학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 생명과학부, 2018. 8. 박상현.Tau is an intracellular microtubule-associated protein that stabilizes microtubules. Under normal physiological conditions, Tau is a naturally unfolded and highly soluble protein. However, in Alzheimers disease, Tau is hyperphosphorylated and aggregated into intracellular neurofibrillary tangles (NFT). Aggregated Tau can transmit into adjacent cells and induce aggregation. This prion-like Tau propagation is an important mechanism for disease progression. Therefore, inhibition of aggregation and transmission of Tau can provide an important strategy for therapeutic agents of Alzheimers disease. In this study, we screened six novel inhibitors for Tau aggregation and designed one novel inhibitor based on the screened compounds structures. A total of 62 compounds were synthesized and 32 compounds efficiently inhibited Tau aggregation. Additionally, we developed a new measuring method for membrane binding of aggregated Tau. To measure aggregation-dependent membrane binding, fluorescence labeled Tau was utilized. Tau bound to living cell membranes by aggregation in a dose-dependent manner. In addition, using aggregation inhibitor, we confirmed that this method could be a convenient cell-based approach for validation or screening of membrane binding inhibitors.
Chapter 1. Screening compounds for Modulating of Tau aggregation 1
INTRODUCTION 2
MATERIALS AND METHODS - 4
Tau K18 purification 4
In vitro compound screening 5
Thioflavin T (ThT) fluorescence assay 5
Bis-ANS fluorescence assay - 5
Transmission electron microscopy (TEM) - 6
MTT reduction assay - 6
RESULTS - 8
Compound screening for Tau K18 aggregation 8
Compound scaffold for analog synthesis 8
Neutral red (NR) analogs - 9
Phneylenediamine (PED) analogs 10
Thiazolidine (Thi) analogs - 10
Dimethylphenzine (DMP) analogs 11
De novo scaffold (DN) analogs 11
Bis-ANS fluorescence assay 12
Cell viability assay 13
Filter-trapping assay 13
Inhibiting β-sheet formation during Tau K18 aggregation 14
Time-dependent disruption of β-sheet structure 15
DISCUSSION 26
Chapter 2. Measurement membrane binding of Tau by using fluorescence 82
INTRODUCTION 83
MATERIALS AND METHODS - 85
Purification and labeling of recombinant Tau K18 - 85
In vitro Tau K18 aggregation - 86
SDS-PAGE analysis 86
Thioflavin T (ThT) fluorescence assay 87
Membrane binding analysis 87
RESULTS - 89
Aggregation with the fluorescence labeled Tau K18 89
Membrane binding of Tau K18 aggregates - 89
Aggregation state and dose-dependent membrane binding of Tau K18 - 91
Binding of Tau K18 in various cell lines - 91
Membrane binding of α-synuclein aggregates - 93
DISCUSSION - 125
REFERENCES 128
국문초록 138Docto
