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EFFECTS OF (+) MK-801 (DIZOCILPINE) AND NON-CODING RNA ON AGGREGATION OF b-AMYLOID PEPTIDES LINKED TO ALZHEIMER’S DISEASE A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Susan E. Coombs December 2018 © 2018 Susan E. Coombs EFFECTS OF (+) MK-801 (DIZOCILPINE) AND NON-CODING RNA ON AGGREGATION OF b-AMYLOID PEPTIDES LINKED TO ALZHEIMER’S DISEASE Susan E. Coombs, Ph. D. Cornell University 2018 RNA aptamers selected for two disparate targets, the nicotinic acetylcholine receptor (Ulrich et al., 1998) and b-amyloid (Ab) peptides (Ylera et al., 2002), share a consensus sequence (Gadalla & Hess, 2006). The RNA aptamers selected for the nicotinic acetylcholine receptors fell into two classes. Class 1 aptamers inhibited the receptor and, in the presence of an activator of the receptor, Class 2 aptamers alleviated the inhibition. The short consensus sequence of Class 1 aptamers retained this activity (Sivaprakasam et al., 2010). Gadalla and Hess (2006) recognized that the same consensus sequence occurred in the RNA aptamers selected for the Ab(1-40) peptide by Ylera et al. (2002). Some organic compounds that bind to the acetylcholine receptor, and affect its function, were assigned to either Class 1 or Class 2 (Hess et al., 2000). (+) MK-801 (dizocilpine) is a Class 1 compound. In the work reported here, the effects of (+) MK- 801 and of memantine on the aggregation pathway of Ab(1-40) and Ab(1-42) peptides have been examined using the Thioflavin T fluorescence assay, nuclear magnetic resonance spectroscopy, and electron microscopy. Memantine is FDA-approved for the treatment of mild-moderate Alzheimer’s Disease. Both compounds accelerated the initial formation of b-sheets by the Ab peptides, (+) MK-801 more rapidly than memantine. But in the presence of either compound a decrease in the fluorescence signal followed the initial rise, a decrease that was not observed when they were absent. The decrease may represent an abruptly formed, insoluble aggregated state, rather than a return to the initial non-b-sheet state. In the absence of either compound, the fluorescence signal indicating b-sheet formation reaches a plateau. NMR spectra of the soluble peptides in the presence and absence of (+) MK-801 were compared to the fluorescence measurements and showed a similar difference for the initial phase. However, no return to the monomeric form was observed, possibly suggesting that the decrease in fluorescence in the Thioflavin T assay represented the formation of insoluble aggregates. Consistent with this interpretation, electron microscopy of the insoluble peptides formed over time showed a more rapid rate of formation of clumped fibrils in the presence of (+) MK-801 than in its absence. Thus, as observed by three different techniques, (+) MK-801 and memantine increase the rate of Ab peptide aggregation as compared to the aggregation that occurs in their absence. This raises a question: What is the effect on patients of long-term treatment with memantine? Is there an explanation for the observation that led to this study, the shared consensus sequence between aptamers selected for two different targets? This cannot yet be answered. In this study, it was recognized that the consensus sequences of the two classes of RNA aptamers targeting the acetylcholine receptor are complementary in the 5’-3’ and 3’-5’ directions. Is this of biological significance? The selection of two classes of aptamers is not limited to this excitatory receptor. Fluorinated RNA aptamers that target the inhibitory GABAA (a1, b2, g2) receptor have been selected (Cui et al., 2004). They also fell into two classes: Class 1 inhibited the receptor and, in the presence of GABA, Class 2 alleviated picrotoxin (Ramakrishan & Hess, 2005) inhibition. Exploration of the observations might lead to an understanding of the shared consensus sequence between the two targets, the nicotinic acetylcholine receptor and the Ab peptides. BIOGRAPHICAL SKETCH Susan Elizabeth Coombs was born in the United Kingdom on October 17, 1937, to Eva Rhoda Minett and Noel Carey Coombs, MRCS, LRCP. Until 1946 she lived for the most part in Romaldkirk, then part of the North Riding of Yorkshire, while her father served in the Royal Army Medical Corps during World War II. From 1943 to 1945 she attended the one-room village school taught by an excellent teacher, Mrs Doris Hart. In 1946 her family moved to Barnard Castle, County Durham, and she went to a Quaker boarding school, Polam Hall School for Girls, in Darlington. Influenced by the medical background of her family, she was always intent on a career in clinical research, but this goal was hindered by her lack of a science education. In her last year at Polam Hall, a teacher, Miss E. M. Harding, brought to her attention an experimental course that was to begin in the autumn of 1956 at Imperial College of Science & Technology, London. This course was designed to increase the number of scientists trained in order to combat the “brain drain” of British scientists to other countries. In English schools at the time, students were tracked from an early age – including choosing science or the arts. The new university course hoped to remedy this by taking applicants who had done well in the arts but were interested in scientific or engineering careers and giving them intensive training for one year in physics, chemistry, and mathematics. Ten students were accepted for the first year; Susan was the only woman. The course was abandoned after a second experimental year, but it had provided her with an eye- vii opening intellectual experience. Susan went on to study plant science at Imperial, and also spent a summer working in a Glaxo Smith & Klein research laboratory. She had always had a wide-ranging interest in deserts, and in 1960 she took up an administrative appointment at Ahmadu Bello University, Zaria, Northern Nigeria, working for Dr Modjubana Dowouna to fill in for an employee who was going to Britain for further training. In Susan’s spare time, she helped with various research projects from architecture to physics, including two that were led by plant science faculty members seconded from Imperial College. When she was returning to England, A. R. Mathieson, the chair of the chemistry department at ABU, who had been a crystallography faculty member at the University of Leeds, suggested she should look for an opening at the MRC Laboratory of Molecular Biology in Cambridge, UK. In 1965 she went to work for Sir John C. Kendrew F.R.S., founding Editor-in- Chief of the Journal of Molecular Biology and co-founder of the European Molecular Biology Laboratory. In 1967 George Hess from Cornell University visited the MRC Laboratory to build a model of a-chymotrypsin, the structure of which had just been determined by a team led by David Blow. Margaret Brown and Susan were recruited by David to help with George’s model-building in their spare time. In 1971 Susan moved to Cornell University, Ithaca, New York, to work for George, enticed by the offer to continue her further education while still earning a living. She entered the Employee Degree Program in 1974. In May 1980 she graduated with an A.B. in Biology (Neurobiology) from Cornell and in July married George. She continued in the Employee Degree Program as a graduate student in the viii Graduate Field of Biochemistry, mentored by Professor Elizabeth Keller. When Professor Keller died, Susan changed Graduate Fields and entered the Graduate Field of Pharmacology, mentored by Professor Robert Oswald. But increasing employment and family responsibilities intervened. George died in September 2015 and early in 2016 she returned to graduate school as a full-time student. She passed her qualifying A-exam in February 2016 and her B-exam in July 2018. ix Dedicated to the memory of George Paul Hess x ACKNOWLEDGMENTS I am truly grateful to my advisor, Robert E. Oswald, for his constant support and help, over the decades of my graduate school adventures. The two years of being a full-time graduate student were exhilarating, even when experiments were not successful or I was wrestling with computer software. Discussions at our weekly meetings and during fluorescence and NMR experiments were always instructive and enjoyable. He was the perfect mentor. I am also deeply grateful to the members of my committee for their wisdom and encouragement: Gerald W. Feigenson, particularly for his advice on fluorescence and on light scattering, Linda K. Nicholson, for her knowledge of the proteins and peptides implicated in Alzheimer’s Disease, and Gregory A. Weiland for his knowledge of receptor mechanisms and for his meticulous proof-reading skills. Robert, Jerry, Linda, and Greg provided a balance between setting the bar high and encouragement to meet it, and they were always available for advice. The members of their groups, past and present, helped in numerous ways, scientific and otherwise, including coffee at East Hill Collegetown Bagels, especially Andrea Acevedo, Ahmed Ahmed, Josephine Gonzalez, Jea Hoo Kwon, Chris Ptak, Ross Resnick, and Jayasri Srinivasan. Andrea and Jea Hoo helped with the OCTET experiments and Ross was always around to kibbitz, particularly at the weekends. I want to mention many others to whom I am also grateful: Elizabeth B. Keller, whose encouragement and support set me on this path; she would have enjoyed the twist that led me to think about non-coding RNAs. Geoffrey W. G. Sharp, who guided xi my subsequent transfer to the Field of Pharmacology and shares my Yorkshire roots, and Maurine Linder and Holger Sondermann, who agreed to my return to graduate studies in pharmacology, and to other faculty members of Molecular Medicine, Rick Cerione, Clare Fewtrell, and Linda Nowak; and to Barbara Baird for orchestrating some funding re-arrangements and for a valued friendship since she was herself a graduate student.

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