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Mechanisms of Synaptic Plasticity Mediated by Clathrin Adaptor-Protein Complexes 1 and 2 in Mice

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Mechanisms of Synaptic Plasticity Mediated by Clathrin Adaptor-Protein Complexes 1 and 2 in Mice Mechanisms of synaptic plasticity mediated by Clathrin Adaptor-protein complexes 1 and 2 in mice Dissertation for the award of the degree “Doctor rerum naturalium” at the Georg-August-University Göttingen within the doctoral program “Molecular Biology of Cells” of the Georg-August University School of Science (GAUSS) Submitted by Ratnakar Mishra Born in Birpur, Bihar, India Göttingen, Germany 2019 1 Members of the Thesis Committee Prof. Dr. Peter Schu Institute for Cellular Biochemistry, (Supervisor and first referee) University Medical Center Göttingen, Germany Dr. Hans Dieter Schmitt Neurobiology, Max Planck Institute (Second referee) for Biophysical Chemistry, Göttingen, Germany Prof. Dr. med. Thomas A. Bayer Division of Molecular Psychiatry, University Medical Center, Göttingen, Germany Additional Members of the Examination Board Prof. Dr. Silvio O. Rizzoli Department of Neuro-and Sensory Physiology, University Medical Center Göttingen, Germany Dr. Roland Dosch Institute of Developmental Biochemistry, University Medical Center Göttingen, Germany Prof. Dr. med. Martin Oppermann Institute of Cellular and Molecular Immunology, University Medical Center, Göttingen, Germany Date of oral examination: 14th may 2019 2 Table of Contents List of abbreviations ................................................................................. 5 Abstract ................................................................................................... 7 Chapter 1: Introduction ............................................................................. 8 1.1 Intracellular protein transport ................................................................... 8 1.1.1 COP coated vesicles in early secretory pathway ...................................... 11 1.1.1.1 COPII mediated anterograde protein transport ..................................... 11 1.1.1.2 COPI mediated retrograde protein transport: ....................................... 13 1.1.2 Clathrin-coated vesicles in late secretory and endocytic pathways: ........... 14 1.1.2.1 Adaptor Protein complexes ....................................................................... 14 1.1.2.1.1 AP2 complex .......................................................................................... 17 1.1.2.1.2 AP1 complex .......................................................................................... 18 1.1.2.2 Clathrin ..................................................................................................... 22 1.1.2.3 Clathrin-Coated Vesicle life-cycle .............................................................. 24 1.1.2.3.2 Uncoating of a CCV ................................................................................ 29 1.1.2.3.2.1 Hsc70 mediated CCV uncoating ........................................................... 29 1.1.2.3.2.2 PI-4,5-P2 dephosphorylation in CCV uncoating .................................... 31 1.2 Synapse and Synaptic Vesicle recycling .................................................... 33 1.2.1 AP1 and AP2 in the synapse ......................................................................... 37 1.2.2. AP1/σ1B functions in synaptic vesicle recycling ........................................... 39 1.2.3. Synaptic Plasticity and AP2 functions in the Synapse ................................. 42 1.2.4. Maturation of early endosomes is regulated by AP1/σ1A and AP1/σ1B ....... 44 Aim of the study ..................................................................................... 46 Chapter 2: Material and Methods ............................................................. 47 2.1. Specific lab Equipments .......................................................................... 47 2.2. Chemical, buffers and solutions:.............................................................. 47 2.2.1. Clathrin Coated Vesicle (CCV) Isolation Buffer: ....................................... 48 2.2.2. SDS Poly-Acrylamide Gel Electrophoresis (PAGE): ................................. 48 2.2.2.1. Stacking gel Buffer: ................................................................................. 48 2.2.2.2. Running gel Buffer: .................................................................................. 48 2.2.2.3. Rotiphorese®-Gel30: Acrylamide-/Bisacrylamide .................................... 48 2.2.2.4. APS: 10% (w/v) Ammonium peroxodisulfate ........................................... 48 2.2.2.5. TEMED: N,N,N′,N′ Tetramethyl ethylenediamine ............................. 49 2.2.2.6. Running Buffer: ....................................................................................... 49 2.2.2.7. 6x reducing sample buffer: ....................................................................... 49 2.2.2.8. Protein molecular weight marker: ........................................................... 49 2.2.2.9. Nitrocellulose membrane ........................................................................ 49 2.2.3. Semi-Dry Western Blots: ...................................................................... 49 2.2.3.1. Anode Buffer: ........................................................................................... 49 2.2.3.2. Cathode Buffer: ....................................................................................... 50 2.2.3.3. TBS (Tris-buffered saline):........................................................................ 50 2.2.3.4. TBST (Tris buffered saline with Tween 20) ............................................... 50 2.2.3.5. Blocking Solution ..................................................................................... 50 2.2.3.6. Chemiluminescence ECL kit ..................................................................... 50 2.2.3.7. Stripping Buffer (for 1L) ........................................................................... 50 2.2.4. MEF Cell Culture Media and Solutions: .................................................. 51 2.2.4.1. PBS (Phosphate-buffered saline): ............................................................ 51 3 2.2.4.2. Trypsin-EDTA Solution: ........................................................................... 51 2.2.4.3. Growth Medium:...................................................................................... 51 2.2.4.4. Freezing Medium: .................................................................................... 51 2.3. Protein extract preparations ................................................................... 53 2.3.1. Preparation of protein extracts from murine brain cortices ......................... 53 2. 3.2. Isolation of synaptosomes from brain cortex extracts ................................ 53 2.3.3. Isolation of synaptic Clathrin-coated vesicles .............................................. 54 2.3.4. Immunoisolation of a sub-fraction of synaptic Clathrin Coated Vesicles ..... 54 2.3.5 Isolation of proteins from adipose tissue ...................................................... 56 2. 4. Mouse Embryonic Fibroblast Cells Culture ............................................... 57 2.4.1. Thawing of the MEF cells: ........................................................................... 57 2.4.2. Culturing of the MEF cells: .......................................................................... 57 2.4.3. Maintenance of the MEF cells culture ......................................................... 58 2.4.4. Long-term storage of MEF cells .................................................................. 58 2.4.5. Protein extraction from MEF cells: .............................................................. 58 2.5. Biochemical analysis of protein extracts: ................................................. 60 2.5.1. Protein concentration determination by Bradford Assay: ........................... 60 2.5.2. Semi-quantitative western blot analysis: .................................................... 60 Chapter 3: Results .................................................................................. 65 3.1 Altered AP1 and AP2 mediated protein transport in σ1B-/- synapses ........... 65 3.1.1 AP2 CCV accumulation in the AP1/σ1B-/- synapses ...................................... 66 3.1.2. AP2 CCV accumulation is synapse specific in AP1/σ1B-/- mice .................... 67 3.2. Existence of stabilized AP2 CCV in σ1B-/- synapses .................................. 68 3.2.1 Upregulated CME in the stabilized AP2 CCV of AP1/ σ1B-/- synapses : ......... 70 3.3 Molecular mechanisms for the formation of stabilized AP2 CCV: ................. 73 3.3.1 Reduced recruitment of Hsp110 to the stabilized AP2 CCV: ......................... 73 3.3.2 Stability of AP2 membrane binding ............................................................. 75 3.3.2.1. Reduced Synaptojanin-1 in stabilized AP2 CCV : ...................................... 75 3.3.2.2 Synaptojanin-1 recruiting proteins EndophilinA1 and Amphiphysin .......... 78 3.3.2.3 Reduction of Intersectin-1 in stabilized AP2 CCV ....................................... 79 3.3.2.4 Increased level of SGIP1 in the stabilized AP2 CCV .................................... 81 3.4. Regulation of stabilized AP2 CCV formation in AP1/σ1B-/-synapses: .......... 83 3.4.1. Increased LRRK2 in the stabilized AP2 CCV of AP1/σ1B-/- synapses............ 83 Chapter 4: Discussion.............................................................................. 86 4.1 Brain-specific AP2 CCV accumulation in the AP1/σ1B-/- synapses ................ 87 4.2. Existence of a sub-pool of stabilized AP2 CCV:........................................
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