Title

Modeling neuronal aging via repetitive neural activation-induced DNA breaks

DGIST Authors

Jaewoo Seok ; Sung Bae Lee ; Jinsoo Seo

Advisor

이성배

Co-Advisor(s)

Jinsoo Seo

Issued Date

2025

Awarded Date

2025-08-01

Type

Thesis

Description

Aging, iPSC-derived neuron, Neuronal activity, DNA double strand breakage

Table Of Contents

List of Contents
Abstract i
List of contents ii
List of Table and Figures iv


Ⅰ. Introduction 1
1.1. Aging and Alzheimer’s disease 1
1.2. Aging model systems 2
1.3. Modeling aged neurons via inducing neuronal activity-mediated DNA breaks 3
1.3.1. KCl treatment to induce neuronal depolarization 4
1.4. Cell-type specific aging markers for neurons 5

II. Materials and Methods 6
2.1. Cell Culture 6
2.1.1. Human induced pluripotent stem cells (hiPSCs) 6
2.1.2. hiPSC-derived cortical neuron (iNEU) differentiation 6
2.1.3. Human dermis fibroblasts (HDFs) 7
2.1.4. Human fibroblast-derived cortical neuron (fNEU) differentiation 7
2.2. Fluorescent-activated cell sorting (FACS) 8
2.3. Lentivirus packaging 8
2.4. Real-time quantitative PCR (RT-qPCR) 9
2.4.1. mRNA extraction and cDNA synthesis 9
2.4.2. RT-qPCR 10
2.4.3. Primer sequences for RT-qPCR 10
2.5. Immunocytochemistry 11
2.5.1. Antibodies 11
2.6. Multielectrode array (MEA) 12
2.7. Senescence associated β-galactosidase (SA-β-gal) assay 12
2.8. Statistical analysis 13

III. Results 14
3.1. Generation of fibroblast-derived cortical neurons (fNEUs) via direct conversion 14
3.2. Induction of cellular senescence in HDFs by replicative exhaustion 19
3.3. Neuronal activity-induced DNA double-stand breaks (DBSs) in hiPSC-derived neurons 23
3.4. Modeling neuronal aging via repeated neuronal activation 27
3.5. Inducing activation-independent DNA double-strand breakages (DSBs) 30

IV. Discussion 34

V. References 37

Abstract in Korean 41


List of Table and Figures iv
Table1. List of primer sequences used in RT-qPCR 10
Figure 1. Generation of fNEUs via direct conversion from human dermal fibroblasts (HDFs). 16
Figure 2. HDFs were aged through replicative expansion, and aged fNEUs were derived from these cells. 20
Figure 3. DNA double-strand breakages (DSBs) were induced in a neuronal activation-dependent manner. 24
Figure 4. Neuronal aging was partially induced by repetitive physiological neuronal activation. 28
Figure 5. I-PpoI induction in human cells generated DNA double-strand breakages (DSBs). 32

URI

https://scholar.dgist.ac.kr/handle/20.500.11750/59810
http://dgist.dcollection.net/common/orgView/200000888205

DOI

10.22677/THESIS.200000888205

Degree

Master

Department

Department of Brain Sciences

Publisher

DGIST

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