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The development of a method to deliver neuroprotective peptides specifically into stroke-affected neurons Lo, Edmund 2007

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THE DEVELOPMENT OF A METHOD TO DELIVER NEUROPROTECTIVE PEPTIDES SPECIFICALLY INTO STROKE-AFFECTED NEURONS  by  EDMUND KWOK-FAI LO B.Sc., The University of British Columbia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007 ©Edmund Kwok-Fai Lo, 2007  ABSTRACT  Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified TatGluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.  ii  TABLE OF CONTENTS Abstract ..................................................................................................................................... ii Table of Contents ..................................................................................................................... iii List of Figures ............................................................................................................................v List of Abbreviations .............................................................................................................. vii Acknowledgements ....................................................................................................................x  CHAPTER 1: INTRODUCTION .......................................................................................1 1.1. Overview ...........................................................................................................................1 1.2. Literature Review..............................................................................................................3 1.2.1. Etiology and Mechanism of Stroke........................................................................3 1.2.2. Cell Death During Stroke.......................................................................................4 1.2.3. NMDA Receptors ..................................................................................................6 1.2.4. Calcium ..................................................................................................................8 1.2.5. Calpain ...................................................................................................................9 1.2.6. AMPA Receptors .................................................................................................12 1.2.7. Therapeutic Treatments Against Stroke...............................................................14 1.2.8. Mechanism of Tat Peptide Delivery ....................................................................16 1.3. Thesis Objectives and Hypothesis ..................................................................................18 CHAPTER 2: MATERIALS AND METHODS ............................................................22 2.1. Materials .........................................................................................................................22 2.1.1. Drugs ....................................................................................................................22 2.1.2. Reagents ...............................................................................................................22 2.1.3. Peptide Construction ............................................................................................23 2.1.4. Animals ................................................................................................................23 2.2. Preparation of Primary Culture of Cortical Neurons ......................................................23 2.3. Experimental Stroke Model In Vitro and In Vivo ...........................................................24 2.3.1. Excitotoxic Insult In Vitro....................................................................................24  iii  2.3.2. Middle Cerebral Artery Occlusion (MCAO) Model ............................................25 2.4. Sample Preparation, SDS-PAGE and Immunoblotting...................................................26 2.5. In Vitro Peptide Cleavability Assay ................................................................................27 2.6. Fluorescent Peptide Detection in Vitro ...........................................................................27 2.7. Histochemistry, Immunohistochemistry and Fluorescence Microscopy ........................28 2.8. Data Analysis ..................................................................................................................29 CHAPTER 3: RESULTS ...................................................................................................30 3.1. Dose- and Time-Dependent Activation of Calpain in In Vitro and In Vivo Stroke Models....30 3.1.1. Dosage-Dependent Calpain Activation In Vitro ...................................................30 3.1.2. Immediate Activation of Calpain Time-Course In Vitro .......................................31 3.1.3. Delayed Activation of Calpain Time-Course In Vitro ..........................................31 3.1.4. Involvement of Caspase in Delayed Activation of Calpain Time-Course In Vitro ......32 3.1.5. Activation of Calpain Time-Course in an In Vivo MCAO Stroke Model ............33 3.2. Designing and Characterizing Tat-GluR2-3Y Peptide Cleavable by Calpain In Vitro .....34 3.2.1. The Insertion of CCS Sequence ............................................................................34 3.2.2. The Insertion of SP-2 or CP-3 Sequences.............................................................36 3.3. Failure to Detect Dansylated Peptides In Vivo ...............................................................38 CHAPTER 4: DISCUSSION .............................................................................................57 4.1. Calpain Activation Time-Course: Reconciliation of Differences Between In Vitro and In Vivo Results ...............................................................................................................57 4.2. Different Peptide Cleavage Profiles................................................................................58 4.3. Advantages of the GluR2/3Y Peptidic Segment in Neuroprotective Schemes ..............60 4.4. Peptide Pharmacokinetics ...............................................................................................60 4.5. Significance in Disease-Specific Delivery of Peptides ...................................................62 4.6. Complications in In Vivo Fluorescence Detection .........................................................64 4.7. Conclusion ......................................................................................................................65  Bibliography ............................................................................................................................66  iv  LIST OF FIGURES  Figure 1.1  The basic structure of NMDA receptors .............................................................20  Figure 1.2  The mechanism of the putative Tat-GluR2/3Y peptide with a calpain consensus cleavage sequence (the CCS peptide) .................................................................21  Figure 3.1  Extent of calpain activation under different doses of NMDA in primary cultured cortical neurons ...................................................................................................40  Figure 3.2  Immediate time-course of NMDA-stimulated calpain activation in primary cultured cortical neurons .....................................................................................41  Figure 3.3  Delayed time-course of NMDA-stimulated calpain activation after returning to Neurobasal medium in primary cultured cortical neurons ..................................42  Figure 3.4  The effect of the caspase inhibitor Q-VD-OPH on the NMDA-stimulated calpain activation after returning to Neurobasal medium in primary cultured cortical neurons ...................................................................................................43  Figure 3.5  The extent of calpain activation in an in vivo MCAO stroke model ..................44  Figure 3.6  The structure and susceptibility to calpain cleavage of the CCS peptide in vitro ......45  Figure 3.7  Comparing the 3Y and CCS peptides on the effect of excitotoxicity in the accumulation of 3Y-containing peptide segments in primary cultured cortical neurons....................46  Figure 3.8  The effect of endogenous calpain on the retainment of 3Y-containing peptide segments in CCS peptide-treated primary cultured cortical neurons..................47  Figure 3.9  The structures and susceptibilities to calpain cleavage of the SP-2 and CP-3 peptides in vitro, and the experimental procedure time-line...............................48  Figure 3.10 The amount of 3Y-containing peptide segments did not change during excitotoxicity in SP-2 peptide-treated primary cultured cortical neurons ..........50  v  Figure 3.11 Changes in the amount of 3Y-containing peptide segments during excitotoxicity in CP-3-treated primary cultured cortical neurons..............................................51 Figure 3.12 Testing the rate of 3Y-containing peptide segment retainment with a modified protocol in CP-3-treated primary cultured cortical neurons ...............................52 Figure 3.13 The extent to which 3Y-containing peptide segments were retained in NMDAtreated conditions under the modified protocol in CP3-treated primary cultured cortical neurons ...................................................................................................53 Figure 3.14 Failure to detect dansylated CCS peptide in vivo via biochemical methods ......54 Figure 3.15 Inability to directly detect dansyl fluorescence in rat brain slices ......................55 Figure 3.16 Non-specificity of the antibody against dansyl in rat brain slices ......................56  vi  LIST OF ABBREVIATIONS  Abbreviation 2+  Definition  [Ca ]i  Intracellular Ca2+ concentration  3Y peptide  Tat-GluR2/3Y  ABP  AMPAR binding protein  AChR  Acetylcholine receptor  AEBSF  4-(2-Aminoethyl) benzenesulfonyl fluoride  AIF  Apoptosis inducing factor  AMPA  Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid  AMPAR  AMPA receptor  ANOVA  Analysis of variance  Apaf-1  Apoptotic protease activating factor-1  APV  2-amino-5-phosphonovaleric acid  ASIC  Acid-sensing ion channel  ASMase  Acid sphingomyelinase  ATP  Adenosine triphosphate  BCL-2  B-cell lymphoma 2  BH3  BCL-2-homology 3  BSA  Bovine serum albumin  CCS  Calpain consensus cleavage sequence  CCS peptide  Tat-CCS-GluR2/3Y  Cdk  Cyclin-dependent kinase  CH  Contralateral hemisphere  CP-3 peptide  Tat-CRMP-3 sequence-GluR2/3Y  CREB  cAMP response element  CRMP  Collapsin Response Mediator Protein  DMEM  Dulbecco modified Eagle's medium  DMSO  Dimethyl sulfoxide  DNA  Deoxyribonucleic acid  ECL  enhanced chemical luminescence  ECS  Extracellular solution  ER  Endoplasmic reticulum  ERK  Extracellular signal-regulated kinase  vii  Fab  Fragment antigen binding  FBS  Fetal bovine serum  FDA  Food and Drug Administration  FRET  Fluorescence resonance energy transfer  GRIP  Glutamate receptor interacting protein  HBSS  Hank's balanced salt solution  HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  HRP  Horseradish peroxidase  IH  Ipsilateral hemisphere  IP3  Inositol triphosphate  LTD  Long-term depression  LTP  Long-term potentiation  MCAO  Middle cerebral artery occlusion  mGluR  Metabotropic glutamate receptor  MOMP  Mitochondrial outer membrane permeabilization  MPT  Mitochondrial permeability transition  MW  Molecular weight  NCX  Na+ / Ca2+ exchanger  NIR  Near infrared  NMDA  N-methyl-D-aspartic acid  NMDAR  NMDA receptor  NO  Nitric oxide  NOS  Nitric oxide synthase  NSF  N-ethyl-maleimide sensitive fusion protein  OGD  Oxygen-glucose deprivation  PARP-1  Poly (ADP-ribose) polymerase-1  PB  Phosphate buffer  PBS  Phosphate-buffered saline  PDL  Poly D-lysine  PFA  Paraformaldehyde  pHLIP  pH-low insertion peptide  PI3K  Phosphoinositide-3 kinase  PICK1  Protein interacting with C-kinase-1  PKB  Protein kinase B  PSD  Postsynaptic density  viii  ROS  Reactive oxygen species  RVG  Rabies viral glycoprotein  SBDP  Spectrin breakdown product  SCOP  Superchiasmatic nucleus circadian oscillatory protein  SDS-PAGE  Sodium dodecyl sulfate polyacrylamide gel electrophoresis  SEM  Standard error mean  siRNA  Short interfering RNA  SP-2 peptide  Tat-α Spectrin II sequence-GluR2/3Y  Tat  Transactivator of Transcription  TBST  Tris-buffered saline with Tween 20  tPA  Tissue plasminogen activator  TRPM  Transient receptor potential melastatin  ix  ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Dr. Yu Tian Wang, for his mentorship over the course of my thesis work. It has been a pleasure to learn my trades from one of the best scientist in the field of neuroscience. I am also deeply appreciative towards my fellow Wang lab colleagues, especially to Dr. Changiz Taghibiglou for the protein biochemistry training he had provided me while I was an undergraduate student in the Wang lab; to Dr. Yitao Liu and Mr. Ted Lai for their help on the in vivo surgical procedures; to Dr. Yuping Li for her assistance in preparing neuronal primary cultures; to Mr. Taesup Cho for his generous help with the immunohistochemical techniques; and to Mr. Steve Van Iderstine for advices in designing peptides as well as other lab-related issues. I would also like to thank Drs. Jie Lu, Joseph Pathakamuri, Tak Pan Wong and Paul Yong for their advices and insights, as well as Misses Helen Chiu and Rachelle Yong for their wonderful assistance in proofreading this thesis. To my dearest family and friends: words cannot even describe how grateful I am for your unceasing Love, prayers and support. Hence, let my words be few.  “In one manner or the other it still remains true that, even in the view of the mere biologist, the human epic resembles nothing so much as a way of the Cross.” — Pierre Teilhard de Chardin SJ  Ad Majorem Dei Gloriam  x  CHAPTER 1: INTRODUCTION  1.1.  Overview Stroke is a debilitating disease that exerts many negative impacts in society. It is the  fourth leading cause of death in Canada (Heart and Stroke Foundation of Canada, 2002); while in the United States it ranks third and is notoriously the leading cause of long-term disability (Rosamond et al., 2007). It also exerts a heavy burden on the economy: the annual cost of stroke-related expenses in Canada was an estimated 2.7 billion (CAD) in 2002 (Heart and Stroke Foundation of Canada, 2002) whereas the cost in the United States in 2007 is projected to be 62.7 billion (USD) (Rosamond et al., 2007). In addition to these statistics, the emotional and mental stresses that stroke patients and their caretakers have to endure are unquantifiable and severely hamper one’s well-being and quality of life. Prospective stroke treatments mainly focus on two strategies: a thrombolytic approach that aims to re-establish blood perfusion in an ischemic brain, or a neuroprotective approach with a priority of protecting and rescuing brain cells that are at risk (Fisher and Bogousslavsky, 1998). The only thrombolytic approach approved by the Food and Drug Administration (FDA) is the tissue plasminogen activator (tPA) but it is only effective when administered within three hours of stroke onset. Neuroprotective drugs have mostly focused on antagonizing the N-methyl-D-aspartate (NMDA) receptors as they are integral in the translation of excitotoxicity to cell death in stroke. However, most of the putative drugs have failed due to severe side effects. Clearly, new therapeutic approaches are needed. Accumulated evidence suggests that NMDAR activation triggers downstream cell death signaling by complex protein-protein interactions, and the disruption of these interactions by short interfering peptides holds promise as a new generation of  1  neuroprotective therapeutics. For instance, a peptide interfering with the association between the NMDAR subunit NR2B and the scaffolding protein PSD-95 is found to be neuroprotective in in vitro and in vivo animal stroke models (Aarts et al., 2002). Among the many deleterious effects that occur during excitotoxicity are the activation of the calciumdependent protease calpain and the endocytosis of the alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors. A previous study from our laboratory has demonstrated the neuroprotective effects of inhibiting AMPAR endocytosis by a synthetic peptide that mimics a short sequence on C-terminus of the AMPAR subunit GluR-2 (3Y) (Wang et al., 2004). This sequence can be delivered into intracellular space when tethered to the cargo-delivering Tat sequence (Brebner et al., 2005). In my thesis, I hypothesize that the delivery of neuroprotective peptides into stroke-affected regions can be enhanced by the insertion of a sequence cleavable by calpain between Tat and 3Y. The rationale is that the therapeutic 3Y segment will be retained in neurotoxic neurons after calpain cleavage, thus concentrating the neuroprotective effects at pathological regions. In order to test my hypothesis, the extent of calpain activation in an excitotoxic stroke model was first examined. In addition to a NMDA dosage-dependent increase, the level of calpain activation gradually elevated over time in both the immediate- and delayed time-course during excitotoxicity in vitro. Calpain activation was also observed in an in vivo stroke model, although the beginning of its activation seemed to be delayed compared to the in vitro models. These served as the first extensive account of temporal calpain activation in animal transient cerebral ischemia models. Three peptides with different cleavage sites recognized by calpain were then tested; while all were susceptible to calpain cleavage in in vitro cleavage assays, neither the CCS nor the SP-2 peptide showed difference between NMDA-treated and control condition in the amount of retained peptide and its exiting rate. The CP-3 peptide was the only  2  candidate that had more retained peptide and a slower peptide-exiting rate compared to the other two peptide candidates during excitotoxicity. An attempt to detect the dansylated peptides in an in vivo animal stroke model was unsuccessful due to technical difficulties. These findings suggest that it is possible to increase the amount of the therapeutic 3Y peptide fragment accumulation in stroke-affected neurons in vitro. This may lead to an enhanced neuroprotection and more importantly, a revolutionary shift in therapeutic drug design.  1.2.  Literature Review  1.2.1. Etiology and Mechanism of Stroke Stroke is a pathological condition where blood supplies to the brain are compromised; the two major types are of ischemic and hemorrhagic nature. Ischemic stroke, or focal cerebral ischemia, refers to a sudden interruption of blood to the brain which can be further categorized into two subtypes: embolic stroke, which occurs when an embolus serves as the source of arterial or blood vessel blockage, and thrombotic stroke, in which no proximal embolic source is involved (Blumenfeld, 2002). Hemorrhagic stroke is a result of artery or blood vessel rupture within the brain (Llinas, 2007). Ischemic stroke is the more prevalent of the two, accounting for over 80% of the reported stroke cases (Heart and Stroke Foundation of Canada, 2002; Llinas, 2007). Another type of stroke, albeit less common, is called global ischemia and it occurs when there is a systemic reduction of blood flow to the brain, the latter of which may be a consequence of cardiac arrests (Kawai et al., 1992). The brain areas damaged by focal cerebral ischemia can be categorized into two subregions: the infarct core and the penumbra. It is widely believed that the infarct core suffers severe damage, which results in rapid and irreversible cell death (Lipton, 1999; Syntichaki and Tavernarakis, 2003). However, the area surrounding the infarct core called the penumbra  3  endures a milder impact due to residual perfusion of proximal blood vessels (Weinstein et al., 2004). These neurons are thus mostly viable but still suffer functional impairment to a certain degree (Ginsberg, 2003; Calabresi et al., 2003). The penumbra also presents itself as an attractive target for neuroprotective drugs, as these damaged cells have the potential for revival. The brain region damaged by stroke is largely dependent upon the location of the blocked artery or vessel. The brain utilizes two molecules for its metabolic use: oxygen (O2) and glucose, both of which are purposefully delivered by blood. Ischemic stroke, then, by its nature deprives the brain of its nutrients. This results in the exhaustion of the energy molecule, adenosine triphosphate (ATP), since its primary substrates are precisely O2 and glucose (Cherubini et al., 2005). This post-ischemic energy depletion triggers depolarization in neurons from strokeaffected regions as well as sustained firings of action potentials given that both mechanisms are regulated by ATP. This leads to the continuous release of neurotransmitters into synaptic clefts, including the excitatory neurotransmitter glutamate. In addition, glutamate reuptake is compromised during focal cerebral ischemia (Lo et al., 2003; Seifert et al., 2006); consequently, the combination of these events exacerbates the over-abundance of glutamate, which in turn overwhelms postsynaptic glutamate receptors. These result in glutamate receptor over-activation and a subsequent influx of excess cations like sodium (Na+) and calcium (Ca2+) ions. This phenomenon, which is appropriately termed excitotoxicity, triggers many harmful downstream signaling pathways and reactions, eventually leading to cell death.  1.2.2. Cell Death During Stroke Brain cells that are severely damaged during excitotoxicity in the infarct core are thought to die by necrosis, a phenomenon characterized by mitochondria swelling,  4  intracellular vacuole formation and eventual cell lysis (Syntichaki and Tavernarakis, 2003). Meanwhile, the cells that are less severely hampered by the excitotoxic insult in the penumbra region do not die immediately but instead undergo a delayed cell death process called apoptosis, which is characterized by chromatin condensation, DNA fragmentation, nuclear pyknosis and the blebbing of plasma membrane (Kerr et al., 1972; Walker et al., 1988; Lo et al., 2003). Caspase-dependent and -independent cell death are the two types of apoptosis that are observed in stroke. The BCL-2 protein family plays a critical role in the caspase-dependent pathway: its family members consist of both pro-apoptotic and anti-apoptotic proteins (Cory and Adams, 2002). The anti-apoptotic protein BCL-2 serves to preserve the integrity of the mitochondrial membrane (Yenari et al., 2002). Pro-apoptotic proteins from the BCL-2 family that have only one BCL-2-homology 3 (BH3) domain sequester anti-apoptotic proteins to neutralize their protective effects (Wang et al., 1996; Cory and Adams, 2002). BH3-only proteins like Bid and Bim are involved in initiating apoptosis during cerebral ischemia (Wang et al., 1996; Shibata et al., 2002; Yin et al., 2002; Kuan et al., 2003; Okuno et al., 2004). Also, a part of the pro-apoptotic sect of the BCL-2 family is Bax, a protein that has more than one BH3 domain: its expression is found to be increased after transient cerebral ischemia and is then translocated to the mitochondria (Krajewski et al., 1995; Gillardon et al., 1996; Hara et al., 1996). This leads to the formation of mitochondrial outer membrane permeabilization (MOMP) and a subsequent release of cytochrome c which serves as the initial step of caspase-dependent apoptosis (Goldstein et al., 2000; Cao et al., 2001a; Green and Kroemer, 2004). It is thought that the generation of reactive oxygen species (ROS) also plays a critical role in priming the mitochondria to release cytochrome c and initiate the apoptotic cascade (Fujimura et al., 1999; Chan, 2001). The cytoplasm-bound cytochrome c  5  binds to the protein apoptotic protease activating factor-1 (Apaf-1) to form a complex called apoptosome (Zou et al., 1997; Zou et al., 1999; Johnson and Jarvis, 2004; Cao et al., 2004). It then binds to the inactive form of the initiator protease procaspase-9 and activates it into caspase-9 through autocatalysis (Srinivasula et al., 1998). The cytochrome c-Apaf-1-caspase9 apoptosome complex serves as a caspase amplification system to activate the effector protease procaspase-3 into caspase-3 (Gill et al., 2002; Ferrer et al., 2003). Caspase-3 is the enzyme that carries out proteolysis of targeted proteins (Namura et al., 1998; Chen et al., 1998b; Manabat et al., 2003), and it also triggers other deleterious events like DNA fragmentation (Liu et al., 1997; Janicke et al., 1998; Cao et al., 2001b; Luo et al., 2002). As aforementioned, the caspase-independent apoptosis pathway also contributes to ischemic cell death. Poly (ADP-ribose) polymerase-1 (PARP-1) is one of the major players: it is a nuclear enzyme that is activated upon DNA damage and is responsible for catalyzing DNA repair (de Murcia et al., 1994; Shall and de Murcia, 2000). Despite its function, PARP-1 activation during excitotoxicity leads to NAD+ reduction which is sensed by the mitochondria, paradoxically triggering the release of apoptosis inducing factor (AIF) (Yu et al., 2002). AIF is a flavoprotein that is found in the intra-mitochondrial space (Susin et al., 1999). AIF then translocates into the nucleus where it mediates DNA fragmentation and cell death (Cregan et al., 2002; Plesnila et al., 2004). Cross-talk between the caspase-dependent and -independent pathways also exists, as the BH3-only protein Bid is found to regulate AIF (Culmsee et al., 2005).  1.2.3. NMDA Receptors Functional NMDARs are mostly heterodimers consisting of two NR1 and two NR2 subunits where different combinations of these yield vastly different pharmacological and  6  kinetic properties (Dingledine et al., 1999; Lau and Zukin, 2007) (Figure 1.1). While there exists multiple slice variants of the NR1 subunit, there are four distinctive NR2 subunits (A to D). NR2B and NR2D are more predominant during the neonatal period. Over the course of development, NR2A, and in some regions, NR2C, becomes the prominent NR2 subunits (Monyer et al., 1994). The trafficking of NR2A-and NR2B-containing receptors is very dynamic in young animals (Bellone and Nicoll, 2007), but NMDARs are believed to be relatively stable in mature nervous systems in general. The functions of NR2A and 2Bcontaining NMDARs are best characterized in the adult brain (Cull-Candy et al., 2001). Compared to NR2B-containing receptors, NR2A-containing ones have faster channel kinetics, less affinity for glutamate, stronger channel open probabilities and are more susceptible to Ca2+-dependent desensitization (Lau and Zukin, 2007). Meanwhile, NR2C and 2D-containing receptors have weaker channel open probabilities (Cull-Candy and Leszkiewicz, 2004). The channel pore of NMDAR is blocked by the divalent cation magnesium (Mg2+) at neuronal resting membrane potential (Mayer et al., 1984). This block is removed upon simultaneous membrane depolarization along with the association of NR2 subunit-binding glutamate and the receptor co-agonist glycine, which itself binds to the NR1 subunit (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Once the Mg2+ block is removed, NMDARs become permeable to various ions like monovalent cations and Ca2+ (Mayer and Westbrook, 1987). NMDARs are critical components in synaptic plasticity, as evident from their leading roles in physiological phenomena such as long-term potentiation (LTP) and long-term depression (LTD), mechanisms that are thought to underlie the process of learning and memory (Bliss and Collingridge, 1993; Collingridge et al., 2004; Malenka and Bear, 2004).  7  In an ischemic environment, NMDARs are activated by excess glutamate (Simon et al., 1984), and the extrasynaptic NR2B-containing receptors are largely responsible for the observed NMDA-mediated cell death (Hardingham and Bading, 2003; Liu et al., 2007). Since NMDARs become highly permeable to Ca2+ upon Mg2+-block removal (MacDermott et al., 1986; Jahr and Stevens, 1987), they are integral in triggering Ca2+-dependent downstream pathways that are detrimental to the cells. In addition to initiating pathological increases in intracellular level of Ca2+ ([Ca2+]i), NMDARs also contribute to excitotoxicity in other ways: for instance, they are coupled to scaffolding proteins in the postsynaptic area called postsynaptic density-95 (PSD-95), which bind to many other different proteins in the PSD (Sheng, 2001). One of its many binding partners is nitric oxide synthase (NOS), an enzyme that synthesizes nitric oxide (NO) under excitotoxic conditions (Brenman et al., 1996). It has been found that disrupting the interactions between the NMDAR subunit NR2B and PSD-95 prevented NMDAR-mediated apoptosis during stroke (Aarts et al., 2002; Cui et al., 2007).  1.2.4. Calcium [Ca2+]i under normal physiological conditions is kept in the 100 nM range in most neurons, and is vigilantly regulated by ATP-dependent Ca2+ pumps in order to maintain a steep Ca2+ concentration gradient. [Ca2+]i is strictly monitored because it is an important second messenger of many intracellular signaling pathways that greatly impact synaptic plasticity (Ghosh and Greenberg, 1995). During stroke in mammalian brains, the resulting excitotoxicity is a Ca2+-dependent process (Choi, 1985), further highlighting its importance. In addition to the aforementioned NMDARs, other membrane-bound channels also contribute to Ca2+ influx. Voltage-sensitive Ca2+ channels are activated by the prolonged depolarization during cerebral ischemia, and the administration of its antagonist is found to  8  rescue the ischemic damages (Germano et al., 1987; Rami and Krieglstein, 1994). Ca2+permeable acid-sensing ion channels (ASICs) are activated by the excess lactic acid and proton that are generated during excitotoxicity (Xiong et al., 2004; Xiong et al., 2006). TRPM7 channels are Ca2+-permeable ion channels that are activated in an in vitro oxygenglucose deprivation (OGD) model of stroke (Aarts et al., 2003). These Ca2+ influxes temporarily deplete the extracellular Ca2+ while further increasing [Ca2+]i. Since these Ca2+ influxes are subject to rapid inactivation, it is likely that they serve as triggers to prime further [Ca2+]i increase through other mechanisms instead of acting as the sole source of Ca2+ elevation (Mody and MacDonald, 1995). Indeed, the inositol triphosphate (IP3) receptors on the endoplasmic reticulum (ER) is activated by the initial rise in [Ca2+]i during excitotoxicity which leads to an efflux of Ca2+ from the ER, resulting in a gradual, sustained rise in [Ca2+]i over time (Tsubokawa et al., 1992; Coulter et al., 1992; Tsubokawa et al., 1994; Tsubokawa et al., 1996; Chae et al., 2004). It has been suggested that the mitochondria also contribute to the sustained increase of [Ca2+]i by the formation of the Ca2+-permeable mitochondrial permeability transition (MPT) pore during excitotoxicity (Kristian and Siesjo, 1998; Matsumoto et al., 1999). An example of the downstream effects of Ca2+ is the activation of acid sphingomyelinase (ASMase) during stroke, an enzyme that cleaves membrane sphingolipids to generate ceramide, a second messenger molecule in apoptosis-signaling (Yu et al., 2000; Yu et al., 2007).  1.2.5. Calpain One of the events triggered by Ca2+ overload during excitotoxicity is the activation of calpain. Calpain is a ubiquitously-expressed protease that is activated by an increase of Ca2+ concentrations (Croall and DeMartino, 1991; Goll et al., 2003). It has many different  9  isoforms (Goll et al., 2003; Croall and Ersfeld, 2007), with the two most prominent ones found in the brain being m-calpain, which activates when Ca2+ level reaches the millimolar (m) range, and μ-calpain, which is activated in the micromolar (μ) range (Cong et al., 1989; Suzuki, 1991). The entire calpain molecule is necessary for its millimolar or micromolar specificity, as deletions or insertions of certain calpain domains do not alter its function (Dutt et al., 2002). Native calpain are heterodimers consisting of a larger (80 kDa) catalytic subunit and a smaller (28 kDa) regulatory subunit (Goll et al., 2003). Domain II from the large subunit of calpain contains the protease domain that is responsible for proteolytic cleavage (Mellor et al., 1993), whereas domain IV and VI inhibit its activity in a Ca2+-free state (Thompson et al., 1990). Prior to activation, domain I and V undergo auto-proteolysis and are cleaved into slightly smaller (76 and 18 kDa) subunits, respectively (Edmunds et al., 1991; Saido et al., 1992). Domain IV and VI then undergo conformational changes upon Ca2+ binding, consequently permitting the proteolytic domain II to become active (Thompson et al., 1990). It has been found that the P2 and P1 of calpain cleavage sites show signs of sequential preferences (Sasaki et al., 1984; Hirao and Takahashi, 1984), but an endogenous consensus recognition sequence seems to be nonexistent. Tompa and colleagues use a statistical approach to devise an 11-amino acid consensus sequence recognized by calpain by averaging the primary amino acid sequences of 106 calpain substrate targets and have found that its cleavability is superior to that of commercially available calpain substrates (Tompa et al., 2004). Others have subsequently use a proteomics approach to devise a different sequences recognized by calpain (Cuerrier et al., 2005). The pathological roles of calpain have been extensively implicated; in particular, its involvement in the induction of necrosis (Syntichaki and Tavernarakis, 2002; Syntichaki and Tavernarakis, 2003; Golstein and Kroemer, 2007) and other caspase-independent cell death  10  pathways (Kroemer and Martin, 2005). Many proteins that are important to neuronal physiology or survival are substrates of calpain, and cleavages of these proteins shift the balance towards cell death. Contrary to other proteases, calpain seems to induce substrate cleavage to trigger cell death pathways rather than destroying protein functions via cleavage per se (Goll et al., 2003). Calpain activation is related to Ca2+-permeable properties of activated NMDARs under excitotoxicity, which are, more specifically, the NR2B-containing NMDARs (Zhou and Baudry, 2006). The membrane-bound Na+ / Ca2+ exchanger, NCX3, is a substrate of calpain, and the inhibition of its calpain-dependent cleavage prevents the occurrence of cell death under excitotoxic conditions (Bano et al., 2005; Bano et al., 2007). The metabotropic glutamate receptor subunit 1 (mGluR1) is associated with the enzyme phosphoinositide-3 kinase (PI3K) in mediating neuronal survival (Rong et al., 2003). The calpain-mediated cleavage of mGluR1 C-terminus prevents the activation of the neuroprotective PI3K-PKB signaling pathway during excitotoxicity while enhancing neurotoxic effects and leaving its normal physiological properties intact (Xu et al., 2007). The enzyme cyclin-dependent kinase 5 (Cdk5) and its activator p35 are involved in neurite outgrowth and other developmental properties in the nervous system (Nikolic et al., 1996; Chae et al., 1997). During excitotoxicity, p35 is cleaved by calpain, yielding the cleavage product p25 and subsequently the prolonged activation of Cdk5; this leads to cytoskeleton collapse and eventually apoptosis (Lee et al., 2000; Kerokoski et al., 2004). A calpaindependent cleavage of the apoptotic protein AIF in triggering caspase-independent apoptosis has also been observed (Cao et al., 2007). Despite the focus on the pathological effects of calpain, it has been discovered recently that calpain is also involved in normal neuron physiological functioning. In stark contrast to the neurotoxic properties of p35 cleavage as mentioned previously, calpain-  11  mediated cleavage of p35 into p25 also regulates Cdk5 activity and the enhancement of LTP (Fischer et al., 2005). Additionally, calpain degrades the superchiasmatic nucleus circadian oscillatory protein (SCOP), which negatively regulates the cAMP response element (CRE)binding protein (CREB) and extracellular signal-regulated kinase (ERK) in facilitating learning and memory in the hippocampus (Shimizu et al., 2007). Other studies have shown that NMDAR subunits (Guttmann et al., 2001; Simpkins et al., 2003; Wu et al., 2005) and the GluR-1 subunit of the AMPA receptors (AMPARs) (Yuen et al., 2007a; Yuen et al., 2007b) are also substrates of calpain, but the exact role of ionotropic glutamate receptor subunit cleavage remains unclear. These seem to suggest that calpain has both regulatory and pathological properties in neurons that are highly dependent on its environment.  1.2.6. AMPA Receptors AMPARs are ionotropic glutamate receptors that have primary roles in mediating rapid excitatory neurotransmission in the mammalian nervous system (Derkach et al., 2007). They are tetramers that exist as a combination of its four subunits, GluR-1 to GluR-4 (Hollmann and Heinemann, 1994). Subunit composition varies across brain regions (Malinow and Malenka, 2002). At mature hippocampal synapses, AMPAR usually consists of a combination of GluR-2 and either GluR-1 or GluR-3 (Wenthold et al., 1996). The GluR2 subunit is subjected to RNA editing, where a genomic glutamine (Q) codon for residue 607 is substituted by an arginine (R) (Burnashev et al., 1992). This modification gives GluR-2 containing AMPARs distinctive single-channel properties like low Ca2+-permeability, channel conductance, open probability and the lack of rectification (Swanson et al., 1997). NMDARs are closely linked to physiological functions of AMPARs. It is thought that activation of synaptic NMDARs leads to AMPAR insertion during LTP (Lu et al., 2001;  12  Pickard et al., 2001). Subsequent researches have found that NR2A-containing NMDARs promote, whereas NR2B-containing NMDARs inhibit, surface insertion of GluR-1 containing AMPARs (Kim et al., 2005). Activation of NMDARs is equally important in the internalization of AMPARs in LTD (Beattie et al., 2000; Carroll et al., 2001). AMPAR endocytosis is a clathrin-dependent process (Wang and Linden, 2000), with the involvement of N-ethyl-maleimide sensitive fusion protein (NSF) and the adaptor protein AP2 (Luthi et al., 1999; Lee et al., 2002), both of which bind to the C-terminus of the GluR-2 subunit and serve to govern the endocytic process. Other proteins like the glutamate receptor interacting protein (GRIP), AMPAR binding protein (ABP) and protein interacting with C-kinase-1 (PICK1) associate with the extreme end of the GluR-2 C terminus and also are part of the regulatory mechanism behind NMDA-mediated AMPAR endocytosis (Dong et al., 1997; Srivastava et al., 1998; Dev et al., 1999; Xia et al., 1999). It is thought that phosphorylations of different residues on the C-terminus protein-binding sites contribute to this process, possibly by altering protein conformations (Chung et al., 2000; Matsuda et al., 2000; Hayashi and Huganir, 2004; Ahmadian et al., 2004). In addition to the physiological functions, AMPAR trafficking is also found to be important in excitotoxic conditions, as the prevention of NMDAR-mediated AMPAR endocytosis has a neuroprotective effect on primary cultured neurons in an in vitro excitotoxicity model (Wang et al., 2004). This is brought about by a synthetic peptide that mimics the short amino acid sequence (Y869KEGYNVYG877) on the AMPAR subunit GluR2 C-terminus. This peptide disrupts interactions between the C-terminus and its associating proteins, which prevents the occurrence of endocytosis (Lin et al., 2000; Ahmadian et al., 2004). The three tyrosine residues in the C-terminus sequence are necessary for this purpose, as a peptide with alanines substituting the tyrosines cannot inhibit endocytosis (Brebner et al.,  13  2005). NMDAR-mediated AMPAR endocytosis is also observed in other pathological conditions such as animal models of amphetamine addiction (Brebner et al., 2005) and Alzheimer’s disease (Hsieh et al., 2006).  1.2.7. Therapeutic Treatments Against Stroke Stroke treatments can be categorized into two main families: a thrombolytic approach or a neuroprotective approach. Most ischemic strokes are of thrombotic or thromboembolic nature in humans (Fieschi et al., 1989), hence it seems logical to develop stroke treatments that gear towards alleviating cerebrovascular deficits. The predominant strategy in the thrombolytic approach is to dissolve the clots that serve as sources of ischemic stroke. Tissue plasminogen activator (tPA) is a fibrinolytic agent that breaks down fibrin meshes of clots or emboli of occluded blood vessels or arteries during stroke (Lijnen et al., 1980; Zivin et al., 1985). The use of tPA has yielded significant recoveries of neurological functions in in vivo animal stroke models (Zivin et al., 1988; Overgaard et al., 1992). To date remains the only drug that has been approved by the FDA for stroke treatments. There is however a major caveat with the use of tPA: it is only effective when administered within three hours of stroke onset in humans (The National institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). This is greatly disadvantageous, as stroke patients often are not diagnosed within the three hour time-window because of various reasons, including unawareness of stroke symptoms, the logistics of an emergency service within the local hospital and the time devoted to ensure an accurate diagnosis of stroke (The American Nimodipine Study Group, 1992; Barber et al., 2001). This is further compounded by numerous reports which suggest neurotoxic properties of tPA that seems to contradict its stroke-alleviating functions (Chen and Strickland, 1997; Nicole et al., 2001; Liu et al., 2004a).  14  Other thrombolytic agents like streptokinase have been tested for their efficacies in treating stroke; however, clinical trials were stopped prematurely due to a high rate of intracranial hemorrhage and mortality (The Multicenter Acute Stroke Trial--Europe Study Group, 1996; Donnan et al., 1996). The endogenous plasminogen activator prourokinase is an effective thrombolytic agent against cerebral ischemia in humans (del Zoppo et al., 1998; Furlan et al., 1999), but it has not been approved by the FDA as a viable stroke treatment (Juttler et al., 2006). The effectiveness of anti-thrombotics has also been tested; these drugs are mainly anti-coagulants that inhibit the development of blood clots, which is a strategy for stroke prevention (Albers et al., 2004). Notwithstanding, the efficacy of anti-thrombotics has been questioned as many clinical trials have shown evidence of an increased likelihood of hemorrhage with their use (Levine et al., 2004). In addition, although the thrombolytic approach strives to restore normal cerebrovascular properties, the subsequent reperfusion process may paradoxically induce more injuries to the brain (Traystman et al., 1991; Aronowski et al., 1997; Jean et al., 1998). In short, tPA remains the only approved thrombolytic drug available for stroke treatments, and while its selective effectiveness cannot be discredited, it is nonetheless worthwhile to explore other therapeutic options that may be more effective. A possibility is to combine the use of tPA with neuroprotective agents. The neuroprotective approach towards stroke treatment is to target the damaged brain cells that are direct consequences of insufficient blood supply to the brain. This approach has mainly focused on therapeutics that target NMDARs since they play a crucial role in stroke pathophysiology. Early research has demonstrated that high-affinity NMDAR antagonists are neuroprotective in in vivo animal stroke models (McCulloch, 1992); however, none of the drugs passed the clinical trial stage due to severe side effects such as hallucinations and catatonia found in human patients (Muir and Lees, 1995). While NMDARs are indeed  15  heavily involved in stroke, they also serve vital functions under normal physiological conditions. Unsurprisingly, robust NMDAR antagonists also inhibit normal receptor functioning in addition to abrogating the pathological effects. Others have tried to target NMDARs at the subunit level: the NR2B-specific antagonist Traxoprodil demonstrated neuroprotective effects in an in vivo animal stroke model (Di et al., 1997). When it entered clinical trials, Traxoprodil seemed to be well-tolerated, as none of the typical NMDAR antagonist-induced side effects were displayed; unfortunately, Traxoprodil failed to exert any neuroprotection at this stage (Saltarelli et al., 2004). The NMDAR antagonist Memantine is a low-affinity, open-channel blocker that does not tamper with normal synaptic transmissions, but nonetheless successfully antagonizes NMDARs during excitotoxicity; consequently, it is neuroprotective without the deleterious effects of its high-affinity counterparts (Lipton, 2006). Memantine has been approved by the FDA as a viable option to treat Alzheimer’s disease. Memantine is also neuroprotective against stroke under an in vivo animal model (Chen et al., 1992; Chen et al., 1998a), so it can potentially serve as a stroke treatment even though it has not been approved by the FDA for this purpose as of yet. In brief, NMDAR-targeting drugs have had mixed results in treating stroke mainly due to their broad strokes of effects. Targeted therapies with a more localized function can potentially alleviate this problem, and a peptide-mediated approach offers much promise in this regard.  1.2.8. Mechanism of Tat Peptide Delivery The pathology of many diseases including stroke can now be targeted at the cellular level with advances in research and technology. It therefore seems promising to develop potential therapeutics on the same plane. With that being said, the cell membrane naturally acts as a barrier to protect the cells from any foreign and potentially harmful objects while  16  only allowing certain molecules to pass through by either passive diffusion or active transport. It was a landmark breakthrough when it was discovered that exogenous proteins chemically attached to a segment of the HIV-1 protein, Transactivator of Transcription (Tat), were able to diffuse in and out of the cell (Fawell et al., 1994). Further investigation has revealed that an arginine-rich segment of Tat corresponding to the amino acid positions 4860 is sufficient for intracellular delivery (Vives et al., 1997). The amino acid arginine is critical in the delivery mechanism as a peptide sequence rich in the equally basic and hydrophobic lysine is unable to replicate the Tat function (Futaki et al., 2001). Subsequently, researchers have also developed a similar delivery method using an arginine-rich sequence with equal success (Tung and Weissleder, 2003; Nakase et al., 2004; Futaki, 2005; Futaki, 2006). The Tat sequence is able to deliver cargos that are up to 200 nm in diameter into cells despite its relatively miniscule size (Torchilin et al., 2001; Torchilin et al., 2003), thus raising the question of the mechanism behind Tat-mediated delivery. It is generally agreed that Tat initially makes contact with the negatively-charged plasma membrane by ionic interactions via their positively-charged amino acids (Mai et al., 2002; Vives, 2003); however, the proceeding mechanism has not been clearly delineated. Some evidence suggests that the membrane surface-bound heparan sulfate proteoglycans associate with cargo-carrying Tat in aiding the internalization process (Tyagi et al., 2001; Ziegler and Seelig, 2004). It has also been proposed that several Tat-mediated delivery mechanisms exist, depending on the types of cells along with the size and nature of the delivered cargo (Brooks et al., 2005; Chauhan et al., 2007). Indeed, Tat-mediated cargos have been found to be delivered via a caveolae- and energy-dependent endocytosis (Lundberg et al., 2003), lipid raft-dependent endocytosis (Jones et al., 2005), and lipid raft-dependent macropinocytosis (Wadia et al., 2004), respectively. Another study demonstrated the cargo-dependence of Tat-mediated delivery, as  17  larger Tat-mediated proteins are found to be endocytosed, whereas smaller Tat-mediated peptides do not seem to be governed by such endocytic mechanisms (Tunnemann et al., 2006). It has been suggested that internalized peptides undergo the endosomal and retrograde pathway from the Golgi apparatus through the ER and are finally delivered into the cytoplasm (Fischer et al., 2004). Despite the differing opinions on the specifics of Tatmediated delivery mechanisms, therapeutic drugs that are tethered onto a Tat segment have demonstrated their effectiveness in targeting excitotoxicity by utilizing this Tat-mediated strategy (Aarts et al., 2002; Cao et al., 2002; Liu et al., 2006; Xu et al., 2007).  1.3.  Thesis Objectives and Hypothesis NMDAR antagonists are neuroprotective in animal stroke models but are ineffective  in clinical trials, a phenomenon that can be partly attributed to the inhibition of normal physiological NMDAR functions, which lead to intolerable side effects. Different attempts have been made to uncouple physiological and pathological functions of NMDARs via peptidic approaches. This includes preventing interactions between NMDARs and PSD-95 (Aarts et al., 2002) and blocking NMDAR-mediated AMPAR endocytosis (Wang et al., 2004). The latter was achieved by using peptides that disrupted protein-protein interactions integral in transmitting cell death cascades; however, it remains possible that these peptides at high concentrations may interfere with normal physiological functions and consequently induce unwanted effects in areas not affected by stroke. This problem can theoretically be alleviated by delivering therapeutic peptides specifically to stroke-affected regions. As noted previously, the protease calpain is activated in a NMDAR-dependent manner under stroke conditions and this may contribute to the delivery process (Adamec et al., 1998; Minger et al., 1998).  18  I hypothesize that the calpain activation during stroke can aid the process of concentrating therapeutic peptides in stroke-affected regions. To test this hypothesis, I have proposed to utilize the Tat-GluR2/3Y (3Y) peptide as the backbone (Brebner et al., 2005), while inserting a calpain consensus cleavage sequence (TPLKSPPPSPR) (CCS) between the Tat and the 3Y sequences (Tompa et al., 2004). Such a peptide will be cleaved by calpain during stroke, resulting in a greater accumulation of the neuroprotective 3Y peptidic segments in stroke-affected regions but not in areas unaffected by stroke. The rationale is illustrated in a cartoon (Figure 1.2). The objectives of my thesis are as listed:  Objective 1: To determine the extent of calpain activation in both in vitro and in vivo excitotoxic stroke models. Objective 2: To examine the cleavabilities and delivery dynamics of the cleavable peptides in both in vitro and in vivo excitotoxic stroke models.  19  Figure 1.1.The basic structure of NMDA receptors. NMDARs exist in combinations of NR1 and NR2 subunits. The glutamate-binding site resides on the NR2 subunit, whereas the binding site of the co-agonist glycine is found on the NR1 subunit along with the modulatory sites of Zinc (Zn2+) and polyamine. Magnesium (Mg2+) binds to the NR1 subunit and blocks the NMDAR channel at resting neuronal membrane potential; this block is removed upon membrane depolarization along with NMDAR activation by both glutamate and glycine.  20  A  B  C  D  E  F  Figure 1.2. The mechanism of the putative Tat-GluR2/3Y peptide with a calpain consensus cleavage sequence (The CCS peptide). A, C and E represent endogenous conditions, whereas B, D and F are of excitotoxic conditions. A and B: NMDAR-dependent AMPAR endocytosis occurs during excitotoxicity which eventually leads to cell death. C and D: The trafficking of the 3Y peptides are not altered during excitotoxicity while the 3Y portions of the peptides block AMPAR endocytosis and exerting neuroprotection. 3Y peptides cannot be cleaved by the Ca2+- activated calpain. E and F: CCS peptides enter and exit the cells in similar fashions as 3Y peptides under endogenous conditions. During excitotoxicity, CCS peptides are cleaved by calpain. Cleaved segments with 3Y are trapped within the intracellular space and subsequently block AMPAR endocytosis. Meanwhile, full-length peptides continue to travel into the cells as peptide cleavages disrupts its equilibrium, resulting in more peptide intake. Consequently, more cleaved segments with the 3Y sequence are retained within cells that suffer excitotoxic insults, resulting in an enhanced neuroprotection.  21  CHAPTER 2: MATERIALS AND METHODS  2.1.  Materials  2.1.1. Drugs All chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Calpain inhibitor III (also named Z-Val-Phe-CHO or MDL 28170) was obtained from Calbiochem (Mississauga, ON); the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) was from Tocris (Ellisville, MO), and the caspase-specific inhibitor Q-VD-OPH was purchased from MP Chemicals (Solon, OH). These chemicals were dissolved in either ddH2O or dimethyl sulfoxide (DMSO), and they were subsequently stored at -20°C.  2.1.2. Reagents Mg2+-free extracellular solution (ECS) was used, and contained the following chemicals dissolved in dH2O with concentrations in mM: 140 NaCl, 5.4 KCl, 1.3 CaCl2, 25 HEPES, 33 glucose. The pH of the ECS was then adjusted to 7.4 and the osmolarity to 310320 mmol/ kg. The buffer used in the in vitro calpain activity assay was made up of 50 mM HEPES and 165 mM NaCl in ddH2O at pH 6.5, as it was reported that the enzymatic activity of calpain is maximized under such conditions in vitro (Maddock et al., 2005). The lysis buffer used in subsequent experiments was based on this optimized assay buffer plus 1% sodium dodecyl sulfate (SDS), 1% NP-40 and 1% Triton X-100, with inhibitors of various concentrations (Aprotinin: 1:100; sodium orthovanadate: 1:1000; AEBSF: 1:1000). 0.1 M of phosphate buffer (PB) with monobasic and dibasic sodium phosphate was used in immunohistological methods. Phosphate buffered saline (PBS) was made by adding 0.9% NaCl into the PB solution.  22  2.1.3. Peptide Construction All peptides (Tat-GluR2/3Y-Dansyl), (Tat-calpain consensus cleavage sequenceGluR2/3Y-Dansyl), (Tat-cleavage site on α-Spectrin-II-GluR2/3Y-Dansyl), (Tat-cleavage site on CRMP-3-GluR2/3Y-Dansyl) were synthesized and their structures were confirmed via mass spectrometry by either AnaSpec (San Jose, CA) or Pepmetric (Vancouver, Canada). The peptides used in in vitro experiments were diluted into stock solutions with ddH2O and were stored at -20°C while wrapped in aluminum foil to prevent any loss of fluorescence. The peptides used in in vivo experiments were stored in its native powdered form at -20°C until use, and they were dissolved in sterile 0.9% saline.  2.1.4. Animals All animal procedures were carried out according to protocols that were approved by UBC Animal Care Committee while adhering to the guidelines provided by the Canadian Council on Animal Care. Animals were purchased from either Charles River (Montreal, QC) or UBC South Campus Animal Science Facilities (Vancouver, BC). Fetuses from female pregnant Wistar rats were used for primary cell culture purposes while male Sprague-Dawley (SD) rats were used for in vivo stroke surgeries. Rats were given ad libitum access to food and water.  2.2.  Preparation of Primary Culture of Cortical Neurons All primary culture dishes were purchased from Nunc and multi-well plates from  Costar. They were coated with poly D-lysine (PDL) in 0.15 M borate buffer prior to use. All chemicals mentioned here were purchased from GIBCO (Invitrogen) unless otherwise specified. E17-E18 rat fetuses from pregnant female Wistar rats were used. After urethane  23  anesthesia, rat fetuses were removed from the womb and their cerebral cortexes were then dissected out. They were submerged in dissection buffer (Hank’s Balanced Salt Solution (HBSS) plus HEPES, sucrose and glucose at pH 7.4, osmolarity 310-320 mOsm/ L) and subsequently in 0.25% Trypsin-EDTA in a 5% CO2-37°C incubator for tissue digestion. Trypsinization was terminated by adding Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). It was followed by multiple centrifugation and subsequent washes with DMEM-FBS to eliminate residual trypsin. After mechanical tritulations it was centrifuged and plating medium (Neurobasal medium with 2% B27, 0.5 mM L-Glutamine and 25 μM Glutamic acid) was added into the cortical neurons suspension. After the number of cells was counted with a haemocytometer, the neurons were then seeded and kept in the 5% CO2-37°C incubator. The media was switched to a maintenance medium (Neurobasal medium with 2% B27 and 0.5 mM L-Glutamine) two days after seeding, and subsequently media was changed every four days. Cell densities used in different conditions were as follows: 8.0 x 106 cells/ dish in 10 cm cell culture dishes for protein biochemistry work and 2.5 x 105 cells/well in 24-well plates for in vitro peptide delivery. Matured neurons (Days in vitro 11-14) were used.  2.3.  Experimental Stroke Models In Vitro and In Vivo  2.3.1. Excitotoxic Insult In Vitro An in vitro stroke model was established by introducing a NMDA-dependent excitotoxic insult with the accompaniment of 10 μM glycine and 1 μM strychnine to primary cultured cortical neurons. In the dosage experiment, various dosages of NMDA were applied (in μM: 5, 10, 25, 50 and 100) for 1 hour. In the immediate time-course experiment, 50 μM of NMDA was applied and samples were collected at various time-points (in minutes: 5, 10,  24  15, 30 and 60); in the delayed time-course experiment, samples were either collected immediately after 1 hour of 50 μM NMDA or at various time-points after returning to Neurobasal medium (in hours: ½, 1, 2, 3 and 6). APV or MDL 28170 were applied to certain samples as negative controls. Cultured neurons were washed twice with ECS before sample collection.  2.3.2. Middle Cerebral Artery Occlusion (MCAO) Model An in vivo stroke model was achieved by the middle cerebral artery occlusion method (Longa et al., 1989). The animal was anaesthetized by inhaled anesthetic isoflurane while its body temperature was monitored by a rectal probe. A longitudinal incision was made on the dorsal surface of the neck, exposing the right common carotid, external carotid and internal carotid arteries. A 4-0 nylon suture, blunted and coated with poly L-lysine, was inserted into the internal carotid artery until it reached the origin of the middle cerebral artery. Proper placement of the suture was determined by two indicators: 1.) The length of suture inserted, which was the distance between the bifurcation of the carotid artery and the origin of the MCA (20 mm ± 0.5 mm for animals weighing 290-310 g), and 2.) A small resistance that could be felt when the suture reached the origin of the MCA. MCAO would partially block the supply of blood to the right hemisphere. Sham surgery without the suture insertion was done on rats in the control condition. The occlusion was maintained for 90 minutes and the suture was removed afterwards. The right external carotid artery was tied off and the incisions were closed with sutures. Thereafter, the animal was returned to its home cage to recover from the surgical procedure. The animal was then sacrificed at certain time-points, its brain taken out, and a small piece of the cortical tissue (approximately 2 x 2 x 1 cm) was dissected out from each of the brain hemispheres.  25  2.4.  Sample Preparation, SDS-PAGE and Immunoblotting Both primary cultured cortical neurons and cortical brain tissues were collected with  lysis buffer. Both were homogenized by a 25G 1½ syringe gauge (BD Bioscience). Brain tissues were broken down by a Dounce glass tissue grinder (7 ml in volume) (Sigma-Aldrich) prior to syringe homogenization. Protein samples were allowed to chill on ice for 20 minutes, centrifuged at 14,000 RPM in room temperature for 15 minutes, and their protein concentration was determined by the BioRad DC Protein Assay which is a modification of the Lowry method. A 2X Laemmli buffer (Laemmli, 1970) was added into samples of equal amount to denature proteins. Samples were agitated for 15 minutes, boiled for 5 minutes and centrifuged at 14,000 RPM at room temperature for 1 minute. They were then loaded into 7% SDS-PAGE and run for 90 minutes at 120 volts (V). After the electrophoresis, proteins were transferred onto PVDF membranes (Millipore) in an overnight transfer. Membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline (TBS) with 0.05% Tween20 detergent (TBS-T). Membranes were incubated with either monoclonal anti α-Spectrin II (Chemicon, 1:1000) or polyclonal anti β-Actin (Sigma-Aldrich, 1:200) for 1 hour, and the corresponding horseradish peroxidase (HRP)-linked secondary antibodies in TBS-T (GE Healthcare, 1:10,000) for 45 minutes. All primary antibodies used in immunoblotting were diluted in TBS-T with 0.1% sodium azide to prevent bacterial contamination. Membranes were visualized by the Fluor-S Max photoimager (BioRad) after the addition of enhanced chemical luminescence (ECL) solution (GE Healthcare). Band densitometries were quantified by the ImageJ software (NIH).  26  2.5.  In Vitro Peptide Cleavability Assay The calpain-sensitive cleavability of the peptides was tested in vitro by mixing 2 μg  of the respective peptides with 5 μg of recombinant calpain I (Calbiochem) in the optimized calpain buffer as aforementioned. 1 mM of Ca2+ solution (CaCl2) was added to activate calpain. The total volume of the reaction mixture was 50 μl. This was carried out under constant temperature (37°C) with gentle shaking at 300 RPM. The reaction was stopped by adding 6X Laemmli buffer which served to denature the proteins. It was then agitated for 15 minutes at 500 RPM, boiled for 5 minutes, centrifuged at 14,000 RPM at room temperature for 1 minute and loaded into 15% SDS-PAGE. The gel was run for 60 minutes at 120V. The respective peptide cleavages were determined by staining the gel with Brilliant Coomassie Blue for 1 hour with overnight de-staining in dH2O.  2.6.  Fluorescent Peptide Detection in Vitro Cultured rat cortical neurons kept in Neurobasal medium in 24-well plates were  washed once and then given a NMDA treatment (50 μM NMDA, 10 μM glycine, 1 μM strychnine) in ECS for various amounts of time. The calpain inhibitor MDL 28170 (1 μM) was pre-incubated with neurons 30 minutes prior to and during the experiment in certain samples. The cultured neurons were returned to Neurobasal medium immediately after the NMDA treatment. Different amounts of peptides (either 2 μM or 10 μM) were then added into the medium for different periods of time. At the end of the incubation period neurons were washed once with ECS and the amount of internalized peptides was detected by the Victor3 V multi-label counter (Perkin Elmer) with fluorescent filter sets (Ex: 355 nm/ Em: 535 nm) that were within the proximity of the dansyl fluorescence (Ex: 340 / Em: 520). Readings were taken once every 15 minutes, with one ECS wash prior to each reading.  27  2.7.  Histochemistry, Immunohistochemistry and Fluorescence Microscopy Peptides were administered intravenously (I.V.) via the right femoral vein of the  animal. The animal was anaesthetized with 25% urethane. The rat brain was then perfused with saline (0.9% NaCl) and fixed with filtered 4% paraformaldehyde (PFA) in 0.1 M PBS. The brain was subsequently immersed in 4% PFA with 30% sucrose at 4°C for cryoprotection until the brain had sunk to the bottom of the container. It was stored in the -80°C freezer for one day while wrapped in aluminum foil. Brain slices (40 μm thick) were cut using a cryostat (Leica Microsystems) and collected in 24-well multi-well plates by the free-floating method. Brain slices that were to be viewed directly without any treatment were mounted onto adhesive-coated glass slides with Premount mounting medium (Fisher Scientific). Brain slices that were to be treated with immunohistological methods were blocked and permeablized in 0.1 M PBS with 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 30 minutes with slight agitation. Brain slices were incubated with polyclonal anti-dansyl antibody (Molecular Probes, 1:200) overnight at 4°C with slight agitation, and subsequently with Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes, 1:1000) at 4°C with slight agitation while being protected from light. All antibodies used in this immunohistochemical procedure were diluted in 0.1 M PBS with 0.5% BSA. These brain slices were subsequently mounted onto adhesive-coated glass slides with mounting medium. The immuno-stained brain slices were viewed with a Leica DM IRE2 fluorescence microscope with the GFP fluorescence cube filter (Ex: 480/ Em: 527) due to the fluorescence properties of the Alexa 488 secondary antibody (Ex: 495/ Em: 519). All images were taken with the same 20X objective lens with the same camera exposure time (100 ms). The unstained brain slices were viewed with the Zeiss Axiovert 200 inverted epifluorescence microscope (Carl Zeiss) with a modified filter set (Ex: 337/ Em: 520) tailored to  28  dansyl fluorescence (Ex: 340 / Em: 520). All photos were taken with the same 5X objective lens with the same camera exposure time (500 ms).  2.8.  Data analysis Data were expressed as Mean ± S.E.M. where appropriate. One-way Analysis of  Variance (ANOVA) was used for comparison among multiple groups, whereas two-way ANOVA was used when there was more than one dependent variable; this was followed by the Holm-Sidak test for comparison between two groups. Student’s t test was used to compare between two distinct groups. Statistical significance was defined as p < 0.05.  29  CHAPTER 3: RESULTS  3.1.  Dose- and Time-Dependent Activation of Calpain in In Vitro and In Vivo  Stroke Models 3.1.1. Dosage-Dependent Calpain Activation In Vitro In order to test the feasibility of our peptide-cleaving hypothesis, it is necessary to determine the extent of calpain activation in both in vitro and in vivo stroke models. Hypothetically, calpain activation can be detected by its autolysis and the consequent changes in its molecular weight (MW); however, antibodies that are able to detect this difference are not readily available. An indirect but more commonly employed detection method is to observe the cleavage of calpain substrates. The protein α-Spectrin II (also known as α-Fodrin in older literature) has been one of the more popular candidates for this purpose. α-Spectrin II is a 240 kDa structural protein that yields fragments with MWs of 150 kDa and 145 kDa upon calpain cleavage (Wang, 2000), and it is suitable for this occasion as Spectrin cleavage has been detected under excitotoxic conditions (Di Stasi et al., 1991). The extent to which calpain is activated under different doses of NMDA was first examined. An increase of Spectrin breakdown products (SBDPs) was observed with the additions of 10 μM, 25 μM, 50 μM and 100 μM of NMDA, whereas 5 μM NMDA stimulation did not have any increases of statistical significance when compared to the control (Control: 100.00 ± 10.47; 5 μM: 117.48 ± 4.29; p > 0.05. 10 μM: 127.89 ± 5.91; p < 0.05. 25 μM: 150.13 ± 11.33; p < 0.001. 50 μM: 140.13 ± 9.73; p < 0.01. 100 μM: 167.29 ± 9.95; p < 0.001; n = 4 in each group) (Figure 3.1A and B). The addition of calpain inhibitor MDL 28170 (1 μM) or NMDAR antagonist APV (10 μM) also abrogated SBDPs accumulation seen in samples that underwent the application of 100 μM NMDA, confirming that the observed SBDPs was NMDAR- and calpain-dependent  30  (MDL 28170: 83.90 ± 7.28; p < 0.001. APV: 111.33 ± 8.31; p < 0.001. n = 4) (Figure 3.1A and B). These results indicated that α-Spectrin II is cleaved by calpain in a NMDARdependent manner at a relatively low concentration under prolonged incubation.  3.1.2. Immediate Activation of Calpain Time-Course In Vitro After determining the dosage effect of NMDA on calpain activation, an immediate time-course of calpain activation was then examined. Cortical neurons were treated with 50 μM of NMDA and collected at various time-points. Immunoblot results showed that SBDPs began to accumulate after 10 minutes of initial NMDA application and continued to increase over time (Control: 100.00 ± 4.21; 5 minutes: 120.08 ± 7.57; p > 0.05. 10 minutes: 133.13 ± 8.66; p < 0.01. 15 minutes: 139.66 ± 9.06; p < 0.001. 30 minutes: 130.77 ± 8.91; p < 0.01. 60 minutes: 143.43 ± 7.76; p < 0.001) (Figure 3.2A and B). SBDPs increases were not seen with the addition of either MDL 28170 or APV when compared to control (MDL 28170: 91.54 ± 3.72; p < 0.001. APV: 111.93 ± 4.10; p < 0.01. n = 4). (Figure 3.2A and B) This data suggested that calpain was activated in a rapid fashion in the presence of excess NMDA.  3.1.3. Delayed Activation of Calpain Time-Course In Vitro The previous experiment suggested a pattern of calpain activation within an hour of excitotoxicity; however, the prolonged activation profile of calpain has yet to be elucidated. Therefore, the prolonged time-course of calpain activation under excitotoxicity was examined. Cortical neurons were treated with 50 μM of NMDA for one hour, returned into Neurobasal medium and were subsequently collected at various time-points. This was to mimic the reperfusion scenario that is commonly observed during stroke. SBDPs dramatically increased under these conditions, with an increase of over 200% at the beginning of the time-course and  31  a continual increase over time (Control: 100.00 ± 15.92; 0 hour in Neurobasal medium: 238.56 ± 21.69; ½ hour: 256.95 ± 19.68; 1 hour: 309.62 ± 24.99; 2 hours: 349.97 ± 20.21; 3 hours: 368.81 ± 18.83) (Figure 3.3A and B). The amount of SBDPs peaked at the end of the conducted time-course (6 hours) with an observed increase of over 1000% (6 hours: 1023.70 ± 110.55. All reperfusion time-points have p < 0.001). The addition of MDL 28170 (103.72 ± 7.51; p > 0.05) or APV (131.25 ± 10.82; p > 0.05) again prevented any significant accumulations of SBDPs (n = 4) (Figure 3.3A and B). The results showed that there was a gradual and robust increase in calpain activation over a long time-course when cortical neurons were returned to Neurobasal medium after excitotoxicity in vitro.  3.1.4. Involvement of Caspase in Delayed Activation of Calpain Time-Course In Vitro In addition to being a calpain substrate, α-Spectrin II is also known to be cleaved by the cysteine protease caspase-3 under apoptosis-triggering conditions, with its breakdown products having MWs of 150 kDa and 120 kDa, respectively (Nath et al., 2000; Williams et al., 2003). Since caspases are generally activated late in the apoptotic pathway, it is possible that the marked increase of SBDPs observed in the later time-points was a combination of both calpain and caspase cleavage. To investigate this possibility, the caspase inhibitor QVD-OPH (1 μM) was applied before, during and after NMDA treatments; samples were collected after 6 hours of post-NMDA Neurobasal medium incubation. Q-VD-OPH caused a slight but insignificant decrease on the observed SBDPs (Control: 100.00 ± 10.71; NMDA: 700.63 ± 60.46; Q-VD-OPH: 530.89 ± 105.36. n = 3), whereas the application of MDL 28170 (186.72 ± 57.97) or APV (116.34 ± 13.71; p < 0.001) dramatically decreased the accumulation of SBDPs (Figure 3.4A & B). Furthermore, the 120 kDa Spectrin breakdown product of caspase could not be found (Figure 3.4A). These results suggested that caspase  32  played an insignificant role in the observed SBDPs, and that these were clear and reliable representations of calpain activation. Taken together, these studies provide an extensive account of NMDA-dependent calpain activation under an in vitro neuronal primary culture model of excitotoxicity.  3.1.5. Activation of Calpain Time-Course in an In Vivo MCAO Stroke Model Calpain activation in an in vivo global ischemia animal model has been welldocumented (Kawai et al., 1992; Neumar et al., 1996; Bartus et al., 1998; Yamashima et al., 2003; Garcia-Bonilla et al., 2006), and there is also a report on calpain activation in a permanent focal ischemia model (Bartus et al., 1995). However, the amount of literature on transient focal ischemia leaves much to be desired. The extent of calpain activation in an in vivo transient focal ischemia model was thus investigated. Middle cerebral artery occlusion (MCAO) was the surgical method of choice for inducing this type of stroke; this technique is advantageous in that stroke is only induced in the right hemisphere of the animal’s brain (the ipsilateral hemisphere (IH)), and the left hemisphere can effectively serve as a negative control (the contralateral hemisphere (CH)). Animals were subjected to 90 minutes of MCAO and were sacrificed after various periods of reperfusion. The levels of SBDPs were measured to estimate the extent of calpain activation. SBDPs were first compared between ipsilateral and contralateral hemispheres of the same brain. SBDPs did not differ between the two hemispheres when animals were subjected to 90 minutes of MCAO with no reperfusion (IH: 113.40 ± 5.96; CH: 108.28 ± 3.57. p > 0.05, n = 4) (Figure 3.5). Increases of SBDPs in ipsilateral hemispheres were seen in rats that were reperfused for different periods of time, respectively: 30 minutes (IH: 114.64 ± 5.78; CH: 81.51 ± 4.62. p < 0.001, n = 4), 60 minutes (IH: 116.44 ± 7.58; CH: 88.84 ± 8.47. p < 0.05, n = 4), 90 minutes (IH: 179.54 ± 17.24; CH:  33  120.54 ± 17.54. p < 0.05, n = 3) and 180 minutes (IH: 217.10 ± 8.17; CH: 113.86 ± 8.77. p < 0.001, Student’s t test, n = 3). When compared to animals treated with sham surgery which were considered to represent a basal level of SBDPs, ipsilateral hemispheres of neither 0, 30 nor 60 minutes of reperfusion showed any significant differences (sham: 0 minutes: 100.00 ± 4.49, 30 minutes: 114.64 ± 5.78, 60 minutes: 116.44 ± 7.58. p > 0.05). Indeed, the IH-CH differences displayed by that of 30 and 60 minute reperfusion were mainly due to surprising decreases of SBDPs in the contralateral hemisphere rather than robust increases from the ipsilateral hemisphere. Animals with 90 and 180 minutes of reperfusion had more profound SBDPs accumulation that reached statistical significance (Sham: 100.00 ± 4.49, n = 10. 90 minutes: 179.54 ± 17.24. p < 0.01; 180 minutes: 217.10 ± 8.17. p < 0.001). These results suggested that the pattern of calpain activation in an animal stroke model differed from that in a primary cultured neurons model as it only began to show differences in SBDPs with 90 minutes of reperfusion after stroke. This also provided the first account of a calpain activation time-course in a rat transient focal ischemia model.  3.2.  Designing and Characterizing Tat-GluR2-3Y Peptide Cleavable by Calpain  In Vitro 3.2.1. The Insertion of CCS Sequence The C-terminal segment of the AMPAR subunit GluR2 (YKEGYNVYG) is neuroprotective in an in vitro neuronal excitotoxicity model by blocking AMPAR endocytosis (Wang et al., 2004). When a segment of the cell-permeable HIV-1 Tat protein (YGRKKRRQRRR) (Tat) was attached to the N-terminus of the GluR2 C-tail polypeptide (Tat-GluR2/3Y, or 3Y), the resulting peptide had both the permeability brought about by the Tat segment and the therapeutic effects of the short GluR2 C-tail sequence (Brebner et al.,  34  2005). As mentioned previously, I hypothesize that introducing a sequence recognized and cleaved by calpain between the Tat and the 3Y will increase the selective delivering of the therapeutic 3Y segment to areas affected by stroke as compared to the unaffected areas, which may potentially reduce the required peptide dosages. To test my hypothesis, a calpain consensus cleavage sequence (CCS) (TPLKSPPPSPR) was chosen. This sequence was derived from averaging 106 sites of cleavage sites recognized by calpain (Tompa et al., 2004). The CCS sequence was inserted between the Tat and 3Y sequence (Tat-CCS-GluR2/3Y, or CCS) (Figure 3.6A). To test its susceptibility to calpain cleavage, an in vitro peptide cleavage assay was performed. In the presence of recombinant μ-calpain, the CCS peptide was cleaved as demonstrated by a band of lowered MW compared to that of the uncleaved peptide in the absence of Ca2+ (Figure 3.6B). The addition of MDL 28170 prevented the CCS peptide cleavage. The 3Y peptide was not cleaved by calpain thus serving as an effective negative control (Figure 3.6B). This indicated that the CCS peptide could be recognized and cleaved by μ-calpain, which confirmed the CCS cleavability from previous findings (Tompa et al., 2004). As the purpose of the CCS peptide was to be cleaved intracellularly during excitotoxicity to accumulate the GluR2-3Y inside neurons in greater numbers, the extent of its proposed entrapment within neurons was then investigated. The experimental procedure time-line is outlined in Figure 3.7A. This was tested by detecting the fluorophore dansyl which was chemically attached to the C-terminus of the synthetic peptides. This method allowed detections of accumulation of cleaved fragments containing the 3Y segments, as well as full-length peptides within the cell. The difference between the 3Y and CCS peptides was first tested. Under NMDAtreated conditions, the intracellular retainment of the combination of cleaved and uncleaved CCS peptides was markedly more than that of the 3Y peptide, as indicated by the stronger fluorescence signals detected over a 90-minute time-course (Figure 3.7B, light red curve).  35  Meanwhile, as expected, the amount of intracellularly-retained 3Y peptides under control conditions did not differ from its NMDA-treated counterparts (Figure 3.7B, deep red curve). Interestingly, CCS peptides under control conditions also did not differ from that of NMDAtreated conditions (Figure 3.7B, black curve: control; grey curve: NMDA-treated. n = 9). A possible explanation is that the CCS peptides were cleaved by basally-active calpain; calpain inhibitors were therefore added to test this possibility. The assumption was that 3Y-containing peptide segment retainment of CCS peptides would be less if the CCS peptides were not cleaved, since there would not be any cleaved 3Y-containing segments retained. When contrasting against both control (Figure 3.8A, n = 3) and NMDA-treated (Figure 3.8B, n = 3) conditions, the retainment of 3Y-containing peptide segments in MDL 28170-treated samples was comparably less. This seemed to suggest that the accumulation of CCS peptides under NMDA-treated conditions did not significantly differ from control conditions was possibly due to endogenous calpain activities. As such, the CCS sequence seemed to be too sensitive to proteolytic activities of calpain. Since it is cleaved under endogenous conditions and does not appear to be diseasespecific, the CCS peptide therefore may not be suitable for the present study.  3.2.2. The Insertion of SP-2 or CP-3 Sequences Since the synthetic peptide with the CCS sequence did not yield any satisfactory results, other endogenous sequences recognized by calpain were tested. Two candidate sequences were selected as both are calpain substrates under excitotoxic conditions: 1.) Amino acid position 1173 to 1182 (QEVYGMMPRD) on structural protein α-Spectrin II (SP-2), and 2.) Amino acid position 72 to 81 (TRLQMPVMGM) on Collapsin Response Mediator Protein (CRMP) -3 (CP-3). CRMP-3 has been known for its role in collapsing growth cones during neuronal development (Wang and Strittmatter, 1996), and it is cleaved  36  by calpain during transient cerebral ischemia in both mice and rats (Hou et al., 2006; Jiang et al., 2007). The SP-2 and CP-3 sequences were similarly inserted between the Tat and 3Y sequence as the CCS (Figure 3.9A). Their susceptibilities to calpain cleavage were put to test through a similar in vitro cleavage assay that was previously done with the CCS peptide (Figure 3.9B). In the presence of recombinant μ-calpain and Ca2+, both SP-2 and CP-3 peptides were cleaved as shown by the lowered MW bands compared to that of their uncleaved versions. The CP-3 peptide cleavage seemed to be less robust than the SP-2, as peptides of the original size were still present, albeit in a lesser amount. This cleavage was inhibited by the addition of MDL 28170. The 3Y and CCS peptides served as effective negative and positive controls, respectively. These results suggested that the SP-2 and CP-3 sequences served as competent sites that are cleavable by calpain under a polypeptide setting. The amount of retained peptides was then tested in a similar fashion compared to the 3Y assays (Figure 3.9C), except that the values were normalized to t = 0 of the control condition. The amount of SP-2 peptide retained in NMDA-treated condition was surprisingly less than that of the control condition, whereas the addition of MDL 28170 in both conditions resulted in a decrease in total retained peptides over time (Figure 3.10A). When the obtained values were normalized to its own t = 0 value, the resulting rates of peptide entrapment did not differ amongst the four conditions (Figure 3.10B). In contrast, there were more retained CP-3 peptides in NMDA-treated condition than that of the control; the differing amount of peptide seemed to be constant throughout the time-course as it hovered around 15%. The presence of MDL 28170 dramatically lowered peptide retainment in both conditions (Figure 3.11A). Surprisingly, when the rates of peptide retainment were examined, NMDA-treated samples showed a statistically insignificant increase in peptide accumulation compared to that of the control whereas MDL 28170-treated samples showed less peptide retainment  37  (Figure 3.11B). This might be due to an over-saturation of peptides as the amount added was 10 μM and samples were incubated in Neurobasal medium for 30 minutes. Since calpain was shown to be activated relatively quickly after returning to the medium (Figure 3.3), it is possible that equilibrium of the entering and exiting of peptides has already been established and that after t = 0 there are not any peptides available to travel in and out of the cell anymore. Hence the CP-3 peptide concentration was reduced to 2 μM and its incubation time reduced to 5 minutes in an attempt to alleviate this problem (Figure 3.12A). The initial fluorescence at t = 0 under these new conditions was similar to its predecessor in that NMDA-treated samples continued to have slightly more fluorescence than control samples; it was statistically insignificant, however (Control: 100.00 ± 7.80; NMDA: 122.91 ± 8.58. Student’s t test, p > 0.05, n = 5) (Figure 3.12B). Under these modified conditions, NMDAtreated neurons allowed significantly more 3Y-containing peptide fragments to be retained intracellularly when compared to that of control conditions. The difference between the two conditions peaked at t = 90 minutes (Figure 3.13; percentage of entrapped peptides: NMDAtreated: 49.11 ± 2.80; control: 30.49 ± 2.67, n = 5). Taken together, there was more CP-3 peptide accumulation observed in NMDA-treated neurons while the same trend was not seen in samples treated with the SP-2 peptide.  3.3.  Failure to Detect Dansylated Peptides In Vivo The efficacy of peptide delivery and accumulation specifically in stroke-affected  areas was then examined in an in vivo animal stroke model. The CCS peptide was initially tested. Peptides were introduced into sham- or MCAO-treated animals via IV injection. The extent of peptide delivery was first examined by detecting dansyl fluorescence in brain homogenate. Surprisingly, there was not any difference in fluorescence intensity between  38  sham- and MCAO-operated animals or when the ipsilateral and contralateral hemispheres were being compared (Figure 3.14A). Other dansyl-detecting methods were tested: the same brain homogenates were taken and an antibody that recognized the chemical group dansyl was used in immunoblotting. The dansyl antibody could not detect any CCS peptides in the brain samples, whereas peptides that were directly applied into the SDS-PAGE (2 μg) as a positive control could be detected (Figure 3.14B). Since dansylated peptides could not be detected via biochemical means, it was subsequently examined with histochemistry methods. Four conditions were tested: sham-saline, sham-peptide, MCAO-saline and MCAO-peptide. These brains were fixated and brain slices were then observed with fluorescence microscopy. When dansyl fluorescence was examined under fluorescence filters compatible to its excitation/ emission wavelengths, there were no observed differences between the four conditions and this trend was consistent across different brain regions (Figure 3.15). It is possible that the thickness of brain tissues (40 μm) or the fixation procedure might have hindered the dansyl fluorescence. Immunohistochemistry methods were thus attempted as to amplify the dansyl signal; however, the antibody against dansyl was found to be non-specific, as immunostained neurons were found in all four conditions and also in multiple brain regions (Figure 3.16: A & B, hippocampus; C & D, striatum; E & F, cerebral cortex; G & H: hippocampus). These results suggested that neither directly observing the brain slices nor immunohistochemistry served as adequate tools in in vivo dansyl detection. This highlighted the technical difficulties in detecting dansyl fluorescence in an in vivo animal model rather than the peptide’s ability to be recognized and cleaved by calpain.  39  A  B  Figure 3.1. Dose-dependent calpain activation by NMDA in cultured cortical neurons. A. Neurons were treated with different amounts of NMDA along with 10 μM glycine and 1 μM strychnine for 1 hour and the amount of calpain-cleaved Spectrin breakdown product (SBDPs) were quantified by immunoblotting. C: control; M: calpain inhibitor MDL 28170; A: NMDAR-antagonist APV. The amount of β-Actin was measured as a loading control. This figure is representative of four independent experiments. B. A bar graph representation of the averaged means obtained from Figure 3A. SBDPs was normalized first to full-length calpain and then to β-Actin; they were subsequently compared to the control. Statistical analysis was performed with one-way ANOVA. *p < 0.05; **p < 0.01; *** p < 0.001. Error bars, S.E.M. (n = 4). 40  A  B  Figure 3.2. Immediate time-course of calpain activation by NMDA in cultured cortical neurons. A. Neurons were treated with 50 μM of NMDA along with 10 μM glycine and 1 μM strychnine for various periods of time and the amount of calpain-cleaved SBDPs were quantified by immunoblotting. C: control; M: calpain inhibitor MDL 28170; A: NMDAR-antagonist APV. The amount of β-Actin was measured as a loading control. This figure is representative of four independent experiments. B. A bar graph representation of the averaged means obtained from Figure 3.2A. Values were obtained in a similar fashion as described in Figure 3. Statistical analysis was performed with one-way ANOVA. *p < 0.05; **p < 0.01; *** p < 0.001. Error bars, S.E.M. (n = 4).  41  A  B  Figure 3.3. Delayed time-course of calpain activation by NMDA after returning to Neurobasal medium in cultured cortical neurons. A. Following treatments with 50 μM of NMDA along with 10 μM glycine and 1 μM strychnine for 1 hour, neurons were returned to Neurobasal medium for various periods of time and the amount of calpain-cleaved SBDPs was then quantified by immunoblotting. C: control; M: calpain inhibitor MDL 28170; A: NMDAR-antagonist APV. The amount of β-Actin was measured as a loading control. This figure is representative of four independent experiments B. A bar graph representation of the averaged means obtained from Figure 3.3A. Values were obtained in a similar fashion as described in Figure 3.1. Statistical analysis was performed with one-way ANOVA. *** p < 0.001. Error bars, S.E.M. (n = 4).  42  A  B  Figure 3.4. The effect of caspase inhibitor Q-VD-OPH on calpain activation by NMDA after returning to Neurobasal medium in cultured cortical neurons. A. Neurons were treated with 50 μM NMDA along with 10 μM glycine and 1 μM strychnine for 1 hour and returned to Neurobasal medium for 6 hours with various inhibitors. The amount of calpaincleaved SBDPs was quantified by immunoblotting. MDL 28170 is a calpain inhibitor, and APV is a NMDAR-antagonist. The amount of β-Actin was measured as a loading control. This figure is representative of three independent experiments B. A bar graph representation of the averaged means obtained from Figure 3.4A. Values were obtained in a similar fashion as described in Figure 3. Statistical analysis was performed with one-way ANOVA. *** p < 0.001. Error bars, S.E.M. (n = 3).  43  Figure 3.5. Calpain activation in an in vivo MCAO stroke model. Animals were subjected to MCAO and reperfusion was allowed for certain periods of time before sacrificing. Equal amounts of brain homogenates from the different treatment groups were analyzed by SDSPAGE and immunoblotting against α-Spectrin II and β-Actin. SBDPs was normalized first to full-length calpain and then β-Actin; they were subsequently compared to the opposite brain hemisphere under the same condition and its corresponding brain hemisphere of the shamoperated animals. The bar graph is a representation of averaged means. Statistical analysis was performed with one-way ANOVA across the different treatment groups, while Student’s t test was performed to analyze differences between ipsilateral and contralateral hemispheres. *p < 0.05; **p < 0.01; *** p < 0.001. Error bars, S.E.M. (Sham: n = 10; 0 min: n = 4; 30 min: n = 4; 60 min: n = 4; 90 min: n = 3; 180 min: n = 3).  44  A  B  Figure 3.6. The structure and susceptibility to calpain cleavage of the CCS peptide in vitro. A. Amino acid sequences of the Tat-GluR2/3Y (3Y) and Tat-calpain Consensus Cleavage Sequence-GluR2/3Y (CCS) peptides. Both peptides have the fluorophore dansyl chemically attached to their carboxyl termini. B. CCS peptide was susceptible to calpain cleavage in vitro. After the in vitro peptide cleavage assay (please refer to experimental procedures), the samples were analyzed by 15% SDS-PAGE and Coomassie-Blue protein staining. The 3Y peptide was added as a negative control. This gel is representative of three independent experiments.  45  A  B  Figure 3.7. Comparing the 3Y and CCS peptides on the effect of excitotoxicity in the retainment of 3Y-containing peptide segments in cultured cortical neurons. A. A time-line of the experimental procedure. B. CCS peptide-treated samples had more 3Ycontaining peptide segments retained over time compared to that of the 3Y peptide in excitotoxicity; however, there were no significant differences observed between the ControlCCS and NMDA-CCS conditions. There was a dramatic increase in the retainment of CCS peptide over time compared to that of the 3Y peptide, suggesting that the CCS peptide might be cleaved by basally active calpain under non-NMDA stimulated conditions. m: minutes. Data points were averaged means relative to the t = 0 value. Statistical analysis was performed with two-way ANOVA. Error bars, S.E.M. (n = 9).  46  A  B  Figure 3.8. The effect of endogenous calpain on the retainment of 3Y-containing peptide segments in CCS peptide-treated cultured cortical neurons. A. Less 3Y-containing peptide segments were retained under control conditions with the calpain inhibitor MDL 28170. B. Less 3Y-containing peptide segments were retained under NMDA-treated conditions with MDL 28170, confirming the role of endogenous calpain actions. For both Figure A and B, data points were averaged means relative to initial values at t = 0. Statistical analysis was performed with two-way ANOVA. Error bars, S.E.M. (n = 3).  47  Figure 3.9. The structures and susceptibilities to calpain cleavage of the SP-2 and CP-3 peptides in vitro, and the experimental procedure time-line. A. Amino acid sequences of the Tat-α-Spectrin II-GluR2/3Y (SP-2) and Tat-CRMP-3-GluR2/3Y (CP-3) peptides. Both peptides have the fluorophore dansyl chemically attached to their carboxyl termini, respectively. B. Both SP-2 and CP-3 peptides were susceptible to calpain cleavage in vitro. After the in vitro peptide cleavage assay (please refer to experimental procedures), the samples were analyzed by 15% SDS-PAGE and Coomassie-Blue protein staining. The 3Y and CCS peptides were added as negative and positive controls, respectively. This gel is representative of three independent experiments. C. The time-line of the experimental procedure in determining the extent to which 3Y-containing peptide segments was retained under excitotoxic conditions in SP-2 or CP-3 peptide-treated cultured cortical neurons. m: minutes.  48  A  B  C  49  A  B  Figure 3.10. The amount of 3Y-containing peptide segments did not change during excitotoxicity in SP-2 peptide-treated primary cultured cortical neurons. A. The amount of 3Y-containing peptide segments was relative to the initial value of control SP-2 condition at t = 0. B. The results from different conditions were relative to its own initial value at t = 0 to obtain the rate. M: MDL 28170. Data points were averaged means. Statistical analysis was performed with two-way ANOVA. Error bars, S.E.M. (n = 5).  50  A  B  Figure 3.11. Changes in the amount of 3Y-containing peptide segments during excitotoxicity in CP-3-treated primary cultured cortical neurons. A. The amount of 3Ycontaining peptide segments was relative to the initial value of control CP-3 condition at t = 0. B. The results from different conditions were relative to its own initial value at t = 0 to obtain the rate. M: MDL 28170. Data points were averaged means. Statistical analysis was performed with two-way ANOVA. Error bars, S.E.M. (n = 5).  51  A  B  Figure 3.12. Testing the extent of 3Y-containing peptide segment retainment with a modified protocol in CP-3-treated cultured cortical neurons. A. The time-line of examining the extent to which the 3Y-containing peptide segments was retained under excitotoxic conditions in cultured cortical neurons. B. Initial fluorescence levels of the two conditions at t = 0. Values were averaged means relative to the initial fluorescence of the control measured at t = 0. m: minutes. Its statistical significance was analyzed by the Student’s t test (p > 0.05; n = 5). Error bars, S.E.M.  52  Figure 3.13. The extent to which 3Y-containing peptide segments were retained in NMDA-treated conditions under the modified protocol in CP3-treated cultured cortical neurons. Data points were averaged means relative to their own initial value at t = 0 to obtain the rate. Statistical analysis was performed with two-way ANOVA. Error bars, S.E.M. (n = 5).  53  A  B  Figure 3.14. Failure to detect dansylated CCS peptide in vivo via biochemical methods. 3 nM/ gram of CCS peptide dissolved in sterile saline were administered via IV injection into either sham- or MCAO-operated rats, and dansyl fluorescence from brain homogenates was subsequently measured. A. Detecting fluorescence intensity by a fluorescence plate reader. Results were shown in its raw fluorescence intensity units (RFU). Equal amounts of brain homogenates from different conditions in quadruplicates were added. I: ipsilateral hemisphere; C: contralateral hemisphere. Statistical analysis was performed with one-way ANOVA. Error bars, S.E.M. B. The same brain homogenates were analyzed by 15% SDSPAGE and subsequent immunoblotting with a polyclonal antibody against dansyl. “CCS only” represented the 2 μg of CCS peptide that were directly applied onto the SDS-PAGE as a positive control. I: ipsilateral hemisphere; C: contralateral hemisphere. This diagram is representative of two independent experiments.  54  Figure 3.15. Inability to directly detect dansyl fluorescence in rat brain slices. Brains from rats treated with different conditions were processed by histochemical methods and were examined by fluorescence microscopy with filters that directly correspond to the Ex/ Em of dansyl. Exposure time and light intensities were consistent throughout. Both contralateral and ipsilateral hemispheres from the same brain slice were showed. Scale bar, 300 μm.  55  A  B  C  D  E  F  G  H  Figure 3.16. Non-specificity of the antibody against dansyl in rat brain slices. Brains from rats treated with different conditions were processed by immunohistochemical methods and stained with anti-dansyl and subsequently the Green Alexa 488 secondary antibody. The brain slices were viewed under a fluorescence microscope. Exposure time and light intensities were consistent throughout. Both contralateral and ipsilateral hemispheres from the same brain slice were showed. Brain regions: A & B: hippocampus; C & D: striatum; E & F: cerebral cortex; G & H: hippocampus. Scale bar, 100 μm. 56  CHAPTER 4: DISCUSSION The main purpose of my thesis is to develop potential therapeutics for stroke that act specifically in stroke-damaged areas in the brain. I hypothesize that therapeutic peptides that are cleavable by the Ca2+-activated protease calpain will be specifically delivered to diseased regions in animal stroke models.  4.1.  Calpain Activation Time Course: Reconciliation of Differences Between In Vitro  and In Vivo Results In order to test my hypothesis, I first characterized the extent of calpain activation under excitotoxic stroke conditions, and subsequently, several candidate peptides on their site-specific delivery. I have found that calpain activation in cultured cortical neurons occurs early in excitotoxicity (Figure 3.2A & B) and continues to increase after returning to culture medium, which represents an in vitro correlate of reperfusion. It begins early, as more than 200% of SBDPs are detected immediately after 60 minutes of excitotoxic insult (Figure 3.3A & B). This is in stark contrast to the delayed activation of calpain in the in vivo animal stroke model, which does not begin until 90 minutes of reperfusion after MCAO (Figure 3.5). This is possibly due to the basic mechanistic difference between in vitro and in vivo disease models, in that in vitro systems are more artificial and easier to manipulate, while in vivo models offer less flexibility in experimental manipulation but are nonetheless closer representations of real-life scenarios. In this case, the direct NMDA application into cultured cortical neurons should trigger excitotoxicity in a rapid fashion, whereas it may take the MCAO surgical procedure longer to induce the effects of stroke in the cerebral cortex of the animal. The same rationale can be implied at reperfusion. This suggests that the observed discrepancy between in vitro and in vivo results must be taken into account when the extent 57  to which the peptides are being cleaved by calpain is being considered. Both sets of data are firsts of their kind as extensive time-courses of calpain activation, both with NMDA-induced excitotoxicity in cultured cortical neurons and with the MCAO animal stroke model.  4.2.  Different Peptide Cleavage Profiles Since calpain can be activated under both endogenous and pathological conditions, it  is then conceivable that endogenous calpain activities may play a role in cleaving the candidate peptides, albeit presumably a minor one. Surprisingly, the CCS peptides are cleaved at the same rate in both endogenous and excitotoxic conditions (Figure 3.7B). The addition of calpain inhibitor MDL 28170 allowed the full-length peptides to exit the cells at a faster rate (Figure 3.8A and B), suggesting that the CCS peptides are not cleaved, nor are they retained intracellularly when calpain activity is inhibited. Since the CCS sequence is a result of an averaged consensus and does not exist endogenously, it is possible this sequence is so sensitive under the experimental conditions that it is even susceptible to basal calpain activities. The rate at which the SP-2 peptides are retained in neurons under excitotoxic conditions does not differ from that of the control condition (Figure 3.10A and B). This may be due to the phosphorylation of the tyrosine on the cleavage site (QEVY1176GMMPRD), a process which is known to hinder calpain-mediated cleavage of α-Spectrin II (Nicolas et al., 2002; Nedrelow et al., 2003). The Y1176 is phosphorylated by the tyrosine kinase Src, an enzyme whose activity is increased during transient cerebral ischemia (Hu et al., 1998; Paul et al., 2001). This seems to agree with the in vitro calpain assay result, where the SP-2 peptide can be cleaved by calpain in the absence of any kinases (Figure 3.9B). The reason why a full-length α-Spectrin II protein can still be cleaved by calpain is possibly due to the fact that its susceptibility to cleavage is also dependent on its secondary and tertiary  58  structures (Stabach et al., 1997). In contrast, while it is evident that the CP-3 peptide is also cleaved under endogenous conditions, significantly more CP-3 peptides are nonetheless retained intracellularly in NMDA-treated conditions than in control: the discrepancy at each time point is at least 10% and this continues to increase over time, with the differences peaking at t = 90 minutes (Figure 3.13; percentage of entrapped peptides: NMDA-treated: 49.11 ± 2.80; control: 30.49 ± 2.67). Therefore it is unlikely that post-translational protein modifications on CP-3 peptides are, if at all, sufficient to hinder its cleavage by calpain. In short, these results suggest that the CP-3 peptide demonstrates the best disease-specific delivery characteristics amongst the three peptides that are examined. Vaslin et al. recently reported that endocytosis is enhanced during NMDA-mediated excitotoxicity in cortical neurons (Vaslin et al., 2007), although the mechanism of the excitotoxicity-mediated endocytosis in their study has yet to be delineated. Whilst it is indeed conceivable that excitotoxicity-mediated endocytosis may play a role in peptide internalization, this is not supported by our observation, as the retainment rate of the uncleavable 3Y peptide during NMDA-treated conditions does not differ from that of the control (Figure 3.7B). This suggests that whatever internalization mechanisms may be involved do not seem to affect the rate of peptide intake. The lack of difference between rates of control-3Y and NMDA-3Y also rules out another potential confound: the compromised and thus “leaky” cellular membranes during excitotoxicity which may provide an avenue for additional peptide entrance. This suggests that the observed results with CP-3 peptides are not byproducts of excitotoxicity-specific phenomena or compromised cellular membranes.  59  4.3.  Advantages of the GluR2/3Y Peptidic Segment in Neuroprotective Schemes As mentioned in the introduction, all clinical trials using NMDAR antagonists as  potential stroke treatments have failed in part due to the inhibition of the receptors’ normal physiological functions (Muir and Lees, 1995). The peptide approach that has been taken in my thesis has evaded this problem as it blocks the endocytosis of AMPARs, a process that is downstream of NMDAR receptor activation and occurs during excitotoxicity (Wang et al., 2004). The 3Y amino acid sequence (Y869KEGYNVYG877) from the C-terminus of the GluR2 subunit is known to play a role in regulated AMPAR endocytosis while leaving constitutive AMPAR endocytosis unperturbed (Ahmadian et al., 2004); furthermore, it does not affect the physiological functions of NMDARs in addition to preventing NMDA-induced apoptotic cell death (Wang et al., 2004). Therefore, the 3Y sequence blocks a harmful phenomenon while leaving normal physiological functions intact, making it more attractive in terms of therapeutic potential. It has been found that the number of membrane-bound Ca2+-permeable AMPARs is increased in the hippocampus during transient focal ischemia (Liu et al., 2004b), and it is possible that the 3Y sequence may play a part in its receptor trafficking. However, most Ca2+-permeable AMPARs lack the GluR-2 subunit, and its trafficking involves associating proteins that do not overlap with the 3Y sequence (Cull-Candy et al., 2006; Kwak and Weiss, 2006). Therefore, it is highly unlikely that the peptide will play any conceivable role in Ca2+-permeable AMPAR trafficking and their respective functions during excitotoxicity.  4.4.  Peptide Pharmacokinetics A common practice in pharmacological studies is to derive the kinetics, that is, rate  constant (k), or half-life (t1/2) of a drug from its rate of decay. Kinetic reactions of drugs often involve only the substrate, enzymes and co-factors required for the reaction so that the  60  yielded results are deduced from an undisturbed, in vitro setting. The peptide-trapping experiments that are described here are different in several ways: 1.) It has not been done in an undisturbed, in vitro setting; in addition to the necessary substrates and enzymes, other factors that may influence the reaction outcome are also present. For instance, proteases that degrade peptides may potentially alter the reaction dynamics. 2.) Contrary to a supposed in vitro reaction where all components are controlled, the substrate of the reaction –the peptides– travel in and out of neurons. More specifically, those cleaved by calpain are retained in cells, whereas full-length peptides continue their in-and-out movements through equilibrium. The consequence is that this does not guarantee a consistent amount of intracellular peptides as substrates. 3.) Data points are measured as a percentage of the total peptide fluorescence at t = 0, and the subsequent values at different time-points are relative to that of t = 0. They are of importance in illustrating relative peptide dynamics, but there is no information regarding the specific amount of fluorescent peptides at the beginning, nor the respective amounts at different time-points. One cannot assume that these relative values can be directly and accurately correlated to the precise amount of peptides on the scale of molarities. 4.) Although one dansyl fluorophore is attached to one peptide, the raw fluorescence values and the amount of peptides do not have a stoichiometric relationship. Dansyl fluorescence thus only serves as a relative measure of the amount of peptides. It is inadequate if accurate quantification data is needed. Therefore, it cannot be used to deduce the reaction rate reactions nor any other parameters. In addition, chemicals that quench dansyl fluorescence or produce auto-fluorescence may serve as confounds in quantification attempts.  61  4.5.  Potential Significance in Disease-Specific Delivery of Peptides In my thesis, intracellular delivery of the therapeutic GluR2/3Y segment is achieved  by coupling it with the Tat sequence. Tat-mediated cargo delivery is a well-documented process with much success (Vives et al., 2003). Despite the efficient delivery scheme, Tatconjugated cargoes are systemically delivered into biological systems, but not specifically to disease-affected regions. This study represents the first attempt to overcome the nonspecificity shortcomings of Tat by taking advantage of the selective activation of calpain in excitotoxic brain regions during stroke. The peptide delivery assay provides evidence that by inserting an amino acid sequence derived from CRMP-3, a substrate of calpain, between Tat and the therapeutic 3Y sequence, more peptidic 3Y fragments are detected under strokeaffected conditions in vitro (Figure 3.13). This suggests that selective delivery is achieved with the CP-3 peptide. With advances in scientific knowledge and technologies, other methods of delivery have been made available. For instance, certain cargo delivery techniques rely on identifying cell surface-expressing marker proteins that are unique to a particular type of cell. Short interfering RNA (siRNA) duplexes can be coupled to the nucleic acid-binding protamine, which is, in turn, fused to a heavy-chain antibody fragment (Fab) that targets surface proteins on the HIV-1 envelope. These siRNAs can then be delivered to and internalized by HIV-infected cells both in vitro and in vivo (Song et al., 2005). siRNAs can also be specifically delivered by using a similar method: a short peptide segment from the rabies viral glycoprotein (RVG) that is chemically linked to a short D-arginine sequence is able to deliver siRNAs specifically to neurons, taking advantage of the reliance of rabies viruses in recognizing membrane-bound, neuron-specific acetylcholine receptors (AChRs) to gain entry (Kumar et al., 2007). Since RVG-mediated peptide delivery is tailored to neurons, this may translate into a lowered required dosage if a therapeutic drug is to be specifically  62  delivered. It is thus worthwhile to examine the differences in peptide delivery efficiency between a RVG-mediated and a Tat-mediated one in the future when a drug of interest is to be delivered to the central nervous system. Peptides with unique characteristics have also been incorporated into drug development. A peptide derived from bacteriorhodopsin termed pH-low insertion peptide (pHLIP) is capable of folding and inserting itself into the cellular membrane under acidic conditions, and fluorescence-tagged pHLIP accumulation is detected in acidic pathological tissues by using in vivo fluorescence imaging techniques (Andreev et al., 2007). In addition to unique peptide folding, specific peptide cleavages have been exploited for diagnostic purposes. By coupling fluorescent proteins to a sequence from calpain substrates like αSpectrin II, calpain activation can be detected via fluorescence resonance energy transfer (FRET) (Vanderklish et al., 2000; Mittoo et al., 2003). Similarly, an amino acid sequence recognized by caspases can be inserted into a peptide where the fluorescence of a fluorophore is kept in check by a chemical quencher; when the sequence is cleaved upon caspase activation, the fluorophore is released from the quencher, consequently allowing the identification of apoptosis by fluorescence in vitro (Bullok and Piwnica-Worms, 2005). Again by utilizing fluorescence techniques, the activity of the protease matrix metalloproteinase-7 (MMP-7) can be monitored by a MMP-7-cleavage-sensitive peptide and its subsequent increase in fluorescence after cleavage (McIntyre et al., 2004). In brief, these novel peptide technologies are mainly targeted towards either delivering cargoes to specific cell types or developing diagnostic tools where a diseased region can be identified, but not for delivering cargoes to specifically disease-affected areas. In addition to disease-specific peptide delivery properties, the peptides that are tested in my thesis also represent the first attempt to combine both specific disease-targeting and  63  therapeutic potential into the same drug. The 3Y peptidic sequence exerts neuroprotection in an in vitro stroke model (Wang et al., 2004), but the rate of Tat-Glu2/3Y accumulation during excitotoxicity is not altered (Figure 3.7B). The CP-3 peptide tested in my thesis should theoretically be more protective given the greater accumulation of 3Y peptidic fragments under excitotoxic conditions in vitro (Figure 3.13). Following this logic, this may possibly lead to a reduced minimum dosage to induce neuroprotection. As part of the future studies, the neuroprotective properties of the CP-3 peptide in both in vitro and in vivo stroke models should be investigated to confirm its therapeutic potential. In addition to the disease-targeting and the dosage-lowering potential of this specific peptide-cleaving method, the greatest advantage it presents is its universal applicability to other spectrums of biology. In neurodegenerative diseases like Alzheimer’s and Huntington’s disease, part of the pathological effects are brought about by proteolytic cleavages of certain protein substrates (Alzheimer’s: β-amyloid and tau; Huntington’s: Huntingtin) (Goldberg et al., 1996; Yan et al., 1999; Gamblin et al., 2003). As well, the roles of caspases and calpain observed in neuronal apoptosis seem to be consistent with the general apoptotic pathways like cancer (Cory and Adams, 2002). This suggests a potential paradigm shift in peptidic drug design, in that three components are necessary: 1.) a segment like Tat or poly-arginine for efficient intracellular delivery; 2.) a cleavable sequence that is recognizable by proteases in a disease-specific manner; and 3.) a therapeutic agent targeted to the disease of interest.  4.6.  Complications in In Vivo Fluorescence Detection Whereas detections of peptides under in vitro systems in my thesis have been  successful, the attempt to detect the dynamics of peptide delivery in vivo has been rendered fruitless due to the inability to reliably detect the chemical fluorophore dansyl that is attached  64  onto the C-termini of the peptides. Dansyl has been a popular fluorophore to attach onto synthetic peptides since it can be incorporated into the peptide synthesis process with ease. However, it has been known to be a weak chemical fluorophore and its fluorescence is highly dependent on experimental conditions. This is especially true under an in vivo environment, as animal tissue autofluorescence is often found within the green-blue range of the spectrum, which directly overlaps with the emission wavelength of dansyl (Em: 520 nm), further dissipating the efficiency of dansyl. It is a common practice in in vivo fluorescence detection studies to use a fluorophore whose emission wavelength is in the near-infrared (NIR) range to evade the autofluorescence problem. This is a point worthy to be taken into consideration if any future in vivo peptide detection studies are to commence.  4.7.  Conclusion In conclusion, the CP-3 peptide has demonstrated disease-specific targeting properties  in an in vitro model of stroke. Possible future studies that are worth pursuing include examining the neuroprotective properties of the CP-3 peptide in both in vitro and in vivo stroke models, comparing delivery efficiencies between Tat-mediated and neuronrecognizing RVG-mediated peptides conjugated to NIR fluorophores in vivo. In addition, it will be intriguing to examine the therapeutic potential of different permutations of peptides by exchanging the calpain-cleaved sequence with other amino acid sequences recognized by different disease-specific proteases, and substituting the 3Y sequence with other sequences that exert neuroprotection.  65  BIBLIOGRAPHY  Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 115:863-877. Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M (2002) Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. 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