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LIMK1 Regulation of Long-Term Memory and Synaptic Plasticity

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LIMK1 Regulation of Long-Term Memory and Synaptic Plasticity LIMK1 Regulation of Long-Term Memory and Synaptic Plasticity by Zarko Todorovski A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology © Copyright by Zarko Todorovski 2012 LIMK1 Regulation of Long-Term Memory and Synaptic Plasticity Zarko Todorovski Doctor of Philosophy Department of Physiology University of Toronto 2012 Abstract The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Jia lab have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 ii lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton. iii Acknowledgments This work could not have been done without the help and support of my supervisor Dr. Zheng Ping Jia and the support and advice of my exceptional committee members: Dr. Sheena Josselyn, and Dr. Michael Salter. The guidance and teaching I have received from Dr. Zhen Ping Jia has been invaluable and I am grateful to have had the opportunity to work in his lab. I am thankful for the guidance, advice of friends and former lab members Dr. Suhail Asrar and Dr. Zikai Zhou. Suhail Asrar was especially helpful with his feedback for this thesis and for his help during my graduate career. I am also thankful for the technical support of Shouping Zhang, Nicole Meng and Guijin Lu. I would also like to thank the members of the Jia lab past and present: Suhail Asrar, Zikai Zhou, Wayne Huang, Ner Mu Nar Saw, Jackie Liu and Celeste Leung, along with the summer students I have had the pleasure of teaching and mentoring: Tommy Pan, Anna Rosen and Alyssa Wang. I am extremely happy to have Anh Nguyen in my life. I thank her for her love, friendship, and support. I would also like to thank my parents, Dime and Persa Todorovski, who have instilled in me the value of education that has made me pursue my goals. I would like to thank them and my brother, Igor Todorovski, for supporting me and being there for me as a family. Lastly, I would like to thank my grandfather, Bogosav Todorovski, who gave me an early start to my education and instilled in me a love for reading and seeking knowledge. All of my academic successes are due to the help of my family and for that I am extremely grateful. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Figures ................................................................................................................................ xi 1 Background ................................................................................................................................ 1 1.1 Learning, Memory and Synaptic Plasticity ......................................................................... 1 1.1.1 Learning and Memory ............................................................................................. 1 1.1.2 The Hippocampus and Its Importance in Learning and Memory ........................... 2 1.1.3 Circuitry of the Hippocampus ................................................................................. 3 1.2 Synapses .............................................................................................................................. 6 1.2.1 Structure of the Synapse and Synaptic Transmission ............................................. 6 1.3 Synaptic Plasticity: A general overview ............................................................................. 7 1.3.1 Long-Term Potentiation .......................................................................................... 8 1.4 The Dendritic Spine and Synaptic Plasticity .................................................................... 18 1.4.1 Dendritic Spine Shape ........................................................................................... 18 v 1.4.2 Molecular Determinants of Dendritic Spine Structure ......................................... 18 1.4.3 The dendritic spine and synaptic plasticity ........................................................... 19 1.5 The Rho family of small GTPases and Synaptic Plasticity .............................................. 23 1.6 Regulation of Actin Dynamics .......................................................................................... 27 1.6.1 Actin and Synaptic Regulation ............................................................................. 30 1.7 LIM-kinase ........................................................................................................................ 31 1.7.1 Importance of LIMK1 in cognition: Williams-Beuren Syndrome ....................... 31 1.7.2 General Properties of LIM-kinase ........................................................................ 32 1.7.3 LIM-kinase signaling and effect on actin dynamics ............................................. 35 1.7.4 LIMK1 in Synaptic Regulation and Cognition in Mice ........................................ 36 2 Hypothesis and Objectives ....................................................................................................... 39 2.1 Rationale ........................................................................................................................... 39 2.2 Hypothesis and Objectives ................................................................................................ 40 3 Methods .................................................................................................................................... 44 3.1 Study animals .................................................................................................................... 44 3.2 Euthanasia and slice preparation ....................................................................................... 44 3.3 Electrophysiology ............................................................................................................. 45 vi 3.3.1 Background ........................................................................................................... 45 3.3.2 General Methods ................................................................................................... 46 3.3.3 Induction of LTP ................................................................................................... 46 3.3.4 Data collection and analysis .................................................................................. 47 3.3.5 Immunochemistry ................................................................................................. 50 3.3.6 Immunoprecipitation ............................................................................................. 55 3.3.7 Western blot .......................................................................................................... 55 3.3.8 Data acquisition and analysis ................................................................................ 55 3.4 Behaviour Tests ................................................................................................................ 56 3.4.1 Background ........................................................................................................... 56 3.4.2 Open field test ....................................................................................................... 57 3.4.3 Fear conditioning test ............................................................................................ 58 3.4.4 Data acquisition and analysis ................................................................................ 59 3.4.5 Morris Water Maze ............................................................................................... 59 3.4.6 Elevated plus maze ............................................................................................... 60 4 Results ...................................................................................................................................... 61 4.1 Electophysiological deficits in LIMK1-/- mice ................................................................ 61 vii 4.1.1 Introduction ..........................................................................................................
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