Protective effects of s propargylcysteine (SPRC) on in vitro neuronal damage induced by amyloid beta (25 35 p 1 3

Figure 24: Effects of pre-treatment of drugs on cystathionine-β synthase CBS expression in cell lysates.. Figure 28: Effects of pre-treatment of drugs on total superoxide dismutase SOD e

PROTECTIVE EFFECTS OF S-PROPARGYLCYSTEINE (SPRC) ON IN VITRO

NEURONAL DAMAGE INDUCED BY AMYLOID-BETA(25-35)

WONG WAN HUI

B.Sc (Hons) National University of Singapore

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2011

DECLARATION

I hereby declare that this thesis is my original work and it has been written

by me in its entirety I have duly acknowledged all the sources of information

which have been used in the thesis

This thesis has also not been submitted for any degree in any university previously

Wong Wan Hui

I would like to thank the following people for making this thesis possible

Firstly, I would like to convey my heartfelt thanks to my supervisor A/P Zhu Yi-Zhun

for his support and guidance the past seven years in his laboratory I have learnt much and grew

to be a better researcher with the independence and freedom he has always granted me Without his encouragement I would never have embarked on this journey of self-discovery and learning

I would also like to thank my mentors Dr Wang Hong, Dr Sonja Koh and Dr Wang Zhong Jing for their patience and guidance Dr Wang Hong had been my mentor in both

research and life I will never forget all the times we spent having lunch together and discussing about a wide range of issues Dr Sonja Koh had made herself always available to answer my doubts Her presence had been inspiring with her creativity and I always felt assured with her around Dr Wang Zhong Jing was the first person who introduced me to the world of hydrogen sulfide and research with his passion He taught me how to think through problems and to

analyse research questions that made me a scientist today For this much guidance from such great scientists, I am most grateful

Thirdly, I would like to extend my gratitude to the Head of Department and his staff from the Department of Pharmacology, Yong Loo Lin School of Medicine I had been in this

department since my undergraduate days I have been nurtured on this unique field of study by wonderful and passionate teaching staff I also had the honour of knowing and working with most of the staff in our department Regardless of their positions, various staff from the

department never failed to make me feel a part of the family, and always ready to extend their

enjoyable with their support

To my fellow seniors and juniors from this laboratory throughout these seven years, it

had been a great honour to know each and every one of them They have given me their

unbridled comments and encouragement through the times at lab meetings They have also inspired me with their perseverance and pulled me along Their friendship and camaraderie accompanied me on the most lonely days

I would also like to thank my family members who have given me their unconditional

support through this arduous time They have never doubted my ability even when I felt lost, and they have given me the motivation to stand up again despite falling down so many times Even through the difficult twelve-hour incubation times when I have to return to office at nights, my family would encourage me whenever I wanted to give up This thesis would not have been possible without them

I want to specially mention my best friends Shi Ping, Shixin and Shu Min for their

encouragement and belief in me throughout this time Even though they might not be in the same field of study, they were willing to lend a listening ear to me whenever I felt down In fact, without Shixin’s encouragement in the form of a bamboo story, I would still be that bamboo farmer who will give up because I cannot see the shoots growing I will also remember the times Shumin and I discuss our research topics over coffee, and how she told me to doubt in order to grow I am most blessed to have these best friends

Lastly, I would like to thank my fiancé Isaac for being with me in this journey He had

brought out the best in me, and allowed me to be myself He was always patient to listen to my

with my difficulties I will never forget the times he painstakingly tried to help me organize my thoughts for this thesis as he struggled to study for his own exams I will always be grateful to his understanding and patience, such that he would wait for me to finish my experiments and accompany me through those rushed dinners I am thankful to have him in this journey and will

be happy to have him for the rest of my life

I am most grateful for this chance to write this thesis and most importantly, a chance to prove myself as a researcher and a person I have grown to be more patient and determined from this experience And most importantly, I have learnt to pick myself up whenever I fall This is something I am sure, will help me through my life

Acknowledgements i

Abstract viii

List of Tables x

List of Figures xi

CHAPTER 1: INTRODUCTION 1

1.1 GENERAL INTRODUCTION 1

1.1.1 Alzheimer’s Disease (AD) 1

1.1.2 Aggregated proteins and diseases 2

1.1.3 Self-aggregation properties of amyloid peptides 2

1.1.4 Factors affecting fibril formation 4

1.2 AMYLOID-BETA PEPTIDE (Aβ) 6

1.2.1 Products of sequential cleavage 6

1.2.2 Neurotoxicity of Aβ25-35 8

1.2.3 Structure of Aβ25-35 8

1.3 GARLIC AND S-ALLY-L-CYSTEINE (SAC) 10

1.3.1 Aged garlic extract 10

1.3.2 S-ally-L-cysteine 10

1.4 S-PROPARGYL-L-CYSTEINE (SPRC) 13

1.4.1 Chemical properties and pharmacokinetics 13

1.4.2 Cardioprotective effects 14

1.4.3 Neuroprotective effects 16

1.5 HYDROGEN SULFIDE (H2S) 17

1.5.1 General properties 17

1.5.2 Synthesis 17

1.5.3 Biological targets of H2S 19

1.5.4 H2S as a neuromodulator 20

1.5.5 H2S in AD 21

1.6 OXIDATIVE STRESS 23

1.6.1 Oxidative balance in the brain 23

1.6.2 Imbalance in the disease state 24

1.6.3 Aβ-induced oxidative stress 25

1.6.4 Antioxidant therapy in AD 29

1.7 INFLAMMATION 31

1.7.2 Role of inflammation in the brain and disease state 32

1.7.3 Inflammatory mediators in AD 35

1.7.4 Therapeutic efficacy of anti-inflammatory drugs 39

1.8 CELL DEATH MECHANISMS 41

1.8.1 Roles of astrocytes in neuroprotection and defense 41

1.8.2 Apoptosis in glial cells 42

1.8.3 The relationship between autophagy and Aβ 47

CHAPTER 2: AIMS AND OBJECTIVES 52

CHAPTER 3: MATERIALS AND METHODS 54

3.1 EXPERIMENTAL PROTOCOLS 54

3.1.1 In vitro study 54

3.1.2 Part II: Oligomeric Aβ25-35 55

3.1.3 Part III: Fibrillar Aβ25-35 55

3.2 IN VITRO STUDY 56

3.2.1 Thioflavin T fluorescence 56

3.2.2 Coomassie Blue Staining 57

3.2.3 Free radical scavenging 57

3.24 Statistical analysis 58

3.3 CELL CULTURE STUDIES 59

3.3.1 Chemicals 59

3.3.2 Cell culture 59

3.3.3 MTT assay 60

3.3.4 H2S pathway 60

3.3.5 Western blot 61

3.3.6 Reactive oxygen species (ROS) generation 62

3.3.7 Inflammation 64

3.3.8 Cell Death Mechanisms 65

3.3.9 Transmission electron microscopy 67

3.3.10 Statistical analysis 67

4.1 RESULTS 68

4.1.1 Aβ25-35 aggregation with time 68

4.1.2 Aβ25-35 aggregation with temperature 69

4.1.3 Effects of SPRC on Aβ25-35 aggregation 70

4.1.4 Effects of SAC on Aβ25-35 aggregation 72

4.1.5 Effects of sodium hydrosulfide (NaHS) on Aβ25-35 aggregation 74

4.1.6 Comparison of equimolar concentrations of drugs 76

4.1.7 Effects on radical scavenging 78

4.2 DISCUSSION 80

4.2.1 Aβ25-35 aggregates with increasing time and temperature 80

4.2.2 Drug treatments disrupt the formation of Aβ25-35 fibrils .81

4.2.3 SPRC reduces Aβ25-35 aggregation more effectively than SAC 84

4.2.4 NaHS confer a Type II protection against aggregation 85

4.2.5 Free radicals were scavenged by drugs in solution 87

4.3 SIGNIFICANCE OF PART I 80

CHAPTER 5: PART II - OLIGOMERIC Aβ .91

5.1 RESULTS 91

5.1.1 Effects of aggregated Aβ25-35 on cell viability 91

5.1.2 Effects of drug pre-treatment on Aβ-induced cytotoxicity 92

5.1.3 Effects on H2S pathway 95

5.1.4 Effects on oxidative stress 98

5.1.5 Effects on inflammation 104

5.1.6 Effects on cell death mechanisms 108

5.1.7 Effects on cell morphology 115

5.2 DISCUSSION 122

5.2.1 Oligomeric Aβ25-35 is toxic to glial cells 122

5.2.2 Pre-treatment of drugs alleviates Aβ-induced injury mediated by the H2S pathway .122

5.2.3 SPRC and SAC can relieve intracellular ROS 125

5.2.4 SPRC restores SOD levels and activities 127

5.2.5 SPRC possesses anti-inflammatory properties different from SAC 129

5.2.6 G1 progression encouraged by SPRC and SAC 133

5.2.8 The autophagic pathway is halted by SPRC and SAC 136

5.2.9 Pre-treated cells had improved cell ultrastructures 137

CHAPTER 6: PART III - FIBRILLAR Aβ 140

6.1 RESULTS 140

6.1.1 Time dependence of aggregated Aβ25-35 on cell viability 140

6.1.2 Effects of drugs on Aβ-induced cytotoxicity 141

6.1.3 Effects on H2S pathway 144

6.1.4 Effects on ROS generation 147

6.1.5 Effects on inflammation 155

6.1.6 Effects on cell death mechanisms 159

6.1.7 Effects on cell morphology 168

6.2 DISCUSSION 176

6.2.1 Higher incubation temperature encourages the formation of Aβ25-35 fibrils 176

6.2.2 SPRC confers protection after longer pre-treatment at higher dose 178

6.2.3 The H2S pathway mediated the cytoprotection by SPRC 180

6.2.4 More oxidative stress was induced by fibrillar Aβ 183

6.2.5 Drug treatments regulated antioxidant enzyme expressions and activities differently .185

6.2.6 SPRC does not involve the H2S pathway its anti-inflammatory effects 190

6.2.7 Fibrillar Aβ25-35 affects autophagy differently from oligomeric Aβ25-35 194

6.2.8 SPRC modifies the autophagic pathway 195

6.2.9 The ultrastructural changes were restored after drug treatments 197

CHAPTER 7: CONCLUSIONS 199

7.1 SIGNIFICANT CONTRIBUTIONS 199

7.2 FUTURE WORK 203

References 205

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by widespread extracellular deposits of amyloid-beta (Aβ) protein in the brain Amongst which, the Aβ25-35peptide is the shortest fragment which retains the toxicity of the full-length protein In this study, this peptide was found to aggregate in a time- and temperature-dependent manner This

aggregation can be slowed with the co-incubation of S-propargyl-cysteine (SPRC), cysteine (SAC) or sodium hydrosulfide (NaHS) in the solution, accompanied with decreased sizes of the Aβ aggregates Aβ radicalizes in solution to cause detrimental damage even outside cells SPRC can scavenge free radicals better than SAC, but less competent than NaHS The triple bond in SPRC is more nucleophilic than SAC that can react with the lone pair of electrons

S-allyl-in free radicals Oligomeric or fibrillar Aβ25-35 were added to the C6 glioma cell line and the cell viabilities were compromised The decline in cell viability was more obvious when treated with fibrillar Aβ25-35, which acted on exacerbating oxidative stress by increasing H2O2 levels,

inflammation and disruption of the autophagic activation Moreover, the aggregated Aβ

decreased the H2S levels produced and the expression of cystathione-β synthase (CBS) in the cells Pre-treatments of SPRC and SAC both restored the Aβ-induced reductions in cell

viabilities, but the doses required to restore the cell viabilities were higher in damage by Aβ fibrils SPRC mimicked the protection by SAC on glioma cells through its antioxidant nature, particularly targeting superoxide dismutase (SOD) and glutathione peroxidase (GPx) in both forms of Aβ injuries SPRC also decreases pro-inflammatory IL-1β and increases anti-

inflammatory IL-10 to reduce the inflammatory responses evoked by Aβ25-35 though differently from SAC SPRC reduces DNA fragmentation and reverses autophagic activation that preserves cellular integrity in a similar manner to SAC These protective effects of SPRC can be due its cysteine backbone similar to SAC and its endogenous H2S-producing nature When compared to

the form of Aβ These promising results can suggest SPRC as a novel therapeutic agent for neurodegenerative diseases

Table 1: Summary of commonly used therapies demonstrating strong antioxidant properties Table 2: Primer sequences for various inflammatory genes used in RT-PCR

Table 3: Summary of effects of SPRC on various aspects affecting cell survival

Table 4: Summary of effects of SAC on various cellular aspects

Figure 1: Aggregation properties of peptides and their mechanisms

Figure 2: Amyloid precursor protein (APP) proteolysis through the non-amyloidogenic or

amyloidogenic pathway

Figure 3: Chemical structure of S-allyl-cysteine (SAC)

Figure 4: Chemical structure of S-propargyl-cysteine (SPRC)

Figure 5: Comparison of metabolic pathways of cysteine and SPRC

Figure 6: The production of hydrogen sulfide (H2S) in mammalian tissues

Figure 7: The reactions of detoxifying oxygen radicals in the body

Figure 8: Release of pro-inflammatory cytokines by the overproduction of Aβ

Figure 9: Experimental protocol for Part II: Oligomeric Aβ25-35

Figure 10: Experimental protocol for Part III: Fibrillar Aβ25-35

Part I: In Vitro Study

Figure 11: Aggregation of Aβ25-35 at 37°C over time

Figure 12: Aggregation of Aβ25-35 with increasing temperature for 96 hours

Figure 13: Effects of different concentrations of SPRC on Aβ25-35 aggregation

Figure 14: Effects of different concentrations of SAC on Aβ25-35 aggregation

Figure 15: Effects of different concentrations of sodium hydrosulfide (NaHS) on Aβ25-35

aggregation

Figure 16: Comparison between treatments with different drugs

Figure 17: Effects of radical scavenging abilities of various drugs

Figure 18: Size-exclusion chromatography carried out by HPLC to separate samples according to molecular size

Part II: Oligomeric Aβ

Figure 19: Effects of aggregated Aβ25-35 on C6 glioma cell viability

Figure 20: Effects of SPRC pre-treatment on Aβ-induced cytotoxicity

Figure 21: Effects of SAC or NaHS pre-treatments on Aβ-induced cytotoxicity

Figure 22: Comparison between equimolar concentrations of drugs on Aβ-induced cytotoxicity Figure 23: Effects of pre-treatment of drugs on H2S concentrations in cell medium

Figure 24: Effects of pre-treatment of drugs on cystathionine-β synthase (CBS) expression in cell lysates

Figure 25: Effects of the CBS inhibitor aminooxyacetate (AOAA) on cell viability

Figure 26: Effects of pre-treatment of drugs on DCF production

Figure 27: Effects of pre-treatment of drugs on DHE fluorescence

Figure 28: Effects of pre-treatment of drugs on total superoxide dismutase (SOD) expression and activities in cell lysates

Figure 29: Effects of pre-treatment of drugs on catalase expression and activities in cell lysates Figure 30: Effects of pre-treatment of drugs on glutathione peroxidase (GPx) expression and activities in cell lysates

Figure 31: Effects of pre-treatment of drugs on IL-1β expression

Figure 32: Effects of pre-treatment of drugs on IL-6 expression

Figure 33: Effects of pre-treatment of drugs on TNF-α expression

Figure 34: Effects of pre-treatment of drugs on IL-10 expression of inflammatory factors

Figure 35: Effects of pre-treatment of drugs on cell cycle analysis

lysates

Figure 38: Effects of pre-treatment of drugs on autophagy using acridine orange staining

Figure 39: Effects of pre-treatment of drugs on LC3 expression in cell lysates

Figure 40: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at lower magnification

Figure 41: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at higher magnification

Part III: Fibrillar Aβ

Figure 42: Effects of different times of incubation with aggregated Aβ25-35 on cell viability Figure 43: Effects of SPRC pre-treatment on Aβ-induced cytotoxicity

Figure 44: Effects of SAC and NaHS pre-treatment on Aβ-induced cytotoxicity

Figure 45: Comparison between equimolar concentrations of drugs on Aβ-induced cytotoxicity Figure 46: Effects of pre-treatment of drugs on H2S concentrations in cell medium

Figure 47: Effects of pre-treatment of drugs on CBS expression in cell lysates

Figure 48: Effects of the CBS inhibitor AOAA on cell viability

Figure 49: Effects of pre-treatment of drugs on DCF production

Figure 50: Effects of pre-treatment of drugs on DHE fluorescence

Figure 51: Effects of pre-treatment of drugs on SOD expression and activities in cell lysates Figure 52: Effects of pre-treatment of drugs on catalase expression and activities in cell lysates Figure 53: Effects of pre-treatment of drugs on glutathione peroxidase (GPx) expression and activities in cell lysates

Figure 54: Effects of pre-treatment of drugs on IL-1β expression

Figure 55: Effects of pre-treatment of drugs on IL-6 expression

Figure 56: Effects of pre-treatment of drugs on TNF-α expression

Figure 57: Effects of pre-treatment of drugs on IL-10 expression

Figure 58: Effects of pre-treatment of drugs on cell cycle status

Figure 59: Effects of pre-treatment of drugs on DNA fragmentation using TUNEL staining Figure 60: Effects of pre-treatment of drugs on PARP and pro-caspase 3 expressions in cell lysates

Figure 61: Effects of pre-treatment of drugs on autophagy using acridine orange staining

Figure 62: Effects of pre-treatment of drugs on LC3 expression in cell lysates

Figure 63: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at lower magnification

Figure 64: Effects of pre-treatment of drugs on the ultrastructure of the cells using transmission electron microscopy at higher magnification

in the occurrence of AD, where there were about 30,000 affected patients as of 2010 (1) The worrying trend of an aging population has been highlighted extensively by the Singapore

government and has been directing future policy-making Amongst which, the dependence of

AD patients on caregivers and long-term care systems is of concern to the healthcare

infrastructure

AD is a progressive disease clinically characterized by episodic memory problems to a slow global decline of cognitive functions End-stage AD patients are usually bedridden and dependent on custodial care until death occurs on an average of nine years after diagnosis (2) There is currently no known cure for AD, although several types of treatments targeting different pathways had been shown to alleviate symptoms

AD patients typically exhibit cerebral atrophy where neuronal death is the main cause of the behavioural deficits Post-mortem analyses of human AD brains reveal senile plaques

composed of extracellular deposits of amyloid-β (Aβ) and neurofibrillary tangles formed by accumulation of abnormal hyperphosphorylated tau in brain regions responsible for memory and

cognition, namely the hippocampus and frontotemporal cortex The severe neuronal cell death in such areas significantly correlated to the distribution of Aβ plagues found However, the extent

of Aβ toxicity does not parallel the clinical severity and thus increases the challenges in drug discovery for AD

1.1.2 Aggregated proteins and diseases

Aggregated proteins have been implicated in several neurodegenerative diseases such as Alzheimer’s, Parkinson’s, prion and motor neuron diseases (3) Protein deposits observed in the brain were first characterized in the 19th century and could confer vastly different symptoms to affected individuals Such deposits may differ largely in their amino acid sequences, but form morphologically similar aggregates - termed amyloid - in tissues most affected by

neurodegeneration, suggesting that some general properties of affected polypeptides steer them towards enhanced self-assembly and hence, toxicity

1.1.3 Self-aggregation properties of amyloid peptides

Amyloid fibrils are rich in β-sheets with a highly ordered cross-β core structure to give strength and stability These fibrils exhibit the interesting phenomenon of self-assembly, or “the spontaneous organization of molecules under thermodynamic equilibrium, towards a state of free energy via non-covalent interactions such as hydrogen bonding, electrostatic, hydrophobic and van der Waals” (4) Amyloid peptides are usually soluble; yet under certain conditions these peptides undergo conformational changes to self-assemble into fibrils All amyloid fibrils are defined by three experimental criteria: i) green birefringence upon staining with Congo Red; ii) fibrillar morphology; and iii) β-sheet secondary structure (4)

Figure 1: Aggregation properties of peptides and their mechanisms (a) Self-aggregation

properties of peptides that undergo a conformational change from the protofibril state to

filaments and fibrils Adapted from Nilsson, Methods 34 (2004) (b) Amyloid-beta peptides can display aggregation by folding into aggregates through different proposed mechanisms Adapted from Hughes and Dunstan, Modern Biopolymer Science (2009)

The formation of fibrils occurs when conformational changes expose hydrophobic groups usually hidden, driving the non-specific aggregation of such peptides The typical self-

aggregation of the amyloid peptide (Figure 1a) starts from the formation of protofibrils (or oligomers), that extents in length to become filaments The filamentous materials aggregate and grow in size to become fibrils Typically, fibrils are linear, unbranched and of variable length dependent on the number of protofilaments that make up the fibril Certain fibrils exhibit helical shapes where protofilaments twist around each other through both electrostatic and hydrophobic interactions (5) Such aggregation that occurs from protein misfolding is proposed to be a generic property of all proteins and peptides (6) The mechanisms of amyloid fibril assembly have been summarized by Hughes and Dunstan (2009) into three pathways:

(b) (a)

2 Nucleation conformational model

This model proposes that the accumulation of disordered polypeptides with a nucleus transits these proteins into stable conformations This conformational conversion to

stability is a rate-limiting step after which monomers could build on quickly

3 Off pathway model

The nucleus is formed in a parallel, irreversible step after the initial refolding phase

where an amyloidogenic intermediate emerges The filaments are elongated by addition

of such intermediates at each end, and further associate to form fibrils

1.1.4 Factors affecting fibril formation

The ideal thermodynamics promoting fibril formation should allow for a small energy barrier between the folded and unfolded states, as well as an optimal distance for efficient

intermolecular interactions between molecules Hence, factors that can achieve such favourable energetics will promote fibril formation (Figure 1b)

The rate of amyloid peptide aggregation is highly dependent on solvent conditions (7) Firstly, conditions that decrease solubility and/or increase the ease of acquiring the required secondary structure for polymerization accelerate the aggregation kinetics Secondly,

aggregation rates are affected by sites available for elongation Initial lag times and threshold

behavior observed in typical aggregation studies suggest the need for available sites, and once present the aggregation proceeds rapidly Factors that affect aggregation processes include temperature, pH, addition of salts, mutations resulting in changes to the amino acid side chains, shear forces, available interfaces for formation etc As such, these suggest the delicate balances necessary to slow the production of amyloid aggregates

1.2 Amyloid-beta peptides are central to Alzheimer’s disease

The amyloid-beta (Aβ) peptide forms aggregates that are a hallmark of Alzheimer’s disease In the brain, both soluble and insoluble Aβ exist at differing concentrations Soluble Aβ (oligomers) is decreased in the cerebrospinal fluid and plasma of AD patients (8) and insoluble

Aβ (fibrils) exists mainly as pathological plagues in postmortem brain sections (9)

1.2.1 Products of sequential cleavage

The amyloid fibrils found in plaques are made of Aβ peptides containing 39-42 amino acid residues The most common Aβ species found in patients are the 40-mer and 42-mer

peptides The sequential cleavage of the amyloid precursor protein (APP) by three enzymes, α-, β- and γ-secretases, produces these peptides of different lengths Of which, the γ-secretase

enzyme has been identified as a complex of enzymes composed of presenilin 1 or 2 (PS1 and PS2), nicastrin, anterior pharynx defective and presenilin enhancer 2 Familial AD is an

autosomal dominant disease with early onset pathogenesis where patients had been identified with mutations of three genes - APP, PS1 and PS2 These mutations resulted in the early-onset

of AD and affected the stability and metabolism of Aβ peptides, directly linking these gene products to the disease

The proteolytic cleavage of APP can be divided into the non-amyloidogenic and

amyloidogenic pathways (Figure 2) (10)

The non-amyloidogenic pathway is the prevalent pathway in cells where APP is cleaved

at a position 83 amino acids from the C terminus by the α-secretase Three putative candidates of the enzyme belongs to the family of a disintegrin and metalloprotease (ADAM) have been

identified: ADAM9, ADAM10 and ADAM17 The resultant products are a large N-terminus

fragment (sAPPα) that is secreted into the extracellular medium and a 83-amino-acid C-terminus (C83) that is retained in the membrane The fragment retained in the membrane is subsequently cleaved by the γ-secretase, producing a short fragment termed p3 Since these cleavages occur out of the range of the Aβ peptides, this pathway does not contribute to the production of the pathological Aβ

Figure 2: APP proteolysis (adapted from LaFerla et al, Nature Rev Neurosci 8, 2007) APP can

undergo proteolytic processing either the non-amyloidogenic or amyloidogenic pathway The amyloidogenic pathway results in the production of pathological Aβ peptides

The amyloidogenic pathway occurs when the initial proteolysis was carried out by secretase (also known as -site APPcleaving enzyme 1; BACE1) at a position 99 amino acids from the C-terminus This 99-mer fragment (C99) is retained in the membrane while the N-terminus (sAPPβ) is released into the extracellular medium Subsequent cleavage of the free fragment by the γ-secretase complex - comprising of presenilin 1 or 2, nicastrin, anterior

β-pharynx defective (APH-1) and presenilin enhancer 2 (PEN2) - liberates the intact Aβ peptide The major species produced is 40 residues in length (Aβ1-40), while a small proportion

(approximately 10%) is the 42 residue variant (Aβ1-42) Aβ1-42 is more hydrophobic and more prone to fibril formation than the shorter variant

1.2.2 Neurotoxicity of Aβ 25-35

Interestingly, different lengths of Aβ peptides display a range of neurotoxicity in vivo (11), and the ability of the full-length peptide to affect cognitive processes is best displayed by the undecapeptide Aβ25-35 (12) This fragment of sequence GSNKGAIIGLM is the shortest segment responsible for the observed neurotoxicity of Aβ and is widely accepted as the

functional domain of the full-length Aβ Administering Aβ25-35 to rats led to impaired spatial working memory and the acquisition of passive avoidance reactions (13) that correlated well with a deficiency in the amount and activity of choline acetyltransferase Aβ25-35 is also observed

in neurons of subiculum and entorhinal cortex of AD brains (14)

The ten amino-acid fragment is also the shortest fragment that can exhibit large β-sheet fibrils in solution Moreover, some in vitro studies have found that the fragment is not only toxic upon aging, it also demonstrates strong toxicity when freshly prepared (15), a property dissimilar

to the full-length peptide

1.2.3 Structure of the Aβ 25-35 peptide

The rapid dissolution of Aβ25-35 in solution has presented difficulties in unraveling its

three-dimensional monomeric structure by NMR either in water or organic solvents The actual monomeric structure is pivotal in the resultant morphology because this affects the conformation

of the intermediate species and subsequent fibril shapes The peptide can adopt different

monomeric conformations depending on the media Generally, it was concluded that the peptide adopts a helical structure in apolar organic solvents and a β-structure (β-turn or β-sheet) in

aqueous buffers But these trends may be affected by pH, concentration, incubation times,

preparation and purification processes (5) Noted by Wei and Shea (16), the

hydrogen-bond-forming groups of the β-turn in Aβ25-35 are more exposed than the other peptide residues and are

possibly the initial points for rapid aggregation In particular, the presence of glycine residues at positions 25, 29 and 33 are proposed to play an important role in formation of β-sheet structures

in the aqueous solution (17) Although the C-terminal hydrophobic portion of the fragment is crucial for aggregation, substitution of methionine35 with alanine (18) disrupted the required β-sheet conformation but surprisingly enhanced toxicity The β-branched residues of isoleucine31 and isoleucine32 have also been implicated in the folding and/or stabilization of the assembled aggregate structure

Much aggregation kinetics had been done on Aβ1-42 and Aβ1-40, but little is known about

Aβ25-35

1.3 Garlic and S-allyl-cysteine

1.3.1 Aged garlic extract

Garlic (Allium sativum) is a condiment used by many peoples in their daily lives and has

been reputed to have strong medicinal uses Amongst these, garlic had shown to prevent

atherosclerosis (19), cancer (20) and ischemia-reperfusion injury (21) by its antioxidant and radical scavenging properties Aged garlic extract is one of the many nutraceuticals that have been in the market as a complementary supplement to our diets (22, 23) The aged garlic extract can most prominently protect the frontal brain morphology in the senescence- accelerated mouse model, most likely through influencing the cognitive and behavioural functions to alleviate neurodegenerative conditions (24)

free-Both aged garlic extract and its main active compound, S-allyl-cysteine (SAC) showed neuroprotective effects in the mouse transgenic model of Alzheimer’s disease (25) Aged garlic extract and SAC effectively reduced the Alzheimer’s cerebral plaques with a concomitant

increase in α-cleaved sAPPα This protection reduced the amyloidogenic pathway while steering towards the secretory pathway, preventing the accumulation of the pathological Aβ In the same study, the authors also found that while aged garlic extract and SAC could reduce inflammation and tau phosphorylation, the efficacy was greater using aged garlic extract

1.3.2 S-allyl-cysteine

S-allyl-cysteine (SAC) is a major organosulfur component of aged garlic extract SAC is

a derivative of the amino acid cysteine, where an allyl group is added to the sulfur atom (Figure 3) (26) As an active compound of garlic extract, SAC exhibits beneficial effects similar to the garlic extract The antiproliferative effects of SAC on human neuroblastoma cells prompted

cancer research using natural active compounds (27) SAC also demonstrates strong antioxidant properties useful in diabetes (28) and ethanol-induced acute liver injury (29) Moreover, SAC alleviated symptoms in a mouse model of acetaminophen-induced hepatotoxicity (30) through anti-inflammatory and anti-oxidative effects

Figure 3: Chemical structure of SAC

Interestingly, SAC has anti-amyloidogenic effects and destabilizes the Aβ fibrils in vitro (31) Rao et al suggested that SAC inhibited Aβ aggregate formation by halting further

oligomerization and breaking preformed Aβ fibrils The -OH group of the carboxylic group in SAC was also postulated to enhance hydrophobic interactions by hydrogen bonding to the donor groups of Aβ, although the actual mechanisms are not known

Furthermore, SAC ameliorated Aβ-induced cytotoxicity in nerve-growth

factor-differentiated PC12 cells (32) Aβ-induced cytotoxicity was not attenuated by a caspase-3

inhibitor, nor did 4-hydroxynonenal (HNE) play a role in the observed cell death, suggesting that apoptosis is not pivotal in Aβ-induced cytotoxicity, and the SAC neuroprotection is not via the anti-apoptotic pathway

As such, the antioxidant potential of SAC was investigated in other models of

Alzheimer’s disease SAC improved learning abilities using the radial maze following

intrahippocampal injection of Aβ25-35 related to its capability to reduce oxidative stress (33) Pretreatment of SAC just thirty minutes before the peptide injection significantly reduced

reactive oxygen species (ROS) and lipid peroxidation markers, although the drug was not found

to alter superoxide dismutase (SOD) or glutathione peroxidase (GPx) - key antioxidant

enzymes - activities

In contrast, SAC can moderate oxidative stress in mice treated with D-galactose (34), a known factor that increases Aβ damage, by maintaining the levels of the endogenous antioxidant reduced glutathione (GSH) and restoring the activities of SOD, GPx and catalase These were in addition to the abilities of SAC to suppress APP and its cleavage enzyme (BACE)

Likewise, in another Part IInvolving an experimental dementia model using streptozocin, pre-treatment with SAC significantly improved cognition accompanied by histopathological alterations (35) Using the Morris water maze, SAC-treated mice used less time and shorter distances to reach the hidden platform At the same time, SAC significantly reduced oxidative stress indicators and increased activities of the antioxidant enzymes, reinforcing the free radical scavenging role of SAC SAC also demonstrated, albeit to a lower extent, the anti-apoptotic nature via reduction of p53 and up-regulation of Bcl-2 proteins

On another note, SAC augmented hydrogen sulfide levels in rats subjected to myocardial infarction (36) In this pivotal study, our lab first demonstrated that SAC served as an

endogenous hydrogen sulfide donor to release the gasotransmitter, in turn achieving its

cardioprotective role SAC has a similar structure to cysteine and undergoes β-elimination while binding to the main H2S-producing enzyme in the heart, cystathionine-γ-lyase (CSE) to augment

H2S levels

As such, the role of SAC and its possible involvement through hydrogen sulfide in Aβ

25-35-induced neurodegeneration is of great interest in this research project

1.4 S-propargyl-cysteine

1.4.1 Chemical properties and pharmacokinetics

S-propargyl-cysteine (SPRC) is a structural analog to its parent compound SAC by exchanging the double bond with a triple bond (Figure 4) Upon the introduction of the triple bond, the compound becomes more polar and hence, less soluble in water However, the

modification can enhance its cardioprotective and neuroprotective roles when compared with SAC, as outlined below

Figure 4: Chemical structure of SPRC

The pharmacokinetics of SPRC was investigated in rats by tracking the plasma

concentration-time-course profiles following single i.g and i.v administrations (37) Both the values of the log transformed Cmax/dose and AUC0-t/dose were not significantly different and the mean Tmax was also dose-independent, suggesting a kinetic linearity of the drug SPRC could be absorbed readily after oral administration of 25, 85 and 225 mg/kg The absolute bioavailability

of oral SPRC of the given doses was calculated to be 96.6%, 97.0%, and 94.7%, respectively

The structural similarities of SPRC to cysteine suggest a similar metabolic pathway to cysteine Cysteine is also a substrate of cystathionine-beta synthase (CBS) - the main H2S-producing enzyme in the brain - that results in the endogenous release of H2S upon metabolism Hence in a similar manner, SPRC can be a potential candidate of CBS that could release prop-2-yne-1-thiol as a product of its metabolism (Figure 5)

Figure 5: Comparison of metabolic pathways of cysteine and SPRC (a) Cysteine is a substrate of CBS that will result in the release of endogenous H2S (b) The structural similarities of SPRC to cysteine suggest a similar metabolic pathway that releases prop-2-yne-1-thiol as a metabolic product

Interestingly, in addition to the structural similarity to cysteine, SPRC demonstrated increased affinity to both CSE and CBS in silico (unpublished data) Both H2S-producing

enzymes can be stimulated after treatment with SPRC to produce the gasotransmitter and to provide additional protection

1.4.2 Cardioprotective effects

The replacement of the allyl moiety with a propargyl group in SPRC resulted in a much more stable compound than the parent SAC, and hence a more plausible candidate as a

therapeutic drug

SPRC was compared with SAC to elucidate its cardioprotective effects in adult rat hearts

and neonatal cardiomyocytes in an ischemic/hypoxia model (38) In both in vivo and in vitro

models, SPRC showed greater protection than SAC in terms of mortality and cell viability Other than reducing the infarct sizes, plasma levels of lactate dehydrogenase (LDH) and creatinine kinase (CK) levels were decreased to a larger extent in SPRC-treated rats Both SPRC- and SAC-treated rats presented lowered ST elevation and antioxidant potentials compared to the controls

BH+

SH(b)

SPRC

Levels of CSE enzyme activities and expressions were also greatly increased upon treatment with SPRC and much higher than in the SAC group, implying an increased effect on CSE Lastly, these cardioprotective effects were abolished when co-treated with the CSE inhibitor

propargylglycine (PAG), confirming the involvement of the H2S pathway

Furthermore, SPRC was compared with SAC in terms of its antioxidant potentials in the same model of ischemic injury (39) Both compounds preserved tissue GSH levels and plasma maldionaldehyde (MDA) levels that were correlated to an observed upregulation of CSE, SOD and GPx SPRC was found to improve manganese SOD (Mn-SOD) activity while SAC could also increase the cytosolic copper/zinc SOD (Cu/Zn-SOD) These were activated by the Akt signaling pathway and more significantly observed in the SPRC group yet abolished with PAG These are strong evidence of the antioxidant properties of SPRC

The anti-inflammatory effects of SPRC were elucidated using a lipopolysaccharide

(LPS)-induced response in H9c2 cardiac myocyte cell line (40) LPS down-regulated CSE

expression and H2S levels which were reversed by SPRC treatment SPRC also attenuated the LPS-induced pro-inflammatory factor TNF-α expression and release, iNOS and iCAM- 1

Down-regulation of iNOS expression suppressed the intracellular ROS generation and signaling cascades activation These anti-inflammatory observations were associated to the inhibition of IκBα degradation, NFκB activation, induction of Akt phosphorylation and thus activation of the

PI3K/Akt pathway

hippocampal Aβ levels Moreover, SPRC administration also decreased TNF-α levels and

expressions of TNFR1 dependent on restoring levels of IκB and activation of NFκB

Similarly, SPRC ameliorated Aβ-induced cognitive impairment using the Morris water maze and neuronal ultrastructure damage (42) Treatment with SPRC inhibited the expressions

of pro-inflammatory TNF-α and COX-2 productions, preventing the exacerbation of Aβ-induced damage to the brain These were mediated by decreasing phosphorylation of ERK1/2, inhibiting the degradation of IκB and subsequent phosphorylation of the p65 subunit of NFκB

From the animal studies, SPRC had shown great potential as a novel therapeutic drug candidate for neurodegenerative diseases, particularly in Alzheimer’s disease However, the underlying mechanisms and cell targets remain to be discovered

1.5 Hydrogen sulfide

1.5.1 General properties

Hydrogen sulfide (H2S) is a water-soluble, colourless, flammable gas with a

characteristic rotten egg smell It is commonly known to be an environmental hazard and is toxic

in large amounts

Until recently, little is known about H2S apart from its toxicity H2S research picked up about three decades ago since the discovery of substantial amounts produced endogenously in mammalian cells Since then, H2S has been implicated in several disease models, and identified

as a third gasotransmitter, alongside nitric oxide (NO) and carbon monoxide (CO)

About one-third of H2S exists as its undissociated form and the rest as the dissociated anion (HS-) under physiological conditions of pH 7.4 Because of its small molecular weight and lipophilicity, H2S easily permeates plasma membrane to enter the cell’s interior It is also a strong reducing agent The physiological concentration of H2S detected in serum and most tissues is about 50 µM, although the concentration found in various regions of the brain can be

up to three-fold higher (43)

1.5.2 Synthesis

Endogenous H2S is produced from three enzymes detected in mammalian tissues:

cystathionine β-synthase (CBS, EC 4.2.1.22), cystathionine-γ-lyase (CSE, EC 4.4.1.1) and mercaptopyruvate sulfurtransferase (3-MST) CBS is mainly localized in the central nervous system, while CSE in the cardiovascular system The recent discovery of 3-MST in the brain and

3-aorta suggested its contribution to the production of H2S levels in the brain (44) However, the

mechanisms of action of these three enzymes differ (Figure 6)

CSE catalyzes the γ-elimination reaction, where cystathionine is degraded into L-cysteine,

α-ketobutyrate and ammonia CSE preferentially recruits L-cystine in the β-elimination reaction

to degrade it into pyruvate, ammonia and thiocysteine The resultant thiocysteine reacts

non-enzymatically with L-cysteine or other thiols to form H2S and corresponding disulfides On the

other hand, CBS is heavily involved in the transsulfuration pathway that yields cystathionine

from L-serine and L-homocysteine in a β-replacement reaction, releasing H2O In a similar

manner, CBS also generates cystathionine from L-cysteine and L-homocysteine, releasing H2S

instead 3-MST generates H2S from L-cysteine only in the presence of α-ketoglutarate, and in

conjunction with cysteine (aspartate) aminotransferase (CAT) The transamination between

cysteine and α-ketoglutarate is catalyzed by CAT, producing 3-mercaptopyruvate and

L-glutamate 3-MST transfers the sulfur in 3-mercaptopyruvate to sulfurous acid, resulting in

pyruvate and thiosulfate In the presence of GSH, thiosulfate can then be reduced to H2S

Figure 6: The production of H2S in mammalian tissues is mainly catalyzed by three enzymes,

cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate

sulfurtransferase (3-MST) Adapted from J Biochem 149(4): 357- 359, 2011

1.5.3 Biological targets of H 2 S

The appreciable amounts of H2S detected in mammalian tissues points to a physiological role of the gas H2S transverses cell membranes without specific transporters and can be effective intracellularly The extensive interest in H2S has elucidated protective roles in various different systems and pinpointed several biological targets as widely reviewed (45)

Many effects of H2S can be mimicked by potassium-opened ATP channel (KATP) openers including pinacidil and diazoxide, leading to the fervent belief that H2S acts primarily on KATPchannels (46) Although found to stimulate the KATP channel opening, the actual mechanisms are unknown Observations of H2S on the cardiovascular system such as vasodilation,

preconditioning and cardioprotective effects had implicated the KATP channel, and even more convincingly when effects are negated by channel inhibitors Similarly, H2S inhibited insulin secretion in pancreatic β-cells - a function heavily dependent on KATP channels (47)

Interestingly, H2S can cross-talk with other gasotransmitters, particularly nitric oxide, to realize smooth muscle relaxation (48) and vasodilation (49) The administration of H2S donors elicits a transient hypotensive response (46), and such blood pressure regulation seemed to involve an interaction with the NO pathway (49) All three gasotransmitters can competitively affect cytochrome c oxidase which in turn affects cellular respiration H2S inhibits the copper centre of the enzyme (50), directly inhibiting the oxygen consumption of cell (51) This has been postulated to be the reason behind its suspended animation effects observed in mice over-

exposed to H2S (52) and its toxic effects (53)

The highly reactive H2S molecule is prone to react with compounds such as superoxide radical anion (54), hydrogen peroxide (H2O2) (55), peroxynitrite (56), and hypochlorite (57) Its

free radical scavenging and antioxidant properties had been widely documented and implicated

in the observed protective effects In fact, the reducing potential of H2S appeared to modulate redox-sensitive thiol groups of the NMDA receptor leading to altered responses, in turn,

induction of hippocampal long-term potentiation (58)

1.5.4 H 2 S as a neuromodulator

In the brain, the endogenous levels of H2S have been measured in the rat, human and bovine and were found with relatively high concentrations of 50-160 µM The high levels of the gas may be altered in a disease state

Kimura et al (59) first proposed the idea of H2S as a neuromodulator when they observed that physiological concentrations of H2S facilitate hippocampal long-term potentiation (LTP) in the presence of a weak titanic stimulation The LTP-facilitating effect of H2S appears only at active synapses and involves the selective increase of NMDA receptor-mediate responses This involves the activation of cAMP pathways (58), where phosphorylation of the receptor subunits

by protein kinase A would alter the NMDA receptor activity However, at high concentrations of

H2S (>320 µM), H2S inhibits synaptic transmission in the hippocampus that may contribute to acute sub-lethal toxicity of the gas The delicate balance of the gas levels may change and

influence the pathology of learning and memory-related diseases

In neurons, H2S increases the production of GSH by enhancing the activity of

γ-glutamylcysteine synthetase and upregulating cysteine transport (60), directly increasing GSH synthesis The larger pool of the antioxidant GSH helps to prevent oxytosis in neurons that is typically caused by oxidative stress In addition, H2S also activates KATP and Cl- channels that can halt oxidative glutamate toxicity, protecting hippocampal cells (61)

Besides neurons, exogenous H2S can trigger calcium waves in both astrocytes and

hippocampal slices (62) Calcium waves in astrocytes are commonly a form of communication between such cells and signify cooperation to accomplish their supportive roles The calcium wave is preceded by an increase in intracellular calcium concentration that may spread amongst the localized astrocyte population Such spikes in intracellular calcium concentration triggered

by H2S were a result of calcium influx through membrane-bound calcium channels, and some extent of calcium release from the cellular stores Likewise, exogenous H2S acts on microglia in

a similar manner and partially dependent on the activation of adenylyl cyclase, instead of the usual phospholipase C-protein kinase C pathway (63)

H2S can also protect against neuroinflammation, as demonstrated in attenuation of induced inflammation in both microglia and astrocytes (64) The significant lowering of LPS-induced nitric oxide and TNF-α secretion by H2S are said to be mediated by the inhibition of iNOS expression and MAPK phosphorylation

LPS-1.5.5 H 2 S in Alzheimer’s disease

The role of H2S in Alzheimer’s disease was unheard of until 2002, when Kimura et al published a study showing a severe decrease in levels of the gas in brains of individuals afflicted with AD (65) Endogenous brain H2S levels were almost halved (55%) in investigated

individuals, compared to age-matched controls with a corresponding decline in the CBS activator S-adenosyl-L-methionine (SAM) The amount of homocysteine found in AD brains were also elevated, signifying a decline in CBS activity that eventually led to decreased H2S

On the other hand, exogenously-treated H2S could confer a dose-dependent degree of protection to the neuronal cell culture model of PC12 treated with Aβ25-35 with a corresponding

inhibition on apoptosis (66) Besides, the effects of H2S were modulated by preserving the mitochondrial membrane potential and attenuating the intracellular ROS levels These imply the neuro-protective role of H2S and hint the plausible mechanisms that may be involved

In a similar manner, exogenous treatment of H2S reduced cytotoxicity in microglial cells exposed to Aβ1-40 (67) This protective effect was mediated by a recovery in cell cycle, anti-inflammatory effects including suppression of nitric oxide and TNF-α production, suppression of JNK and p38 activities as well as preservation of mitochondrial functions

The plethora of cell functions modified by exogenous H2S in Aβ-induced injury of microglia substantially demonstrates the significance of H2S in AD pathology

1.6 Oxidative Stress

The common late onset of sporadic AD calls for the need to understand the relationship between aging and the disease development Indeed, aging associates with free radical generation that adversely mutates mitochondrial DNA and accelerates senescence (68) This free radical theory of aging indicates the importance of oxidative stress in AD

1.6.1 Oxidative balance in the brain

The oxidative homeostasis in cells is tightly regulated with a delicate balance between pro-oxidants and antioxidants Under physiological conditions, the balance is maintained with a surplus of antioxidative mechanisms to prevent the overload of radicals

The production of radicals in the cell is an inevitable event of cellular respiration

Aerobic cells use molecular oxygen to generate energy; but oxygen can also become a powerful oxidant in the presence of unpaired electrons of the molecule Superoxide anion (O2•-) is

produced by taking one electron from oxygen and is a strong radical initiating oxidative chain reactions The superoxide anion can be dismutated spontaneously or catalytically by endogenous SOD to produce H2O2 This can be reduced to water or to a hydroxyl radical (•OH) under partial

reduction catalysed by reduced-transition metals including copper and iron (69) The superoxide anion can attack other radicals including nitric oxide (NO) to produce a peroxynitrite (ONOO-), a reactive nitrogen species capable of oxidative stress

The site of free radical generation occurs in the electron transport chain in the

mitochondria During respiration, electrons are transferred to molecular oxygen with a small percentage lost in the process churning out the detrimental superoxide anion Usually, such

radicals produced are detoxified by SOD into H2O2 and subsequently converted to the benign

H2O by either GPx or catalase (Figure 7)

Figure 7: The reactions of detoxifying oxygen radicals in the body Superoxide anions are

neutralized into oxygen and H2O2 by superoxide dismutase (SOD) The resultant H2O2 is then

reduced into water by either catalase or glutathione peroxidase (GSH peroxidase) Adapted from http://www.terry-oberley.net/research.htm

The brain is particularly vulnerable to oxidative stress with the high consumption of oxygen (about 20% of the body’s total oxygen) and the relatively lower levels of antioxidant activities (70) The free radicals produced inevitably accumulate as age increases and eventually incur damage to the brain architecture In addition, the cells experiencing oxidative stress are prone to further insults, such as those from Aβ

1.6.2 Imbalance in the disease state

The oxidative homeostasis is changed in the disease state Alterations in oxidation

markers are often noticed even before manifestation of the symptoms, implying that oxidative stress precedes the disease conditions

Clinically, increased markers of lipid peroxidation and nucleic acid oxidation in mortem AD brains, cerebrospinal fluid and plasma link oxidative stress to the disease In

post-particular, levels of isoprostanes - products of fatty acid oxidation - were reportedly higher in

patients diagnosed with mild cognitive impairment compared to age-matched controls (71) A profile of oxidized proteins in patients with early symptoms of AD showed that the oxidatively modified proteins are involved in energy metabolism, mitogenesis/proliferation and

neuroplasticity, likely contributing to the even more oxidative imbalance (72) Similarly, mutant APP transgenic mice were found to have heightened oxidative stress well before deposits of Aβ deposition can be detected (73)

Several groups have reported elevated oxidative stress and subsequent cellular

dysfunctions linked to AD Increased protein oxidation, DNA damage, lipid peroxidation and

increased levels of 4-HNE are consequences of the Aβ (reviewed in 74) Peroxynitrites produced

by Aβ also caused nitration of tyrosine residues in AD brain samples (75) On the other hand, compensatory mechanisms set up to defend the cells against radical attacks were reduced in AD patients An analysis of cerebrospinal fluid in AD patients displayed marked reduction of SOD activities, although the declines were not well-correlated with the severity of dementia (76) Another Part IInvestigating autopsy brains from AD patients found that the SOD activity was significantly lower in disease patients compared to control with little changes in catalase and GPx activities (77) SOD activities were much reduced in the cerebellum by 27%, frontal cortex

by 27% and hippocampus by 35% The total antioxidant capacity including GSH, ascorbic acid and bilirubin amongst others, was also decreased by 24% in plasma samples from AD patients (78)

1.6.3 Aβ-induced oxidative stress

Oxidative stress had been detected in senile plagues made up of Aβ (79), suggesting that such plagues typical in AD patients are a source of free radicals Using multi-photon imaging,

two fluorogenic compounds that fluoresce upon oxidation were applied to both sections of transgenic mice and human AD brains The coincidence of the oxidation marker (DCF-DA/ Amplex Red) with the thioflavin-S probe strongly implies the area of aggregated Aβ as a

location of heightened oxidative stress

i) Aβ as a peptidyl radical

Aβ peptides readily precipitate in the physiological solution and radicalize to inactivate endogenous antioxidant enzymes like glutamine synthetase (80) In particular, Aβ25-35 quickly forms radicals in solution compared to a lag time observed in Aβ1-40 Aβ25-35 forms free radicals

in the presence of oxygen and can act as a peptidyl radical capable of attacking biological

molecules Such radical formation is thought to be dependent on the presence of methionine on position 35 (Met35) of the peptide

Methionine is an amino acid prone to oxidation and sometimes acts as protection against oxidation in proteins (81) There are two main forms of oxidized methionine: methionine

sulfoxide and methionine sulfone Met35 contributed to the neurotoxicity observed in Aβ

peptides (82) Firstly, the substitution of the S atom of Met35 by a methylene group (norleucine) attenuates the oxidative stress and neurotoxicity of Aβ1-42 while no change was observed in the morphology of aggregated Aβ fibrils Secondly, shorter Aβ fragments that do not include Met35 (e.g Aβ1-28) demonstrated no oxidative stress or neurotoxicity Thirdly, while Aβ25-34 is not neurotoxic, Aβ25-35 containing a C-terminal Met35 is very neurotoxic These evidences amplify the importance of Met35 in the induction of oxidative stress by Aβ peptides