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Table of contents
Summary
Index of Figures
Index of Tables
Abbreviations
1. Introduction
Alzheimer’s Disease and Amyloid Precursor Protein processing
1.1.1 The Amyloid Precursor Protein (APP)
1.1.2 Amyloidogenic Pathway
1.1.2.1 ß-Secretase
1.1.2.2 Amyloid ß
1.1.3 Non-Amyloidogenic Pathway
1.1.3.1 α -Secretase
1.1.3.2 P3
1.1.4 γ-Secretase
1.1.5 η-Secretase Pathway
1.1.5.1 η -Secretase
1.1.5.2 Amyloid η-α and Aη-β
1.1.6 Soluble APP
1.1.6.1 sAPP-α
1.1.6.2 sAPPβ
1.1.6.3 sAPP-η
1.1.7 Carboxyl-Terminal Fragment
1.1.7.1 AICD
1.1.8 Other APP Processing Pathways
The Hippocampus
1.2.1 Anatomy of the Hippocampus
1.2.2 Functional Role of the Hippocampus
1.2.3 Hippocampus and Memory
1.2.3.1 Definition of Memory
1.2.3.2 Memory formation
1.2.3.3 Working memory
1.2.4 Spatial Memory
1.2.5 Anxiety
1.2.6 An Object to Study in Neurobiology
Methods to Study Physio-Pathological Role of Proteins
1.3.1 Electrophysiology
1.3.1.1 The Synapse
1.3.1.1.1 Glutamate
1.3.1.1.1.1 mGluRs
1.3.1.1.1.2 iGluR
1.3.1.1.2 AMPAR
1.3.1.1.3 NMDAR
1.3.1.2 Long-Term Synaptic Plasticity
1.3.1.2.1 LTP
1.3.1.2.1.1 Mechanism of LTP
1.3.1.2.2 LTD
1.3.1.2.2.1 Mechanisms of LTD
1.3.1.3 Short-Term Synaptic Plasticity
1.3.1.3.1 Facilitation
1.3.1.3.2 Depression
1.3.1.4 Synaptic plasticity reflects behavioral outcome
1.3.2 Behavioral Studies
1.3.2.1 Experimental Design
1.3.2.1.1 Husbandry
1.3.2.2 Behavior
1.3.2.2.1 Spatial Navigation Tasks
1.3.2.2.2 Aversive Learning
1.3.2.2.3 Recognition Memory
1.3.2.2.4 Anxiety
2. Objectives
3. Material and Methods
Animal Model
3.1.1 Acute Effects of Aη on Synaptic Plasticity in Electrophysiology Field Recordings
3.1.2 Acute Effects of Synthetic Aη-α Injections into the CA1 Hippocampal Region or Lateral Ventricle
3.1.3 MISEPA2: A Transgenic Mouse Line Overexpressing Aη-α in the Brain
3.1.4 MISEPA4: A Transgenic Mouse Line Overexpressing Aη-α in the Brain with an Elevated Expression Level
Compared to MISEPA2 Line
3.1.5 APPΔEta: A Mouse Model Without η-Secretase Processing of APP due to Deletion the Enzymatic Recognition
Site on APP
3.1.1 Immunofluorescence and Western Blot Verify Expression of Aη-α in MISEPA2 Mice and Absence of Aη-α in APPΔEta Mice
3.1.2 Housing Conditions
3.1.3 Genotyping
3.1.3.1 Lysis
3.1.4 Polymerase-Chain Reaction Protocol for MISEPA2 and MISEPA4 Mouse Line
3.1.5 Polymerase-Chain Reaction Protocol for APPΔEta Mouse Line
3.1.6 Gel Electrophoresis
Electrophysiology
3.2.1 Peptides
3.2.2 Solutions
3.2.3 Harvesting and Slicing of Mice Hippocampi
3.2.4 Rig Set-Up
3.2.5 Field Recordings
3.2.5.1 Long-Term Synaptic Plasticity Recordings
3.2.5.2 Short-Term Synaptic Plasticity Recordings
3.2.5.2.1 Paired-Pulse Ratio
3.2.5.2.2 Synaptic Fatigue
3.2.5.2.3 Input/ Output
Acute M108 Injection
3.3.1 Surgery
3.3.2 Injection Volume and Concentration
3.3.3 Verification of Injection Site and Distribution of M108
3.3.3.1 Microscopy imaging for injection side in bilateral hippocampal injections
3.3.3.2 Blue Evans Staining to Verify Correct Placement of Canula Guides and Distribution after Injection
3.3.3.2.1 Perfusion Surgery
3.3.3.2.2 Perfusion
3.3.3.3 Western Blot
3.3.3.3.1 Brain Harvesting and Storage
3.3.3.3.2 RIPA Extraction Protocol
3.3.3.3.3 Immunoblotting
Behavioral Testing
3.4.1 Experimental Design for MISEPA2 and MISEPA4 Lines
3.4.2 Experimental Design of M108 Injected Mice Submitted to Behavioral Tasks
3.4.3 Experimental Design for APPΔEta Mice
3.4.4 Morris Water Maze
3.4.5 Novel Object Recognition
3.4.6 Contextual Fear Conditioning
3.4.6.1 CFC Protocol for the M108 Mice
3.4.6.2 CFC Protocol for APPΔEta Mice
3.4.7 T-Maze
3.4.7.1 Injection of M108 During T-Maze Testing
3.4.7.2 Familiar versus New Arm
3.4.7.2.1 Alterations of the T-Maze Task for Testing APPΔEta Mice
3.4.7.3 Forced Alternation
3.4.8 Open Field
3.4.9 Light-Dark Box
3.4.10 3-Chambers Social Interaction Task
3.4.11 Actimeter
Statistical Analysis
3.5.1 Electrophysiology
3.5.2 Behavioral Testing
4. Results
Consequences of Elevated Aƞ Levels on Synaptic Plasticity and Behavior
4.1.1 Effect of Acute Increase of Aη-α Levels on Synaptic Function and Behavior
4.1.1.1 Impact of Acutely Elevated Aƞ-α Levels on Synaptic Plasticity
4.1.1.1.1 Aƞ-α, the secreted APP fragment processed by ƞ- and α-secretases, acutely modulates post-synaptic plasticity mechanisms shifting the balance towards depression of synaptic st
4.1.1.2 Impact of Acute in vivo Injection of M108 into the Brain
4.1.1.2.1 Optimization of Protocol for in vivo Delivery of M108 Peptide
4.1.1.2.2 Presence of M108 in the Hippocampus Post-Injection Confirmed via Blue Evans Dye and Western Blot
4.1.1.2.3 Impact of Acute Injection of M108 into the Hippocampus on CFC
4.1.1.2.3.1 Acute Injection of M108 Prior to Conditioning Session in CFC Does Not Impact Memory Formation but Increases Memory Extinction During Secondary Downstream Retrieval
4.1.1.2.3.2 Contextual Memory Formation Is Not Impacted by Acute in vivo M108 Injection Irrespective of Time-point of Injection
4.1.1.2.4 Effect of Acute Injection of M108 into the Right Lateral Ventricle on Performance in T-Maze
4.1.1.2.4.1 Times of Injections Rather Than Delay of Retrieval Leads to Performance Impairment in M108 Injected Mice in a Familiar versus New Arm T-maze Task
4.1.1.2.4.2 Performance in M108 Injected Mice Is Not Impaired in a Forced Alternation T-Maze
4.1.1.3 Discussion
4.1.2 Effect of Chronic Enrichment of Aη-α Levels on Synaptic Function and Behavior
4.1.2.1 Impact of Chronically Elevated Aƞ-α Levels in a MISEPA2 Mouse Line on Synaptic Plasticity and Behavior
4.1.2.1.1 Influence of Chronically Elevated Aƞ-α Levels in a MISEPA2 Mouse Line on Synaptic Plasticity
4.1.2.1.1.1 Impaired LTP in MISEPA2 Mice
4.1.2.1.1.2 Short-Term Synaptic Plasticity and Basal Transmission Unaltered in MISEPA2 Mice
4.1.2.1.2 Influence of Chronically Elevated Aη-α Levels in a MISEPA2 Mouse Line on Behavior
4.1.2.1.2.1 No Impairment of Performance in a Spatial Memory Dependent MWM Task for MISEPA2 Mice
4.1.2.1.2.2 Indication of External Factors Influencing Contextual Memory of MISEPA2 MICE in CFC
4.1.2.1.2.3 Unaltered Diurnal Activity in MISEPA2 Mice
4.1.2.2 Impact of Chronically Elevated Aƞ-α Levels in a MISEPA4 Mouse Line on Synaptic Plasticity and Behavior
4.1.2.2.1 Synaptic Plasticity in MISEPA4 Mouse Line
4.1.2.2.1.1 LTP is Normal in MISEPA4 Mice
4.1.2.2.1.2 Impaired Basal Transmission in MISEPA4 Mice but No Alterations in Short-Term Synaptic Plasticity
4.1.2.2.2 Impact of Chronically Elevated Aη-α Levels in a MISEPA4 Mouse Line on Behavior
4.1.2.2.2.1 No Effect on Recognition Memory in NOR for MISEPA4 Mice
4.1.2.2.2.2 No Impairment of Performance in a Spatial Memory Dependent MWM Task for MISEPA4 Mice
4.1.2.2.2.3 Insufficient CFC Task Set-Up to Examine an Effect on Contextual Learning for MISEPA4 Mice
4.1.2.2.2.4 Normal Diurnal Activity in MISEPA4 Mice
4.1.2.3 Discussion
Consequences of Inhibition of the APP Processing η-Secretase Pathway on Synaptic Plasticity and Behavior
4.2.1 Impact of Deficiency in η-Secretase Processed APP in an APPΔEta Mouse Line on Synaptic Plasticity
4.2.1.1 No Alterations of LTP in APPΔEta Mice
4.2.1.2 Deficiency in ƞ-Secretase Processed APP Prevents LTD, a Phenotype Rescued by Acute Application of M108
4.2.1.3 Short-Term Synaptic Plasticity and Basal Transmission are Normal APPΔEta Mice
4.2.2 Impact of Loss of η-Secretase-dependent Cleavage of APP on Behavior
4.2.2.1 Indication of Reduced Anxiety for HOMO in Open Field
4.2.2.2 Indication of Reduced Anxiety for APPΔEta Mice in the Light-Dark Box
4.2.2.3 Regular Social Interaction Observed for APPΔEta Mice in the 3-Chambers Social Interaction Task
4.2.2.4 Impaired Spatial Memory in HOMO in T-Maze Task
4.2.2.5 APPΔEta Mice Display Loss of Spatial Memory in the MWM
4.2.2.6 APPΔEta Mice Display Normal Contextual Fear Memory
4.2.2.7 Diurnal Activity is Altered in HOMO Mice (Preliminary Data)
4.2.3 Discussion
Synaptic Plasticity, Under Acute and Chronic elevated Aη-α Conditions
Behavioral Studies, Under Acute and Chronic elevated Aη-α Conditions
Comparing the Acute and Chronic elevated Aη-α Conditions
Depletion of Aη-α levels
APPΔEta and Alternatives: A Comparison
Comparing the Outcomes of Aη-α Level Modification
Probing the η-Secretase Pathway: Prospective Approaches
6. Conclusion
Bibliography
Supplementary
