Therapeutic Role of miRNAs in Alzheimer’s Disease = 마이크로RNA를 이용한 알츠하이머병 치료 연구

다국어 초록 (Multilingual Abstract)

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ) accumulation and tau hyperphosphorylation, which lead to synaptic dysfunction and cognitive decline. Despite remarkable research efforts, effective disease-modifying therapies remain limited. MicroRNAs (miRNAs), as post-transcriptional regulators of gene expression, have emerged as critical modulators of neuronal differentiation, survival, and signaling pathways, thereby offering potential therapeutic opportunities in AD. In this study, I investigated the functional roles of miR-937-3p, miR-4536-3p, miR-4650-5p, and miR-4284 in neuronal differentiation and AD pathogenesis by using in vitro (Aβ-treated SH-SY5Y cells) and in vivo (5xFAD mice) systems. The upregulation of neuronal markers (tubulin beta 3 class III, microtubule-associated protein 2, neurofilament heavy chain, neurofilament medium chain, neuronal nuclei, and synaptophysin) demonstrated that the inhibition of miR-937-3p, miR-4536-3p, and miR-4650-5p enhanced neuronal differentiation; it also activated Wnt and mitogen-activated protein kinase signaling pathways. Mechanistically, Netrin1, Drebrin1, and Netrin-G1 were individually identified as direct targets of these miRNAs, individually. In Aβ-induced models, miR-937-3p inhibition reduced apoptosis, elevated the anti-apoptotic marker Mcl-1, and suppressed caspase activation, indicating a neuroprotective effect. Furthermore, miR-4536-3p inhibition restored DBN1 expression, reduced Aβ plaque deposition and tau phosphorylation, and improved cognitive performance in 5xFAD mice through activation of the phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3-beta pathway. In parallel, miR-4284 inhibition increased the expression of amyloid beta precursor protein binding family A Member 1, reduced neuroinflammation and apoptosis, and improved neuronal survival and cognition by modulating the Janus kinase/signal transducer and activator of transcription 3 pathway. Collectively, these findings highlight the therapeutic potential of targeting specific miRNAs to promote neuronal differentiation, restore synaptic plasticity, and attenuate neurodegeneration in AD. My work provides novel mechanistic insights into the miRNA-mediated regulation of AD pathogenesis and identifies miR-937-3p, miR-4536-3p, miR-4650-5p, and miR-4284 as promising candidates for the development of miRNA-based therapeutics for AD.

국문 초록 (Abstract)

알츠하이머병은 아밀로이드-β 축적, 타우 단백질의 과인산화, 시냅스 기능 장애, 인지 기능 저하를 특징으로 하는 진행성 신경 퇴행성 질환이다. 지금까지 많은 연구가 이루어졌음에도 불구하고 효과적인 질병 조절 치료법은 여전히 제한적이다. 마이크로RNA는 유전자 발현을 전사 후 단계에서 조절하는 인자로, 신경 분화, 생존, 신호 전달 경로의 핵심 조절자로 부각되며 알츠하이머병 치료의 잠재적 표적으로 주목받고 있다.
본 연구에서는 miR-937-3p, miR-4536-3p, miR-4650-5p, miR-4284의 신경 분화 및 알츠하이머병 치료 효과를 in vitro (SH-SY5Y 세포, Aβ 처리 모델)와 in vivo (5xFAD 유전형질 전환 마우스) 시스템을 통해 규명하였다. 그 결과, miR-937-3p, miR-4536-3p, miR-4650-5p 억제는 신경 분화를 촉진하였으며, 이는 TUBB3, MAP2, NEFH, NEFM, NeuN, SYP 등 신경세포 표지자의 발현 증가와 Wnt/MAPK 신호 경로의 활성화로 확인되었다. 또한, Netrin1 (NTN1), Drebrin1 (DBN1), Netrin-G1 (NTNG1)이 miR-937-3p, miR4536-3p, miR-4650-5p의 직접적 표적 유전자로 규명되었다.
Aβ 유도 세포 모델에서 miR-937-3p 억제는 세포사멸을 감소시키고, 항apoptotic 인자인 Mcl-1 발현을 증가시키며, caspase 활성을 억제하여 신경 보호 효과를 나타냈다. 또한 알츠하이머병 동물 모델에서 miR-4536-3p 억제는 DBN1 발현을 회복시키고, Aβ 침착과 타우 인산화를 감소시켰으며, PI3K/Akt/GSK3β 경로 활성화를 통해 5xFAD 마우스의 인지 기능을 개선하였다. 한편, miR-4284 억제는 APP 결합 단백질 APBA1의 발현을 증가시키고, 신경 염증과 세포사멸을 억제하여 JAK/STAT3 경로를 조절함으로써 신경 생존과 인지 기능을 향상시켰다.
종합적으로, 본 연구는 특정 마이크로RNA의 억제가 알츠하이머병 모델에서 신경 분화 촉진, 시냅스 가소성 회복 및 신경 퇴행 억제 효과를 나타냄을 성공적으로 규명하였다. 이는 알츠하이머병에서 마이크로RNA에 의해 매개되는 신경 기능 조절 기전에 대한 깊은 통찰을 제공하며, 해당 질환의 치료적 접근에 대한 새로운 가능성을 제시한다. 특히 본 연구를 통해 발굴된 miR-937-3P, miR-4536-3P, miR-4650-5P, miR-4284는 알츠하이머병 치료를 위한 마이크로RNA 기반 신약 개발에 있어 매우 유망한 치료 후보임을 시사한다.

목차 (Table of Contents)

• ABSTRACT (in English) 1
• CHAPTER 1 General Introduction 3
• 1.1 Alzheimer’ disease 4
• 1.2 MicroRNA 6
• 1.3 Relationship between AD and miRNAs 8
• 1.4 Target genes 9
• 1.5 Neuronal differentiation 11
• 1.6 Apoptosis 12
• 1.7 Inflammation 14
• 1.8 Rationale 16
• 1.9 Hypothesis 17
• CHAPTER 2 Materials and Methods 18
• 2.1 Cell culture and reagents 19
• 2.2 Demonstration of Aβ1-42 in the in vitro AD model 20
• 2.3 Biological analysis 21
• 2.4 Luciferase assay 21
• 2.5 RNA isolation and qPCR 22
• 2.6 Fluorescence-activated cell sorting 25
• 2.7 Animals 25
• 2.8 miRNA inhibitor transfection in the 5xFAD mouse brain 26
• 2.9 Morris water maze 27
• 2.10 Antibody array 27
• 2.11 Western blotting analysis 28
• 2.12 Immunocytochemistry 33
• 2.13 Immunohistochemistry 33
• 2.14 Screening of miRNAs from NI-hADSCs 34
• 2.15 Statistical analysis 35
• CHAPTER 3 Results 36
• 3.1 Inhibition of miR-937-3p, miR-4536-3p, and miR-4650-5p promotes neuronal differentiation via the Wnt/MAPK pathway 37
• 3.2 Therapeutic role of miR-937-3p inhibition in Aβ-induced neuronal cell death through NTN1 regulation 62
• 3.3 Inhibition of miR-4536-3p reduces Aβ accumulation and tau hyperphosphorylation in Alzheimer’s disease 89
• 3.4 Inhibition of miR-4284 attenuates apoptosis and neuroinflammation via APBA1/JAK1/STAT3 signaling in Alzheimer’s disease 131
• CHAPTER 4 Discussion 158
• 4.1 miRNA inhibition promotes neuronal differentiation 159
• 4.2 miRNAs previously implicated in neuronal differentiation 159
• 4.3 Therapeutic potential of miRNAs in AD 160
• 4.4 Biological functions of miR-937-3p and miR-4284 and its implications in neurodegeneration 161
• 4.5 Functional roles of target genes: NTN1, DBN1, NTNG1, and APBA1 163
• 4.6 Complexity of miRNA regulatory networks in AD pathology 166
• 4.7 Discrepancies between mRNA and protein levels 167
• 4.8 Activation of the PI3K/Akt/GSK3β Pathway as a Neuroprotective Mechanism 168
• 4.9 Neuroprotective function of STAT3 activation in AD 169
• 4.10 Limitations and future directions 170
• CHAPTER 5 Conclusions 173
• REFERENCES 177
• ABSTRACT (in Korean) 195
• ACKNOWLEDGEMENTS 197