Development of a novel cell based assay for tau aggregation
- 주제(키워드) tau aggregation , tau , Neurodegeneration
- 발행기관 고려대학교 대학원
- 지도교수 백자현
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위구분 석사
- 학과 일반대학원 생명과학과
- 원문페이지 78 p
- 실제URI http://www.dcollection.net/handler/korea/000000049733
- 본문언어 영어
- 제출원본 000045793137
초록/요약
Abnormal protein aggregation characterizes many neurodegenerative disorders. The microtubule-associated protein, tau forms intraneuronal filaments in a spectrum of neurological disorders collectively called tauopathies. In a healthy neuron, tau stabilizes microtubules promoting axonal outgrowth and neuronal plasticity. When pathologically hyperphosphorylated, tau molecules are dissociated from microtubules and become insoluble aggregates. This leads microtubule disruption and results in neuronal degeneration in brain. Due to its pathological significance, tau researches have been focused on the formation of the filamentous tau aggregates. However, there has been no single method that able to visualize and quantify the tau aggregation processes in living cells. I developed a cell-based sensor to monitor the early stage of tau aggregation in a living cell. By introducing bimolecular fluorescence complementation (BiFC) technique, I could achieve spatial and temporal information of tau dimerization and aggregation eliminating background fluorescence from monomeric tau. Recent studies have showed that tau oligomers, not a monomer nor aggregates, induce memory impairment and neuronal degeneration. Now it has become widely accepted that the intermediate oligomeric species might actually be toxic to neuronal cells. The larger inclusions might form as a survival strategy to protect neuronal cells by decreasing the number of toxic oligomers. Our fluorescence “Turn-on” sensor would be a useful tool to investigate tau pathology and to discover method to prevent and reverse the process.
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Table of contents
Table of contents---------------------------------------i
List of figures----------------------------------------- iii
List of tables-------------------------------------------v
Abstract-----------------------------------------------vi
I. Introduction-----------------------------------------1
1.1 Neurodegenerative diseases-----------------------1
1.2 Tau protein and tauopathies------------------------3
1.2.2 Tau expression of tau isoforms and mutations-----6
1.3 Tau aggregation and Tau hyperphosphorylation-----9
1.4 Difficulties in studying tau aggregation in cells-------14
II. Materials and Methods------------------------------17
2.1 DNA vector construction----------------------------17
2.2 Cell culture----------------------------------------18
2.3 Cell transfection------------------------------------18
2.4 Cell treatments-------------------------------------19
2.5 Western blot analysis-------------------------------19
2.6 Image anaylsis-------------------------------------20
III. Results and Discussion-----------------------------21
3.1 Biomolecular Fluorescence Complementation (BiFC)-21
3.2 Preparation of Tau-BiFC constructs-----------------24
3.3 Establishment of Tau-BiFC sensor-----------------33
3.4 Validadtion of Tau-BiFC sensor--------------------40
3.5 Application of Tau-BiFC sensor--------------------40
3.5.1 Adjustment of Tau-BiFC assay condition----------40
3.5.2 Screening of small molecule inhibitor for tau aggregation-44
IV. Conclusion----------------------------------------49
V. References----------------------------------------50
VI. Abstract in Korean---------------------------------54
VII. Appendix------------------------------------------56
