WWOX (WW domain-containing oxidoreductase) 為一腫瘤抑制基因，且其功能在先前研究中與阿茲海默症Tau蛋白有關。Tau 為一種微管結合蛋白，其對於tubulin之聚合及微管穩定扮演重要的角色，而GSK3β藉由磷酸化來調控Tau蛋白。高度磷酸化之Tau蛋白會降低與tubulin之親和力，進而喪失微管之穩定性。藉由生物資訊的預測與分析，我們發現在WWOX的序列中具有三個與GSK3β結合域相似之區域FXXXLI/VXRLE，而利用免疫沈澱法證明WWOX會與GSK3β相結合，並由免疫螢光分析發現二具有colocalization之情形。進一步利用in vitro之pull down assay證明，WWOX藉由ADH/SDR domain與GSK3β相結合，並利用定點突變法確此結合區域。而在in vitro kinase assay的實驗中，可觀察到WWOX可藉由結合GSK3β而抑制Tau在Ser396及Ser404之磷酸化，此結果亦可在in vivo實驗中觀察到，因此WWOX對於GSK3β之調控可能參與在neurite outgrowth。在先前的MT formation assay的結果中顯示，經由GSK3β高度磷酸化之Tau蛋白會喪失Tau蛋白對於tubulin polarization之能力。結合上述結果，WWOX之功能可能參與於神經分化中。而有趣的是，在以維生素A(retinoic acid)誘導分化之SH-SY5Y的細胞中，隨著細胞分化的比例愈高，WWOX蛋白表現隨之升高，且磷酸化Ser396 Tau 之表現量則隨之下降。以RNAi 抑制WWOX的表現，可觀察到磷酸化Ser396 Tau蛋白之增加，且SH-SY5Y的分化減緩。進一步使用GSK3抑制劑Bio可促使SH-SY5Y細胞之分化，而過量表現GSK3β則會減低SH-SY5Y之分化。綜合以上結果，WWOX藉由調控GSK3β活性而減低Tau的高度磷酸化，而此可能包含在神經分化中促進neurite之outgrowth。

WW domain-containing oxidoreductase WWOX, a putative tumor suppressor, has been suggested to be involved in the hyperphosphorylation of Alzheimer’s tau. Tau is a microtubule-associated protein which plays an important role in microtubule assembly and stability. GSK-3b regulates tau by phosphorylation as well as by regulating splice variance. Hyperphosphorylated tau has less affinity toward the microtubules and disrupting microtubule stability. By bioinformatics analysis, we found that WWOX has three GSK3β binding motifs, FXXXLI/VXRLE. The results of immunoprecipitation demonstrated that WWOX interacted physically with GSK3β, which was supported by the colocalization of WWOX and GSK3β in immunofluorescence study. Using in vitro GST pull down assay, we demonstrated that WWOX can bind directly to GSK3β through its ADH/SDR domain. The site-direct mutagenesis was performed to locate the GSK3β binding sequences within the ADH/SDR domain precisely. By in vitro kinase assay, we found that WWOX can inhibit Ser396 and Ser404 phosphorylation of Tau by GSK3β in a dose- and time- dependent manner, indicating that WWOX may be involved in regulating GSK3β activity in cells. It has been demonstrated that inhibition of GSK3β plays an essential role during neuron differentiation. By in vitro MT formation assay, we showed that GSK3β hyperphospohorylated Tau failed to promote tubulin polymerization. Together with the Tau hyperphosphorylation inhibited its function to promote microtubule formation and neurite outgrowth, we investigated the effect of WWOX in neuron differentiation. Interestingly, overexpression of WWOX enhanced the SH-SY5Y differentiation with or without retinoic acid treatment. SH-SY5Y cells increase expression of WWOX and decrease expression of pTau S396 as they differentiate in culture. RNAi-mediated knockdown of WWOX in RA-differentiated SH-SY5Y cells caused a decrease in neurite outgrowth. Furthermore, inhibition of GSK3 by Bio enhanced SH-SY5Y differentiation and overexpression of GSK3β caused a decrease in neurite outgrowth. We concluded that WWOX may be involved in regulating GSK3β activity to reduce the level of phosphorylated Tau that subsequently promoted the neurite outgrowth during neuron differentiation.

Table of Contents

1. 中文摘要............................................2
2. Abstract............................................3
3. Abbreviation........................................5
4. Introduction........................................6
5. Purpose............................................13
6. Materials and Methods..............................14
7. Results............................................22
8. Discussion.........................................30
9. References.........................................35
10. Figures............................................40
11. Appendix...........................................62

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