Title

Functional analyses of Collybistin mutants found in neurodevelopmental disorders

Alternative Title

신경 발달 장애에서 발견되는 Collybistin 단백질의 변이에 대한 기능적인 연구

DGIST Authors

Hyojeong Lee ; Ji Won Um ; Jaewon Ko

Advisor

엄지원

Co-Advisor(s)

Jaewon Ko

Issued Date

2023

Awarded Date

2023-02-01

Citation

Hyojeong Lee. (2023). Functional analyses of Collybistin mutants found in neurodevelopmental disorders. doi: 10.22677/THESIS.200000655705

Type

Thesis

Description

Collybistin, Gephyrin clustering, GABAA receptor, GEF, Autism

Table Of Contents

I. Introduction 1
1.1 Synapses are Important for Transmitting Information and Communication with Neurons. 1
1.2 Excitatory and Inhibitory Synaptic Proteins Are Associated with Neurotransmission. 2
1.3 Various Cb isoforms and Domains of Cb 3
1.3.1 SH3 Domain Has Autoinhibitory Roles by Adopting a Closed Conformation. 3
1.3.2 DH Domain Activates GTPases by functioning as specific GEF. 4
1.3.3 PH Domain Mediates Gephyrin Clustering by Binding with PI3P. 5
1.4 Missense Mutations of Collybistin Are Related with Neurodevelopmental Disorders. 5
II. Materials and Methods 8
2.1 Plasmids 8
2.2 Antibodies 8
2.3 Generation of Collybistin KO mice 9
2.4 Animals 9
2.5 Culture, Transfection, Immunostaining, and Imaging in COS-7 Cells 10
2.6 Culture, Infection, Transfection, Immunostaining, and Quantitative Analyses of Cortical and Hippocampal Neurons 11
2.7 Preparation of Brain Lysates and Immunoblot Analysis 11
2.8 Preparation of Lentiviruses 12
2.9 Preparation of Adeno-associated Viruses and Titration 12
2.10 Stereotaxic Injection 13
2.11 Mouse Behavioral Tests 13
Open-Field Test 14
Marble Burying Test 14
Elevated Plus Maze Test 14
Three-Chamber Sociability Test 15
Prepulse Inhibition 15
Ultrasonic Vocalization Test 16
2.12 Statistics 16
III. Results 17
3.1 Cb Isoforms Are Identified by Alternative Splicing of Cb mRNAs Depending on Its Species. 17
3.2 Collybistin Missense Variants Which Are Associated with Neurodevelopmental Disorders Express at the Same Level in Western Blotting. 17
3.3 CbII(SH3-) Mutation Transfected with EGFP-Gephyrin Showed Distinct Gephyrin Clustering Patterns Compared with CbII(SH3-) WT. 18
3.4 hCbIII Transfected With EGFP-Gephyrin and Neuroligin-2 (NL2) in COS-7 Cell Showed Various Gephyrin Cluster Forms Depending on Its Mutations. 19
3.5 Inhibitory Somatic Puncta Density Increased in Hippocampal Cultured Neurons Transfected with CbII(SH3-) WT. 20
3.6 CbII(SH3-) Missense Variants Transfected in Transfected Mouse Cultured Neurons to Identify the Function of Cb Domains. 20
3.7 Cb KO mice in the mPFC region Showed Behavioral Phenotypes in Behavioral Assays. 21
IV. Discussion and Conclusion 24
Figures 27
References 43
요약문 49

URI

http://hdl.handle.net/20.500.11750/45695
http://dgist.dcollection.net/common/orgView/200000655705

DOI

10.22677/THESIS.200000655705

Degree

Master

Department

Department of Brain Sciences

Publisher

DGIST

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