And Non-Coding Rna on Aggregation of Β-Amyloid

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

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-

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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

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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.

Dedicated to the memory of George Paul Hess

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

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. Another valued friend is David Holowka, who enabled me to use initially the Baird/Holowka plate reader and conducted collaboratively a critical fluorescence experiment on an SLM instrument.

I am especially grateful to Eric Alani and then Bill Brown, Chairs of the

Department of Molecular Biology and Genetics, for enabling me to remain in the

Biotechnology Building and helping in many other ways, to all members of the Alani

Lab., particularly for the cups of tea and associated chats, and to all the departmental staff for much help. Warren Zipfel, Rebecca Williams, and Carol Bayles in the

Imaging Facility of the Cornell Institute of Biotechnology provided much helpful advice, particularly relating to the PTI Spectrofluorometer. Andy Clark generously allowed me to use the Molecular Devices plate reader in his laboratory, and Grace

Chi, Yassi Hafezi, and Jackson Champer in the Clark group provided guidance.

Throughout, Anne Dunford was a constant source of wise counsel, and her memories of her father kept me going. Dr Michael J. Dunford completed a Cornell Ph.D. under similar circumstances. When I found myself straying into the RNA world, conversations with Daniel Barbash, Andrew Grimson, Ailong Ke, Jun Kelly Liu, and

Marianna Wolfner at Cornell really helped.

Outside the Departments of Molecular Biology and Genetics and of Molecular

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Medicine at Cornell, discussions with Praveen Sethupathy (Biomedical Sciences,

Cornell), Priya Rajasethupathy (Rockefeller University), Jayant Udgaonkar (Indian

Institute of Science Education and Research, Pune), and Hua Shi (SUNY Albany) were much appreciated.

The initial NMR experiment was done at Rensselaer Polytechnic Institute in a collaboration with Chunyu Wang and Nicolina Clemente, and I am indebted to both of them. The electron microscopy was done in collaboration with Sudeep Banjade in

Scott Emr’s group, a collaboration I really appreciated and enjoyed. Bryan Conn of

Molecular Devices, and Mikah Tran of ANASpec, both answered my numerous questions patiently and promptly in detail.

The project was funded by awards from The Camille & Henry Dreyfus

Foundation and a Podell Emeriti Award for Research and Scholarship, both to George

P. Hess, and by a grant from the Cornell Center for Advanced Technology to George

P. Hess that was later transferred to Robert E. Oswald. I was very grateful for an earlier award from the President’s Council of Cornell Women, which provided a timely boost to my confidence, and I was particularly grateful for the encouragement by one of their members, Elsie Dinsmore Popkin. Initial encouragement I received from the late Jennie Farley, one of the Cornell Eleven, in locker room conversations was invaluable.

Much encouragement was happily received from extended family and friends, especially Margaret Coombs-Tate and Janet and Pierre Dupont (“allez, allez, hop, hop, hop”), who all started post-graduate degrees later than me and finished earlier, and

Peter Hess, Natalie Mahowald, Rowan and Lyndon Hess for family dinners and

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concerts, which were enjoyable and sustaining breaks. My parents, Eva Rhoda Minett

Coombs and Noel Carey Coombs, and my grandmother, Nina May Matthews

Coombs, exposed me to many interesting things, including the world of medicine, for which I am grateful.

I am also grateful to three Cornell undergraduates, Ksenia Kriksunov, Ling

Shen, and Yan Ma, with whom I obtained the initial results suggesting that this was a problem that could be solved and with whom it was fun to work. And above all, I thank two people, the late George Hess, and the late Moataz Gadalla. As an undergraduate in George’s group, Moe made the original observations and, after he joined the MD/PhD program at The John Hopkins School of Medicine, continued to provide helpful suggestions to Ksenia, Ling, Yan, and me.

I have been very lucky in working for the people who provided my unorthodox education, many at ABU – an experience I would not have missed for anything, and

John Kendrew, and George Hess. I benefitted enormously from being a “fly on the wall” at editorial meetings for the Journal of Molecular Biology, and many other opportunities John gave me. There is no way I can adequately recognize the depth of experience George enabled me to gain, from the initial model building, to growing chymotrypsin crystals that could be used to identify a tetrahedral inhibitor complex by laser Raman scattering, setting up cell culture techniques in his laboratory, manually measuring single-channel current recordings in Peter Lauger’s laboratory at the

University of Konstanz, and working with Lambda Physik AG to convert a laser so that it could be used at two different wavelengths, for photochemically characterizing caged compounds and using them in single-cell experiments; and more mundane

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things like learning what a purchase order number is and how to get one. And much else besides, but above all for constantly encouraging me to obtain a Ph.D. (and for always being able to make me laugh).

TABLE OF CONTENTS

Biographical Sketch ……………………………………………………………….... vii Acknowledgements ………………………………………………………………….. xi List of Figures ……………………………………………………………………... xvii List of Tables ……………………………………………………………………... xviii List of Abbreviations ………………………………………………………………. xix List of Symbols ……………………………………………………………………… ix Chapter One INTRODUCTION: THE HISTORY OF ALZHEIMER’S DISEASE …………………………………………………………….. 1 Section 1.1 Introduction ……………………………………………………... 1 Section 1.2 Model Organisms …………………………………………….... 19 Section 1.3 Hereditary Amyloidosis ……………………………………….. 23 Chapter Two BETA-AMYLOID PEPTIDE ……………………………………... 28 Section 2.1 Introduction ……………………………………………………. 28 Section 2.2 Thioflavin T Assay to Quantify b-sheet Formation …………… 37 Section 2.3 Soluble States of Ab Peptide …………………………………... 38 Section 2.4 Ab Interactions with Ligands ………………………………….. 40 Section 2.5 Nicotinic Acetylcholine Receptor Interactions with Inhibitors .. 41 Section 2.6 RNA Aptamers ………………………………………………… 45 Chapter Three SMALL MOLECULE INHIBITORS OF SOME NEUROTRANSMITTER RECEPTORS AFFECT THE AGGREGATION OF b-AMYLOID PEPTIDES ……………... 49 Section 3.1 Introduction ……………………………………………………. 49 Section 3.2 Materials ……………………………………………………….. 52 Section 3.3 Methods ………………………………………………………... 57 Section 3.4 Results …………………………………………………………. 61 Section 3.5 Discussion ……………………………………………………... 97 Chapter Four RNA APTAMERS AND NON-CODING RNAS ………………. 101 Section 4.1 Introduction …………………………………………………... 102 Section 4.2 Materials and Methods ……………………………………….. 109 CONCLUSION …………………………………………………………………… 126 REFERENCES …………………………………………………………………… 129

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LIST OF FIGURES

Figure 1.1 Alois Alzheimer and his coworkers …………………………………….. 4 Figure 1.2 Sequential secretase cleavage of transmembrane amyloid precursor protein (APP), thus generating Ab peptide………………………………. 9 Figure 2.1 Sequence of Ab peptides (1-40) and Ab(1-42) ………………………… 30 Figure 2.2 Spectra of Thioflavin T in the absence and presence of b-sheet structure 33 Figure 2.3 Proposed mechanism of inhibition by cocaine or (+) MK-801 of nAChRs43 Figure 2.4 Cartoon depicting the SELEX method …………………………………. 47 Figure 3.1 Structures of ligands used in this study………………………………..... 55 Figure 3.2 Thioflavin T assay in a cuvet………………………………………….... 65 Figure 3.3 Thioflavin T assay in a cuvet…………………………………………… 68 Figure 3.4 Thioflavin T assay in a 96-well plate…………………………………… 72 Figure 3.5 Thioflavin T assay in a 96-well plate …………………………………... 75 Figure 3.7 Two-dimensional NMR spectrum of 15N-labeled Ab(1-40) in HFIP/phosphate buffer in the absence and presence of (+) MK-801…... 83 Figure 3.8 Normalized integrated methyl peaks over time obtained by nuclear magnetic resonance spectroscopy compared to normalized and inverted results of a Thioflavin T fluorescence assay over time………………… 87 Figure 3.9 Normalized integration of methyl peaks obtained by nuclear magnetic resonance spectroscopy compared to normalized and inverted results of Thioflavin T assay……………………………………………………… 90 Figure 3.10 Electron microphase of 25-µM Ab(1-42) after 3 hours in the (A) absence or (B) presence of 2-mM (+) MK-801 …………………………………. 94

Figure 4.1 Ab(1-40) RNA aptamer B55 inhibits the nAChRs in a BC3H1 cell ….. 104 Figure 4.2 Sandwich ELISA assay………………………………………………... 107 Figure 4.3 Ab(1-40) in 100-mM NaCl, 50-mM sodium phosphate (monobasic and

dibasic) and 0.02 % NaN3, pH 7.4…………………………………….. 112 Figure 4.4 The effect of 25-µM consensus sequence (UUCACCG) of Class 1 RNA aptamers in the aggregation of the Ab(1-40) peptide over time………. 115 Figure 4.5 “A working model: the integrative action of small RNAs during synaptic plasticity (Rajasethupathy et al., 2012)” ……………………………... 123

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LIST OF TABLES

Table 1.1 Targets that have been used in the development of potential therapies for Alzheimer’s Disease …………………………………………………… 17 Table 1.2 Sporadic amyloid diseases and the system affected by each …………… 22 Table 1.3 Genes and their mutations, or allelic polymorphisms, implicated in hereditary amyloid diseases and the function affected ………………… 24 Table 1.4 Neurodegenerative diseases that are not affected by amyloidogenesis … 26 Table 3.1 Samples for electron microscope imaging were taken 3 hours and 24 hours after the start of a parallel Thioflavin T assay …………………………. 93 Table 4.1 Consensus sequence of 14 nAChR-binding Class 1 RNA aptamers that noncompetitively inhibit the receptor (Ulrich et al., 1998) and 18 Ab(1- 40)-binding RNA aptamers (Ylera et al., 2002) ………………………. 103 Table 4.2 Potential hybridization between the consensus sequence of Class 1 and Class 2 of the RNA aptamers targeting the nAChR when aligned 5’ to 3’ and 3’ to 5’ ……………………………………………………………. 117

Table 4.3 Consensus sequence of 11 GABAA Receptor Class 1 RNA aptamers that noncompetitively inhibit the receptor (Cui et al., 2004) and of 5 GABAA receptor Class 2 RNA aptamer (Cui et al., 2004) …………………….. 118

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LIST OF ABBREVIATIONS

5-HT3 ……… Serotonin receptor sub-type 3 AD ………… Alzheimer‘s Disease APOE-e2 ….. Allele of apolipoprotein type2 APOE-e4 ….. Allele of apolipoprotein type 4 APP ……….. Amyloid Precursor Protein Ab …………. Beta-amyloid peptide BSE ……….. Bovine spongiform encephalopathy CAA ……… Cerebral amyloid angiopathy CJD ……….. Creutzfeldt-Jakob disease CTE ……….. Chronic traumatic encephalopathy FAP ……….. Familial amyloid polyneuropathy FFI ………… Fatal familial insomnia GWAS …….. Genome-wide association studies HFIP ...... 1,1,1,1, 3,3,3-hexafluoro-2-propanol LCDD …….. Light-chain deposition disease MME ………. Metalloendopeptidase, MSA ………. Multiple system atrophy nAChR …….. Nicotinic acetylcholine receptor PET ………... Positron-emission tomography PIB ………… Pittsburgh compound B PrPC ………... Prion proteins in cells PrPs ………… Prion proteins in certain misfolded protease-resistant conformations PSI …………. Presenilin PTI …………. Photon Technology Instrument r.p.m. ……….. Revolutions p[er minute RNA ………... Ribonucleotide SAA ………… Serum amyloid A protein SAR ………… Structure-activity relationship TSE ………… Transmissible spongiform encephalopathy TTR ………… Transthyretin vCJD ……….. Variant Creutzfeldt-Jakob disease NMDA ...... N-methyl-D-aspartate NMR ...... Nuclear magnetic resonance a7 …………... Nicotinic acetylcholine receptor subtype a7 b2M ………… Beta-2 microglobulin

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LIST OF SYMBOLS

F-1 ...... Channel-opening equilibrium constant

CHAPTER 1

INTRODUCTION: THE HISTORY OF ALZHEIMER’S DISEASE

Section 1.1 Introduction

In 1901, Alois Alzheimer, a neuropathologist in Frankfurt, saw a 50/51

(sources vary)-year old patient, generally known as Auguste D. and in some accounts as Auguste Deter, suffering from progressive paranoia, memory deficits, disorientation, disordered sleep, and language and behavioral changes. She died in

April 1906 in the Municipal Institution for the Mentally Ill and Epileptics in Frankfurt.

Perhaps the best description of a person with Alzheimer’s Disease is Alzheimer’s own clinical report of the initial patient (Alzheimer, 1907; translated by Stelzman et al.,

1998), “a peculiar severe disease process of the cerebral cortex”. Alzheimer was then a lecturer at the University of Frankfurt.

“The first symptom the 51-year-old woman showed was the idea that she was jealous of her husband. Soon she developed a rapid loss of memory. She was disoriented in her home, carried things from one place to another and hid them, sometimes she thought somebody was trying to kill her and started to cry loudly. In the institution her behavior showed all the signs of complete helplessness. She is completely disoriented in time and space. Sometimes she says that she does not understand anything and that everything is strange to her. Sometimes she greets the attending physician like company and asks to be excused for not having completed the household chores, sometimes she protests loudly that he

intends to cut her, or she rebukes him vehemently with expressions which imply that she suspects him of dishonorable intentions. Then again she is completely delirious, drags around her bedding, calls her husband and daughter and seems to suffer from auditory hallucinations. Often she screamed for many hours. Unable to understand the situation, she starts screaming as soon as an attempt is made to examine her. Some more detailed observations were possible only after repeated efforts. Her memory is seriously impaired. If objects are shown to her, she names them correctly, but almost immediately afterwards she has forgotten everything. When reading a test, she skips from line to line or reads by spelling the words individually, or by making them meaningless through her pronunciation. In writing, she repeats separate syllables many times, omits others, and quickly breaks down completely. In speaking, she uses gap-fills and a few paraphrased expressions (“milk-pourer’’ instead of cup); sometimes it is obvious that she cannot go on. Plainly, she does not understand certain questions. She does no longer remember the use of some objects. Her walk is unimpeded, she uses her hands equally well. Her patellar reflexes are normal. Her pupils react normally. There is a slight hardening of the radial arteries, no noticeable prolongation of the systolic pulse, no albumen. As the illness progressed, these phenomena which are to be interpreted as complex symptoms appear sometimes stronger, sometimes weaker. But they are never severe. On the other hand, the imbecility of the patient increased in general. Her death occurred after four and a half years of illness. At the end, the patient was lying in bed in a fetal position completely pathetic, incontinent.“ (Alzheimer, 1907. Translation by Stelzman et al., 1995).

What caused these symptoms? An autopsy was performed by Dr Alzheimer in the anatomical laboratory of the Royal Psychiatric Clinic at the University of Munich

(where Dr Alzheimer had taken up a post as lecturer). The post-mortem examination revealed widespread atrophy in the cortex of the brain. Microscopy was performed, using the silver histological method newly developed by Max Bielschowsky (1902).

This revealed the presence in the cerebral cortex of neurofibrillary tangles of fibrils, both singly and in bundles, and these remained even where a cell had disintegrated

(Alzheimer, 1907). Alzheimer noted the fibrils were stained by “dyes to which normal neurofibrils do not react” and thought that this was caused by a chemical change.

On November 3, 1906, Alzheimer described his accumulated observations, at the 37th meeting of Society of Southwest German Psychiatrists in Tuebingen, and an abstract of his presentation was published (Alzheimer, 1907a). The chair of the session (Alfred Hoche, a prominent psychiatrist) in which Alzheimer spoke was singularly unenthusiastic about the presentation. Dr Alzheimer died of bacterial endocarditis on December 19, 1915. Emil Kraepelin, a colleague of Alzheimer’s, included a description of Alzheimer’s Disease (AD) in his textbook Psychiatrie

(1910). Auguste D. became the index case for the disease, which Kraepelin eponymously named Alzheimer’s Disease (Hoff & Hippius, 1989). Kraepelin was interested in a nosological approach to psychiatry, trying to classify diseases as distinct entities, and was influential in Alzheimer’s development of his ideas. In 1910 another colleague of Alzheimer’s, Gaetano Perusini, reported four cases of AD, including that of Auguste D., and another patient Johann F. (Perusini, 1910). The clinical record for Johann F. is interesting, and more detailed than that for August D.

Figure 1.1: Alois Alzheimer and his coworkers. Alzheimer is fourth from the left in the top row. Lewy (Lewy Body dementia) is at the far end of the same row. Mrs

Gombrich is first on the left of the bottom row, the person in the middle of the bottom row is unamed, and Perusini is the last person on the bottom row. Photograph taken in

Alzheimer’s Laboratory in Munich in 1909 or 1910. (From B. Lucca, M.D. and F.

Vinci, M.D) (Boller, 2008)

And in 1911 Alzheimer reviewed the current literature (e.g., Fischer, 1907), and the cases that had been reported, in an attempt to separate AD from other senile dementias based on clinical and histological evidence (Alzheimer, 1911). This was going to be a long-lasting problem that continues, to some degree, to this day.

Although plaques were seen in Johann F.’s brain, neurofibrillary tangles were not, but

Alzheimer laid out the arguments for classifying the case as one of AD.

Recently (Maurer, 1997) Maurer and colleagues have uncovered an epicrisis written by Alzheimer or a colleague. It contains fascinating illustrations demonstrating the histology and pathology that he had observed. From Alzheimer’s original Johann

F. histological slides, which accompanied the text, almost a hundred years later

Graeber and colleagues were able to show (Koesel & Graeber, 1993) that mutations in exon 17 of the gene encoding the Amyloid Precursor Protein (APP) were absent and the apolipoprotein E risk factor was absent! The genotype was e3/e3 (see below for further information about the implications of this finding). Further studies were not done on this recovered tissue, in order to preserve the limited material available until

AD is better understood.

Alzheimer also presented two other observations: the presence of a core within a plaque surrounded by a halo, and changes in glial cells (Masters et al., 1985).

Further details had to wait for the development of the electron microscope in 1931 by

Kroll and Ruska (Ruska & Kroll, 1931) when it became possible to characterize the components of the extracellular plaques and neurofibrillary tangles. The major components were abnormal filaments (Kidd, 1963; Terry et al., 1964). In 1959,

Cohen and Calkins (Cohen & Calkins, 1959) used electron microscopy to define

amyloidosis that occurs spontaneously, apparently, or as a result of disease. Electron microscopy overcame a problem that had plagued previous studies, namely the insolubility of amyloids. Cohen and Calkins (1959) showed that non-branching amyloid fibrils occurred in the kidneys of rabbits with casein-induced amyloidosis, in skin tissue of a human patient who was known to have primary amyloidosis, and in the kidney of a human patient known to have parenchymal amyloidosis. The width of the fibrils ranged from 50 to 140 Angstroms and the length from 1200 to 16,000

Angstroms. Their previous studies had indicated amyloids were proteinaceous but were not formed from collagen or g-globulin. Amyloid fibrils have a common cross- b-core structure, in which the polypeptide chain is perpendicular to the long axis of the fibril (Sunde et al., 1997).

The clinical phenomenon that was revealed is amyloidosis, in which ”insoluble abnormal fibrils, derived from aggregation of misfolded, normally soluble protein”, occur extracellularly (Pepys, 2006). The misfolding occurs locally or systemically in

23 (to date) proteins, in a variety of tissues, not just in the nervous system.

The extracellular plaques contain aggregates of 39-42 amino-acid Aβ peptides.

The amyloidogenic peptides arise from sequential cleavage by β- and g-secretase of a transmembrane glycoprotein, the Amyloid Precursor Protein (APP). The peptides are normally soluble; but the soluble monomers with an a-helical structure can form soluble oligomers with b-sheet structure, and from there an insoluble cross-b-sheet aggregate of fibrils can form. The soluble b-sheet oligomers seed (nucleate) the formation of amyloid fibrils (Jarrett & Lansbury, 1993). The plaques are formed from tangled, aggregated fibrils. It should be noted that in the more prevalent non-amyloidogenic

pathway APP is cleaved by a- and b-secretases.

Figure 1.2: Sequential secretase cleavage of transmembrane amyloid precursor protein (APP), thus generating amyloidogenic Ab peptides. (From Wikipedia.org)

Ab is removed from organs in four ways: (1) endocytosis by astrocytes (glia in the CNS) and microglia (macrophages in the CNS); (2) neprilysin (membrane metalloendopeptidase, MME) or insulysin (a metalloprotease that degrades insulin) enzymatic degradation; (3) via the blood-brain barrier into the cerebrospinal fluid or blood plasma; and (4) drainage into periarterial spaces.

The extracellular deposits, neuritic amyloid plaques, contain not only the aggregated amyloid protein but also proteoglycans and glycosaminoglycans (heparin sulfate and dermatan sulfate) and serum amyloid P component (SAP) (Tennent et al.,

1995). These compounds bind tightly to the fibrils and may stabilize them, protecting them from degradation and / or an inflammatory reaction. The plaques may also contain apolipoprotein E (ApoE), remnants of reactive glial cells, a non-b-amyloid component (NAC), and parts of the complement cascade (Iwatsubo, 2000).

The extracellular plaques are not the only histopathological indicators of

Alzheimer’s disease. Intracellular (in the neuronal cell bodies) neurofibrillary tangles of hyperphosphorylated tau protein (a microtubule-associated protein important for the neuron cytoskeleton) (Grunde-Iqbal et al., 1986b) are also a distinguishing feature.

Both the extracellular fibrils and the intracellular tau occur in the hippocampus, entorhinal complex, and “association” areas of the neocortex (Arnold et al., 1991), but how their relative formation occurs is not well understood.

Before death, the patients suffer from a progressive presenile (before 65 years) dementia. Until the 1960’s Alzheimer’s Disease was considered relatively rare, but then a late-onset (after age 65) form with the same symptoms (reviewed by Cummings

& Benson, 1992) was recognized. In 1968 at the University of Frankfurt, a scale was

developed to measure the progressive loss of cognitive function in Alzheimer’s and

Parkinson’s (another neurodegenerative disease) Diseases, Braak Staging (Braak &

Braak, 1991; Braak et al., 2003). The scale was based on a histopathological examination of 83 brains from patients diagnosed with dementia and from controls

(Braak & Braak, 1991). Twenty-one of the brains were from individuals who had been diagnosed with AD, including four from Down’s Syndrome patients. Eight were from individuals who had been diagnosed with AD but on autopsy the diagnosis was not sustained. The extracellular amyloid plaques varied widely in size and shape.

Plaques were found in 6 of the individuals who were not diagnosed with dementia and had no neurofibrillary tangles (Braak & Braak, 1991). This is a common finding, but neurofibrillary tangles, and neuropil threads, were used to delineate 6 stages of disease. The Braaks draw a distinction between amyloid and neuritic plaques (Braak et al., 1989). Several generations of a large family in Columbia with a mutation

E280A in the gene that encodes presenilin-1, are actively being studied (Lopera et al.,

1994). In 1976 Katzman described AD as the most common cause of dementia.

AD is now recognized by a progressive loss of memory, particularly recently acquired information, language (anomia, aphasia, echolalia) and personality changes, visuospatial deficits, lack of judgement, and delusions and agitation, but not by loss of motor function (Cummings & Benson, 1992). A response to familiar music often remains much longer than speech or comprehension. A change that is sometimes not mentioned, but may be important, particularly to care givers, is a change in sleeping patterns. Another change which might provide an early diagnostic test is the sense of smell. The rate of the progressive changes is widely variable but generally patients

live 7-10 years after the initial diagnosis. The changes that occur can be grouped into three stages, stage I which lasts for one to three years and stages II and III for two to ten years.

It has always been a problem to diagnose AD definitively ante-mortem.

Gradually other diseases were separated, often, unsurprisingly, by scientists working at research institutions where the emphasis was on a pathological understanding of nervous diseases, as opposed to a psychoanalytical approach to mental illness. Blocq and Marinesco (1892) were early major contributors, at Charcot’s Institute at

Salpetriere, France, to the delineation of Parkinson’s Disease. They were first to identify senile plaques in a patient, post-mortem, who had had epilepsy. Pick (Pick,

1892) had described a different form of inclusion in neurons in which the atrophy is asymmetrical. The inclusion came to be known as a Pick body. In 1911 Alzheimer

(Alzheimer, 1911) included Pick’s disease in the group of frontotemporal lobe degenerative diseases. A hiatus in AD research occurred during the period of World

Wars I and II, while other clinical needs became more urgent.

But in the latter half of the twentieth century, AD research again increased. In

1963 Kidd (Kidd, 1963), using electron microscopy, identified the intracellular tangles seen in Alzheimer’s disease as being paired helical filaments that could be stained with Congo Red. In 1986 the intracellular tau protein was identified as forming the neurofibrillary tangles (Grunde-Iqbal, 1986a) that are the second pathological component of AD. The tau protein was also identified as being hyperphosphorylated

(Grunde-Iqbal, 1986b). And in 1984 Glenner and Wong identified the main component of the extracellular plaques as b-amyloid protein and suggested it caused

the nerve cell damage typical of AD. Since then, these two proteins have been the focus of much research in the field.

A further development has been the result of increasing human life span, leading to an increase in the number of patients with the disease. In a survey of studies from 1945 to 1985, Jorm and colleagues (Jorm et al., 1987) reported that the prevalence of AD doubles every 5.1 years from the age of 60 years onward, with a tendency to be more common among women, but it is not clear if this is attributable to their longer life span or a hormonal difference. Among men, multi-infarct dementia is more common.

In ~ 10 % of cases of AD, primarily early-onset (even as early as 20-30 years of age), a familiar form of AD is inherited and leads to AD in 50 % of the children of patients. In many cases, the familiar form is due to mutations in genes on chromosomes 1, 14, or 21; the mutation in the gene on chromosome 21 affects the

Amyloid Precursor Protein.

Approximately 90% of cases, primarily late-onset, are sporadic. A variety of risk factors have been reported. One is the APOE-e4 allele of apolipoprotein type 4

(Corder et al., 1993; Genin et al., 2011) on chromosome 19. In 42 families with late- onset AD, the risk for an individual increased from 20 % to 90 % with an increase in the number of APOE-e4 alleles. Concomitantly, the onset age fell from age 84 to 68.

Homozygosity for the allele almost guaranteed the patient would have AD by age 80 years. APOE-e4 allele is present in 13.7 % of the global human population. In later studies, the APOE and Alzheimer Disease Meta Analysis Consortium (Farrer et al.,

1997; Genin et al., 2011) show that this correlation held true for Caucasian, African

American, Hispanic, and Japanese populations, with the same distributions of age and sex.

The link with AD is not seen for apoliprotein types 2 and 3; indeed the APOE- e2 allele is believed to be protective. Four early-onset families were tested. Two had the APP mutation on chromosome 21 and two were considered to have a mutation on chromosome 14, but none had the increased APOE-e4. A database has been established connecting brain protein pathways in mouse models showing changes that correlate with the development of AD-like changes (Savas et al., 2017). It reveals an increase in ApoE levels associated with an increase in Ab levels.

There are other diseases with some similar features: vascular dementia, frontal lobe degeneration, Jakob-Creutzfeldt disease, metabolic, HIV, or toxic encephalopathies. With recent increasing understanding, a careful differential diagnosis can be made ante mortem, including cognitive tests, exclusionary laboratory tests and neuroimaging (X-ray computed tomography, magnetic resonance imaging, and single photon emission computed tomography). In 2004 Pittsburgh Compound B

(PIB) was reported as an agent that can be used in positron emission tomography

(PET). PIB can cross the blood-brain barrier and bind to the b-amyloid plaques. But a more satisfactory specific biomarker for AD is still urgently needed.

b-Amyloid peptides and tau are not limited to the brain. They are also found normally in cerebrospinal fluid and blood serum, but at much lower concentrations. b-amyloid peptides have been detected in the cytoplasm of lens fiber cells of the eye

(Goldstein et al., 2003) (see below) and are reported to be linked to Down’s Syndrome and AD.

Therapies can be designed to target many different aspects of a disease. They may prevent the disease, for example a vaccine or a lifestyle change, treat symptoms but not the cause, or modify the disease by increasing the function of the remaining cells, restoring a physiological mechanism to normal levels, or encouraging the regeneration of lost cells. The chances of success are much greater if the nature of the disease is truly understood, or at least if a reliable bio-marker of the presence and progress of the disease is available. At present, this is not the case with Alzheimer’s

Disease.

A little progress has been made with symptomatic treatments but not with preventative or disease modifying treatments. The range of targets shown below reflects, to some extent, the lack of knowledge about the cause of the disease.

Table 1.1: Targets that have been used in the development of potential therapies

for Alzheimer’s Disease

Immune system, for example beta-amyloid immunization. b-secretase inhibitors Ab aggregation inhibitors, for instance tramiprosate (Neurochem - Alzhemed) Ab production inhibitors

Glutamate/N-methyl-D-aspartate receptor inhibitors Selective Ab(1-42) lowering agents, such as nonsteroidal anti-inflammatory drugs, for instance an inhibitor of cyclooxygenases. An example is R-flurbiprofen (Myriad Genetics – Flurizan). Although the NSAIDs carry cardiovascular problems. Anti-Ab neuroprotectants. For example chelating agents, and also GABA receptor inverse agonists Increasing cholinergic function by inhibiting acetylcholine esterase provides some temporary and limited relief of cognitive symptoms in some patients.

b-Amyloid peptide and tau are thought to build up 15-20 years before AD symptoms appear (reviewed Ising et al., 2015). Nevertheless, there has been an active search for therapies and early diagnostic tools. In 1987 a coalition of the Alzheimer’s

Association (formed in 1980), the National Institute for Ageing (founded in 1974 as part of the National Institutes of Health), and Pfizer (then Warner-Lambert

Pharmaceutical Company) initiated a clinical trial of tacrine for treating symptoms of

AD. Tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate) reversibly inhibits acetylcholine esterase, which hydrolyses acetylcholine. The idea was that by blocking the action of the enzyme, more acetylcholine would be available

to bind to its receptors at cholinergic synapses in the central nervous system.

Acetylcholine is an excitatory neurotransmitter. Tacrine also inhibits monoamine oxidase, and possibly serotonin uptake. It may reduce levels of interleukin-1 beta in the brain and blood. In 1993 tacrine (CognexTM) became the first drug approved by the US Food and Drug Administration (FDA) for treating symptoms of AD, but approval was later withdrawn over concerns about the drug’s effectiveness and hepatotoxicity.

Currently there are five drugs with FDA approval. They target either the cholinergic or the glutaminergic neurotransmitter systems. Some may provide modest benefits for a time, particularly in the early stages after diagnosis, but none halt the inexorable progression of the disease. Three are centrally acting cholinesterase inhibitors, AriceptTM (donepizl), ExelonTM (galantamine), and RazadyneTM

(rivastigmine). A fourth is an inhibitor of the excitatory glutamate/NMDA (N-methyl-

D-aspartate) receptor, NamendaTM (and is marketed under many other names)

(memantine). NamzancTM is a combination of donepezil and memantine. A fifth is approved for use elsewhere. Donepezil (2-((1-benzylpiperidin-4-ylmethyl)-5,6- dimethoxy-2,3-dihydro-1H-inden-one) is a racemic mixture of R- and S-donepizil. In addition to its inhibitory effect on acetylcholine esterase, galantamine (4aS,6R,8aS)-

4a,5,9,10,11,12-hexhydro-3-methoxy-11-methyl-6H-benzofuro [3a,3,2- ef][2]benzazepine-6-ol) exerts allosteric affects on some nicotinic acetylcholine receptors, inducing a conformational change in the receptors. Rivastigmine [(S)-N- ethyl-N-methyl-3-(1-(dimethylamino)ethyl-phenyl carbamate hydrogen-(2R,3R)- tartrate] inhibits both butyryl- and acetylcholine esterases. At doses above

therapeutic levels, memantine acts as a dissociative anesthetic, but it is rarely used recreationally. It reduces the excitotoxicity of the glutamate receptor, postulated to be involved in AD, by displacing magnesium ions, thus prolonging the movement of calcium ions into neurons. Memantine also inhibits serotonin 5-HT3 receptors, although it is not clear what role they play in AD, and nicotinic acetylcholine receptors, particularly the a7-subtype. In contrast, memantine is an agonist of dopamine D2 receptors and sigmaergic s1 receptors.

In view of the lack of progress in developing effective biomarkers to diagnose

AD in living patients, or compounds to reverse, slow, or halt the progression of the disease, it may be worth considering information coming from traditional medicine

(Howes & Houghton, 2003). Many drugs in current use in the western world were originally isolated from plants. An example is galantamine (see above), which was isolated from plants such as Lycoris radiata, the red spider lily, and Galanthus nivalis, the snow drop. The drug is approved in the UK to treat AD and other CNS and muscle diseases. Curcumin, extracted from Curcuma longa, protects PC12 cells from

Ab damage in vitro (Kim & Kim, 2001; Park & Kim, 2002), perhaps through its antioxidant effects. A Ginkgo biloba extract, EGb 761, is neuroprotective in vitro against Ab and nitric oxide toxicities (Bastianetto et al., 2000 a, b).

Section 1.2 Model Organisms

There was a critical need for model organisms expressing molecules that play a role in AD, and some whole-organism models have been developed: Caenorhabditis

elegans (Ab) (Link et al., 2003; Kaletta & Hengartner, 2006), Drosophila melanogaster (e.g., human Ab42, APP+b-secretase+Drosophila g-secretase presenilin) (Pruessing et al., 2013), Saccharomyces cerevisiae, and Mus musculus

(APP) (Games et al., 1995). Research on the rare neurodegenerative Gerstman-

Straeussler-Scheinker syndrome provided a basis for developing mouse models of AD, and with this objective Games et al. (1995) reported that a human APP gene had been inserted into mice. The transgenic mice displayed pathology typical of AD. In many studies cells in culture have been used.

In 1987, Gajdusek (e.g. Goldhaber et al., 1987), amongst others, identified the first gene, on chromosome 21, associated with the familial form of AD and coding for

APP. Trisomy of chromosome 21 occurs in Down’s syndrome patients, many of whom have AD by the age of 60 years.

In 2010 the Centers for Disease Control and Prevention reported that AD had become the sixth leading cause of death in the United States. In 2012 clinical trials

(DIAN-TU and A4 trials) were begun to determine whether Gantemerumab or

Solanezumab could prevent AD symptoms in patients with an autosomal dominant mutation. An international collaboration performed a meta-analysis of genome-wide association studies (GWAS) to find genetic variations associated with an increased risk of AD in people of European ancestry (Lambert et al., 2013). They found 19 loci with genome-wide significance, of which 11 had not previously been associated with

AD. Interestingly, in light of the proposal by some scientists that AD involves an immunological response, some of the 11 may be linked to the immune system.

Amyloidosis characterizes numerous diseases. Other proteins that can form

amyloid structures include transthyretin (TTR), serum amyloid A protein (SAA), and beta-2 microglobulin (b2M). Aggregation of misfolded proteins can occur intracellularly or extracellularly. Such aggregation may coincide with diseases, the amyloidoses. Many amyloidosic diseases are neurological in nature, notably Prion

Disease (including transmissible spongiform encephalopathies), Alzheimer’s,

Parkinson’s, and amyotrophic lateral sclerosis (Lou Gehrig’s disease). The transmissible spongiform encephalopathies include scrapie of sheep and goats, and chronic wasting disease of deer. Creutzfeldt-Jacob disease in humans or bovine spongiform encephalopathies (mad cow disease), kuru (laughing disease), Gerstmann-

Straeussler-Scheinker disease, and fatal insomnia of humans are other examples.

Table 1.2: Sporadic amyloid diseases and the system affected by each. *A prion disease: A transmissible spongiform encephalopathy and can be acquired by exposure to affected brain or nervous tissue from another individual.

Sporadic or acquired amyloid System Affected Organism disease known to be affected Alzheimer’s disease Brain H. sapiens Bovine spongiform encephalopathy Nervous system, digestive tract, Cattle (BSE) blood and other body fluids H. sapiens “Mad cow” disease

Cerebral amyloid angiopathy (CAA) Ab deposits in leptomeningal and H. sapiens cerebrovascular vessel walls Also known as Congophilic angiopathy b-protein Chronic traumatic encephalopathy Brain H. sapiens (CTE) including Dementia Pugilista DP) Neurodegenerative disease occurring in those who receive repetitive (although may be mild) brain trauma, such as athletes and military veterans. Creutzfeld-Jakob disease (CJD) (TSE) Brain and nervous system, H. sapiens * including spinal cord

Dialysis-associated amyloidosis Amyloid fibrils of b2- H. sapiens microglobulin deposited in bone, Incidence falling since ~1980 with periarticular and abdominal improvements of dialysis techniques structures of dialysis patients with chronic kidney disease Fatal familial insomnia (FFI) (TSE) * Thalamus H. sapiens Sleep problems Feline encephalopathy (TSE) * Brain and nervous system Cats, both wild and domesticated Kuru (CJD) (TSE) * Brain and nervous system H. sapiens Identified in Papua New Guinea but has now virtually disappeared. Mink encephalopathy (TSE) * Brain and nervous system Mustela vison (Mink)

Monoclonal immunoglobulin light Respiratory system, urogenital H. sapiens chains. tract, or skin Scrapie (TSE) * Brain and nervous system Sheep and goats Senile cardiac amyloidosis – see Wild H. sapiens type TTR Transthyretin-related hereditary Nervous system H. sapiens amyloidosis (Wild type TTR) Two forms exist: Heart 1.Familial amyloid polyneuropathy (FAP) or Corino de Andrade’s disease 2. Familial amyloid cardiomyopathy

Section 1.3 Hereditary Amyloidosis

More 80 mutations have so far been linked to amyloidogenic diseases, and

several mutations and polymorphisms are associated specifically with Alzheimer’s

Disease. These discoveries led to efforts to treat the diseases, ranging from surgery,

transplants, and mechanistic drug discovery.

Table 1.3: Genes and their mutations, or allelic polymorphisms, implicated in

hereditary amyloid diseases and the function affected.

Hereditary amyloid disease System(s) affected Organism affected Creutzfeld-Jakob disease (CJD) * Brain and nervous system H. sapiens Fatal familial insomnia (FFI) (TSE) * Thalamus H. sapiens Sleep problems Gerstman-Straeussler-Scheinker Nerve and muscle H. sapiens syndrome (TSE)* Autosomal dominance. Hereditary fibrinogen amyloidosis Fibrinogen A a-chain deposits in H. sapiens Autosomal dominance the renal system Heredity cerebral hemorrhage with Ab deposits in cerebral vessel walls H. sapiens amyloidosis Dutch form (HCHWA-D) Autosome dominance. Mutation in APP gene Heredity cerebral hemorrhage with Nystatin C H. sapiens amyloidosis Icelandic form Primary systemic amyloidosis Apolipoproteins AI and AII H. sapiens A G/A polymorphism in promoter of apo A1 gene affects age at which AD occurs in patients. Interaction between a-tocopherol and Apo A1 gene. APOA1 mRNA regulated by endogenous antisense RNA. Primary systemic amyloidosis. Gelsolin H. sapiens Actin-modulating protein that interacts with the amyloid precursor protein. Gene defects cause Familial amyloidosis Finnish type. Lysozyme amyloidosis Lysozyme deposits, particularly in H. sapiens Autosomal dominance salivary glands, intestinal tract, vasculature Transthyretin-related hereditary TTR gene H. sapiens amyloidosis (variant TTR) Nervous system Two forms exist: Heart 1. Familial amyloid polyneuropathy (FAP) or Corino de Andrade’s disease 2. Familial amyloid cardiomyopathy

Protein affected Chromosome Number Function to Reference Disease number on of which which mutations mutation mutation identified has been occurs linked

Amyloid precursor 21 10 Goate et al., HCWA protein (APP) 1991 Dutch Arctic Piedmont Iowa Flemish Italian Presenilin 1 (PS1) 14 60 Cleavage of Sherrington APP by b- et al., 1995 secretase Presenilin 2 (PS2) 1 2 Cleavage of Levy-Lehad APP by g- et al., 1995 secretase (a, b). Sisodia et al., 2000

ITM2B gene product 13 2 No stop HCWA 1. Abri British variant signal (1) or British and Ter267Arg / X267R extra DNA Danish 2. Adan Danish variant added, types 795 lengthening 796insTTTAATTTGT the gene product

Gerstman-Straeussler- 20 Mutation in Scheinker syndrome prion protein (TSE) * gene codon Autosomal dominance. 102 from proline to leucine

CST gene product 20 HCWA cystatin C, which Icelandic inhibits cathepsins. type High levels in CSF.f Cystatin amyloidosis Apolipoprotein E e4 allele of Saunders et ApoE al., 1993

Although amyloids are a major factor in many proteins, not all neurodegenerative diseases result from amyloidogenesis.

Table 1.4: Neurodegenerative diseases that are not affected by amyloidogenesis

Neurodegenerative but non- Affected system Organism known amyloidogenetic disease to be affected Light-chain deposition disease Respiratory, urogenital H. sapiens (LCDD) tract , skin, blood, brain Non-amyloid monoclonal immunoglobulin light chain deposits.

As described previously, Alzheimer’s disease is an age-related

neurodegenerative disorder characterized by progressive loss of memory and

deteriorating higher cognitive function (Kar et al., 2004). As life expectancy increases

globally, and comcomitantly birth rates fall, AD poses a societal challenge. In 2000

an estimated 4.5 million Americans had Alzheimer’s disease; by 2050 this number is

expected to be more than 11.3 million (Hebert et al., 2003). In 2006 the world-wide

prevalence was ~26 million people in early and late stages of the disease. This

prevalence is estimated to quadruple by 2050 (Brookmeyer et al., 2007). No FDA-

approved drug has a clear effect on progression of the disease. Although much has

been learned about the disease, and about the involvement of Ab peptides, there is still

a black hole at the center of our understanding. And the same is true of the other

amyloid-related diseases, with their high economic costs.

In summary, the brain of an individual with Alzheimer’s Disease contains

extracellular plaques of aggregated b-amyloid (Ab) peptides, and also intracellular (in

the neuronal cell bodies) neurofibrillary tangles of hyperphosphorylated tau protein (a microtubule-associated protein important for the neuron cytoskeleton). The research to be described in the main part of this dissertation addresses the question of whether drugs designed as inhibitors of some neurotransmitter receptors may affect the aggregation of Ab peptides. In the next chapter the Ab-peptides will be described.

CHAPTER 2

AGGREGATION OF b-AMYLOID PEPTIDES: CURRENT STATE OF THE FIELD

Section 2.1 Introduction

As was noted in Chapter 1, the brain of an individual with Alzheimer’s disease contains extracellular plaques of aggregated b-amyloid (Ab) peptides. The peptides are formed by β- and g-secretase degradation of a transmembrane glycoprotein, the

Amyloid Precursor Protein (APP). (In a more prevalent nonamyloidogenic pathway

APP is cleaved by a- and b-secretases, leading to amorphous aggregates.) The peptides are normally soluble; but the soluble monomers with an a-helical structure can form soluble oligomers with b-sheet structure, and from there an aggregate of insoluble cross-beta-sheet of fibrils forms. The soluble b-sheet oligomers nucleate

(seed) the formation of amyloid fibrils (Jarrett & Lansbury, 1993). Plaques are formed from the aggregated fibrils.

The conformation of soluble Aβ monomers is variable and disordered, but they oligomerize and eventually form the highly ordered, insoluble unbranched amyloid fibrils (about 10 nm long) with a b-sheet structure (Selkoe, 2003; Morgado &

Fändrich, 2011). Time is needed for the process in which ordered Aβ oligomers form, and this is the rate-determining step. The lag phase is characteristic of a polymerization that may be dependent on nucleation occurring. The nucleation is specific for the fibrils formed.

Aβ oligomers misfold and self-propagate (Stohr et al., 2012), as do prions

(Bucciantini et al., 2002; Selkoe, 2003; Chi et al., 2010). But there is a difference, at least as presently understood. Prion proteins (Prusiner, 1982) and Ab peptides both occur in healthy individuals, prion proteins in cells as PrPC, and the peptides as soluble entities in cerebral spinal fluid and blood plasma. But prions in certain misfolded protease-resistant conformations, PrPs, are infectious. That is to say, when

PrPs are introduced to a new individual the PrPC already present in the normally folded state adopt, and replicate, the misfolded structure. The highly ordered amyloid fibrils aggregate. The misfolded proteins affect the nervous system and cause progressive, fatal diseases, such as scrapie, BSE, CJD and vCJD, fatal insomnia, and kuru. Misfolded a-synuclein causes multiple system atrophy (MSA). Prion “strain” refers to prions with same sequence but resulting in differing diseases, each characterized by a specific incubation period and distinct profile (Aguzzi, 2008). Ab peptides share some of these properties but transmission of a misfolded Ab peptide to another individual has not been observed.

The concentration of intermediate-stage structures – Aβ oligomers that are still soluble in aqueous solution – is now widely thought to be a better predictor of eventual synaptic loss (Walsh et al., 2002; Selkoe, 2003) than the concentration of fibrils. In contrast to earlier work, Aβ oligomers are reported to be substantially more toxic than the mature, insoluble fibrils (Walsh et al., 2002; Selkoe, 2003; Zhao et al.,

2012), and have multiple toxic effects on cells. Aβ exerts toxic effects on a wide variety of cells in the nervous system.

The Aβ peptides occur in two main isoforms that differ not only in length but also in behavior.

Figure 2.1: Sequence of Ab peptides (1-40) and Ab(1-42). The last two residues, which are underlined, are only present in Ab(1-42).

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-

Phe-Ala-GluAsp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-

Val-Val-Ile-Ala

The slightly longer (with a C-terminal dipeptide underlined in the sequence above) Aβ(1-42) assembles into amyloid fibrils at a higher rate than does Aβ(1-40).

However, Aβ(1-40) is more common overall. Both isoforms are thought to be neurotoxic, especially when soluble (Bitan et al., 2003), although it is still not clear which forms truly are the toxic species. The presence of plaques correlates poorly with the severity of Alzheimer’s disease. Soluble extracellular Ab may impair memory before plaques accumulate. Consequently, much recent research over the last two decades has focused on the soluble oligomeric form of Aβ (Klein et al., 2001;

Hardy & Selkoe, 2002; Kirkitadze et al., 2002). Additionally, it is not yet clear that

Ab(1-42) follows an aggregation pathway that is mechanistically the same as the pathway followed by Ab(1-40).

Individual steps on the pathway to plaque formation reflect oligomerization of monomers, fibril nucleation, and fibril extension. The widely used Thioflavin T fluorimetric assay is the general method of choice to follow this pathway. A red shift in the emission spectrum of the dye indicates the presence of b-sheet present in the compound examined.

Figure 2.2: Spectra of Thioflavin T in the absence and presence of b-sheet structure. Fluorescence spectra of free and Ab(1-42)-bound thioflavin (ThT): 5-µM

ThT in 50-mM glycine-NaOH, pH 8.5; 2-µM peptide when present.

Excitation/emission fluorimeter slits = 3/10 nm. Solid line, excitation; dotted line, emission spectra. (Top) 5-µM ThT alone. (Bottom) 5-µM ThT + 2-µM Ab(1-42) amyloid fibrils. (From LeVine, 1999).

The early symptoms appear to correlate with dysfunction of cholinergic and glutamatergic synapses (Selkoe, 2002). Non-aggregated, soluble Aβs inhibit many subtypes of nicotinic acetylcholine receptors (nAChR) (Pettit et al., 2001; Pym et al.,

2005). Aβ peptides alone, in the absence of an agonist and without pre-incubation, are reported to activate both α7 and non-α7 nAChRs (Dineley et al., 2002; Fu et al.,

2003). However, preincubation with Aβ peptides for 3-12 min., followed by co- application of an agonist, results in a dose-dependent reduction in the peak current.

This has been demonstrated for both α7- and α4β2-nAChRs (Pettit et al., 2001; Liu et al., 2001; Wu et al., 2004). Circular dichroism, NMR, and UV spectroscopic results indicate that (S)-(-)-nicotine, the iconic ligand for nAChRs, and its metabolite (S)-(-)- cotinine, slow the rate of aggregation of Ab(1-42) (Salomon et al., 1996), perhaps by preventing the structural transition from a-helix to soluble b-sheet.

The ionotropic glutamate/N-methyl-D-aspartate (NMDA) receptor plays a role in learning and memory processes and in excitotoxic mechanisms (Dingledine et al.,

1999), all of which are impaired in the brain of Alzheimer’s patients (Harkany et al.,

2000; Walsh et al., 2002). The soluble monomeric and oligomeric forms of Ab inhibit long-term potentiation with (Chen et al., 2002) and without (Raymond et al., 2003;

Nomura et al., 2005) involvement of the NMDA receptor. The effects of Aβ on the

NMDA receptor-mediated excitatory postsynaptic currents are controversial. Early reports suggested that Aβ(1-40) selectively augments NMDA receptor-mediated synaptic transmission in the rat hippocampus (Wu et al., 1995). In contrast, Raymond et al. (2003) reported that Aβ(1-40) moderately reduces the same process in the same tissue. Moreover, Nomura et al. (2005) showed there is no apparent effect of Aβ(1-

42) on NMDA currents in pyramidal neurons. But Ab(1-42) is reported to enhance

NMDA responses in rat hippocampal neurons (Molnar et al., 2004). And Ab itself may bind to the agonist-binding sites of the receptor (Cowburn et al., 1997).

Application of Ab to cultured cortical neurons decreases surface expression of NMDA receptors, promotes their endocytosis, and depresses NMDA-evoked currents (Snyder et al., 2005). In other words, the mechanism of how Aβ peptides interact with the

NMDA receptor remains unclear. Some of this confusion may be due to the type of

Aβ peptide used, and its aggregation state (Ye et al., 2004).

As indicated in Chapter 1, no good drug is available to treat Alzheimer’s disease. Drugs with limited effect fall into two classes, cholinesterase inhibitors

(donepezil, rivastigmine and galantamine) or NMDA receptor antagonists

(memantine). Memantine, at therapeutically relevant concentrations (steady-state plasma concentration of 2.34 µM), can protect against neuronal degeneration induced by Ab (Miguel-Hidalgo et al., 2002). It is also a non-competitive antagonist of the serotonin 5HT3 receptors and nAChRs, and an agonist of dopamine D3 receptors.

Harkany et al. (2000) reported that both in vivo and in vitro application of Aβ(1-42) triggered excitotoxicity via the NMDA receptor, and that such Aβ neurotoxicity can be effectively prevented by the antagonist MK-801. (+) MK-801 is also an inhibitor of nAChRs, although the affinity is about 40-fold higher for the NMDA receptor

(Amador & Dani, 1991).

Section 2.2: Thioflavin T Assay to Quantify b-Sheet Formation

LeVine made a major contribution to the armentarium with which AD can be attacked. In 1997 he used Thioflavin T and a stopped-flow kinetic approach to delineate the phases in which Thioflavin T binds Ab(1-40) (LeVine, 1997). He followed this up in 1999 by providing a detailed protocol (LeVine, 1999; LeVine &

Scholten, 1999) that could be used to quantify b-sheet formation by Ab(1-40) and

Ab(1-42) and to screen for compounds that affected the process. But there are caveats to be considered.

LeVine reviewed some of the problems that are encountered with the new method (LeVine, 2002). He believes that the inhibition of Ab fibril formation is a potential therapeutic target, but that seeking inhibitors with high affinity for the

Ab peptide is difficult. The problem is the large interface between units with which a small molecule would have to interact. This large interface poses a large free energy barrier (van der Waals bonds, hydrogen bonds, and electrostatic bonds, each of which has characteristic geometry, strength, and specificity) which would have to be overcome by a small molecule inhibitor. Some polypeptides or peptidomimetic inhibitors that can do this have been developed (see below); but for a variety of reasons they are not useful as drugs. But some small molecule inhibitors of fibrillation have been generated. How do they exert their effect?

LeVine postulates that small amyloidogenic peptides and small molecule inhibitors interact differently than do larger proteins with small molecule inhibitors.

In the latter case, only a small number of protein conformations are involved. In the

former case there may be many conformations and he references some reports of other molecules that form fibrils, for example tau (Scheers et al., 1994) and a-synuclein

(Serpell et al., 2000.)

Section 2.3: Soluble States of Ab Peptides

Since the Thioflavin T assay was developed, the emphasis has shifted to earlier states of the Ab molecule, namely the monomers and early oligomers. An answer is needed to the question: What initiates the formation of oligomers from monomers?

Fibrillogenic molecules have been termed “natively denatured”. LeVine invokes the term “conformationally ambiguous” for the molecules that form random-coil structures in solution and b-strands as they become insoluble. The term originated with studies of some better understood amylogenic systems, such as tau (Scheers et al., 1994) and a-synuclein (Serpell et al., 1994). If a solution contains a peptide that exists in many different interconvertible conformations, LeVine suggests that the chance of two monomers in the same conformation, with the same properties, assembling is low. And that while formation of a dimer is of low probability, a dimer can assemble with monomers, dimers, or oligomers.

An explanation for the observed lag phase in the pathway of Ab through assembly/fibrillation to aggregation is sought. Application of biophysical techniques

(e.g. light scattering, electron microscopy, and atomic force microscopy) led to the suggestion that oligomers first increase in length to become protofibrils rather than increasing in width, and then the protofibrils associate with other protofibrils in a

helical structure. a-Synuclein, amongst other molecules, behaves this way (Conway et al., 2000). To find effective inhibitors it may be better to look for something that stops the reaction before fibrillization has occurred.

Two alternative models are frequently invoked. One is that amyloid formation depends on a seeding step, similar to polymerization of actin or tubulin (Oosawa &

Higashi, 1967; Oosawa & Akura, 1975; Jarrett et al., 1993). In this model the observed lag phase represents the formation of nuclei, a rate-limiting step. The other is a micellar model (Lomakin et al., 1996, 1997; Figure 6), in which Ab nucleates in micelles, or on heterogeneous seeds.

Micelles form above a critical concentration (~ 0.1-mM Ab). Below this concentration the initial elongation rate varies with the Ab concentration, and the fibrils formed are longer than if they were present in the initial solution at higher concentrations. Above an Ab concentration of ~ 0.1 mM, the initial elongation rate and the eventual length are not concentration-dependent. The monomers of Ab irreversibly bind to the end of the fibrils. In this model the observed lag phase represents a slowing of fibril formation above the critical Ab concentration of ~ 0.1 mM. Ferrone et al. (1985) provides a helpful discussion of the process as it occurs in sickle-cell hemoglobin polymerization. The concentration of the peptide/protein used and the temperature at which the study is made are prime factors to be considered.

The lag phase can also be explained by the existence of a template, as is the case with the prion protein (Cohen & Prusiner, 1998; Serio et al., 2000).

Elongation of fibrils differs depending on whether the origin was fibril fragments or endogenous seeds. For instance, growth from the former is virtually

unaffected by 700-µM Congo Red but growth from the latter is inhibited by 0.2-µM

Congo Red. When first associated with a fibril, the peptide is exchangeable, but an apparent conformation change occurs with time. This is known as the Dock-and-Lock

Mechanism (Esler et al., 2000). It occurs in two steps, a reversible step with a t1/2 of

10 minutes, and two hours later a step in which the peptide enters a very slowly dissociable (t1/2 > 1000 minutes). The fibrils become stiffer and the periodicity of their structure changes (Harper et al., 1997, 1999).

Section 2.4: Ab Peptide Interaction with Ligands

What of an Ab interaction with a ligand, given that the formation of fibrils from monomers and oligomers is stochastic or random, and the outcome is not predictable. And the dynamic range of the assay used, usually Thioflavin T fluorescence, is limited. These facts lead (Broadway, 2012) to a shallow and narrow

(flat) structure-activity relationship (SAR) that is difficult to explore (Dollar &

Thurston, 2016). LeVine contends that the flat SAR allows the peptide to specifically interact with multiple inhibitors with certain substitutions. It may make it possible for such inhibitors to be effective against more than one protein that misfolds.

One way of dealing with this problem is to use several different assays to measure the same step in the reaction (LeVine & Scholten, 1999). For instance, one can test the effect of an inhibitor on a rate-limiting step of peptide aggregation by measuring the size of the aggregates, the reaction with Thioflavin T, and sensitivity to a protease (Norstedt et al., 1994). One could compare assays in which spontaneous

fibril formation occurs versus seeding with fibrils, for example in Dock and-Lock experiments (Findeis & Molineaux, 1999; Esler et al., 2000).

In addition to a flat SAR, another problem is that IC50 values for fibrillization inhibitors are usually micromolar rather than nanomolar. This because relatively high peptide concentrations are used, and so events in which a solution in which monomers predominate are not easily observed. Peptidyl compounds have been synthesized in the hope that they would be effective inhibitors. Some did function effectively as inhibitors but were problematic in biological cell and tissue studies. Can aggregation of Ab be reversed, not just stopped in its tracks? Extracellular Ab plaques generally cannot be dissociated except by trifluoroacetic or formic acid.

Section 2.5: Nicotinic Acetylcholine Receptor Interactions with Inhibitors

In the nervous system signals are regulated by, amongst other things, neurotransmitters binding to specific receptors. The receptors may be excitatory or inhibitory. The binding reaction can lead to conformational changes in the protein, and the transient opening of a receptor channel across the plasma membrane of a cell.

The channel remains open for approximately a millisecond, and then becomes desensitized or transiently inactive, before the receptor reverts to its original closed state. Early kinetic studies of nicotinic acetylcholine receptors (nAChR) revealed that movement of inorganic ions through the channel, and subsequent desensitization, was much faster than previously thought (Hess et al., 1975; Hess et al., 1979).

This led to the development of transient kinetic methods by which the

reactions of the membrane-bound receptors could be studied with µs-ms time resolutions (Udgaonkar & Hess, 1987). With the ability to measure the rate and equilibrium constants of the reactions, especially eventually the channel-opening rate constant, the effect of inhibitors on the constants could be determined. However, the time resolution of the original stopped-flow technique was not sufficient to measure the channel-opening rate constant over a wide range of concentrations. To achieve this, caged compounds were synthesized Milburn et al., 1989), and used in a laser- pulse photolysis technique with single cells expressing the neurotransmitter receptor of interest. The values obtained for the constants were compared with the values obtained in single-channel measurements, albeit over a limited concentration range.

Hess and colleagues (Hess et al., 2000, 2003) used the transient kinetic techniques to measure the rate and equilibrium constants for the opening and closing of the channel-forming nAChR, and proposed a cyclical mechanism to describe inhibition of the protein. From this information, the relationship between the

___ dissociation constants of an inhibitor from the closed- (RL2) and open- (RL 2) channel

__ forms, KI and KI in the absence and presence of an inhibitor respectively, and the

-1 -1 channel-opening equilibrium constants F (= kop/kcl) and F* (= kop*/kcl*) was established. The results of investigations of the rapidly equilibrating site are consistent with the cyclical mechanism shown in Figure 2.1.

Figure 2.1: Proposed mechanism of inhibition by cocaine or (+) MK-801 of nAChRs (Hess & Grewer, 1998; Hess et al., 2000). R represents the unliganded

__ receptor, RL2 the liganded receptor in the closed-channel conformation, RL2 the liganded open-channel form of the receptor, and the subscript 2 the number of neurotransmitter (L) molecules bound to the receptor. K1 represents the receptor:neurotransmitter dissociation constant. I represents the inhibitor. RIL2 and

___

RIL2 represent the liganded receptor with inhibitor bound, in the closed- and open-

__ channel conformation respectively. KI and KI are the dissociation constants of an inhibitor from the closed- and open-channel form respectively. kop and kcl are the rate constants for channel opening and closing respectively. kop* and kcl* are the rate constants for the opening and closing, respectively, of the receptor channel when the inhibitor is bound to the open-channel form. The channel-opening equilibrium constants for the receptor in the absence (F-1) and presence (F*-1) of inhibitor are defined by the ratios kop/kcl and kop*/kcl* respectively. On a longer timescale, the receptors become transiently desensitized.

K1 kop R + I + L RL2 RL2(open) kcl

KI KI * kop RIL RIL (open) 2 kcl* 2

With the application of the principle of microscopic reversibility, two predictions (Hess et al., 2000) arise from the cyclical mechanism. (i) Compounds that bind with higher affinity to the closed-channel form than to the open-channel one will shift the equilibrium towards the closed-channel form, inhibiting the receptor. (ii)

Compounds that bind to the open-channel form with equal or higher affinity than to the closed-channel form will not decrease the channel-opening equilibrium constant.

They will not inhibit the receptor, but they can displace inhibitors.

Section 2.6: RNA Aptamers

To confirm, or not, the predictions, nucleotide polymers (aptamers) that specifically bind to nAChRs were combinatorially selected from RNA libraries

(Ulrich et al., 1998), using a modified version of the Systematic Evolution of Ligands by Exponential Enrichment SELEX technique (Tuerk & Gold, 1990; Ellington &

Szostak, 1990). The starting point of the technique is a very large library of randomly generated oligonucleotides of fixed length, a variable region is flanked by constant primer regions. The sequences that bind most strongly to the target are eluted and then amplified by PCR, with increasing selection stringency. The selected sequences provide the starting point for the next round. After multiple rounds, aptamers, single- stranded oligonucleotides (RNA or DNA) that specifically bind to a target, are obtained. The two modifications introduced by Ulrich and colleagues were the use of phencyclidine (PCP) to extract the RNA-bound receptor and a gel-binding step to remove loosely bound RNA, thus increasing the specificity of the selected aptamers.

Figure 3.2: Cartoon depicting the SELEX method. (From http://www.molmed.uni- luebeck.de/)

The effects of the selected individual aptamers on nAChRs were then tested, using the newly developed transient kinetic techniques. The individual aptamers fell into two classes, Class 1 and Class 2, each with a different consensus sequence (Ulrich et al., 1998). Class 1 aptamers bound with higher affinity to the closed-channel form and inhibited the nAChRs. Class 2 aptamers bound with approximately equal affinity to the closed- and open-channel forms and alleviated receptor inhibition by certain small organic molecules.

When the aptamers were truncated to their consensus sequences, their biological activity, retested kinetically, was retained, although to a lesser degree

(Sivapraksam et al., 2010). Armed with this information, small organic molecules

(cocaine analogs) that alleviate cocaine and (+) MK-801 inhibition of nAChRs were identified (Hess et al., 2003; Chen et al., 2004). Examples of cocaine-alleviating agents are the nAChR alleviatory RNA aptamers (Hess et al., 2000), ecgonine methyl ester (Chen et al., 2004), RTI-4229-70, RCS-III-140A, RCS-III-143A, RCS-III-202A, and RCS-III-218 (Hess et al., 2003).

Gadalla (Gadalla & Hess, 2006) identified a possible link between the Class I

RNA aptamers and Ab(1-40). In preliminary experiments he observed that cocaine,

(+) MK-801, ecgonine methyl ester, and PCP affected Ab(1-40) aggregation, with

PCP being the most effective. Ecgonine methyl ester, a metabolite of cocaine, stands out in this group because in the kinetic studies with nAChRs it alleviates inhibition by cocaine. This will be considered in Chapter 3.

CHAPTER 3

SMALL MOLECULE INHIBITORS OF SOME NEUROTRANSMITTER

RECEPTORS AFFECT THE AGGREGATION OF b-AMYLOID PEPTIDES

Section 3.1 Introduction

The serendipitous observation by Gadalla (Gadalla & Hess, 2006) that there might be a link between ligands that bind the nAChR and Ab peptides led to this project. Amongst the nAChR ligands that were used in the studies that led to the link,

(+) MK-801 is only one which can be purchased for research purposes without a drug license. So in the current chapter, the focus will be on the possible interactions of (+)

MK-801 [dizocilpine; (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5-

10-imine maleate] with Ab(1-40) and Ab(1-42). The ligand was originally developed in 1983 (US Patent 4399141) by Merck & Co. as a non-competitive antagonist of the glutamate/N-methyl-D-aspartate receptor. It also antagonizes the nAChR (Ramoa et al., 1990; Amador & Dani, 1991; Briggs & McKenna, 1996), and inhibits the serotonin (Iravani et al., 1999) and dopamine (Clarke et al., 1995) transporters. (+)

MK-801, phencyclidine (PCP), and ketamine, are all dissociative anesthetics, with shared binding sites on the glutamate receptor that can induce psychosis. Ketamine remains FDA-approved in the U.S. Chronic use of PCP (Burns & Lerner, 1978) and ketamine (Jansen, 1990; Krystal et al., 1994) can lead to cognitive and memory impairment. The properties of (+) MK-801 are more “constrained”. But it has cardiovascular side effects, and in pigeons, rats, and rhesus monkeys it has behavioral

effects similar to those of PCP (Kock et al., 1988). PCP and (+) MK-801 increase both glucose metabolism and blood flow (Nehls et al., 1990) in the brain. In the case of (+) MK-801 this was particularly evident in the entorhinal and cingulate cortex, hippocampus, and dentate gyrus. Increased glucose metabolism induced by (+) MK-

801 was also seen in the olfactory tubercle (Kurumaji & McCulloch, 1990), but only in the conscious brain.

Although there are disadvantages to the clinical use of noncompetitive glutamate/NMDA antagonists, they are neuroprotective after ischaema, hypoglycemia, or epilepsy, probably because they reduce the excitotoxity effects of glutamate (Choi,

1990). But (+) MK-801, PCP, and ketamine have another unfortunate neurotoxic property, namely causing the appearance of vacuoles in the cytoplasm of neurons

(Olney et al., 1989). (+) MK-801 is no longer used clinically in the U.S.A. for this reason. However, the neurons are reported to be protected from (+) MK-801-linked vacuolization by a range of compounds, including scopolamine, benzotropine, tritexyphenidyl (anticholinergics), diazepam and barbiturates (potentiators of g- aminobutyric acidA receptors) (Olney et al., 1991). When given for four days, together with ethanol, (+) MK-801 induced degeneration in the dentate gyrus, entorhinal cortex, and other olfactory areas (Corso et al., 1992). Ellison (1994) also saw this degeneration in the parahippocampal, hippocampal, and olfactory regions, after repeated, frequent doses of (+) MK-801. Ab(25-35) and substance P both increase (+) MK-801 binding to rat cortical membranes in the presence of glutamate or glycine, but the binding did not appear to be related to glutamate/NMDA receptors

(Calligaro et al., 1993). In contrast, Cowburn et al. (1994) reported that Ab peptides

did affect the glutamate/NMDA receptor. The entorhinal cortex is an area of the brain in which AD is manifested early and persistently (Braak & Braak, 1991).

Degeneration in this area, and in the hippocampus, aligns with the results in a variety of tests (Kessslak et al., 1991). Indicators of the olfactory aspect of AD are widely reported (Feldman et al., 1991; Serby et al., 1991; Doty, 1991; Buchsbaum et al.,

1991; Reyes et al., 1993; Struble & Clark, 1992; Davies et al., 1993; Kesslak et al.,

1991) and correlate with results of odor identification and matching tests (Kesslak et al., 1991). Evidence for loss of specific neurotransmitter receptors, in specified brain regions, has been reported by many investigators (Kowall & Beal, 1991), for example glutamate /AMPA receptors in the entorhinal cortex (Armstrong et al., 1994).

As discussed in Chapter 1, there are gender differences in the rate of occurrence of Alzheimer’s Disease. Vacuolation in response to the administration of

(+) MK-801 or PCP in the retrosplenial cortex of adult female rats is about twice that seen in males (Olney et al., 1989), and there are similar differences in the behavioral response (Nabechimsa et al., 1984; Blanchard et al., 1992; Hoenack & Loescher,

1993). Hormonal treatment or deprivation recapitulates these differences.

The observation that (+) MK-801 might affect both the aggregation of Ab peptides and inhibition of neurotransmitter receptor proteins, prompted this investigation. Proteins and peptides fold into characteristic thermodynamically favorable conformations, the native states. In this state a protein usually presents its predominantly hydrophilic amino acids to the external milieu and protects the predominantly hydrophobic ones in the core of the structure. If a protein is misfolded it may aggregate, the exposed hydrophobic regions of one molecule interacting with

the hydrophobic region of another. The aggregates so formed may be amorphous, oligomeric, and fibrillar. For a long time, as seen in the previous chapter, it was believed that the final form of the aggregates, amyloid plaques, was most toxic to the cell, but more recently this has been the subject of debate. Now many people believe that a structure on the path to the final aggregation state is the more toxic form (Zhu et al., 2000; Nilsbeth et al., 2001).

The aggregation may be due to a genetic mutation, an error in transcription or translation, environmental stress (for instance, oxidative stress), or aging. In mammalian cells, misfolded proteins in the aggregates may be refolded correctly, sometimes with the involvement of heat shock factors, marked by ubiquitination for subsequent degradation by a proteasome, or sent to a lysosome for autophagy.

Misfolding and aggregation could lead to neurodegeneration due to the loss of the normal function of the protein, which will be discussed in the next chapter, or to toxicity of the aggregate itself or a species on the pathway to aggregation.

In this project, the effects of (+) MK-801 on the aggregation of Ab(1-40) and

Ab(1-42) were examined. The behavior of the Ab (1-40) and Ab(1-42) peptides over a range of concentrations (3.75, 7.5, 10, 15, 20, 25, or 30 µM) in the absence or presence of 2-mM (+) MK-801 were observed by complementary techniques. The effect of a second ligand, memantine, was also examined in some experiments.

Memantine is FDA-approved for the treatment of Alzheimer’s Disease, especially in those patients who cannot be treated with acetylcholine esterase inhibitors. However, it is only moderately effective. It binds to, and inhibits, the glutamate/NMDA receptor with low affinity in a voltage-dependent manner (Parsons et al., 1993; Pelligrini &

Lipton, 1993; Lipton & Chen, 2004; Miguel-Hidalgo et al., 2002). It also inhibits the serotonin 5-HT3 receptor (Rammes et al., 2001), some nAChRs (Buisson & Bernard,

1998; Gotti & Clementi, 2004; Aracava et al., 2005), and dopaminergic D2 receptors

(Seeman et al., 2008). Inclusion of memantine allowed me to compare my results obtained with (+) MK-801 to those of an inhibitor that has been used in other laboratories. The primary method I utilized is the Thioflavin T assay (LeVine, 1999).

Secondary methods used include Octet Bilayer Interferometry (BLI) assays, nuclear magnetic resonance spectroscopy, and electron microscopy.

Section 3.2: Materials

All materials used were commercially available. Their quality was critical to the success of the project. The peptide content of the product varies with the supplier; in the supply from some sources residual solvent from the lyophilization process is a problem. For the initial studies, rPeptide (https://www.rpeptide.com/) was used as the main source for unlabeled Aβ(1-40) peptide in purified, HFIP-treated form and as the

NaOH salt. Use of HFIP as the solvent is believed to ensure that the Ab peptide is present initially as monomers. Consequently, peptides were initially prepared in HFIP and phosphate buffer, but later in the optimization they were prepared in a proprietary

(putatively Tris) buffer without HFIP. Ab(1-40) in HFIP/phosphate buffer was used in the Octet experiments. The HFIP/phosphate buffer measurements were mostly made in cuvets, and the Tris buffer measurements in 96-well plates.

Unlabeled and 15N uniformly labeled Aβ(1-40) peptides were obtained from

rPeptide. A scrambled Aβ(1-40) available from rPeptide was used as a control. For the later studies, SensolyteR Thioflavin T Beta-Amyloid (1-40) and (1-42)

Aggregation Fluorimetric kits were purchased from ANASpec (Fremont, CA) and the manufacturer’s protocol followed.

The Super-streptavidin-coated probes and 96-well plates used in the Octet instrument were supplied by Pall ForteBio. Thioflavin T, (+) MK-801 hydrogen maleate (dizocilpine), memantine, and High Pressure Liquid Chromatography grade

1,1,1,1, 3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma Aldrich (St.

Louis, MI). Morin was supplied by ANASpec. Greiner non-binding, black 96-well plates with flat bottoms for use in all the experiments except the Octet and some other preliminary experiments were purchased from E & K Scientific (Santa Clara, CA), deuterium oxide and NMR tubes from Sigma-Aldrich, and Formvar/Carbon 200 mesh

Copper microscope grids were from Electron Microscopy Sciences (Hatfield PA). All other chemicals used were reagent grade and purchased from Sigma-Aldrich. Water from a BarnsteadTM MicroPureTM Water Purification System, courtesy of Professor

Gerald Feigenson, Cornell University, was used to prepare all solutions. The structures of the ligands used in the study are shown in Figure 3.1.

Figure 3.1: Structures of the ligands used in this study. The effect of (+) MK-801 and memantine were tested on Ab peptides, morin is an inhibitor of peptide aggregation. Thioflavin T is the dye used in the fluorescence assay.

Section 3.3: Methods

Octet Red96e System Measurements. The Super-streptavidin-coated sensors were inserted into the instrument and stored in buffer at 4 oC. A solution of 25-µM biotin-labeled Ab(1-40) (lyophilized peptide in NaOH; Lot # 2061240N from rPeptide) was prepared in 10% HFIP/20-mM potassium phosphate buffer, pH 7.2.

This solution was allowed to incubate overnight with shaking at 100 rpm at ambient temperature. The Ab(1-40) peptide labeled with biotin was loaded on the Super

Streptavidin sensor tips. Replicates containing HFIP, buffer, and Biocytin were run as controls. These were placed in a 96-well plate, with or without 2-, 1.0-, 0.5-, or

0.25-mM (+) MK-801, and the plate inserted into the instrument. The solutions in the plate were allowed to equilibrate for 2 min. with shaking before readings began. The reaction was followed for four hours.

Thioflavin T Assays. Before beginning the measurements, the solutions were allowed to come to the ambient temperature, and the fluorimeter lamp allowed to warm up, both for 30 min..

A. Thioflavin T assays in a cuvet and preliminary plate exceperiments. A 10-µM

Thioflavin T solution was prepared in a 50-mM glycine buffer, adjusted to pH 8.5 with 5 N sodium hydroxide. In the assay mixture the Thioflavin T was further diluted to ~ 5 µM. The solution was stored at 4 oC in amber Eppendorf tubes, protected from the light.

The reaction buffer was 100-mM NaCl, 0.02% sodium azide, 50-mM sodium phosphate (monobasic and dibasic), adjusted to pH 7.4 with 5N sodium hydroxide. A

10-mM solution of (+) MK-801 was prepared in the reaction buffer. All solutions were stored at 4 oC. In the reaction mixture in which aggregation was followed over time, the 10-mM solution of (+) MK-801 was diluted to 2 mM. In the aggregation reaction mixture, the (+) MK-801 was diluted to 2-mM and was ~0.15 mM in the final assay.

Measurements were made in a Photon Technology Instrument (PTI)

(HORIBA) fluorimeter using a Versafluor microcuvet. A constant volume of 240 µL for all measurements was used (maximum volume of the microcuvet was 300 µL).

The excitation and emission slit widths were set to 5 nm.

Reaction mixtures of 25-, 20-, 15-, 10-, or 3-µM peptide alone, or in the presence of 2-mM (+) MK-801, were prepared and aliquots removed at intervals from

0 to 28 hours. Before the nominally zero-hour time point, an excitation scan of two replicates of peptide with and without (+) MK-801 was run, and the peak wavelength recorded (415-430 nm on the PTI instrument). The excitation wavelength was set to the peak value, and emission was scanned from 460 – 550 nm. The recorded emission peak was between 470 and 490 nm. At each time point a control solution containing Thioflavin T and buffer but without peptide or (+) MK-801 was read.

Between time points the samples were stored at ambient temperature and shaken orbitally at 100 rpm on a New Brunswick G10 gyrotatory shaker.

B. ANAspec SensolyteR Thioflavin T Beta-Amyloid (1-42) Aggregation

“Fluorimetric”Kit. A proprietary assay buffer optimized for fibrillization is provided

with the kit. From the pH of the buffer (7.2), and from the information provided, it is probably a 50-mM Tris buffer containing 150-mM sodium chloride, pH 7.2. 100 µL of the assay buffer were added to a 20-mM Thioflavin T solution (2-mM final). 20- mM morin and 20-mM Phenol Red solutions, provided as inhibitor controls, were diluted with the assay buffer to a final concentration of 100 µM. Appropriate amounts of each of these solutions, and of the compounds to be tested, are added to replicate wells in a Greiner 96-well plate. Four to six wells for each condition were prepared as replicates. The next steps were done as rapidly as possible.

The kit included the lyophilized peptide pretreated to be monomeric. One milliliter of the cold assay buffer was added to 0.25 mg of lyophilized Ab peptide, and the peptide allowed to hydrate, for 3 minutes for Ab(1-40) and for 5 minutes for Ab(1-

42). The solution must be clear at this stage and, if so, was transferred to a Lo-Bind

Eppendorf tube and spun in an Eppendorf 5417C centrifuge for 10,000 r.p.m. for 5 minutes. Appropriate amounts of the peptide solution were added to the wells, with a total volume of 100 µL in each well. The plate was then inserted into a Molecular

Devices Spectromax M2 reader, where the excitation wavelength had been set to 440 nm and the emission wavelength to 485 nm. Fluorescence values were usually recorded every 5 minutes, with 15 seconds of orbital shaking prior to each reading.

Readings were usually recorded for an initial period of 3 hours, and then repeated at various times over the next 24 hours. The temperature was ambient and varied less than one degree centigrade. Between the long reading periods, the plate was shaken on a New Brunswick G10 gyrotatory orbital shaker at 100 r.p.m. at ambient temperature. During this time, to limit evaporation and to avoid unnecessary exposure

to light, the plate was covered with a ParafilmTM sheet and the cover provided with the plate, then wrapped in aluminum foil, sealed in a Ziplock plastic bag and taped shut.

This proved to be more successful than using the commercial available sealing film frequently used, particularly in PCR work. Evaporation that had been noted earlier during the optimization no longer occurred using this protocol.

Two-dimensional Nuclear Magnetic Resonance Spectroscopy of the

Monomeric Species. 15N,1H HSQC spectra of a 15N-labeled Ab(1-40) from rPeptide, in the presence or absence of (+) MK-801, were recorded on a Bruker 800 MHz (18.8

Tesla) instrument at the NMR Facility at Rensselaer Polytechnic Institute at Albany,

New York, in collaboration with Chunyu Wang and Nicola Clemente.

One-dimensional Nuclear Magnetic Resonance Spectroscopy. Ab(1-42) peptide with 13C and 15N labeling on arginine at position 5 and lysines at positions 16 and 28 was purchased from ANAspec. Each vial contained 0.25 mg lyophilized peptide. 25 µL of deuterium oxide solution, and 195 µL of assay buffer (from the

Thioflavin T kit), were prepared in a Lo-Bind Eppendorf tube. Some of this mixture was used to dissolve the lyophilized peptide in the vial, and then 220 µL of the peptide reaction mixture was transferred to an NMR tube and placed in the Varian Inova 500-

MHz spectrometer (11.7 Tesla). One-dimensional proton spectra were recorded for 1 hour. 30 µL of the inhibitor being tested were then added to the reaction mixture in the NMR tube, which was returned to the spectrometer and spectra were recorded for

24 hours. Methyl peaks were integrated and normalized to the first point. The

Thioflavin T data were transformed by inversion in order to compare results obtained using the two different techniques.

Electron Microscopy. Electron microscopy was done in collaboration with

Sudeep Banjade, Weill Institute for Cell and Molecular Biology, Cornell University.

A Thioflavin T assay and a duplicate set of wells without Thioflavin T were run in parallel. After 3 hours, 10 µL were removed from one well for each experimental condition that did not contain Thioflavin T. The plate was then returned to the plate reader and fluorescence readings continued. The samples that were removed were applied to an electron microscopy grid, stained with 2% ammonium molybdate, and imaged on an FEI Morgnani transmission electron microscope.

Section 3.4 Results

Octet Red96e System Measurements. The Octet system was used in a trial to determine whether (+) MK-801 bound specifically to 30-µM Ab(1-40) peptide. Three replicates were run with no peptide adhered to the Streptavidin-coated sensors to provide a baseline. When the peptide was in the presence of (+) MK-80 at concentrations of 2, 1, 0.5, and 0.25 mM, the ligand did indeed appear to bind specifically to the peptide, and in a concentration-dependent manner. Not only association between a ligand and a protein/peptide events can be measured with the

Octet system but also dissociation of a ligand. Although (+) MK-801 association with the peptide was seen, no dissociation was observed under the conditions of these experiments. Although the peptide had been prepared in HFIP it was not known in what state (monomeric or polymeric) the peptide was in during the time course of the experiment. Close examination of the start of all the traces suggested that the initial

association event, when the peptide would have been in a monomeric state, had already occurred before the initiation of the trace recordings. Biotin-labeled scrambled peptide was not available as a control so Biocytin was used since it binds tightly and in high amounts to streptavidin. In the trial of the instrument, it was concluded that (+) MK-801 probably binds specifically to 30-µM Ab(1-40) peptide under the experimental conditions used, but that the approach would not be further pursued.

Thioflavin T Assays. Having established that (+) MK-801 may specifically bind to the Ab(1-40) peptide, the Thioflavin T assay (LeVine, 1999) was adopted to illuminate which steps, if any, in the pathway from monomers to fibrils and plaque formation were affected by this. Thioflavin T was introduced for use in histology by

Vassar and Culling (1959), with subsequent development by Naiki et al. (1989) and

LeVine (1993) for in vitro characterization of amyloid fibrils. When the dye binds fibrils the excitation maximum shifts from 385 nm to 450 nm and the emission maximum from 445 nm to 482 nm. The exact wavelength of the peak varies slightly from instrument to instrument.

Amyloid fibrils are long, generally unbranched, structures that form b-sheets, the chains of which are perpendicular to the long axis of the fibril. The fibril structure forms a Thioflavin T-binding site. It is believed (reviewed by Biancalana & Koide,

2010) that the change in Thioflavin T fluorescence is caused by a change from a state in which its rings rotate freely and quench excited states to one in which they are immobilized and trap the fibrils, thus increasing the fluorescence. Interestingly, a crystal structure of acetylcholine esterase with Thioflavin T bound (Harel et al., 2008)

revealed that the binding occurred at a site at which a-helices predominated and not b-sheet. Three aspects of the assay are particularly relevant (Xue et al., 2017), the optimal concentration of the dye to use, the effect of the dye on peptide aggregation, and its usefulness as a quantitative probe of fibril concentration. Between 0.2- and

500-µM Thioflavin T, there is a linear correlation of fluorescence with amyloid concentration; with 10 µL of 2-mM stock solution in each assay well. At 20-

µM Thioflavin T or lower the dye has little or no effect on peptide aggregation. For the three proteins studied by Xue and colleagues (Xue et al., 2017) the maximum fluorescence was seen with 10- to 50-µM Thioflavin T and was dependent on

Thioflavin T concentration rather than the peptide concentration. Ab(-40) and Ab(1-

42) were two of the peptides studied. They found that with a linear regression fit to the data, the slope reflected what they termed the Fluorescence per Amyloid

Concentration. They also suggested that when analyzing the results of an assay it is important to subtract the background Thioflavin T fluorescence. When comparing data obtained by different groups it is better to present it as the fold change in intensity over background, thus allowing for differences in the instruments used.

Thioflavin T Assay /Cuvet Initial results indicated that the Thioflavin T assay could be used with the peptides prepared in HFIP / phosphate buffer at concentrations ranging from 5 to 50 µM. The results of an experiment over a 10-25 µM range are shown in Figure 3.2. The effect of an inhibitor at 10-µM peptide would seem to be undetectable; the lag phase is barely distinguishable from the rising phase. Between

15- and 25-µM peptide the lag, rising, and plateau phases of the traces were clearly

distinguishable and characteristic. The highest concentration of peptide examined under these experimental conditions was 50 µM. 25 µM was chosen as the standard peptide concentration for most future experiments.

Figure 3.2: Thioflavin T assay in a cuvet. Aggregation of four concentrations (10-,

15-, 20-, and 25-µM) of Ab(1-40) in HFIP/phosphate buffer over 100 hours. n = 3 for each data point. Error bars are the standard deviations. (Arbitrary Units) (pH 7.2, ambient temperature). (Ksenia Kriksunov and Susan E. Coombs)

The next optimization step was to select a concentration of (+) MK-801 that could be used with 25-µM Ab(1-40) peptide and still maintain the linearity of the assay. It was found that 2-mM (+) MK-801 met this requirement (Figure 3.3). I recognize that this is higher than normally used in pharmacological studies in vitro but it gives consistent results with the Thioflavin T assay.

Figure 3.2: Thioflavin T assay in a cuvet. Aggregation of 25-µM Ab(1-40) in the absence and presence of 2-mM (+) MK-801 in HFIP/phosphate buffer over 140 hours. n = 3 for each data point. Error bars are the standard deviations. (Arbitrary Units) (pH

7.2, ambient temperature). (Ksenia Kriksunov and Susan E. Coombs)

Thioflavin T assay with Ab(1-40) cuvet and 96-well plate studies. To refine the Thioflavin T assay, in collaboration with David Holowka, and using a sensitive

SLM fluorimeter, the excitation and emission peaks of the Ab(1-40) peptide in the

HFIP/buffer solution with and without (+) MK-801 were determined. The peaks were reproducible and agreed with those obtained by LeVine (1999). But a 1 x1 cm glass cuvet with 1 mg of peptide had to be used, and the peptide is very expensive. A smaller cuvet gave inconsistent results.

To solve this problem, Corning black 96-well plates were used on a Tecan plate reader in the Baird/Holowka laboratory. The aggregation of Ab(1-40) in

HFIP/buffer in both the absence and presence of (+) MK-801 increased over 24 hours.

The reproducibility obtained with more replicates was thus improved.

ANAspec SensolyteR Thioflavin T Beta-Amyloid (1-42) Aggregation

“Fluorimetric”Kit / Plates. At this point, two changes in the protocols were made.

The first change was to a Ab(1-40) peptide preparation that did not include HFIP.

Consistent results (e.g. Figures 3.2 and 3.3) were obtained in the Thioflavin T assay with the peptide prepared in HFIP and buffer, but it was preferable to run the experiments with no organic solvent present. And there was some evidence that HFIP can affect aggregation of the peptides (Ryan et al., 2013). This was later seen to be an important change. The choice of solvent is very important since solvent polarity affects the formation of the aggregated Ab b-sheet (Barrow & Zagorski, 1991).

The second change was to use non-binding 96-well plates rather than a microcuvet or regular black plates. The high cost of the peptide obtained

commercially prohibited the use of large amounts. The advantages of using 96-well plates were that the experiments could be done with less peptide, 100 µL rather than

300 µL, and more replicates could be read in less time (4, 5, or 6 for each condition or control). Fortuitously, a respected company, ANAspec, now provides a kit in which human Ab(1-40) or Ab(1-42) and other components, except the drug to be tested, are included. A Molecular Devices Spectromax M2 plate reader was kindly made available by Professor Andrew Clark, Cornell University. An advantage of this instrument is that both cuvets and plates can be used, making comparison between the cuvet studies and the plate studies more robust than would otherwise be the case.

The results were greatly improved with the adoption of the new protocol with

Ab(1-40) (Figure 3.4). More replicates could be done for each data point, and more time points could be recorded. Morin provided a good control for inhibition, and the

Thioflavin T alone gave a consistent background. The experiment was repeated twice.

The third time the experiment failed. Eventually the problem was traced to the lot, and the kit was replaced. Thereafter, efforts were made to use, as far as possible, the same lot number for similar experiments. In both successful experiments, (+) MK-801 accelerated the formation of the b-sheet by Ab(1-40), followed by a decrease in the amount of b-sheet. When the peptide was in the absence of (+) MK-801 the trace reached a plateau, as had been seen previously.

Figure 3.4: Thioflavin T assay in a 96-well plate. Aggregation of 25-µM Ab(1-40) in the assay buffer in the absence (black) or presence of 2-mM (+) MK-801 (blue) or

2-mM morin (red), over 300 minutes. The control was Thioflavin T and assay buffer alone (green). Five replicates in all conditions were measured, except for the morin control where four were measured. Error bars show standard deviations. n = 5, except for morin where n = 4, for each data point. (Arbitrary Units) (pH 7.2, ambient temperature).

Does the same thing happen with Ab(1-42)? Four serial dilutions of Ab(1-42), ranging from 3.25-µM to 30-µM, were examined with the new protocol. Assays with the peptide alone or in the presence of 2-mM (+) MK-801 were conducted. With 7.5-,

15-, and 30-µM Ab(1-42), 2-mM (+) MK-801 the same acceleration of aggregation was seen, with a subsequent drop, as observed with Ab (1-40). The “cross-over” point was dependent on the peptide concentration. At higher concentrations it occurred earlier in the time course and at lower concentrations later. The initial point of both pairs of peptide with or without (+) MK-801 was also dependent on the peptide concentration. In subsequent experiments, it was found that (+) MK-801 always caused an acceleration in the aggregation of the peptide, but whether the peptide alone points were above or below the values in the presence of (+) MK-801 appeared to be dependent on the initial time point of each.

Figure 3.5: Thioflavin T assay in a 96-well plate. Aggregation of 3.25-(green), 7,5-

(beige), 15- (grey) and 30- (black) µM Ab(1-40) in assay buffer in the absence of (+)

MK-801, and aggregation of 3.25-(orange), 7,5- (purple), 15- (cyan) and 30- (blue)

µM Ab(1-40) in assay buffer in the presence of 2-mM (+) MK-801, over 400 minutes.

The control was Thioflavin T (green) alone in assay buffer. n = 4 for each data point.

Error bars show standard deviations. Fluorescence is measured in Arbitrary Units. (pH

7.2, ambient temperature).

The improvement made by making the measurements in a 96-well plate instead of a cuvet is seen in Figures 3.5 and 3.6, partly due to the increased number of replicates. The use of 96-well plates rather than a cuvet was recommended by

Professor Gerald W. Feigenson, Cornell University. The advantage lies in the short light path that reduces scattering artifacts, as well the advantage of smaller volumes.

Measurements were made every 5 minutes instead of every several hours. In the presence of 2-mM (+) MK-801 the peptide clearly behaves differently than in its absence. Although the focus was on (+) MK-801 interactions with Ab peptides, morin supplied with the Sensolyte R kit provided a positive control for inhibition. Morin [2-

(2,4-dehydroxyphenyl)-3,5,7 trihydroxchromen-4-one, a flavonol] inhibits formation of, and disaggregates, amyloids fibrils by amylin (islet amyloid polypeptide) (Noor et al., 2012). It is not considered as a treatment for AD because it is toxic. This was normally used as an inhibition control.

Phenol red (phenolsulfonphthalein), an inhibitor of aggregation (and also a pH indicator frequently used in the range pH 6.8 to 8.2), was also provided. It is thought to increase the solubility of the protofibrils. It was used in the earliest experiments,

(data not shown), and then no longer included. It did not provide additional information and not including it allowed for more replicates of the peptide alone and in presence of the other compounds. However, its inclusion in early experiments confirmed that no changes of pH were occurring during the measurements.

The drug approved for treatment of mild-medium AD, memantine, at 2 mM was used in some experiments (Figure 3.6). It displayed a similar acceleration and subsequent drop as (+) MK-801 but the drop was slower and later in the time course

(Figure 3.6).

Figure 3.6: Thioflavin T assay in a 96-well plate. Aggregation of 25-µM Ab(1-42) in the assay buffer in the absence (black) or presence of 2-mM (+) MK-801 (blue), 2- mM memantine (beige), or 2-mM morin (red), over 400 minutes. The control was

Thioflavin T and assay buffer alone (green). Error bars show standard deviations. n =

5, except for morin which was n = 4, for each data point. Fluorescence is shown as

Arbitrary Units. (pH 7.2, ambient temperature).

The experiment with Ab (1-42) was repeated 11 times, with minor variations, each experiment with 4-5 replicates for each condition at each time point. In general, readings were recorded every 5 minutes, with 15 sec. linear shaking preceding each reading, for 3-5 hours. This was followed by various periods with no readings but continuous orbital shaking at 100 r.p.m. Readings were resumed for 1 or 2 hours during the next 14-16 hours, with 15 sec. orbital shaking preceeding each reading.

Final readings for 1 hour were done at 19 and 24 hours after the initiation of the experiment.

Shaking is frequently used in experiments with amyloidogenic compounds to encourage amyloid formation. Lindberg et al. (2015) reported that the effect of shaking is greater with Ab(1-40) than with Ab(1-42), with the difference being in the intensity of the fluorescence. The effect of shaking or not between readings (when the plate was not in the plate reader) was examined in one experiment with Ab(1-42); although there was an effect it was very small.

Several techniques have been used to try and find one that would confirm the results obtained with the Thioflavin T assays, possibly extending the measurements to the soluble, pre-fibrillary state of the peptide. These approaches included NMR spectroscopy, electron microscopy, conductivity measurements, light scattering measurements, and gel chromatography. Dynamic light scattering and gel chromatography are still being explored, but to date NMR and electron microscopy have been the most successful. Solution and solid-state NMR have been extensively used in the field to examine the structure of Ab peptides, b6-macroglobulin, and other amyloidogenic-forming molecules (e.g., Bayro et al., 2011; Eichner et al., 2011; Lu et

al., 2013) despite the problems posed by the heterogeneity of samples as the peptides move along the pathway from monomers (and indeed the difficulty of even knowing what the initial state of the sample is) to oligomers, to fibrils. Each state is transient.

In some states the peptide is soluble, in some insoluble, and each sample may contain a variable mixture of these two conditions. The aim here was not to obtain structures of the peptides with and without (+) MK-801, but to further delineate the pathway to fibril formation.

Two-dimensional Nuclear Magnetic Resonance Spectroscopy of the

Monomeric Species. Professor Chunyu Wang, at the Rensselaer Polytechnic Institute in Troy, NY, has extensive experience with NMR of Ab peptides (Yan & Wang, 2006;

Rosenman et al., 2018). His group usually prepares their own recombinant peptides.

In a collaboration with Professor Chunyu Wang and Nicolina Clemente, a 15N-labeled

Ab(1-40) sample from rPeptide was used, with and without (+) MK-801, and spectra recorded on their Bruker 800 MHz (18.8 Tesla) instrument. Two-dimensional spectra

(Figure 3.7) of very good quality were obtained. Scrupulous attention was paid to maintaining a constant pH. The only shifts over time and increasing concentrations of

(+) MK-801that were seen occurred in the three histidines in the peptide, the peaks of which are very pH-sensitive. Clearly (+) MK-801 did not affect Ab(1-40) in the soluble monomeric state, supporting a conclusion that (+) MK-801 does not bind to the peptide in its monomeric state.

Figure 3.7: Two-dimensional nuclear magnetic resonance spectrum of 15N- labeled Ab(1-40) in HFIP/phosphate buffer in the absence (blue) and presence of

(red) (+) MK-801. For clarity, spectra at intervening concentrations, which were recorded, are not shown. (pH 7.2, ambient temperature). (Nicolina Clemente, Susan

E. Coombs, and Chunyu Wang)

Could NMR still be useful to support the results obtained with the Thioflavin T assays? It was decided to try using one-dimensional spectra integrating the methyl peaks recorded over 24 hours. Spectra of the peptide in the absence and presence of 2- mM (+) MK-801 were obtained. The changes were compared to the results of a

Thioflavin T assay run in parallel.

One-dimensional Nuclear Magnetic Resonance Spectroscopy.

2-mM (+) MK-801 was added initially to a 10 % deuterated solution of labeled Ab(1-

42) peptide. The control was a 10 % deuterated solution of labeled Ab(1-42) peptide without (+) MK-801. The one-dimensional protein NMR spectra were recorded for 24 hours. In Figure 3.8, the decrease in the integrated methyl peaks over time is displayed. The decrease is due to aggregation and line broadening; (+) MK-801 accelerates the line broadening. The conclusion from this result is that (+) MK-801 accelerates the transition of the peptide away from the monomeric state.

The NMR result obtained was then compared to the Thioflavin T result obtained at the same time with the second vial containing 0.25 mg Ab(1-42) peptide from the same Sensolyte kit. The NMR trace with (+) MK-801 was noisy because the lower control signal was normalized, but the maximum (time zero) was normalized to

1. The data from the Thioflavin T assay in which the peptide was in the presence of

(+) MK-801 was normalized and transformed (inverted) to the same scale as the NMR data.. The time course obtained with the two different techniques did indeed agree.

The conclusion was that the NMR spectrum of the peptide in the presence of (+) MK-

801 reflects a transition away from the monomeric state, and this is the same as the initial acceleration of b-sheet formation by the peptide, as seen in the Thioflavin T

experiment.

Figure 3.8: Normalized integrated methyl peaks over time obtained by NMR spectroscopy compared to normalized and inverted results of a Thioflavin T fluorescence assay over time. Aggregation of 25-µM Ab(1-42) in assay buffer in the absence (black) or presence of 2-mM (+) MK-801 (blue) as observed in one- dimensional spectra, compared to aggregation of 25-µM Ab(1-42) in assay buffer in the presence of 2-mM (+) MK-801 (red) as observed in a Thioflavin T fluorescence assay (pH 7.2).

Next, I compared one-dimensional spectra of the peptide before and after addition of 2-mM (+) MK-801 and, separately, 2-mM morin. In Figure 3.9, one- dimensional proton NMR spectra were recorded from a 10 % deuterated solution of

Ab(1-42) in the absence of (+) MK-801 for one hour. The spectra were identical.

Then (+) MK-801 was added to the samples and spectra were collected for 23 hours.

Figure 3.9 gives the integral of the methyl peak over a 24-hour period. The relative intensities decreased over time.

Figure 3.9: Normalized integration of methyl peaks obtained by nuclear magnetic resonance spectroscopy compared to normalized and inverted results of

Thioflavin T fluorescence assay. Aggregation of 25-µM Ab(1-42) in phosphate buffer in the absence (black) or presence of 2-mM (+) MK-801 (blue) (pH 7.2).

Electron microscopy is widely used to visualize fibril formation (Sunde et al.,

1997; Sunde & Blake, 1997). Here it was used as a simple assay to see if fibril formation occurred at the same or different times when the peptide was alone and in the presence of (+) MK-801, memantine, or morin. A fluorescence assay and an identical experiment except that Thioflavin T was replaced with an equal volume of assay buffer were run in parallel. After 3 and 24 hours one sample was removed from each well in which Thioflavin T was absent, and immediately applied to a microscope grid.

Electron Microscopy. The Ab(1-42) samples that were in the presence of the ligands, (+) MK-801, memantine, and morin, had formed fibrils after three hours

(Table 3.1; Figure 3.10). After three hours no fibrils were seen in the Ab(1-42) sample without an inhibitor, but 24 hours later fibrillization had occurred. In wells in which (+) MK-801, memantine, or morin were present fibrils were still observed at 24 hours. A Thioflavin T assay was run in parallel.

Table 3.1: Samples for electron microscope imaging were taken 3 hours and 24

hours after the start of a parallel Thioflavin T assay. The samples were

taken from solutions of 25-µM Ab(1-42) in the absence or presence of 2-

mM (+) MK-801, 2-mM memantine, or 2-mM morin in assay buffer (pH

7.2), in which Thioflavin T was absent. The absence or presence of fibrils

in the sample was determined. (Sudeep Banjade and Susan Coombs

Ab(1-42) Ligand Fibrils present Fibrils present present present at 3 hours at 24 hours + None No Yes

+ (+) MK-801 Yes Yes

+ Memantine Yes Yes

+ Morin Yes Yes

Figure 3.10: Electron micrographs of 25-µM Ab(1-42) after 3 hours in the (A) absence or (B) presence of 2-mM (+) MK-801. Bar scale is 50 nm.

Section 3.5: Discussion

So what have we learned? Several simple, robust assays were optimized so that the observation of Gadalla and Hess could be explored, using small amounts of commercially available but costly peptide. The assumption was that during the first part of the time course, the peptide would be in a soluble form. Later in the time course, when the peptide was decreasingly soluble, a different assay would be needed.

Throughout, the inherent, transient heterogeneity of the samples had to be considered.

Initially, the Ab(1-40) peptide was used because there were reports in the literature that it aggregated more slowly than did the Ab(1-42). But there are also reports that solutions of Ab(1-40) are more heterogeneous than those of Ab(1-42), due to the lack of the C-terminal dipeptide which renders the peptide more flexible. Accordingly, the

Ab(1-42) peptide was used in most of the experiments reported here. The widely used

Thioflavin T assay was the best choice to follow the formation of b-sheet by the peptide, particularly when the change from using a cuvet to a 96-well plate was made.

How can the fluorescence results be explained? The current hypothesis is that

(+) MK-801 accelerates the pathway of Ab peptides away from a monomeric state towards the formation of fibrils. The subsequent drop in fluorescent signal is very reproducible. It may reflect the Ab peptides in the presence of (+) MK-801 forming an insoluble aggregate that then is not measured by the Thioflavin T assay. When memantine was used, the drop also occurred but later and less precipitously. The explanation and significance of this aggregate formation may have to wait until more details of the progression from monomers through oligomers to fibrils are elicited.

The availability of the Octet instrument, albeit for a limited period, made it possible to see, for the first time, evidence that (+) MK-801 bound to the Ab(1-40) peptide in a concentration-dependent manner, although it was not known in which monomeric or polymeric state(s) the peptide was. Similar experiments have been described by Frenzel and Willbold (2014) and Cheng et al. (2014). Frenzel and

Willbold immobilized Ab(1-42) biotinylated at the C-terminus coupled to the Super-

Streptavidin probes and examined the interaction with a ligand scFvIC16. In that case, unlike the results reported here, the ligand did dissociate from the peptide within 300 seconds. Using interferometry, Cheng and colleagues examined the binding of a polyphenol in green tea, (-)-epigallocatechin-3-gallate, to Ab(1-40) and found that it promoted the formation of off-pathway oligomers. They, therefore, used it as a model inhibitor in their studies of Fe(m), Cu(II), Zn(II), and Al(m). These metal ion species were found to accelerate the formation of plaques, in agreement with other reports, particularly as concerns Zn(II).

The Ab peptides have been extensively studied by both solution and solid-state

NMR. The hope was that NMR spectra might provide further information about the specific interaction of (+) MK-801 with soluble Ab peptides. Were there any differences in the NMR structures of free and (+) MK-801-bound Aβ(1-40) peptides?

Were there any peak shifts when the peptide is in the presence of the ligand as compared to the spectrum recorded in its absence? NMR spectroscopy might illuminate the first part of the pathway when the peptide is still soluble and before a b- sheet structure forms. A complete structure was not the aim.

From the two-dimensional NMR experiment, (+) MK-801 was found to not

bind to the soluble monomers of the Ab(1-40) peptide. Similarly, in the 1H 13C

HSQC experiment with Ab(1-42) labeled on the arginine and lysines, no difference in the peptide was detected between the absence or presence of (+) MK-801. However, the presence of (+) MK-801 did appear to accelerate the pathway by which the Ab(1-

42) peptide progressed from monomers towards fibrils. With the peptide alone, in the absence of (+) MK-801, no such acceleration was seen. Nor was any acceleration observed with the control solutions containing Thioflavin T and (+) MK-801 but no peptide.

In the one-dimensional proton spectra of the Ab (1-42) peptide labeled on the arginine and lysines, no chemical shift changes were seen in these residues. This is consistent with the result obtained with Ab(1-40) using two-dimensional NMR spectroscopy. Again, (+) MK-801 did not appear to bind to monomeric Ab peptides.

When the integration and normalization of methyl peaks obtained by one-dimensional

NMR spectroscopy were compared to the normalized results of a Thioflavin T fluorescence assay, it was seen that (+) MK-801 accelerates the transition away from a monomeric state of the peptide on the same time scale in the two methods. This is also consistent with the results with Ab(1-40) that were seen in the preliminary experiments using HFIP. The electron microscopy supports these findings. After incubation for 24 hours Ab(1-42) alone had formed fibrils, whereas Ab(1-42) in the presence of (+) MK-801 had not only formed fibrils but the fibrils were wider and clumped together. In a further experiment, fibrils are seen earlier, at 3 hours, when

Ab(1-42) was in the presence of (+) MK-801 (or memantine or morin). At that time

no fibrils were observed with Ab(1-42). At 24 hours, Ab(1-42) fibrils were seen in the absence and presence of the ligands.

So where does that leave us? Ab peptides progressively pass from monomers to oligomer to fibrils. The effect of (+) MK-801 and memantine on the aggregation pathway of Ab(1-40) and Ab(1-42) peptides has been examined. Preliminary experiments suggested that (+) MK-801 would reduce fibril formation, but further testing revealed otherwise. Both compounds accelerated the formation of b-sheets by the Ab peptides, (+) MK-801 doing so more rapidly than memantine.

A plausible hypothesis is that in the presence of either (+) MK-801 or memantine, there is an accelerated transition of Ab(1-42) away from the monomeric state. The subsequent decrease is not a return to the monomeric state, but more likely a transition to a larger, possibly insoluble, complex.

The putative connection between RNA aptamers and Ab-peptides will be discussed in detail in Chapter 4.

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CHAPTER 4

RNA APTAMERS AND NON-CODING RNAs

Section 4.1: Introduction

In this chapter we turn to the convergence of information obtained in two different research areas. Small molecule inhibitors and Class 1 and 2 RNA aptamers target the neurotransmitter receptors; both the small molecule inhibitors and the Class

1 RNA aptamers may also affect the aggregation of Ab peptides that are implicated in

Alzheimer’s disease. Some effects on Aβ peptide aggregation of a representative

Class 1 ligand, (+) MK-801, that inhibits the opening of nAChR channels were presented in Chapter 3. In this chapter the role non-coding RNAs may play in

Alzheimer’s disease will be explored. Why did selective pressure in the SELEX procedure produce two classes of consensus sequences with different effects on neurotransmitter receptors? And is there a biological functional basis for the difference? A casual search of a Drosophila database indicated there might be something worth pursuing.

Ylera and colleagues (Ylera et al., 2002) had selected aptamers (containing a

70-nucleotide variable region) that bind with high affinity (Kd ~ 29 - 48 nM) specifically to the 40-amino-acid long b-amyloid peptide [Aβ(1-40)]. Moataz Gadalla

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(Gadalla & Hess, 2006) noticed that aptamers selected in the two independent studies

(Ulrich et al., 1998; Ylera et al., 2002) contained similar consensus sequences. He used the online software Teiresias (Rigoutsos & Floratos, 1998) to confirm the observation. The first seven nucleotides in the consensus sequence of the Aβ(1-40) aptamers are similar to the consensus sequence of the nAChR inhibitory Class I aptamers (Table 4.1). In addition, the fidelities of the different nucleotides share a similar pattern. The high-fidelity portion of the sequence, namely ACCG, is strongly conserved in the aptamers selected for the two different targets.

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Table 4.1: Consensus sequence of 14 nAChR-binding Class 1 RNA aptamers that

noncompetitively inhibit the receptor (Ulrich et al., 1998) and 18

Aβ(1-40)-binding RNA aptamers (Ylera et al., 2002). The number

below each nucleotide indicates the number of times that nucleotide

occurs in the same position in the total number of aptamers selected.

nAChR Class 1 RNA U U C A C C G aptamer consensus sequence Occurrence in 14 9 9 9 10 14 13 14 selected aptamers

Ab(1-40) RNA aptamer U U U A C C G U U U G G consensus sequence Occurrence in 18 14 12 10 16 18 18 18 18 18 18 17 12 selected aptamers

To explore the extent of similarity between the full length RNA aptamers selected for binding to Aβ(1-40) and those selected for binding to nAChRs, the aptamer with the highest affinity for Aβ(1-40) (aptamer B55) was synthesized by

Henning Ulrich and Antonio H. Martins at the University of Sao Paulo, Brazil

(Martins & Ulrich, unpublished). (Aptamer B55 was not available from Ylera and colleagues.) Aptamer B55 inhibited carbamoylcholine-induced currents from nAChRs

(Martins, H., Ulrich, H., Gadalla, M., & Hess, G. P., unpublished). Carbamoylcholine is a stable analog of acetylcholine, an endogenous ligand of the receptors. Aptamer

B55 can thus be considered a Class 1 ligand of the nAChR.

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Figure 4.1: Ab(1-40) RNA aptamer B55 inhibits the nicotinic acetylcholine receptors in a BC3H1 cell. 100-µM carbamoylcholine-induced currents in the absence (yellow line) or presence (red line) of 0.75-µM aptamer B55. The rising phases record the opening of the receptor channels. The upper flat yellow and red lines are the currents after they have been corrected for receptor desensitization

(recorded by the falling phases). (pH 7.4, -60 mV, 22 oC) (Martins, H., Ulrich, H.,

Gadalla, M., & Hess, G. P., unpublished)

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Nucleotides interfere with the Thioflavin T assay to some degree, so a different assay was needed to measure the effect of truncated RNA aptamers on aggregation of

Ab. An ELISA assay could potentially be used, but which one? In a direct ELISA assay, the antigen to be tested is added to wells on a plate and over time adheres by charge interaction to the internal surface of the wells. Bovine serum albumin or casein is added to cover any part of the surface to which the antigen did not adhere. The next addition is a primary antibody, to which a conjugated enzyme is bound, and this binds specifically to the antigen. Finally, a substrate for the conjugated enzyme is added and the ensuing change in signal, usually colorimetric, indicates the concentration of the primary antibody. A disadvantage is that the binding of the antigen to the surface of the well is not specific. In an indirect ELISA assay this problem is overcome by using a capture antibody that is specific for the antigen being tested. The optical density

(OD) of the sample is compared to a standard curve prepared by measuring the OD of serial dilutions of the target.

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Figure 4.2: Cartoon of a sandwich ELISA Assay. (1) Wells in a multiwell plate are coated with a capture antibody. (2) The sample is added and any antigen present binds to the capture antibody. (3) A detecting antibody is added and binds to the antigen.

(4) An enzyme-linked secondary antibody is added and binds to the detecting antibody. (5) The substrate is added and is converted to a detectable form by the enzyme. (From: Wikipedia.org)

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A sandwich ELISA assay includes a further modification, which is illustrated in Figure 4.2. A known amount of a capture antibody is added to the wells. Non- specific binding to the surfaces is then blocked. The antigen is then added and captured by the antibody. The wells are washed well to removed unbound antigen. A specific antibody is then added and binds the antigen, being sandwiched between the capture and specific antibodies. A secondary antibody conjugated to an enzyme is added and binds specifically to the FC region (non-specifically). Unbound antibody- enzyme complexes are removed by washing. Finally, the substrate for the enzyme is added and the resulting reaction signal provides a measure of the amount of antigen present. In this method there is a risk of false positives. Commercial ELISA kits are available for quantifying aggregated forms of amyloid peptides from Invitrogen /

ThermoFisher Scientific and from ANAspec. They differ and so one must obviously choose the one that will measure the property one wants to highlight.

Section 4.2: Materials and Methods

ANAspec sells a Sensolyte Anti-Mouse/Rat b-Amyloid (1-40 and 1-42)

Quantitative ELISA Colorimetric kit. (A similar kit is not available for the human

Ab.) One assumes that the peptides provided with this kit are the same as the ones in the fluorimetric kit that were discussed in Chapter 3. In the ELISA kit the antibody that detects a horse radish peroxidase-conjugated antibody is added together with the samples and standards. However, then incubation must be overnight at 4 oC and early

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events would be missed.

Invitrogen / ThermoFisher Scientific ELISA Assay. The Invitrogen /

ThermoFisher Scientific ELISA Assay kit does not have this problem and was used in preliminary experiments conducted by Ling Shen and Susan Coombs. Takahashi and colleagues (Takahashi et al., 2009) had also selected RNA aptamers that bound to

Ab(1-40), and found they inhibited the formation of fibrils. Two of the 6 selected aptamers, N2 and E2, bound with the highest affinity to a gold-labeled Ab(1-40) peptide. Fluorescently labeled N2 and E2 aptamers bound Ab(1-40) with dissociation constants of 21.6 and 10.9 µM, respectively. But the selection process did not converge to a consensus sequence, although guanosine and cytidine did predominate heavily in the full-length sequences. Takahashi and colleagues had devised an ELISA assay (not commercially available) to determine the degree of aggregation of the peptide, and their results provided a guide for us.

The amount of an aggregated amyloid peptide in a sample as a function of time can be measured, although there is an inevitable delay before the first time point is measured. 96-Well plates coated with monoclonal capture anti-Ab (1-40 or 1-42) antibodies are provided by the manufacturer. They were washed three times with a provided working wash buffer. The peptide was added to the wells and incubated at room temperature for two hours, before washing again three times. Human, aggregated Ab, biotin-conjugated antibodies (provided by the manufacturer) were added and incubated at room temperature for one hour, and then washed three times.

Streptavidin-conjugated horse radish peroxidase (provided by the manufacturer) was added to the wells and incubated at room temperature for 30 minutes. After another

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washing step, stabilized chromogen tetramethylbenzidine (provided by the manufacturer) was added. This reacts with the peroxidase to provide a signal that can be read. The absorbance is measured at 450 nm.

For the Invitrogen ELISA assay that was conducted, rPeptide was the supplier of the Ab peptides, as for the cuvet Thioflavin T assay described in Chapter 3.

Peptides from other suppliers were tested but the rPeptide materials were most consistent. The Ab peptides were available with different counterions, including HFIP and NaOH. HFIP was initially used for the ELISA assay but gave abnormal results, further supporting the decision described in Chapter 3, to abandon its use. Ab(1-40) peptide with a counterion of NaOH gave consistent results.

Several phosphate and trifluoroacetic acid (TFA) buffers were tested (see an example in Figure 4.3) to find one that gave results that fell within the concentration range over which the readings are valid (0 – 5.7 ng/mL). A phosphate buffer [100- mM NaCl, 50-mM sodium phosphate (monobasic and dibasic), and 0.02% NaN3; pH

7.4] that we were already using was satisfactory. The dilution of the incubated Ab(1-

40) peptide solution was optimized; a dilution of 1:100 gave results with the required range but not 1:100,000. This 1:100 dilution factor was also used by Takahashi and colleagues. In an attempt to correlate the results of the Thioflavin T and the ELISA measurements, the Ab(1-40) peptide was incubated with (+) MK-801, but the drug induced false positives in a dose-dependent manner.

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Figure 4.3: Elisa assay of Ab(1-40) aggregation in two different buffers and at different concentrations. Trifluoroacetic buffer and phosphate buffer, pH 7.4, were used. The peptide was incubated in these buffers at dilution factors of 1:100 and

1:100,000. A standard curve was established, following the manufacturer’s protocol, to determine the concentration (ng/mL) of aggregated Ab(1-40). (Ling Shen and

Susan E. Coombs)

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Using the experimental conditions thus determined, the effect of the consensus sequence (UUCACCG) of Class I RNA aptamers on the aggregation of Ab(1-40) peptide over time was examined. The sequence decreased (Figure 4.4) the amount of aggregated peptide occurring over time as compared to the amount in its absence.

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Figure 4.4: The effect of 25-µM consensus sequence (UUCACCG) of Class I

RNA aptamers on the aggregation of 100-µM Ab(1-40) peptide over time. Ab(1-

40) peptide in 100-mM NaCl, 50-mM sodium phosphate (monobasic and dibasic) and

0.02% NaN3, pH 7.4. at room temperature. (Ling Shen and Susan Coombs). pH 7.4.

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The existence of consensus sequences that defined Class 1 and Class 2 RNA aptamers, with clearly defined differences in mechanistic effects on a neurotransmitter receptor was intriguing. This interest was strengthened when the shared identity between Class 1 aptamers that targeted the receptor and aptamers that targeted the peptide was recognized. During the planning of the ELISA assays with Ab(1-40) peptide it was recognized that the Class 1 and Class 2 consensus sequences could potentially complement each other if they were aligned 5’ to 3’ and 3’ to 5’ (Table

4.2).

Table 4.2: Potential hybridization between the consensus sequences of Class 1

and Class 2 of the RNA aptamers targeting the nAChR when aligned

5’ to 3’ and 3’ to 5’.

nAChR Class 1 aptamer U U C A C C G consensus sequence 5’ to 3’ nAChR Class 2 aptamer A A G U G consensus sequence 3’ to 5’

Was the occurrence of two classes of RNA aptamers with different specific consensus sequences peculiar to the excitatory nAChR? No. Cui and colleagues

(2004) had selected two classes of fluorinated RNA aptamers for binding to the inhibitory g-gamma aminobutyric acid A (GABAAR) neurotransmitter receptor.

Fluorination renders the aptamers more resistant to RNA degradation. Class 1 aptamers inhibited the receptor and Class 2 aptamers alleviated picrotoxin inhibition of the receptor. The consensus sequence of the Class 1 aptamers also occurs in miR-

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7642. The a1-subunit of the GABA receptor is targeted by miR-181, -23, and -213.

Table 4.3: Consensus sequence of 11 GABAA receptor Class 1 RNA aptamers

that noncompetitively inhibit the receptor (Cui et al., 2004) and of 5

GABAA receptor Class 2 RNA aptamers (Cui et al., 2004). The

number below each nucleotide indicates the number of times that

nucleotide occurs in the same position in the total number of aptamers

selected.

GABAAR Class 1 RNA aptamer U C U U G U consensus sequence

Occurrence in 11 11 11 11 11 11 8 selected aptamers GABAA R Class 2 RNA aptamer U U C A C C C G U consensus sequence

Occurrence in 5 5 5 5 5 5 5 5 5 5 selected aptamers

Single-stranded RNA can form stem-loops in hairpin structures resulting from intramolecular base pairing. During the development of aptamers that targeted neurotransmitter receptors the question was raised as to whether binding was occurring at the loop. During the ELISA experiments, it was noticed that the consensus sequences of the Class 1 and Class 2 RNA aptamers raised against nAChRs could hybridize, with one mismatch, if the Class 1 sequence would run 5’ to 3’ and the

Class 2 sequence 3’ to 5’. Was this just a coincidence? Did it result from the presence of more guanine-cytosine base pairs than adenine-uracil (or adenine-thymidine), and, therefore, a more stable structure? Stem-loops occur in several non-coding RNAs

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groups, including pre-microRNAs and at the prokaryotic 5’ untranslated region

(UTR). Might the discovery of microRNAs be relevant? A naïve search of the miR database, and the literature, found some sequences that might be interesting. But were they just random? The Class 1 sequence was apparently in a small non-coding

RNA, the miR-124 miRNA precursor [www.(mirbase.org/)] (Coombs, unpublished). miR-124 may be involved in neuronal development and also in neurodegeneration. A change in the expression of glutamate receptor subtypes (Arrant & Roberson, 2014;

Gascon et al., 2014) is associated with some neurodegenerative diseases. Are specific miRs involved in this change? There are at least two others miRs of interest – miR-

990 and hsa-miR-197.

A preliminary search of the miR database (www.mirbase.org) for the consensus sequences was done. But nothing of significance was found – the only thing that might be relevant was that the Class 2 sequence had a p value of 0.03. So this line of investigation was abandoned for a time. But on returning to it later, it was found that if the search was for a 7-nucleotide stretch there were no hits but if one increased the length to 8 or 9 nucleotides something of potential interest emerges.

Searches were begun using the sequence as it occurs in individual aptamers and not the consensus sequence. This aspect of the research project is ongoing.

Micro-RNAS (MiRs). But what are microRNAs? mi-RNAs are defined

(Lagos-Quintana et al., 2003) as hairpin precursor structures, 20-30 base pairs long.

They are phylogenetically conserved in many species. “They regulate transcription and stability of target RNAs based on (partial – in animals) sequence complementarity.” They are tissue-specific. Mature miRNAs are excised from 60-80

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nucleotide, double-stranded RNA hairpins by an enzyme, Dicer RNase III. Usually only one of the strands accumulates. The original mi-Rs, lin-4 and let-7, were identified in C. elegans because they blocked protein stability during development.

Lin-4 recognizes complementary sequences in 3’-UTR of targets by forming an imperfect or bulged RNA duplex structure. Let-7 has partial sequence complementary with a targeted 3’-UTR. The 5’ prime ends of some D. melanogaster mi-RNAs are complementary to 3’UTR sequence motifs that mediate negative post-transcriptional regulation (Lai, 2002).

Expression of immediate-early gene proteins and brain-derived neurotrophic factor mRNA in rat cingulate and retrospinial cortices is reduced by inhibiting central muscarinic acetylcholine receptors (Hughes et al., 1993a; Fix et al., 1994). The expression of heat shock proteins induced by PCP, ketamine, or MK-801 can be countered by some antipsychotics (Farber et al., 1993). Do miRs play a role in AD?

Reports in the scientific literature would suggest so. miR-107 targets beta-secretase-1

(BACE-1), a membrane-bound aspartic acid-protease that catalyzes cleavage of the

Ab precursor protein (APP). Nelson, Wang, and colleagues (Wang et al., 2008;

Nelson & Wang, 2010) analyzed 19 brain samples from the University of Kentucky

Alzheimer’s Disease Center Brain Bank and reported that there was a correlation between a decreased level of miR107 expression and an increased presence of plaques and tangles in the temporal cortex.

MicroRNAs can be secreted by cells through a microvesicle-dependent pathway or a microvesicle-free dependent protein-dependent pathway. MicroRNAs are more stable under some conditions than many other RNA types when circulating

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outside a cell. The microRNAs can be associated with proteins, such as Argonaute-2.

Sadeghizadeh (Javidia et al., 2014) has reviewed reports of microRNAs circulating in body fluids in cancer. They may result from damaged cells, secreted microvesicles or exosomes, or a protein-dependent pathway. Could the same occur in neurodegenerative diseases, and, if so, would they provide a biomarker as a much- needed diagnostic tool? Kumar and Reddu (2016) reviewed the evidence. Six miRNAs (miR9, 125b, 146a, 181c, 191-5p, and let-7-5b) have been reported by several groups to occur in exosomes found in extracellular fluids (in the CSF and blood serum).

Pi-RNAs. For many years, Kandel has dissected the processes of memory, in both invertebrates such as Aplysia, and mammals. More recently, he and his colleagues have examined the role molecular biology plays in these processes

(Kandel, 2012), especially the role of some specific transcription factors such as

CREB (cAMP Response Element Binding protein) (e.g. Dash et al., 1990). miR-124 and miR-22 are reported to be brain specific and to be linked to CREB1 function and to serotonin, a modulatory neurotransmitter which functions during learning (reviewed by Landry et al., 2013).

Small non-coding RNAs were mentioned earlier, but piRNAs were not. Piwi- interacting RNAs are short 26-32-nucleotide non-coding RNAs that interact with

Argonaute proteins and can silence germline transposons. They were originally thought to be limited to germline functions but are now known to play a role in neuronal function (Nandi et al., 2016). And transposons have been implicated in neurodegenerative disease (Li et al., 2012).

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Rajasethupathy et al. (2009) used parallel sequencing to identify the small

RNAs of A. californica. They found 170 miRs, 9 of which were highly expressed in the brain, and several were down-regulated in the presence of serotonin. The most abundant was miR-124, which plays a role in synapse plasticity. From this data they then found (Landry et al., 2013) a high expression of piRNAs in the A. californica

CNS. The same group has drawn on their studies of long-term memory and proposed a fascinating working model (see Figure 4.5) (Landry et al., 2013), bringing together bidirectional regulation by small non-coding RNAs, transcription factors, and a neurotransmitter (serotonin).

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Figure 4.5: “A working model: the integrative action of small RNAs during synaptic plasticity (Rajasethupathy et al., 2012). A model of bidirectional small

RNA function during synaptic plasticity. In Aplysia piRNAs and miRNAs are transcriptional and translational repressors at baseline. With exposure to serotonin, piRNAs become activated, which leads to further transcriptional repression of its targets (inhibitors of synaptic facilitation) and the eventual enhancement of synaptic strength. By contrast, serotonin downregulates miRNAs, which leads to translational upregulation of its targets (enhancers of synaptic facilitation) and the eventual enhancement of synaptic strength. The two classes of small RNAs in Aplysia, therefore, may act in a coordinated fashion to regulate long-term memory processes.

Abbreviations: 5HT-5-hydroxytryptamines or serotonin; miRNA, microRNA; piRNA, piwi-interacting RNA; UTR, untranslated region “. (Figure 4 From Landry et al.,

2013)

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It would appear that at least two groups of small, noncoding RNAs regulate gene expression to maintain, or not, neuronal memory. What might be productive next steps to testing this hypothesis? This will have to be left for another time. But one can speculate. Perhaps the complementarity of the bidirectional consensus sequence is the tip of iceberg. Perhaps the working model proposed by Landry and colleagues (2013) is generally applicable. This will have to be left for another time.

But one can speculate. Do neurotransmitters upregulate and downregulate piRNAs and miRNAs, leading to turning off transcriptional repressors or turning on translational activators? These are all questions for the future.

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CONCLUSION

The unexpected observation that RNA aptamers selected for two disparate targets, the nicotinic acetylcholine receptors (Ulrich et al., 1998) and Ab peptides

(Ylera et al., 2002), shared a consensus sequence led to the project described here.

The RNA aptamers selected for the nicotinic acetylcholine receptors fell into two classes. Class 1 aptamers inhibited the receptor and, when in the presence of an activator of the receptor, Class 2 aptamers alleviated the inhibition. The consensus sequence of Class 1 aptamers alone retained this functional activity, although to a lesser degree. Some small organic compounds that bind to the receptor could similarly be assigned to Classes 1 and 2. One of the Class 1 compounds is (+) MK-

801. The most effective of the aptamers selected for binding to the Ab peptides, aptamer B55 (Ylera et al., 2002), also inhibited the receptor (Martins, Ulrich, Gadalla,

& Hess, unpublished), thus behaving like a Class 1 compound.

Ab peptides progressively pass from monomers, to oligomers, to fibrils, and eventually to the plaques that are associated with Alzheimer’s Disease, transitions that are companied by a change from soluble to insoluble forms. The effects of (+) MK-

801 on the aggregation pathway of Ab(1-40) and Ab(1-42) peptides have been examined using the Thioflavin T assay, nuclear magnetic resonance spectroscopy, and electron microscopy. Preliminary Thioflavin T experiments suggested that (+) MK-

801 would reduce fibril formation, but further testing, including using additional techniques, revealed otherwise. (+) MK-801 accelerated the initial formation of b- sheets by the Ab peptides, (+) MK-801. But a precipitous decrease in the fluorescence signal followed, a decrease that was not observed when it was absent.

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The Thioflavin T assay is not applicable when the Ab peptides are not in solution. Analysis of NMR spectra of the soluble peptides in the presence and absence of (+) MK-801 replicated the fluorescence measurements. Electron microscopy of the insoluble peptides formed indicated a more rapid rate of formation of fibril aggregates in the presence of (+) MK-801 than in its absence.

A second compound, memantine, was tested in the Thioflavin T assay in parallel with (+) MK-801. Like (+) MK-801, memantine accelerated the initial formation of b-sheets by the Ab peptides, but not as rapidly. A decrease in the fluorescence signal also followed the initial formation but not as precipitously as was seen with (+) MK-801. Memantine is FDA-approved for the treatment of mild- moderate Alzheimer’s Disease, and has some limited efficacy before the disease reaches a severe stage. The result with memantine raises a concern that should be evaluated. Does chronic treatment with memantine cause the severe phase of the disease to appear earlier than it does without the treatment? But if the neurotoxic form of Ab peptides is the soluble oligomers, then perhaps the formation of fibrils is protective.

The existence of two classes of RNA aptamers targeting the nicotinic acetylcholine receptor, with different consensus sequences and different effects, is not limited to this excitatory receptor. Fluorinated RNA aptamers that target the inhibitory GABAA (a1, b2, g2) receptor were selected (Cui et al., 2004). They too fell into two classes, as defined by consensus sequence and function. Class 1 inhibited the receptor and, in the presence of GABA, Class 2 alleviated inhibition by picrotoxin.

The two consensus sequences appear to be present in some microRNAs; evaluating

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this will be the topic of future work. Is there a biological significance to these findings? During the current work, it was recognized that the consensus sequences of the Class 1 and Class 2 RNA aptamers against the nicotinic acetylcholine receptors were complementary when aligned 5’ to 3’ and 3’ to 5’. Again, this is a topic to be evaluated in the future.

The apparent convergence of some seemingly unrelated observations in two different research areas has been described. The work has defined several questions that need to be answered in the future.

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REFERENCES

Aguzzi, A. & Polymenidou, M. (2004) Cell 116, 313-327. Mammalian prion biology: One century of evolving concepts.

Aguzzi, A. (2008) Proc. Nat. Acad.Sci. USA 105, 11-12. Unraveling prion strains with cell biology and organic chemistry.

Alberti, S., Halfmann, R., King, O., Kapla, A., & Lindquist, S. (2009) Cell 137, 146- 158. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.

Alzheimer, A. (1906) Neurol. Central. 25, 1134. Ueber einen eigengarten schwere Erkrankungsprozess der Hirnrinde.

Alzheimer, A. (1907a) Allg. Z. Psychiatr. Und Psych.ger. Medizin 64, 146-148. Ueber eine eigenartige Erkrankung der Hirnrinde. (Translated into English by Stelzmann et al., 1995).

Alzheimer, A. (1907b) Zbl Nervenheilkd Psychiatr.30 (Neue Folge 18 bd), 177-179. Ueber eine eigenartige Erkrankung der Hirnrinde.

Alzheimer, A. Z. (1911) Zbl. Ges. Neurol. Psychiat. 4, 356-385. Ueber eigenartige Krankenheitsfaewlle des spaeteren Alters. (Translated by Foerstl, H. & Levey, R., 1991).

Amador, M. & Dani, J. A. (1991) Synapse 7, 207-215. MK-801 inhibition of nicotinic acetylcholine receptor channels.

Aravin, A. A., Sachidanandam, R., Girard, A., Fejes-Toth, K., & Hannon, G. J. (2007) Science 316, 744-747. Developmentally regulated piRNA clusters implicate MILI in transposon control.

Arcava, Y., Pereiora, E. F., Maelicke, A., & Albuquergue, E. X. (2005) J. Pharmacol. Exp. Ther. 312, 1195-1205. Memantine blocks alpha7* nicotinic acetylcholine receptors more potently than N-methyl-D-aspartate receptors in rat hippocampal neurons.

Armstrong, R. A. (1994) Neurosci Lett. 169, 133-136. Differences in beta-amyloid (beta/A4) deposition in human patients with Down’s syndrome and sporadic Alzheimer’s Disease.

129

Arnold, F. H., Schofield, S. A., & Blanch, H. W. (1986) J. Chromatogr. 355, 1-27. Analytical affinity chromatography.

Arnold, S.E., Hyman, B.T., Flory, J., Damasio, A.R., & Van Hoesen, G.W. (1991) Cereb Cortex. 1, 103-106. The topographical and neuroanatomical distribution of neurofibrillary tangles and neurotic plaques in the cerebral cortex of patients with Alzheimer’s disease.

Arrant, A. E. & Roberson, E. D. (2014) Nat. Med. 20, 1381-1383. MicroRNA-124 modulates social behavior in frontotemporal dementia.

Arzensek, D. (2010) Dynamic light scattering and application to proteins in solution. Fourth year seminar, University of Ljubljana. http://www- f1.ijs.si/~rudi/sola/Dynamic_light_scattering_and_application_to_proteins_in_solu tions.pdf

Azizeh, B., Head, E., Ibrahim, M. A., Torp, R., Tenner, A. J., Kim, R. C., Lott, I. T., & Cotman, C. W. (2000) Exp. Neurol. 163, 111-112. Molecular dating of senile plaques in the brains of individuals with Down syndrome and in aged dogs.

Barrow, C. J. & Zagorski, M. G. (1991) Science 253, 179-182. Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition.

Bastianetto, S., Ramassamy, C., Dore, S., Christen, Y., Poirer, J., & Quirion, R. (2000a) Eur. J. Neurosci. 12, 1882-1859. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by b-amyloid.

Bastianetto, S., Zheng, W. H., & Quirion, R. (2000b) J. Neurochem. 74, 2268-2277. The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity; involvement of its flavonoid constituents and protein kinase C.

Batchelor, G. K. (2000) An Introduction to Fluid Dynamics. (Cambridge University Press)

Bayro, M. J., Debelouchina, G. T., Eddy, M. T., Birkett, M. T., MacPhee, C. E., Rosay, M., Maas, W. E., Dobson, C. M., & Griffin, R. G. (2011) J. Am. Chem. Soc. 113, 13967-13974. Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR.

Berhanu, W. M. & Masunov, A. E. (2014) Polyphenols in Human Health and Disease 1, 59-70. Chapter 6 – The atomic level interaction of polyphenols with the Ab

130

oligomer aggregate, a molecular dynamic guidance for rational drug design.

Bertolotti, A. (2018) Current Opinion in Neurobiology 51, 127-133. Importance of the subcellular location of protein deposits in neurodegenerative disease.

Biancalana, M. & Koide, S. (2010) Biochim. Biophys. Acta 1804, 1405-1412. Molecular mechanism of Thioflavin T binding to amyloid fibrils.

Bielschowsky, M. (1902) Neurologisches Zentralblatt (Leipzig) 13, 579-584. Die Silberimpreaegation der Achsen-zylinder.

Bird, T. D. (2005) N. Engl. J. Med. 352, 862-864. Genetic factors in Alzheimer’s disease.

Bitan, G., Tarus, B., Vollers, S. S., Lashuel, H. A., Condron, M. M., Straub, J. E., & Teplow, D. B. (2003) J. Am. Chem. Soc. 125, 15359-15365. A molecular switch in amyloid assembly: Met35 and amyloid beta-protein oligomerization.

Blocq, P. & Marinesco, G. (1892) Semaine. Med. 12, 445-446. Sur les lesions et la pathogenese de l’epilepsie dite essentielle.

Boller, F. (2008) Handbook of Clinical Neurology 89, 3-13. History of dementia.

Bolton, D. C., McKinley, M. P., & Prusiner, S. B. (1982) Science 218, 1309-1311. Identification of the protein that purifies with the scrapie protein.

Booth, D. R., Tan, S. Y., Booth, S. E., Hsuan, J. J., Totty, N., Nguyen, O., Hutton, T., Vigushin, D. M., Tennent, G. A., Hutchinson, W. L. et al. (1995) QJM. 88, 695- 702. A new apolipoprotein Al variant, Trp50Arg, causes hereditary amyloidosis.

Braak, H. & Braak, E. (1991) Acta Neuropathologica 82, 239-259. Neuropathological staging of Alzheimer-related changes.

Braak, H., Braak, E., Ohm, T., & Bohl. J. (1989) Neurosci Letter. 103, 24-28. Alzheimer’s disease: mismatch between amyloid plaques and neuritic plaques.

Braak, H., Tredici, K. D. Udo, R., Vios, R. I., Steur, E. N. H., & Braak, E. (2003) Neurobiology of Aging 24, 197-211. Staging of brain pathology related to sporadic Parkinson’s disease.

Briggs, C. A. & McKenna, D, G. (1996) Neuropharmacology 35, 407-414. Effect of MK-801 at the human alpha 7 nicotinic acetylcholine receptor.

131

Broadway, N. (2012, edited 2016) Mater. Methods 2, 121. How to develop assays: 5 considerations and 8 fundamentals.

Brookmeyer, R., Johnson, E., Ziegler-Graham, K., & Arrighi, H. M. (2007) Alzheimers Dement. 3, 186-191. Forecasting the global burden of Alzheimer's disease.

Buccianti, M., Giannoni, E., Chiti, F., Barone, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., & Stefani, M. (2002) Nature 416, 507–511. Inherent toxicity of aggregates implies a common mechanism for protein folding diseases.

Buchsbaum, M., Kesslak, J. P., Lynch, G., Chui, H., Wu, J., Sicotte, N., Hazlertt, E., Teng, E., & Cotman, C. W. (1991) Arch. Gen. Psychiatry 46, P144-151. Temporal and hippocampal metabolic rate during an olfactory memory task assessed by positron emission tomography in patients with dementia of the Alzheimer type and controls. Preliminary studies.

Buisson, B. & Bertrand, D. (1998) Mol. Pharmacol. 53, 555-563. Open-channel blockers at the human alpha4beta2 neuronal nicotinic acetylcholine receptor.

Burns, R. S. & Lerner, S. E. (1978) Clin Toxicol. 12, 463-481. Causes of phencyclidine-related deaths.

Calligaro, D. O., O’Malley, P. J., & Monn, J. A. (1993) J. Neurochem. 60, 2297-2303. b-amyloid (25-35) or Substance P stimulates [3H]MK-801 binding to rat cortical membranes in the presence of glutamate and glycine.

Chen, Q. S., Wei, W. Z., Shimahara, T., & Xie, C. W. (2002) Neurobiol. Learn. Mem. 77, 354-371. Alzheimer amyloid beta-peptide inhibits the late phase of long-term potentiation through calcineurin-dependent mechanism in the hippocampal dentate gyrus.

Chen, Y., Banerjee, A., & Hess, G. P. (2004) Biochemistry 43, 10149-10156. Mechanism-based discovery of small molecules that prevent non-competitive inhibition by cocaine and MK-801 mediated by two different sites on the nicotinic acetylcholine receptor.

Cheng, X. R., Daabout, G. G., Unlu, M. S., & Kerman, K. (2014) Analyst 139, 59-65. LED-based interferometric reflectance imaging sensor for the detection of amyloid-b aggregation.

132

Chi, E. Y., Frey, S. L., Winans, A., Lam, K. L., Kjaer, K., Majewski, J., & Lee, K. Y. (2010) Biophys. J. 98, 2299-22308. Amyloid-beta fibrillogenesis seeded by interface-induced peptide misfolding and self-assembly.

Choi, N.-H., Tobe, T., Hara, K., Mazda, T., & Tomita, M. (1990) J. Immunol. Methods 131, 159-163. Sandwich ELISA assay for quantitative measurement of SP-40, 40 in seminal plasma and serum.

Clark, C. M. & Karwalish, H. T. (2003) Ann. Intern. Med. 138, 400-410. Alzheimer disease: Current concepts and emerging diagnostic and therapeutic strategies

Clarke, R. T. & Oprysa, A. (2004) J. Chem. Ed. 81, 705-707. Fluorescence and light scattering.

Cohen, A. S. & Calkins, E. (1959) Nature 183, 1202-1203. Electron microscopic observations on a fibrous component in amyloid of diverse origins.

Cohen, F. E. & Prusiner, S. B. (1998) Annu. Rev. Biochem. 67, 793-819. Pathological conformations of prion proteins.

Conway, K. A., Lee, S. J., Rochet, J. C. Ding, T. T., Williamson, R. E., & Lansbury, P. T. (2000) Proc. Natl. Acad. Sci. USA 96, 571-576. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations, lined to early-onset Parkinson’s disease: implications for pathogenesis and therapy.

Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., & Pericak-Vance, M. A. (1993) Science 261, 921-923. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families.

Corso, E. A., Campo, G., Triglio, A., Napoli, A., Reggio, A., & Lanaia, F. (1992) Ital. J. Neurol. Sci. 13, 215-219. Prevalence of moderate and severe Alzheimer dementia and multi-infarct dementia in the population of southeastern Sicily.

Cowburn, R. F., Messamora, E., Li Li, M., Winblad, B., & Sundstrom, E. (1994) NeuroReport 5, 405-408. b-Amyloid related peptides exert differential effects on [3H]MK-801 binding to rat cortical membranes.

Cowburn, R. F., Wiehager, B., Trief, E., Li-Li, M., & Sundstrom, E. (1997) Neurochem. Res. 22, 14371442. Effects of beta-amyloid-(25-35) peptides on radioligand binding to excitatory amino acid receptors and voltage-dependent

133

calcium channels: evidence for a selective affinity for the glutamate and glycine recognition sites of the NMDA receptor.

Cui, Y., Rajasethupathy, P., & Hess, G. P. (2004) Biochemistry 43, 16442-16449. Selection of stable RNA molecules that can regulate the channel-opening equilibrium of the membrane-bound gamma-aminobutyric acid receptor.

Cummings, J. L. & Benson, D. F. (1992) Dementia: A Clinical Approach. (Second edition). Butterworth & Heinemann.

Dash, P. K., Hochner, B., & Kandel, E. R. (1990) Nature 345, 718-721. Injection of cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation.

Datki, Z., Juhasz, A., Galfi, M., Soos, K., Papp, R., Zadori, D., & Penke, B. (2003) Brain Res. Bull. 62, 223-229. Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells.

De Ferrari, G. V., Mallender, W. D., Inestrosa, N. C., & Roseberry, T. L. (2001) J. Biol. Chem. 276, 23282-23287. Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites.

Dineley, K. T., Bell, K. A., Bui, D., & Sweatt, J. D. (2002) J. Biol. Chem. 277, 25056- 25061. Beta amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Dingledine, R., Borges, K., Bowie, D., & Traynelis, S. F. (1999) Pharmacol. Rev. 51, 7-61. The glutamate receptor ion channels.

Doller, D. & Thurston, D. (2016) Allosterism in Drug Discovery 1st Ed. (RSC Drug Discovery series) Royal Society of Chemistry, London, UK. p. 17.

Doty, R. L. (1991) Ann. N.Y. Acad. Sci. 640, 20-27. Olfactory capacities in aging and Alzheimer’s disease. Psychophysical and anatomic considerations.

Dravid, S. M., Erreger, K., Yuan, H., Nicholson, K., Le, P., Lyuboslavsky, P., Almonte, A., Murray, E., Mosley, C., Barber, J., French, A., Balster, R., Murray, T. F., & Traynelis, S. F. (2007) J. Physiol. 581, 107–128. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block.

Drolle, E., Hane, F., Lee, B., & Leononko, Z. (2014) Drug Metabolism Reviews 1-17.

134

Atomic force microscopy to study molecular mechanisms of fibril formation and toxicity in Alzheimer’s disease

Ege, C. & Lee, K. Y. (2004) Biophys. J. 87, 1732-1740. Insertion of Alzheimer's Aβ40 peptide into lipid monolayers.

Egensperger, R., Koesel, S., Schnabel, R., Mehraein, P., & Graeber, M. B. (1995) Acta Neuropathol. (Berl.) 90, 257-265. Apolipoprotein E genotype and neuropathological phenotype in two members of a German family with chromosome 14-linked early onset Alzheimer’s Disease.

Eichner, T., Kalverda, A. P., Thompson, G. S., Homans, S. W., & Radford, S. E. (2011) Molecular Cell 41, 129-131. Conformational conversion during amyloid formation at atomic resolution.

Einstein, A. (1906) Annalen der Physik 19, 289-306. Eine neue Bestimmung der Molekueldimensionen.

Ellington, A. D. & Szostak, J. W. (1990) Nature 346, 818-822. In vitro selection of RNA molecules that bind specific ligands.

Ellison, G. & Switzer, R. (1993) NeuroReport 5, 17-20. Dissimilar patterns of degeneration in brain following four different addictive stimulants.

Ellison, G. (1994) NeuroReport 5, 2688-2692. Neuronal degeneration following both competitive and non-competitive NMDA antagonists.

Ellison, G. (1995) Brain Res. Rev. 20, 250-267. The N-methyl-D-aspartate antagonists phencyclidine, ketamine and dizocilpine as both behavioral and anatomical models of the dementias

Esler, W. P., Stimson, E. R., Jennings, J. M., Vintners, H. V., Ghilardi, J. R., Lee, J. P., Mantyh, P. W., & Maggio, J. E. (2000) Biochemistry 39, 6288-6295. Alzheimer’s Disease amyloid propagation by a template-dependent dock-lock mechanism.

Evans, K. C., Berger, E. P., Cho, C.-G., Weusgraber, K. H., & Lansbury, P. T. (1995) Proc. Natl. Acad. Sci. USA 92, 763-767. Apolipoprotein E is a kinetic not a thermodynamic inhibitor of amyloid formation: Implications for the pathogenesis and treatment of Alzheimer disease.

Farber, N. B., Price, M. T., Labruyere, J., Nemnich, J., St Peter, H., Wozniak, D. F., &

135

Olney, J. W. (1993) Biol. Psychiatry 34, 119-121. Antipsychotic drugs block phencyclidine receptor-medicated neurotoxicity.

Farrer, L. A., Cupples, L. A., Haines, J. L., Human, B., Kukill, W. A., Mayeux, R., Myers, R. H., et al. (1997) JAMA 278, 1349-1356. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.

Farrar, C. T., William. C. M., Hudry, E., Hashimoto, T., & Hyman, B. T. (2014) PLoS One 9, e89901. RNA aptamer probes as optical imaging agents for the detection of amyloid plaques.

Feldman, J. L., Murphy, C., Davidson, T. M., Jalowayski, A. A., & de Jaime, G. G. (1991) Laryngoscope 101, 1198-1201. The rhinologic evaluation of Alzheimer’s disease.

Ferrone, F. A., Hofrichter, J., & Eaton, W.A. (1985) J. Mol. Biol. 183, 591-610. Kinetics of sickle hemoglobin polymerization. I. Studies using temperature-jump and laser photolysis techniques.

Findeis, M. A. & Molineaux, S. M. (1999) Methods Enzymol. 309, 475-488. Design and testing of inhibitors of fibril formation.

Findeis, M. A. (2000) Biochim. Biophys. Acta 1502, 76-84. Approaches to discovery and characterization of inhibitors of amyloid b-peptide polymerization.

Fischer, O. (1907) Monatsschr. Psychiatr. Neurol. 22, 361-372. Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmaessige Veranderung der Hirnrinde bei seniler Demenz.

Fisher, G. H., Payan, I. L., Chou, S. J., Man, E. H., Cerwinski, S., Martin, T. Emory, C., & Frey, W. H. (1992) Brain Res. Bull. 28, 127-131. Altered aspartate in Alzheimer neurofibrillary tangles.

Fix, A. S., Horn, J. W., Truex, L. L. Smith, R. A., & Gomez, E. (1994) Acta Neuropathol. 88, 511-519. Neuronal vacuole formation in the rat posterior cingulate/retrosplenial cortex after treatment with the N-methyl-D-aspartate (NMDA) antagonist MK-801 (dizocilpine maleate).

Foerstl, H. & Levy, R. (1991) History of Psychiatry II, 71-101. On certain peculiar diseases of old age.

136

Frenzel, D. & Willbold, D. (2014) PLoSONE 9, e106882 (and references therein). Kinetic titration series with biolayer interferometry.

Fried, M. G. (1989) Electrophoresis 10, 366-376. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.

Fu, W. & Jhamandas, J. H. (2003) J. Neurophysiol. 90, 3130-3136. Beta-amyloid peptide activates non-alpha7 nicotinic acetylcholine receptors in rat basal forebrain neurons.

Furey, T. S. & Sethupathy, P. (2013) Science 342, 705-706. Genetics driving epigenetics.

Gadalla, M. M. & Hess, G. P. (2006) Alzheimer's & Dementia S594, P4-263: Discovery of a regulatory site on beta-amyloid peptides.

Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., et al. (1995) Nature 373, 523-527. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein.

Gascon, E., Lynch, K., Ruan, H., Almeida, S., Verhevden, J. M., Seeley, W. W., Dickson, D. W., Petrucelli, L., Sun, D., Jiao, J., Zhou, H., Jakovcevski, M., Akbarian, S., Yao, W.-D., & Gao, F.-B. (2014) Nat. Med. 20, 1444-1451. Alterations in microRNA-124 and AMPA receptors contribute to social behavioral deficits in frontotemporal dementia.

Genin, E. Hannequin, D., Walton, D., Sleegers, K., Hiltunen, M. et al. (2011) Mol. Psychiatry 16, 903-907. APOE and Alzheimer disease: a major gene with semi- dominant inheritance.

Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res. Comm. 120, 885-890. Alzheimer’s disease: Initial report of the purification and chacterization of a novel cerebrovascular amyloid protein.

Glenner, G. G. (1980) N. Engl. J. Med. 302, 1283-1292. Amyloid deposits and amyloidosis. The beta-fibrilloses (first of two parts).

Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Luis Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M., & Hardy, J. (1991) Nature 349, 704–706. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's

137

disease.

Goldhaber, G., Lerman, M. I., McBride, O. W., Saffiotti, U., & Gajdusek, D. C. (1987) Science 235, 877-880. Characterization and chromosomal localization of a cDNA encoding the brain amyloid of Alzheimer’s disease.

Goldstein, L. E., Muffatt, J. A., Cherny, R. A., Moir, R. D., Erisccon, B. S., Huang, X., Mavros, C., Coccia, J. A., Faget, K. Y., Fitch, K. A., Masters, C. L., Tanzi, R. E., Chylack, L. T., & Bush, A. I. (2003) The Lancet 361, 1258-1265. Cytosolic b-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer’s disease.

Gotti, C. & Clementi, F. (2004) Prog. Neurobiol. 74, 363-393. Neuronal nicotinc receptors: from structure to pathology.

Graeber, M. B., Koesel, S., Egensperger, R., Banati, R. B., Mueller, U., Bise, K., Hoff. P. Moeller, H. J., Fujisawa, K,. & Mehraein, P. (1995) Neurogenetics 1, 73-80. Rediscovery of the case described by Alois Alzheimer in 1911: historical, histological and molecular genetic analysis.

Graeber, M. B., Koesel, S., Egensperger, R., Schnopp, N. M., Bise, K., & Mehraein, P. (1995) J. Neuropathol.Exp. Neurol. 54, 527-528. Strategies for using archival brain tissue in neurogenetic research.

Grewer, C. & Hess, G. P. (1999) Biochemistry 38, 7837-7846. On the mechanism of inhibition of a nicotinic acetylcholine receptor by the anticonvulsant MK-801 investigated by laser-pulse photolysis in the microsecond-to-millisecond time region.

Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.C., Zaidi, M. S., & Wisniewski, H. M. (1986a) J. Biol. Chem. 261, 6084-6089. Microtubule-associated protein tau. A component of Alzheimer paired helical filaments.

Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H. M., & Binder, L. I. (1986b) Proc. Natl. Acad. Sci. U S A. 83, 4913-4917. Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.

Harel, M., Sonoda, L. K., Silman, I., Sussamn, J. L. & Rosenberry, T. L. (2008) J. Am. Chem. Soc. 130, 7856-7861. Crystal structure of thioflavin T bound to the peripheral site of Torpedo californica acetylcholinesterase reveals how thioflavin T

138

acts as a sensitive fluorescent reporter of ligand binding to the acylation site.

Hardy, J. A. & Higgins, G. A. (1992) Science 256, 184-185. Alzheimer’s disease: the amyloid cascade hypothesis.

Hardy, J., & Selkoe, D. J. (2002) Science 297, 353-356. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics.

Hargreave, F. E., Wong, B. J., Popov, T., & Dolovich, J. (1993) Agents Actions Suppl. 43, 291-295. Noninvasive methods to examine the anti-inflammatory effects of drugs.

Harkany, T., Abraham, I., Timmerman, W., Laskay, G., Toth, B., Sasvari, M., Konva, C., Sebens, J. B., Korf, J., Nvakas, C., Zarandi, M., Soos, K., Penke, B., & Luiten, P. G. (2000) Eur. J. Neurosci. 12, 2735-2745. Beta-amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis.

Harper, J. D. & Lansbury, P. T. (1997) Ann. Rev. Biochem. 66, 385-407. Models of amyloid seeding in Alzheimer’s Disease and Scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins.

Harper, J. D., Lieber, C. M., & Lansbury, P. T. (1997) Chem. Biol. 4, 951-959. Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer’s disease amyloid-beta protein.

Harper, J. D., Wong, S. S., Lieber, C. M., & Lansbury, P. T. (1999) Biochemistry 38, 8972-8980. Assembly of A beta amyloid protofibrils: an in vitro model for a possible early event in Alzheimer’s disease.

Hebert, L. E., Scherr, P. A., Bienias, J. L., Bennett, D. A., & Evans, D. A. (2003) Archives of Neurology 60, 1119-1122. Alzheimer disease in the U.S. population: prevalence estimates.

Hess, G. P. & Grewer, C. (1998) Methods in Enzymology 291, 443-473. Development and application of caged ligands for neurotransmitter receptors in transient kinetic and neuronal circuit mapping studies.

Hess, G. P. (2003) Biophysical Chemistry 100, 493-506. Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors).

Hess, G. P., Andrews, J. P., Struve, G. E., & Coombs, S. E. (1975) Proc. Natl. Acad.

139

Sci. USA 72, 4371-4375. Acetylcholine-receptor-mediated ion flux in electroplax membrane preparations.

Hess, G. P., Cash, D. J., & Aoshima, H. (1979) Nature 282, 329-331. Acetylcholine receptor-controlled ion fluxes in membrane vesicles investigated by fast reaction techniques.

Hess, G. P., Gameiro, A. M., Schoenfeld, R. C., Chen, Y., Ulrich, H., Nye, J. A., Sit, B., Carroll, F. I., & Ganem, B. (2003) Biochemistry 42, 6106-6114. Reversing the action of noncompetitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor)-mediated reaction.

Hess, G. P., Ulrich, H., Breitinger, H.G., Niu, L. Gameiro, A. M., Grewer, C. Srivastava, S., Ippolito, J. E., Lee, S., Jayaraman, V., & Coombs, S. E. (2000) Proc. Natl. Acad. Sci. USA 97, 13895-13900. Mechanism-based discovery of ligands that prevent inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801.

Hoff, P. & Hippius, H. (1989) Nervenarzt 60, 332-337. Ein Ueberblick ueber Leben und Werk anlaesslich seines 125.

Hoenack, D. & Loescher, W. (1993) Eur. J, Pharmacol. 242, 99-104. Effects of the novel 5-HT1A receptor antagonist, (+)-WAY 100135, on sterotyped behavior induced by the NMDA receptor antagnost dizocilipine in rats.

Horváth, Z. C., Czopf, J., & Buzsáki, G. (1997) Brain Res. 753, 181-195. MK-801- induced neuronal damage in rats.

Hou, L. & Zagorski, M. G. (2006) J. Am. Chem. Soc. 128, 9260-9261. NMR reveals anomalous copper(II) binding to the amyloid Abeta peptide of Alzheimer’s disease.

Howes, M-J. R. & Houghton, P. J. (2003) Pharmacology, Biochemistry and Behavior 75, 513-527. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function.

Iravani, M. M., Muscat, R., & Kruk, Z. L. (1999) Synapse 32, 212-224. MK-801 interaction with the 5-HT transporter: a real-time study in brain slices using fast cyclic voltammetry.

Ising, C., Stanley, M., & Holtzman, D. M. (2015) Clin. Pharm, & Therapeutics 98, 469-471. Current thinking on the mechanistic basis of Alzheimer’s and

140

implications for drug development.

Iwatsubo, T. (2000) In Neurodegenerative Dementias (Eds. Clark, C.M., Trojanowski, J.Q. Lee, M-Y.L.) McGraw-Hill, New York. pp. 145-157. Amyloid deposits and plaque formation.

Jansen, K. L. (1990) Int. J. Addict. 25, 133-139. Ketamine – can chronic use impair memory?

Jarret, J. T., Berger, E. P., & Lansbury, P. T. (1993) Biochemistry 32, 4693-4697. The carboxy terminus of the b amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s Disease.

Jarrett, J. T. & Lansbury, P. T. (1993) Cell 73, 1055-1059. Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer’s disease and Scrapie? Minireview

Javidia, M. A., Ahmadi, A. H., Bakhshinejad, B., Nouraee, N., Babashah, S., & Sadeghizadeh, M. (2014) Med. Oncol. 31, 295-306. Cell-free microRNAs as cancer biomarkers: The odyssey of miRNAs through body fluids. Review.

Johnston, R. F., Pickett, S. C., & Barker, D. L. (1990) Electrophoresis 11, 355-360. Autoradiography using storage phosphor technology.

Jorm, A. F., Korten, A. E., & Henderson, A. S. (1987) Acta Psychiatrica Scandinavica 76, 465-479. The prevalence of dementia: A quantitative integration of the literature.

Kaletta, T. & Hengartner, M. O. (2006) Nature Reviews Drug Discovery 5, 387-389. Finding function in novel targets: C. elegans as a model organism.

Kandel. E. R. (2012) Mol. Brain 5, 14. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB.

Kar, S., Slowikowski, S. P., Westaway, D., & Mount, H. T. (2004) J. Psychiatry Neurosci. 29, 427-441. Interactions between β–amyloid and central cholinergic neurons: Implications for Alzheimer’s disease.

Kasowski, M., Kyriazopoulou-Panagfiotopoulou, S., Grubert, F., Zaugg, J. B., Kundaje, A., Liu, Y., Boyle, A. P., Zhang, Q. C., Zakharia, F., Spacek, D. V., Li, J., Xie, D., Olarerin-George, A., Steinmetz, L. M., Hogenesch, J. B., Kellis, M., Batzoglou, S., & Snynder, M.(2013) Science 342, 750-752. Extensive variation in

141

chromatin states across humans.

Katzman, R. (1976) Arch. Neurol. 33, 217-218. The prevalency and malignancy of Alzheimer Disease. A major killer.

Kesslak, J. P., Nalcioglu, O., & Cotman, C. W. (1991) Neurology 41, 51-54. Quantification of magnetic resonance scans for hippocampal and parahippocampal atrophy in Alzheimer’s Disease.

Khurana, R., Uversky, V. N., Nielsen, L., & Fink, A. L. (2001) J. Biol. Chem. 276, 22715-22721. Is Congo Red an amyloid-specific dye?

Kidd, M. (1963) Nature 197, 192-193. Paired helical filaments in electron microscopy of Alzheimer’s Disease.

Kilpinen, H., Waszak, S. M., Gschwind, A. R., Raghav, S. K., Witwicki, R. M., Orioli, A., Migliavacca, Wiederkehr, M., Gutierrez-Arcelus, M., Panousis, N. I. Yurovsky, A., Lappalainen, T., Romano-Palumbo, L. Planchon, A., Bielser, D., Bryois, J., Padioleau, I., Udin, G., Thurnheer, S., Hacker, D., Core, L. J., Lis, J. T., Hernandez, N., Reymond, A., Deplancke, B., & Dermitzakis, E. T. (2013) Science 342, 744-747. Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.

Kim, D. S. H. L. & Kim, J. Y. (2001) Bioorg. Med. Chem. Lett. 11, 2541-2543. Total synthesis of calebin-A, preparation of its analogues, and their neuronal cell protectivity against b-amyloid insult.

Kirkitadze, M. D., Bitan, G., & Teplow, D. B. (2002) J. Neurosci. Res. 69, 567-577. Paradigm shifts in Alzheimer’s disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.

Klein, W. L., Krafft, G. A., & Finch, C.E. (2001) Trends Neurosci. 24, 219-224. Targeting small Abeta oligomer: the solution to an Alzheimer’s disease conundrum?

Klunk, W. E., Jacob, R. F., & Mason, R. P. (1999) Methods Enzymol. 309, 285-305. Quantifying amyloid by congo red spectral shift assay.

Koesel, S. & Gaeber, M. B. (1993) Brain Pathol. 3, 421-424. Non-radioactive direct sequencing of PCR products amplified from neuropathological specimens.

Kowall, N. W. & Beal, M. F. (1991) Ann. Neurol. 29, 162-167. Glutamate,

142

glutaminase- and taurine-immunoreactive neurons develop neurofibrillary tangles in Alzheimer’s disease.

Kowall, N. W., Beal, M. F., Busciglio, J., Duffy, L. K., & Yankner, B. A. (1991) Proc. Natl. Acad. Sci. USA 88, 7247-7251. An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P.

Kraepelin, E. (1910) Psychiatrie. Ein Lehrbuch fuer Studierene und Aerzte. Band 2. (Barth Verlag, Leipzig).

Krystal, J. H., Karper, L. P., Seibyl, J. P., Freeman, G. K., Delaney, R., Brenberm J. D., Heninger, G. R., Bowers, M. B., & Charney, D. S. (1994) Arch. Gen. Psychiatry 51, 199-214. Subanesthetic effects of the noncompetititve NMDA antagonist, ketamine, in humans. Pyschotomimetic, perceptual, cognitive, and neuroendocrine responses.

Kumar, J., Namsechi, R., & Sim, V. L. (2015) PLos One 10, e0129087. Structure- based peptide design to modulate amyloid beta aggregation and reduce cytotoxicity.

Kumar, S. & Reddu, P. H. (2016) Biochem. Biophys. Acta 1862, 1617-1627. Are circulating microRNAs peripheral biomarkers for Alzheimer’s disease?

Kurumaji, A. & McCulloch, J. (1990) Brain Res. 518, 342-346. Effects of unilateral intrahippocampal injection of MK-801 upon local cerebral glucose utilisation in conscious rats.

Kurumaji, A. & McCulloch, J. (1990) Brain Res. 531, 72-82. Effects of MK-801 upon local cerebral glucose utilization in conscious rats following unilateral lesion of caudal entorhinal cortex.

Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., & Tuschl, T. (2003) RNA 9, 175-179. New microRNAs from mouse and human.

Lai, E. C. (2002) Curr. Biol. 12, R200-202. Notch cleavage: Nicastin helps presenilin make the final cut.

Lambert, J. C., Ibrahim-Verbaas, C. A., Harold, D., Naj, A. C., Sims, R. et al. (2013) Nat. Genetics 45, 1452-1458. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease.

Landau, L. D. & Lifschitz, E. M. (1987) Fluid Mechanics. (Pergamon)

143

Landry, C. D., Kandel, E. R., & Rajasethupathy, R. (2013) Trends in Neurosciences 36, 535-542. New mechanisms in memory storage: piRNAs and epigenetics.

Lansbury, P. T. (1997) Curr. Opin. Chem. Biol. 1, 260-267. Inhibition of amyloid formation: a strategy to delay the onset of Alzheimer’s disease.

LeVine, H. & Scholten, J. D. (1999) Methods Enzymol. 309, 467-476. Screening for pharmacologic inhibitors of amyloid fibril formation.

LeVine, H. (1997) Arch. Biochem. Biophys. 342, 306-316. Stopped-flow kinetics reveal multiple phases of Thioflavin T to Alzheimer b(1-40) amyloid fibrils.

LeVine, H. (1999) Methods Enzymol. 309, 274-84. Quantification of beta-sheet amyloid fibril structures with thioflavin T.

LeVine, H. (2002) Current Medicinal Chemistry 9, 1121-1133. The challenge of inhibiting Ab polymerization.

Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, W. H., Yu, C. E., Jondro, P. D., Schmidt, S. D., Wang, K., et al. (1995) Science 269, 973-977. Candidate gene for the chromosome 1 familial Alzheimer's disease locus.

Levy-Lahad, E., Wijsman, E. M., Nemens, E., Anderson, L., Goddard, K. A., Weber, J. L., Bird, T. D., & Schellenberg, G. D. (1995) Science 269, 970-973. A familial Alzheimer's disease locus on chromosome 1.

Li, W., Jin, Y., Prazak, I., Hammell, M., & Dubnau, J. (2012) PLoS One 7, e44099. Transposable elements in TDP-43-mediates neurodegenerative disorders.

Lindberg, D. J., Wranne, M. S., Gatty, M. G., Westerlund, F. & Esbjorner, E. K. (2015) Biochem. Biophys. Res. Comm. 458, 418-423. Steady-state and time- resolved Thioflavin T fluorescence can report on morphological differences in amyloid fibrils formed by Ab(1-40) and Ab(1-42).

Link, C. D., Taft, A., Kapulkin, V., Duke, K., Kim, S., Fei, Q., Wood, D. E., & Sahagen, B. G. (2003) Neurobiology of Aging 24, 397-413. Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model.

Lipton, S. A., Choi, Y-B., Pan, Z-H., Lei, S. Z., Chen, H-S. V., Sucher, N. J., Singel, D. J., Loscalzo, J., & Stamler, J. S. (1993) Nature 364, 626–632. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and

144

related nitroso-compounds.

Lipton, S. A. & Chen, H-SV. (2004) Cell Death and Differentiation 11, 18-20. Paradigm shift in neuroprotective drug development: clinically tolerated NMDA receptor inhibition by memantine.

Liu, Q., Kawai, H., & Berg, D. K. (2001) Proc. Natl. Acad. Sci. U S A. 98, 4734-4739. Beta-amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons.

Liu, Y. & Schubert, D. (1997) J. Neurochem. 69, 2285-2293. Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis.

Lomakin, A., Chung, D. S., Benedek, G. B., Kirschner, D. A., & Teplow, D. B. (1996) Proc. Natl. Acad. Sci. USA 94, 7942-7947. On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

Lomakin, A., Teplow, D. B., Kirschner, D. A., & Benedek, G. B. (1997) Proc. Natl. Acad. Sci. USA 93, 1125-1129. Kinetic theory of fibrillogenesis of amyloid beta- protein.

Lomakin, A., Teplow, D. B., & Benedek, G. B. (2005) Methods Mol. Biol. 259, 153- 174. Quasielastic light scattering for protein assembly studies.

Lopera, F., Arcos, M., Madrigal, L., Kosik, K., Cornerjo, W., & Ossa, J. (1994) Acta Neurologica Colombiana 10, 173-187. Demencia de tipo Alzheimer con agregacin familiar en Antioquia, Colombia.

Lu, J.-X., Qiang, W., Yau, W.-M., Schwieters, C. D., Meredith, S. C., & Tycko, R. (2013) Cell 154, 1257-1268. Molecular structure of b-amyloid fibrils in Alzheimer’s disease brain tissue.

Masters, C. L., Simms, G., Weinman, N. A., Maulthaup, G., McDonald, B. L., & Beyreuther, K. (1985) Proc. Natl. Acad. Sci., USA 82, 4245-4249. Amyloid plaque core protein in Alzheimer disease and Down syndrome.

Maurer, K., Volk, S., & Gerbaldo, H. (1997) Lancet 349, 1546-1549. Auguste D. and Alzheimer’s disease.

McVicker, G., van de Geijn, B., Degner, J. F., Cain, C. E., Banovich, N. E., Raj, A., Lewellen, N., Myrthil, M., Gilad, Y., & Pritchard, J. K. (2013) Science 342, 747-

145

749. Identification of genetic variants that affect histone modifications in human cells.

Miguel-Hidalgo, J. J., Alvarez, X. A., Cacabelos, S., & Quack, G. (2002) Brain Res. 958, 210-221. Neuroprotection by memantine against neurodegeneration induced by beta-amyloid (1-40).

Milburn, T., Matsubara, N., Billington, A. P., Udgaonkar, J. B., Walker, J. W., Carpenter, B. K., W. W., Marque, J., Denk, W. & Hess. G. P. (1989) Biochemistry 28, 49-55. Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand.

Minnema, M. C., Kimby, E., D’Sa, S., Fornecker, L-M., Poulain, S., Snijders, T. J., Kastritis, E., Kremer, S., & Fitsiori, A., (2017) Haematologica 102, 43-51. Guideline for the diagnosis, treatment, and response criteria for Bing-Neel syndrome.

Moeller, H.-J. & Graeber, M. B. (1998) Eur. Arch Psychiatry Clin Neurosc. 248, 111- 122. The case described by Alois Alzheimer in 1911. Historical and conceptual perspectives based on the clinical record and neurohistological sections.

Molnar, Z., Soos, K., Lengyel, I., Penke, B., Szegedi. V., & Budai, D. (2004) Neuroreport 15, 1649-1652. Enhancement of NMDA responses by b-amyloid peptides in the hippocampus in vivo.

Morgado, I. & Faendrich, M. (2011) Curr. Opin. Colloid Interface Sci. 16, 508-514. Assembly of Alzheimer Ab peptide into nanostructured amyloid fibrils.

Nabeshima, T., Yamaguchi, K., Hiramatsu, M., Amano, M. Furukawa, H., & Kameyama, T. (1984) Pharmacol. Biochem. Behav. 21, 401-408. Serotonergic involvement in phencyclidine-induced behaviors.

Naiki, H. & Nakakuki, K. (1996) Lab. Invest. 74, 347-383. First-order kinetic model of Alzheimer’s beta-amyloid fibril extension in vitro.

Naiki, H., Higuchi, K., Hosokawa, M., & Takeda, T. (1989) Analyt. Biochem. 177, 244-249. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T.

Nandi, S., Chandramohan, D., Fiorita, L., Melnick, A. M., Hebert, J. M., Mason, C. E., Rajasethupasy, P., & Kandel, E. R. (2016) Proc. Nat. Acad. Sci., USA 113, 12697-12702. Roles for small noncoding RNAs in silencing of retrotransposons in

146

the mammalian brain.

Nehls, D. G., Park, C. K., MacCormack, A. G., & McCulloch, J. (1990) Brain Res. 511, 271-279. The effects of N-methyl-D-aspartate receptor blockade with MK- 801 upon the relationship between cerebral blood flow and glucose utilization.

Nelson, P. T. & Wang, W. X. (2010) J. Alzheimer’s Dis. 21, 75-79. MiR-107 is reduced in Alzheimer’s disease brain neocortex: validation study.

Nelson, R. & Eisenberg, D. (2006) Curr. Opin. Struct. Biol. 16, 260-265. Recent atomic models of amyloid fibral structure

Nilsbeth, C., Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K., Forsell, C., Stemh, C., Luthman, J., Teplow, D. B., Younkin, S. G., Naslund, J., & Lannfelt, L. (2001) Nat. Neurosci. 4, 887-893. The ‘Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced Abeta protofibril formation.

Nomura, I., Kato, N., Kita, T., & Takechi, H. (2005) Neurosci. Lett. 391, 1-6. Mechanism of impairment of long-term potentiation by amyloid β is independent of NMDA receptors or voltage dependent calcium channels in hippocampal CA1 pyramidal neurons.

Noor, H., Cao, P., & Raleigh, D. P. (2012) Protein Science 21, 373-382. Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers.

Norstedt, C., Naslund, J., Tjernberg, L. O., Karlstrom, A. R., Thyberg, J., & Terenius, L. (1994) J. Biol. Chem., 269, 30773-30776. The Alzheimer Ab peptide develops protease resistence in association with its polymerization into fibrils.

Olney, J. W. (1989) Drug Devel. Res. 17, 299-319. Excitotoxicity and N-methyl-D- aspartate receptors.

Olney, J. W., Ikonomidou, C., Mosinger, J. L. & Frierdich, G. (1989) J. Neurosci. 9, 1701-1704. MK-801 prevents hypobaric-ischemic neuronal degeneration in infant rat brain.

Olney, J. W., Labruyere, J., Wang, G., Wozniak, D. F., Price, M. T., & Sesma, M. A. (1991) Science 254, 1515-1518. NMDA antagonist neurotoxicity; mechanism and prevention.

Ono, K, L. L., Takamur, Y., Yoshiike, Y., Zhu, L., Han, F., Mao, X., Ikeda, T.,

147

Takasaki, J., Nishijo, H., Takashima, A., Teplow, D. B., Zagorski, M. G., & Yamada, M. (2012) J. Biol. Chem. 287, 14631-14643. Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site- specific binding.

Oosawa, F. & Akakura, S. (1975) Statistical Thermodynamics of Polymerization (Oosawa, F. & Akakura, S. Eds.) Academic Press, New York., pp. 41-45.

Oosawa, F. & Higashi. S. (1967) Statistical Thermodynamics of Polymerization and Polymorphism of Protein (Oosawa, F. & Higashi, S., Eds.) Academic Press, New York., pp. 79-164.

Osawa, Y. & Davila, J. C. (1993) Biochem. Biophys. Res. Commun. 194, 1435-1439. Phencyclidine, a psychomotor agent and drug of abuse, is a suicide inhibitor.

Park, S. Y. & Kim, D. S. H. L. (2002) J. Nat. Prod. 65, 1227-1231. Discovery of natural products from Curcuma longa that protect cells from beta-amyloid insult: a drug discovery effort against Alzheimer’s disease.

Parsons, C. G., Gruner, R., Rozental, J., Millar, J., & Lodge, D. (1993) Neuropharmacology 32, 1337-1350. Patch clamp studies on the kinetics and selectivity of N-methyl-D-aspartate receptor antagonism by memantine (1-amino- 3,5-dimethyladamantan).

Pelligrini, J. W. & Lipton, S. A. (1993) Ann. Neurol. 33, 403-407. Delayed administration of memantine prevents N-methyl-D-aspartate receptor-mediated neurotoxicity.

Perusini, G. (1910) In Nissl, F., Alzheimer, A. Eds. Anat. Der Geisteskrankheiten. Jena 3, 297-352. (Fischer, Germany) Histol. u. histopathol. Arbeiten ueber die Grosshirnrinde mit besondered Beruecksichtigung der path.Ueber klinish und histologisch eigenartige psychische Erkrankungen des spaeteren Lebensalters.

Pepys, M. B. (2006) Annu. Rev. Med. 57, 223-241. Amyloidosis.

Pettit, D. L., Shao, Z., & Yakel, J. L. (2001) J. Neurosci. 21, RC120. Beta-amyloid(1- 42) peptide directly modulates nicotinic receptors in the rat hippocampal slice.

Pick, A. (1892) Prager Med. Wochenschr. 17, 165-167. Ueber die beziehungen der senilen hirnatrophie zur aphasie.

Pruessing, K., Voigt, A., & Schulz, J. B. (2013) Molecular Neurodegeneration 8, 35-

148

46. Drosophila melanogaster as model organism for Alzheimer’s disease.

Prusiner, S. B. (1982) Science 216, 136-144. Novel proteinaceous infectious particles cause scrapie.

Pym, L., Kemp, M., Raymond-Delpech, V., Buckingham, S., Boyd, C. A., & Sattelle, D. (2005) Br. J. Pharmacol. 146, 964-971. Subtype-specific actions of beta- amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4) expressed in Xenopus laevis oocytes.

Rajasethupathy, P., Fiumara, F., Sheridan, R., Betel, D., Puthanveettil, S. V., Russo, J. J., Sander, C., Tuschl, T., & Kandel, E. (2009) Neuron 63, 803-817. Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.

Rajasethupathy, P., Antonov, I., Sheridan, R., Frey, S., Sander, C., Tuschl, T., & Kandel, E. R. (2012) Cell 149, 693-707. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity.

Ramakrishnan, L. & Hess, G. P. (2005) Biochemistry 44, 8523-8532. Picrotoxin

inhibition mechanism of a g-aminobutyric acidA receptor investigated by a laser- pulse photolysis technique.

Ramakrishnan, L. & Hess, G. P. (2010) Biochemistry 49, 7892-7901. Mechanism of

potentiation of a dysfunctional epilepsy-linked mutated GABAA receptor by a neurosteroid (3a,21-dihydroxy-5a-pregnan-20-one): Transient kinetic investigations.

Rammes, G., Rupprecht, R., Ferrari, U., Zieglgansberger, W., & Parons, C. G. (2001) Neuroscience Letters 306, 81-84. The N-methyl-D-aspartate receptor channel blockers memantine, MRZ 2/579 and other amino-alkyl-cyclohexanes antagonize 5-HT(3) receptor currents in cultured HEK-293 and NIE-115 cell systems in a non-competitive manner.

Ramoa, A. S., Alkondon, M., Aracava, Y., Irons, J., Lun. G. G., Deshpande, S. S., Wonnacott, S., Aronstam, R. S., & Albuquerque, E. X. (1990) J. Pharmacol. Exp. Ther. 254, 71-82. The anticonvulsant MK-801 interacts with peripheral and central nicotinic acetylcholine receptor ion channels.

Ramon y Cahal, S., (1910-1911) Histologie du Systeme Nerveux de l’Homme & des Vertebres 2, pp. 854-858. Instituto Ramon y Cahal, Madrid. Consejo superior de investigaciones cientfices.

149

Raymond, C. R., Ireland, D. R., & Abraham, W. C. (2003) Brain Res. 968, 263-272. NMDA receptor regulation by amyloid-beta does not account for its inhibition of LTP in rat hippocampus.

Reyes, P. F., Deems, D. A., & Suarez, M. G. (1993) Brain Res. Bull. 32, 1-5. Olfactory-related changes in Alzheimer’s disease: a quantitative neuropathologic study.

Rigoutsos, I. & Floratos, A. (1998) Bioinformatics 14, 55-67. Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm.

Rosenman, D. J., Clemente, N., Ali, M., Garcia, A. E., & Wang, C. (2018) Chem. Commun. (Camb.) 54, 4609-4612. High pressure NMR reveals conformational perturbations by disease-causing mutations in amyloid b-peptide.

Ruska, E. & Knoll, M. (1931) Z. Tech. Phys. 12, 389-400, and 448. Die magnetische Sammespule fuer schnell Elektronenstrahlen. (The magnetic concentrating coil for fast electron beams.)

Ryan, T. M., Caine, J., Mertens, H. D. T., Kirby, N., Nigro, J., Breheney, K., Waddington, L. J., Streltsov, V. A., Curtain, C., Masters, C. I., & Roberts, B. R. (2013) PeerJ 1;e73; DOI 10.7717/peerj.73 Ammonium hydroxide treatment of Ab produces an aggregate free solution suitable for biophysical and cell culture characterization.

Salomon, A. R., Marcinowski, K. J., Friedland, R. P., & Zagorski, M. G. (1996) Biochemistry 35, 13568-13578. Nicotine inhibits amyloid formation by the b- peptide.

Sambrook, J., Fritsch, E., & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY).

Sato, T., Kienlen-Campard, P., Ahmed, M., Liu, W., Huilin, L., Elliott, J. I., Aimoto, S., Constantinescu, S. N., Octave, J.-N., & Smith, S. O. (2006) Biochemistry 45, 5503-5516. Inhibitors of amyloid toxicity based on b-sheet packing of Ab40 and Ab41.

Saunders, A. M., Strittmatter, W. J., Schmechel, D., George-Hyslop, P. H., Pericak- Vance, M. A., Joo, S. H., Rosi, B. L., Gusella, J. F., Crapper-MacLachlan, D. R., Alberts, M. J., et al. (1993) Neurology 43, 1467-1472. Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's

150

disease.

Savas, J. N., Wang, Y-Z., DeNardo, L. A., Martinez-Bartolome, S., McClatchy, D. B., Hark, T. J., Shanks, N. F., Cozzolino, K. A., Lavalluee-Adam, M., Smukowski, S. N., Park, S. K., Kelly, J. W., Koo, E. H. Nakagaswa, T., Masliah, E. Ghosh, A., & Yates, J. R. (2017) Cell Reports 21, 2614-2627. Amyloid accumulation drives proteome-wide alterations in mouse models of Alzheimer’s disease-like pathology.

Schmittschmitt, J. P. & Scholtz, J. M. (2003) Protein Sci. 2, 2374-2378. The role of protein stability, solubility, and net charge in amyloid fibril formation.

Schweers, O., Schonbrunn Hanebeck, E., Marx, A., & Mandelkow, E. (1994) J. Eur. Chem. 269, 234290-234297. Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for b-structure.

Selkoe, D. J. (2000) Ann. N.Y. Acad. Sci, 924, 17-25. Toward a comprehensive theory for Alzheimer's disease. Hypothesis: Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein.

Selkoe, D. J. (2002) Science 298, 789-791. Alzheimer’s disease is a synaptic failure.

Selkoe, D. J. (2003) Nature. 426, 900-904. Folding proteins in fatal ways.

Seeman, P., Caruso, C., & Lasaga, M. (2008) Synapse 62, 143-153. Memantine agonist action at dopamine D2 high receptors.

Serby, M, Larson, P., & Kalkstein, D. (1991) Am. J. Psychiatry 148, 357-360. The nature and course of olfactory deficits in Alzheimer’s disease.

Serio, T.R., Cashikar, A. G., Kowai, A. S., Sawicki, G. J., Moshlehi, J. J. Serpell, L., Arnsdorf, M. F., & Lindquist, S. (2000) Science 289, 1317-1320. Nucleated conformational conversion and the replication of conformational information by a prion determinant.

Serpell, L. C., Berriman, J., Jakes, R., Goedert, M., & Crowther, R. A. (2000) Proc. Nat. Acad. Sci. USA 97, 4897-4902. Fiber diffraction of synthetic a-synuclein filaments shows amyloid-like cross-b conformation.

Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., L,i G., Holman, K., Tsuda, T., Mar, L., Foncin, J. F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L., Chumakov, I., Pollen, D., Brookes, A., Sanseau, P., Polinsky, R. J., Wasco, W., Da Silva, H. A., Haines,

151

J. L., Perkicak-Vance, M. A., Tanzi, R. E., Roses, A. D., Fraser, P. E., Rommens, J. M., & St George-Hyslop, P. H. (1995) Nature 375, 754-760. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease

Simon, L., Fitsion, A., Lemal, R., Dupuis, J., Carpenter, B., Boudin, L., Corby, A. Aurran-Schlienz, T., Gastaud, L., Talbot, A. Lepretre, S., Mahe, B., Payet, C., Soussain, C., Bonnet, C., Vincent, L., Lissandre, S., Herbrecht, R., Kremer, S., Leblond, V., & Formecker, L. M. (2015) Haematologica 100, 1587-1594. Bing- Neel syndrome, a rare complication of Waldenstroem macroglobulinemoa: analysis of 44 cases and review of the literature. A study of the Franch innovative leuikemia organization (FILO).

Sisodia, S. S. (2000) Science 289, 2296-2297. Neuroscience. An accomplice for gamma-secretase brought into focus.

Sivaprakasam, K., Pagán, O. R., & Hess, G. P. (2010) J. Membr. Biol. 233, 1-12. Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

Snell, L. D., Yi, L., S. J., & Johnson, K. M. (1988) Eur. J. Pharmacol. 145, 223-226. Comparison of the effects of MK-801 and phencyclidine on catecholamine uptake and NMDA-induced norepinephrine release.

Snyder, E. M., Nong, Y., Almeida, C. G., Paul, S., Moran, T., Choi, E. Y., Nairn, A. C., Salter, M. W., Lombroso, P. J., Gouras, G. K., & Greengard, P. (2005) Nature Neuroscience 8, 1051-58. Regulation of NMDA receptor trafficking by amyloid- b.

Stelzmann, R. A., Schnitzlein, H. N., & Murtagh, F. R. (1995) Clinical Anatomy 8, 429-431. An English translation of Alzheimer’s 1907 paper, “Ueber eine eigenartige Erkrankung der Hirnrinde”.

Stine Jr., W. B., Dahlgren, K. N., Krafft, G. A., & LaDu, M. J. (2003) J. Biol. Chem. 278, 11612-22. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.

Stöhr, J., Watts, J. C., Mensinger, Z. L., Oehler, A., Grillo, S. K., DeArmond, S. J., Prusiner, S. B., & Giles, K. (2012) Proc. Natl. Acad. Sci. U S A. 109,11025-11030. Purified and synthetic Alzheimer's amyloid beta (Aβ) prions.

Stromer, T. & Serpell, R. C. (2005) Microscopy Research and Technique 67, 210-217. Structure and morphology of the Alzheimer’s amyloid fibril.

152

Strozyk, D., Launer, L. J., Adlard, P. A. Cherny, R. A., Tsatsanis, A., Volitakis, I., Blennow, K., Petrovitch, H., White, L. R., & Bush, A. I. (2009) Neurobiol. Aging 30, 1069–1077. Zinc and copper modulate Alzheimer Aβ levels in human cerebrospinal fluid.

Struble, R. G. & Clark, H. B. (1992) Neurobiol. Aging 13, 469-473. Olfactory bulb lesions in Alzheimer’s disease.

Sunde, M. & Blake, C. F. (1997) Advances in Protein Chemistry 50 (Protein Misassembly Ed. Richards, F. M., Eisenberg, D. S., & Kim, P. S.), 125-159. The structure of amyloid fibrils by electron microscopy and X-ray diffraction.

Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B., & Blake, C. F. C. (1997) J. Mol. Biol. 273, 729-739. Common core structure of amyloid fibrils by synchrotron X-ray diffraction.

Szendrai, G. I., Fabian, H., Mantsch, H. H., Lovas, S., Nyeki, O., Schon, I., & Otvos, L. (1994) Eur. J. Biochem. 226, 917-924. Aspartate-bond isomerization affects the major conformations of synthetic peptides.

Takahashi, T., Tada, K., & Mihara, H. (2009) Mol. Biosyst. 5, 986-999. RNA aptamers selected against amyloid beta-peptide (Abeta) inhibit the aggregation of Abeta.

Tennent, G. A., Lovat, L. B., & Pepys, M, B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4299-4303. Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.

Terry, R.D., Gonatas, N. K., & Weiss, M. (1964) Am. J. Pathol. 44, 269-297. Ultrastructural studies in Alzheimer’s presenile dementia.

Thunecke, M., Lobbia, A., Kosciessa, U., Dyrcks, T., Oaklyey, A. E., Turner, J, Saenger, W., & Georgalis, Y. (1998) J. Pept. Res. 52, 509-517. Aggregation of A beta Alzheimer’s disease-related peptide studied by dynamic light scattering.

Tuerk, C. & Gold, L. (1990) Science 249, 505-510. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.

Udgaonkar, J. B. & Hess, G. P. (1987) Proc. Nat. Acad. Sci., U.S.A. 84, 8758-8762. Chemical kinetic measurements of a mammalian acetylcholine receptor by a fast- reaction technique.

153

Ulrich, H., Ippolito, J. E., Pagan, O. R., Eterovic, V. A., Hann, R. M., Shi, H., Lis, J. T., Eldefrawi, M. E., & Hess, G. P. (1998) Proc. Natl. Acad. Sci. USA 95, 14051- 14056. In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic receptor.

Valentine, J. J., Arnold, L. W., & Owens, S. M. (1994) J. Pharmacol Exp., Ther. 269, 1079-1085. Antiphencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats.

Vandersteen, A., Hubin, E., Sarroukh, De Baets, G., Schymkowitz, J., Rousseau, F., Subramanian, V., Raussens, V., Wenschuh, H., Wildemann, D., & Broersen, K. (2012) FEBS Letters 586, 4088-4093. A comparative analysis of the aggregation behavior of amyloid-b peptide variants.

Vassar, P. S. & Culling, C F. (1959) Arch. Pathol. 68, 487-498. Fluorescent stains, with special reference to amyloid and connective tissues.

Velazquez, P., Cribbs, D. H., Poulos, T. L., & Tenner, A. J. (1997) Nat. Med. 3, 77-79. Aspartate residue 7 in amyloid beta protein is critical for classical complement pathway activation: implications for Alzheimer’s disease pathogenesis.

Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., & Selkoe, D. J. (2002) Nature 416, 535-539. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.

Wang, R. X., Rajeev, B. W., Stromberg, A. J., Ren, N., Tang, G., Huang, Q., Rigoutsos, I., & Nelson, P. T. (2008) J. Neurosci. 28, 1213-1223. The expression of miR-107 decreases early in Alzheimer’s Disease and may accelerate disease progression through regulation of beta-site amyloid precursors protein-cleaning enzyme 1.

Wu, J., Anwyl, R., & Rowan, M. J. (1995) Neuroreport 6, 2409-2413. beta-Amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus.

Wu, J., Kuo, Y. P., George, A. A., Xu, L. Hu, J., & Lukas, R. J. (2004) J. Biol. Chem. 279, 37842-37851. Beta-amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells

Xue, C., Lin, T. Y., Chang, D., & Guo, Z. (2017) R. Soc. open sci. 4, 160696. Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and

154

effect on aggregation.

Yan, Y. & Wang, C. (2006) J. Mol. Biol. 364, 853-862. Ab42 is more rigid than Ab40 at the C terminus: Implications for Ab aggregation and toxicity.

Ye, C., Walsh, D. M., Selkoe, D. J., & Hartley, D. M. (2004) Neurosci. Lett. 366, 320- 325. Amyloid beta-protein induced electrophysiological changes are dependent on aggregation state: N-methyl-D-aspartate (NMDA) versus non-NMDA receptor/channel activation.

Ylera, F., Lurz, R., Erdmann, V. A., & Furste, J. P. (2002) Biochem. Biophys. Res. Commun. 290, 1583-1588. Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.

Zeeberg, B. (1980) J. Biol. Chem. 255, 3062-3067. New approach for studying macromolecular ligand binding. Determination of the dissociation constant for macromolecule-bound ligand by gel filtration.

Zhang, S. Z., Zhao, F. L., Li, K. A., & Tong, S. Y. (2001) Analytica Chimica Acta 431, 133-139. Determination of glycogen by Rayleigh light scattering.

Zhao, L. N., Long, H., Mu, Y., & Chew, L. Y. (2012) Int. J. Mol. Sci. 13, 7303-7327. The toxicity of amyloid β oligomers.

Zhu, Y.J., Lin, H., & Lal, R. (2000) FASEB J. 14, 1244-1254. Fresh and nonfibrillar amyloid beta protein (1-40) induces rapid cellular degeneration in aged human fibroblasts: evidence for Abeta P-channel mediated cellular toxicity.

155