Optical Detection of Interaction between Alzheimer s Disease Biomarkers and Carbon Nanotubes

Transcription

1 Optical Detection of Interaction between Alzheimer s Disease Biomarkers and Carbon Nanotubes by Amina Zaheer A thesis submitted in conformity with the requirements for the degree of Master of Science Chemistry Department University of Toronto Copyright by Amina Zaheer 2014

2 Optical Detection of Interaction between Alzheimer s Disease Biomarkers and Carbon Nanotubes Abstract Amina Zaheer Master of Science Department of Chemistry University of Toronto 2014 Alzheimer s disease (AD) is a debilitating neurodegenerative disorder characterized by cognitive impairment. During AD, Amyloid Beta (Aβ) is deposited extracellularly and forms plaques, disrupting neuronal communication. Acetylcholinesterase (AChE) and carbon nanotubes (CNTs) modulate amyloid fibrillation pathways. We explored effect of multi-walled CNTs (MWCNTs) and Aβ 42 on AChE and Butyrylcholinesterase (BuChE) activities using Ellman s method. CNTs inhibited AChE activity significantly at high concentrations and hydrophobicity. Non-functionalized MWCNTs showed greatest inhibition of AChE followed by NH 2 - and COOH-functionalized MWCNTs. Adsorption of AChE on MWCNTs was believed to be the main mechanism for inhibition and was unaffected by time of interaction between the two molecules. By forming complexes, Aβ 42 promoted activities of AChE and BuChE. Greater facilitation of enzyme activities was observed for soluble Aβ 42 oligomers. Our results provided insights into development of novel drugs that would be transported across the blood-brain barrier by CNTs and target AChE and BuChE towards AD therapy. ii

3 Acknowledgments First and for most, I would like to deeply thank my supervisor Dr. Kagan Kerman for giving me this opportunity and for all of his support throughout my studies. He has continuously been very encouraging and inspiring. My research has greatly benefited from his expertise in analytical chemistry and I really appreciate him for challenging me and for motivating me during times of struggle. I would also like to thank my lab mates: Anthony Veloso, Vinci Hung, Xavier Cheng, Nan Li, Amy Liu, Han Su, Meisam Rahemi-Pour and Suria Jahan for all of their support and motivation. They have provided me with great advice and suggestions throughout my studies. I am glad to have met and worked with them. I want to express my gratitude to Bob Temkin, electron microscopy technician at the Centre of Neurobiology of Stress at UTSC, for all of his support and hard work in helping me obtain my TEM images. Furthermore, I would like to sincerely thank Dr. Michael Thomson for being my external reader. I truly appreciate all of his time and feedback. The Department of Chemistry at UofT have done an excellent job in handling administrative affairs of my studies and for keeping me informed of important deadlines. Last but not least, I would like to thank my parents, Samina Akhtar and Zaheer Ahmeed Raja and my siblings, Salaha, Rabia, Waqas, Hussain and Hashim for always being so supportive throughout my studies. iii

4 Table of Contents Acknowledgments....iii Table of Contents... iv List of Tables... vii List of Figures... iix List of Appendices... xiii Chapter 1 Introduction to Alzheimer s Disease & Key Biomolecules Alzheimer s Disease Overview Amyloid Beta (Aβ) Aβ Formation Aβ Fibril Formation Pathway Aβ Fibril Structure Cholinergic System Cholinesterases (ChE) Acetylcholinesterase (AChE) Butyrylcholinesterase (BuChE) Alzheimer s Disease Hypotheses Cholinergic Hypothesis Amyloid Cascade Hypothesis Metal Hypothesis Oxidative Stress Hypothesis Alzheimer s Disease Treatment Chapter 2 Introduction to Carbon Nanotubes & their Interactions with Acetylcholinesterase and Amyloid-β Carbon Nanotubes Methods of Production iv

5 2.2 Functionalization of Carbon Nanotubes Thermally Activated Functionalization of CNTs Functionalization of CNTs using Electrochemistry CNTs and Amyloid Fibrillation Pathways Proposed Mechanism of Promotion/Inhibition of Amyloid Fibrillation by CNTs Carbon Nanotubes and AChE Chapter 3 Enzyme Kinetics Michelis-Menten Lineweaver-Burk Plots Chapter Ellman s Photometric Method Research Objectives Chapter Experimental Reagents Preparation of Samples Time-Dependence Studies of MWCNTs Incubated with AChE Aggregation State Experiments of Aβ 42 Incubated with AChE and BuChE Ellman s Photometric Method Determination of Kinetic Parameters for the Interaction of COOH-MWCNTs with AChE Transmission Electron Microscopy (TEM) Chapter 6 Results and Discussion Optimum Enzyme Concentration Effect of MWCNTs on AChE Activity Effect of Concentration of MWCNTs v

6 6.1.2 AChE Activity in Presence of COOH- & NH 2 -Functionalized MWCNTs Time-Dependence Studies of AChE-CNT Samples Kinetic Parameters for AChE-MWCNT Interaction Effect of Amyloid-β on AChE activity Effect of Aggregation State of Aβ TEM Images for Enzyme Samples that were Incubated with CNTs and Aβ Conclusions Chapter Future Directions References Appendices Appendix A : Lineweaver-Burk Plots for the Interaction of AChE with COOH-MWCNT Copyright Acknowledgements vi

7 List of Tables Table 1: Activities (a.u./min) of AChE and BuChE at various concentrations to hydrolyze substrates (ATChI and BuTChI). AChE activity was measured only using ATChI. Activities were calculated by finding slopes of absorbance at 410 nm over a period of 180s...37 Table 2: Percentage of AChE activity observed in the presence of various concentrations of MWCNTs...39 Table 3: Percentage AChE activity observed in presence of various types of MWCNTs (nonfunctionalized, COOH- and NH 2 functionalized.41 Table 4: AChE activity (a.u./min) detected in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs at time 0. Each activity is an average of three trials.43 Table 5: AChE activity (a.u./min) observed in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs for 24 h. Each activity is an average of three trials trials...44 Table 6: AChE activity (a.u./min) observed in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs for 72 h. Each activity is an average of three trials...45 Table 7: Percentage AChE activity observed in various fractions incubated with COOH- MWCNTs for different time periods (0, 24 and 72 h) (results from Table 4, 5, 6). Average column indicates AChE activity observed across the three time points..46 Table 8: AChE activity (a.u./min) observed in various fractions incubated with COOH- MWCNTs as a function of substrate concentration (56, 112, 225 and 550) µm. SN, W1, W2 and W3 represent supernatant, wash 1, wash 2 and wash 3 fractions, respectively.48 vii

8 Table 9: Kinetic parameters, K m (µm ) and V max (a.u./min) for the complex of AChE with COOH-MWCNT, observed in various fractions. Values were obtained from linear regression of Lineweaver-Burk plots shown in appendix...49 Table 10: Activities of AChE and BuChE in (a.u./min) and percentage for various fractions after 5 days of incubation with Aβ 42. Each activity represents an average of three trials..52 viii

9 List of Figures Figure 1: Amyloid Precursor Protein (APP) cleavage pathways (Reprinted with permission from Salminen et al., 2013 [10] Elsevier).3 Figure 2: The nucleation-dependant amyloid fibrillation pathway (Reprinted from Kumar et al., 2011 [12] Kumar and Walter)..5 Figure 3: Cross-β structure of peptide GNNQQNY, a yeast prion that has a high propensity to form amyloid fibrils (Reprinted from Lührs et al., 2005 [14] The National Academy of Sciences)..6 Figure 4: Cholinergic neurotransmission and its elements in a normal human brain (Reprinted with permission from Nordberg et al., 2013 [16] Physicians Postgraduate Press, Inc.)..8 Figure 5: A schematic representation of AChE binding sites (Reprinted from Colović et al., 2013 [22] Bentham Science Publishers) 10 Figure 6: Mechanism of acetylcholinesterase hydrolysis at the esteratic subsite of AChE (Adapted from Colović et al., 2013 [22] Bentham Science Publishers) 11 Figure 7: Structures of four FDA approved AD drugs: Tacrine, Donepezil, Rivastigmine and Galantamine that act by increasing ACh content (Reprinted with permission from Jann et al., 2002 [42] Springer) 17 Figure 8: Structure of Memantine (Namenda ), a FDA approved NMDA receptor antagonist for AD treatment (Reprinted from Tomek et al., 2013 [44] Creative Commons Attribution License, Figure 9: Structure of Memoquin (MQ) (adapted with permission from Prati et al., 2014 [47] Royal Society of Chemistry)..19 Figure 10: Schematic representation of two types of CNTs (MWCNTs & SWCNTs) and the possible arrangements of SWCNTs (Reprinted from Madani et al., 2013 [53] Creative Commons Attribution License, ix

10 Figure 11: Two step functionalization of CNTs through thermal activation (Reprinted with permission from Balasubramanian et al., 2005 [62] John Wiley and Sons)..22 Figure 12: Without SWCNTs, random Aβ (16-22) chains form β-sheet rich oligomers (Reprinted with permission from Li et al., 2011 [69] Elsevier).25 Figure 13: Hydrolysis of acetylcholine by AChE and oxidation of choline using choline oxidase (ChO) (Reprinted with permission from Liu et al., 2006 [72] American Chemical Society)...26 Figure 14: Michaelis-Menten kinetics equation (Reprinted from Berg et al., 2002 [77] W. H. Freeman and Company).27 Figure 15: A representative Lineweaver-Burk plot (adapted from Berg et al., 2002 [77] W. H. Freeman and Company).28 Figure 16: Representative Lineweaver-Burk plots for competitive, mixed and non-competitive inhibitors (adapted from Berg et al., 2002 [77] W. H. Freeman and Company)..29 Figure 17: A representation of Ellman s photometric method. Hydrolysis of ATCh by AChE produces ATCh and acetate (Adapted from Pohanka et al., 2011 [17] Creative Commons Attribution License, Figure 18: Absorbance (a.u.) at 410 nm over a time period of 180s for various AChE concentrations (0.060, 0.125, and U/mL). Each plot represents an average of three trials (n=3)..35 Figure 19: Absorbance (a.u.) at 410 nm measured over a time period of 180s for various BuChE concentrations (0.060, 0.125, and 0.500) U/mL. Each plot is an average of three trials (n=3)...36 Figure 20: Absorbance (a.u) at 410 nm over a period of 180 s for conditions: enzyme alone, enzyme with 4 µg/µl MWCNTs and blank. Each plot represents an average of three trials (n=3)...38 x

11 Figure 21: Absorbance (a.u.) at 410 nm over a period of 180 s in the presence of various concentrations of MWCNTs. Each plot represents an average of three trials (n=3)...39 Figure 22: Absorbance (a.u.) at 410 nm over a period of 180 s in presence of various types of MWCNTs (non-functionalized, COOH- and NH 2 -functionalized). Each plot is an average of three trials (n=3).40 Figure 23: TEM image of AChE B (0.5µg/µL) on nickel formvar mesh grid obtained using Hitachi H-7500 transmission electron microscope. Magnification is indicated by the scale bar..55 Figure 24: Crystal structure of Torpedo californica AChE in complex with 20 MM thiocholine obtained from Protein Data Bank (PDB #: 2C5G) 56 Figure 25: TEM image of AChE obtained on formvar-coated grids and negative-stained with 2% uranyl acetate. Scale bar represents 100 nm (Adapted with permission from Inestrosa et al., 1996 [89] Elsevier).57 Figure 26: TEM image of BuChE (0.5 µg/µl) on nickel formvar mesh grid obtained using Hitachi H-7500 transmission electron microscope. Magnification is indicated by scale bar 58 Figure 27: Crystal structure of human BuChE in complex with tacrine obtained from Protein Data Bank (PDB #: 4BDS) 59 Figure 28: TEM image of AChE (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate AChE adsorbed on COOH-MWCNTs and scale bar indicates magnification.60 Figure 29: TEM image of AChE B (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate AChE adsorbed on COOH-MWCNTs and scale bar indicates magnification.61 xi

12 Figure 30: TEM image of BuChE from human serum (0.5µg/µL) incubated with COOH- MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate BuChE adsorbed on COOH-MWCNTs and scale bar indicates magnification...62 Figure 31: TEM image of AChE B (0.5 µg/µl) incubated with Aβ 42 (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrow indicates AChE-Aβ 42 complexes and scale bar indicates magnification 63 Figure 32: TEM image of BuChE from human serum (0.5 µg/µl) incubated with Aβ 42 (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrow indicates BuChE- Aβ 42 complexes and scale bar indicates magnification.64 Figure 33: Crystal structure of Aβ 40 (Alzheimer s disease amyloid A4 peptide, PDB #: 1AML) obtained from the Protein Data Bank.65 Figure 34: TEM image of Aβ 42 (5µM) in the presence of Cu (II) (10 µm) (Adapted from [91] Macmillan Publishers Limited)..66 Figure 35: Absorbance (a.u.) at 410 nm over a period of 180 s for AChE samples incubated with MWCNTs and Aβ..68 xii

13 List of Appendices Appendix A: Lineweaver-Burk Plots for the interaction of AChE with COOH-MWCNTs...81 xiii

14 1 Chapter 1 Introduction to Alzheimer s Disease & Key Biomolecules 1. Alzheimer s Disease Overview Alzheimer s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive impairment, memory loss and a decline in language [1]. It was first described in 1906 by a German neuropathologist named Alois Alzheimer [1]. Currently, it is the leading cause of dementia and fourth leading cause of death in people over the age of 65 years worldwide and impacts more than 18 million people. By the year 2050, this number is expected to rise as high as 70 million [2]. Two forms of AD exist today: early-onset AD (EOAD) also known as familial AD and late-onset AD (LOAD) also referred to as sporadic AD [3]. EOAD is associated with an overproduction of toxic/amyloidogenic forms of amyloid-β (Aβ) peptide and is caused by mutations in one of following three genes: Amyloid Precursor Protein (APP), Presenilin-1 (PS1) or Presenilin-2 (PS2) [3]. APP is a transmembrane protein from which Aβ peptide is derived and PS1 or PS2 can be catalytic subunits involved in this pathway, discussed in section 1.1. Unlike EOAD, LOAD is the most common form of AD (accounting for approximately 95% of the cases) and results from a combination of environmental and genetic factors, which cannot be explained by the aforementioned mutations [3]. The most important risk gene for EOAD is apolipoprotein E (Apoε) [3]. The corresponding protein of this gene is synthesized by astrocytes and mircroganglia in the brain and is involved in cholesterol and phospholipid metabolism. APoε has three isoforms in humans known as ε2, ε3 and ε4, which differ in their preferential binding to various types of lipid. It is the ε4 isoform that is associated with increased risk for AD [4]. In addition to psychological symptoms, AD also presents several neuropathological characteristics. Some of which are selective neuronal loss (cholinergic neurons of basal forebrain projecting to hippocampus and neocortex), acetylcholine (ACh) depletion, senile plaques (amyloid-β protein deposits) and neurofibrillary tangles composed of hyperphosphorylated tau protein [5]. Another characteristic observed in AD is the loss of acetylcholinesterase (AChE) activity both from cholinergic and non-cholinergic neurons and an increase in its activity around

15 2 senile plaques [5-6]. AChE is an enzyme involved in cholinergic neurotransmission within the autonomic and somatic nervous system and promotes assembly of Aβ peptides into fibrils [5]. Due to increased incidence rates caused by lack of effective treatment strategies and an increase in life expectancy, many research efforts have been devoted to study AD progression. Despite these efforts, the exact cause of AD is still unknown. The factors proposed in research are the various characteristics observed among AD patients discussed above such as Aβ, deposits but none of these has yet been able to individually account for all aspects of the disease [7]. This chapter will provide a detailed background on AD including its development, progression and current available therapeutic strategies. 1.1 Amyloid Beta (Aβ) Amyloid Beta (Aβ) is a 4.2 kda peptide derived from cleavage of Amyloid Precursor Protein (APP), a transmembrane protein which is ubiquitously expressed in most cell types but whose function is currently unknown. Aβ was first isolated as the principal component of amyloid deposits (senile plaques) in AD and Down s syndrome patients [7]. Later it was found that Aβ is produced during normal cellular metabolism and is found in extracellular milieu of the brain and the cerebrospinal fluid (CSF). Aβ ranges from 39 to 43 residues in length and exists in various forms (monomeric, oligomeric and fibrillar) owing to its ability to self-associate [7-9]. In its monomeric form, Aβ peptide is composed mainly of α-helical and/or unordered structure that is soluble and non-toxic. Oligomeric and fibrillar forms, however, are key to its biological effect/neurotoxicity with oligomeric species being more neurotoxic [8-9]. This section will discuss Aβ formation and its transition from monomeric to fibrillar species Aβ Formation APP can be cleaved via two pathways known as amyloidogenic and non-amyloidogenic (Figure 1). In both pathways, APP is sequentially cleaved by two membrane bound endoprotesases. The main difference between the two pathways is the secretase doing the initial cleavage. In the amyloidogenic pathway, β- secretase initially cleaves APP generating a membrane bound C- terminal fragment (C99) and releasing an extracellular soluble fragment/ectodomain (APPβ). The C-terminal fragment then generates Aβ peptide following cleavage by γ- secretase. In the non-amyloidogenic pathway, APP is first cleaved by α- secretase and produces a C-terminal

16 3 fragement (C83) which is then cleaved by γ- secretases producing a benign peptide known as P3. (Figure 1) [7-8, 10]. Figure 1: Amyloid Precursor Protein (APP) cleavage pathways. In the non-amyloidogenic pathway (left), APP is first cleaved by α-secretase (ADAM 10) releasing a soluble ectodomain (sappα) and generating a membrane bound C-terminal domain of APP (C83). C-terminal domain is then cleaved by γ-secretase (presenilin) and releases intracellular APP domain (AICD) in the cytosol and P3 peptide into the lumen side of the membrane. In the amyloidogenic pathway (right), APP is first cleaved by β-secretase (BACE1) releasing a soluble APP fragment (sappβ) and creating membrane bound fragment (C99). Aβ peptide is then created by subsequent cleavage of C99 by γ-secretase (presenilin). Upon formation, in normal brain, Aβ is degraded by various enzymes such as insulin-degrading enzyme (IDE). In AD patients, it starts to accumulate abnormally (Reprinted with permission from Salminen et al., 2013 [10] Elsevier). Being involved in the amyloidogenic pathway, β- and γ-secretases are believed to be prime targets for development of anti-ad drugs and have been widely studied. As can be observed in Figure 1, β-secretase cleaves APP and its other substrates outside of the bilayer. β-secretase exits in two major forms BACE1 and BACE2, which are 65% homologous. BACE1 is the major form responsible for Aβ production and is highly expressed in the brain. While, BACE2 is found in low levels in the brain and is highly present in most peripheral tissues. The activity of β-secretase is the rate limiting step in the amyloidogenic pathway and it processes approximately 10% of the

17 4 total cellular APP, while 90% of it is processed via the non-amyloidogenic pathway. β-secretase activity is increased in sporadic AD [8]. Unlike β-secretase, γ-secretase cleaves APP and its other substrates within the lipid bilayer and can only process substrates, which have first been cleaved by another endoprotease (ie. have their ectodomain region removed). In addition, γ-secretase does not detect its substrate by a specific sequence but rather by factors such as length of the transmembrane domain. Hence, cleavage by γ-secretase is somewhat imprecise and results in a heterogenous population of Aβ peptides, which differ in the number of amino acids at the C-terminal. Aβ40 is the most abundant (~80-90%) followed by Aβ 42 (~5-10%). Aβ 42 is more hydrophobic and fibrillogenic and it is the principal species deposited in the brain [8] Aβ Fibril Formation Pathway As discussed earlier, Aβ peptide exists in a variety of forms due to its propensity to aggregate in solution. This section will discuss formation of Aβ fibrils from monomeric species. Aβ aggregation occurs via a nucleation-dependent polymerization pathway, which is believed to consist of a lag phase (nucleation phase) and an elongation phase (Figure 2). In the nucleation phase, monomers undergo conformational changes and attach to one other to form larger complexes ranging from dimers to heptamers, which eventually grow into oligomers. The nucleation phase is a slow process, as it is thermodynamically unfavourable [11-12].

18 5 Figure 2: The nucleation-dependent amyloid fibrillation pathway. Amyloid fibril formation involves a slow nucleation/lag phase (thermodynamically unfavoured) and a rapid elongation phase (thermodynamically favoured). In the nucleation phase, monomers undergo conformational change/misfolding and self-associate to form dimers and oligomers. In the elongation phase, the oligomers grow by further addition of monomers and form mature fibrils (green curve). The rate limiting step in the formation of amyloid fibrils is the formation of nuclei/seeds. Addition of preformed seeds can speed up amyloid fibril formation by reducing lag time and inducing faster aggregate formation (red curve) (Reprinted from Kumar et al., 2011 [12] Kumar and Walter). The elongation process however is much more favourable and occurs rapidly. During elongation, oligomeric nuclei grow into protofibrils with the addition of monomers [12]. Protofibrils predominately contain β sheets and are regarded as intermediates in the fibrillation process as they may lead to fibrils or disassemble into smaller oligomers [11, 13]. The kinetics of amyloid fibril formation are well represented by a sigmoidal shape curve consisting of a nucleation phase/lag phase and a rapid elongation phase. Research suggests that

19 6 the lag phase is determined by critical concentration of nuclei/seeds and that it can be shortened by the addition of preformed seeds [12] Aβ Fibril Structure In native form, Aβ peptide has an α-helix conformation or exists as a random coil. X-Ray diffraction analysis, circular dichroism spectroscopy and fourier transform infrared spectroscopy have shown that Aβ fibrils have a cross-β structure consisting of ribbon-like-β-sheets running parallel to and β-strands running perpendicular to fibril axis [14] (Figure 3). The β-sheets are held together by hydrogen bonds between back-bone atoms running parallel to fibril axis [11, 14]. Figure 3: Cross-β structure of peptide GNNQQNY, a yeast prion that has a high propensity to form amyloid fibrils. Cross- β structure of amyloid fibrils consists of extended β-sheets stabilized by intermolecular hydrogen bonds (Reprinted from Lührs et al., 2005 [14] The National Academy of Sciences) Contrary to conventional protein structures, which are robust to modest changes in sequence and solution conditions, Amyloid fibrils are polymorphic. They are insoluble, filamentous structures longer than 1 µm and have a diameter within 8-12 nm. A given sequence will give rise to different amyloid morphologies depending on the conditions in which the monomer is incubated

20 7 such as ionic strength, ph, temperature and concentration. Amyloid polymorphism can be due to differences in molecular structure and to variations in packing of extended β-sheets and/or protofilaments in amyloid fibrils. This polymorphism is likely due to generic interactions that stabilize the cross-β structure of amyloid fibrils [11]. 1.2 Cholinergic System Cholinergic neurotransmitter systems are widely distributed in the human brain and play an important role in regulating processes such as memory, learning and behavior. They consist of neurotransmitter acetylcholine (ACh), vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), ACh receptors and cholinesterases (acetylcholinesterase and butyrylcholinesterase) discussed in section [15]. All of these elements play important roles in the brain and their proper functioning is essential for cholinergic neurotransmission. For instance, VAChT mediates accumulation of ACh in synaptic vesicles of cholinergic neurons and ChAT synthesizes ACh [15]. In the normal human brain, neurons communicate by transmitting nerve impulses. This involves fusion of ACh-containing vesicles with presynaptic membrane, release of ACh into synaptic cleft, diffusion of ACh across synaptic cleft and lastly interaction of ACh with cholinergic receptors on postsynaptic neuron [16]. Nerve impulses are terminated by hydrolysis of ACh into choline and acetate by cholinesterases. This reaction allows the neurons to return to their resting state. Choline is then transported back to presynaptic neuron and is used as a substrate to synthesize ACh (Figure 4) [16].

21 8 Figure 4: Cholinergic neurotransmission and its elements in a normal human brain. Neurotransmitter, ACh containing vesicles fuse with membrane of presynaptic neuron and release ACh into the synaptic cleft, which then interacts with cholinergic receptors on the postsynaptic neuron. AChE is involved in termination of nerve impulses by hydrolyzing ACh into choline and acetate. Synaptically released ACh can also be hydrolyzed by glial BuChE (Reprinted with permission from Nordberg et al., 2013 [16] Physicians Postgraduate Press, Inc.) In an AD patient s brain, elements of cholinergic system are altered, which causes a disturbance in cholinergic neurotransmission. For instance, there is a significant loss of ChAT activity and ACh levels. In addition, cholinergic neurons are degenerated and there are changes in activities and levels of AChE and BuChE, which are discussed below [15] Cholinesterases (ChE) As discussed in section 1.2, cholinesterases (ChE) are a family of enzymes that catalyze hydrolysis of acetylcholine (ACh) into choline and acetate. Two types of cholinesterases exist in the central and peripheral nervous system: AChE and BuChE, which are responsible for

22 9 cholinergic neurotransmission [17]. In addition to their catalytic activities both are involved in roles such as cell differentiation and development. Immunohistochemical and in situ hybridization studies suggest that there is a distinct distribution of AChE and BuChE in the brain with respect to amounts and location. AChE is expressed at high levels in the hippocampus as well as the motor, premotor and neocortical areas of cerebral cortex [16]. BuChE is also found in hippocampus and temporal neocortex but at lower levels. Hippocampal and neocortical AChE is localized in the axons while BuChE is associated with glial cells [16]. In the amygdala, the number of BuChE-positive neurons exceeds AChE-positive neurons and BuChE resides predominately in neurons and their dendritic extensions, while AChE resides in the neutrophils [16]. In the healthy human brain, AChE is believed to be the predominant cholinesterase. Specifically a ratio of 4:1 is observed with respect to AChE and BuChE [18]. In AD patients, however, AChE activity is decreased by 85% in certain brain regions, while BuChE activity increases or remains unchanged [18-19]. Research suggests that most of the cortical AChE activity present in AD brain is associated with the amyloid core of the senile plaques rather than the neuritic component found at the periphery [19]. Increased levels of glial-derived BuChE and decrease in synaptic AChE result in a relative increase in the ratio of BuChE to AChE in cortical regions [16]. In addition to changes in activities, changes in AChE and BuChE protein expression are also observed in AD [20]. Both AChE and BuChE exist in six polymeric forms divided into two classes: asymmetric and globular based on presence or absence of a collagen-like tail [21]. The globular forms of AChE and BuChE are known as G1, G2 and G4 [21]. The G4 is the most predominant form expressed in the CNS and is responsible for degradation of ACh at the cholinergic synapse. The G1 form is found in smaller amounts in the brain and as AD progresses, there is an increase in this form of both AChE and BuChE and a decrease in the G4 form of AChE [16] Acetylcholinesterase (AChE) AChE is a serine-protease mainly found at neuromuscular junctions and cholinergic brain synapses. It plays a key role in cholinergic neurotransmission by regulating concentration of

23 10 ACh at the synapses via its hydrolysis. AChE has a high specific catalytic activity such that each molecule degrades about molecules of ACh per second [16, 22]. AChE has an ellipsoidal shape with dimensions 45 Å by 50 Å by 65 Å and consists of a 20 Å deep and narrow gorge, which penetrates halfway into the enzyme [23]. It has two binding sites, catalytic active site (CAS) and a peripheral anionic site (PAS) [16, 22-23]. The CAS further consists of two subsites: anionic subsite and an esteratic subsite. The anionic subsite contains the catalytic machinery/acyl pocket while the esteratic subsite contains the choline binding pocket and the catalytic triad (Figure 5). The catalytic triad (Ser200-His440-Glu327) along with nearby tryptophan residue (Trp84) play an important role in catalysis as they orient the substrate as needed [23]. Figure 5: A schematic representation of AChE binding sites. AChE consists of two binding sites: peripheral anionic site (PAS) and a catalytic active site (CAS). CAS further consist of two subsites: anionic subsite containing the acyl pocket and an esteratic subsite containing the catalytic triad and choline binding site. Upon entry into the enzyme, ACh binds to both the catalytic triad and the choline binding site (Reprinted from Colović et al., 2013 [22] Bentham Science Publishers).

24 11 The hydrolysis reaction of ACh forms an acyl enzyme and a free choline. The acyl enzyme then undergoes nucleophilic attack by a water molecule. This releases acetic acid and regenerates the free enzyme (Figure 6) [22]. The PAS is involved in modifying the catalytic activity (substrate inhibition) and mediating inhibitor interactions of AChE. It is also believed to be involved in binding of AChE to Aβ peptides as discussed in section Figure 6: Mechanism of acetylcholinesterase hydrolysis at the esteratic subsite of AChE. The catalytic triad (Glu-327, His-440 and Ser-200) assist in the process by orienting the substrate as needed. Upon hydrolysis, an acyl-enzyme is generated and free choline is released. Acylenzyme then undergoes nucleophilic attack by water with assistance from His-440 to liberate acetic acid and the free enzyme (Adapted from Colović et al., 2013 [22] Bentham Science Publishers).

25 12 Various forms of AChE exist in the brain due to alternative splicing and interactions with other gene products [24]. The two most common forms are known as the T-form and the R-form [24]. T-form also known as synaptic form is the variant expressed in mammalian brain and muscles [24]. It consists of a 40 residue C-terminal tail domain (determines the functional localization of synaptic AChE) and a cysteine by which dimerization might occur [24-25]. The second most common variant is AChE-R [25]. This form consists of a 26 amino acid C-terminal, which lacks a cysteine, hence it remains monomeric. AChE-R is also believed to be stress induced and is thought to increase with aging [24-25] Acetylcholinesterase and Amyloid-beta In addition to the cholinergic role discussed in section 1.2.2, AChE binds to Aβ peptides and promotes their deposition as insoluble fibrils. This binding takes place at enzyme s PAS and induces a conformational transition of Aβ into an amyloidogenic form [1-2]. Research suggests that AChE may act as a chaperone for assembly of peptides into oligomers by two possible mechanisms. It may increase the seeds necessary for nucleation step (decreasing the lag phase) or it may stimulate fibril elongation [26]. More support has been found for the former mechanism [26]. The incorporation of AChE into the amyloid complex is thought to be an early event during fibril formation (ie. Nucleation phase) [26]. Research suggests that promotion of Aβ aggregation by AChE is thermodynamically favoured because only a small amount of enzyme is required. In addition, AChE-Aβ complex is very stable and held together by strong intermolecular bonds that can only by broken by chaotropic agents and SDS detergent, which are known to disassemble amyloid fibrils. While, high ionic strength buffers are incapable of disrupting the complex [9, 26]. The formation of AChE-Aβ complex changes properties of both the peptide and the enzyme. In addition to the fibrillogenic effect, AChE-Aβ complex also boosts the neurotoxicity of Aβ fibrils [9]. Similarly, histochemical studies have shown that AChE associated with senile plaques has a different properties than AChE associated with normal fibers and neurons [9]. AChE associated with Aβ is more resistant to low ph conditions and inhibition by anti-cholinesterase agents and by excess acetylthiocholine (substrate) [9]. This is due to fibrils establishing a physical barrier hindering access of the substrate to the enzyme active site [19, 26].

26 13 Similar to AChE promoting aggregation of Aβ peptides and increasing their toxicity, Aβ has different properties than AChE and was found to promote its activity [9, 27]. This is supported by findings of an increase in AChE activity surrounding amyloid plaques [27]. Increase in AChE s activity induced by Aβ peptide is proposed to be due to disruption of calcium homeostasis and is mediated by oxidative stress [5, 27]. Disruption of calcium homeostasis, enhancement of excitotoxic mechanisms and oxidative stress are widely reported as mechanisms by which Aβ causes neurotoxicity [26-27]. For instance, Aβ increases release of nitric oxide (NO), which has free radical properties and activates intracellular signaling mechanisms [26]. In addition, generation of ROS induced by Aβ causes cell membrane lipid peroxidation. This results in formation of several aldehydes, which impair function of membrane proteins such as ion-motive ATPases and voltage-ca 2+ channels and result in disruption of ion homeostasis. Lipid peroxidation also results in alteration of cell membrane order [26] Butyrylcholinesterase (BuChE) BuChE, also known as pseudocholinesterase is an enzyme closely related to AChE and serves as co-regulator of cholinergic neurotransmission (by hydrolyzing ACh). BuChE gets its name as it hydrolyzes butryrylcholine more quickly than ACh [11]. BuChE is synthesized in the liver and is found mainly in peripheral tissues including plasma where its levels exceed those of AChE [11]. In the muscle tissue and brain, however, AChE is the dominant cholinesterase [11, 28]. Studies using AChE-knockout mice and human brain tissue treated with AChE inhibitors show that BuChE can compensate when AChE levels are depleted [16]. BuChE activity is increased in elderly (60-90 years) and in AD patients brain mainly in the hippocampus and temporal cortex in agreement with observation of episodic memory loss in dementias and cognitive decline in AD respectively [18-19]. Similar to AChE, BuChE also has a 20 Å deep gorge into with ACh enters. However, in BuChE, several aromatic residues lining the base of this gorge in AChE are replaced by hydrophobic residues. The acyl binding pocket of BuChE also replaces two Phe residues of AChE with Leu and Val, which allow the binding of bulkier substrates to BuChE. Similarly, although both enzymes have a PAS, the PAS of BuChE lacks three of the four residues found in PAS of AChE [29-30].

27 14 In addition to these structural variations, the response of BuChE upon ligand binding to the PAS differs from that of AChE. Unlike for AChE, where ligand binding to PAS is associated with substrate inhibition, the binding of a molecule onto PAS of BuChE results in substrate activation [29-30]. Inconclusive findings exist in literature regarding interaction between BChE and Aβ peptides [30]. Although, BuChE is found in senile plaques and is believed to promote plaque maturation, some studies suggest that it attenuates amyloid fibril formation by prolonging the lag phase [29]. While others suggest that unlike AChE, it incorporates itself into Aβ fibrils at a late phase of their formation and binds to soluble peptides rather than fibrils [31]. Still others suggest that it transforms Aβ peptides from a benign form to a malignant one associated with neuritic tissue degeneration and clinical dementia [30]. In addition to this enzymatic role, BuChE can associate with Aβ proteins and may delay the onset and rate of neurotoxic Aβ fibril formation in vitro [16]. Over 65 genetic variants exist for BuChE gene [25]. K variant (BChE-K, 1615 A, rs ) is the most studied as a risk factor for AD and is associated with a 33% reduction of BuChE molecules in plasma [25]. As described earlier in section 1.2.1, four molecular forms of BuChE are present in serum: G1 (monomer), G1-ALB (monomer linked to albumin), G2 (dimer) and G4 (tetramer) [24]. 1.3 Alzheimer s Disease Hypotheses The key biomolecules involved in AD development and progression have been introduced in sections 1.1 and 1.2. The goal of this section is to discuss the main hypotheses proposed in literature regarding the development of AD Cholinergic Hypothesis Cholinergic hypothesis is the classical hypothesis of AD [32-33]. It suggests that low levels of ACh and associated loss of cholinergic neurotransmission are responsible for cognitive and memory deterioration observed in AD patients [32]. This is thought to occur due to degeneration of cholinergic neurons in cerebral cortex and other areas of the brain and hyperactivity of AChE and BuChE [32].

28 15 Cholinergic hypothesis gains its support from studies in humans and non-human primates showing an involvement of ACh in memory and learning [33-34]. Specifically, patients in these studies show a loss of three main markers of cholinergic neurons and synapses. There is a reduction in 1) activity of choline acetyltransferase (enzyme responsible for synthesizing ACh) 2) depolarization induced ACh release and 3) choline uptake in nerve terminals (responsible for replenishing ACh synthetic machinery) [33-34]. In addition to explaining cholinergic deficits observed in AD patients, cholinergic hypothesis also proposes that sustaining/recovering ACh levels can alleviate some of these symptoms [32]. This led to development of AChE inhibitors, which are currently the only drugs approved by FDA to provide symptomatic relief of AD (discussed in section 1.4) [35]. Although these drugs show improvements in memory and cholinergic neurotransmission, which supports the hypothesis, the exact role of cholinergic system in causing cognitive deterioration is still a controversial topic [33] Amyloid Cascade Hypothesis Amyloid hypothesis, in contrast, has recently gained research interest and is currently the most widely investigated model explaining the etiology of AD [36]. Based on convergent biochemical and genetic evidence, this hypothesis suggests that accumulation of Aβ peptide (oligomers and fibrils) initiates a sequence of events, which results in formation of neurofibrillary tangles, neuronal cell death and dementia [36]. These events include development of large swellings by axons passing near amyloid plaques, alterations in local and long-distance neuronal signaling, neurons becoming hyperactive and astrocytes developing spontaneous calcium waves uncoupled from local network activity [37]. Similarly in vitro application of naturally secreated oligomeric preparations results in rapid loss of dendritic spines and deficits in synaptic plasticity. Intracranial secretion of these preparations results in memory and learning deficits [37]. Based on this hypothesis, overproduction of Aβ is a result of disruption of homeostatic processes that regulate the proteolytic cleavage of APP or is due to the interaction of the peptide with pathological chaperones such as Apoε, AChE and BuChE [38]. In addition to the events

29 16 described above, the amyloidogenic processing of Aβ peptides (described in section 1.1) is also thought to disturb metabolism of tau proteins and evoke their aggregation [38] Metal Hypothesis Metal hypothesis suggests that elevated levels of transition metals such as Fe, Cu and Zn (which are involved in many physiological processes) in the brain, is associated with AD [39-40]. These metals play important roles in Aβ aggregation and may modify AChE activity, most importantly may induce formation of reactive oxygen species (ROS) [39]. The hypothesis further suggests that lowering concentration of these metals by chelating them could prove to be a great therapeutic strategy for halting AD pathogenesis [39] Oxidative Stress Hypothesis Oxidative stress refers to an imbalance between production of free radicals and reactive metabolites and the cells ability to neutralize them by antioxidant defences [26]. The main source of ROS is the electron transport chain (ETC) at the inner mitochondrial membrane. During the formation of ATP in the ETC, some electrons leak from the inner mitochondrial membrane and react with oxygen forming radicals such as superoxide anions (O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH ) and hydroxyl ions (OH ) [26, 41]. Other sources of reactive metabolites are astrocytes and microglia that produce these species when activated and in reactions catalyzed by metal ions such as Cu and Fe [26, 41]. ROS are capable of irreversibly damaging and modifying several macromolecules within cells such as DNA, RNA, lipids and proteins [26]. For instance, OH is one of the most aggressive radical generated during AD. It is able to induce lipoperoxidation, protein oxidation and oxidation of DNA and RNA. In AD, OH radicals may also affect amino acid residues of AChE and hence modify the CAS and decrease its catalytic activity [41]. 1.4 Alzheimer s Disease Treatment Currently, no treatments are available to modify the progression of AD. However, five FDA approved drugs are available to treat clinical symptoms. These are tacrine (Cognex ), donepezil (Aricept ), galantamine (Reminyl ), rivastigmine (Exelon ) and memantine (Namenda ) [19, 42]. The four former drugs are AChE inhibitors, which act by increasing ACh content (Figure 7). They differ from each other with respect to their pharmacological properties. Donepezil and

30 17 galantamine are short-acting and reversible competitive inhibitors. While, rivastigmine is an intermediate-acting or pseudo-irreversible inhibitor, as it is actively metabolized by cholinesterases. The primary target of these inhibitors is AChE (donepezil and galantamine), rivastigmine, however, shows equal affinity for both AChE and BuChE [42]. In addition to blocking the catalytic function of cholinesterase, only donepezil (from these four) has been able to also block/prevent the interaction of AChE with Aβ peptides (~ 20%) [19]. Figure 7: Structures of four FDA approved AD drugs: tacrine, donepezil, rivastigmine and galantamine that act by increasing ACh content (Reprinted with permission from Jann et al., 2002 [42] Springer). Memantine is an N-methyl-D-aspartate (NMDA) receptor antagonist that acts by preventing glutamate excitotoxicity (Figure 8) [43].

31 18 Figure 8: Structure of Memantine (Namenda ), a FDA approved NMDA receptor antagonist for AD treatment (Reprinted from Tomek et al., 2013 [44] Creative Commons Attribution License, Although these five drugs do not stop disease progression, clinical studies show that they are capable of stabilizing cognitive impairment and help maintain global function, delaying the need for a patient to be placed in nursing homes for several months [43,45]. As discussed in section 1, AD is a multifactorial disease [43]. Although there are drugs under development that treat factors of AD such as APP pathogenic cleavage, metal ion accumulation, protein misfolding and oxidative stress, none of them individually has been able to modify disease progression. Based on this, many researchers suggest that a polypharmacological approach involving simultaneous modulation of multiple drug targets is needed (46-47]. Polypharmacological approach refers to either one drug binding to multiple targets (ie. MTDs) or multiple drugs binding to different targets (ie. drug combination). MTDs are thought to be superior than combinations due to the possibility of undesirable drug-drug interactions such as conflicting bioavailabilities, pharmacokinetics and metabolism [47-49]. These approaches gained popularity based on findings suggesting that use of one-target specific drugs in combination leads to better results than treatment with each one separately for diseases such as cancer [46]. This is proposed to occur due to alteration of several interconnected pathways involved in disease progression [46]. Similarly, combination studies involving AChE inhibitors have also shown a synergistic enhancement of therapeutic effects compared to single drug therapies [49].

32 19 These studies lead to an era of research geared towards development of multi-target directed ligands for treatment of AD [46-49]. Bivalent compounds, are a recent innovation that address the issue of undesired effects of drug combinations and have been explored in AD research as potential multi-target directed ligands [49]. They refer to small molecules consisting of two identical pharmacophores joined by an appropriate spacer. Bivalent compounds have also been used for a variety of diseases and have shown improved biological profile with respect to their corresponding monovalent counterparts [50]. Considering the molecular similarity between the PAS and CAS of AChE, bivalent compounds capable of binding these two sites simultaneously have been explored in AD therapeutic development [47]. Memoquin (MQ) is one of the first rationally designed multi-target drug candidates against AD, which in vitro shows a free radical scavenger action and inhibition of Aβ aggregation and AChE activity (Figure 9) [46, 51]. MQ is a quinone-bearing polyamine compound [46, 51]. MQ has a symmetrical structure consisting of two 2-methooxybenzyldiamino moieties connected by a benzoquinone spacer [52]. Figure 9: Structure of Memoquin (MQ) (adapted with permission from Prati et al., 2014 [47] Royal Society of Chemistry). Similarly, Veloso et al. [49] synthesized a small library of novel sym-triazine-derived compounds that are capable of parallel modulation of Aβ aggregation and AChE/BuChE hydrolytic activity in our laboratory [49].

33 20 Chapter 2 Introduction to Carbon Nanotubes & their Interactions with Acetylcholinesterase and Amyloid-beta 2. Carbon Nanotubes Carbon Nanotubes (CNTs) are one dimensional macromolecules consisting of single (SWCNTs) or multiple (MWCNTs) concentric sheets of graphene (two-dimensional sheet of sp 2 -hybridized carbon atoms) rolled up into cylinders [53]. Their structural arrangement and order gives CNTs a wide range of unique properties such as ultra-light weight, high surface area as well as thermal and electrical conductivity [53-56]. These properties allow them to have applications in electronics, catalysis, chemical sensing, drug/vaccine delivery, tissue engineering and novel biomaterials [53, 55]. SWCNTs have a diameter within a range of nm, which can be expanded by using higher synthesis temperatures. They exist in four possible arrangements: chiral, armchair, helical and zigzag arrangements [53, 56] (Figure 10). MWCNTs consist of central cylinder tubes with an average diameter ranging between 1-3 nm and an external cylinder with a diameter between nm. Depending on arrangement of graphite layers, MWCNTs can be split into two categories [53-56]. One with graphene sheet rolled up around itself and another with layers of graphene sheets arranged within a concentric structure [53].

34 21 Figure 10: Schematic representation of two types of CNTs (MWCNTs & SWCNTs) and the possible arrangements of SWCNTs (Reprinted from Madani et al., 2013 [53] Creative Commons Attribution License, ). 2.1 Methods of Production Chemical vapor deposition (CVD), electric arc-discharge and laser evaporation are three methods most commonly used to synthesize CNTs [53]. CVD also known as thermal CVD or catalytic CVD is the dominant method used for high-volume production [57-58]. While, laser evaporation and arc-discharge are most widely applied for the production of CNTs for experimental purposes. As its name suggests, CVD involves the thermal decomposition of a hydrocarbon vapor in presence of a metal catalyst. Compared to arc-discharge and laser-ablation methods, CVD synthesizes CNTs at low temperatures and ambient pressure [53, 57-58]. Laser evaporation involves generation of carbon through laser irradiation of graphite [53, 59]. Specifically, a pulsed laser is used to strike graphite in a high temperature reactor in the presence of an inert gas such as helium [59-60]. The inert gas vaporizes the graphite, which upon condensation forms CNTs on the cooler surface of the reactor [59-60]. Electric arc-discharge involves growing CNTs at the negative end of a carbon electrode [53, 61]. This method involves igniting an electric arc discharge by making a pair of graphite electrodes contact one another [57-59]. As the laser evaporation technique, this method also takes place in a reactor consisting of an inert gas such as hydrogen or helium. During the arc discharge, carbon atoms sublimate from the anode and are deposited at the cathode surface where CNTs are produced [61].

35 Functionalization of Carbon Nanotubes Functionalization of CNTs is often performed to increase their solubility in water and organic solvents. Two main approaches for functionalization of CNTs are thermally activated chemistry and electrochemical modification [62-63] Thermally Activated Functionalization of CNTs Thermally activated functionalization is used to produce short CNTs with a high density of oxygenated functions (such as carbonyl, carboxyl and hydroxyl) on their sides or ends [62-64]. This method involves ultrasonic treatment of CNTs in a mixture of concentrated nitric and sulfuric acid and results in release of carbon dioxide. In this treatment, CNT caps are opened and holes are formed on the sidewalls where oxidative groups (mostly COOH) can be etched. These carboxyl groups can then be further modified to other functional groups by creation of amide or ester bonds (Figure 11) [62]. Figure 11: Two step functionalization of CNTs through thermal activation. Oxidatively introduced carboxyl groups can then be converted into other groups by formation of amide or ester linkages (Reprinted with permission from Balasubramanian et al., 2005 [62] John Wiley and Sons).

36 23 In addition, the covalent attachment of groups on their sides or ends, CNTs may also be functionalized with groups directly coupled onto their π-conjugated carbon framework. Reactive species such as radicals, carbenes and nitrenes are attached to CNTs by thermally activated reactions [62] Functionalization of CNTs using Electrochemistry The second most common method for functionalization of CNTs is electrochemistry [62, 65]. This method involves applying either a constant potential or current to a CNT modified electrode immersed in a solution containing a suitable reagent. The transfer of electrons between the electrode and the reagent results in the formation of reactive species, which would have a tendency to self-polymerize generating a polymeric coating on the CNT [62]. The extent of film deposition on the CNTs can be controlled by changing electrochemical conditions such as magnitude and duration of current application. A variety of functional groups can be added to the nanotube surface by using reagents containing the desired substituents [60, 62]. 2.3 CNTs and Amyloid Fibrillation Pathways Contrasting views exist in literature regarding effect of various types of carbon nanoparticles (such as fullerenes, graphenes and CNTs) on amyloid fibrillation [66-69]. Promotion or inhibition of fibril formation depends on intrinsic properties of both peptide and nanoparticle (such as surface area and curvature) as well as the nature of interaction between them [66]. For instance, Linse et al. [67] found that hydrophobic copolymeric N-isopropylacrylamide and N- tert-butylacrylamide (NiPAM: BAM) nanoparticles accelerate fibrillation of β2-microglobulin (β-sheet rich in its native state), but inhibit that of Aβ 40 (random coils in monomeric state) [67]. Similarly, SWCNTs were found to also accelerate fibrillation of β2 microglobulin but prevent aggregation of human acidic fibroblast growth factor (hfgf-1) and Aβ peptide [68-69]. Similar to amyloid forming proteins (β2 microglobulin and hfgf-1), Aβ is believed to be a good representation of Aβ 40 (full length of Aβ), because it includes central hydrophobic core (LVFFA), which is essential for Aβ fibrillation. Moreover, Aβ is capable of forming fibrils with antiparallel-β strands [69].

37 Proposed Mechanism of Promotion/Inhibition of Amyloid Fibrillation by CNTs Amyloid fibrillation may be promoted by increasing local protein concentration and accelerating rate of nucleation on nanoparticle surface. While inhibition of fibril formation may occur due to tight binding between the molecules (CNTs and peptides) or a large protein/particle surface area [66]. A variety of studies using CNTs and various analytical methods have provided a deeper understanding of the impact of CNTs on amyloid fibrillation [70]. For instance, circular dichroism and Thioflavin-T studies have shown that SWCNTs promote nucleation but inhibit subsequent growth of peptides into amyloids. NMR studies have shown that SWCNTs perturb hydrophobic and charged residues in Aβ peptides (although SWCNTs are hydrophobic themselves and do not interact with charged resides). These studies have shown that SWCNTs promote the conversion of Aβ peptides from random coils into a parallel β-sheet conformation. However, upon binding to SWCNTs, this conformation is stabilized and prevents rearrangement of the peptide into the cross- β strand conformation [70]. Similarly, Li et al. [69] found that the presence of SWCNTs destabilizes already formed β-sheet structures and induces the formation of disordered coil aggregates (Figure 12). In other words, this suggests that fibrillation of Aβ can be reversed by SWCNTs [69].

38 25 Figure 12: Without SWCNTs, random Aβ (16-22) chains form β-sheet rich oligomers. In the presence of SWCNTs, the formation of β-sheets is inhibited and disordered coil aggregates are formed. In addition, the introduction of SWCNTs to prefibrillar β-sheet bilayers destabilizes the β-sheet and induces the formation of disordered coil aggregates (Reprinted with permission from Li et al., 2011 [69] Elsevier) Computational studies of Aβ peptides found that CNTs promoted the formation of beta-barrels around the nanotube, which were reported to suppress further aggregation and reduced the population of monomers/oligomers available for fibril growth and hence resulted in inhibition of fibrillation [68]. Studies which found an inhibitory effect of CNTs on amyloid fibrillation suggested that nanoparticles disrupted the intra-peptide hydrophobic and π-π stacking interactions. This was supported by the presence of mainly hydrophobic and aromatic residues in the interaction between amyloid peptides and CNTs [66]. Despite contrasting views on the role of CNTs on fibrillation of amyloid proteins, there is a consensus in research that a given carbon nanoparticle promotes or inhibits amyloid fibril formation by decreasing or increasing the lag time for nucleation, but they all leave the elongation phase unaffected [71]. Furthermore, it is believed that effect of CNTs on aggregation occurs by a surface-modulated nucleation mechanism, which involves modification of protein

39 26 conformation and is dependent on protein s intrinsic stability as well as dimensionality and degree of curvature of nanoparticle [68]. 2.4 Carbon Nanotubes and AChE The interaction between CNTs and AChE has been explored extensively in the field of biosensors [63, 72-75]. Modified electrodes prepared with the immobilization of AChE on CNTs have been widely used to detect organophosphate and carbamate pesticides as well as nerve agents [72-75]. These compounds inhibit AChE activity by diminishing phosphorylation of the serine residue (part of the catalytic triad). In these biosensors, amperometric detection was commonly used and was based on degree of inhibition. Residual enzyme activity was compared to initial activity [72-75]. In general, amperometric biosensors either use AChE alone or combined with choline oxidase (ChO). Inhibition of enzyme s activity is monitored by measuring oxidation current of the product of the reaction: choline in the case of enzyme alone or hydrogen peroxide in bienzyme systems, as shown below [72, 75]: Figure 13: Hydrolysis of acetylcholine by AChE and oxidation of choline using choline oxidase (ChO) (Reprinted with permission from Liu et al., 2006 [72] American Chemical Society). The most significant advantage of using CNTs in AChE-based biosensors is the ability of CNTs to reduce working potential by catalyzing electrochemical oxidation of the enzymatic product, which significantly amplifies th sensitivity of the amperometric response [72].

40 27 Chapter 3 Enzyme Kinetics 3. Michelis-Menten Michaelis-Menten kinetics is a model used to determine activity of enzymes that have one substrate [76-77]. It describes rate of enzymatic reactions by the following equation (which relates reaction velocity to substrate concentration): Figure 14: Michaelis-Menten kinetics equation. V max represents maximum rate achieved by the system at saturating substrate concentrations. K m represents the substrate concentration at which reaction rate is at half its maximum (Reprinted from Berg et al., 2002 [77] W. H. Freeman and Company). The Michaelis constant, K m, represents two things. 1) It is the substrate concentration at which the reaction rate is at half-maximum and 2) it is an inverse measure of substrate s affinity for the enzyme. In other words, a small K m indicates high affinity for substrate and means that reaction rate will approach V max more quickly [76-77]. 3.1 Lineweaver-Burk Plots Lineweaver-Burk plots also known as double reciprocal plots are a graphical method to analyze the Michaelis-Menten equation (Figure 15). They are used to determine the two important parameters of the Michaelis-Menten equation (V max and K m ) as discussed above. These plots are obtained by plotting the reciprocal of initial velocity against reciprocal of substrate concentration. A straight line is fitted to the points and V max is calculated as reciprocal of the y-intercept (line intersecting 1/v axis). K m can either be obtained by multiplying slope of the line (K m /V max ) by V max or by extrapolating line to the 1/S axis (where intercept will be -1/K m ) [76-78].

41 28 Figure 15: A representative Lineweaver-Burk plot. V max can be determined from the inverse of the y-intercept. K m is the negative reciprocal of the x-intercept (adapted from Berg et al., 2002 [77] W. H. Freeman and Company). In addition to determining K m and V max, these plots can also be used to distinguish between different types of inhibitors: competitive, non-competitive and uncompetitive. Competitive inhibitors would have the same y-intercept (ie. V max ) as uninhibited enzyme, but they would have a different x-intercept (ie. K m ). Non-competitive inhibitors have the same x-intercept as uninhibited enzyme, but a different y-intercept. Lastly, mixed inhibition leads to both different K m and V max values compared to those obtained from uninhibited enzyme (Figure 16) [77, 79-80].

42 29 Figure 16: Representative Lineweaver-Burk plots for competitive, mixed and non-competitive inhibitors. Compared to uninhibited enzyme, competitive inhibitors change K m but leave V max unaffected. Mixed inhibitors change both K m and V max. Non-competitive inhibitors change V max but leave K m unaffected (adapted from Berg et al., 2002 [77] W. H. Freeman and Company). In general, competitive inhibitors would bind to the enzyme. Mixed inhibitors would bind to the enzyme-substrate complex and non-competitive inhibitors would bind to both (enzyme alone and the enzyme-substrate complex) [79-80].

43 30 Chapter 4 4. Ellman s Photometric Method Ellman s photometric method is the most commonly used assay for studying AChE activity. It involves using a psedosubstrate acetylthiocholine (ATCh) and 5,5 -dithio-bis-2-nitrobenzoic acid (DTNB), also known as Ellman s reagent. The psedosubstrate s mechanism of hydrolysis is similar to that of the natural substrate ACh [81]. Upon hydrolysis by AChE, ATCh produces thiocholine (TCh), which reacts with DTNB to produce 5-thio-2-nitrobenzoate (TNB). TNB is a yellow anion that absorbs strongly at 410 nm. As this reaction is rapid, the enzyme activity can be obtained from the graphs of absorbance at 410 nm over time (finding the slope of the line for ~ 2 min) [81]. The same method can be used to measure BuChE activity using substrate butyrylthiocholine iodide (BuTCh) [82]. Figure 17: A representation of Ellman s photometric method. Hydrolysis of ATCh by AChE produces ATCh and acetate. ATCh reacts with DTNB and produced a yellow anion (TNB), which absorbs strongly at 410 nm (Adapted from Pohanka et al., 2011 [17] Creative Commons Attribution License,

44 Research Objectives Motivated by appearance of AChE and BuChE in association with Aβ plaques, many studies have monitored the interaction between these enzymes and Aβ peptide [1-2, 5,9,16, 26-27, 29-31]. Specifically, these studies have focused on the role of enzymes on peptide aggregation [1-2, 16, 26-27, 29-31]. However, few studies have focused on effect of Aβ on AChE activity [5, 9] and no study has been reported on effect of Aβ on BuChE s activity, to the best of our knowledge. In addition, although CNT-AChE interactions have been explored in the field of biosensors, little research has been done on the effect of CNTs on activities of AChE and BuChE. The goal of our research is to study the impact of Aβ and CNTs on AChE and BuChE activities using Ellman s photometric method. To further understand the nature of interaction of Aβ and CNTs with AChE and BuChE, Michaelis-Menten parameters, K m and V max were determined. Findings from this study would enhance our understanding of interaction between Aβ and enzymes (AChE and BuChE) and would have implications for the development of novel AD therapeutics targeting these biomarkers.

45 32 Chapter 5 5. Experimental 5.1 Reagents Aβ 42 was purchased from BioBasic (Markham, ON). Wild-type AChE B of Nippostrongylus brasiliensis was obtained from Invitrogen (Karlsruhe, Germany). MWCNTs with an outer diameter between nm, length between 1-12 µm and a purity of approximately 99 wt%, were purchased from Cheap Tubes Inc. (VT, USA). DTNB, BuChE from human serum, substrates (ATChI and BuTChI) were purchased from Sigma-Aldrich (Oakville, ON). 5.2 Preparation of Samples MWCNTs were prepared at desired concentrations using DMSO and were dispersed in solution by sonication for 15 min. Solutions of COOH- and NH 2 -functionalized MWCNTs were prepared using ultrapure water. Desired concentrations of AChE and BuChE were prepared by using 0.1 PBS buffer at ph 8 and measuring OD at 280 nm (ε280=93820 M-1 & M-1) and mw (64.7 and 55) kda respectively using NanoDrop 2000 (Thermoscientific, ON). ATChI and BuTChI solutions were prepared in ultrapure water. DTNB was prepared in 0.01 M in PBS at ph 7 with 0.15% w/v NaHCO 3 and was aluminum foiled to prevent reactivity with light. Aβ 42 monomers were prepared by dissolving Aβ powder in HFIP at a ratio of 1 mg: 1 ml and leaving it overnight sealed. HFIP was then removed by nitrogen bubbling. Samples were stored at -20 C until needed and were reconstituted in DMSO. Desired concentrations of peptides were obtained by diluting them with 50 mm phosphate buffer saline (PBS) at ph 7.4 and measuring OD at 280 nm (ε280= 1280 M-1) using NanoDrop 2000 (Thermoscientific, ON) [49].

46 Time-Dependence Studies of MWCNTs Incubated with AChE AChE (0.5 µg/µl) in 0.1 PBS buffer ph 8 was incubated individually and with COOH modified MWCNTs (500 µg/µl) (ratio of 1000:1, MWCNTs: AChE) at 37 C with stirring for various time periods (0, 24 and 72 h). Following protocol described by Alvarez et al. (1998) [9], after incubation for desired time, samples were centrifuged at rpm for 30 min using Microcentrifuge 5418 (Eppendorf, Canada). Supernatants were removed and pellets (AChE-MWCNT complexes) were resuspended in 0.1 PBS buffer at ph 8. To remove non-complexed AChE, pellet fraction was washed three times by centrifugation and resuspension in PBS buffer using vortex. AChE activity was measured in all supernatants and final pellet using Ellman s photometric method described in section Aggregation State Experiments of Aβ 42 Incubated with AChE and BuChE AChE and BuCHE (0.5 µg/µl) were incubated individually and with (500 µg/µl) Aβ for 5 days. Following incubation, various fractions (Supernatant, washes 1,2,3 and pellet) were collected and their activities were measured as described in section 5.2 [9]. 5.5 Ellman s Photometric Method Depending on molecules under study MWCNTs/Aβ, samples were added to a 96- microwell plate followed by addition of the enzyme (AChE/BuChE) and DTNB (340 µm). Samples were mixed well by stirring for 15 min. Depending on enzyme under study, substrate, ATChI/BuChE (550 µm) was added simultaneously using a multi-channel pipette. Absorbance at 410 nm over time was measured for about 2-3 min. Enzyme activity in (a.u./min) was determined from slope of graph of absorbance over time. Percentage enzyme activity in presence of MWCNTs and Aβ was calculated using the following formula [49, 82]: % enzyme activity = enzyme activity in presence of desired sample (MWCNTs/Aβ) enzyme alone

47 Determination of Kinetic Parameters for the Interaction of COOH-MWCNTs with AChE To determine Michaelis-Menten parameters (V max and K m ), AChE activity was measured in various fractions of samples (incubated with COOH-MWCNTs described in section 5.2) at different ATChI concentrations (56, 112, 225 and 550 µm). The activity was measured by Ellman s method as described above. Lineweaver-Burk plots were created by plotting 1/v (inverse of initial velocity) as a function of the inverse of substrate concentration. 5.7 Transmission Electron Microscopy (TEM) TEM images were obtained for AChE and BuChE (enzymes alone) and for samples containing complexes of COOH-MWCNTs with AChE, BuChE and Aβ. An aliquot (6 µl) of desired sample was spotted onto a nickel formvar mesh grid (Electron Microscopy Sciences, Hatfield, PA) for 5 min and blot dried. TEM grids were then stained using 6 µl of 2% uranyl acetate for 1 min followed again by blot drying. Samples were imaged using Hitachi H-7500 transmission electron microscope at the Centre for the Neurobiology of Stress at UTSC.

48 35 Chapter 6 Results and Discussion 6. Optimum Enzyme Concentration To evaluate the effect of MWCNTs and Aβ 42 on AChE and BuChE activities, enzyme concentrations, which would give a desired signal (intial velocity between a.u./min) were determined. Figures 18 and 19 show plots of absorbance (a.u.) over time (s) for various concentrations of AChE and BuChE using substrate ATChI. Figure 18: Absorbance (a.u.) at 410 nm over a time period of 180s for various AChE concentrations (0.060, 0.125, and U/mL). Blank condition (control) contains ATChI, DTNB and PBS. Other experimental conditions include contents of blank in addition to indicated concentrations of AChE. Each plot represents an average of three trials (n=3).

49 36 Figure 19: Absorbance (a.u.) at 410 nm measured over a time period of 180s for various BuChE concentrations (0.060, 0.125, and 0.500) U/mL. Blank consists of DTNB, ATChI and PBS. Experimental conditions include contents of blank as well as indicated concentrations of BuChE. Each plot is an average of three trials (n=3). Table 1 shows activities of AChE and BuChE with ATChI and BuTChI (determined from slopes in Figures 18 and 19) at various enzyme concentrations. At all concentrations, BuChE showed higher activities for substrate BuTChI compared to ATChI. Comparing across enzymes, BuChE showed higher activities to hydrolyze ATChI. This effect was more visible for greater concentrations of ATChI (0.500 and 0.250) U/mL. For both enzymes, the concentration of U/mL gave an activity in the desired range ( a.u./min). This concentration was selected for subsequent experiments.

50 37 Table 1: Activities (a.u./min) of AChE and BuChE at various concentrations. Activities were calculated by using the slopes of absorbance at 410 nm over a period of 180 s. [enzyme] (U/mL) Substrate ATChI BuTChI ATChI BuTChI ATChI BuTChI ATChI BuTChI AChE Activity 0.25 ± ± ± ± (a.u./min) BuChE Activity 0.76 ± ± ± ± ± ± ± ± (a.u./min) Two important results were obtained from our experiments observing the hydrolysis of ATChI and BuTChI by AChE and BuChE. 1) For a given concentration of enzyme, BuChE showed a greater activity (faster reaction rate) for BuTChI compared to ATChI. 2) For a given ATChI concentration, BuChE showed a greater reaction rate compared to AChE and this effect was more visible at higher ATChI concentrations. At lower ATChI concentrations, activities of AChE and BuChE were fairly similar. Both of these results are in agreement with literature. The preferential hydrolysis of BuTChI over ATChI by BuChE has been widely reported (29-30, 82-83). Similarly, under normal conditions, where enzyme activity is only controlled by diffusion of substrate to its active site, the K m of ACh hydrolysis by BuChE is higher than that of AChE. This suggests that BuChE was more effective at higher substrate concentrations, while AChE is more active at lower substrate concentrations [20, 83]

51 Effect of MWCNTs on AChE Activity After determining enzyme concentrations which provided a desired signal/initial velocity, effect of MWCNTs on AChE was explored. Figure 20 shows absorbance at 410 nm over time for three experimental conditions: 1) blank 2) AChE alone 3) AChE in the presence of 4 µg/µl MWCNTs. The condition with MWCNTs gave the lowest slope/activity, indicating the inhibitory activity of the MWCNTs on AChE. Figure 20: Absorbance (a.u) at 410 nm over a period of 180 s for conditions: enzyme alone, enzyme with 4 µg/µl MWCNTs and blank. The contents of blank included DTNB, ATChI in PBS in the absence of AChE. As expected, AChE gave the highest slope by itself compared to the other two conditions. Each plot represents an average of three trials (n=3) Effect of Concentration of MWCNTs Figure 21 shows absorbance (a.u.) at 410 nm over time for samples with various concentrations of MWCNTs (1, 2 and 4 µg/µl). Table 2 shows the percentage of AChE activity observed in the presence of various MWCNT concentrations.

52 39 Figure 21: Absorbance (a.u.) at 410 nm over a period of 180 s in the presence of various concentrations of MWCNTs. AChE showed the highest slope, followed by 1 µg/µl, 2 µg/µl and 4 µg/µl MWCNTs conditions. Each plot represents an average of three trials (n=3). Highest slope/percent AChE activity was observed in the absence of MWCNTs. As the concentration of MWCNTs increased a lower slope/percent AChE activity was observed. Table 2: Percentage of AChE activity observed in the presence of various concentrations of MWCNTs. Percentage activity was determined with respect to enzyme alone using the following equation: % activity = activity in presence of MWCNTs (desired concentration) enzyme alone Concentration of non-functionalized % AChE activity MWCNTs (µg/µl)

53 AChE Activity in Presence of COOH- & NH 2 -Functionalized MWCNTs To further understand the nature of interaction between MWCNTs and AChE, AChE activity was determined using functionalized (carboxylated and amine-modified) MWCNTs. Figure 22 shows plots of absorbance (a.u.) at 410 nm over time for samples incubated with various types of MWCNTs. Table 3 shows the percentage of AChE activities observed under each condition represented in Figure 22 with respect to enzyme alone. Highest slope/activity was observed for the enzyme itself followed by COOH- and NH 2 -functionalized MWCNTs. Lowest enzyme activity was observed in the presence of the non-functionalized MWCNTs. Figure 22: Absorbance (a.u.) at 410 nm over a period of 180 s in presence of various types of MWCNTs (non-functionalized, COOH- and NH 2 -functionalized). Highest slope was obtained for the enzyme by itself followed by COOH- and NH 2 -functionalized MWCNTs. Each plot is an average of three trials (n=3).

54 41 Table 3: Percentage AChE activity observed in presence of various types of MWCNTs (nonfunctionalized, COOH- and NH 2 -functionalized). Percentage activity was calculated with respect to enzyme activity using the following equation: % activity = activity in presence of MWCNTs (desired type) enzyme alone Type of MWCNTs % AChE activity NH 2 -functionalized 72 COOH-functionalized 38 Non-functionalized 10 Our results indicated that MWCNTs inhibited AChE activity. As the concentration of MWCNTs increased in AChE samples, less enzyme activity (indicated by a lower slope) was observed. Non-functionalized MWCNTs were found to inhibit AChE the most followed by NH 2 - functionalized MWCNTs. While, COOH-MWCNTs showed the lowest inhibition of AChE activity. Our results were in agreement with previous studies monitoring the interaction between CNTs and AChE. These studies suggested that CNTs adsorbed on AChE and inhibited its activity [84-85]. For instance, a study by Cabral et al. [84] observing the behavior of immobilized AChE on COOH-MWCNTs in the presence of various inhibitors found that following immobilization, AChE activity (indicated by V max ) decreased approximately by a factor of 45. Furthermore, our observation of strongest AChE inhibition by non-functionalized MWCNTs and lowest inhibition by COOH-MWCNTS may be explained by the computational studies of Cabral et al. [63] involving electrodes modified with AChE covalently bound to CNTs. This study suggested that the crystal structure of AChE consisted of 57% hydrophobic residues and 23% hydrophilic residues. They also reported that interaction between MWCNTs and AChE involved seven residues (involving both hydrophobic and hydrophilic residues) with hydrophobic residues being dominant [63]. Based on this, as non-functionalized MWCNTs were the most hydrophobic compared to other types used in our experiment, they would be expected to have a stronger binding/interaction with AChE. Hence, they reduced its activity more than functionalized MWCNTs (COOH- and NH 2 -functionalized), which were more polar.

55 42 Our results were also in agreement with studies reporting the impact of CNTs on Aβ fibril formation where hydrophobic interactions were found to be responsible for the affinity between these two molecules [66-71]. It was proposed that CNTs inhibited the fibrillation by disrupting intra-peptide hydrophobic interactions [69-70] Time-Dependence Studies of AChE-CNT Samples In addition to exploring concentration and type of MWCNTs, their effect on AChE activity was also studied by varying incubation time of the two samples. Tables 4, 5 and 6 show AChE activities observed in various fractions (supernatant, washes 1, 2, 3 and pellet) after incubation with COOH-MWCNTs for 0, 24 and 72 h, respectively. As can be observed in Table 4, at time 0, similar activities with respect to enzyme alone were observed in supernatant (7%) and washes 1,2 and 3 (6%). While, 12% AChE activity was observed in the pellet. After 24 h of incubation of COOH-MWCNTs with AChE, fairly similar enzyme activities with respect to enzyme alone were observed in supernatant (6%) and in wash 1 (6%), wash 2 (9%) and wash 3 (6%) (Table 5). While the pellet fraction displayed only 5% AChE activity.

56 43 Table 4: AChE activity (a.u./min) detected in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs at time 0. Each activity is an average of three trials. Percentage AChE activity was determined using the following equation: % activity = activity in presence of MWCNTs (desired fraction) enzyme alone Enzyme Fraction AChE activity ± SD (a.u./min) % AChE activity Enzyme alone 2.81 ± Supernatant ± Wash ± Wash ± Wash ± Pellet ±

57 44 Table 5: AChE activity (a.u./min) observed in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs for 24 h. Each activity is an average of three trials. Percentage AChE activity was determined using the following equation: % activity = activity in presence of MWCNTs (desired fraction) enzyme alone Enzyme fraction AChE activity (a.u./min) ± SD % AChE activity Enzyme alone ± Supernatant ± Wash ± Wash ± Wash ± Pellet ± As can be observed in Table 6, similar enzyme activities were obtained for supernatant (11%), wash 1(12%), wash 2 (12%) and wash 3 (11%) after 72 h of incubation with COOH-MWCNTs. While, 30% AChE activity was observed in the pellet fraction. Table 7 displays the percentage AChE activity from Tables 4, 5 and 6 as a function of incubation time with COOH-MWCNTs.

58 45 Table 6: AChE activity (a.u./min) observed in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) of samples incubated with COOH-MWCNTs for 72 h. Each activity is an average of three trials. Percentage AChE activity was determined using the following equation: % activity = activity in presence of MWCNTs (desired fraction) enzyme alone AChE fraction AChE activity ± SD (a.u./min) % AChE activity Enzyme alone ± Supernatant ± Wash ± Wash ± Wash ± Pellet ±

59 46 Table 7: Percentage AChE activity observed in various fractions incubated with COOH- MWCNTs for different time periods (0, 24 and 72 h) (results from Tables 4, 5 and 6). Average column indicates AChE activity observed across the three time points. Percentage AChE activity was determined using the following equation: % activity = activity in presence of MWCNTs (desired fraction) Enzyme alone AChE Fraction % AChE activity Incubation time Time 0 24 hours 72 hours Average Enzyme alone Supernatant Wash Wash Wash Pellet Our results for time-dependence studies of AChE incubated with COOH-MWCNTs were fairly similar. This suggested that time of interaction between these two molecules was not a factor in inhibition by MWCNTs. It also suggested to the following two possibilities. 1) Adsorption was a rapid process that occurred quickly upon contact between the two molecules (was not timedependent). 2) A factor other than adsorption had a greater impact on the interaction between MWCNTs and AChE. This second factor could be the size or concentration of MWCNTs as shown in section 6.1 [63]. Due to their large size, CNTs might be blocking the substrate from

60 47 entering enzyme s active site. Hence, despite the long interaction time, no change in AChE activity could be observed. Our results for various fractions of AChE (Supernatant, Wash 1, Wash 2 and Wash 3) had lower activities compared to the pellet. This confirmed our earlier concentration effect results suggesting that higher concentration of MWCNTs caused a greater inhibition in AChE activity Kinetic Parameters for AChE-MWCNT Interaction Table 8 shows AChE activity (a.u./min) in various fractions (supernatant, wash 1, wash 2, wash 3 and Pellet) as a function of substrate concentration after incubation with COOH-MWCNTs. Supernatant, wash 1, wash 2, wash 3 and pellet showed fairly similar AChE activities at all substrate concentrations studied (56, 112, 225, 550 µm), while the enzyme alone showed increasing AChE activity with increasing substrate concentrations.

61 48 Table 8: AChE activity (a.u./min) observed in various fractions incubated with COOH- MWCNTs as a function of substrate concentration (56, 112, 225 and 550) µm. SN, W1, W2 and W3 represent supernatant, wash 1, wash 2 and wash 3 fractions, respectively. Enzyme Fraction Substrate Concentration (µm) Enzyme alone SN W1 W2 W3 Pellet ± ± 0.15 ± 0.14 ± 0.14 ± 0.21 ± ± ± 0.14 ± 0.15 ± 0.14 ± 0.24 ± ± ± 0.15 ± 0.15 ± 0.14 ± 0.24 ± ± ± 0.16 ± 0.16 ± 0.16 ± 0.25 ± Table 9 shows K m and V max values for the interaction of AChE with COOH-MWCNTs determined from Lineweaver-Burk plots (shown in appendix section) using data presented in Table 8. The highest K m and V max values (67 µm and 0.77 ± 0.2 a.u./min respectively) were obtained for enzyme alone. The lowest K m and V max values (0.25 µm and 0.15 ± a.u./min respectively) were obtained for the pellet. Roughly similar K m and V max values respectively were obtained for supernatant (8.3 µm, 0.16 ± a.u./min), wash 1 (4.3 µm, 0.16 ± a.u./min), wash 2 (9.1 µm, 0.16 ± a.u./min) and wash 3 (8 µm, 0.15 ± a.u./min).

62 49 Table 9: Kinetic parameters, K m (µm ) and V max (a.u./min) for the complex of AChE with COOH-MWCNT observed in various fractions. Values were obtained from linear regression of Lineweaver-Burk plots shown in appendix. Condition K m (µm) V max (a.u./min) Enzyme alone ± 0.2 Supernatant ± Wash ± Wash ± Wash ± Pellet ± For supernatant and washes 1, 2 and 3, fairly similar AChE activity was observed with increasing substrate concentrations. Although for all these fractions, an overall pattern of increasing enzyme activity with an increasing substrate concentration was observed. A similar effect was observed for enzyme alone and the pellet fractions, but it was more pronounced for the conditions with enzyme alone in solution. Our results were in agreement with our earlier studies observing the effect of MWCNT concentration on AChE activity. As all fractions (supernatant, wash 1, wash 2, wash 3 and pellet) included MWCNTs, for a given substrate concentration, activity observed in any fraction was less than that for enzyme alone. In addition, although the concentration of substrate was changed similarly in all fractions (ie. same quantity of change), increase in substrate concentration showed a greater increase in AChE activity for enzyme alone then for other conditions/fractions. This provided support for our earlier suggestion that due to their size, CNTs might be blocking the entry of substrate into the enzyme s active site and that concentration of

63 50 substrate mediates the extent of AChE inhibition by MWCNTs. In other words, a given concentration of CNTs could inhibit AChE activity less at high substrate concentrations. However, as for the enzyme alone, we would expect this effect to not hold at very high substrate concentrations, where an opposite effect was observed. Many studies have reported the inhibition of AChE activity at very high substrate concentrations [9, 22, 74]. For instance, in their studies involving immobilization of AChE onto polyacrylamide/carbon nanotube nanocomposite nanofibers, Amini et al. [74] reported that up until a substrate concentration of 0.6 mm, AChE activity increased with an increase in substrate concentration. They suggested that a reverse trend was observed after this concentration. The inhibition of AChE at high substrate concentration was proposed to be due to some substrate binding PAS of the enzyme [74]. In our studies, we obtained the highest K m and V max 67 µm and 0.77 ± 0.2 a.u./min, respectively for enzyme alone and the lowest K m and V max values of 0.25 µm and 0.15± a.u./min for the pellet fraction. The K m and V max values for other conditions were fairly similar and were significantly lower than those of the enzyme alone. Comparing the K m and V max values of enzyme alone with the pellet fraction, both values were significantly less in the pellet fraction. This was attributed to a mixed inhibition mechanism of AChE activity by MWCNTs, where the inhibitor would bind to enzyme-substrate complex only [77, 79-80]. Our results were also in agreement with those found by Cabral et al. [63], who studied kinetic parameters for covalent immobilization of AChE onto COOH-MWCNTs. Compared to free enzyme, the AChE/MWCNTs suspensions also found V max values to decrease significantly. However, in their experiments the K m value for enzyme alone and that of AChE/MWCNTs was the same suggesting a non-competitive inhibition mechanism, where the inhibitor was bound to both free enzyme and the enzyme-substrate complex. One possible explanation for this difference could be that our experiments were carried out in solution, while in the experiments of Cabral et al. [63], AChE was immobilized onto CNTs. Cabral et al. [63] proposed that at MWCNTs affected the degree of freedom of AChE, hence the enzyme might not be working under diffusion control with respect to substrate concentration. They also suggested that there might be a competition between CNTs and AChE for electrostatically attracting the substrate. Furthermore, V max may be reduced due to substrate being unable to reach the active site and getting adsorbed on CNTs [63].

64 51 From our time-dependence experiments averaged across the three time points, we observed an average of 32% AChE activity in the pellet fractions of COOH-MWCNTs. We assumed that some AChE adsorbed on MWCNTs might be retaining their enzymatic activity [84]. As adsorption is believed to be the main mechanism of inhibition by CNTs, it can cause inactivation of enzyme due to conformational changes and kinetic enhancement due to increased concentration of substrate inside the CNTs. In other words, when the local concentration of the substrate was increased [ie. high substrate concentration], this could have led to the inhibition of the enzyme [9, 22, 74, 85]. 6.2 Effect of Amyloid-β on AChE activity As both AChE and BuChE were reported to interact with Aβ peptides discussed in Chapter 1, we explored the effect of Aβ 42 in various stages (soluble, aggregated) on their activities Effect of Aggregation State of Aβ 42 Table 10 shows activities of AChE and BuChE in various fractions (supernatant, wash 1, wash 2, wash 3 and pellet) after incubation with Aβ 42 for 5 days. Supernatant and washes 1, 2 and 3 were hypothesized to contain soluble peptide fractions, while the pellet contained aggregates [9].

65 52 Table 10: Activities of AChE and BuChE in (a.u./min) and percentage for various fractions after 5 days of incubation with Aβ 42. Each activity represents an average of three trials. Percentage activities for various fractions of AChE and BuChE (with respect to the respective enzyme alone) were determine using the equation % activity = activity in the presence of Aβ 42 (desired fraction) Enzyme alone AChE BuChE Enzyme fraction Enzyme activity % enzyme Enzyme activity ± % enzyme ± SD (a.u./min) activity SD activity Enzyme alone 0.14 ± ± Supernatant 0.13 ± ± Wash ± ± Wash ± ± Wash ± ± Pellet 0.10 ± ± For AChE, with respect to enzyme alone, 98% activity was observed in supernatant fraction. Fairly similar activities 79%, 75%, 83% and 75% were observed for wash 1, wash 2, wash 3 and pellet respectively. For BuChE, with respect to enzyme alone fairly similar activities, 94%, 101%, 103% 95% and 102 % were observed for supernatant, wash 1, wash 2, wash 3 and pellet fractions, respectively. Fairly high levels of AChE activity observed in supernatant, wash 1, wash 2, wash 3 and pellet fractions, suggested that unlike COOH-MWCNTs, Aβ did not significantly inhibit AChE

66 53 activity. Our results were in agreement with existing literature suggesting that Aβ promotes AChE activity [5, 9]. For instance, Alvarez et al. [9] suggested that formation of AChE-Aβ complexes changed the biochemical and pharmacological properties of AChE. They also found that AChE bound to Aβ was resistant to low ph, high substrate concentration and had a lower sensitivity to its inhibitors. Furthermore, in their pellet fractions, a 78% AChE activity with respect to enzyme alone was observed [9]. This was very similar to our pellet fraction, which showed an average activity of 75%. Similarly, Melo et al. [5] found that retinal cells incubated with Aβ showed an increase in AChE activity. They suggested that this effect was mediated by oxidative stress as antioxidants such as α-tocopherol acetate and nitric oxide synthase inhibitors were capable of preventing this effect [5]. As discussed in section 7.1, AChE has widely been reported to be less inhibited by high substrate concentrations [9, 22, 74, 85]. This is thought to be due to excess substrate binding to PAS, which further prevents its ability of binding to substrates at its CAS [74]. However, results from our studies in agreement with Melo et al. [5] and Alvarez et al. [9], suggested that binding of Aβ to PAS resulted in enhancing the enzyme s activity. Hence, this inhibition effect of high substrate concentration would be specific to the substrate. Our observation of a greater activity in supernatant fraction compared to pellet and the three washes suggested that soluble forms of the peptide were better at enhancing AChE activity than the fibril/aggregated forms. One explanation for this might be that as observed in the MWCNTs, Aβ fibrils due to their large size could have blocked the active site of AChE. From the various fractions of BuChE incubated with Aβ, the following percentages of enzyme activity were observed for supernatant, washes 1,2,3 and the pellet respectively 94%,101%, 103%, 95% and 102%. As these values were fairly close to uninhibited enzyme, similar to the case for AChE, Aβ peptide did not affect BuChE s activity. The difference in the values, however, was attributed to the interaction between two factors. 1) The structural differences between the enzymes such as their different molecular size, channel of substrate molecule to the active site being wider in BuChE and the gorge cavity in BuChE being less confined and having a large volume than AChE [86]. 2) Due to research showing that BuChE inhibits and AChE promotes fibrillation of Aβ, the pellet of AChE would have contained more fibrils compared to the BuChE condition [29].

67 54 Our results were in agreement with those of Diamant et al. [29], who studied the effect of BuChE on Aβ aggregation. Upon incubation of the two molecules, BuChE activity was present in all fractions (soluble and pellet) of samples incubated with Aβ. Diamant et al. [29] also found that BuChE bound the soluble (monomeric and oligomeric) species of Aβ and slowed down their conversion to larger β-sheet rich species [29]. 6.3 TEM Images for Enzyme Samples that were Incubated with CNTs and Aβ This section shows TEM images of enzymes alone (AChE and BuChE) and also the ones that were incubated with Aβ 42 and COOH-MWCNTs (pellet fractions). All images were obtained by spotting desired sample onto a nickel formvar mesh grid and were viewed using Hitachi H-7500 transmission electron microscope as described in the Experimental section 5.7. Figure 23 shows the TEM image of AChE (0.5 µg/µl) alone. Using the crystal structure of Torpedo californica AChE in complex with 20 MM thiocholine obtained from the Protein Data Bank (PDB #: 2C5G) shown in Figure 24, we approximated the size of AChE to be between 4-7nm. Our results were in agreement with this finding and were similar to those obtained by Inestrosa et al. [89] (Figure 25).

68 55 Figure 23: TEM image of AChE B (0.5µg/µL) on nickel formvar mesh grid obtained using Hitachi H-7500 transmission electron microscope. Magnification is indicated by the scale bar.

69 56 Figure 24: Crystal structure of Torpedo californica AChE in complex with 20 MM thiocholine obtained from Protein Data Bank (PDB #: 2C5G).

70 57 Figure 25: TEM image of AChE obtained on formvar-coated grids and negative-stained with 2% uranyl acetate. Scale bar represents 100 nm (Adapted with permission from Inestrosa et al., 1996 [89] Elsevier). Figure 26 shows the TEM image of BuChE (0.5 µg/µl) alone. Similar to AChE, the crystal structure of human BuChE complexed with tacrine was obtained from the Protein Data Bank (PDB #: 4BDS) (Figure 27). Based on the crystal structure we estimated the size of BuChE to be between 3-5 nm. Our TEM image was in agreement with this finding.

71 58 Figure 26: TEM image of BuChE (0.5 µg/µl) on nickel formvar mesh grid obtained using Hitachi H-7500 transmission electron microscope. Magnification is indicated by scale bar.

72 59 Figure 27: Crystal structure of human BuChE in complex with tacrine obtained from Protein Data Bank (PDB #: 4BDS). Figures 28 and 29 show TEM images of AChE (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl) at different magnifications. Figure 30 shows the TEM image of BuChE (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl). The diameters and lengths of COOH-MWCNTs observed in our TEM images were in agreement with the ranges of nm and 1-12 µm respectively reported by Cheap Tubes Inc. In addition, the adsorption of AChE and BuChE on MWCNTs can also be observed in our images supporting our earlier results (indicated by arrows).

73 60 Figure 28: TEM image of AChE (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate AChE adsorbed on COOH-MWCNTs and scale bar indicates magnification.

74 61 Figure 29: TEM image of AChE B (0.5µg/µL) incubated with COOH-MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate AChE adsorbed on COOH-MWCNTs and scale bar indicates magnification.

75 62 Figure 30: TEM image of BuChE from human serum (0.5µg/µL) incubated with COOH- MWCNTs (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrows indicate BuChE adsorbed on COOH-MWCNTs and scale bar indicates magnification. Figures 31 and 32 show the TEM images of AChE and BuChE (0.5µg/µL) respectively incubated with Aβ 42 (500 µg/µl). Figure 33 shows the crystal structure of Aβ 40 obtained from the Protein Data Bank (Alzheimer s disease amyloid A4 peptide, PDB #: 1AML). Based on this structure we estimated the size of Aβ 42 to be between 4-6 nm. However, our TEM images showed an approximate size of nm. This can be explained by Aβ 42 being in its aggregated form formed by clumping of many monomers. Our TEM images were in agreement with those obtained by Mold et al. [91] of Aβ 42 incubated with Cu (II), shown in Figure 34.

76 63 Figure 31: TEM image of AChE B (0.5 µg/µl) incubated with Aβ 42 (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrow indicates AChE-Aβ 42 complexes and scale bar indicates magnification.

77 64 Figure 32: TEM image of BuChE from human serum (0.5 µg/µl) incubated with Aβ 42 (500 µg/µl) on nickel formvar mesh grid. Image was obtained using Hitachi H-7500 transmission electron microscope. Arrow indicates BuChE- Aβ 42 complexes and scale bar indicates magnification.

78 65 Figure 33: Crystal structure of Aβ 40 (Alzheimer s disease amyloid A4 peptide, PDB #: 1AML) obtained from the Protein Data Bank.

79 66 Figure 34: TEM image of Aβ 42 (5 µm) in the presence of Cu (II) (10 µm) (Adapted from [91] Macmillan Publishers Limited). 6.4 Conclusions In our studies, MWCNTs were found to inhibit AChE activity in a concentration dependent manner (higher concentrations of MWCNTs led to a greater inhibition). We also found that hydrophobicity played a role in this interaction such that non-functionalized MWCNTs interacted strongly to AChE and inhibited it to a greater extent compared to COOH- and NH 2- functionalized MWCNTs. Furthermore, we found that incubation time of AChE and MWCNTs had no effect on inhibition of enzyme activity. With respect to our Aβ experiments, in agreement with previous literature, we found that Aβ promoted the activities of both AChE and BuChE. However, further experiments are needed to understand the mechanism by which this occurs particularly for BuChE, as its PAS differs from that of AChE. The change in the levels of AChE and BuChE activity upon binding to Aβ has implications for AD, as it could help explain the devastated low levels of ACh observed in the disease. Future work involving studying activities of AChE and BuChE incubated in the presence of both MWCNTs and Aβ combined would provide insights into MWCNTs as potential vehicles of future AD therapeutics.