알츠하이머 환자 유래 유도만능줄기세포를 이용한 3D 뇌 뉴로스피어 모델의 개발 = Development of 3D brain neurosphere models using induced pluripotent stem cells derived from Alzheimer’s disease patient

국문 초록 (Abstract)

알츠하이머 질병은 가장 흔한 뇌 신경 퇴행성 질환 중 하나로, 치매 환자의 약 75%가 이에 해당하는 것으로 알려져 있다. 알츠하이머 질병의 명확한 원인은 아직 밝혀지지 않았으나 병리학적 증상으로는 ‘amyloid beta(Aβ)’와 ‘phosphorylated tau(p-tau)’가 뭉쳐서 각각 ‘senile plaques(SP)’, ‘neurofibrillary tangles(NFT)’라는 비정상 단백질 응집체들이 뇌 신경 주변에 과도하게 축적되어 독성 물질로 작용하여 주변의 뇌 신경 세포의 사멸을 일으키는 것으로 알려져 있다. 알츠하이머 치료제를 개발하는 노력은 지난 30여 년 동안 꾸준히 진행되었으며 특히 알츠하이머 대표 증상인 Aβ를 표적으로 하는 치료제 개발이 지속되었으나 현재까지 완치 방법 및 완치제를 찾아내지는 못했다. 약물 스크리닝을 위한 동물 질병 모델 및 2D 체외배양 기술들이 비약적인 발전을 이뤘으나 이들은 인간의 신체 장기가 지닌 3D 구조의 복잡성과 기능들을 효과적으로 재현하지 못한다는 큰 한계점을 지니고 있다. 최근에는 인간 체세포에서 유래된 유도만능 줄기세포(iPSC) 기술을 응용한 3D 오가노이드 기술이 이러한 한계점을 지닌 기존 방식들을 대체할 것으로 기대받고 있고 이를 약물 스크리닝 기술에 접목시키려는 노력들이 활발히 진행되고 있다.
본 연구에서는 3D 오가노이드 약물 스크리닝 플랫폼 개발을 위해서 기존에 본 연구실에서 분석했던 Preseniln 1-S170F mutation(PS1) 환자로부터 유래된 iPSC의 colony line을 선별하고 해당 세포로부터 만들어진 3D 오가노이드가 약물 스크리닝에 적합한지 확인하기 위해서 3D neurosphere를 분화시킨 후 알츠하이머 치료약물에 반응하는지 확인하였다. PS1 iPSC로부터 선별된 5가지 colony line 1, 3, 6, 9, 10을 10주 동안 cortical neuron으로 분화시킨 후 알츠하이머 증상인 Aβ를 측정하여 가장 많이 발현됨가 동시에 LY-2886721(BACE 1 inhibitor)를 처리했을 때 Aβ가 가장 크게 감소하는 colony line 3, 6번을 선별했다. 두 colony line 중 어느 것이 가장 p-tau 발현량이 높고 Rapamycin(mTORC1 inhibitor) 처리 시 p-tau 발현량이 크게 감소하는지 비교하여 최종적으로 colony line 3(C3)이 알츠하이머 약물 스크리닝에 적합할 것이라는 판단을 내렸다. 또, PS1 3D 오가노이드가 알츠하이머 치료약물에 잘 반응하는지 확인하기 위해서 3D neurosphere로 10주간 분화해 서 LY-2886721, Rapamycin, Memantine(NMDA inhibitor), Donepezil(Ache inhibitor)를 처리한 결과 LY-2886721에서 Aβ 및 p-tau 발현량의 감소가 나타났고 Rapamycin에서 p-tau 발현량이 감소한 것이 나타났으며 나머지 약물에서도 Aβ와 p-tau 발현량이 감소한 것을 확인할 수 있었다. 이를 통해, 본 연구에서 제작한 PS1 3D neurosphere가 알츠하이머 치료제에 반응해서 알츠하이머의 병리학적 특성인 Aβ와 p-tau 발현량이 감소하는 모습이 재현되는 것을 확인할 수 있었고, 새롭게 구축된 PS1 colony line 3(C3)을 해당 3D neurosphere에 접목해서 추후 다양한 알츠하이머 치료약물 후보군을 선별하는 실험들에 적용할 수 있을 것으로 기대하고 있다.

다국어 초록 (Multilingual Abstract)

Alzheimer's disease is one of the most common neurodegenerative diseases of the brain, and it is known that about 75% of dementia patients correspond to it. Although the cause of Alzheimer's disease is unknown, pathological symptoms include aggregation of 'amyloid beta (Aβ)' and 'phosphorylated tau (p-tau)'. It accumulates and acts as a toxic substance, causing the death of surrounding brain nerve cells. Efforts to develop a treatment for Alzheimer's have been steadily progressing for the past 30 years, and in particular, development of a treatment that targets Aβ, the representative symptom of Alzheimer's, has continued, but no cure or cure has been found so far. Although animal disease models and 2D in vitro culture technologies for drug screening have made rapid progress, they have a major limitation in that they cannot effectively reproduce the complexities and functions of 3D structures of human organs. Recently, 3D organoid technology using induced pluripotent stem cell (iPSC) technology derived from human somatic cells is expected to replace existing methods with these limitations, and efforts are being made to graft it into drug screening technology. In this study, for the development of a 3D organoid drug screening platform, colony lines of iPSCs derived from patients with Presenilin 1-S170F mutation (PS1), which were previously analyzed in our laboratory, were selected, and 3D organoids made from the cells are suitable for drug screening. To check whether the 3D neurosphere is differentiated, it was checked whether it responds to the Alzheimer's drug. Five colony lines 1, 3, 6, 9, and 10 selected from PS1 iPSCs were differentiated into cortical neurons for 10 weeks, and then Aβ, a symptom of Alzheimer’s, was measured and expressed the most, and LY-2886721 (BACE 1 inhibitor) was treated at the same time. colony lines 3 and 6, in which Aβ decreased the most, were selected. By comparing which of the two colony lines had the highest p-tau expression level and a significant decrease in p-tau expression level upon treatment with Rapamycin (mTORC1 inhibitor), it was finally determined that colony line 3 would be suitable for screening for Alzheimer's disease drugs.
In addition, to check whether the PS1 3D organoid responds well to the Alzheimer's drug, it was differentiated into 3D neurosphere for 10 weeks and treated with LY-2886721, Rapamycin, Memantine (NMDA inhibitor), and Donepezil (Ache inhibitor). As a result, Aβ in LY-2886721 and p-tau expression decreased. Rapamycin showed decreased p-tau expression level, and it was confirmed that Aβ and p-tau expression levels were also decreased in other drugs. Through this, it was confirmed that the PS1 3D neurosphere produced in this study reproduced the decreased expression of Aβ and p-tau, which are pathological characteristics of Alzheimer's in response to Alzheimer's treatment, and the newly constructed PS1 colony line 3 By grafting it to the 3D neurosphere, it is expected that it can be applied to experiments that select various Alzheimer's drug candidates in the future.

목차 (Table of Contents)

• ACKNOWLEDGEMENTS·········ⅰ
• TABLE OF CONTENTS·············ⅱ
• LIST OF FIGURES····················ⅳ
• ABSTRACT·····························ⅴ
• Ⅰ. INTRODUCTION·······································7
• Ⅱ. MATERIALS AND METHODS···················9
• 1. Patient derived-iPSC culture··················9
• 2. NPC culture··································10
• 3. Brain cortical neuron differentiation··················11
• 4. Brain cortical neurosphere differentiation··········11
• 5. Drug treatment······················12
• 6. ELISA assay·····························12
• 7. Cryosection····························13
• 8. Immunocytochemistry···········13
• 9. Western blot··························14
• 10. Statistical analysis················14
• Ⅲ. RESULTS···························15
• 1. Maintenance and characterization of PS1 iPSC colony lines·············15
• 2. NPC induction of PS1 iPSC colony lines···············17
• 3. AD-pathological analysis of PS1 colony line-derived neurons·············17
• 4. Reduction of Amyloid beta by drug treatment in PS1 colony line iPSC-derived neurons·············21
• 5. Reduction of phosphorylated tau level by drug treatment in PS1 colony line iPSC-derived neurons···25
• 6. Comparison continuity of MAP2 and GFAP in PS1 colony line iPSC-derived neurons·····31
• 7. PS1 cortical neurosphere induction & differentiation··················34
• 8. Amyloid beta and p-tau expression in PS1 neurosphere···············37
• 9. Control drugs treatment in PS1 neurosphere···············37
• Ⅳ. DISCUSSION······························40
• Ⅴ. CONCLUSION····························43
• REFERENCES··································44
• ABSTRACT IN KOREAN··················47