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SNAP-25 Is a Component of a Ubiquitous SNARE Complex Required for Evoked Neuroexocytosis in Gabaergic Neurons Lawrence C

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SNAP-25 Is a Component of a Ubiquitous SNARE Complex Required for Evoked Neuroexocytosis in Gabaergic Neurons Lawrence C University of New Mexico UNM Digital Repository Biomedical Sciences ETDs Electronic Theses and Dissertations 5-1-2011 SNAP-25 is a component of a ubiquitous SNARE complex required for evoked neuroexocytosis in GABAergic neurons Lawrence C. R. Tafoya Follow this and additional works at: https://digitalrepository.unm.edu/biom_etds Recommended Citation Tafoya, Lawrence C. R.. "SNAP-25 is a component of a ubiquitous SNARE complex required for evoked neuroexocytosis in GABAergic neurons." (2011). https://digitalrepository.unm.edu/biom_etds/33 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Biomedical Sciences ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. i ii Dedication I dedicate this dissertation to my beautiful wife Charlene and my two precious daughters, Isabella and Gabriella. It is through their unconditional support, undying patience, and infinite understanding that I was able to reach this important milestone. I cannot begin to repay them for the numerous sacrifices they gracefully made over the years. Individually, I would like to thank my oldest daughter, Isabella, for her inquisitive nature, serving as a continual reminder that knowledge belongs to those who seek it and, at any age, one should never stop questioning the world around us. For Gracie, I owe my gratitude for the moments of levity she provides, which extinguish panic, neutralize anxiety, and put into perspective the true priorities in life. Lastly, I pay tribute to my wife, who single-handedly has galvanized my success as a father, husband, physician, researcher, and a man. To have such special people in my life is truly a blessing and I cannot begin to enumerate the many contributions they have made. However, I can say that it is with seemingly uncanny perception and impeccable timing that they supply the hugs, cheers, and kisses that not only help me maintain my successes, but also propel me to a higher level in search of new goals. Perhaps more importantly, my family gives the encouragement that, during times of seemingly crushing failure, replenishes my spirit and reinforces my resolve. They are everything I will ever need in this life, and their unconditional love and iii unrestrained laughter will continue to eliminate my notions of impossible and perpetually redefine my potential. For my Dissertation Committee, I will never be able to express the gratitude I have for their invaluable guidance and support during this journey, especially during times when my mentor was physically unable to fufill his role due to unfortunate medical circumstances. Word by word I composed this dissertation, but without the guidance of my committee members, the quality of the following manuscript would have never truly reflected the heart, soul, and determination I poured into the project over my five years of training. For that reason, I wholeheartedly thank Donald Partridge, Lee Anna Cunningham, Dusanka Deretic, and Fernando Valenzuela. Lastly, for my mentor, friend, and brother, Michael C. Wilson, there will never be enough words or enough time to express the impact of your influence in my life. Sir, I humbly thank you for not only allowing me the priviledge to work in your laboratory, but also for caring enough to challenge me at every turn, demanding performance at the capacity I had yet to discover, but one you knew I possessed all along. With every publication I write, surgery I perform, and patient I treat, there will be the knowledge that my skills and professionalism began as a seed that germinated in the Wilson Labs. You have changed my life in profound ways and as I carry your teachings forward, you will continue to change the lives of many. iv v SNAP-25 is a Component of a Ubiquitous SNARE Complex Required for Evoked Neuroexocytosis in GABAergic Neurons By Lawrence C. R. Tafoya B.S., Biology, University of New Mexico, 2001 Ph.D., Biomedical Sciences, University of New Mexico School of Medicine 2011 April 3, 2011 vi Abstract The processes of vesicular trafficking and membrane fusion are fundamental to nervous system development and communication among neurons within integrated circuits. The regulated release of several neurotransmitters is dependent on synaptosomal-associated protein 25 kDa (SNAP-25)-containing soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complexes. The requirement for this fusogenic machinery in evoked neurosecretion has been repeatedly demonstrated through the use of mutant mouse models and neurotoxin blockades. However, the existence of a functional role for a SNAP-25-containing SNARE complex has not been shown in the major inhibitory neurotransmitter system of the brain, gamma-aminobutyric acid (GABA). To determine whether SNAP-25 participates in the evoked GABAergic neurotransmission, we investigated the expression and function of this protein in inhibitory terminals. In addition, we identified the major SNAP-25 isoform expressed by mature GABAergic neurons. The results presented here provide compelling evidence that SNAP-25 is critical for evoked GABA release and is expressed in the presynaptic terminals of mature GABAergic neurons, consistent with its function as a component of a fundamental core SNARE complex required for stimulus-driven neurotransmission. Furthermore, we conclude that SNAP- 25b is the predominant isoform expressed in central inhibitory neurons of the adult brain. vii Table of Contents 1. INTRODUCTION ..................................................................................................................................... 1 1.1 THE DISCOVERY OF COMPONENTS WITHIN THE NEURAL SNARE COMPLEX ................................... 2 1.2 STRUCTURAL PROPERTIES OF NEURAL SNARE PROTEINS ............................................................... 4 1.3 NEURAL SNARE COMPLEX ASSEMBLY AND STRUCTURE ................................................................... 8 1.4 MEMBRANE FUSION ........................................................................................................................... 13 1.5 REGULATED EXPRESSION OF SNAP-25 ........................................................................................... 15 1.6 SNAP-25 EXPRESSION AND FUNCTION IN DIVERSE NEUROTRANSMITTER SYSTEMS ...................... 19 1.7 THE ROLE OF SNAP-25 IN INHIBITORY NEUROTRANSMISSION ........................................................ 21 1.8 DOES THE EXOCYTOTIC MACHINERY IN GABAERGIC NEURONS HAVE UNIQUE PROPERTIES? ....... 24 1.9 GOALS OF THIS INVESTIGATION:........................................................................................................ 25 2. EXPRESSION AND FUNCTION OF SNAP-25 AS A UNIVERSAL SNARE COMPONENT IN GABAERGIC NEURONS ......................................................................................................................... 30 2.1 ABSTRACT.......................................................................................................................................... 31 2.2 INTRODUCTION .................................................................................................................................. 33 2.3 MATERIALS AND METHODS ............................................................................................................... 36 2.4 RESULTS ............................................................................................................................................ 47 2.5 DISCUSSION ....................................................................................................................................... 62 2.6 FIGURE LEGENDS .............................................................................................................................. 69 2.8 ACKNOWLEDGEMENTS: ..................................................................................................................... 90 3. THE ROLE OF THE T-SNARE SNAP-25 IN ACTION POTENTIAL-DEPENDENT CALCIUM SIGNALING AND EXPRESSION IN GABAERGIC AND GLUTAMATERGIC NEURONS ........... 91 3.1 ABSTRACT.......................................................................................................................................... 92 3.2 BACKGROUND .................................................................................................................................... 94 3.3 RESULTS ............................................................................................................................................ 98 3.4 DISCUSSION ..................................................................................................................................... 109 3.5 CONCLUSIONS ................................................................................................................................. 116 3.6 METHODS ........................................................................................................................................ 117 3.7 FIGURE LEGENDS ............................................................................................................................ 125 3.8 FIGURES .......................................................................................................................................... 133 3.9 TABLES ...........................................................................................................................................
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