathway from activating the genes downstream of β-catenin……….………29
Reference……………………………………………………………………………..30
Figures…………………………………………………………………………..……37
Figure 1. Adhesion molecules of cadhrin/catenin complex……………………..37
Figure 2. Multiple β-catenin tyrosine phosphorylation sites that regulated by tyrosine kinases or phosphatases……………………………………….39
Figure 3. Tyrosine phosphorylation on β-catenin Y142 residue was reduced in the NMDA-treated neuron…………………………………………….…...40
Figure 4. The spine density was not altered in the β-catenin Y142F overexpressed neurons, but the stubby and mushroom spines were increased after NMDA treatment……..…………………………………………….….43
Figure 5. NMDAR activity caused increase of synaptic β-catenin………..……46
Figure 6. NMDA induces β-catenin translocation to spines…………………….48
Figure 7. β-catenin Y142 infected neurons inhibit genistein-induced β-catenin translocation……………………………………………………………50
Figure 8. Surface N-cadherin and membrane-bound β-catenin were not increased upon NMDA or genistein treatment……………………………………52
Figure 9. β-catenin was not in the nuclei upon NMDA or LiCl treatment in the mature hippocampal neurons…………………..………………………55
Tables………………………………………………………………………………...58
Table 1. Primers for site-directed mutagenesis……………………………………….58
Table 2. Primers for checking the sequence………………………………………….58
Table 3. Plasmid list……………………………………………………………...…..59

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