在先前的文獻描述過PKA/GSKIP/GSK3β複合物作為一個平台，用於磷酸化Drp1錨定，影響粒線體動力學以提供神經保護作用。最近，一種已知PKA/GSK3β基底Tau蛋白質已被揭示，在Tau蛋白Ser409的位置上磷酸化與阿茲海默氏症病中的神經元細胞終止有相關。在本研究中，探討PKA/GSKIP/GSK3β複合物介導Tau蛋白磷酸化。我們的數據顯示，在SH-SY5Y的細胞中過度表達GSKIP-WT基因增加了Tau ser409位點的磷酸化(並非S214; S262; S396; S404; T205 和 T212位點)，PKA與GSK3β結合的位點(GSKIP V41/L45和L130)，表明PKA與GSK3β結合位點與Tau ser409磷酸化位點有相關。而令人注意的是，在SH-SY5Y細胞中過度表達GSKIP-WT這個基因，使用活化劑(Foskion)處理，磷酸化Tau ser409位點表現量增加。然而移除GSK3β會導致Tau s409磷酸化降低，揭示GSKIP和GSK3β都是PKA/GSKIP/GSK3β/Tau複合物的關鍵。此外在SH-SY5Y細胞中表達GSK3β K85R(保留與GSKIP的結合能力)與K85M(喪失與GSKIP結合能力)基因，在表達GSK3β K85R的條件下，Tau ser409位點磷酸化明顯增加，確保GSK3β是作為錨定結合蛋白。綜合起來，我們的數據表明GSKIP和GSK3β作為錨定蛋白，促進cAMP/PKA/Tau複合物信號作用，再者由於文獻指出Protein Kinase會使Tau多個位點磷酸化，因此我進行了In vitro kinase assay實驗，得知兩個結果(1) .這些結果與以前研究的結果相同，PKA確實是可以使Tau Ser409、Ser214、Ser262這三個位置磷酸化。(2) 在PKA Kinase 與GSK3B kinase共同存在的情形下，使PKA構型產生變化，而使PKA磷酸化位點被遮蔽了，而使Tau Ser409這個磷酸位點無法被偵測。 由此結果而更進一步在誘導多能幹細胞中將基因利用CRISPR/Case9方式將帶有APP WT/D678H基因型病人，轉譯成APP WT/D678H、APP WT/WT與APP D678H/D678H、三種，結果顯示在Tau Ser214、Ser262、Ser409這三個磷酸位點在阿茲海默氏症患者腦中磷酸化，特別是Tau Ser409這個位置表現更強烈，而這三個磷酸位點屬於PKA相關，也證實PKA/GSKIP/GSK3β/Tau複合物，在阿茲海默氏症發病過程中，預期扮演一重要角色。

We previously described that PKA/GSKIP/GSK3β complex serves as a platform to anchor and phosphorylate Drp1 affecting mitochondria dynamics to provide neuroprotection. Recently, a known PKA/GSK3β substrate, Tau, has been revealed its S409 phosphorylation is associating with the termination of neuron cells in Alzheimer disease. In this study, we attempt to extend that Tau phosphorylation is also mediated by PKA/GSKIP/GSK3β working complex. Our data showed that GSKIP-WT overexpression in SH-SY5Y cells increased phosphorylation of Tau S409 site (not S214; S262; S396; S404; T205 and T212 sites) over that of PKA- and GSK3β binding-defective mutants (V41/L45 and L130) under forskolin challenge, indicating that both PKA and GSK3β bindings may be associated to phosphorylate Tau at S409 site. Surprisingly, treatment with foskion in GSKIP-WT-overexpressing SH-SY5Y cells were significantly increase Tau phosphorylation at S409, suggesting that only PKA kinase activity, but not GSK3β, is required in the GSKIP-mediated Tau phosphorylation. However, silencing of GSK3β resulted in a dramatic decrease in phosphorylation of Tau S409, revealing both GSKIP and GSK3β are crucial of PKA/GSKIP/GSK3β/Tau complex. Further, overexpressed kinase-dead GSK3β K85R (retains capacity to bind GSKIP) in SH-SY5Y cells, but not K85M (loss of capacity to bind GSKIP), has a higher Tau S409 phosphorylation, ensures that GSK3β acts solely as an anchor binding protein rather than its kinase activity in this signaling axis. Due to previous studies showed several different residues of Tau can be phosphorylated by PKA, we conducted In vitro kinase assay and provided two clear results: (1) As similar to early findings, PKA played a phosphorylation role on Ser409, Ser214 and Ser262 residues of Tau. (2) GSK3β provided a conformational shelter in PKA/GSKIP/GSK3β/Tau complex to harbor Tau Ser409 residue so that PKA is failed to phosphorylate Tau Ser409 residue. Furthermore, by using CRISPR/Cas9 system to produce APP WT/D678H and APP WT/WT 、 APP D678H/D678H multifunctional stem cells (modified from an APP patient WT/D678H genotype), the results of analysis showed phosphorylation in Tau Ser262 and Ser214 residues and the Tau Ser409 intense phosphorylation in the brain of Alzheimer’s patients.. Coupling with previous findings of PKA suggested that the PKA/GSKIP/GSK3β/Tau complex may plays a key role on the development of Alzheimer’s disease. Taken together, our data provide compelling evidence to implicate that both GSKIP and GSK3β function as anchoring proteins to enhance cAMP/PKA/Tau axis signaling during Alzheimer pathogenesis.

致謝 ……………………………………………………. ……… i
中文摘要 ………………………………………………………. ii
英文摘要………………………………………..……………… iv
英文縮寫表…………………………………………………….. vi
一、前言 ……………………………………………………….... 1
二、目的 ………………………………………………………… 5
三、材料與方法 ………………………………………………… 6
四、結果 …………………………………………………………17
五、討論 ……………………………………………..................21
六、文獻 ……………………………………………....................23
七、圖表 ……………………………………………........... …...25
八、附錄 ……………………………………………........ ….......30

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27. Virginia M-Y Lee,1 Michel Goedert,2 NEURODEGENERATIVE
TAUOPATHIES Annu. Rev. Neurosci. 2001. 24:1121–159 Copyright°c
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