Publications
Detailed Information
Study on the characterization of caspase-cleaved tau transgenic mice and genetic modifier POLDIP2 in tauopathy : Tau에 의한 퇴행성뇌질환 모델로서 caspase 절단 형태의 tau를 과발현하는 쥐 분석과 Tau 질환을 조절하는 새로운 유전자 POLDIP2의 기능 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 정용근 | - |
| dc.contributor.author | 김영두 | - |
| dc.date.accessioned | 2017-07-14T00:50:22Z | - |
| dc.date.available | 2017-07-14T00:50:22Z | - |
| dc.date.issued | 2015-08 | - |
| dc.identifier.other | 000000049856 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/121423 | - |
| dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 8. 정용근. | - |
| dc.description.abstract | In Alzheimers disease (AD) and other tauopathy, Tau proteins form intracellular aggregates and Tau filaments. These tau oligomers are known as one of major cause of neurotoxicity in tauopathy. However, the mechanisms that regulate Tau aggregation are not fully understood. In this study, I approached animal model to better understand the mechanism by generating tau transgenic mouse model showing tau oligomers and by isolating tau aggregation regulator.
In the AD brain, activated caspases cleave tau at the 421th Asp, generating a caspase-cleaved form of tau, TauC3. Although TauC3 is known to assemble rapidly into filaments in vitro, a role of TauC3 in vivo remains unclear. Here, I generated a transgenic mouse expressing human TauC3 using a neuron-specific promoter. In this mouse, I found that human TauC3 was expressed in the hippocampus and cortex. Interestingly, TauC3 mice showed drastic learning and spatial memory deficits and reduced synaptic density at a young age (1.3?3 months). Notably, tau oligomers as well as tau aggregates were found in TauC3 mice showing memory deficits. Further, i.p. or i.c.v. injection with methylene blue or Congo red, inhibitors of tau aggregation in vitro, and i.p. injection with rapamycin significantly reduced the amounts of tau oligomers in the hippocampus, rescued spine density, and attenuated memory impairment in TauC3 mice. Together, these results suggest that TauC3 facilitates early memory impairment in transgenic mice accompanied with tau oligomer formation, providing insight into the role of TauC3 in the AD pathogenesis associated with tau oligomers. Also, in this study, we show that POLDIP2 is a novel regulator of Tau aggregation. From a cell-based screening using cDNA expression library, we isolated POLDIP2 which increased Tau aggregation. Expression of POLDIP2 was increased in neuronal cells by the multiple stresses, including A?, TNF-? and H2O2. Accordingly, ectopic expression of POLDIP2 enhanced the formation of Tau aggregates without affecting Tau phosphorylation, while down-regulation of POLDIP2 alleviated ROS-induced Tau aggregation. Interestingly, we found that POLDIP2 overexpression induced impairments of autophagy activity and partially proteasome activity and this activities were retained in DUF525 domain of POLDIP2. In a drosophila model of human tauopathy, knockdown of the drosophila POLDIP2 homolog, CG12162, attenuated rough eye phenotype induced by Tau overexpression. Further, the lifespan of neural-TauR406W transgenic files were recovered by CG12162 knockdown. Together, these observations indicate that POLDIP2 plays a crucial role in Tau aggregation via the impairment of autophagy activity, providing insight into mechanism that regulates Tau aggregation in Tau pathology. | - |
| dc.description.tableofcontents | CONTENTS
ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .i CONTENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii LIST OF FIGURES AND TABLES. . . . . . . . . . . . . .vi ABBREVIATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . xiii CHAPTER 1. Caspase-cleaved tau exhibits rapid memory impairment associated with tau oligomers in a transgenic mouse model 1-1 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 1-2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . .4 1-3 Material and methods. . . . . . . . . . . . . . . . . . . .7 Generation of TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Cell culture, DNA construction, and DNA transfection. . . . . .9 Dendritic spine density analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 In vitro Tau fibrillation and thioflavin T fluorescence assay. . . . . . 10 Filter trap assay. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .11 Histology and immunohistochemistry . . . . . . . . .. . . . . . . . .11 Behavior tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Y-maze. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .13 Novel object recognition test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Passive avoidance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Wire-hang test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Intracerebroventricular injection of Aβ oligomers. . . . . . . .15 1-4 RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Generation of a neuron-specific TauC3 transgenic mouse.. . . .16 Impaired memory function and reduced synaptic density in TauC3 mice at an early age. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Appearance of Tau oligomers in TauC3 mice showing memory deficit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Accumulation of detergent-insoluble Tau aggregates in TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .22 Methylene blue improves memory function and synaptic density with reduced Tau oligomers in TauC3 mice...............................................23 Rapamycin attenuates memory deficits in TauC3 mice.. . . . . . .25 1-5 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 1-6 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87 CHAPTER 2. Essential role of POLDIP2 in Tau aggregation and neurotoxicity via Autophagy / proteasome inhibition 2-1 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 2-2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 101 2-3 Material and methods. . . . . . . . . . . . . . . . . . 104 Cell culture and DNA transfection. . . . . . . . . . . . . . . . . . . . . . . . . 104 Western blotting and antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . 104 Filter trap assay for Tau aggregates. . . . . . . . . . . . . . . . . 105 DNA construction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Proteasome Activity Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Reverse transcription-PCR. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 106 Small interfering RNA (siRNA) transfection. . . . . . . . . . . . . . . 106 Transgenic Drosophila. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Longevity assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 2-4 RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Identification of POLDIP2 as an inducer of Tau aggregation. . 108 The expression of POLDIP2 is increased by reactive oxygen stress for Tau aggregation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Increased POLDIP2 impairs autophagy activity. . . . . . . . . . . . . . 110 The POLDIP2 regulates Tau aggregation and autophagy via DUF525 domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 POLDIP2 knockout rescues human Tau-mediated neural degeneration in drosophila. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2-5 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 2-6 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . 136 2-7 ABSTRACT IN KOREAN/국문 초록. . . . . . . . 143 LIST OF FIGURES AND TABLES Fig. 1-1. TauC3 mice display neuronal-specific expression of human tau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28 Fig. 1-2. Expression of human caspase-cleaved Tau in the brain region of cortex, hippocampus, cerebellum and striatum of transgenic mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 Fig. 1-3. Human TauC3 is expressed in cortex and hippocampus of TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 Fig. 1-4. Rapid memory impairment in TauC3 mice. . . . . . . . . . . . . . . . . .34 Fig. 1-5. TauC3 mice does not show severe behavior abnormality. ……....36 Fig. 1-6. Reduced synaptic proteins in the hippocampus CA3 region of TauC3 mice at young age. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38 Fig. 1-7. Occurrence of TUNEL-positive cell death in aged TauC3 mice. . 40 Fig. 1-8. Hematoxylin staining showing cell numbers in the hippocampus and cortex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42 Fig. 1-9. Lifespan analysis of TauC3 mice.. . . . . . . . . . . . . . . . . . . . .44 Fig. 1-10. Appearance of Tau hyperphosphorylation in TauC3 mice. . . . . .46 Fig. 1-11. Detection of high molecular weight of Tau oligomers in TauC3 mice.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48 Fig. 1-12. Immunohistochemical analysis showing Tau oligomers in the hippocampal tissue of TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Fig. 1-13. The treatment with Aβ oligomers induces the formation of T22-positive tau oligomers in a caspase-dependent manner in cultured neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52 Fig. 1-14. Caspase inhibitor blocks Tau cleavage and oligomer formation, and attenuates memory deficit in the Aβ oligomers-injected mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54 Fig. 1-15. Detection of detergent (NP-40)-insoluble Tau aggregates in TauC3 mouse brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56 Fig. 1-16. Detection of silverstainig positive Tau aggregates with filamentous structure in TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . .58 Fig. 1-17. Effects of methylene blue and Congo red on the filament formation of Tau protein in vitro and in cell model. . . . . . . . . . . . . . . .60 Fig. 1-18. i.p. injection of Methylene blue or i.c.v. injection of Congo red retards the memory impairment in TauC3 mice. . . . . . . . . . . . . . . .62 Fig. 1-19. Memory impairment of TauC3 mice is not attenuated by i.p.-injection of Congo red. . . . . . . . . . . . . . . . . . . . . . . . . . . . .64 Fig. 1-20. Methylene blue decreases the amounts of 170kDa Tau oligomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Fig. 1-21. Methylene blue preferentially decreases the amounts of TauC3 and PHF-1-positive tau. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68 Fig. 1-22. Immunohistochemical analysis showing the decrease of human TauC3 and tau oligomers by methylene blue in TauC3 mice. . . . . . 70 Fig. 1-23. Methylene blue rescues the loss of spine density and synaptic proteins in TauC3 overexpressed primary neuron and TauC3 mice. .72 Fig. 1-24. Rapamycin rescues the spatial and recognition memory impairment in TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74 Fig. 1-25. Reduction of the phosphorylated tau and detergent-insoluble tau aggregates by rapamycin in TauC3 mice. . . . . . . . . . . . . . . . . . . . .76 Fig. 1-26. Enhanced autophagy markers in the rapamycin-treated hippocampal tissues of TauC3 mice. . . . . . . . . . . . . . . . . . . . . . . . . . .78 Fig. 1-27. Effect of rapamycin on the level of tau oligomers in TauC3 mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80 Fig. 2-1. Isolation of POLDIP2 as a regulator of Tau aggregation. . . . . . .114 Fig. 2-2. POLDIP2 expression is increased by oxidative stress in SH-SY5Y cells and primary neuron. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .116 Fig. 2-3. Oxidative stress enhance Tau aggregation and knockdown POLDIP2 protein expression inhibit oxidative stress induced Tau aggregation in the SH-SY5Y cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118 Fig. 2-4. POLDIP2 expression impairs autophagy flux. . . . . . . . . . . . . . . 120 Fig. 2-5. Increased POLDIP2 impairs proteasome activity. . . . . . . . . . . . .122 Fig. 2-6. POLDIP2 regulates Tau aggregation and autophagy flux through its DUF525 domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124 Fig. 2-7. POLDIP2 does not regulates proteasome activity through its yccv-like or DUF525 domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .126 Fig. 2-8. Knockdown of POLDIP2 expression rescues Tau toxicity in a Drosophila model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .128 Fig. 2-9. Deficiency of Drosophila POLDIP2 homologue CG12162EY08866 flies does not alter Tau phosphorylation in the flies expressing tau. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130 | - |
| dc.format | application/pdf | - |
| dc.format.extent | 4624342 bytes | - |
| dc.format.medium | application/pdf | - |
| dc.language.iso | en | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | tau | - |
| dc.subject.ddc | 570 | - |
| dc.title | Study on the characterization of caspase-cleaved tau transgenic mice and genetic modifier POLDIP2 in tauopathy | - |
| dc.title.alternative | Tau에 의한 퇴행성뇌질환 모델로서 caspase 절단 형태의 tau를 과발현하는 쥐 분석과 Tau 질환을 조절하는 새로운 유전자 POLDIP2의 기능 연구 | - |
| dc.type | Thesis | - |
| dc.description.degree | Doctor | - |
| dc.citation.pages | xiii, 145 | - |
| dc.contributor.affiliation | 자연과학대학 생명과학부 | - |
| dc.date.awarded | 2015-08 | - |
- Appears in Collections:
- Files in This Item:
Item View & Download Count
Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.