Exploring postsynaptic CLSTN3 and mitochondrial CLPB: Insights into synapse regulation and neurodegenerative diseases
- Title
- Exploring postsynaptic CLSTN3 and mitochondrial CLPB: Insights into synapse regulation and neurodegenerative diseases
- Author(s)
- Hyeonho Kim
- DGIST Authors
- Hyeonho Kim ; Ji Won Um ; Jaewon Ko
- Advisor
- 엄지원
- Co-Advisor(s)
- Jaewon Ko
- Issued Date
- 2024
- Awarded Date
- 2024-02-01
- Type
- Thesis
- Description
- Synapse formation;hippocampus;CLSTN3;NRXNs;proteostasis;ClpB;Huntington’s disease;striatum
- Table Of Contents
-
General Introduction 1
Part 1. Calsyntenin-3 Regulates Excitatory Synapse Formation in the Mouse Hippocampus by
Interacting with α- and β-Neurexins
I. Introduction 6
1.1 Synapse and Synaptic adhesion molecules 6
1.2 Role of Neurexins as presynaptic hub molecules 7
1.3 Synaptic functions of Calsyntenins 9
1.4 Role of Calsyntenins in Alzheimer's disease 11
II. Materials & Methods 15
2.1 Materials 15
2.1.1 Animals 15
2.1.2 Recombinant DNA 16
2.1.3 Antibodies 17
2.2 Methods 18
2.2.1 Animal husbandry and handling 18
2.2.2 HEK293T cell culture 18
2.2.3 Primary neuron culture 19
2.2.4 Preparation of recombinant adeno-associated viruses (AAV) 19
2.2.5 Titer measure of recombinant adeno-associated viruses 20
2.2.6 Stereotaxic surgery and virus injections 20
2.2.7 Preparation of solubilized proteins 21
2.2.8 Cell Surface-binding assay 21
2.2.9 Pulldown assays 22
2.2.10 Direct protein binding assays 22
2.2.11 Preparation of protein samples (Crude synaptosomal (P2) fractionation) 23
2.2.12 Immunohistochemistry (IHC) 23
2.2.13 Nissl staining 24
2.2.14 Mouse behavioral test 25
2.2.14.1 Open field test 25
2.2.14.2 Novel object recognition test 25
2.2.14.3 Y-maze test 25
2.2.14.4 Elevated plus maze test 25
2.2.14.5 Light and dark transition test 26
2.2.15 Statistical analysis 26
III. Results 27
3.1 Clstn3 exhibits direct interaction with Nrxn1α and Nrxn1β 27
3.2 Generation of anti-CLSTN3 antibodies and Clstn3 localization in the hippocampus 28
3.3 Generation and characterization of Clstn3 Knockout mice 29
3.4 Clstn3 cadherin domains are required for excitatory synapse formation in the hippocampal CA1 region of Clstn3 knockout mice 29
3.5 The generation and characterization of Nestin-Clstn3 mice 31
3.6 Clstn3 cadherin domains are necessary for excitatory synapse formation in the hippocampus CA1 regions of Nestin-Clstn3 mice 31
3.7 Nestin-Clstn3 mice exhibit elevated bright light-induced anxiety 32
IV. Discussion & Conclusion 54
Part 2. ClpB Inhibits Huntingtin Aggregation and Modulates Inhibitory Synapse Formation in a
Mouse Model of Huntington’s Disease
I. Introduction 57
1.1 The Role of Heat Shock Proteins in Maintaining Proteostasis 57
1.2 The heat-shock protein (HSP) family 58
1.3 The role of the Hsp100/Clp subfamily 59
1.4 The role of Hsp104/ClpB in eukaryotes 60
1.5 The role of ClpB (Skd3) in mammals 62
1.6 The structure of Hsp104/ClpB 64
1.7 Neuro-aggregates near mitochondria 65
1.8 Molecular chaperones and neurodegeneration 69
1.9 Disaggregase activity in ClpB-mutant patients 71
II. Materials & Methods 76
2.1 Materials 76
2.1.1 Animals 76
2.1.2 Recombinant DNA 76
2.1.3 Antibodies 77
2.2 Methods 78
2.2.1 HEK293T cell culture 78
2.2.2 Primary neuron culture 78
2.2.3 Preparation of competent cell 79
2.2.4 Preparation of recombinant adeno-associated viruses (AAV) 79
2.2.5 Titer measure of recombinant adeno-associated viruses 80
2.2.6 Preparation of lentiviruses 80
2.2.7 Stereotaxic surgery and virus injections 81
2.2.8 Preparation of protein samples 81
2.2.8.1 Protein extraction from various tissues 81
2.2.8.2 Mitochondrial fractionation 81
2.2.9 Immunohistochemistry (IHC) 82
2.2.10 LDH release assay 82
2.2.11 Mitochondrial membrane potential measurement 82
2.2.12 Quantitative reverse transcription-PCR 83
2.2.13 Statistical analysis 83
III. Results 85
3.1 The role of ClpB in the disaggregation of neurodegenerative disease-associated proteins 85
3.2 ClpB knockout causes Htt aggregation and cytotoxicity in HEK293T cells 86
3.3 Decreased ClpB levels are associated with increased mitochondrial fission and cytotoxicity in cultured neurons 87
3.4 Characterization of ClpB expression in the mouse brain 88
3.5 ClpB regulates WT and mHTT aggregation in the striata of WT mice 89
3.6 ClpB regulates WT and mHTT aggregation in the striatum of a mouse model of HD 90
3.7 ClpB regulates inhibitory synapse formation in the striatum of a mouse model of HD 91
IV. Discussion & Conclusion 114
V. References 117
- URI
-
http://hdl.handle.net/20.500.11750/47998
http://dgist.dcollection.net/common/orgView/200000731445
- Degree
- Doctor
- Department
- Department of Brain Sciences
- Publisher
- DGIST
- Related Researcher
-
-
Um, Ji Won
- Research Interests Molecular and cellular mechanisms underlying synapse elimination; Key synaptic mechanisms associated with Alzheimer's disease and autism spectrum disorders; Synaptic homeostasis
-
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- Appears in Collections:
- Department of Brain Sciences Theses Ph.D.
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