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  • Presynaptic Protein Interactions that Regulate Synaptic Strength at Crayfish Neuromuscular Junctions

    by

    Rene Christopher Prashad

    A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

    Department of Physiology University of Toronto

    Copyright by Rene Christopher Prashad, 2012

  • ii

    Presynaptic Protein Interactions that Regulate Synaptic Strength

    at Crayfish Neuromuscular Junctions

    Rene C. Prashad

    Doctoral of Philosophy

    Department of Physiology University of Toronto

    2012

    Abstract

    Synapses vary widely in the probability of transmitter release. For instance, in response to an

    action potential the phasic synapses of the crayfish have a 100-1000-fold higher release

    probability than tonic synapses. The difference in release probability is attributed to differences

    in the exocytotic machinery such as the degree of zippering of the trans-SNARE (Soluble N-

    ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and

    molecular approaches to determine if the zippered state of SNAREs associated with synaptic

    vesicles and the interaction between the SNARE complex and Complexin influence the

    probability of release at the synapse.

    I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to

    determine whether these sites were occluded by SNARE interaction (zippering) or open to

    proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B

    (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and

    inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-

    terminal half of crayfish VAMPs SNARE motif (Vc peptide) designed to interfere with SNARE

    complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of

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    VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes

    at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-

    terminal end exposed under resting conditions.

    I used a peptide containing part of the crayfish Complexin central -helix domain to interfere

    with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic

    evoked release and inhibited tonic evoked release under low stimulation but attenuated release at

    both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual

    function under low synaptic activity but only promoted release under high synaptic activity.

    The results showed that the zippered state of the SNARE complex does not determine initial

    release probability as a similar zippered SNARE complex structure under resting conditions is

    common to both phasic and tonic synapses. However, Complexin may have a role in influencing

    the initial release probability of a synapse. Therefore, the interaction between the SNARE

    complex and Complexin is important for release but other factors contribute more significantly to

    synaptic strength.

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    Acknowledgments On the path of completing my thesis, there are a number of people I had the pleasure of

    meeting along the way that I owe many thanks for their help.

    First, I would like to thank my supervisor, Dr. Milton Charlton, for everything that he has

    done to make this thesis possible. Through his mentorship, I was given the opportunity to explore

    my research potential with the freedom to expand my creativity and curiosity, and test my own,

    and often wild, ideas. All of this has helped in my transformation from a nave graduate student

    to a research scientist. It was a privilege to be under the guidance of someone of his calibre and I

    am honoured to be his last graduate student.

    I would like to thank my Ph.D supervisory committee members (Dr. Shuzo Sugita, Dr.

    Zhong-Ping Feng, and Dr. Lu-Yang Wang) for their insightful discussions and guidance over the

    years that helped to shape this thesis. I am also grateful to Dr. Elise Stanley for her comments

    and suggestions as a member of my Final Supervisory Committee, and to my Final Defense

    Committee (Dr. A. Joffre Mercier, Dr. Peter Carlen, Dr. Melanie Woodin, Dr. Shuzo Sugita, Dr.

    Zhengping Jia, and Dr. Tom Paus) for devoting their time to see me through the final stage of

    my graduate study.

    Many thanks go to the members of the Charlton lab (Dr. Lorelei Silverman-Gavrila, Dr.

    Alex Smith, and Dr. Jeffrey Dason) for their advice, guidance, and friendship over the years, and

    special thanks to the late Dr. Guotang Wang for introducing me to various molecular biology

    techniques. He was a great teacher and friend who will be greatly missed. I am also thankful for

    the work by Hui Zhang for her help with the cloning and sequencing experiments. I am also

    grateful for the assistance provided by Dr. Zhong-Ping Feng and her lab members, especially Dr.

    Kwokyin Hui and Qing Li, with the cloning and sequencing of crayfish VAMP and Complexin.

    In addition, I would like to thank Dr. Masami Takahashi for the Syntaxin 6D2 clone antibody,

    Dr. Clifford C. Shone for the VAMP antibody, Dr. J. Troy Littleton for the Drosophila

    Complexin antibody, and Dr. Andrew Christie for the shrimp Complexin sequences.

    Finally, special thanks to my entire family, especially my parents and sister, Nina, for

    their kind support and guidance.

  • v

    Table of Contents

    ABSTRACT .................................................................................................................... II

    ACKNOWLEDGMENTS................................................................................................ IV

    TABLE OF CONTENTS ................................................................................................. V

    LIST OF TABLES........................................................................................................ XIII

    LIST OF FIGURES ......................................................................................................XIV

    LIST OF APPENDICES................................................................................................XX

    LIST OF APPENDIX FIGURES...................................................................................XXI

    ABBREVIATIONS ......................................................................................................XXII

    1 INTRODUCTION...................................................................................................1

    1.1 Neuronal communication.................................................................................................... 1

    1.1.1 Calcium and exocytosis .......................................................................................... 1

    1.1.2 Vesicle pools ........................................................................................................... 5

    1.1.3 Modes of vesicle fusion: Full collapse fusion vs. kiss-and-run .............................. 7

    1.2 Exocytotic machinery ....................................................................................................... 11

    1.2.1 SNARE proteins.................................................................................................... 11

    1.2.2 The SNARE complex ........................................................................................... 13

    1.2.2.1 Structure ................................................................................................. 13

    1.2.2.2 SNARE complex assembly .................................................................... 17

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    1.2.2.2.1 Zippering hypothesis ............................................................ 19

    1.2.2.3 Is a single trans-SNARE complex sufficient for fusion? ....................... 22

    1.2.3 The importance of SNAREs in exocytosis as demonstrated by various

    experimental tools................................................................................................. 23

    1.2.3.1 Clostridial neurotoxins ........................................................................... 24

    1.2.3.2 Interfering peptides................................................................................. 25

    1.2.3.3 Genetic manipulations ............................................................................ 25

    1.2.4 Post-docking role of the trans-SNARE complex.................................................. 26

    1.2.5 SNARE-associated proteins.................................................................................. 27

    1.2.5.1 Synaptotagmin-1..................................................................................... 29

    1.2.5.2 Complexin .............................................................................................. 31

    1.2.5.3 Proposed mechanism of vesicle docking and fusion .............................. 36

    1.3 Clostridial neurotoxins ..................................................................................................... 38

    1.3.1 Mechanism of action............................................................................................. 40

    1.3.1.1 Syntaxin is the target of BoNT/C1 ......................................................... 41

    1.3.1.2 SNAP-25 is the target of BoNT/A/E/C1 ..........................................

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