Novel allosteric modulators of the M1 muscarinic acetylcholine receptor provide new insights into M1-dependent synaptic plasticity and receptor signaling By Sean P Moran Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Neuroscience December 14, 2019 Nashville, Tennessee Approved: Sachin Patel M.D., Ph.D. Colleen Niswender, Ph.D. Danny Winder, Ph.D. P. Jeffrey Conn, Ph.D. ACKNOWLEDGMENTS Throughout my scientific career, there have been many people instrumental in my scientific journey. I would first like to thank Jeff Conn for providing a very supportive research atmosphere, for always making me laugh, for his insight and constructive criticism in our one-on-one meetings and for occasionally getting my name correct. I would also like to thank my thesis committee of Sachin Patel, Colleen Niswender and Danny Winder for their unwavering support during my time here at Vanderbilt. Additionally, I have very much enjoyed collaborating with Jerri Rook on many projects and would like to thank her for her sage behavioral pharmacology advice over the years. Also, thanks to the many members of the VCNDD including: Zixiu, Craig, Jon Dickerson, Dan Foster, Max, Nicole, Mark and Carrie who provided intellectual contributions, technical training or research support throughout my graduate career. Thank you to the many other members of the VCNDD, past and present, that have contributed to my various projects over the years. Thanks to the better half of Shames (James). I will our miss our long winded, highly pedantic and semantic discussions that occasionally touched on pharmacology topics…and thanks for making me not homeless. Thank you to my previous mentor Anthony Chan who introduced me to translational neuroscience research, allowed me to drive independent projects forward and provided the unique opportunity to both create and then play with baby monkeys. Overall, my family has been incredibly supportive of my scientific career and I could not have gotten through graduate school without them. I will be forever thankful for my mom and dad who provided endless education opportunities that were not afforded to ii them. Thank you to my sister and brother-in-law who have always provided support. Thank you, David, for providing endless laughs throughout graduate school but since you missed my thesis defense, I am not that thankful. I will miss living with you and conversing exclusively in LOTR quotes. Lastly, thank you to my incredibly smart and supportive girlfriend Carolyn, who has been my partner in crime over these many years. Our many fun vacations always provided a great getaway from science. I look forward to living in the same time zone for once. Lastly, and most importantly, thank you to the Conn Lab coffee. Without you, none of this would have been possible. iii TABLE OF CONTENTS Page Acknowledgments......................................................................................................... ii LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES........................................................................................................ vii Chapter I. Introduction ................................................................................................................ 1 Background .............................................................................................................. 1 Cognitive disruptions in schizophrenia and Alzheimer’s disease ............................. 2 Targeting muscarinic acetylcholine receptors for the treatment of AD and schizophrenia ........................................................................................................... 4 Allosteric modulators of mAChRs ............................................................................. 6 Potential cognition enhancing effects of M1 PAMs ................................................... 7 II. Diverse M1 PAM pharmacology .............................................................................. 11 Agonist activity in addition to PAM activity .......................................................... 13 MK-7622 and PF-06764427 display robust agonist activity in an in vitro calcium mobilization assay .................................................................................................. 14 VU0453595 and VU0550164 act as highly selective PAMs lacking agonist activity in the M1 in vitro calcium mobilization assay .......................................................... 16 Ago-PAMs but not PAMs lacking agonist activity increase sEPSC in layer V mPFC pyramidal neurons .................................................................................................. 19 Ago-PAMs but not PAMs lacking agonist activity induce robust depression of fEPSP slopes in the mPFC .................................................................................... 21 MK-7622, but not VU0453595, induces behavioral convulsions in rodents ............ 26 VU0453595 but not MK-7622 can enhance rodent object recognition ................... 27 Agonist activity is dependent in part on receptor reserve ................................... 29 Characterization of M1 PAM PF-06827443 ............................................................ 29 PF-06827443 displays robust agonist activity in the mPFC ................................... 35 PF-06827443 increases sEPSC frequency in layer V prelimbic mPFC neurons .... 38 PF-06827443 induces behavioral convulsions in mice ........................................... 39 Discussion ............................................................................................................... 41 Overactivation of the M1 mAChR may be detrimental to M1 PAM efficacy ............. 41 III. Multiple modes of PAM activity............................................................................. 44 M1 PAMs that display bias can have differential effects in the CNS ....................... 44 M1 receptor activation leads to PLD activity in hM1-CHO cells ............................... 47 PLD1, but not PLD2, is required for M1 receptor–mediated LTD in the mPFC ........ 50 Biased M1 receptor PAMs fail to potentiate M1 receptor–dependent LTD in the mPFC ..................................................................................................................... 54 iv PLD is not necessary for the M1 receptor–dependent increase of layer V sEPSC in the mPFC ............................................................................................................... 56 PLD is not necessary for the effects of the M1 receptor on the excitability of striatal SPNs ...................................................................................................................... 60 Discussion ............................................................................................................... 63 IV. Summary & Discussion ......................................................................................... 67 Summary & Discussion ........................................................................................... 67 Overactivation of the M1 mAChR may be detrimental to M1 PAM efficacy ............. 67 M1 PAMs that display bias can have differential effects in the CNS ....................... 68 M1 PAMs with low α-values may also minimize adverse effects ............................ 77 Future Directions ..................................................................................................... 69 Characterization of M1 signaling pathway to phospholipase D ............................... 69 Role of PLD downstream of other canonical Gαq GPCR signaling ......................... 71 Development of the next generation of biased M1 PAMs ....................................... 75 Characterization of the molecular mechanisms involved in conferring bias ........... 76 Potential utility of M1 PAMs for the treatment of other CNS disorders .................... 80 Conclusions ............................................................................................................. 81 V .................................................................................................................................... 83 Materials and Methods ............................................................................................ 83 Calcium mobilization assay .................................................................................... 83 Animals .................................................................................................................. 85 Behavioral manifestations of seizure activity .......................................................... 86 Extracellular field electrophysiology ....................................................................... 86 Whole cell electrophysiology .................................................................................. 88 Novel object recognition task ................................................................................. 90 In vivo pharmacokinetic analysis ............................................................................ 91 Western blot analysis of PLD1 protein .................................................................... 92 Statistical analyses ................................................................................................. 92 References