Amyloid Aβ Peptide: In-Cell Studies And

AMYLOID Aβ PEPTIDE: IN-CELL STUDIES AND

MECHANISM OF POLYPHENOL-BASED

INHIBITION TO AGGREGATION

by FANG HAN

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Thesis Advisors: Dr. Michael G. Zagorski

Department of Chemistry CASE WESTERN RESERVE UNIVERSITY August, 2014

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

Fang Han ______

Candidate for the ______Ph.D______degree*

James Burgess ______(chair of the committee)

Mary Barkley ______

Paul Carey ______

Hyoung-Gon Lee ______

Michael Zagorski ______

______

(Date) _07/02/14______

*We also certify that written approval has been obtained for any proprietary material contained therein.

This thesis is dedicated to my parents and my husband

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TABLE OF CONTENTS

Table of Contents iv

List of Tables vi

List of Figures vii

Acknowledgements xiii

List of Abbreviations and Acronyms xv

Abstract xviii

1 Introduction 1

1.1 Alzheimer’s Disease and Its Impact on Public Health 2

1.2 Causes of AD 4

1.3 Treatments for AD 5

1.4 Amyloid beta (Aβ) peptides and AD 6

1.5 Objective and Significance of the Research 17

2 Studies of Introduction of Aβ Peptide Inside the Living Cells 19

2.1 Introduction 20

2.2 Materials and Methods 37

2.3 Results 41

2.4 Discussion 45

3 In-cell NMR Studies of the Aβ Peptide 49

3.1 Introduction 50

3.2 Materials and Methods 54

3.3 Results 60

3.4 Discussion 66

4 Mechanism of Polyphenol-Based Inhibition of Aβ Aggregation 71

4.1 Introduction 72

4.2 Materials and Methods 81

4.3 Results 86

4.4 Discussion 109

5 Conclusions and Future Directions 116

Bibliography 121

LIST OF TABLES

Table 1.1 Food and Drug Administration (FDA) approved AD drug. http://www.alz.org/research/science/alzheimers_disease_treatments.as p...... 5

Table 1.2 Proposed factors for Aβ self-aggregation. Picture and caption taken from [67] ...... 15

LIST OF FIGURES

Figure 1.1 Projected numbers of people aged >65 years in the U.S. population with AD using the U.S. Census Bureau estimates of population growth. Numbers indicate middle estimates per decade. Shaded area indicates high and low estimates per decade. Figure and caption taken from [15] ...... 4

Figure 1.2 Sequence of the Aβ(1-40) and Aβ(1-42). It contains two hydrophobic domains comprising residues 17-21 and 29-40/42...... 6

Figure 1.3 Amyloid cascade hypothesis of AD. Figure and caption taken from [29]...... 8

Figure 1.4 Formation and toxicity mechanisms of extracellular Aβ oligomers. Picture and caption taken from [39]...... 11

Figure 1.5 Formation and toxicity mechanisms of intracellular Aβ oligomers. Picture and caption taken from [39]...... 12

Figure 2.1 Schematic representation of principle of Confocal Laser Scanning Microscopy...... 31

Figure 2.2 Schematic FCM. http://qbab.aber.ac.uk/aberinst/mcytmain.html ...... 32

Figure 2.3 Schematic representation of forward-scattered light (FSC) and side- scattered light (SSC). Figure and caption taken from http://qbab.aber.ac.uk/aberinst/mcytmain.html...... 34

Figure 2.4 (A) Schematic representation and (B) real graph of FSC versus SSC histogram. (A) Gating cells in different regions could help us calculate viability of cells. (B) Cells inside of the circle are considered as live or normal while the others are considered as dead or abnormal...... 35

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Figure 2.5 (A) Schematic dual-color fluorescence histogram. Cells in region1 (R1) are those with negative fluorescent signals. Cells in the R2 are those with only PI signals. Cells in R3 have both PI and DyLight signals. Cells in R4 have only DyLight signals. (B) Real dual-color fluorescence histogram...... 36

Figure 2.6 Schematic illustrations of incorporation of the target protein within HeLa cells by SLO permeabilization and resealing of the target cells...... 39

Figure 2.7 Confocal microscope and (B) differential interference contrast images of SLO treated HeLa cells incubated with Dylight488-labeled Aβ (1- 40)...... 42

Figure 2.8 (A) Flow cytometry (FCM) analysis of HeLa cells, with or without the SLO-treatment (100 ng/ml), subsequently incubated with Dylight488- labeled Aβ(1-40), and resealed by 1mM Ca2+. Prior to the FCM analysis, the resealed cells were incubated with propidium iodide (PI) to stain the cells with compromised plasma membranes. (B) Schematic illustration of 1, DyLight-488 negative cells, 2, DyLight 488-positive but PI-negative cells and 3, DyLight 488-positive and PI-positive cells...... 43

Figure 2.9 Efficiency of the pore-formation and resealing of HeLa cells, treated with various concentrations of SLO, ranging from 0-150 ng/ml. The DyLight-positive and PI-negative cell population (dark bars) indicates the cells with successful pore formation and ...... 44

Figure 3.1 Cell suspension preparation and NMR experiment (a) Schematic illustrations of incorporation of the target protein within HeLa cells by SLO permeabilization and resealing of the target cells. (b) Supernatant preparation...... 57

Figure 3.2 (a) The 1H–15N HSQC spectrum of the Ala2 and Ala21 15N labeled Aβ (1-40) in-cell NMR sample immediately after sample preparation. (b) The 1H–15N HSQC spectrum of the supernatant of the in-cell sample...... 60

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Figure 3.3 (a) The 1H–15N HSQC spectrum of the uniformly 15N labeled Aβ (1- 40) in aqueous solution at neutral pH. (b)The 1H–15N HSQC spectrum of the uniformly 15N...... 62

Figure 3.4 (a) The 1H–15N HSQC spectrum of the lysate of the harvested cells after 20 hours NMR measurement. (b) The 1H–15N HSQC spectrum of the nuclease treated lysate of the harvested cells after 20 hours NMR measurement...... 64

Figure 3.5 (a) The 1H–15N HSQC spectrum of the harvested cells for 20 hours NMR measurement. (b) The 1H–15N HSQC spectrum of the harvested cells for 8 hours NMR measurement...... 65

Figure 3.6 (a) The 1H–15N HSQC spectrum of the nuclease treated lysate of the harvested cells after 20 hours NMR measurement. (b) The 1H–15N HSQC spectrum of the nuclease treated lysate of the harvested cells after 0 hour NMR measurement...... 66

Figure 4.1 Scavenging of reactive oxygen species (R•) by flavonoid. The free radical Fl-O• may react with a second radical, acquiring a stable quinone structure. Figure and caption taken from [185]...... 73

Figure 4.2 Pro-oxidant chemistry of flavonoids. (Auto)oxidation of the flavonoid generates the flavonoid semiquinone radical, which may be (auto)oxidized to produce quinone, which may be scavenged by GSH not by means of chemical reduction but rather by conjugate formation. Figure and caption taken from [191] ...... 75

Figure 4.3 Circular Dichroism Spectrum of poly-L-lysine in α-helix, β-sheet and random confirmation. [193]...... 77

Figure 4.4 Scheme of the STD-NMR experiment. In the difference spectrum, only the binding molecules that received saturation transfer from the protein show signals. Non-binding small molecule will not receive any saturation transfer; their signals will be of...... 80

Figure 4.5 Time-dependent Effects of Myricetin (different concentrations) on the β-Aggregation Rates of Aβ (1–42) (25uM). (A) Aβ (1–42) alone. (B) Aβ (1–42) with 25...... 89

Figure 4.6 Representative EM images of Aβ(1-42) aged for (A) 0 h without myricetin, (B) 48 h without myricetin, (C) 0 h with myricetin and (D) 48 h with myricetin at 22°C...... 91

Figure 4.7 Time-dependant Effects of Myricetin (Myr) on the β-Aggregation Rates of Aβ(1–42). A, flow chart summarizing the partitioning of a single Aβ(1–42) solution (25 uM, pH 7.3) into four equivalent fractions. For fractions 1, 2, and 3 the time at which oxidation was commenced is also provided. Each fraction was monitored over time by ThT. B, The ThT fluorescence data shows that Myricetin causes a significant reduction in β-sheet production...... 93

Figure 4.8 SDS-PAGE of Aβ (1-42) aggregates before and after adding Myr. The Aβ (1-42) was incubated at 6, 24, 48 and 72 h (lane A, C, E and G, respectively, labeled with Myr ‘-’). Portion of 6, 24, 48, 72 h aged samples were incubated with Myr (4h) (lines B, D, F and H, respectively, labeled with Myr ‘+’). Molecular standards are indicated in the lane on the left...... 94

Figure 4.9 Hydrogen peroxide formation during Myricetin oxidation. (A)The

kinetics of H2O2 formation during the auto-oxidation of Myricetin in the absence and in the presence of Aβ (1–42). (B) The kinetics of Aβ (1–42) (25µM) fibril formation in the absence and in the presence of Myricetin, by ThT fluorescence assays were shown for comparison...... 96

Figure 4.10 Effect of catalase on Aβ (1–42) fibrillation. Data for the Aβ (1–42) fibrillation in the presence of myricetin and catalase is similar the data of Aβ (1–42) fibrillation in the presence of myricetin...... 98

Figure 4.11 Effect of oxygen on Aβ (1–42) fibrillation. (A) Data for the Aβ (1– 42) fibrillation in the presence of myricetin and oxygen is different (B) the data of Aβ (1–42) fibrillation under anaerobic condition...... 99

H2O:D2O) with 0.50 mM Na2EDTA and 0.05 mM NaN3. On the left, Aβ (1-42) 0 hour (black peaks) and Aβ (1-42) 72 hours were superimposed. The cross-peaks represent intraresidue couplings between the HR and NH. One the right, Aβ (1-42) 0 hour (black peaks) and Aβ (1-42) 8 days (green peaks) were superimposed...... 101

Figure 4.13 Time-dependant effect of myricetin (500µM) on the heteronuclear single quantum coherence (15N HSQC) of uniformly 15N labeled Aβ (1-42) (25 µM, 25 °C, pH 7.3). The 15NH peaks showing different chemical shifts between 0 hour and 72hours/8days are labeled. These data show that myricetin induced the oxidation of the Met35 side chain that promotes shift changes of the Met35-Val36-Gly37-Gly38-Val39 in Aβ (1-42)...... 102

Figure 4.14 Time-dependant heteronuclear single quantum coherence (13C HSQC) of selectively 13C labeled Aβ (1-42) (25 µM, 25 °C, pH 7.3). All the spectra (900 MHz) were obtained at 5 °C using 25 µM samples in buffered (10 mM sodium phosphate, pH 7.3) aqueous solution (9:1,

H2O:D2O) with 0.50 mM Na2EDTA and 0.05 mM NaN3. (A) Aβ (1- 42) 0.5 hour (black peaks) and Aβ (1-42) 72 hours were superimposed. The cross-peaks represent intraresidue couplings between the HR and CH. (B) Aβ (1-42) 0 hour (black peaks) and Aβ (1-42) 8 days were superimposed...... 105

Figure 4.15 Time-dependant effect of myricetin (500µM) on the heteronuclear single quantum coherence (13C HSQC) of uniformly 13C labeled Aβ (1-42) (25 µM, 25 °C, pH 7.3). The 13CH peaks showing different chemical shifts between 0 hour and 72hours/8days are labeled. These data show that myricetin induced the oxidation of the Met35 side chain...... 106

Figure 4.16 Effect of myricetin (500µM) on the electrospray ionization mass spectra (ESI-MS) of Aβ (1-42) (25µM) incubated at the room temperature (25°C) for 8 days...... 107

Figure 4.17 Upfield 1H NMR spectral regions of Αβ40 alone (25 µM, pH 7.2, 5°C) and plus Myr. (A) Reference spectrum of Aβ40 alone obtained with off-resonance irradiation at 30 ppm, (B) STD spectrum obtained by subtracting spectrum in (A) from that obtained...... 108

Figure 4.18 Representative EM images of Aβ(1-42) Met35red (100 µM, pH 7.3) aged for (A) 24 h, (B) 48 h, and (C) 96 h at 22◦ C and after the treatment with H2O2 for 24h (D, E, and F, respectively.). At 24h, the heights were within 2.3–12.5 nm, while at 48 and 96h the average

heights are 16.7 and 24.9 nm. After H2O2 addition and aging for 24 h, the heights became 3.7–7.9 nm...... 113

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ACKNOWLEDGEMENTS

I would like to take this opportunity to thank all the individuals who have supported me during my PhD work.

First and foremost, I would like to sincerely thank Dr. Michael Zagorski for his generous support, guidance and encouragement. He has been a tremendous mentor for me and allows me to grow as a research scientist. I would also like to thank all the members of Zagorski’s group: Lei, Lijun, Megan, Colin for their support and encourgement, especially to Lei for his help with my in-cell NMR project and Lijun for her help with my polyphenol project.

I would also like to thank the Cytometry & Imaging Microscopy Core Facility of the

Case Comprehensive Cancer Center. Dr Mike Sramkoski has introduced me Confocal

Microscopy and Flow Cytometry. I really appreciate his patience for answering my numerous questions.

I would like to thank Dr. Xian Mao for help with NMR experiments and for introducing me to this amazing technique with his ample knowledge and skills. I would like to thank Dr. Hisashi Fujioka and Dr. Midori Hitomi for teaching me EM.

I would like to thank Dr. Barkley ’s group and Dr. Tolbert’s group for their help with

SDS-PAGE experiments and Dr. Solomon’s group for help with sonication cells. I would like to thank Dr. Geneviève Sauvé’s group for teaching me AFM.

I would also like to thank my committee members, Dr. Mary Barkley, Dr. James

Burgess, Dr. Paul Carey and Dr. Hyoung-gon Lee for serving as my committee members

xiii and for their valuable time, efforts and suggestions on my thesis. I would like to thank all people in the Department of Chemistry who helped me during my studies.

I would like to acknowledge NIH for their financial support of this project.

Last, but by no means least, I would like to thank my parents, grandparents and my husband. This work could not be finished without their unconditional love and support.

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LIST OF ABBREVIATIONS AND ACRONYMS

Abbreviations and Acronyms Equivalent

Aβ amyloid β

AD Alzheimer’s disease

APOE apolipoprotein E

APP amyloid precurser protein

CCD charge-coupled device

CD circular dichroism

CHO Chinese hamster ovary

CLSM or LSCM confocal laser scanning microscopy

CPPs cell-penetrating peptides

E.coli Escherichia coli

EGCG epigallocatechin gallate

EM electron microscopy

EMEM Eagle's Minimum Essential Medium

ESI MS electrospray ionization mass spectroscopy

FBS fetal bovine serum

FCM flow cytometry

FPRL1 formyl peptide receptor-like protein 1

FSC forward-scattered light

HBSS Hank’s balanced salt solution

HPLC high performance liquid chromatography

HSQC heteronuclear single quantum coherence

LDD low-density lipoprotein

MALDI-MS matrix-assisted laser desorption/ionization

time-off light mass spectrometry

MCI mild cognitive impairment

MTSs membrane translocating sequences

MW molecular weight

Myr myricetin

NHS N-hydroxysuccinimide

NMDAR NMDA-type glutamate receptor

NMR nuclear magnetic resonance

PBS phosphate buffered saline

PI propidium iodide

PMT photodetector

PrPC prion protein

PTDs protein transduction domains

RAGE receptor for advanced glycation end-

products

ROS reactive oxygen species

SEM scanning electron microscope

SLO Streptolysin-O

SSC side-scattered light

STD saturation transfer difference

Tβ4 thymosin β4

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TEM transmission electron microscope

ThT thioflavin-T

WT wild type

α7nAChR α7 nicotinic acetylcholine receptor

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Amyloid Aβ Peptide: In-Cell Studies and Mechanism of

Polyphenol-Based Inhibition to Aggregation

ABSTRACT

Abstract

FANG HAN

The two major goals of my research work are to obtain in-cell nuclear magnetic resonance (NMR) spectra of the Aβ peptide, and to elucidate the molecular mechanism of the polyphenol-based inhibition to Aβ aggregation.

To characterize the Aβ structure inside living cells, optimum conditions were established to get the Aβ peptide inside living cells using steptolysin-O (SLO) reversible membrane permeabilization and Ca2+ resealing of the target cells. This was confirmed by multi-complementary techniques, including confocal microscopy, flow cytometry, and

NMR. The confocal images demonstrated that Aβ(1-40) was evenly distributed throughout the cell cytoplasm and the nucleus, indicating that the pore formation and resealing occurred properly. To assess the efficiency of the pore formation and resealing, flow cytometry (FCM) analysis was performed. The FCM showed that 100 ng/ml SLO yielded optimal resealing efficiency (86%). By comparing the cells without SLO treatment, FCM results also indicated that the pore formation and resealing occurred properly. Based on the 2D NMR hetero-single quantum coherence (HSQC) spectra of the cells and supernatant with Ala2 and Ala21 15N labeled Aβ (1-40), two peaks were observed from the cell samples, while no signals were observed from the supernatant,

xviii establishing that all of the NMR signals originated from the Aβ protein within the cells.

Next, the HSQC spectrum of the uniformly 15N labeled Aβ(1-40) inside the cells showed only 5 peaks, and none had identical chemical shifts with control spectra. In an effort to elucidate the reasons for the peak disappearances and shifts, cells were lysed and further

NMR studies were conducted with cell lysate. These studies involved HSQC of cell lysate and cell lysate treated with nuclease. Analysis of these spectra showed that the Aβ peptide did not bind to cell membranes during and after delivery into the cells and that the nuclease does not change the viscosity to alter the conformation and/or mobility of the peptide. To examine the influence of survival rate of cells on the structure of Aβ(1-

40) inside cells, NMR experiments were performed for different time spans. Comparison of the results suggested that no obvious changes occurred in the cellular environment during 8-20 hr after the cells were resealed.

In regard to the second project with elucidating the molecular mechanism of polyphenol-based inhibition to the Aβ(1-42) aggregation, circular dichroism (CD), thioflavin-T (ThT), electron microscopy (EM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and NMR were carried out. The CD and EM data established that the polyphenol, myricetin, inhibits Aβ(1-42) aggregation while the ThT and SDS-PAGE data demonstrated that myricetin also disaggregates preformed aggregates. HSQC showed that oxidation of the Met35 sulfur side chain to the sulfoxide occurred in Aβ(1-42) after incubating with myricetin, which was also confirmed by

ESM-mass spectrometry (MS). However, when catalase (an H2O2 scavenger) was present in the mixture, the myricetin-induced inhibition of Aβ(1–42) fibrillation was not altered, suggesting that oxidation of the Met35 side chain by H2O2 was not the only factor to alter

xix

Aβ(1–42) fibrillation. CD experiments carried out under anaerobic conditions suggested that auto-oxidation of myricetin was a prerequisite for the myricetin-induced inhibition of the protein fibril. Moreover, the overlaid HSQC spectrum of selectively 13C-labeled

Aβ(1-42) with and without myricetin is essentially the same, which indicates that Schiff base formation between the quinone section of myricetin, and the Aβ(1-42) did not occur.

However, saturation transfer difference (STD) NMR studies demonstrated that myricetin weakly interacts with monomeric Aβ(1-40).

In conclusion, the first part of this thesis work established optimal conditions for in-cell NMR studies of the Aβ peptides and constitutes the first step for additional work that may determine the structure inside living cells. The second part of this work established that myricetin oxidizes the Met35 side chain of the Aβ peptide which in part inhibits aggregation.

CHAPTER 1

INTRODUCTION

1 Introduction

1.1 Alzheimer’s Disease and Its Impact on Public Health

Alzheimer’s disease (AD) is a common neurodegenerative disease in the world, and accounts for an estimated 60%–80% of cases. AD can be divided into two forms: a familial form (FAD, early onset before 65 years old) and a sporadic late-onset form.

Currently, the three stages of AD are often described as mild/early stage, moderate/mid stage, or severe/late stage. However, the newer criteria describe the three stages of AD as preclinical AD, mild cognitive impairment (MCI) due to AD, and dementia due to AD, because the new criteria propose that AD begins before the mild/early stage and that new technologies have the potential to identify AD-related brain changes that occur before mild/early stage disease.[1] The new criteria[2-4], however, state that additional biomarker research is needed before the preclinical stage of AD can be diagnosed.

The greatest risk factor for AD is advancing age, but AD is not a normal part of aging.[5] Other factors include family history[6], Apolipoprotein E ε4[7], mild cognitive impairment,[2-4] cardiovascular disease risk factors,[8-11] social engagement,[12] diet,[13] head injury and traumatic brain injury[14].

Early symptoms of AD include difficulty remembering names and recent events, as well as apathy and depression. Later symptoms include impaired judgment, disorientation, confusion, behavior changes, and difficulty speaking, swallowing, and walking.[1]

People with developed AD require all aspects of personal care. The following are warning signs of AD(www.alz.org/10signs):

• Memory loss that disrupts daily life.

• Challenges in planning or solving problems.

• Difficulty completing familiar tasks at home, at work, or at leisure.

• Confusion with time or place.

• Trouble understanding visual images and spatial relationships.

• New problems with words in speaking or writing.

• Misplacing things and losing the ability to retrace steps.

• Decreased or poor judgment.

• Withdrawal from work or social activities.

• Changes in mood and personality.

AD is the sixth leading cause of death in the United States and the fifth leading cause of death in Americans age >65 years. In America, 5.4 million people were diagnosed with AD by 2012,[1] including 5.2 million people aged >65 years (Chicago Health and

Aging Project ), and 200,000 individuals aged <65 years who have younger-onset AD.

An additional 10 million people will be diagnosed with AD in the coming decades because of the aging of baby boomers.[1] Today, someone in America develops AD every 68 seconds. By 2050, there is expected to be one new case of AD every 33 seconds, or nearly a million new cases per year, and AD prevalence is projected to be 11 million to

16 million. [1]

140 Alzheimer’s Association / Alzheimer’s& Dementia 8 (2012) 131–168

Fig. 3. Projected changes between 2000 and 2025 in AD prevalence by state. Created from data from Hebert et al. Neurology 2004;62:1645 [79].A10

3.4. Looking to the future As the number of older Americans grows rapidly, so too will the numbers of new and existing cases of AD, as shown The number of Americans surviving into their 80s and in Fig. 4.A11 90s and beyond is expected to grow dramatically owing to advances in medicine and medical technology, as well as so- In 2000, there were an estimated 411,000 new cases of cial and environmental conditions [80]. Additionally, a large AD. For 2010, that number was estimated to be segment of the American population—the baby boomer gen- 454,000 (a 10% increase); by 2030, it is projected to eration—is reaching the age of greater risk for AD and other be 615,000 (a 50% increase from 2000); and by dementias. In fact, the ﬁrst baby boomers reached age 65 2050, it is expected to be 959,000 (a 130% increase years in 2011. By 2030, the segment of the U.S. population from 2000) [76]. aged 65 years is expected to double, and the estimated 71 By 2025, the number of people aged 65 years with AD ! million! older Americans will make up approximately 20% is estimated to reach 6.7 million, a 30% increase from of the total population [81]. the 5.2 million aged 65 years currently affected [47]. !

Fig. 4. Projected numbers of people aged 65 years in the U.S. population with AD using the U.S. Census Bureau estimates of population growth. Numbers indicateFigure middle estimates 1.1 Projected per decade. Shaded numbers! area indicates of people high and low aged estimates >65 per years decade. Createdin the from U.S. data frompopulation Hebert et al. Archwith Neurol AD 2003;60:1119– 22 [47].A11 using the U.S. Census Bureau estimates of population growth. Numbers indicate middle

estimates per decade. Shaded area indicates high and low estimates per decade. Figure

and caption taken from [15]

Aggregate payments for health care, long-term care, and hospice are projected to

increase from $200 billion in 2012 to $1.1 trillion in 2050 (in 2012 dollars). (www.

alz.org/trajectory.) Moreover, more than 15 million Americans provide unpaid care for a

person with AD or other dementias.[1] Unpaid caregivers are primarily family members,

but they also may be other relatives and friends. In 2011, these people provided an

estimated 17.4 billion hours of unpaid care, a contribution to the nation valued at more

than $210 billion. [1]

1.2 Causes of AD

The cause or causes of AD are not yet known. It is believed that AD is a multifactorial

syndrome rather than a single disease.[16] Most experts agree that AD, like other

common chronic diseases, develops as a result of multiple factors rather than a single

4 cause. One important factor is a variety of brain changes, including Amyloid beta peptide, plaques, Tau protein, tangles, neurotransmitter deficiencies, dysfunction in brain cell communication, effects of inflammation and oxidative stress.

Another known cause for AD is genetic mutations. A small percentage (<1%) of AD cases are caused by three known genetic mutations and inheriting any of these genetic mutations guarantees that an individual will develop AD. In such individuals, the disease tends to develop before age 65 years, sometimes in individuals as young as 30 years.(www.alz.org)

1.3 Treatments for AD

The U.S. Food and Drug Administration (FDA) has approved five medications (Table

1.1) to treat the symptoms of AD. The effectiveness of these drugs varies across the population, and none of the treatments available today alters the underlying course of this terminal disease. However, researchers around the world are studying dozens of treatment strategies that may have the potential to change the course of the disease. [1]

Table 1.1 Food and Drug Administration (FDA) approved AD drug. http://www.alz.org/research/science/alzheimers_disease_treatments.asp

1.4 Amyloid beta (Aβ) peptides and AD

1.4.1 Introduction

AD is named after Alois Alzheimer, who over 100 years ago first described the symptoms, the presence of neurofibrillary tagles and extracellular deposits of senile plaques in the brain and blood vessels of his patient Auguste D.[17] Ultrastructural observations have revealed that within the neurofibrillary tangles are paired helical filaments [18] and within the core of senile plaques are the closely packed, radiating fibrils [19, 20].

Based on extensive research, it has been determined that the major chemical constituent of senile plaques is Aβ peptide [21] and the principal component of fibrillary tangles is the microtubule-associated phosphoprotein tau [22, 23]. It is thought that the plaque deposits in AD are caused by progressive aggregation of the Aβ to form oligomers, protofibrils and fibrils, and that these aggregated Aβ species contribute to neurotoxicity

[24, 25] and that Aβ peptide is considered an important pathologic marker of AD.[26]

Figure 1.2 Sequence of the Aβ(1-40) and Aβ(1-42). It contains two hydrophobic domains comprising residues 17-21 and 29-40/42.

1.4.2 Biogenesis of Aβ

Aβ is cleaved from the C-terminal region of a much larger, ubiquitously-expressed, type I membrane-spanning glycoprotein, the amyloid precurser protein (APP), whose function remains unknown.[27] The APP is normally cleaved by an enzyme called “α- secretase” by a pathway that does not lead to the production of Aβ. However, abnormal cleavage by two other “secretases”, at the N-terminal end by β -secretase and at the C- terminus, by γ-secretase yields Aβ.[28] The cleavage site of γ-secretase is not precise and

Aβ peptides of lengths 39 to 43 are formed.

Aβ is a 4kDa polypeptide and varies in length (39 to 43 residues), with the heterogeneity at the carboxyl terminus. The Aβ (1-40) and Aβ (1-42) (in a ratio of about

10:1) are the two predominant species that exist in the senile plaques, and their sequences are shown in Figure 1.2.

1.4.3 Aβ Aggregation and Neurotoxicity

The “amyloid cascade” hypothesis (Figure 4) suggests that accumulation and aggregation of the Aβ is

Figure 1.3 Amyloid cascade hypothesis of AD. Figure and caption taken from [29]

The primary cause of AD, inducing an inflammatory response followed by neuritic injury, hyperphosphorylation of tau protein and formation of fibrillary tangles, eventually leading to the neuronal dysfunction and cell death.[30] This hypothesis is supported by the observation that mutations, which increase the aggregation propensity of Aβ, cause early onset forms of AD. The APP gene resides on chromosome 21. Down’s syndrome people, who have an extra copy of chromosome 21, will almost invariably have contracted AD by the time they are 40 years of age[31] and express APP level approximately 1.5 times that of normal individuales[32]. Total Aβ is distributed in an

AD brain at a concentration of about 31 µg /g, compared to 2 µg /g in age-matched non-

AD brains.[33] Plasma and cerebrospinal fluid levels of Aβ (1–42) are about 20 pg/ml

[34] and 800pg/ml, [35] respectively. However, both of the levels are lower in individuals with AD.

Although Aβ is predominantly produced at plasma membranes and extruded into the extracellular space where it is deposited as the senile plaques that are characteristic of

AD,[27] it is also produced in membranes within neurons and is extruded into the cytosol.[36] There is a dynamic equilibrium between intra- and extracellular Aβ, at least in part by active transport via attachment to the a7 nicotinic acetylcholine receptor

(a7nAChR) [37]and the receptor for advanced glycation end products.[38]

As a monomer, Aβ is generally thought to be inherently nontoxic to neurons or neuron-like cell lines. Actually, Aβ, at low concentration, is a normal constitution of human biological fluids. Under certain conditions, Aβ will self aggregate and become neurotoxic. Based on their morphology, Aβ aggregates can be classified as fibrillar and amorphous and both forms exist in AD brains.

Which form, however, plays more significant roles in the production of dementia?

Insoluble amyloid β-peptide (Aβ) fibrillar aggregates found in extracellular plaques have long been thought to cause the neurodegenerative cascades of AD. However, the number of senile plaques in a particular region of the AD brain correlates poorly with the local extent of neuron death or synaptic loss, or with cognitive impairment.[39] Present studies established that the soluble Aβ oligomer levels correlate robustly with the extent of synaptic loss and severity of cognitive impairment.[36, 40] It has also been reported that soluble Aβ oligomers inhibit many critical neuronal activities, including long-term potentiation (LTP), a classic model for synaptic plasticity and memory loss in vivo and in culture [25, 41, 42] Thus, strong evidences suggest that soluble Aβ oligomers are the

9 causative agents of AD. The term ‘soluble’ refers to any form of Aβ that is soluble in aqueous buffer and remains in solution after high-speed centrifugation. [39] Aggregates ranging from dimers to 24-mers, or even those of higher molecular weight (MW), have been reported as Aβ oligomers. [40, 43, 44]

The mechanism of formation of soluble Aβ oligomer in vivo remains unclear.

However, it has been proposed that the oligomer formation mechanism is complicated since multiple Aβ oligomer conformations were produced via different pathways. [43]

The mechanisms of formation may also be different for extracellular and intracellular oligomers. [39] The proposed mechanisms for extracellular and intracellular oligomers are shown in Figure 1.4 and Figure 1.5. For extracellular mechanism, Aβ is released extracellularly as a product of proteolytically cleaved amyloid precursor protein (APP). It has been suggested that extracellular Aβ oligomers toxicity is induced by several receptors. Besides interacting with receptors, Aβ oligomers induce toxicity also by forming the membrane pores, allowing abnormal flow of ions, such as Ca2+, which causes cellular dysfunction. [39]

Figure 1.4 Formation and toxicity mechanisms of extracellular Aβ oligomers. Picture and caption taken from [39]

For intracellular oligomer toxicity mechanism, Aβ can be localized intracellularly by the uptake of extracellular Aβ or by the cleavage of APP in endosomes generated from the ER or the Golgi apparatus. The presence of GM1 ganglioside meditates the oligomers formation on the cell membrane and induces Aβ oligomer-induced neuronal cell death with the help of nerve growth factor (NGF) receptors. The aB-crystallin and ApoJ meditate the production of toxic non-fibrillar Aβ. A cellular prion protein (PrPC) acts as an Aβ oligomer receptor with nanomolar affinity, mediating synaptic dysfunction.

Furthermore, Aβ oligomers form the membrane pores and allow abnormal flow of ions, such as Ca2+, which causes cellular dysfunction. Binding of Aβ oligomers to the

NMDA-type glutamate receptor (NMDAR) also causes abnormal calcium homeostasis and thus increased oxidative stress and synapse loss. Frizzled (Fz) receptor can inhibit

Wnt signaling, leading to cell dysfunctions such as tau phosphorylation and neurofibrillary tangles, by binding with Aβ oligomers. Moreover, Aβ oligomer can induce insulin receptor loss. [39]

Figure 1.5 Formation and toxicity mechanisms of intracellular Aβ oligomers. Picture and caption taken from [39]

Aβ can be localized intracellularly by the uptake of extracellular Aβ or by the cleavage of APP in endosomes. Extracellular Aβ is internalized through various receptors and transporters, such as formyl peptide receptor-like protein 1 (FPRL1) or scavenger receptor for advanced glycation end-products (RAGE). These receptor-Aβ complexes are internalized into early endosomes. Most Aβ in the endosome is degraded by the endosome ⁄ lysosome system. However, Aβ in the lysosome can leak into the cytosol by destabilization of the lysosome membrane. The cytosolic Aβ can be degraded by the

12 proteasomal degradation system, but the cell death caused by Aβ oligomers inhibits the proteasome function. Suppression of protein aggregation by interactions with various cellular proteins, may cause the formation of Aβ oligomers.

1.4.4 Fibrillization of Aβ

The predominant structure that pathological Aβ peptides adopt is β-sheet conformation, because the hydrogen bonding in β-sheet is formed between on strand and another, which can come from a different region or the same molecule or from a different molecule. This structure feature, therefore, promotes the protein oligomerizaiton and aggregation, by allowing the formation of β-pleated sheets. In α-helix, however, the hydrogen bonding is formed within the same strand between residues.

Synthetic Aβ peptides adopts different conformation depending on different solution conditions, such as solvent pH and solvent properties. [45] The Aβ monomer favors a α- helix structure in a membrane or membrane-mimicking environment such as ionic detergents. [45-48] In contrast, Aβ adopts mainly a random coil with few components of

α-helix and or β-sheet conformations in aqueous soluble state. [49-53] However, it has been indicated by x-ray diffraction measurements on oriented fibril bundles that an extended β-sheet structure for Alzheimer’s β-amyloid fibrils, with peptide chains in β- strand conformations running approximately perpendicular to the fibril axis and hydrogen bonds between peptide chains in each layer occurring roughly parallel to the fiber axis.

[54, 55]

These different dominant structure of Aβ in different surrounding solutions leads to a hypothesis that conformational conversion from an α-helix or random coil to a β-sheet

13 occurs during (or before) the aggregation of Aβ. [56-58] In addition, recent SS-NMR and

EM data show that a spherical Aβ, β-sheet intermediate of 15–35 nm is neurotoxic, and that its toxicity decreases with a transition to a fibrillar form. [59] In another word, the neurotoxicity was the result of Aβ undergoing a conformational transition to a predominantly β-sheet structure accompanied by peptide aggregation. Proteins of other diseases, such as α-Synuclein of Parkinson’s disease and PrPSC of Prion Diease, undergo similar conformational conversions. [60-62] Thus, a rigorous study of how to impede or slow the conformational transition is critical to the development of therapeutic approaches for the conformational disorders in general, and AD in particular. [63]

1.4.5 Possible Factors for Aβ Self-aggregation

It has been proposed that the accumulation of Aβ might result from the increase of production, aggregation, or from its decreased clearance. Increased production of Aβ is a feature of early onset AD while late onset AD cases is characterized by reduced degradation of Aβ by neprilysin and insulin-degrading enzyme[64, 65] and reduced perivascular drainage [66] which might elevate the levels of Aβ in the AD brain, though it does not show signs of increased Aβ production.

Table 1.2 Proposed factors for Aβ self-aggregation. Picture and caption taken from [67]

A considerable number of environmental factors as well as some intrinsic properties of

Aβ that can work in concert to cause amyloidogenesis and cause AD are listed in Table

1.2. [67] These factors can influence the thermodynamic stability of the various accessible conformations of the protein potentially causing amyloidogenesis. [67]

Although, protein aggregation and amyloid formation have been thought to be cytotoxic, recent studies have identified novel biological functions for amyloidogenic protein fibrils in bacteria, fungi and even mammals.[68] More and more studies have shown that rather than amyloidogenic aggregates, the oligomeric intermediates could be the toxic entities.[69, 70]

1.4.6 Structure of Aβ Oligomers

A growing body of literature has implicated the prefibrillar oligomers, and not the fibrillar form, as the primary pathological species of AD. [25, 71, 72] Though Aβ oligomers in intracellular environment have been shown to cause neuronal dysfunction, disrupt long-term potentiation and impair memory in mouse models of AD, considerably limited is known about the molecular structure of Aβ oligomers. [25, 41, 71]

Several types of natural and synthetic Aβ oligomers of different sizes and shapes have been reported. [39] SDS-stable dimers and trimers have been found in the soluble fractions of human brain and amyloid plaque extracts, which suggests that these low-MW

Aβ oligomers could be the fundamental building blocks of larger oligomers or insoluble amyloid fibrils. [73-75] It has also been reported that Aβ dimers are threefold more toxic than monomers, and that Aβ tetramers are 13-fold more toxic. [76] Moreover, nonamers and dodecamers have been suggested to be associated with deleterious effects on cognition. [25] However, it is unlikely that these oligomers alone cause brain dysfunction.

[39] Actually, natural Aβ oligomers with a wide-ranging MW distribution (from < 10 kDa to > 100 kDa) have been found in the AD brain. [77]

There are also many reports of toxic oligomers from synthetic Aβ, which forms fibrillar aggregates similar to AD plaques in the AD brains. The formation of soluble Aβ assemblies has been detected by using an analytical ultracentrifugation technique. [78]

Globular oligomers [79], doughnut-like oligomers [25], and disc-shaped pentamers [71] have been reported and suggestions of how they correlate with AD were made respectively. It is also demonstrated that oligomers having heights of 4–5 nm can be reduced to heights of 2–3 nm in the presence of designed peptide inhibitors that bind the

16 hydrophobic C terminus of Aβ (1-42) and inhibit neurotoxicity. [80] These studies suggest that the pentamer and hexamer oligomers may be the building blocks of the more toxic decamer and dodecamer complexes. [71]

1.4.7 Aβ (1-42) and Aβ (1-40)

The two predominant species of Aβ peptides are Aβ (1-42) and Aβ (1-40). One of the major puzzles in the AD field has been how the difference of two residues between Aβ

(1-42) and Aβ (1-40) can so markedly change the toxicity and aggregation properties of the peptide. It has long been proposed that Aβ42 has a greater propensity to aggregate owing to two additional hydrophobic residues (Ile41 and Ala42) at the C-terminus (Jarrett et al., 1993). However, the actual mechanism of how the small structural difference between Aβ40 and Aβ42 (the C-terminal Ile-Ala sequence) causes the significantly different behaviors in vitro and in vivo remains unknown. Recent structural studies have hypothesized that the hydrophobic C-terminal residues in Aβ(1-42) stabilize the neurotoxic low-order oligomers in a non-sheet secondary structure and that the conversion to protofibrils and fibrils having β-sheet secondary structure reduces toxicity.

[71]

1.5 Objective and Significance of the Research

In this thesis, a new method to introduce Aβ peptides into cells and obtain its in-cell

NMR spectra is established and confirmed by Confocal Microscopy and Flow

Cytrometry. The NMR spectra indicate binding of the Aβ with other macromolecules in cells. The optimum condition of introducing AB into cells has been established and these

17 results suggested the feasibility of structural analysis of Aβ (1-40) in HeLa cells by NMR.

The second project showed that a wine related polyphenol, myricetin, is able to inhibit conformation conversion and fibril formation. The structural properties of Aβ (1-42) were investigated in aqueous solution at neutral pH by NMR, circular dicroism (CD),

Thioflavin T (ThT), Electron Microscopy (EM), and sodium dodecyl sulfate

Polyacrylamide gel electrophoresis (SDS PAGE). The results established that the oxidation of Met35 by myricetin and weak interactions between Aβ and myricetin inhibit the conformation conversion. These studies can serve as a model for the development of therapeutic drugs in combating Alzheimer’s disease.

CHAPTER 2

STUDIES OF INTRODUCTION OF Aβ PEPTIDE INSIDE THE LIVING CELLS

2 Studies of Introduction of Aβ Peptide Inside the Living Cells

2.1 Introduction

2.1.1 Significance of Biophysical Studies of Peptides Within Cells

In many ways, the importance of studies of peptides and proteins inside cells are obvious, in that it allows them to be studied “at work” in a native and living environment.

The cellular environment is difficult to replicate in vitro [81], because in living cells, proteins function in an environment in which they interact specifically with other proteins, nucleic acids, co-factors and ligands, and are subject to extreme molecular crowding [82].

No one could deny the valuable contribution of in vitro methods of structure determination to understanding the functions of many proteins; however, in vivo observations of 3D structures, structural changes, dynamics or interactions of proteins, are the prerequisites for the explicit understanding of the structural basis of their functions inside cells.[81] NMR spectroscopy is ideally suited for the task, due to its non- invasive character and ability to provide data at atomic resolution.[81] Indeed, so-called in-cell NMR has been used to study proteins inside living cells by heteronuclear multidimensional NMR due to improvements in instrumentation and the related sensitivity. [81,

83, 84]

Another important application of delivery peptides into cells is to develop therapeutic strategy for a variety of human diseases. In Eukaryotic cells, several thousand proteins have been selected to play specific roles in maintaining virtually all cellular functions.[85]

Correct expression of these proteins then, is tremendously important to every cell and the organism as a whole. Mutations or deletions in the amino acid sequence, or through changes in expression to cause overexpression or suppression of protein affect a particular protein’s function, and invariably lead to abnormal cellular function. In the end,

20 these undesired modifications lead to a wide variety of genetic and acquired disorders.

Consequently, manipulating cell biology at the protein level, without the use of DNA based methods, would provide us with a powerful tool for understanding and affecting complex biological processes and would likely be the basis for the treatment of a variety of human diseases including cancer.[85] For instance, the reconstitution of tumor- suppressor function following the mutation or deletion of tumor-suppressor proteins, such as p53, in cancer therapy or the replacement of defective proteins in genetic disease such as cystic fibrosis or Duchene’s muscular dystrophy is often considered the goal of effective treatment.[86]

The poor permeability of the plasma membrane of eukaryotic cells constitutes the major barrier for the introduction of peptides into cells. Several strategies, such as microinjection[87, 88], overexpression, electroporation[89, 90], and liposome and viral- based vectors have been developed to permit the introduction of macromolecules across cellular membranes. However, these methods have different drawbacks, including low efficiency, poor specificity, high toxicity, and penurious bioavailability, and limit their applications.[91] Recently, cell-penetrating peptides (CPPs) or protein transduction domains (CPDs) have emerged as an alternative strategy to traverse the impermeable phospholipid bilayer of the cell membrane, in a manner of low toxicity and low specificity. [92]

2.1.2 Importance of In-cell Studies of the Aβ peptides

The extracellular deposition of the Aβ peptides as amyloid plaques is one important neuropathological feature of Alzheimer’s disease (AD) [93] and this has long been thought to be a primary cause of Alzheimer’s disease (AD). However, detection of

21 intracellular neuronal Aβ(1–42) accumulation before extracellular Aβ deposits questions the relevance of intracellular peptides in AD. The intracellular Aβ builds up in two ways: a portion of the Aβ generated in the cell is not secreted, and consequently remains intracellular [94-98], and alternatively, secreted Aβ is taken back up by cells and internalized to form intracellular pools.[99-101]

In AD brains, the intracellular Aβ(1–42) accumulates as aggregates or granules in the cytoplasm of neurons (Gouras et al., 2000; D’Andrea et al., 2001). The accumulation of

Aβ precedes the appearance of senile plaques [102, 103] and is observed in regions affected early in AD: the hippocampus and entorhinal cortex [104]. It has been demonstrated by numerous studies that intraneuronal Aβ accumulation results in synaptic dysfunction [105-111].

Notably, intraneuronal Aβ(1-42) is observed in the brains of young (3–4 years old)

Down’s syndrome patients, years before plaque development [112]. Similarly, synaptic loss and intraneuronal Aβ were observed before the deposition of Aβ plaques and formation of tangles in transgenic mice [72, 102, 109, 113, 114]. Interestingly, the transgenic expression of Aβ(1-42) targeted to the lumen of the endoplasmic reticulum resulted in intraneuronal accumulation and caused significant neuro-degeneration [115].

Taken together, these studies suggest that intraneuronal Aβ(1-42) may be considered as an early pathogenic event preceding the formation of extracellular plaques [116].

Aβ binds to proteins, lipids and DNAs [117]. Actually, it is through interacting with various receptors and transporters that Aβ can be taken up by cells and internalized into intracellular pools and can induce early pathogenic events. One example is that that Aβ binds to the α7 nicotinic acetylcholine receptor (α7nAChR) with high affinity, resulting

22 in receptor internalization and accumulation of intracellular Aβ [37, 118]. Several studies have shown that apolipoprotein E (APOE) receptors, one of the low-density lipoprotein

(LDD) receptors, modulate Aβ production and cellular uptake [99], by directing binding between Aβ and LDL receptor-related protein or through ligands such as APOE [99]. It has also been reported that the binding of Aβ to RAGE in neurons initiated a cascade of events that produces oxidative stress and nuclear factor- κB (NF-κB) activation, inducing the production of macrophage colony-stimulating factor and an enhanced microglial response [38, 119].

On the other hand, accumulation of Aβ has been observed in mitochondria [120, 121], resulting mitochondrial dysfunction accompanied by energy reduction and an elevated production of reactive oxygen species (ROS). Aβ monomers and oligomers have been observed in transgenic mice and AD brains. Injection of Aβ into cells caused severe axonal damage which is accompanied by the entrance of Aβ into the cell, alterations in the ionic gradient across the inner mitochondrial membrane which is accompanied by disruption of subcellular structure, oxidative stress, and a significant reduction in both the respiratory control ratio and in the hydrolytic activity of ATPase. [120, 122] It has also been demonstrated that voltage-dependent anion channel 1 protein VDAC1 interacts with

Aβ and may in turn block mitochondrial pores, leading to mitochondrial dysfunction in

AD pathogenesis. [123]

23 limited to cytotoxicity studies with mostly mutant transgenic mice, antibodies and in vitro approaches. No studies have been performed to elucidate the in-cell structure of the Aβ monomer and oligomers, and understanding these early events associated with β- amyloidosis is especially important since many recent reports suggest that early-formed, soluble β-sheet aggregates are neurotoxic [25, 44]. A major advantage of the NMR approach is that it can provide atomic level aspects of the structures and dynamics in solution that are not available with other low-resolution techniques.

2.1.3 Methods to Deliver Peptides Into Cells

2.1.3.1 Streptolysin-O (SLO) Permeabilization

SLO is a thiol-activated, membrane-damaging protein toxin with molecular weight of

69,000 and is produced by most strains of β-hemolytic group A streptococci. SLO is a native, primarily water-soluble toxin molecule and binds to cholesterol-containing target membranes to assemble into supramolecular curved rod structures to form rings and arcs that penetrate into the apolar domain of the bilayer. [127] Electron microscopic analyses of toxin polymers in their native and reconstituted membrane-bound form indicate that the convex surface of the rod structures is a hydrophobic, lipid-binding domain, whereas the concave surfaces appear to be hydrophilic.[127] Large transmembrane slits or pores of up to 35-nm diameter are generated by the embedment of the rings and arcs and can be directly visualized by negative staining and freeze-fracture electron microscopy.[128]

The pore formation by SLO can be used to reversibly permeabilize adherent and non- adherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol.

105–106 molecules were induced to each cell using FITC-labeled albumin[128], and Ca2+ was used to repair the lesions on intact microtubules.

Methods for delivering proteins to the cytosol under retention of cell viability are scarce. SLO provides a new and simply method bring exogenous proteins into living cells.

It is applicable to any kind of cell and does not require any special equipment or artificial modification of the molecule of interest. Reversible permeabilization by SLO has been used to obtain the in-vivo NMR structure of thymosin β4 (Tβ4).[129]

2.1.3.2 Protein Over-expression

Expression systems are normally referred to the host and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria

(such as E.coli, B. subtilis), yeast or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses, plasmids, artificial chromosomes and bacteriophage.

The best expression system of choice depends on the gene involved.

Escherichia coli (E.coli) is one of the most widely used hosts for production of heterologous protein. The genetic of E.coli is better characterized than those of any other microorganisms. Also, bacterial systems remain most attractive due to low cost, high productivity, and rapid use.[130] However, bacteria, as prokaryotes, are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. As a result, multi-domain eukaryotic proteins expressed in bacteria often are non-functional.[131] Also, many proteins become insoluble because inclusion bodies are very difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding procedures.

Therefore, there is no guarantee that the recombinant gene product will accumulate in

E.coli at high levels, in a full-length and biologically active form. If the posttranslational modification is essential for bioactivity, alternative hosts such as yeasts, filamentous

25 fungi, or insect and mammalian cell cultures are available for this application.[130] For example the Saccharomyces cerevisiae is often preferred for proteins that require significant posttranslational modification and Insect or mammal cell lines are used when human-like splicing of the mRNA is required.

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Many of these applications involve complex glycoproteins and antibodies with relatively high production needs. [132] The development of a variety of improvements in protein expression technology, particularly involving mammalian and microbial culture systems has been driven by these needs. The majority of therapeutic proteins have been produced in either mammalian cell-culture systems, with Chinese hamster ovary (CHO) cells representing the most common system, or in Escherichia coli.

[133, 134]

Also, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance

(NMR) experiments for structure determination. For example, overexpression in E.coli system was used to calculated 3D protein structure exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. [81] The instability and low sensitivity of living E. coli samples was solved by rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned. [81]

2.1.3.3 Microinjection

Microinjection is a process of using a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The recipient is often a living cell and intercellular space, like cytosol, nuclei, and mitchodial. The use of microinjection as a biological procedure began in the early twentieth century, though even through the 1970s it was not commonly used. By the 1990s, however, its use had escalated significantly and it is now considered a common laboratory technique for introducing a small amount of a substance into a small target.

One of the most widely used recipient cells for microinjection is Xenopus oocytes, which have long served as important laboratory tools in the disciplines of developmental and cellular biology. For example, isolated oocytes in an in vitro setting can be executed by the external addition of hormones, which renders this system an important laboratory tool for studying signaling events during cell cycle progression. [84] Another example is that cellular extracts from Xenopus oocytes or from Xenopus eggs are easily obtained in a virtually undiluted form and similarly recapitulate most of the intact cells’ biological activities. They are frequently used as alternative, cell-free systems for ex vivo analyses of cellular processes.[135] Moreover, X. laevis oocytes were used to provide a detailed structural analysis of the conformational in vivo properties of a small, biologically inert protein domain by in-cell NMR.[83]

Stage VI oocytes are conveniently manipulated by microinjection, which permits the direct deposition of defined quantities of exogenous compounds into the cellular environment. Sophisticated setups and protocols for manipulating Xenopus oocytes have been devised over the years. These devises include a fully automated injection setup[136],

27 which is used to routinely introduce labeled protein samples into large numbers of

Xenopus oocytes for in-cell NMR measurements.

2.1.3.4 Cell-Penetrating Peptides

The poor permeability of the plasma membrane of eukaryotic cells constitutes the major barrier for the introduction of peptides into cells. In 1988, the HIV TAT transactivating factor was discovered,[91] and a few years later, the Drosophila

Antennapedia transcription factor [137] proteins were shown to be able to translocate cell membranes and enter cells. Later, short sequences of these proteins including the 16-mer peptide derived from the third helix homeodomain of Antennapedia (later named penetratin) and the 11-mer derived from TAT protein, were found to have membrane- crossing properties. Based on TAT and penetratin, a new type of molecular vector able to promote the delivery of a variety of cargos: cell-penetrating peptides (CPPs), was developed. [91] Generally, CPPs are short peptides (generally not exceeding 30 residues) that are able to ubiquitously transverse cellular membranes with low toxicity and low specificity, which is caused by chiral recognition by specific receptors.[92] The mechanisms could be either energy-dependent and/or independent. Most common CPPs are positively charged peptides.[92]

Cell-Penetrating Peptides (CPPs), also known as protein transduction domains (PTDs), membrane translocating sequences (MTSs), and Trojan peptides are short peptides (≤40 amino acids), with the ability to gain access to the interior of almost any cell by means of various mechanisms, primarily endocytosis and direct translocation across the plasma membrane, and also are able to facilitate the intracellular delivery of covalently or

28 noncovalently conjugated bioactive cargos (including nucleic acids, proteins, low molecular weight drugs and even 200 nm liposomes.) [91]. They are highly cationic and usually rich in arginine and lysine amino acids. The main characteristics of the CPPs are low cytotoxicity, their ability to be taken up by a variety of cell types, dose-dependent efficiency, and no restriction with respect to the size or type of cargo.[138]

2.1.4 Basic principles of Techniques Related to Peptides Introduction

2.1.4.1 SLO Permeabilization

To introduce Aβ (1-40) protein into the target cells, we utilized the bacterial toxin

SLO, which can form a 35 nm diameter pore on the cholesterol-containing plasma membrane that allows the entry and exit of molecules with sizes up to 150 kDa. Since the pore formed by SLO can be repaired by the addition of Ca2+ into the cytosol, the SLO- permeablized cells can be resealed after they assimilate the molecule of interest2.

Although the SLO permeabilization and resealing methods have previously been used for the incorporation of small amounts of proteins or nucleotides into many kinds of cells, sample preparation on the Aβ (1-40) is unprecedented.

2.1.4.2 Confocal Microscopy

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity.[139] The most important feature of a confocal microscope is that it could isolate and collect a plane of focus for a sample and thus eliminate the out of focus “haze” normally seen with a fluorescent sample (Figure

2.1). This advantage is due to the fact that confocal microscope uses one or more small

29 apertures through which the light pass to remove out-of-focus haze, resulting a thin, highly focused plane. The light from this focused plane can be digitized and stored on a computer. When the distance between the specimen and the microscope objective is changed, a new focal plane is formed, digitized and stored. The computer can reconstruct the whole specimen as a three-dimensional volume after a series of planes has been collected. [139] Most confocal microscopes use an intense laser light to scan the specimen because this intense light source is necessary to compensate for the light loss, which occurs as the light passes through small apertures.

To understand how Confocal Microscopy works, fluorescence and fluorochromes must be introduced. Fluorochromes are dyes, which accept light energy (e.g. from a laser) at a given wavelength (exitation) and re-emit it at a longer wavelength (emission). The process of emission follows extremely rapidly, commonly in the order of nanoseconds, and is known as fluorescence. Fluorescent probe, such as a fluorochrome-conjugated antibody, can directly target an epitope of interest and to allow its biological and biochemical properties to be measured more easily by the facilities like flow cytometer or fluorescent microscope.[128, 139] The specific fluorescent probe I used in my experiment is DyLight 488- Aβ (1-40). Fluorescent probes widely used in identifying and quantifying distinct populations of cells, cell sorting; immunophenotyping; calcium influx experiments; determining nucleic acid content; measuring enzyme activity, and for apoptosis studies. Several parameters of the sample can be analyzed at one time by using more than one fluorochrome.

DyLight 488 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of

30 lysine residues to form stable amide bonds. The DyLight 488 Amine-Reactive Dye is an

NHS ester-activated derivative of high-performance DyLight 488 dye and is able to label antibodies and other proteins to be used as molecular probes for cellular imaging and other fluorescence detection methods. DyLight 488 has high fluorescence intensity over a broad pH range (pH 4-9) and high water solubility and is more photostable than traditional fluoresein-based dyes like FITC, Cy2*, and Alexa Fluor 488* in many applications. DyLight 488 has wide application in fluorescence microscopy, flow cytometry, Western blotting, ELISA, high-content screening and other array platforms.

(http://www.piercenet.com/guide/overview-dylight-488-fluorophore)

Figure 2.1 Schematic representation of principle of Confocal Laser Scanning Microscopy. http://sbio.uct.ac.za/Webemu/training/EM_for_biologists/clsm/

2.1.4.3 Flow Cytometry

The physical and chemical properties of cells or cellular components can be measured by flow cytometry (FCM), in which cells are measured individually but in large numbers.

Unlike microscopists visualize cells based on their morphology and staining characteristics, flow cytometrists measure cells based on similar characteristics. Hence, cells can be measured both qualitatively and quantitatively by flow cytometry. Flow cytometry is a rapid technique; measurements are typically made at rates of 1000 cells.s-1.

[140] This means that many thousands of cells can be measured in a realistic time scale.

There are four general components of a flow cytometer: fluidics, optics, detectors and electronics (Figure 2.2). The cell sample is injected into a stream of sheath fluid. The cells in the sample are accelerated and individually pass through a laser beam for interrogation.[140]

Figure 2.2 Schematic FCM. http://qbab.aber.ac.uk/aberinst/mcytmain.html

Light emitted from the interaction between the cell particle and the laser beam is collected by lens. The light moves through a system of optical mirrors and filters.

Specified wavelengths are then routed to optical detectors. Because more than one laser is focused on the sample stream in modern flow cytometry, cells can be measured based on not only their size and internal complexity but also their fluorescent signal intensities.

The laser beam excites the fluorochrome at a specific wavelength (absorption) and the fluorochrome emits light at a separate wavelength (emission). The emission wavelength of a fluorochrome is optically separated from other confounding light by optical filters, and then is detected by a photodetector (PMT), generating an electrical pulse. An electrical pulse (the voltage pulse) is then processed by the signal processing electronics of the flow cytometer. When a cell passes through the laser beam, it deflects incident light (Figure 2.3). Forward-scattered light (FSC) is proportional to the surface area or size of a cell. Side-scattered light (SSC) is proportional to the granularity or internal complexity of a cell. Live cells and dead cells or subcellular debris have different physical characteristics and thus can be distinguished from each other by size, estimated by forward scattered light. Also, dead cells have higher side scattered light. [140]

Figure 2.3 Schematic representation of forward-scattered light (FSC) and side-scattered light (SSC). Figure and caption taken from http://qbab.aber.ac.uk/aberinst/mcytmain.html

An important principle of flow cytometry data analysis is to select the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating. Figure 2.4A is the schematic representation of SSC versus

FSC histogram while Figure 2.4B represents one real graph for SSC versus FSC histogram. Fluorescence is typically given to a cell by using fluorescent dyes, like FITC,

PI, DyLight. The schematic and real dual-color fluorescence histogram presented below as Figure 2.5 A and B respectively. Cells in region1 (R1) are those with negative fluorescent signals. Cells in the R2 are those with only PI signals. Cells in R3 have both

PI and DyLight signals. Cells in R4 have only DyLight signals.

Figure 2.4 (A) Schematic representation and (B) real graph of FSC versus SSC histogram. (A) Gating cells in different regions could help us calculate viability of cells.

(B) Cells inside of the circle are considered as live or normal while the others are considered as dead or abnormal.

Figure 2.5 (A) Schematic dual-color fluorescence histogram. Cells in region1 (R1) are those with negative fluorescent signals. Cells in the R2 are those with only PI signals.

Cells in R3 have both PI and DyLight signals. Cells in R4 have only DyLight signals. (B)

Real dual-color fluorescence histogram.

2.2 Materials and Methods

2.2.1 Experimental Design

2.2.2 Cell Preparation

HeLa cells were stored in an incubator at 37°C and 5% CO2 and maintained in culture flasks (Corning, Catalog # 430641). Media was prepared by combining 500 mL Eagle's

Minimum Essential Medium (EMEM), 50 mL Fetal Bovine Serum (FBS) and 5 mL

Penicillin streptomycin. The stock solution contains 10,000 units of penicillin-G, and

10mg of streptomycin per mL. The final concentration in our media is ~ 100U/mL penicillin and 100ug/mL streptomycin. All the reagents above are commercially available at ATCC.

To split cells, cell media, Dulbecco’s Phosphate Buffered Saline (Ca+2 and Mg+2 - free salt solution, (PBS), ATCC), and trypsin were brought to room temperature. Cell media was aspirated off and washed with 5 mL PBS. PBS was removed by aspiration. 2 mL of

Trypsin/EDTA was added to just cover the surface of the dish. Cells were returned to incubator for ~2 minutes and 8 mL of cell media was added to the dish. The FBS quenched the trypsinization. Media was pipetted up and down and was squirted around

37 the surface of the dish to remove adherent cells from the surface. 1 mL of cells above was transferred to new flask with 9 mL media.

2.2.3 Peptide and Buffer Solution

Wild type (WT) Aβ (1-40) was synthesized and purified in our laboratory. Synthetic

Aβ peptides were prepared using standard Fmoc chemistry on an automated synthesizer

(Applied Biosystems 433A). The primary amino acid sequence for the amyloid Aβ (1–40)

+ 1 5 10 15 20 peptide is the following: H3N -D -A-E-F-R -H-D-S-GY -E-V-H-H-Q -K-L-V-F-F -A-

E-D-V-G25-S-N-K-G-A30-I-I-G-L-M35-V-G-G-V-V40-COO-. Peptide mass and purity were determined by a combination of matrix-assisted laser desorption/ionization time-off light mass spectrometry (MALDI-MS), electrospray ionization mass spectroscopy (ESI), analytical and preparative high performance liquid chromatography (HPLC). Purified peptides were aliquoted, lyophilized, and stored at -20 °C until used. Aβ (1-40) was also obtained from Bachem (Torrance, CA). Site-specific or uniformly (>95%) 15N-labeled peptides were prepared by chemical synthesis using 15N-labeled Fmoc protected amino acids (Cambridge Isotopes).

Lyophilized Aβ (1-40) was dissolved in sodium borate buffer (0.05M, pH 8.5, Thermo

Scientific, Product No. 28384). 200 µL protein solution was transferred to 50 µg

DyLight 488 (Thermo Scientific, Product No. 46403). The solution was vortexed until it was completely dissolved and was incubated at room temperature for 1 hour. Non-reacted reagent was removed from the protein by dye removal column (Thermo Scientific,

Product No. 46403).

2.2.4 Confocal Microscopy

To introduce Aβ(1-40) protein into the target cells, we utilized the bacterial toxin streptolysin O (SLO), which can form a 35 nm diameter pore on the cholesterol- containing plasma membrane that allows the entry and exit of molecules with sizes up to

150 kDa. Since the pore formed by SLO can be repaired by the addition of Ca2+ into the cytosol, the SLO-permeablized cells can be resealed after they assimilate the molecule of interest2. Although the SLO permeabilization and resealing methods have previously been used for the incorporation of small amounts of proteins or nucleotides into many kinds of cells, sample preparation on the Aβ (1-40) is unprecedented.

We chose adherent HeLa cells as the host cells and an amyloid protein, Aβ (1-40), as the protein to be delivered. The experimental scheme of our in-cell study is presented in

Figure 2.6.

Figure 2.6 Schematic illustrations of incorporation of the target protein within HeLa cells by SLO permeabilization and resealing of the target cells.

First, we prepared a large amount of cells (2.4 × 107 cells, 60% at confluency) for

NMR measurements in medium (Eagle's Minimum Essential Medium, ATCC, with 10%

FBS, 100U/ml penicillin and 100ug/ml streptomycin). Medium was removed from culture dish(s) by aspiration and the monolayer of cells was washed with Ca+2 and Mg+2 - free salt solution (Dulbecco’s Phosphate Buffered Saline (PBS), ATCC) to remove all traces of serum.

PBS was removed and before pore-formation by SLO, 4% formaldehyde was added to monolayer of cells for better images and was incubated for 10 minutes at room temperature. After formaldehyde was removed, SLO (100ng/ml, Bioacademia) and Aβ

(1-40) (10µM) were added to cover the monolayer of cells. The cells were placed in 37

°C incubator for 30 minutes. The dish(es) and cells were transferred to 4°C refrigerator for 1 hour and 15 minutes. Then SLO and Aβ solution was removed by aspiration and cells layer was washed with PBS for three times.

Then ice cold CaCl2 (2mM in PBS) was added to cover the cells for at least 2 hours to reseal the pores on the membranes permeablized by SLO. CaCl2 solution was removed by aspiration and the cells layer was washed with PBS for three times.

All procedures including pore formation, peptide introduction and resealing were all performed in 35-mm non-coated glass bottom dishes (MatTek) for better resolution. No trypsonization was performed before fluorescent imaging. All microscopy images were acquired with a Zeiss LSM 510 confocal system with a 63x /1.4 lens.

2.2.5 FCM Analysis of Peptide’s Entry, Pore Formation Efficiency and Cell

Viability

Aβ(1-40) was conjugated with Dylight 488 (Thermo Scientific) and then was incorporated into the cells as described above. Prior to FCM analysis, Hank’s balanced salt solution (HBSS, Thermo Scientific) buffer containing 50 ug/ml propidium iodide (PI), which stains only the cells with damaged plasma membranes, was added to resealed cell suspension. The resealed HeLa cells were analyzed with EPICS XL-MCL flow cytometry.

All procedures including pore formation, peptide introduction and resealing were all performed in 12 well plates (Corning).

2.3 Results

2.3.1 Confocal Microscope and Differential Interference Contrast image of

the Treated HeLa Cells

The flrorescent images of the cell indicate that Aβ (1-40) was evenly distributed throughout the cytoplasm as well as the nucleus (Figure 2.7A). Cells on the fluorescent image generally coincide with those on the contrast image, except the cell without fluorescent signals (Figure 2.7A and B, arrowed). This cell is without pore formation during SLO treatment since no fluorescence dispersed throughout the cell. The fact that the fluorescent cells adhered on the surface indicates that the cells treated with SLO are still viable (Figure 2.7B).

Figure 2.7 Confocal microscope and (B) differential interference contrast images of SLO treated HeLa cells incubated with Dylight488-labeled Aβ (1-40).

2.3.2 FCM Analysis of HeLa Cells With or Without SLO Treatment

To assess the efficiency of the pore formation and resealing, FCM analysis was performed. By comparing the cells without SLO treatment, the cells treated with SLO exhibited increased DyLight-positive and PI-negative populations (lower right of the

FCM density plots in Figure 2.8), indicating that the pore formation and resealing occurred properly. Also, Dylight-positive and PI-positive cells were also observed, which could provide unwanted signals because of DyLight 488 –labeled Aβ (1-40) leaking from unsealed pores. The DyLight-negative cells indicate that the pore formation did not occur.

Schematic illustration of 1, DyLight-488 negative cells, 2, DyLight 488-positive but PI- negative cells and 3, DyLight 488-positive and PI-positive cells.

2.3.3 Efficiency Analysis of the Pore Formation and Resealing of the HeLa

Cells

The optimal concentration of SLO for cell permeabilization was examined. HeLa cells were treated with various concentrations of SLO, ranging from 0-150 ng/ml. The

DyLight-positive and PI-negative cell population (dark bars) indicates the cells with

43 successful pore formation and resealing. The DyLight-positive and PI-positive cell population (light bars) indicates the cell with unrepaired pores.

The FCM analysis proved that the higher SLO concentration, the more cells were

DyLight-positive. The PI-positive, however, also increased at higher SLO concentration, indicating that the more pores were formed, the less could be resealed (Figure 2.9). From the SLO concentration we tested, a 100 ng/ml SLO yielded optimal resealing efficiency

(86%).

Figure 2.9 Efficiency of the pore-formation and resealing of HeLa cells, treated with various concentrations of SLO, ranging from 0-150 ng/ml. The DyLight-positive and PI- negative cell population (dark bars) indicates the cells with successful pore formation and

44 resealing. The DyLight-positive and PI-positive cell population (light bars) indicates the cell with unrepaired pores.

2.4 Discussion

The extracellular deposition of Aβ peptides as amyloid plaques is one important neuropathological feature of AD [93] and they have long been thought to be a primary cause of AD. However, detection of intracellular neuronal Aβ accumulation before extracellular Aβ deposits questions the relevance of intracellular peptides in AD.

In AD brains, the intracellular Aβ accumulates as aggregates or granules in the cytoplasm of neurons (Gouras et al., 2000; D’Andrea et al., 2001). The accumulation of

Aβ precedes the appearance of senile plaques [102, 103] and is observed in the hippocampus and entorhinal cortex [104], resulting in synaptic dysfunction [105-111].

Taken together, these studies suggest that intraneuronal Aβ may be considered as an early pathogenic event preceding the formation of extracellular plaques [116].

Moreover, it has been reported that Aβ binds to proteins, lipids and DNAs [37, 38, 99,

117-119].

Despite the extensive studies focused on intracellular Aβ and related cytotoxicity, in cell Aβ structure, size, conformation, and interrelationships with other amyloid aggregates, as well as the exact mechanism of intracellular Aβ-induced neurotoxicity, remain elusive [124-126]. Studies of intracellular Aβ accomplished so far basically limited on cytotoxicity studies via mostly mutant transgenic mice, antibodies and in vitro approaches. There is no study has been performed to elucidate the in-cell structure of Aβ monomer and oligomers. However, understanding these early events associated with β-

45 amyloidosis is especially important since many recent reports suggest that early-formed, soluble β-sheet aggregates are neurotoxic [25, 44]. We are the first group performed the in cell structure study of Aβ.

The two predominant species that exist in the plaques are Aβ(1-40) and Aβ(1-42).

Monomeric Aβ is inherently non-toxic and is a normal constitution of human biological fluids produced in low levels by most cell types.[141] Aβ, however, is highly aggregation-prone under certain conditions (e.g. high concentration) and Aβ aggregates are the toxic moieties. Compared to Aβ(1-40), Aβ(1-42) is more aggregation-prone and thus is believed to be more toxic. We chose Aβ(1-40), in the first place, for our in-cell study.

To obtain the in cell NMR spectrum of Aβ peptides, which is our final goal, Aβ peptides have to be introduced to the cells first. Common methods to induce heterologous peptides into cells include overexpression, microinjection and CPP. However, Aβ peptide is not flexible enough for all of the methods above due to its inherent characteristics.

Escherichia coli cells are used to overexpress proteins within the bacterial cytosol, where the total protein concentration exceeds 200 mg/mL. This high concentration of Aβ will decrease the cell viability because of the cytotoxicity of Aβ. In-cell NMR analysis using eukaryotic cells, which is our final destination, is technically more challenging because overexpression of the isotopically labeled target proteins is impossible.[129] It has been showed by several groups that isotopic labeled protein microinjected into Xenopus laevis oocytes yields high-resolution spectra that can be used to analyze protein structures.[142,

143] However, considering the small injection volume per cell (50 nL) compared to cell

46 volume (1 µL) of an oocyte, a final Aβ concentration of 5 µM in an oocyte cell requires the start Aβ concentration of 100 µM, which will decrease the cell viability.

More recently, CPP has been reported as a vector, which attaches to target protein and delivers the protein to the cell as a fusion. Although this method can be used for any kind of cell, including mammalian cells, the method has the limitations that the target protein has to be fused with the Tat tag and the tag must be cleaved off after translocation to the cytoplasm for further analysis. [129]

We chose the new method to successfully introduce Aβ (1-40) by SLO permeabilization and Ca2+ resealing of the target cells, mainly because it is a simple method to bring exogenous proteins into living cells. It is applicable to any kind of cell and does not require any special equipment or artificial modification of the molecule of interest. The confocal images of the cell indicate that Aβ (1-40) was evenly distributed throughout the cytoplasm as well as the nucleus (Figure 2.7). To assess the efficiency of the pore formation and resealing, flow cytometry (FCM) analysis was performed. By comparing the cells without SLO treatment, the cells treated with SLO exhibited increased DyLight-positive and PI-negative populations (lower right of the FCM density plots in Figure 2.8), indicating that the pore formation and resealing occurred properly. It was also tested by FCM that a 100 ng/ml SLO yielded optimal resealing efficiency (86%).

(Figure 2.9) FCM results also prove that the 100 ng/ml SLO and 10 µM Aβ (1-40) yielded optimal cell viability as well as resealing efficiency.

In conclusion, we have established the optimal conditions for the in cell NMR experiments. Compared to the other existing methods to introduce peptides into cells, this method does not require any modification of the target protein or any specialized

47 equipment and that it is applicable to a wide range of cells. In fact, the purpose to deliver

Aβ into cells is to study its in-cell structure by in-cell NMR. Detailed study of the in cell structures of peptides is the first step to understand the actual structure Aβ in human body and the present in-cell NMR technique opens up a multitude of different applications, such as the study of protein dynamics, investigations of amyloid-forming or intrinsically unstructured proteins in neuronal cells, and molecular diagnostics by observing probe proteins in biopsy specimens.[144] In-cell NMR is suitable not only because the task for it provides information at atomic level but also because recent advances in measurement sensitivity.[83, 84, 145] In next chapter, we delivered fully isotopic labeled Aβ into cells and obtained its HSQC structure, based on the studies in this chapter.

CHAPTER 3

IN-CELL NMR STUDIES OF THE Aβ PEPTIDE

3 In-cell NMR Studies of the Aβ Peptide

3.1 Introduction

3.1.1 Importance of In-cell NMR Study of the Aβ Peptides

The cellular environment in living cells is subject to molecular crowding and difficult to replicate in vitro,[82] because in this environment proteins interact specifically with other proteins, nucleic acids, macromolecules and lipids.[37, 38, 81, 99, 117, 119]

Although in vitro methods of structure determination have made very valuable contributions to understanding the functions of Aβ, in vivo observations of 3D structures, structural changes, dynamics or interactions of proteins and importantly, the aggregation

Aβ, are the prerequisites and play a significant role in the pathogenesis of AD. In vitro studies have reported that monomeric Aβ undergoes conformation transitions and proceeds to form low molecular oligomers (dimer/trimer), and then soluble high molecular aggregates and progress to form spherical oligomers which are composed of 12 to 24 monomers, which prolong to protofibrils and finally become insoluble fibrils [43].

These various structures differ not only in aggregation state, but also in their toxic effects.

Recently, many have reported that fibrils are actually second in toxicity to intermediate aggregates of Aβ (spherical oligomers and protofibrils) [126, 146, 147].

In AD brains, the intracellular Aβ accumulates as aggregates or granules in the cytoplasm of neurons (Gouras et al., 2000; D’Andrea et al., 2001). The accumulation of

Aβ precedes the appearance of senile plaques [102, 103] and is observed in regions affected early in AD: the hippocampus and entorhinal cortex [104]. Moreover, it has been demonstrated by numerous studies that intraneuronal Aβ accumulation results in synaptic dysfunction [105-111]. Taken together, these studies suggest that intraneuronal Aβ may

50 be considered as an early pathogenic event preceding the formation of extracellular plaques [116]. Despite the extensive studies focused on intracellular Aβ and related cytotoxicity, in cell Aβ structure, size, conformation, and interrelationships with other amyloid aggregates, as well as the exact mechanism of intracellular Aβ-induced neurotoxicity, remain elusive [124-126]. However, understanding these early events associated with β-amyloidosis is especially important since many recent reports suggest that early-formed, soluble β-sheet aggregates are neurotoxic [25, 44].

Detailed study of the in cell structures of peptides is the first step we reach to understand the actual structure Aβ in human body and the present in-cell NMR technique opens up a multitude of different applications, such as the study of protein dynamics, investigations of amyloid-forming or intrinsically unstructured proteins in neuronal cells, and molecular diagnostics by observing probe proteins in biopsy specimens. [144] In-cell

NMR is suitable not only because the task for it provides information at atomic level but also because recent advances in measurement sensitivity.[83, 84, 145]

Two features of NMR make it the ideal tool to investigate the interaction of biological macromolecules with binding ligands. The first feature is that it is able to provide information about molecules under physiological or at least “near-physiological” conditions. [148]Secondly, it provides the sensitivity of the chemical shift of an NMR- active nucleus to changes in its chemical environment. In fact, In-cell NMR experiments aim not only at determining structures directly in the cellular environment, but also at using the sensitivity of the chemical shift towards changes in the environment to obtain information about the state of a macromolecule in its natural surrounding. Changes in this

51 environment might indicate interactions occurred, such as post-translational modifications, conformational changes, or binding events. [148, 149]

In-cell NMR has been used to detect protein–protein interactions inside E. coli cells[150] as well as the behavior of intrinsically disordered proteins [151, 152]. In eukaryotic cells, in-cell NMR studies have been performed by injecting proteins into

Xenopus laevis oocytes or eggs[142] and, more recently, cell-penetrating peptides and

SLO have been used to deliver proteins that can be observed in living cells[129, 148].

Moreover, Chemical-shift differences observed in HSQC spectra can also be widely used as a screening tool in the pharmaceutical industry, by testing and characterizing interactions between peptides and drugs. Protein-drug interactions in vitro are not necessarily the same as in vivo. For example, the inability of a drug molecule to cross the cellular membrane, its fast metabolization, or differences in the target-protein conformation between its in vitro and in vivo states.[148] In vivo testing, for example, in- cell NMR experiments for screening, can at least overcome these disadvantages of in vitro screens.

3.1.2 Factors affecting In-cell NMR Signals

There are two major difficulties with in-cell NMR spectroscopy. First, the macromolecule must be able to tumble freely. Second, the cells have to survive the conditions inside the NMR tube at least for the time period of the experiment without significant changes of their metabolic state.

3.1.2.1 Viscosity

It is required by high-resolution liquid-state NMR spectroscopy of biological macromolecules that these molecules have to tumble in solution with a sufficiently short correlation time. Short rotational correlation times lead to slow relaxation and, therefore, sharp peaks. Since the rotational correlation time is proportional to the surrounding viscosity, the intracellular viscosity is an important parameter for the observation of macromolecules inside living cells.[148] Investigation of the rotational correlation time has shown that the intracellular rotational correlation time is only twice as long as the rotational correlation time of the same molecule in pure water. [153, 154]

The relatively low viscosity of the cellular cytoplasm, combined with TROSY and similar techniques extending NMR to larger macromolecules with weights of 100 kDa and more[155, 156], makes the cytoplasmic viscosity not a major limitation for the observation of proteins inside living cells. However, viscosity differs among the individual cellular organelles. The viscosity of nucleus is significantly greater than cytoplasm and endosomes.[157] In addition, the viscosity of the organelles and the cytoplasm can also change during different states of the cell, for example, different phases of the cell cycle. [148]

When proteins bind to cellular components, it is difficult to observe the protein in cell because this significantly increases the rotational correlation time of the protein.

Binding to large components especially, such as chaperones and nucleic acids, leads to the disappearance of a protein’s resonances due to extensive line broadening.

3.1.2.2 Cell Survival Rate

The second critical parameter that strongly influences the applicability of in-cell NMR experiments is the survival rate of the cells in the NMR tube. Specifically, oxygen starvation and limiting the amount of available nutrients caused by the high cellular density lowers the cell viability. If the concentration of the peptides (in the overexpression system) is high enough, the NMR spectra can be measured relatively quickly (less than an hour). Lower concentration of peptides in cell, however, requires longer experiments or series of experiments, during which significant changes in the cellular status can occur. These changes include shifts of the intracellular pH and cell death observed, for example. In order to obtain the peptide structure close to its actual conformation, cell cultures have been kept alive for long times. Modified NMR sample tubes which enables continuous exchange of media and low-melting agarose which encapsulates cells help increase the cell viability in long NMR experiments. [148]

3.2 Materials and Methods

3.2.1 Experimental Design

3.2.2 Peptides and Reagents

Uniformly (>95%) 15N-labeled peptides were prepared by chemical synthesis using

15N-labeled Fmoc protected amino acids (Cambridge Isotopes) ands tandard Fmoc chemistry on an automated synthesizer (Applied Biosystems 433A). Peptide mass and purity were determined by a combination of matrix-assisted laser desorption/ionization time-off light mass spectrometry (MALDI-MS), electrospray ionization mass spectroscopy (ESI), analytical and preparative high performance liquid chromatography

(HPLC). Purified peptides were aliquoted, lyophilized, and stored at -20 °C until used.

Aβ (1-40) was also obtained from Bachem (Torrance, CA).

Lyophilized Aβ (1-40) was dissolved in sodium borate buffer (0.05M, pH 8.5, Thermo

Scientific, Product No. 28384). 200 µL protein solution was transferred to 50 µg

Product No. 46403).

Lyophilized Aβ (1-40) peptide was first thoroughly dissolved in dilute NaOH solution

(10 mM) with sonication for 1 min to disaggregate. The basic pH solution of Aβ (1-40)

(10 mM) was then combined directly with a potassium phosphate-buffered solution (10 mM, pH 7.3) to yield a final stock solution (8 ml) with a peptide concentration of 50 µM, which was later combined with cell and made the concentration of Aβ (1-40) during cell permeabilization and peptide introduction to be 10 µM.

3.2.3 Cell Permeabilization and Resealing

SLO was used to permeabilize cells as we introduced in Chapter 2. Although the SLO permeabilization and resealing methods have previously been used for the incorporation of small amounts of proteins or nucleotides into many kinds of cells,[128, 158, 159] sample preparation for Aβ peptides on the NMR scale is unprecedented.

We chose adherent HeLa cells as the host cells and an amyloid protein, site- specifically labeled and uniformly 15N labeled Aβ (1-40), as the protein to be delivered for NMR observation. The experimental scheme of our in-cell NMR experiments is presented in Figure 3.1.

A large amount of cells (2.4 × 107 cells) for NMR measurements were prepared in dish(es). Medium was removed from culture dish(s) by aspiration and the monolayer of cells was washed with Ca+2 and Mg+2 - free salt solution to remove all traces of serum.

PBS was removed and SLO (100ng/ml, Bioacademia) and Aβ (1-40) (10 uM) were added to cover the monolayer of cells. The cells were placed in 37 °C incubator for 30 minutes. The dish(es) and cells were transferred to 4°C refrigerator for 1 hour and 15 minutes. Then SLO and Aβ solution was removed by aspiration and cells layer was washed with PBS for three times. Then ice cold CaCl2 (2mM in PBS) was added to cover the cells for at least 2 hours to reseal the pores on the membranes permeablized by SLO.

CaCl2 solution was removed by aspiration and the cells layer was washed with PBS for three times.

Enough trypsin (0.05% (w/v) in PBS, ATCC) was dispensed into culture dish to completely cover the monolayer of cells, which was later placed in 37 °C incubator for approximately 2 minutes. Trypsin solution was removed by aspiration and then the culture dish(es) was transferred to incubator. The coated cells were allowed to incubate until cells detached from the surface. Progress could be checked by examination with an inverted microscope. Since trypsin causes cellular damage, time of exposure should be kept to a minimum. When trypsinization process was completed, PBS was added to make the cells in suspension. The suspension was centrifuged and washed for 3 times. The resealed cells were transferred to a Shigemi tube as a suspension in PBS containing 5%

D2O and 1mg/ml glucose and NH4Cl to support the living of HeLa cells during the NMR measurement. All experiments were performed on a Bruker 900 or an 800 MHz spectrometer, both with cryoprobes. The sample temperature was kept at 5 degree C. 1H-

15N HSQC data were acquired with 1024 points in the direct dimension and 48 points in the indirection dimension. Typical instrumental time for a HSQC experiment was 20-22 hours. Water resonance was suppressed either by coherence selection using pulse field gradients or by Water-Gate technique. Spectra were processed by zero-filling the indirectly detected dimension into 128 or 256 points before Fourier transformation.

Square sine-bell window function with pi/2 shift was applied for processing the FID in both dimensions.

3.2.4 Lysate Treated With or Without Nuclease

Probe sonicator was used to turn cells into cell lysate. Sonication uses high frequency sound waves to agitate and lyse cells, during which excessive heating forms and denatures protein. To avoid excessive heating, ultrasonic treatment was applied in multiple short bursts to a sample immersed in an ice bath. To protect the integrity of proteins from a broad range of proteases during the protein extraction and purification process, protease inhibitor was added to cell suspension before sonication. (cOmplete

Protease Inhibitor Cocktail Tablets, Roche) After cell lysis, cell lysate was transferred to a Shigemi tube and examined by NMR.

To reduce the viscosity of cell lysate, nuclease (Pierce* Universal Nuclease for Cell

Lysis, Thermo Scientific) was added to cell lysate. Nuclease is a group of enzyme that cleaves nucleic acids. When cells are lysed, proteins, organelles, DNA and RNA are all released from the cell membrane and enter into the cell lysate, therefore increasing the viscosity of cell lysate.

3.3 Results

3.3.1 2D HSQC Spectra of Cell and Supernatant with Ala2 and Ala21 15N

labeled Aβ (1-40)

After the measurement, the suspension of the resealed cells was collected and examined by NMR spectroscopy. Two peaks were observed (Figure 3.2a), while almost no signals were observed from the supernatant (Figure 3.2b), indicating that almost all of the NMR signals originated from the Aβ protein within the cells. These results also suggest that structural analysis of Aβ(1-40) in HeLa cells will be feasible.

3.3.2 2D HSQC Spectra of Cell and Supernatant with fully 15N labeled Aβ

(1-40)

After the measurement and by comparison with the in vitro spectrum of the same peptides (Figure 3.3a), only 5 peaks were observed on the 2D HSQC spectrum (Figure

3.3b) of the uniformly 15N labeled Aβ (1-40) of the in-cell NMR sample immediately after sample preparation, which indicates that the structure or mobility of Aβ (1-40) changes after the peptides were transferred into the cells or during the NMR experiment or that Aβ (1-40) peptides bond to cell membrane or organelles. For in-cell NMR, it is crucial to make sure that the peptides that provide the NMR spectra are actually inside the cells, so the suspension of the resealed cells was collected and examined by NMR spectroscopy. Almost no signals were observed from the supernatant (Figure 3.3c), indicating that almost all of the NMR signals originated from the Aβ protein within the cells. Moreover, the lysate spectrum of the collected cells showed no obvious new crosspeaks (Figure 3.3d), establishing that Aβ peptide did not bind to cell membranes during and after delivery into the cells. These results also demonstrated that the contribution of extracellular protein to the observed signals is negligible.

Figure 3.3 (a) The 1H–15N HSQC spectrum of the uniformly 15N labeled Aβ (1-40) in aqueous solution at neutral pH. (b)The 1H–15N HSQC spectrum of the uniformly 15N

62 labeled Aβ (1-40) of the in-cell NMR sample immediately after sample preparation. (c)

The 1H–15N HSQC spectrum of the supernatant of the in-cell sample. (d) The 1H–15N

HSQC spectrum of the lysate of the harvested cells after 20 hours NMR measurement.

3.3.3 Effects of Viscosity on NMR Signals

A major limitation for the application of high-resolution liquid state NMR spectroscopy of biological macromolecules is the requirement that these molecules have to tumble in solution with a sufficiently short correlation time. Long rotational correlation times lead to broad peaks. Diffusion measurements have indeed shown that the translational diffusion of a macromolecule inside cells can be severely restricted relative to an in vitro system with the purified molecule. Since the rotational correlation time is proportional to the surrounding viscosity, the intracellular viscosity is an important parameter for the observation of macromolecules inside living cells.[148] The major reason for the high viscosity of cell lysate is the liberation of large quantity of DNA during cell lysis. To reduce the viscosity of cell lysate, nuclease (Pierce Universal

Nuclease for Cell Lysis) was used to treat cell lysate.

Cell lysate treated without and with nuclease were examined by NMR. By comparison with the spectrum (Figure 3.4a) of cell lysate not treated by nuclease, the spectrum

(Figure 3.4b) of the cell lysate treated by nuclease showed no obvious new peaks. A new strong peak at (7.97 ppm, 121.84 ppm) and a peak stronger at (6.66 ppm, 113.15 ppm) were from the nuclease background.

3.3.4 Effects of Cell Survival Rate on NMR Signals

Another critical parameter that strongly influences the applicability of in-cell NMR experiments is the survival rate of the cells in the NMR tube. In particular, the high cellular density can cause problems like oxygen starvation and limiting the amount of available nutrients.[148] Due to the relatively low concentration of uniformly 15N labeled

Aβ (1-40) (lower than 10uM), NMR experiment takes at least 5 hours, during which significant changes in the cellular status can occur, such as the shifts of the intracellular pH in cells.

To examine the influence of survival rate of cells on the structure of Aβ(1-40) inside cells, NMR experiments were performed for different time spans. Comparison of the results suggested that no obvious changes occurred in the cellular environment during 8-

20 hr after the cells were resealed.

Figure 3.5 (a) The 1H–15N HSQC spectrum of the harvested cells for 20 hours NMR measurement. (b) The 1H–15N HSQC spectrum of the harvested cells for 8 hours NMR measurement.

To examine the influence of time on the NMR structure of Aβ (1-40) in cell lysates, resealed cells after 0 hour and 20 hours NMR experiments were collected repectively and treated with lysis buffer and nuclease. The spectra of both samples are shown in Figure

3.5. By comparison of the spectrum of cell lysate after 20 hours NMR experiment, the 65 spectrum of Aβ (1-40) in cell lysate after 0 hour NMR experiments showed several new peaks (in boxes) at about 7 ppm and a weakened peak (in box) at about 8.4 ppm.

3.4 Discussion

Synthetic Aβ peptides have been used extensively to study the mechanism and factors involved in the amyloid fibril formation by the methods of Tinctorial analysis, X-ray diffraction analysis, CD analysis, NMR, FTIR and so forth.[160-162] However, in vivo study of structure of Aβ peptides is surprisingly limited yet significantly important, because Aβ peptides is extremely sensitive to its surrounding environment and thus its in

66 vitro structure is not necessarily the same as its in vivo structure. Experiments with synthetic Aβ peptides showed that Aβ peptides can adopt either mainly random coil, α- helix or β sheet secondary structures depending on solution conditions, such as pH and solvent properties.[45]

Here we used in-cell NMR to study the in vivo structure of Aβ (1-40). To obtain the

NMR spectra of Aβ (1-40), Ala 15N labeled Aβ (1-40) was first induced into HeLa cells by SLO permeabilization and calcium resealing, which was proved to be effective in

Chapter 2. Two peaks were observed (Figure 3.2a) in cell sample, while almost no signals were observed from the supernatant (Figure 3.2b), indicating that almost all of the NMR signals originated from the Aβ protein within the cells. These results also suggest that structural analysis of Aβ (1-40) in HeLa cells will be feasible.

Later, fully 15N labeled Aβ(1-40) was induced into HeLa cells and the sample was examined by 2D HSQC. After the measurement and by comparison with the in vitro spectrum of the same peptides (Figure 3.3a), only 5 peaks were observed on the 2D

HSQC spectrum (Figure 3.3b) of the uniformly 15N labeled Aβ(1-40) of the in-cell NMR sample immediately after sample preparation and none of the 5 peaks has the same chemical shift as the peaks in vitro spectra, which indicates that the structure of Aβ(1-40) changes after the peptides were transferred into the cells or during the NMR experiment.

Binding of the proteins to other cellular components might cause the disappearance of peaks, since this significantly increases the rotational correlation time of the protein. In particular, binding to large components, such as chaperones and nucleic acids, leads to the disappearance of a protein’s resonances due to extensive line broadening.[148] And it

67 has been demonstrated that Aβ binds to proteins, lipids and nucleic acids in cell. [37, 38,

99, 117, 119]

For in-cell NMR, it is crucial to make sure that the peptides that provide the NMR spectra are actually inside the cells, so the suspension of the resealed cells was collected and examined by NMR spectroscopy. Almost no signals were observed from the supernatant (Figure 3.3c), indicating that almost all of the NMR signals originated from the Aβ protein within the cells. The confocal images of the cell indicate that Aβ(1-40) was evenly distributed throughout the cytoplasm as well as the nucleus (Figure 2.7). This further confirms that all NMR signals are from the Aβ inside the cells.

Did Aβ(1-40) bind to cell membranes when it was induced into HeLa cells or after it was in the HeLa cells? HeLa cells, which collected after in-cell NMR experiment, were lysed and the cell lysate was then examined by NMR. The lysate spectrum (Figure 3.3d) of the collected cells showed one peak (6.62 ppm, 113.11 ppm) became weaker and one peak (7.38 ppm, 113.21) disappeared. These changes might be cause by the increased viscosity. Meanwhile, several new weak peaks appeared, which might be caused by Aβ

(1-40) being released from the membrane. The possibility of background has to be ruled out before reaching this conclusion. Much sharper cross-peaks (Figure 3.3d), demonstrating that Aβ protein did not bind to cell membranes during and after delivery into cells.

As we discussed early in this chapter, although intracellular rotational correlation time is only twice as long as the rotational correlation time of the same molecule in pure water

[153, 154] and the cytoplasmic viscosity is not a major limitation for the observation of proteins inside living cells, the viscosity of nucleus is significantly greater than cytoplasm

68 and endosomes. [157] Therefore, the viscosity of the surrounding environment of Aβ(1-

40) after cell lysis might be higher than the cytoplasmic viscosity when Aβ(1-40) was in the cells. This increased viscosity in peptides’ surrounding environment might cause the change in NMR spectra. To reduce the viscosity of cell lysate, nuclease was added into collected cells before cell sonication. Cell lysate treated without and with nuclease were examined by NMR. By comparison with the spectrum (Figure 3.4a) of cell lysate not treated by nuclease, the spectrum (Figure 3.4b) of the cell lysate treated by nuclease showed no obvious new peaks or shifts after viscosity was reduced. (A new strong peak at (7.97 ppm, 121.84 ppm) and a peak stronger at (6.66 ppm, 113.15 ppm) were cause by nuclease.) These results indicate that the nuclease makes no enough difference in viscosity that could change the conformation or mobility of the peptides.

Besides environment viscosity, cell viability of the cells in NMR tubes is another major limitation in obtaining in-cell NMR structures. Due to the relatively low concentration of uniformly 15N labeled Aβ(1-40) (about 1 uM), NMR experiment takes at least 5 hours, during which significant changes in the cellular status can occur, such as the shifts of the intracellular pH in cells. To examine the influence of survival rate of cells on the structure of Aβ(1-40) inside cells, NMR experiments were performed for different time spans. By comparison of the spectrum (Figure 3.5a) of harvested cells for

20 hours NMR experiment, the spectrum (Figure 3.5b) for 8 hours showed no obvious new peaks or peak shifts but only weaker signals due the shorter experiment span. These results suggest that no obvious changes occurred in the cellular environment during 8-20 hours after the cells were resealed.

To examine the influence of time on the NMR structure of Aβ(1-40) in cell lysates, resealed cells after 20 hours NMR experiments and after 0 hours NMR experiments were collected and treated with lysis buffer and nuclease. The spectra of both samples are shown in Figure 3.6. By comparison of the spectrum of cell lysate after 20 hours NMR experiment, the spectrum of Aβ(1-40) in cell lysate after 0 hour NMR experiments showed several new peaks (in boxes) at about 7 ppm and a weakened peak (in box) at about 8.4 ppm. These new peaks, alone with the previous results indicate that the decreased cell viability during the time span of 20 hours changed lysate properties, which in turn caused the structural changes on Aβ(1-40).

CHAPTER 4

MECHANISM OF POLYPHENOL-BASED INHIBITION OF Aβ AGGREGATION

4 Mechanism of Polyphenol-Based Inhibition of Aβ Aggregation

4.1 Introduction

4.1.1 Properties of Polyphenols

AD is the most common form of dementia, resulting in deterioration of cognitive function and behavioral changes.[163] One of the pathological hallmarks of AD is characterized by the aggregation of the Aβ peptide as insoluble amyloid plaques

(amyloidosis), rich in cross-β-sheet structure.[164] Aβ is a normally secreted soluble peptide that under certain environmental conditio11ns can produce β-sheet aggregates that eventually precipitate as amyloid fibrils. Numerous studies have established that the deposition of high levels of fibrillar Aβ in the AD brain is associated with loss of synapses, impairment of neuronal functions, and loss of neurons.[165-168] Additional studies suggest that the longer, 42-residue Aβ (1-42) is more pathogenic than the shorter,

40-residue Aβ (1-40) because of its greater in vitro tendency to aggregate and precipitate as amyloid.[169] These findings set the stage for the proposal that Aβ aggregation, especially Aβ (1-42) aggregation, is the primary event in AD pathogenesis and that anti-

Aβ aggregation is a strategy for AD therapy.

Nature itself may have created useful therapeutic agents for AD, such as French and

Danish epidemiological studies suggest that moderate wine drinking may protect against

AD. [170-172] The flavonoid constituents of red wine and red grapes are factors of particular interest.

Flavonoids have been reported to exhibit antioxidant properties and other multiple biological effects, such as antiviral [173], antibacterial [174], anti-inflammatory [175], vasodilatory [176], anticancer [177], and anti-ischemic [178]. Moreover, they are able to inhibit lipid peroxidation and platelet aggregation and improve increased capillary

72 permeability and fragility [179]. The term of antioxidant refers to any substance that prevents damage to cellular components arising as a consequence of chemical reactions involving free radicals [180]. Flavonoids can prevent injury caused by free radicals by direct scavenging of reactive oxygen species (ROS) (Figure 4.1), activation of antioxidant enzymes [181], metal chelating activity [182], inhibition of oxidases [183], and etc. Nowadays, their possible health benefits have driven more and more focuses owing mostly to their potent antioxidant and free radical scavenging activities observed in vitro. However, the antioxidant efficacy of flavonoids in vivo is less documented.

Among various flavonoids, myricetin has been reported as the most effective radical scavengers in the aqueous phase due to its structural features [184]. D. Procházková et al. / Fitoterapia 82 (2011) 513–523 515

Fig. 3. 2,3-Double bond in conjugation with a 4-oxo function in the C ring [52].

fl 2.1.2. Ability to activate antioxidant enzymes Fig. 1. Scavenging of reactive oxygen species (R•) by avonoid. The free fl Figure 4.1 Scavengingradical of reactive Fl-O• may react oxygen with a second species radical, (R•) acquiring by a stableflavonoid. quinone The freeOther radical possible Fl mechanism- by which avonoids act is structure [38]. (The figure is presented with the kind permission of Prof. through interaction with various antioxidant enzymes. Pietta.) Furthermore, some effects may be a reset of a combination O• may react with a second radical, acquiring a stable quinone structureof radical. Figure scavenging and and the interaction with enzyme [42]. The glycosylation of flavonoids reduces their in vitro functions [30]. caption taken from [185]antioxidant activity when compared to the corresponding Flavonoids are able to induce phase II detoxifying enzymes aglycons [43–47]. Comparison of TEAC values of quercetin (e.g. NAD(P)H-quinone oxidoreductase, glutathione S- (4.42 mM) and rutin (2.02 mM), quercetin-3-O-rutinoside, transferase, and UDP-glucuronosyl transferase), which are shows that glycosylation of the 3-OH group has strongly the major defense enzymes against electrophilic toxicants and suppressive effect on the antioxidant activity [43].Similar oxidative stress. Regulation of this protective gene expression results were observed also for other pairs of flavonoid aglycon can be mediated by an electrophile responsive element On the other hand,and flavonoids glycoside (e.g. can hesperetin act –ashesperidin, prooxidants luteolin– luteolin and, hence,(EpRE), promote which is a the regulatory sequence of a number of genes 4′-glucoside; luteolin–luteolin 7-glucoside; baicalein–baicalin; encoding these phase II enzymes [53,54]. The ability of oxidation of other compoundsand quercetin under–quercitrin) certain[43,44] circumstances. Quercetin glycosylation [185] also. Prooxidantflavonoids activity to activate of the EpRE-mediated response correlates significantly reduced its superoxide scavenging ability [48], with their redox properties. Lee-Hilz et al. [55] observed hypochlorite scavenging activity [49] and power to reduce Fe activation of firefly luciferase reporter gene in Hepa-1c1c7 flavonoid is thought to(III) be to directly Fe(II) (determined proportional by FRAP t assay)o the[50] total. number of hydroxylmouse hepatoma groups cellsin upon induction with flavonoids of The main structural features of flavonoids required for different structure. The most effective inducers were flavo- fi a flavonoid molecule ef andcient f radicallavonoid scavenging prooxidant could be summarized properties as seem follows to benoids concentration containing a hydroxyl- group at the 3-position in the ring [51,52]: C (quercetin and myricetin), whereas flavonoids without this hydroxyl group only (luteolin and galangin) were low a) an ortho-dihydroxy (catechol)73 structure in the B ring, for luciferase inducers. Therefore, flavonoids with a higher electron delocalization (Fig. 2): intrinsic potential to generate oxidative stress and redox b) 2,3-double bond in conjugation with a 4-oxo function in cycling are the most potent inducers of EpRE-mediated gene the C ring provides electron delocalization from the B ring expression. It can be concluded that the prooxidant activity of (Fig. 3): flavonoids can contribute to their health-promoting activity c) hydroxyl groups at positions 3 and 5 provide hydrogen by inducing important detoxifying enzymes, pointing to a bonding to the oxo group (Fig. 4): beneficial effect of a supposed toxic chemical reaction [55]. Nagata et al. [56] investigated cytoprotective effect of According to the previously stated criteria, flavonols quercetin and catechin against hydrogen peroxide cytotoxi- quercetin and myricetin should be the most effective radical city in cultured rat hepatocytes BL-9, which are cells highly scavengers in the aqueous phase, which has been confirmed expressing cytosolic glutathione peroxidase (GPx). The experimentally [43].

Fig. 2. An ortho-dihydroxy (catechol) structure in the B ring [52]. Fig. 4. Hydroxyl groups at positions 3 and 5 [52]. dependent [186]. The generation of superoxide anion radical and products of lipid peroxidation (measured as thiobarbituric acid reacting substances) increased with

increased concentration of flavonoids. It was reported that hydrogen peroxide (H2O2) is formed during incubation of polyphenols (Figure 4.2), such as myricetin [187] and epigallocatechin gallate (EGCG). [188] Moreover, they were able to induce DNA strand breakage in concentration-dependent manner as was determined by sensitive comet assay

[185]. For example, in rat liver microsomes, gossypol, quercetin and myricetin effectively inhibited lipid peroxidation at low micromolar concentrations (IC50 ≤ 1.5

µM). However, all three compounds at 100 µM concentration greatly enhanced hydroxyl radical formation up to eight-fold [189]. However, it is misleading to view their prooxidant properties only as toxic ones. Their prooxidant properties could be associated with a cell signaling. By imposing a mild degree of oxidative stress, the levels of antioxidant defences and biotransformation enzymes might be raised, resulting in overall cytoprotection [190].

I.M.C.M. Rietjens et al. / En6ironmental Toxicology and Pharmacology 11 (2002) 321–333 329

Figure 4.2 Pro-oxidant chemistry of flavonoids. (Auto)oxidation of the flavonoid

generates the flavonoid semiquinone radical, which may be (auto)oxidized to produce

quinone, which may be scavenged by GSH not by means of chemical reduction but rather

by conjugate formation. Figure and caption taken from [191]

Fig. 7. Pro-oxidant chemistry of catechol-type ﬂavonoids. this possibleNowadays, pro-oxidant interest toxicity in ittheir is of possible interest health to benefitsto show has carcinogenic increased activity owing inmost the of kidney all to of male notice that the mutagenic properties of the ﬂavonoid F344/N rats. quercetintheir have beenpotent demonstrated antioxidant in a and variety free of bacte- radical scavengingThe relevance activities for the human observed in vivo in situation vitro. as well rial and mammalian mutagenicity tests, and have been as the mechanism behind this quercetin-mediated toxic related to its quinone/quinone methide chemistry (Mac- effect remains a matter of debate (Ito, 1992). Ito (1992) Gregor andNeve Jurd,rtheless, 1978; the Brown, antioxidant 1980; Middleton efficacy andof flavonoidssuggested in vivo a possible is less factor documented of special and interest their to be the

Kandaswami, 1994). Interestingly, the structural re- role of a2u globulin nephropathy in chemically induced quirementsprooxidant for good properties antioxidant have activity been match actually the describedrenal carcinogenicity, in vivo. The a nephropathyextent to which which are is observed requirements essential for pro-oxidant action and qui- selectively in male rats only. Such a hypothesis would none methideflavonoids formation. able to act as anti- or prooxidants bein invivo line is with still the poorly observations understood that increased and this numbers Based on these positive mutagenicity results in a of benign tumors are often observed in male but not variety oftopic bacterial clearly as wellrequires as mammalian further studies. test systems, [185] female rats (NTP, 1991; Dunnick and Haily, 1992). several studies have investigated the possible carcino- Another mechanism which may be of importance for genicity of especially quercetin. Several animal studies carcinogenicity upon exposure to quercetin is the hy- Myricetin, which is found in various foods, including onions, berries, and grapes, as reported no tumor initiating activity (Hirono et al., pothesis that overloading the organism with quercetin 1981; Morino et al., 1982; Stoewsand et al., 1984; may deplete the cofactor for catechol O-methyltrans- Hirose etwell al., as 1983; red Ito wine, et al., was 1989). reported In contrast, to be Pa- a candidateferases, therapeS-adenosyl-utic L molecule-methionine for (SAM), inhib becauseiting cate- mukcu et al. (1980) reported induction of intestinal and chol O-methyltransferase metabolism represents an bladder tumorsAβ(1–42) by quercetin fibrillation. in male[172, and 192] female In rats. thisA chapterimportant, the molecular metabolic mechanisms pathway forunderlying catechol type study from the National Toxicology Program (NTP, ﬂavonoids (Zhu et al., 1994; Williamson et al., 2000). 1991) reportedthe inhibiti someon evidence of Aβ(1 of-42) carcinogenic fibrillation activity by myricetinCofactor was further depletion investigated. may affect the methylation of cate- of quercetin in male F344/N rats, based on an increased chol estrogens, thereby providing increased possibilities incidence of renal tubular cell carcinomas. Ertu¨rk et al. for estrogen mediated carcinogenesis, because accumu- (1985) reported bladder tumors in rats exposed to lation of catechol-type estrogens in the kidney may quercetin. Dunnick and Haily (1992) reported quercetin 75 stimulate their oxidation to DNA alkylating elec-

4.1.2 Basic Techniques Used to Study the Effects of Myricetin on the Aβ (1-

42) Aggregation.

4.1.2.1 Circular Dichroism

Circular Dichroism (CD) is a widely used spectroscopic tool to probe the conformational properties of polypeptides and proteins. The CD bands for peptides and proteins are in the near UV (250-310 nm) and far UV region (190-250), while only the latter region indicates primarily electronic transitions of the backbone and provide information about the secondary structure.

Standard CD curves of the polylysine in three most common elements of secondary structures are shown in Figure 4.3. [193] The three conformations are α-helix, β-sheet and random structure. The α-helix gives rise to a strong positive band at 190-195 and two weaker minimum bands at 222 nm and 208-210 nm. The β-sheet shows a maximum band around 195 nm and a minimum at 217. A random coil is characterized by a strong negative band at 197 nm.

Figure 4.3 Circular Dichroism Spectrum of poly-L-lysine in α-helix, β-sheet and random confirmation. [193]

4.1.2.2 Thioflavin-T

Thioflavin-T (ThT) is one of the most commonly used dyes to identify and quantify amyloid beta-sheet structure. [194] ThT shows characteristic alterations on binding to amyloid fibrils. Upon binding to ThT, amyloid fibrils give rise to a large fluorescence excitation spectral shift that does not occur with binding to the precursor monomer or amorphous aggregates. The mechanism by which ThT binds to fibrils is still unknown.

4.1.2.3 Electron Microscopy

The technique of electron microscopy (EM) uses a beam of electrons to create an image of the specimen. It is capable of high magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail.

All electron microscopes, use electromagnetic and/or electrostatic lenses to control the path of electrons and there are two kinds of EM: the transmission electron microscope

(TEM) and the scanning electron microscope (SEM). The TEM involves transmitting electrons through a sample that carries information about the structure of it and then magnified by a series of magnetic lenses. In the end, this information is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a charge- coupled device (CCD) camera.

The TEM produces two-dimensional, black and white images and uses a staining agent to enhance the contrast of the specimen. The staining agents contain heavy metal salts that deflect the electron beam away from the film. Positive stains increase the density of the specimen when compared to the background while negative stains decrease the density of the specimen when compared to the background[195].

Resolution of the TEM is also limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome or limit these aberrations. It allows the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.089 nm and atoms in silicon at 0.078 nm at magnifications of 50 million times. In the life sciences, however, specimen preparation limits the resolution of what we can see in the electron microscope.

4.1.2.4 AmplexRed Hydrogen Peroxide/Peroxidase

In the presence of peroxidase, the Amplex® Red reagent (10-acetyl-3,7- dihydroxyphenoxazine) reacts with H2O2 in a 1:1 stoichiometry to produce the red- fuorescent oxidation product, resorufn. Resorufn has excitation and emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (58,000 ± 5,000 cm–1 M–1), experiments can be performed fuorometrically or spectrophotometrically. This reaction has been used to detect as little as 10 picomoles of

H2O2 in a 100 µL volume (50 nM). http://www.lifetechnologies.com/us/en/home/brands/ molecular-probes/key-molecular-probes-products/amplex-red-enzyme-assays.html

4.1.2.5 Saturation Transfer Difference (STD)

NMR spectroscopy is a unique tool to study molecular interactions in solution, and has become an essential technique to study molecular recognition and interactions of small ligands with biologically relevant macromolecules.[196] Ligand-based NMR screening is especially important for drug-discovery process. The saturation transfer difference NMR

(STD-NMR) experiment has been developed as an essential tool for some years to characterize ligand receptor complexes in pharmaceutical industry. The STD-NMR experiment is based on the nuclear Overhauser effect and in the observation of the ligand resonance signals. The term binding epitope refers to hydrogens of the ligand that are closer to the protein upon binding.[197]

For a weak- binding ligand (dissociation constant, KD, ranging from 10-8 mol L-1 to

10-3 mol L-1), there is exchange between the bound and the free ligand state. Based on this fact, STD experiment is performed and involves the subtraction between two spectra:

on-resonance spectrum and off-resonance spectrum. On-resonance spectrum obtained by

irradiating at a region of the spectrum that contains only resonances of the

receptor/protein such as 0 ppm to -1 ppm, with signal intensities ISAT. Off-resonance

spectrum indicates the one obtained without protein saturation, with signal intensities I0.

In the difference spectrum (I = ISAT - I0) only the signals of the ligand(s) that received

saturation transfer from the protein (via spin diffusion, through the nuclear Overhauser

effect) will remain (Figure 4.4). The difference in intensity due to saturation transfer can

be quantified (I = ISAT - I0) and indicates of binding. For a molecule that binds to the

receptor, only the hydrogen that are in close contact to the protein (≤5 Å) and receive

magnetization transfer will have signals in the difference spectrum and those closer to the

protein will have more intense signals, because of a more efficient saturation transfer.

[198] Journal of Chemical Education LABORATORY EXPERIMENT

Figure 1. Scheme ofFigure the STD-NMR 4.4 Scheme experiment. of the The STD exchange-NMR between experiment. free and bound In the ligand diff allowserence intermolecular spectrum, transferonly the of magnetization from the receptor to the bound small molecule. binding molecules that received saturation transfer from the protein show signals. Non- to characterize molecular interactions in a biological context. The inverse 5 mm probehead with z-gradients at 25 °Cusingstandard STD-NMR experiment and data analysis are currently part of the Bruker pulse programs. Spectral acquisition and processing parameters NMR practice in structuralbinding small analysis molecule and structural will not biochemistry receive any saturationare included transfer; in the Supportingtheir signals Information, will be sectionsof 3.2 and 3.3. courses for organic chemistry and biochemistry master degree The STD-NMR Ligand Based-Screening Experiment students, respectively. The experiments were adapted from Wang and co-workers.3 The general setup of the STD-NMR experiment was followed 80 using sample B (HSA 20-fold excess of 6-CH3-Trp and 7-CH3- As reported in the literature, 6-CH3-Trp is used as an example of þ Trp). Details are in the Supporting Information, section 3.3.2 weak ligand (KD = 37 μM) and 7-CH3-Trp is used as an example 3 5 of a nonbinding compound. À Using this system as a model, we The STD Build-Up Experiment and Ligand Mapping will demonstrate the application of STD-NMR to The general setup of the STD-NMR experiment was followed • A ligand-based NMR screening experiment to determine in and a set of 10 STD-NMR experiments were performed using a qualitative manner which compound binds to the protein sample C (200 μL of 6-CH3-D,L-tryptophan was added to 200 μL 2,6,7 in the context of drug discovery. of HSA stock solution and 100 μL of buffer solution). The • Ligand mapping: a more advanced example of the use of saturation times were 0.50, 0.75, 1.00, 1.25, 1.50, 2.00, 2.50, 3.00, NMR for a direct characterization of protein ligand inter- 4.00, and 5.00 s. Details are in the Supporting Information, À actions at the molecular level through the identification of section 3.3.3 important ligand moieties.2,8,9 Determination of KD • The determination of the dissociation constant (KD) between the protein and the ligand. The protein was titrated with 6-CH3-Trp and the general setup for the STD-NMR experiment was followed with samples One of the many advantages of this experiment is that it does D1 to D7 (10 200 μL of 6-CH -D,L-tryptophan was added to not require the use of high-field spectrometers (400 MHz was 3 200 400 μLHSAand290À 0 μLbuffer solution), corresponding used in this work) and therefore should be possible to implement to aÀ ligand excess from 5- toÀ 100-fold. Details are in the Supporting in most colleges and universities with access to a NMR facility. Information, section 3.3.4 The explanation of the STD-NMR experiment was kept very simple to be accessible to different student levels. More details about the outcome and limitations of the STD-NMR experiment ’ HAZARDS can be found in the Supporting Information, section 2.2. There are no significant hazards in running this laboratory. Local safety rules in the NMR lab should be followed. ’ EXPERIMENTAL PROCEDURE ’ RESULTS AND DISCUSSION Preparation of the Samples The STD-NMR Ligand Based-Screening Experiment HSA was purchased from Fluka; 6-methyl-D,L-tryptophan and 7-methyl-D,L-tryptophan were purchased from Sigma. A 50 μM The STD-NMR spectrum obtained for the mixture of HSA, ff 6-CH3-Trp, and 7-CH3-Trp and the reference spectrum for the HSA stock solution was prepared in a phosphate bu er in D2O (75 mM potassium phosphate, 150 mM sodium chloride at pH mixture under study are shown in Figure 2. The interpretation of this experiment is straightforward; in the STD-NMR spectrum, 7.5). For both ligands, 5 mM stock solutions in DMSO-d6 were prepared. Samples for NMR analysis were prepared from the strong STD signals from 6-CH3-Trp are readily observable, stock solutions as described in the Supporting Information, which indicate that this is an active ligand, whereas the absence section 3.1 of STD signals from 7-CH3-Trp is in accordance with the fact that this compound does not interact with the protein. This NMR Experimental Details and General Setup of the STD- example shows how easily the detection of low-affinity com- NMR Experiment pounds can be achieved with STD-NMR in a screening context, NMR spectra were acquired in a Bruker Avance III spectrometer analogous to the one used to screen compound libraries for operating at a proton frequency of 400 MHz with a conventional ligands in the drug-discovery process.

991 dx.doi.org/10.1021/ed101169t |J. Chem. Educ. 2011, 88, 990–994 equal intensity on the on-resonance and the off-resonance spectra and on signal will be oberved after subtraction. Figure and caption taken from [198]

4.2 Materials and Methods

4.2.1 Experimental Design

Shown below is a summary of various steps of study the effects of myricetin on the aggregation of Aβ (1-42).

4.2.2 Synthesis and Purification of Wild Type Aβ(1-42)

Wild type (WT) Aβ(1-42) was synthesized and purified in our laboratory.

Synthetic Aβ peptides were prepared using standard Fmoc chemistry on an automated synthesizer (Applied Biosystems 433A). The primary amino acid sequence for the amyloid Aβ(1–42) peptide is the following: H3N+-D1-A-E-F-R5-H-D-S-GY10-E-V-H-H-

Q15-K-L-V-F-F20-A-E-D-V-G25-S-N-K-G-A30-I-I-G-L-M35-V-G-G-V-V40-I-A42-COO-.

Peptide mass, purity were determined by a combination of matrix-assisted laser

82 desorption/ionization time-off light mass spectrometry (MALDI-MS), electrospray ionization mass spectroscopy (ESI), analytical and preparative high performance liquid chromatography (HPLC). Purified peptides were aliquoted, lyophilized, and stored at -20

°C until used. Aβ(1-42) was also obtained from Bachem (Torrance, CA). Site- specific15N-labeled peptides were prepared by chemical synthesis using 15N-labeled

Fmoc protected amino acids (Cambridge Isotopes) and uniformly (>95%) 15N-labeled peptides were obtained from R-Peptide (Bogart, GA).

4.2.3 Preparation of Aβ (1-42) Solutions

Lyophilized Aβ (1-42) peptide was disaggregated by thoroughly dissolving the peptide in dilute NaOH solution (10 mM) with sonication for 1 min.[199] This disaggregation procedure, which removes potential interference from small aggregates (“seeds”), is vital for obtaining consistent results and overcomes many of the lot-to-lot discrepancies frequently encountered with Aβ(1-42) peptides. [200] The basic pH solution of Aβ(1-42)

(10 mM) was then combined directly with a potassium phosphate-buffered solution (10 mM, pH 7.3) to yield a final stock solution (8 ml) with a peptide concentration of 50 µM.

Myricetin was dissolved in NaOH (10mM) to make a stock solution at a concentration of

0.5~2.5mM and the stock solution was later combined with Aβ(1-42) to make a final solution of Aβ (1-42) and myricetin. The pH of the final solution was checked and, if needed, carefully adjusted to pH 7.3 with dilute NaOH or trifluoroacetic acid solutions.

All of the peptide final solutions contained 0.05mM ethylenediamine tetraacetic acid

(Na2EDTA-d12) and 0.05mM NaN3 to remove metal contaminants and avoid bacteria growth, respectively.

4.2.4 Circular Dichroism Spectroscopy

The CD spectra were obtained at room temperature (22°C) on a J-810 spectropolarimeter (Jasco) using a 1-mm path length cell (Hellma). Five scans were acquired and averaged with 0.2-nm resolution, a 2-s response time, and 50nm/min scan speed and from 190 to 250 nm.

4.2.5 Thioflavi-T Binding

Myricetin and ThT were obtained from Sigma-Aldrich. The peptide solutions with or without myricetin were incubated at room temperature. During incubation, the peptide solutions were prepared at 25-50 µM, with the majority of experiments done at 25 µM.

Myricetin was prepared at 100-500 µM, with the majority of experiments done at 500

µM. ThT fluorescence measurements were performed on a Cary Eclipse spectrophotometer (Varian) using excitation and emission slit widths of 5 nm.

Fluorescence intensity was measured at excitation and emission wavelengths of 450 and

482 nm, respectively. [201] At specific time intervals, aliquots (20 µl) were removed from the Aβ(1–42) peptide solutions, thoroughly mixed with a ThT solution (1 ml, 4 µM) containing potassium phosphate buffer (10 mM, pH 7.4), followed by immediate measurement of the fluorescence.

4.2.6 Electron Microscopy

For negative staining, 5 ml aliquots of the undiluted samples and 45 ml phosphate buffer (20 mM) were adhered to Formvar/carbon coated 400 nickel grids, stained with

2% uranyl acetate for a minute, air-dried over night and examined in a JEOL 1200EX electron microscope.

4.2.7 NMR Spectroscopy

All NMR spectra were acquired at 5°C using Bruker Advance-II 900 MHz spectrometer or Bruker DRX-800 spectrometers with a TXI cryoprobe (Bruker BioSpin,

Inc., Billerica, CA). Stock solutions of myricetin (2.5 mM), and monomeric, uniformly

15N-labelled Aβ(1-42) peptide (0.25 mM) (rPeptide, Bogart, GA) were prepared by dissolution (with sonication) in aqueous basic solution (pD 11, 1 ml, 10 mM NaOD).[202]

Aliquots of the Aβ(1-42) and phenolic solution were combined and mixed with cold (5°C) phosphate buffer solution (0.5-1.0 ml, 5 mM, pH 7.5) that contained 0.5 mM predeuterated ethylenediamine tetraacetic acid (Na2EDTA-d12), and 0.05 mM NaN3. The aliquots were varied so that the final peptide:myricetin concentration ratio was 25 µM :

500 µM. To prevent aggregation, peptide solutions were kept cold (5°C) and standard 1H-

15N HSQC spectra were obtained within 30-min of the sample preparation. Spectra were obtained at 5oC and were processed with the NMRPipe and CARA (http://cara.nmr.ch) programs using a PC computer. The primary amino acid sequence for the site-specific 13C labeled Aβ(1–42) peptide is

The saturation transfer difference (STD) experiments were obtained with the

Aβ(1-40) (25µM) alone or with Myr (50µM) at pH 7.5 and 5 °C. All experiments were

85 performed on a Bruker 900 spectrometer with a cryoprobe tuned at 900.18 MHz using the pulse program (selective irradiation)-(non selective excitation)-(watergate)-(acquisition)

[203, 204]. The selective irradiation used a 3.9-s long and weak pulse that was alternatively applied at -0.2 ppm (where there was a peak) and at 30 ppm (where there was no peak). And the latter constituting the reference spectra. The non-selective excitation was achieved with a 90° pulse at the water position (4.7 ppm). For the

Watergate, 3-9-19 pulse sequence was used to suppress the water signal. Each spectrum was acquired with 128 scans (12 min) and stored separately. The STD was obtained by subtracting the reference spectra from that obtained with irradiation at -0.2 ppm. All

NMR spectra were processed with the NMRPipe [205], Mnova, or CARA programs using a PC computer.

4.3 Results

4.3.1 Secondary Structural Analysis of Aβ (1-42) and Myricetin

CD was used to determine the structural change of Aβ(1-42) in the presence of the myricetin. Samples of Aβ(1-42) incubated without or with myricetin were taken at 0, 48,

72, 96, 120, and 168 h of incubation. Because the Aβ(1-42) structure and aggregational properties are highly concentration-dependent, a 25µM peptide concentration was selected to allow easy monitoring of conformational changes by CD. [202] To ensure that the two solutions were identical, they were prepared from an identical stock solution.

As shown in Figure 4.5, after aging for 0 h both peptides adopted a predominantly random structure as indicated by the major negative bands at 198 nm. After 96 h of aging, the CD spectra of Aβ(1-42) demonstrated that a conformational change occurred in which

86 the 198 nm band disappeared and positive and negative bands at 195 and 214 nm appeared, consistent with β-sheet structure. This random to β-sheet conversion agrees with previous observations.[206-208] By contrast, after 168 h Aβ(1-42) incubated with myrcetin remained predominantly random, with a modest reduction in the intensity of the

198 nm band. These results suggested that myricetin hinders the random to β-sheet conversion associated with the Aβ-aggregation process and Aβ neurotoxicity. [209]

Figure 4.5 Time-dependent Effects of Myricetin (different concentrations) on the β-

Aggregation Rates of Aβ (1–42) (25uM). (A) Aβ (1–42) alone. (B) Aβ (1–42) with 25

89 uM myricetin. (C) Aβ (1–42) with 50 uM myricetin. (D) Aβ (1–42) with 100 uM myricetin. (E) Aβ (1–42) with 250 uM myricetin.

4.3.2 Morphological Studies of Aβ(1-42) and Myricetin Solutions

To test whether myricetin affects Aβ(1-42) fibril morphology, we used EM, a well- established technique for elucidating the fibril structures. As shown in Figure 4.6A and

Figure 4.6C, at 0 h, the Aβ(1-42) solution with and without myricetin gave virtually identical EM data, with essentially no detectable fibrils but small aggregates. However, samples aged for 48 h gave a distinct set of data: Aβ(1-42) without myricetin produced fibrils (Figure 4.6B). These fibrils had heights that varied from 5.3-8.7 nm. The Aβ(1-42) with myricetin produced no fibrils but only small globular structures (Figure 4.6D).

These globular structures had smaller dimensions and variable heights (0.9–3.1 nm).

Figure 4.6 Representative EM images of Aβ(1-42) aged for (A) 0 h without myricetin,

(B) 48 h without myricetin, (C) 0 h with myricetin and (D) 48 h with myricetin at 22°C.

4.3.3 Time-dependent Effects of Myricetin on the β-Aggregation Rates of Aβ

(1–42)

To determine if myricetin slows down β-aggregation not only at an early (random to

β-sheet) or/and later stage (β-sheet (soluble) to β-sheet (amyloid fibril)), we undertook more detailed studies. These involved adding myricetin to Aβ(1-42) at different time points during sample aging and monitoring the secondary structure and β-aggregation rates using ThT binding assay.[210] Shown in Figure 4.7A is an outline of the overall

91 procedure. A freshly prepared Aβ (1–42) solution was split into five equal fractions, and these solutions were then monitored by ThT for a period of up to 72 h. Fraction 4 represents the control, whereas myricetin was added to fractions 2, 3, and 4 at 0, 36, 72 h of aging, respectively. ThT results for all four fractions are shown in Figure 4.7B.

The ThT data shows comparable sigmoidal β-aggregation curves (Figure 4.7B) in which the samples with myricetin have reduced fluorescence because of their lower fibril content. These data establish that myricetin hinders β-amyloidosis (random to β-sheet

(soluble) to β-sheet (fibril)), regardless of the predominant solution conformation.

Figure 4.7 Time-dependant Effects of Myricetin (Myr) on the β-Aggregation Rates of

Aβ(1–42). A, flow chart summarizing the partitioning of a single Aβ(1–42) solution (25 uM, pH 7.3) into four equivalent fractions. For fractions 1, 2, and 3 the time at which oxidation was commenced is also provided. Each fraction was monitored over time by

ThT. B, The ThT fluorescence data shows that Myricetin causes a significant reduction in

β-sheet production.

The change of aggregation state by myricetin was ascertained by SDS-PAGE. Figure

4.8 shows the SDS-PAGE of the samples with and without myricetin. SDS stable Aβ oligomers with molecular weights ranging from 8 to 20 kDa were detected in the three samples without My. By Contrast, minor oligomers were detected with the samples with

Myr, and therefore Myr removes the SDS stable aggregates.

· Figure 4.8 SDS-PAGE of Aβ (1-42) aggregates before and after adding Myr. The Aβ (1-

42) was incubated at 6, 24, 48 and 72 h (lane A, C, E and G, respectively, labeled with

Myr ‘-’). Portion of 6, 24, 48, 72 h aged samples were incubated with Myr (4h) (lines B,

D, F and H, respectively, labeled with Myr ‘+’). Molecular standards are indicated in the lane on the left.

4.3.4 Aβ (1–42) Oxidation by Hydrogen Peroxide Produced in the Presence

of Myricetin

Most of polyphenols including myricetin are potent antioxidants, but why does the presence of myricetin induce the oxidation of the protein under the incubation conditions?

It was reported that hydrogen peroxide (H2O2) is formed during incubation of polyphenols, such as myricetin[187] and epigallocatechin gallate (EGCG). [188] Previously, our group has demonstrated that the Met35 side chain would be oxidized by the addition of H2O2, and the Met35red ! Met35ox conversion prevents aggregation by reducing both

94 hydrophobic and electrostatic association and that the Aβ (1-42) Met35red and Aβ (1-42)

Met35ox [211] may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.[211] Thus, hydrogen peroxide produced during the auto oxidation process of myricetin is a possible intermediate that directly oxidizes methionine residue in Aβ (1-42), and the auto oxidation of myricetin is possibly a reason in the process of inhibiting the nucleation and early fibrillation of Aβ (1-42) aggregation.

Therefore, the AmplexRed hydrogen peroxide/peroxidase assay kit was used to monitor the hydrogen peroxide formation during the incubation of inhibitory flavonoids in the absence and the presence of Aβ (1–42).

As shown in Figure 4.9A, 100 µM myricetin alone generated H2O2 rapidly during the first few hours of incubation. The amount of produced H2O2 was stable during the remaining incubation time. On the other hand, for 25 µM Aβ (1–42) alone, none H2O2 was generated. For the sample of 100 µM myricetin in the presence of 25 µM Aβ (1–42), the kinetics of H2O2 formation was similar to that of the flavonoid alone. ThT fluorescence results were shown 4for comparison. (Figure 4.9B)

Figure 4.9 Hydrogen peroxide formation during Myricetin oxidation. (A)The kinetics of

H2O2 formation during the auto-oxidation of Myricetin in the absence and in the presence of Aβ (1–42). (B) The kinetics of Aβ (1–42) (25µM) fibril formation in the absence and in the presence of Myricetin, by ThT fluorescence assays were shown for comparison.

4.3.5 The Effect of Catalase on the Myricetin-Induced Inhibition of Aβ (1–

42) Fibrillation

To investigate whether Met35 oxidation is a prerequisite of flavonoid-induced inhibition of Aβ (1–42) fibrillation, catalase (a well-known H2O2 scavenger) was added during the incubation of Aβ (1–42) with myricetin. The presence of 1 µM catalase did not significantly affect either the kinetics of Aβ (1–42) fibrillation or the myricetin-induced inhibition of Aβ (1–42) fibrillation (Figure 4.10). However, this amount of catalase was sufficient for both the successful prevention of the H2O2 formation during the incubation of myricetin and for the prevention of the Met oxidation of Aβ (1–42) (confirmed by MS, data not shown). Since the presence of catalase did not affect myricetin-induced inhibition of Aβ (1–42) fibrillation, Met oxidation in Aβ (1–42) was not a prerequisite for the myricetin-induced inhibition of the protein fibrillation.

4.3.6 The Effect of Auto-oxidation of Myricetin on the Myricetin-Induced

Inhibition of Aβ (1–42) Fibrillation

To investigate whether the auto-oxidation of myricetin was required for inhibiting Aβ

(1-42) aggregation, the CD experiment was carried out under the anaerobic conditions, at which the auto-oxidation is mostly prevented. As shown in Figure 4.11A, after 168 h Aβ

(1-42) incubated with myrcetin remained predominantly random, with a modest reduction in the intensity of the 198 nm band. By contrast, after 96 h of aging, the CD spectra of Aβ

(1-42) under anaerobic condition demonstrated that a conformational change occurred.

These results suggested that auto-oxidation of myricetin was a prerequisite for the myricetin-induced inhibition of the protein fibril.

Figure 4.11 Effect of oxygen on Aβ (1–42) fibrillation. (A) Data for the Aβ (1–42) fibrillation in the presence of myricetin and oxygen is different (B) the data of Aβ (1–42) fibrillation under anaerobic condition.

4.3.7 Examination of Aβ (1-42)-Myricetin Interaction by NMR

To investigate the effect of myricetin on the inhibition of Aβ (1-42) fibrillation, 500

µM myricetin was added to the 25 µM Aβ (1-42) solution, and was explored using NMR spectroscopy, a well-accepted tool for obtaining atomic level aspects of protein structure and ligand binding. The sample preparation protocol (see Experimental Procedures) and lower NMR probe temperature (5°C) ensured that the Aβ (1-42) remained monomeric during the entire data acquisition period.[202, 212] Standard heteronuclear single quantum coherence (HSQC) spectra were obtained with uniformly 15N-labeled Aβ (1-42).

The HSQC experiment detects 1H signals that are directly bonded to the 15N atoms, and thus provides a fingerprint of the amide-NH backbone atoms.

100

Figure 4.12 Time-dependant heteronuclear single quantum coherence (15N HSQC) of uniformly15N labled Aβ (1-42) (25 µM, 25 °C, pH 7.3). All the spectra (900 MHz) were obtained at 5 °C using 25 µM samples in buffered (10 mM sodium phosphate, pH 7.3) aqueous solution (9:1, H2O:D2O) with 0.50 mM Na2EDTA and 0.05 mM NaN3. On the left, Aβ (1-42) 0 hour (black peaks) and Aβ (1-42) 72 hours were superimposed. The cross-peaks represent intraresidue couplings between the HR and NH. One the right, Aβ

(1-42) 0 hour (black peaks) and Aβ (1-42) 8 days (green peaks) were superimposed.

101

Figure 4.13 Time-dependant effect of myricetin (500µM) on the heteronuclear single quantum coherence (15N HSQC) of uniformly 15N labeled Aβ (1-42) (25 µM, 25 °C, pH

7.3). The 15NH peaks showing different chemical shifts between 0 hour and

72hours/8days are labeled. These data show that myricetin induced the oxidation of the

Met35 side chain that promotes shift changes of the Met35-Val36-Gly37-Gly38-Val39 in

Aβ (1-42).

Shown in Figure 4.12 are HSQC spectra recorded at different aging time intervals for the Aβ(1-42). Figure 4.12A is superimposed 15N HSQC spectra of the Aβ (1-42)

102 alone in 0 hours (black crosspeaks) and 72 hours (red crosspeaks). Figure 4.12B is superimposed 15N HSQC spectra of the Aβ (1-42) alone in 0 hours (black crosspeaks) and 8 days (green crosspeaks). Due to the sample aggregation and precipitation, the intensity of all the HSQC signals decreased with longer incubation time and changes on the NH chemical shift were not observed. By contrast, the HSQC spectra of Aβ (1-42) with myricetin showed some NH chemical shift movements (labeled peaks in Figure

4.13) indicative of changes. The most pronounced movements (0.02-0.05 ppm) were double NH peaks at residues Met35-Val36-Gly37-Gly38-Val39 after 72 hours of aging, and the intensities of the two peaks for Met35-Val36-Gly37-Gly38-Val39 showed altered intensities after 8 days incubation, with one peak becoming weaker and eventually disappearing, while the other peak grew stronger in intensity. Our suspicion was that the

Met35 side chain might become oxidized in the present of myricetin. The Met side chain is very sensitive to oxidation, as even atomospheric oxygen can oxidize its side chain from the reduced thioether (-CH2-S:-CH3) to the sulfoxide [-CH2-(S=O)-CH3].[213] In addition, our group previously has observed the oxidation of Met side chain by directly adding hydrogen peroxide (H2O2, 30% wt.) into the Aβ (1-42) NMR sample (0.6ml) and

1 15 running H- N HSQC after the addition of H2O2. The Met35 side chain oxidized peak came out, and the shift changes of the Leu34-Met35-Val36-Gly37-Gly38-Val39 segment located next to Met35 were also observed. The overlaid HSQC spectrum of 15N-labeled

Aβ (1-42) with reduced and oxidized states (by adding H2O2) is essentially the same as the HSQC spectrum in Figure 4.13. In Figure 10A, Met 35 side chain is partial oxidized, while in Figure 4.13B, Met 35 side chain is totally oxidized.

103

To investigate whether Met35 oxidation is the only flavonoid-induced modification of Aβ (1–42) and aggregates, Aβ (1-42) was selectively 13C labeled at

Lysine (K16, K28), Methionine, Glycine (G9, G25, G29, G33, G27, G38), and Alanine

(A2, A21, A30), and was explored by 1H-13C HSQC spectroscopy, a well-accepted tool for obtaining atomic level aspects of protein structure and ligand binding. The overlaid

HSQC spectrum of selectively 13C-labeled Aβ (1-42) with and without myricetin is essentially the same as the HSQC spectrum in Figure 4.14 and Figure 4.15, which indicates that Schiff base did not form between quinone and Aβ (1-42).

104

7.3) aqueous solution (9:1, H2O:D2O) with 0.50 mM Na2EDTA and 0.05 mM NaN3. (A)

Aβ (1-42) 0.5 hour (black peaks) and Aβ (1-42) 72 hours were superimposed. The crosspeaks represent intraresidue couplings between the HR and CH. (B) Aβ (1-42) 0 hour

(black peaks) and Aβ (1-42) 8 days were superimposed.

105

Figure 4.15 Time-dependant effect of myricetin (500µM) on the heteronuclear single quantum coherence (13C HSQC) of uniformly 13C labeled Aβ (1-42) (25 µM, 25 °C, pH

7.3). The 13CH peaks showing different chemical shifts between 0 hour and

72hours/8days are labeled. These data show that myricetin induced the oxidation of the

Met35 side chain.

106

Figure 4.16 Effect of myricetin (500µM) on the electrospray ionization mass spectra

(ESI-MS) of Aβ (1-42) (25µM) incubated at the room temperature (25°C) for 8 days.

To confirm Met35 was the only modification by incubating with myricetin, sample from NMR tube was analyzed by mass spectroscopy (MS). The ESI MS spectrum on the mixture of the non-treated (Figure 4.16) of 15N-labeled Aβ (1-42) showed a difference of

15.5Da in the m/z of the two peptides.

The possibility of weak binding or interaction between myricetin and Aβ were further explored using Saturation Transfer Difference (STD) experiments, which is a well established homonuclear NMR technique that permits detection of transient binding of small molecule ligands to macromolecular receptors. Notably, the STD has been used to discriminate ligand binding to monomeric or oligomeric states of the Aβ that co-exist in solution[204, 214, 215]. In the STD, two separate spectra are obtained and then subtracted, where one spectra involves saturation of a resonance that belongs to the receptor (in this case the Aβ), whereas the second spectra involves saturation in a far- removed region that does not contain signals. The presence and strength of signals in the

STD is indicative of binding.

107

Shown in Figure 15 are two groups of spectral data: Aβ (1-40) alone (Figure 4.17, A and B), Aβ40 with Myr (Figure 4.17, C and D). The STD spectrum of Aβ (1-40) alone

(Figure 4.17B) has no signals. In contrast, spectra containing Myr (Figure 4.17D) has weak peaks. Because solution NMR detects only monomeric Aβ [202, 204], these data demonstrated that Myr binds weakly to monomeric Aβ.

108 with irradiation at -0.2 ppm. (C) Reference spectrum for Aβ40 and Myr (50 µM), and (D)

STD spectrum for Aβ40 and Myr. Picture and caption taken from [216].

4.4 Discussion

Recent epidemiological studies have documented the consistent protective effect of polyphenol rich diet on AD. [217] Studies by our collaborators Dr. Ono and colleagues at the Kanazawa University in Japan have demonstrated that certain naturally occurring polyphenols from grapes and red wine prevent aggregation of Aβ peptide.[218] It was also reported by Dr. Ono and colleagues that Myr reduced the amount of Aβ oligomers in the brain of Tg2576 mice and prevented the development of AD pathology.[219]

Ono and colleagues believe that some interesting structure-activity relationships of polyphenols could be considered. Firstly, the structure of the superior polyphenols

(myricetin, morin and quercetin) is not chiral. Their two rings, benzopryn ring and hydroxyphenyl ring, are able to be located on the same plane by rotation. Secondly, myricetin, morin and quercetin have both hydroxyl and hydrophobic and both properties are important for the anti-fibrilization and fibril-destabilization activity because the number of hydroxyl groups in the molecule is proportional to the activity and because hydrophobic interactions (binding) block associations between Aβ molecules leading to

Aβ fibril formation. [220, 221]

Another reason for polyphenols’ anti-fibrilization and fibril-destabilization activity is their antioxidant activity. Polyphenols have been reported to have antioxidant activity

[222]. It has been reported that major red wine–derived polyphenols are capable of both

109 protecting and rescuing cultured rat hippocampal cells against nitric oxide-induced toxicity. [223]

Also, Virgili and Contestabile et al., showed that chronic administration of resveratrol to young-adult rats significantly protects from the damage caused by systemic injection of the excitotoxin kainic acid, in the olfactory cortex and the hippocampus.[224]

Moreover, it has been demonstrated that flavonoids and some metabolites are able to traverse the blood-brain barrier. [225] Ono and his colleagues demonstrated that cell culture experiments with HEK 293 cells suggested that Aβ fibril treated by myricetin might be less toxic than intact Aβ fibril. [218] Thus, it is speculated that red wine-derived polyphenols could prevent the development of AD, not only through scavenging reactive oxygen species, but also through directly binding to specific sites of Aβ and Aβ aggregates in the brain. The exact underlying mechanism of their anti-fibrilization and fibril-destabilization activity, however, remains unknown.

In previous study, myricetin showed prominent anti-amyloidogenic and fibril- destabilizing activity among the polyphenols examined, so we choose myricetin as our subject to study the underlying mechanism of polyphenol-induced inhibition of Aβ (1-42)

Fibrillation. The CD results (Figure 4.5) suggested that myricetin hinders the random to

β-sheet conversion associated with the Aβ-aggregation process and Aβ neurotoxicity.[209] The EM results (Figure 4.6) demonstrated the same conclusion, but in morphology level. To investigate that Myricetin slows down β-aggregation not only at an early event (random to β-sheet) but also at later stages (β-sheet (soluble) to β-sheet

(amyloid fibril)), we undertook more detailed studies. The ThT data shows comparable sigmoidal β-aggregation curves (Figure 4.7B) in which the samples with myricetin have

110 reduced fluorescence because of their lower fibril content. These data establish that myricetin hinders β-amyloidosis (random to β-sheet (soluble) to β-sheet (fibril)), regardless of the predominant solution conformation. SDS-PAGE further confirmed this result.

Polyphenols have antioxidant properties and they are easily oxidized to the corresponding quinones with dioxygen, during which, H2O2 is formed. [187, 188] Figure

4.9 shows that 100 µM myricetin alone generated H2O2 rapidly during the first few hours of incubation. The amount of produced H2O2 was stable during the remaining incubation time. For the sample of 100 µM myricetin in the presence of 25 µM Aβ (1–42), the kinetics of H2O2 formation was similar to that of the flavonoid alone.

It has been demonstrated that oxidation of the Met-35 side chain to a methionine sulfoxide (Met35ox) significantly hinders the rate of fibril formation for the Aβ (1–42) at physiological pH and that Met35ox also alters the characteristic Aβ fibril morphology and prevents formation of the protofibril, which is a key intermediate in β-amyloidosis and the associated neurotoxicity. [211] On the other hand, Met oxidation promotes the disruption of Aβ aggregates. [226] Figure 4.18 showed that Met oxidation modulates fibril formation and aggregation of Aβ. More importantly, the Met oxidation promotes the morphological changes of plaques from human brains, indicating that the stabilizing interactions of in vitro and in vivo fibril structures are comparable. [227, 228]

The present data demonstrate that the Met35 oxidation by myricetin not only hinders

β-aggregation, but also disrupts formed aggregates. Similarly, fibril formation and aggregation of other amyloid forming proteins, including transthyretin, α-synuclein, prion and apolipoprotein C, is modulated by methionine oxidation. It has been

111 demonstrated that the locations of the Metred and Metox side chains are different and can vary with aggregation state. Methionine has been observed as solvent exposed in the

NMR solution structure of monomeric Aβ bound to an engineered affibody protein [229], while Metred has been observed as packed in a hydrophobic interior in a NMR-derived models of early-formed aggregates (preglobulomer and globulomers) [230] and solid state NMR data of the amyloid fibrils [231, 232]. When Methionine is oxidized, bond polarity and dipole moment are promoted and sulfur atom is allowed to act as a hydrogen bond acceptor [233]. All these changes can weaken hydrophobic interactions, which drives the β-aggregation. Therefore, the more polar Met35ox which favors inclusion of water molecules near the hydrophobic interior [234], might in turn break apart hydrophobic interactions such as the intermolecular Met35-Met35 hydrophobic contacts among the β-sheets of the fibrils [235]. A similar example is that the oxidation of a methionine residue in the hydrophobic core in apolipoprotein C-II inhibited amyloid fibril assembly [236].

112

Figure 4.18 Representative EM images of Aβ(1-42) Met35red (100 µM, pH 7.3) aged for

(A) 24 h, (B) 48 h, and (C) 96 h at 22◦ C and after the treatment with H2O2 for 24h (D, E, and F, respectively.). At 24h, the heights were within 2.3–12.5 nm, while at 48 and 96h the average heights are 16.7 and 24.9 nm. After H2O2 addition and aging for 24 h, the heights became 3.7–7.9 nm.

113 of myricetin and for the prevention of the Met oxidation of Aβ (1–42) (confirmed by MS, data not shown). Since the presence of catalase did not affect myricetin-induced inhibition of Aβ (1–42) fibrillation, Met oxidation in Aβ (1–42) was not a prerequisite for the myricetin-induced inhibition of the protein fibrillation.

To investigate the underlying mechanism of myricetin on the inhibition of Aβ (1-42) fibrillation, myricetin was added to the Aβ (1-42) solution, and was explored using NMR spectroscopy, a well-accepted tool for obtaining atomic level aspects of protein structure and ligand binding. Series of HSQC spectrum (Figure 4.12, Figure 4.13, Figure 4.14,

Figure 4.15) showed that, at the atomic level, Myr does not bind to the monomer Aβ, indicating that Myr could prevent aggregation by binding with non-NMR detectable early formed oligomers (dimmer, trimer, etc). [237] Another possible explanation is that Myr binds to distinct monomer structures that in turn inhibit oligomerization. [238]

Related work reported a number of factors that lead to inhibition of α-synuclein fibrillation have been identified, such as chemical modifications of the protein through methionine oxidation.[239] It was also proposed that the stabilized soluble species were a result of the oxidized baicalein (probably the quinone(s)) leading to the inhibition of fibrillation by Schiff Base formation.[240] Amyloid fibrils made of α-synuclein are the main protein constituents of Lewy bodies accumulated inside of dopaminergic neurons in the brains under pathological conditions such as Parkinson’s disease.[241] The α-

Synuclein fibrils have similarity to all amyloid fibrils core made of a cross-beta structure.[242, 243]

The 1H-13C HSQC HSQC showed that Met35 oxidation is the only flavonoid-induced modification of Aβ (1–42) monomers. The overlaid HSQC spectrum of selectively 13C-

114 labeled Aβ (1-42) with and without myricetin (by H2O2) is essentially the same as the

HSQC spectrum in Figure 4.14 and Figure 4.15, which indicates that Schiff base did not form between quinone and Aβ (1-42). The ESI-MS spectra confirmed that Met 35 was the only residue modified by incubating with myricetin. ESI MS spectrum on the mixture of the non-treated (Figure 4.16) of 15N-labeled Aβ (1-42) showed a difference of 15.5Da in the m/z of the two peptides.

STD studies demonstrated that Myr, which does not bind with monomer Aβ, however weakly interacts with monomer Aβ. It is also possible that Myr inhibits aggregation by stabilizing oligomers. However, doing so could produce peptide populations of enhanced toxicity. So the observation that Myr interacts weakly with Aβ monomer and prevent its oligomerization is a more feasible feature. [244]

All of these factors rather than a single one contribute to the inhibition of Aβ fibrillation induced by the myricetin. Taken together our data suggested that the oxidation by myricetin to Met35 and weak interactions between myricetin quinone and Aβ hinder

Aβ aggregation and induces fibril disaggregation as revealed by mostly CD, ThT, ESI

MS, EM, SDS-PAGE and NMR. The myricetin inhibiting Aβ fibrillation can serve as a model for the development of therapeutic drugs in combating Alzheimer’s disease.

115

CHAPTER 5

CONCLUSIONS AND FUTURE DIRECTIONS

5 Conclusions and Future Directions

116

The work described in this thesis is to obtain in-cell nuclear magnetic resonance

(NMR) spectra of the Aβ peptide, and to elucidate the molecular mechanism of the polyphenol-based inhibition to Aβ aggregation.

NMR studies were conducted with cell lysate. These studies involved HSQC of cell lysate and cell lysate treated with nuclease. Analysis of these spectra showed that the Aβ

117 peptide did not bind to cell membranes during and after delivery into the cells and that the nuclease does not change the viscosity to alter the conformation and/or mobility of the peptide. To examine the influence of survival rate of cells on the structure of Aβ(1-

ESM-mass spectrometry (MS). [245]However, when catalase (an H2O2 scavenger) was present in the mixture, the myricetin-induced inhibition of Aβ(1–42) fibrillation was not altered, suggesting that oxidation of the Met35 side chain by H2O2 was not the only factor to alter Aβ(1–42) fibrillation. CD experiments carried out under anaerobic conditions suggested that auto-oxidation of myricetin was a prerequisite for the myricetin-induced inhibition of the protein fibril. Moreover, the overlaid HSQC spectrum of selectively 13C-labeled Aβ(1-42) with and without myricetin is essentially the same, which indicates that Schiff base formation between the quinone section of

118 myricetin, and the Aβ(1-42) did not occur. However, saturation transfer difference (STD)

NMR studies demonstrated that myricetin weakly interacts with monomeric Aβ(1-40).

All of these factors rather than a single one contribute to the inhibition of Aβ fibrillation induced by the myricetin.

Myricetin has antioxidant, anti-inflammatory, anticarcinogen, and antiviral ativities

[246], and can likewise act as a β-secretase inhibitor that reduces Aβ production in a cell cultures [247]. In fact, it has been now established firmly that all polyphenols have related biological activities, most of which are beneficial in vivo.[216] It has been reported that polyphenols also protect neurons against Aβ-induced oxidative stress and neurotoxicity in vitro [248], and long term administration protects mice against Aβ- induced learning and memory deficits in vivo. It is possible that the beneficial effects may not come from the polyphenols themselves but rather from polyphenol metabolites, which can be generated by microbial enzymes in the colon [249]. Once produced, these metabolites could enter the brain and reduce the Aβ aggregation associated with AD.[216]

It would be necessary and important to study the inhibition effects of the other polyphenols on the aggregation of Aβ and associated detailed mechanisms. These studies can serve as a model for the development of therapeutic drugs in combating Alzheimer’s disease.

In conclusion, the first part of this thesis work established optimal conditions for in- cell NMR studies of the Aβ peptides and constitutes the first step for additional work that may determine the structure inside living cells. The second part of this work established

119 that myricetin oxidizes the Met35 side chain of the Aβ peptide which in part inhibits aggregation.

120

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