Mechanism of Carbamathione As a Therapeutic Agent For

MECHANISM OF CARBAMATHIONE AS A THERAPEUTIC AGENT FOR

STROKE

Jigar Modi

A Dissertation Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Florida Atlantic University

Boca Raton, FL

December 2017

ACKNOWLEDGEMENTS

I want to express my sincerest gratitude to my supervisor, Dr. Jang-Yen Wu, for his great patience, advice, and guidance, as well as giving me extraordinary experiences throughout the work. He provided me unflinching encouragement and support for allowing me the room to work in my own way. Without his mentoring, this dissertation would not have been possible. One simply could not wish for a better or friendlier supervisor.

The author wishes to express his sincere thanks and love appreciation to Dr. Jang

Yen Wu for his assistance and guidance in preparation of this thesis. Many thanks to my committee members, Dr. Howard Prentice, Dr. Jianning Wei and Dr. Rui Tao, for their contribution on finalizing this thesis. I would like to thank Hongyuan Chou, Dr. Janet

Menzie, and Dr. Payam Gharibani for helpfulness on this thesis.

At last but not the least, I want to thank my family. My wife Dr. Bhumika Tandel and my sister’s family, Dr. Dipali Mevawala and Dr.Dharmesh Mevawala are always there to encourage and support me. A special thought is devoted to my parents, Nishaben Modi,

Pravinchandra Modi and Dr. Binaben Tandel for their unconditional love and endless support.

iv ABSTRACT

Author: Jigar Modi

Title: Mechanism of Carbamathione as a therapeutic agent for Stroke.

Institution: Florida Atlantic University

Dissertation Advisor: Jang-Yen Wu, Ph.D.

Degree: Doctor of Philosophy.

Year: 2017

Stroke is the third leading cause of mortality in the United States, and so far, no clinical interventions have been shown completely effective in stroke treatment. Stroke may result in hypoxia, glutamate release and oxidative stress. One approach for protecting neurons from excitotoxic damage in stroke is to attenuate receptor activity with specific antagonists. Disulfiram requires bio-activation to S-methyl N, N-diethylthiolcarbamate sulfoxide (DETC-MeSO). In vivo, DETC-MeSO is further oxidized to the sulfone which is carbamoylated forming Carbamathione, a glutathione adducts. Carbamathione proved to be useful as a pharmacological agent in the treatment of cocaine dependence with the advantage that it lacks ALDH2 inhibitory activity. Carbamathione is a partial NMDA glutamate antagonist. The purpose of this dissertation study is to evaluate the neuroprotective effects of Carbamathione drug on PC-12 cell line and to understand the protective mechanisms underlying in three stroke-related models: excessive glutamate, hypoxia/reoxygenation and bilateral carotid artery occlusion (BCAO). Carbamathione was

v administered 14 mg/kg subcutaneously for 4 days with the first injection occurring 30 min after occlusion in the mouse BCAO stroke model. Mice were subjected to the locomotor test, and the brain was analyzed for infarct size. Heat shock proteins, key proteins involved in apoptosis and endoplasmic reticulum (ER) stress, were analyzed by immunoblotting.

Carbamathione reduced both cell death following hypoxia/reoxygenation and brain infarct size. It improved performance on the locomotor test. The level of pro-apoptotic proteins declined, and anti-apoptotic, P-AKT and HSP27 protein expressions were markedly increased. We found that Carbamathione suppresses the up- regulation of Caspase-12,

Caspase-3 and significantly declined ER stress protein markers GRP 78, ATF4, XBP-1, and CHOP. Carbamathione can downregulate ATF 4 and XBP1 expression, indicating that Carbamathione inhibits the ER stress induced by hypoxia/reoxygenation through suppressing PERK and IRE1 pathways. Carbamathione elicits neuroprotection through the preservation of ER resulting in reduction of apoptosis by increase of anti-apoptotic proteins and decrease of pro-apoptotic proteins. Carbamathione can suppress the activation of both

PERK and IRE1 pathways in PC-12 cell cultures and has no inhibitory effect on ATF6 pathway. These findings provide promising and rational strategies for stroke therapy.

vi DEDICATION

This dissertation is dedicated to my beloved guru Param pujya Pramukh Swami Maharaj and Pragat guru Mahant Swami Maharaj, and my family, particularly my mother, Nishaben

Modi, and my father, though no longer with me, Pravinchandra Modi, who have put up with these many years of research. These two people are/ have been the most supportive and caring parents I am/was blessed to have in my life. I also dedicate this work to my father in law, though no longer with us, Dr. Mahesh Tandel all of whom believed in the pursuit of dreams. And finally, to my present BAPS family (Miami Temple), who prayed for me constantly, especially when the going was tough.

MECHANISM OF CARBAMATHIONE AS A THERAPEUTIC AGENT FOR

STROKE

LIST OF FIGURES ...... xi

LIST OF ABBREVIATIONS ...... xiv

1 INTRODUCTION ...... 1

1.1 Stroke ...... 1

1.2 Model of stroke ...... 4

1.3 Endoplasmic reticulum (ER) stress...... 6

1.3.1 The unfolded protein response (UPR): ...... 7

1.4 Carbamathione ...... 10

1.5 Summary of My Research Project ...... 12

2 MATERIALS AND METHODS ...... 13

2.1 Materials ...... 13

2.2 In Vitro Study ...... 13

2.2.1 PC-12 cell line culture ...... 13

2.2.2 Glutamate toxicity ...... 14

2.2.3 Hypoxia/reoxygenation ...... 14

2.2.4 Carbamathione concentration ...... 14

2.2.5 Measurement of cell viability by ATP assay ...... 15

2.3 In Vivo Study ...... 15

2.3.1 Animal preparation ...... 16

viii 2.3.2 Bilateral common carotid artery occlusion (BCAO) ...... 16

2.3.3 Corner tests ...... 17

2.3.4 Locomotion (Force-plate actometer) test ...... 17

2.3.5 Mice Groups and Treatment Schedules ...... 18

2.3.6 2, 3, 5-Triphenyltetrazolium chloride (TTC) assessment of lesion size ...... 19

2.4 Sample collection for western blot analysis ...... 19

2.5 Data and Statistical analysis ...... 20

3 EXPERIMENTAL RESULTS ...... 22

3.1 Glutamate excitotoxicity is dose-dependent in PC-12 cell culture ...... 22

3.2 Carbamathione protects PC-12 cells against glutamate-induced excitotoxicity ...... 23

3.2.1 Carbamathione modulates expression of Heat shock protein (Hsp) and AKT

induced by glutamate toxicity ...... 25

3.2.2 Carbamathione can decrease apoptosis by down-regulation of apoptotic

markers ...... 28

3.2.3 Carbamathione protects neuronal cells against glutamate excitotoxicity by

suppressing the expression of GRP 78, CHOP ...... 31

3.3 Carbamathione demonstrates robust protective activity against

hypoxia/reoxygenation on PC-12 cell cultures ...... 35

3.3.1 Effect of Carbamathione on expression of Heat shock protein and AKT induced

by hypoxia/reoxygenation ...... 37

3.3.2 Carbamathione inhibits the expression of GRP 78, CHOP and Caspase-12

induced by hypoxia/reoxygenation ...... 41

3.3.3 PERK and IRE1 pathways were inhibited by Carbamathione under

ix hypoxia/reoxygenation, but there was an increaseing in ATF6 cleavage ...... 44

3.3.4 Effect of Carbamathione on the hypoxia/reoxygenation induced change in Bcl2,

Bax and Caspase-3 expression ...... 48

3.4 Carbamathione treatment attenuates infarction volume, and neurological deficits ...53

3.4.1 Carbamathione can modulate unfolded protein response and decrease apoptosis

by down-regulation of apoptotic markers ...... 60

4 DISCUSSION ...... 64

Conclusion ...... 74

REFERENCES ...... 81

x LIST OF FIGURES

Figure 1: Cascade of events occurred in Ischemia [12] ...... 4

Figure 2: The signaling pathways in ER stress [26]...... 8

Figure 3: Metabolism of Disulfiram [35]...... 11

Figure 4: Dose-dependence of glutamate-induced cell injury in PC-12 cells measured

by ATP assay...... 23

Figure 5: Effect of Carbamathione on glutamate (10 mM)-induced cell injury in PC-

12 cells...... 24

Figure 6: Hsp 27 expression after glutamate treatment with Carbamathione...... 26

Figure 7: Effect of Carbamathione on expression of AKT, P-AKT after glutamate

(10 mM) treatment...... 27

Figure 8: Effect of Carbamathione on glutamate-induced elevated expression of Bax. .. 29

Figure 9: Effect of Carbamathione on glutamate-induced decreased expression of

Bcl2...... 30

Figure 10: Cleaved Caspase-3 expression after treatment of Glutamate (10 mM)...... 31

Figure 11: Effect of Carbamathione on glutamate-induced elevated expression of

GRP 78...... 33

Figure 12: Effect of Carbamathione on glutamate-induced elevated expression of

CHOP...... 34

Figure 13: Protective effects of Carbamathione on PC-12 cell culture under

hypoxia/reoxygenation condition...... 36

xi Figure 14: The effects of Carbamathione on Hsp 27 expression after exposure to

hypoxia/reoxygenation ...... 38

Figure 15: Effects of Carbamathione on Hsp 70 expression induced by

hypoxia/reoxygenation...... 39

Figure 16: Effect of Carbamathione on expression of AKT, P-AKT induced by

hypoxia/reoxygenation...... 40

Figure 17: Effect of Carbamathione on expression of GRP 78 and CHOP induced by

hypoxia/reoxygenation...... 42

Figure 18: Effect of Carbamathione on expression of Caspase-12 and Cleaved

Caspase-12 induced by hypoxia/reoxygenation...... 43

Figure 19: Effect of Carbamathione on expression of ATF4 induced by

hypoxia/reoxygenation...... 45

Figure 20: Effect of Carbamathione on expression of ATF6 induced by

hypoxia/reoxygenation...... 46

Figure 21: Effect of Carbamathione on expression of XBP1 induced by

hypoxia/reoxygenation...... 47

Figure 22: Effects of Carbamathione on hypoxia/reoxygenation induced changes of

Bcl2/Bax ratio...... 50

Figure 23: Carbamathione prevented hypoxia/reoxygenation induced activation of

Caspase-3...... 52

Figure 24: Morphological analysis of effect of Carbamathione on ischemia- induced

brain injury in BCAO stroke model...... 55

Figure 25: Corner test results of BCAO mice...... 57

xii Figure 26: Effect of Carbamathione on locomotor activity test after BCAO ...... 59

Figure 27: Effect of Carbamathione on expression of UPR (GRP 78) and CHOP

protein in BCAO stroke model...... 61

Figure 28: Effect of Carbamathione on expression of Bcl2 and Bax protein in BCAO

stroke model...... 62

Figure 29: Bcl2/Bax ratio expression are in right and left part of BCAO brain...... 63

Figure 30: Scheme for protective effects of Carbamathione against activation of ER

stress pathways...... 77

xiii LIST OF ABBREVIATIONS

AKT/PKB: Protein kinase B

ALDH2: Aldehyde dehydrogenase

ALS: Amyotrophic lateral sclerosis

AMPA: Amino-3-hydroxy-5-methyl-4-isoxazolepropionate

ANOVA: Analysis of variance

APAF1: Apoptotic protease activity factor-1

ATF4: Activating transcription factor 4

ATF6: Activating transcription factor 6

ATP: Adenosine 5 triphosphate

BAK: Bcl-2 antagonist/killer

BAX: Bcl-2 associated protein X

BCAO: Bilateral common Carotid Artery Occlusion

BCL2: B cell lymphoma 2

BCL2-xL B cell lymphoma 2 extra long

BDNF: Brain-derived neurotrophic factor

BFGF: Basic fibroblast growth factor

BH: Bcl-2 homology

BH3: Bcl-2 homology domain 3

BiP: Binding immunoglobulin protein

Carb: Carbamathione

xiv Caspase: Cysteine aspartic acid protease

CCA: Common Carotid Artery

CHOP: C/EBP homologous protein

Cyt-c: Cytochrome-c

DA: Dopamine

DETC-MeSO: S-methyl N, N-diethythiolcarbamate sulfoxide

DSF: Disulfiram

EIF2α: Eukaryotic translation initiation factor 2 alpha

ER: Endoplasmic reticulum

ERAD: Endoplasmic reticulum associate degradation

ERAD: ER-associated protein degradation

FBS: Fetal bovine serum

GABA: Gamma (γ)-Aminobutyric acid

GADD153: Growth arrest and DNA damage-inducible 153

GAPDH: Glyceraldehayde-3-phosphade dehydrogenase

GRP 78: Glucose-regulated protein 78

GSH: Reduced glutathione

HD: Huntigton's disease

HS: Horse serum

HSP: Heat shock protein

Hyp: Hypoxia

IRE1α: Inositol-requiring protein-1alpha

JNK: cJUN NH2-terminal kinase

xv KA: Kainic acid mPFC: medial Prefrontal cortex

NAc: Nucleus Accumbens

NMDA: N-methyl-D-aspartate

NOS: Nitric oxide synthase

OMM: Outer mitochondrial membrane

P38MAPK: Mitogen-activated protein kinase

PC: Pheochromocytoma

PD: Parkinson's disease

PERK: Protein kinase RNA (PKR)-like ER kinase

PI3K: Phosphatidylinositol-3-kinase

PMDs: Protein misfolding disorders

PUMA: p53-upregulated modulator of apoptosis

STAT3: Signal transducer and activator of transcription

TRAF2: Tumor necrosis factor-α receptor-associated factor 2

TTC: 2, 3, 5- Triphenyl tetrazolium chloride

UPR: Unfolded protein response

VOCC: Voltage operated calcium channel

XBP-1: X-Box-binding protein

xvi INTRODUCTION

1.1 Stroke

When blood supply is blocked to part of the brain or when a blood vessel in the brain ruptures, it is defined as stroke. In both cases, parts of the brain become damaged or die. Permanent brain damage, long-term disability, or even mortality is known to cause by stroke [1]. In the US, Stroke is 3rd leading cause of death and disability, with one person dying every 4 minutes [2]. Leading cause of severe long-term disability is stroke. Stroke decrease movement in more than half of stroke survivors age 65 and over. Approximately

800,000 people have a stroke each year; almost one every 40 seconds. About 87% of all strokes are ischemic strokes, in which blood flow to the brain is constrained and vessels are clogged [3].

A trial study has publicized that more than 60% of patients acquire hypoxia within the first 60 hours after stroke [4]. Stroke or ischemia leads to a rise in the extracellular concentrations of excitatory amino acids, and in particular glutamate [5, 6]. This rise in glutamate could be related to increasing release from neurons, developing from energy failure, or to lowering clearance of glutamate by glial transporters. An augmented production of free radicals and other reactive species in stroke leads to oxidative stress [7].

Stroke (ischemia) causes a series of events that can provoke the glutamate release and increase free radical production via numerous different pathways (shown in Figure 1).

During ischemia, the principle factor affecting neurons is the collapse of the high-energy phosphate compound, ATP, due to the lack of the substrates for its manufacture, i.e.,

1 oxygen and glucose. The energy failure roots membrane depolarization, due to the condensed activity of ATP-dependent ion pumps, such as Na+/K+-ATPase. This damage in turn compromises transmembrane ionic gradients, causing an influx of extracellular Ca²⁺ through voltage-sensitive Ca²⁺ channels and uncontrolled release of excitatory amino acids, such as glutamate in the extracellular space [8]. Glutamate is the most commonly released excitatory neurotransmitter. In low amounts, it is crucial for neuronal function. In high amounts, this is a neuronal poison, a toxin, and is known as excitotoxin. The large release of glutamate is also mediated by Ca²⁺ induced activation of presynaptic terminals and the interruption of the uptake and inactivation system of glutamate, which is ATP and voltage dependent. Glutamate stimulates both N methyl- d-aspartic acid (NMDA) and non-

NMDA-type ionotropic receptors, so further increasing the concentration of Ca²⁺ in the cytoplasm [8]. A few glutamate receptors are non-selective cation-permeable ion channels.

Primarily, over-activation of these channels triggers a passive influx of Cl- (and Na+) into cells producing osmotic (cytotoxic) edema and rapid death. Glutamate activation of AMPA and kainite receptors and exposure to the raised extracellular K⁺ levels elicits depolarization of neurons. Constant glutamate stimulation of NMDA receptors with synchronized membrane depolarization leads to a prolonged opening of NMDA receptor channels, authorizing massive influx of Ca²⁺ through membrane. Depolarization is also thought to cause extra Ca²⁺ entry into the cell through voltage operated Ca²⁺ channels

(VOCC). The result of ischemia is intracellular Ca²⁺ accumulation from many causes inducing the stimulation of a mixture of Ca²⁺ stimulated enzymes, such as proteases, lipases, nucleases, protein kinases, and nitric oxide synthase (NOS). This is a vital

2 mechanism by which the damage to cellular and subcellular structures leading to neuronal death is caused [9].

The principle reason of the death of neurons in hypoxic–ischemic brain injury is intense stimulation of N-methyl-D-aspartate (NMDA) receptors by glutamate and It is known as calcium-dependent neurotoxicity [10]. Stroke (ischemia) causes a series of events that can provoke glutamate release and increased free radical manufacture via some diverse pathways [8]. As these events begin, including increased excitotoxicity, calcium overload, creation of free radicals and inhibition of protein synthesis all involved in stroke

[10], there is a major overarching to aim to cure stroke by calcium antagonists, glutamate antagonists and antioxidants, etc. Although excellent work in stroke research and major improvements in stroke care took place within the last decade, therapy is still insufficient.

Stroke, characterized as an undertreated disease, is in need of improvement of the current therapy and an urgent search for new remedies [11].

Figure 1: Cascade of events occurred in Ischemia [12]

1.2 Model of stroke

The most central mechanisms of tissue damage in stroke are scarcity of oxygen and glucose, excitotoxicity (excess release of glutamate from affected cells) and production of injurious free radicals. in vitro model for stroke offers approach to all these mechanisms involved in stroke. Primary cultures offer fast access to study molecules of interest frankly

4 in related cell types. Primary cultures are actual cells present in tissue rather than immortalized cell lines and are good tools for key molecule identification. The possible applications include [13]:

1. Oxygen-glucose deprivation

2. Glutamate-induced excitotoxic insult

3. Hydrogen peroxide-induced free radical insult

In vivo models have allowed a huge insight into the pathophysiology of human disease and have been analytical in our understanding of stroke. Rodents are frequently the selected species, because of accessibility of genetically improved strains [13]. Animal models of stroke and brain ischemia accessible for practice in studies include [14]:

1. Transient middle cerebral artery occlusion (tMCAO)

2. Permanent middle cerebral artery occlusion (MCAO)

3. Endothelin-1-induced middle cerebral artery occlusion (EMCAO)

4. Global brain ischemia (2 vessel or 4 vessel occlusion)

5. Hind limb ischemia peripheral model

6. Photothrombotic stroke model

To induce cerebral ischemia, animal models of stroke are used. The goal is the study of essential processes or likely therapeutic interventions in this disease, and the addition of the pathophysiological knowledge on and/or the progress of medical treatment of human ischemic stroke. Complete and partial models of global ischemia, tend to be simpler to operate. However, they are less closely related to human stroke than the focal stroke models, because global ischemia is not a usual mark of human stroke.

5 In this study, we used an in vitro model for stroke namely Glutamate excitotoxicity and

Hypoxia- reoxygenation injury and for the in vivo study, we used Bilateral Common

Carotid Artery Occlusion (BCAO) model.

1.3 Endoplasmic reticulum (ER) stress.

A chief place for folding and handling of freshly produced proteins is the endoplasmic reticulum (ER). The ER has been recognized for years to be a sub-cellular compartment playing a key role in cellular calcium storage and signaling [15]. These are firmly calcium dependent processes that involve high calcium activity for correct functioning [16, 17].Buildup of unfolded proteins in the ER is an extreme form of stress that will prompt apoptosis if ER functioning cannot be fixed. Deficiency of ER functioning can be passed about in several ways including depletion of ER calcium stores, blocking the proteasome that is needed to lower unfolded proteins, or genetic mutations resulting in proteins that cannot be correctly folded [18].

The homeostasis of the ER can be restored by a chain of ailments including calcium depletion from its lumen, oxidative stress, and mutations in proteins that traffic through the secretory pathway, among other events. These worries can affect in disturbance of the folding process in the ER, managing to the aggregation of misfolded/ unfolded proteins

(ER stress). ER stress stimulates the UPR (The unfolded Protein Response), a complex signal-transduction pathway that facilitates cellular adaptation to reestablish ER homeostasis. Under chronic ER stress the UPR produces cell death by apoptosis, reducing injured cells [19]. There is increasing signal that ER stress plays a fundamental role in hypoxia/ischemia-induced cell dysfunction [20–22]. Hypoxia initiates the collecting of unfolded proteins in the ER, leading to the unfolded protein response (UPR) and ER-

6 associated protein degradation (ERAD) [23]. The accretion of misfolded proteins in the brain is a prominent feature of most general neurodegenerative diseases including

Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), in addition to PD (Parkinson’s disease)[6]. These diseases are now categorized as protein misfolding disorders (PMDs)[19].

1.3.1 The unfolded protein response (UPR):

The chief purpose of UPR is to repair ER functions by lowering the burden of proteins that need to be folded and managed in the ER lumen and by increasing protein folding capacity. The UPR is started in mammalian cells by the activation of three definite types of stress sensors located at the ER membrane: double-stranded RNA-activated protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring kinase 1a (IRE1a). The method of stress sensing by these proteins is inadequately understood, but one of the best usually accepted models contains the detection of unfolded proteins by the ER chaperone GRP 78/Bip, leading to its dissociation from the sensors. Once activated, these stress sensors transducer evidence about protein-folding status at the ER to the nucleus by adjusting the expression of certain downstream transcription factors [19].

PERK, ATF6, and IRE1 activities are constrained in the neuronal homeostasis, depending upon the extend that glucose regulated protein 78 (GRP 78), an ER chaperone.

GRP 78 detaches from PERK, ATF6, and IRE1 (Figure 2). ER dysfunction, induces the dimerization and phosphorylation of PERK and IRE1, and cleavage of ATF6 (P90) to

ATF6 (P50). Stimulated PERK in turn phosphorylates eukaryotic translation initiation

7 factor 2 subunit a (eIf2a), which represses global protein synthesis [24]. Also, phosphorylation of eIF2a causes to the paradoxical rise in translation of ATF4 [25].

Figure 2: The signaling pathways in ER stress [26].

IRE1a is a serine–threonine kinase and endoribonuclease that, upon activation, initiates the unconventional splicing of the mRNA encoding the transcription factor Xbox binding protein-1 (XBP1). This splicing event shifts the coding reading frame and leads to the expression of a more stable and active transcription factor, termed XBP1s. XBP1s regulates a subset of UPR target genes related to folding, ER/Golgi biogenesis, and ER- associated degradation (ERAD)[23]. IRE1a also signals to the cytosol by binding adapter proteins, which triggers the activation of alarm pathways (i.e., JNK, ASK1, and NF-kB).

These signaling events impact diverse processes such as autophagy, apoptosis, and

8 inflammatory responses. In addition, IRE1a can selectively degrade mRNAs encoding for proteins that are predicted to be difficult to fold and micro- RNAs [23].

ATF6 is a membrane-spanning protein localized to the ER. Upon dissociation from Bip,

ATF6 traffics to the Golgi and undergoes subsequent proteolytic processing to release the cytosolic domain, ATF6f, an active transcription factor. Cytosolic ATF6f is then imported into the nucleus and can induce expression of protein quality control genes, either independently of or synergistically with XBP1s[6].

Activated PERK phosphorylates eukaryotic initiation factor 2a (eIF2 a), resulting in a general attenuation of protein translation, which is one mechanism that decreases the overload of proteins at the ER. eIF2a phosphorylation allows the specific translation of activating transcription factor 4 (ATF4), which upregulates many important genes that function in redox control, metabolism, and folding under persistent or severe ER stress,

ATF4 contributes to the induction of cell death by controlling the transcription of proapoptotic BCL2 family members including PUMA and BIM, in addition to GADD34 and

CHOP [15]. Thus, the UPR is a global stress network that integrates information about the intensity and kinetics of protein misfolding at the ER, controlling the decision on cell fate through a variety of complementary mechanisms [27].

All of these three pathways up-regulate the transcription factor C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible gene 153

(GADD153) [28]. All upstream signals, such as the activation of transcription factors, kinase pathways and the regulation of BCL2 family members, eventually lead to caspase activation, resulting in the ordered and sequential dismantling of the cell [26]. Caspase-12

9 is known to be essential for this ER stress-induced apoptosis [29]. Caspase-12 has been proposed as a key mediator of ER stress-induced apoptosis (Figure 2) [30].

1.4 Carbamathione

Carbamathione is a metabolite of disulfiram [31], a drug that has been used for over

65 years for the treatment of alcohol abuse since the discovery that disulfiram sensitized individuals to ethanol [32]. The pharmacological basis for disulfiram’s action is its inhibition of liver low Km mitochondrial aldehyde dehydrogenase (ALDH2). In patients treated with disulfiram, unpleasant symptoms characterized by headache, nausea, and vomiting are produced if ethanol is ingested. Disulfiram requires bio-activation to S-methyl

N, N-diethylthiolcarbamate sulfoxide (DETC-MeSO) for its inhibition of ALDH2 [33, 34].

In vivo, DETC-MeSO is further oxidized to the sulfone which is carbamoylated forming

Carbamathione, a glutathione adducts (Figure 3). Carbamathione is a partial NMDA glutamate antagonist and does not inhibit ALDH2 [31, 35]. More recently, disulfiram has been found to be effective in the treatment of cocaine dependence [36], and this effect is independent of ethanol use [37]. We have hypothesized that Carbamathione is responsible for disulfiram’s efficacy in cocaine dependence, whereas the disulfiram metabolite DETC-

MeSO, an ALDH2 inhibitor, is responsible for the aversive action of disulfiram.

Carbamathione proved to be useful as a pharmacological agent in the treatment of cocaine dependence with the advantage that it lacks ALDH2 inhibitory activity.[35].

Figure 3: Metabolism of Disulfiram [35]

In Previous studies, Faiman et. al., 2013 showed that the iv administration of

Carbamathione to mice irreversibly blocks glutamate binding to mouse brain synaptic membranes [31], and speculated that Carbamathione may affect both the N-methyl-D- aspartate (NMDA) and non-NMDA glutamate receptor subtypes. NMDA receptor antagonists are known to increase brain dopamine [38]. Noncompetitive NMDA receptor antagonists [39] also increase the release of glutamate from the medial prefrontal cortex

(mPFC) [40–42] and nucleus accumbens (NAc) [43], and dopamine from the PFC [40, 42].

11 The glutamatergic system also modulates dopamine in the NAc [44]. Pharmacological modulation of the GABAergic system in cocaine treated rats affects extracellular dopamine in the NAc. In addition, several studies reported that GABA-transaminase inhibition increase GABA and dopamine [27, 45, 46]. Both dopamine and glutamate also have been implicated in cocaine dependence [47].

Carbamathione (20, 50, 200 mg/kg iv) in a dose-dependent manner increased DA, decreased GABA, and had a biphasic effect on glutamate, first increasing and then decreasing glutamate in both the NAc and mPFC [44, 48]. After Carbamathione administration, NAc and mPFC Carbamathione, as well as Carbamathione in plasma, were rapidly eliminated within 4 min, while the changes in DA, GABA, and glutamate in the

NAc and mPFC persisted for approximately two hours [44, 49].

1.5 Summary of My Research Project

In our laboratory, we followed guidelines for animal model of stroke. Models of transient global ischemia are commonly used to test free radical scavengers and anti- inflammatory agents. My project is to see the effects of Carbamathione in animal model of stroke (BCAO). The Hypothesis of study is that “Carbamathione provides neuroprotection via inhibiting apoptosis and ER stress in brain in animal model of stroke”. Therefore, the aim of this study is to investigate the protective effect of Carb against ER stress apoptosis in (1) a stroke-like cell culture model involving glutamate-induced or hypoxia/reoxygenation induced cell death and (2) a mouse model of global cerebral ischemia (BCAO)

12 MATERIALS AND METHODS

2.1 Materials

F-12K media, trypsin-EDTA solution, horse serum and rat pheochromocytoma

(PC) PC-12 cell line were purchased from ATCC (Manassas, VA, USA). Fetal bovine

serum, poly-D-lysine and other chemicals like Glutamate were purchased from Sigma (St.

Louis, MO, USA). Adenosine 5’-triphosphate (ATP) Bioluminescent Assay Kit was

purchased from Promega (Madison, WI, USA).

2.2 In Vitro Study

In vitro study was designed to determine the efficacy of Carb to exert a protective

function against cell death. Glutamate was used to simulate glutamate-induced toxicity in

the rat pheochromocytoma (PC12 cell) line and Hypoxia/reoxygenation also used to

simulate ischemia like condition in the PC-12 cells. Then the effect of Carb against

glutamate-induce toxicity (First phase) or hypoxia/reoxygenation injury (Second phase)

was determined by measuring cell viability.

2.2.1 PC-12 cell line culture

PC-12 cells were cultured in petri dish for one week prior to cell use. PC-12 cells were maintained at 37oC /5% CO2 in incubator and Cells were fed every other day using

F12-K medium supplemented with 5% (v/v) fetal bovine serum (FBS), 10% (v/v) heat- inactivated horse serum (HS) and 1% (v/v) penicillin-streptomycin solution. All experiments were performed on undifferentiated cells plated in 96-well plates at a density of approximately 5×10⁴ cells/ml for the ATP assay [27, 50, 51]. On first day of use, cells

13 were harvested by first adding 2 mL Trypsin and incubated for 15 minutes. 2mL fresh medium was then used as a wash and cells were spun, re-suspended. Cell density was determined by cell counting using a hemocytometer and a tissue culture microscope. After cell density was determined, 96-well plates, 6 – well plates were plated with 2.5 x 104 cells per well and 5 x 105 cells per well for Adenosine 5’-triphosphate (ATP) assay and Western blot analysis, respectively. Cells incubated for 24 hours and varying Carbamathione concentrations were added along with 10 mM Glutamate / Hypoxia 24 and reoxygenation

24 hours.

2.2.2 Glutamate toxicity

For glutamate-induced toxicity, Cell culture in vitro were preincubated with 0.5 µM

to 500 µM concentration of Carbamathione for one hour. Then the neurons were treated

with glutamate (10 mM) for 24 hour [27, 50–52].

2.2.3 Hypoxia/reoxygenation

To generate hypoxic conditions, PC-12 cells in 6 or 96 well plates were placed in a

hypoxia chamber with oxygen levels maintained at 0.3-0.4 % [10, 27]. The level of oxygen

was continuously monitored using an oxygen electrode. PC-12 cell cultures in the absence

or presence of Carbamathione were subjected to 24 hours of hypoxia. Reoxygenation was

performed by removing cultured plates from the hypoxic chamber and transferring them

into normal culture incubator remaining for another 24 hours.

2.2.4 Carbamathione concentration

Carbamathione 0.15 M was prepared in 0.15M sodium bicarbonate solution as

described in Kaul et al., 2010.

14 2.2.5 Measurement of cell viability by ATP assay

PC-12 cells in 96-well plates were treated with or without Carbamathione for

1 hour, and then cells were subjected to Glutamate / hypoxia–re-oxygenation conditions to

induce cell death. ATP solution was added to each well, and cells were incubated for 10 min

after which the amount of ATP was quantified through a luciferase reaction. The

luminescence intensity was determined using a luminometer with lysates in a standard

opaque-walled multi-well plate. The ATP content was determined by running an internal

standard and expressed as a percentage of untreated cells (control) [10, 50].

2.3 In Vivo Study

The experiments carried out in vitro provide relatively quick data that are both important and critical in understanding a specific area of research, they tend to be less exact than experiments done in vivo because they do not exhibit the actual biological conditions of a living organism [53]. In addition, the response of the brain to stroke and effects from other systems make it impossible to emulate stroke using only in vitro systems

[54]. For instance, studies in animal models of stroke enables the control over the severity, duration, location and cause of the ischemia and allows for the monitoring of physiological parameters, such as body temperature [55]; all of which presents a more similar scenario to human conditions. PC12 cells may not fully replicate the phenotype of primary cultured neurons, and for this reason our work may need to use in vivo models. Hence data obtained from in vivo studies are more easily extrapolated to human conditions than those obtained from in vitro studies.

15 2.3.1 Animal preparation

Male Swiss Webster mice (20 weeks of age) were obtained from Charles River laboratory. Access to water only before surgery and kept fasting overnight for surgery.

Mice were anesthetized with ketamine (100 mg/kg, i.p.) plus xylazine (10 mg/kg, i.p.). For induction of anesthesia, mice were exposed to a gaseous mixture consisting of 30% oxygen,

70% N2O and 0.5% isoflurane using a vaporizer. For maintenance of anesthesia, isoflurane concentration was used to 0.5%. Mice were breathing spontaneously via breathing mask throughout the surgical procedure. A rectal temperature probe was inserted. During surgery, mice were resting on a thermostat-controlled heating pad, ensuring a constant core temperature of 37.0 ± 0.5 °C.

2.3.2 Bilateral common carotid artery occlusion (BCAO)

Anesthesia was maintained as described above. Mouse was placed on its back. The animal's tail and paws were fixed to the heating pad using adhesive tape. A sagittal ventral midline incision (~ 1cm length) was performed. Both salivary glands were carefully separated and mobilized to visualize the underlying both Common Carotid Artery (CCA).

Both CCA's were carefully separated from the respective vagal nerves and accompanying veins without harming these structures. Manipulations of the vagal nerves might lead to transient or permanent dysfunction of the parasympathetic nerve system, which has the potential for the occurrence of significant cardiac arrhythmia or even irreversible cardiac arrest. Therefore, it is crucial to avoid any manipulations of the vagal nerves. Both common carotid arteries (CCAs) were isolated, freed of nerve fibers, and occluded using non- traumatic aneurysm clips. Complete interruption of blood flow was confirmed under an operating microscope. After 30 min of ischemia, the aneurysm clips were removed from

16 both CCAs. Restoration of blood flow (reoxygenation) was observed directly under the microscope. Sham-operated controls were subjected to the same surgical procedures except that CCAs were not occluded [56]. The body temperature was monitored and maintained at 37°C ± 0.5°C during surgery and during the immediate postoperative period until the animals recovered fully from anesthesia.

2.3.3 Corner tests

The corner test, which determines an animal’s asymmetric direction of turning when encountering a corner, is used as an indicator of brain injury. We used an experimental corner setup composed of two boards (with dimensions of 30 × 20 × 1 cm3) arranged to form a 30° corner; a small opening was left along the joint between the two boards. The mouse was placed 12 cm from the corner and allowed to walk into the corner, so that the vibrissae on both sides of the animal’s face contacted the two boards simultaneously. Before BCAO procedure, we conducted behavior tests (stratification) on all mice to screen for mice with no turning asymmetry (n ≥ 18). Each mouse took part in ten trials, after which we calculated the percentage of turns to each side, recording only those turns involving full rearing along one of the boards [56]. This stratification procedure excludes mice with 80–100% asymmetric turns (n=4); we included mice that turned in either direction (n=14) with a pretest score of 0.50 ± 0.08. Each mouse took part in ten trials for up to 4 days after BCAO.

2.3.4 Locomotion (Force-plate actometer) test

The force-plate actometer is an ensemble of mechanical, electronic, and computing elements that embody mathematical and physical principles to produce measurement of whole-organism behavioral attributes of relevance to basic neuroscience research. Methods

17 of calibration and details of data acquisition have been described elsewhere [57, 58].

Briefly, the force-plate actometer purchased from BASi Corp (model FPA-I; West

Lafayette, IN, USA) consists of a force-sensitive plate at a resolution of 200 Hz, a sound attenuation chamber, a computerized data acquisition board, and an analysis system software (FPA 1.10.01). A newly-developed force-plate actometer was utilized to measure locomotor activity. Animals were placed on the force plate actometer for one separate 60 min sessions. Locomotor activity of BCAO mice with and without Carbamathione treatment were done after 4 days. Between each test, the plate was thoroughly cleaned with paper towels followed by a deodorant treatment (70 % ethanol, 1 % acetic acid, and then water) to remove animal waste (i.e., feces, urine, saliva, and furs) and odor. Trace data of movements were automatically stored on the hard drive for off-line analysis. Changes in locomotion were revealed through power spectral analysis and expressed as arbitrary distance. The unit for changes in the power force was arbitrary.

2.3.5 Mice Groups and Treatment Schedules

Animals were randomly assigned for sham, control, and experimental groups.

Thereafter, in experimental group (Carbamathione treated group, N = 9), Carbamathione

(14 mg/kg in 0.3 mL saline 0.9 %) was injected subcutaneously 30 min after occlusion and continue daily until the animal sacrificed. In control groups (vehicle-treated group, N = 9), vehicle (0.3 ml saline 0.9 %) was injected subcutaneously 30 min after occlusion. mice were received vehicle for 4 days before sacrifice. Sham-operated group (N = 6) received the same surgical procedure without occlusion of common carotid artery. After surgery, animals could recover from the anesthesia and given food and water ad libitum. The

18 animals were daily examined for body temperature and weight, and those who had body temperature more than 39 °C after 24 hour were excluded from the experiment [59].

2.3.6 2, 3, 5-Triphenyltetrazolium chloride (TTC) assessment of lesion size

Mice were euthanized 4 days after surgery. The brain was quickly removed and sectioned into 2 mm thick slices starting at the frontal pole using a Brain Matrix Slicer

(Zivic instruments, PA, USA). Slices were immersed in 2% TTC (J.C. Baker, India) in a

Petri dish and incubated at 37˚C for 5 minutes. TTC, a water-soluble salt, is reduced by mitochondrial dehydrogenases to formazan, a red, lipid-soluble compound that turns normal tissue deep red [60, 61]. Thus, reduced TTC staining identifies regions of diminished mitochondrial function in the ischemic tissue [62]. To assess lesion volume,

TTC-stained slices (2, 4, 6, 8, and 10 mm from the frontal pole) were scanned using a scanner and analyzed by Image-J analysis software [63] (public domain software developed at NIH and available on the Internet at http://rsb.info.nih.gov/nihimage/). Lesion volume was determined as the percent of the total both hemispheric volume and was calculated as:

[(Vc - VL) / Vc] 100

Where VC is the volume of the both hemisphere (Compare the value with whole brain of sham) and VL is the volume of non-lesioned tissue in the lesioned hemisphere [63, 64].

Then, these sections were compared in different treated and untreated animal groups.

2.4 Sample collection for western blot analysis

Animals were deeply anesthetized by isoflurane (Phoenix) and decapitated, and then brains were rapidly removed. After sacrifice, the while the brain was on ice, the left hemisphere [64] and the right hemisphere (identical parts) were quickly dissected on dry

19 ice and homogenized in Lysis buffer consisting of 50mM Tris–HCl, 150mM NaCl, 2mM

EDTA, pH 8.0, 1% Triton-X-100, 1:100 dilution of mammalian protease inhibitors

(Sigma-Aldrich, MO, USA) and protease inhibitor [65] for immunoblotting. Protein concentrations of each sample solution were determined with a Bradford protein assay, and samples were stored at -80⁰ C until use. Protein samples were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Western blot was done as described previously [63] with the following primary antibodies overnight: abcam: GRP 78, HSP70, HSP 27, activating transcription factor 4 (ATF4), and X-box-binding protein 1 (XBP-1) (1:500); Cell Signaling: GAPDH

(1: 3,000), Bax, BCL2 (1: 1,000), AKT, phosphorylated AKT (p- AKT); Santa Cruz:

CHOP/GADD153); Imgenex: ATF6 (1: 1,500). The membranes were washed three times with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and incubated with secondary antibodies for 1 hour at room temperature. Secondary antibodies used were goat

IRDye 800-conjugated anti-rabbit (1:15,000) and IRDye 680 conjugated anti-mouse (1:

15,000) antibodies (LI-COR Biosciences, Lincoln, NE, USA). Fluorescent signals were detected with a LI-COR Odyssey Fc system and the images were quantified with the provided either Image Studio 2.0 software or image J software [66].

2.5 Data and Statistical analysis

All data were expressed as the mean ± SEM. A computer program (SPSS 15.0,

Chicago, IL, USA and Prism Graph Pad 7) was used for statistical analysis. The statistical significance of the data was determined with t-test or one-way ANOVA combined with

Dunnett post-hoc or Tukey test for comparison between groups. Differences of P<0.05

20 were considered statistically significant. At least three independent replicates were performed for each experiment.

21 EXPERIMENTAL RESULTS

3.1 Glutamate excitotoxicity is dose-dependent in PC-12 cell culture

PC12 cells represent a commonly established neuronal model system. This cell model is also generally used to study cellular glutamate toxicity [67]. PC12 cells are very sensitive to glutamate or hypoxia/reoxygenation injury, therefore, we assumed that it is appropriate for PC12 to examine whether Carb offers protection against glutamate or hypoxia induced cytotoxicity.

The PC-12 cells were exposed to different concentrations of glutamate in a range of 0.01 mM to 150 mM for 24 hours, then the ATP assay was performed (Figure 4). As expected, the survival of PC-12 cells decreased with the increasing of concentrations of glutamate. We chose the optimal doses of 10 mM glutamate for cell viability test [50, 67].

At 10 mM glutamate, about 45% of survival PC-12 cells was observed. from 76% at 10

µM glutamate to 18% at 150 mM glutamate.

Figure 4: Dose-dependence of glutamate-induced cell injury in PC-12 cells measured by ATP assay.

Undifferentiated PC-12 cells were exposed to glutamate concentration range from 0.01 to 150 mM for 24 hours. (All data are presented as mean+/-SEM, where *p<0.05, **p<0.01, ***p<0.001, *significant with control group).

3.2 Carbamathione protects PC-12 cells against glutamate-induced excitotoxicity

In our previous research, it was revealed that preincubation with 25 µM DETC

MeSO resulted in maximal recovery from neuronal injury induced by glutamate [10]. To determine the protective effect, we used 0.5 µM to 500 µM concentration of

Carbamathione on 10 mM glutamate-induced excitotoxicity, cell viability was examined using the ATP assay as shown in Figure 5. It showed that 10 mM glutamate significantly decreased the survival of PC-12 cells about 50-55%. The protective effect of

Carbamathione is up to 75-80%, as shown in Figure 4 after treatment with 25 µM

Carbamathione for 1 hour and 24 hours, following exposure to 10 mM glutamate for 24 hours.

Figure 5: Effect of Carbamathione on glutamate (10 mM)-induced cell

injury in PC-12 cells.

Cell viability was measured by ATP assay. 0.5 µM to 500 µM Carbamathione was preincubated for 1 hour following by 10 mM glutamate treatment for 24 hours and 0.5 µM to 500 µM Carbamathione was incubated for 24 hours with 10 mM glutamate treatment. (All data are presented as mean+/-SEM, where #/*/‡/ ¥ p<0.05, ‡‡/**p<0.01, */ ‡ significant with glutamate 10 mM group, #/ ¥ statistical significance with control group).

After establishing the glutamate-induced toxicity model (stroke-related condition) in undifferentiated PC12 cells, our next step was to investigate the potential protective effect of Carb against glutamate-induced cytotoxicity in this model. I hypothesized that in glutamate-induced stress PC12 cells, Carb will downregulate the ER stress apoptotic marker CHOP, cell death marker cleaved caspas-3 and Bax. I predict that the Carb will increase the survival of PC12 cells against glutamate-induced apoptosis by upregulating

Bcl2, P-AKT, GRP 78 and Hsp 27

24 3.2.1 Carbamathione modulates expression of Heat shock protein (Hsp) and

AKT induced by glutamate toxicity

In many cell types Hsp 27 has been shown to control apoptosis by manipulate of

AKT activation. A previous inquiry has identified Hsp 27 as an AKT substrate that detaches from AKT upon phosphorylation [68]. Carbamathione resulted in increased expression of Hsp 27 in Carbamathione-treated groups (1 hour and 24 hours) versus glutamate (10 mM) treated groups (Figure 6b). AKT, also known as protein kinase B (PKB; is a serine/threonine-specific protein kinase) plays a chief part in modulating survival and apoptosis. In our experiments, P-AKT (is the activated form of AKT) showed a dramatic up-regulation in Carbamathione-treated groups (1 hour and 24 hours) versus glutamate (10 mM) treated groups (Figure 7b), whereas AKT expression showed no changes when

Carbamathione was used (Figure 7c).

Figure 6: Hsp 27 expression after glutamate treatment with Carbamathione.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Hsp 27 densitometric scanning are presented. 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

Figure 7: Effect of Carbamathione on expression of AKT, P-AKT after glutamate (10 mM) treatment.

a) Results from western blot analysis as described in material and methods. b) and c) Average results (n=5) of AKT and P-AKT densitometric scanning are presented. d) Quantification of P-AKT: AKT ratio from Western blot (from five independent blots). 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

27 3.2.2 Carbamathione can decrease apoptosis by down-regulation of apoptotic

markers

Cell survival pathways are initiated by the Bcl2 protein, what in turn inhibits mitochondrial permeability transition pore (MPTP) opening, while Bax, BAD, and/or Bak, all proapoptotic proteins, can translocate from the cytosol into the outer mitochondrial membrane after glutamate toxicity to cause MPTP opening and Cyt-c release. Cyt-c triggers caspase-3, which is assumed to be at the last step of apoptosis [69]. Our results show that Carbamathione can down-regulate proapoptotic proteins Bax in drug-treated groups (1 hour and 24 hours) versus glutamate (10 mM) treated groups (Figure 8b). On the other hand, measurement of anti-apoptotic protein Bcl2 showed an increase in the ratio of

Bcl2/Bax in the Carbamathione treated (1 hour and 24 hours) versus glutamate (10 mM) treated group (Figure 9b). Up-regulation of Bcl2 could decrease Cleaved Caspase-3 in

Carbamathione treated (1 hour and 24 hours) group by 3- and 2-fold, respectively, in comparison to the Glutamate (10 mM) treated group (Figure 10b).

Figure 8: Effect of Carbamathione on glutamate-induced elevated expression of Bax.

a) Results from western blot analysis as described in material and methods. b) and c) Average results (n=8) of Bax densitometric scanning are presented. 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

Figure 9: Effect of Carbamathione on glutamate-induced decreased expression of Bcl2.

a) Results from western blot analysis as described in material and methods. b) Average results (n=8) of Bcl2 densitometric scanning are presented. c) Quantification of Bcl2: Bax ratio from Western blot (from eight independent blots). 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

Figure 10: Cleaved Caspase-3 expression after treatment of Glutamate (10 mM).

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Cleaved Caspase-3 densitometric scanning are presented. 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

3.2.3 Carbamathione protects neuronal cells against glutamate excitotoxicity by

suppressing the expression of GRP 78, CHOP

To examine if ER stress can be caused by glutamate and then inhibited by

Carbamathione, specific ER stress effector proteins were analyzed by western blot. 31 Glucose regulated protein-78 (GRP 78) is an ER-associated chaperone, which enhances protein folding in ER [70]. Experimental evidence has indicated that up-regulation of GRP

78 stops neuronal damage induced by ER stress, and the increase in GRP 78 expression may relate to the degree of neuroprotection [63]. The expression of GRP 78 protein was up-regulated in primary neurons after treatment with 10 mM glutamate for 24 hours.

However, Carbamathione restored the level of GRP 78 to control levels, as shown in Figure

11 b. C/EBP homologous protein (CHOP), also recognized as growth arrest and DNA damage inducible protein 153 (GADD153), is a chief ER stress marker [71]. Figure 11b shows that the expression of CHOP was up-regulated by glutamate. Carbamathione treatment restored CHOP expression to the control level (Figure 12b).

Figure 11: Effect of Carbamathione on glutamate-induced elevated expression of GRP 78.

a) Results from western blot analysis as described in material and methods. b) and c) Average results (n=5) of GRP 78 densitometric scanning are presented. 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

Figure 12: Effect of Carbamathione on glutamate-induced elevated expression of CHOP.

a) Results from western blot analysis as described in material and methods. b) and c) Average results (n=5) of CHOP densitometric scanning are presented. 1- Control, 2- Glutamate (10 mM for 24 hours), 3- Carbamathione (25 µM for 1 hour) followed by glutamate (10 mM for 24 hours), 4- Carbamathione (25 µM for 24 hours) with glutamate (10 mM for 24 hours). (All data are presented as mean+/-SEM, Where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3, 2 and 4).

34 3.3 Carbamathione demonstrates robust protective activity against

hypoxia/reoxygenation on PC-12 cell cultures

This in vitro study was designed to determine the efficacy of Carb to exert protection against hypoxia/reoxygenation model of stroke. The following hypotheses would be tested:

(1) Carb would exert protection against ER stress induced apoptosis in the hypoxia/reoxygenation model of stroke. (2) Carb would reestablish ER homeostasis in the hypoxia/reoxygenation. After prolonged ER stress (the accumulation of unfolded proteins in the ER), the three ER stress transmembrane sensors; PERK, IRE1α and ATF6 are activated, leading to the consequential initiation of intracellular pathways that upregulate the ER stress proapoptotic transcription factor, CHOP. CHOP contributes to apoptosis by inhibiting the transcription of the antiapoptotic molecule, Bcl-2. In addressing the first hypothesis, I predict that Carb will negatively modulate the PERK, IRE1α and ATF6 pathways. I also predict that Carb will downregulate or attenuate CHOP, thereby protecting the cells against ER stress apoptosis. Hypoxia/reoxygenation leads to a reduction of the calcium level in ER, resulting in the disruption of the ER’s homeostasis and the consequential accumulation of unfolded/missed folded proteins. Increased levels of unfolded protein results in the upregulation of protein chaperons, such as HSP 27 and HSP

70. I predict that Carb treatment will downregulate GRP78 in the hypoxia induced cells, which would reflect the reestablishment of the ER homeostasis.

In our study, we first tested the effect of Carbamathione on the PC-12 cell culture in hypoxia/reoxygenation condition. To determine the appropriate concentration of

Carbamathione in cultures, PC-12 cells were exposed to hypoxia and reoxygenation in the presence or in the absence of 5 – 100 µM Carbamathione as shown in Figure 13. After

35 hypoxia and reoxygenation, ATP levels for cells without Carbamathione treatment dropped to about 47% (percentage of control). Carbamathione treatment dramatically increases the cell viability. The presence of 25 µM Carbamathione clearly improved the cell viability to greater than 70%. With increasing Carbamathione concentrations up to 100 µM cell viability decreased to 30%. Our data showed that 25 μM Carbamathione can attenuate cell death in hypoxia/reoxygenation.

Figure 13: Protective effects of Carbamathione on PC-12 cell culture under hypoxia/reoxygenation condition.

Cell culture was exposed to 0.3 % oxygen for 24 hours followed by reoxygenation for 24 hours. In hypoxia + 5-100 µM Carbamathione, cells were pre-incubated with Carbamathione (5-100 μM) for 1 hour before hypoxia. Cell viability was measured by ATP assay. Normoxia values were fixed at 100 % (n=5, *significant with hypoxia group, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. *significant with Hypoxia/reoxygenation group, # statistical significance with control group (Normoxia)).

36 3.3.1 Effect of Carbamathione on expression of Heat shock protein and AKT

induced by hypoxia/reoxygenation

Mammalian cells can react to a range of stresses such as heat, cold, oxidative stress, metabolic disturbance, and environmental toxins through necrotic or apoptotic cell death, while increased expression and phosphorylation of heat shock proteins such as Hsp 27 can guard cells against cellular stress. Heat shock proteins (Hsp) generally display molecular chaperone activity and act together with a broad range of proteins to employ specific effects. Previously Settler et al., 2008 showed that Hsp 27 overexpression confers long- term neuroprotection against cerebral ischemia. Hsp 27 and Hsp 70 showed greater overexpression in the Carbamathione treated group compared to Hypoxia-reoxygenation group alone (Figure 14b and 15b). In this trial, P-AKT (is the activated form of AKT) indicated up-regulation in Carbamathione-treated groups versus hypoxia-reoxygenation treated groups (Figure 16b), whereas AKT expression showed no changes when Carbamathione was used (Figure 16d).

Figure 14: The effects of Carbamathione on Hsp 27 expression after exposure to hypoxia/reoxygenation

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Hsp 27 densitometric scanning are presented. Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 - hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 15: Effects of Carbamathione on Hsp 70 expression induced by hypoxia/reoxygenation.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Hsp 70 densitometric scanning are presented. Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 16: Effect of Carbamathione on expression of AKT, P-AKT induced by hypoxia/reoxygenation.

a) and c) Results from western blot analysis as described in material and methods. b) Average results (n=5) of P-AKT densitometric scanning are presented. d) Average results (n=5) of AKT densitometric scanning are presented. e) Quantification of P-AKT: AKT ratio from Western blot (from five independent blots). Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

3.3.2 Carbamathione inhibits the expression of GRP 78, CHOP and Caspase-

12 induced by hypoxia/reoxygenation

To observe the effect of Carbamathione on ER stress induced by hypoxia/reoxygenation, we preincubated 25 µM Carbamathione for 1 hour, followed by hypoxia and reoxygenation. The expression of GRP 78 and CHOP was measured by

Western blot analysis, as shown in Figure 17a and 17c. The expression of GRP 78 and

CHOP was up-regulated after exposure to hypoxia/reoxygenation (Figure 17b and 17d).

Western blot analysis shows that the levels of both Caspase-12 (50 kDa) and Cleaved

Caspase-12 (35 kDa) are up-regulated after hypoxia/ reoxygenation (Figure 18b and 18c).

Carbamathione significantly reduced the expression of GRP 78, CHOP, Caspase-12 and

Cleaved Caspase-12, demonstrating that Carbamathione has ability to inhibit the apoptosis induced by ER stress in hypoxia/reoxygenation.

Figure 17: Effect of Carbamathione on expression of GRP 78 and CHOP induced by hypoxia/reoxygenation.

a) and c) Results from western blot analysis as described in material and methods. b) Average results (n=5) of GRP 78 densitometric scanning are presented. d) Average results (n=5) of CHOP densitometric scanning are presented Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 18: Effect of Carbamathione on expression of Caspase-12 and Cleaved Caspase-12 induced by hypoxia/reoxygenation.

a) Results from western blot analysis as described in material and methods. b) and c) Average results (n=5) of Caspase-12 (50 kDa) and Cleaved Caspase-12 (35 kDa) densitometric scanning are presented. d) Quantification of Cleaved Caspase-12: Caspase- 12 ratio from Western blot (from five independent blots). Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

43 3.3.3 PERK and IRE1 pathways were inhibited by Carbamathione under

hypoxia/reoxygenation, but there was an increaseing in ATF6 cleavage

It is fully known that there are three ER stress-induced signaling pathways: PERK,

ATF6 and IRE1 pathways. Since Carbamathione can protect cells against ER stress induced by hypoxia, we aimed to further classify which signaling pathway is implicated in the protective process. ATF4 are highly expressed after hypoxia/reoxygenation, ATF 4 increased by approximately 3.0-fold over control. After treatment with Carbamathione, followed by hypoxia/reoxygenation, however, the levels of ATF 4 in cells are decreased

(Figure 19b) (*3.0 times that of control), indicating that Carbamathione inhibit the initiation of the PERK pathway under this condition. This result indicated that

Carbamathione has observable effects on PERK pathway activation. We next examined the effect of Carbamathione on the ATF6 pathway in cells induced by hypoxia/reoxygenation. Treatment with Carbamathione reduced the level of ATF6 (90 kDa) (Figure 20b). However, the cleaved ATF6 (50 kDa) in cells treated with

Carbamathione increased relative to cells under hypoxia/reoxygenation without

Carbamathione as shown in Figure 20c and 20d. These results demonstrate that

Carbamathione cannot prevent the activation of the ATF6 pathway in hypoxia/reoxygenation. To determine if Carbamathione affects the IRE1 pathway induced by hypoxia/reoxygenation, we tested the expression of XBP1 in PC-12 cells with and without Carbamathione treatment under hypoxia/reoxygenation conditions by Western blot analysis (Figure 21b). The results show that XBP1 is highly expressed in cells under hypoxia/reoxygenation. Carbamathione reverses the expression of XBP1 to its normal

44 condition, demonstrating that Carbamathione significantly inhibits the IRE1 pathway in

ER stress induced by hypoxia/ reoxygenation.

Figure 19: Effect of Carbamathione on expression of ATF4 induced by hypoxia/reoxygenation.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of ATF4 densitometric scanning are presented. Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 20: Effect of Carbamathione on expression of ATF6 induced by hypoxia/reoxygenation.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of ATF6 (90 kDa) densitometric scanning are presented. c) Average results (n=5) of Cleaved ATF6 (50 kDa) densitometric scanning are presented. d) Quantification of Cleaved ATF6: ATF6 ratio from Western blot (from five independent blots). Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 21: Effect of Carbamathione on expression of XBP1 induced by hypoxia/reoxygenation.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Cleaved XBP1 densitometric scanning are presented. Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

47 3.3.4 Effect of Carbamathione on the hypoxia/reoxygenation induced change in

Bcl2, Bax and Caspase-3 expression

The Bcl2 family members, Bcl2 and Bax, are key regulators of apoptosis. Pro- apoptotic proteins like BAX initially limit to cytosol [72, 73]. The death signal origins proapoptotic proteins to feel conformational modification and target mitochondrial membrane to form homodimers of BAX, which target mitochondria and initiate release of cytochrome c. [73, 74]. Antiapoptotic proteins including BCL2 are primarily integral membrane proteins [73, 75, 76] and can form heterodimers with pro-apoptotic proteins [77]) and avoid the increase of homodimers of BAX resulting in inhibiting the release of cytochrome c from mitochondria. Thus, a rise of the ratio of Bcl2: BAX would be protective. Cells were treated with hypoxia/reoxygenation or with hypoxia/reoxygenation in the presence of 25

µM Carbamathione and then gathered for determination of levels of Bcl2 and Bax protein expression by Western blot analysis. As shown in Figure 22a, top lane 2, and Figure 22b, lane 2; hypoxia/reoxygenation caused a down-regulation of Bcl2. When cells were preincubated with Carbamathione, this reduction in Bcl2 was abated (Figure 22a, top lane

3, and Figure 22b, lane 3). In contrast, hypoxia/reoxygenation treated cells show an up- regulation of Bax when compared with control groups, and this up-regulation was prevented in cells preincubated with Carbamathione (Figure 22c, middle lanes 1–3, and

Figure 22d, lanes 1–3). As shown in Figure 22e, lane 2, hypoxia/reoxygenation induced changes in Bcl2 and Bax levels resulted in a decrease in the Bcl2: Bax ratio. In contrast, in cells preincubated with Carbamathione, the Bcl2: Bax ratio was approximately 60% higher, confirming the protective nature of Carbamathione exposure (Figure 22e, lane 3). Up regulated Bcl2 decreased cytochrome c release that would decreased Cleaved Caspase-3 in

48 Carbamathione treated group compared to control group (Figure 23b lane 3 compare to lane 2).

Figure 22: Effects of Carbamathione on hypoxia/reoxygenation induced changes of Bcl2/Bax ratio.

50 a) and c) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Bcl2 densitometric scanning are presented. d) Average results (n=5) of Bax densitometric scanning are presented. e) Quantification of Bcl2: Bax ratio from Western blot (from five independent blots). Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

Figure 23: Carbamathione prevented hypoxia/reoxygenation induced activation of Caspase-3.

a) Results from western blot analysis as described in material and methods. b) Average results (n=5) of Cleaved Caspase-3 densitometric scanning are presented. Hypoxia = hypoxia (0.3% O2) for 24 hours, reoxygenation for 24 hours; Carb + Hypoxia: cells were treated with 25 µM Carbamathione for 1 hour, then hypoxia for 24 h, reoxygenation for 24 hours. 1- Control (Normoxia 48 hours); 2 -hypoxia/reoxygenation (48 hours); 3 - hypoxia/reoxygenation (48 hours) after 1-hour, 25 µM Carbamathione preincubation. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2; *statistical significance between groups 2 and 3).

52 3.4 Carbamathione treatment attenuates infarction volume, and neurological

deficits

This in vivo study was designed to determine the efficacy of Carb to exert protection against global ischemia in a mouse model. The following hypotheses would be tested: (1) Carb would exert protection against ER stress induced apoptosis in the global ischemic brain by down regulating CHOP, GRP 78 and Bax.

In our in vivo study, in addition to evaluation of the overall advancement in reducing the brain infarct size, we also analyzed the infarct size at 2, 4, 6, 8, and 10 mm from the frontal pole to finalize the effect of ischemia and Carbamathione at different brain sections as shown in Figure 24. TTC staining in mice subjected to BCAO in the vehicle- treated group versus Carbamathione treated groups is shown in Figure 24a–c. The infarct is noticeably reduced in the Mice treated with Carbamathione. Figure 24c represents mean infarct volumes resulting from 30 min of transient BCAO in groups. Carbamathione markedly reduced the volume of the lesion of the sections 2, 4, 6, 8 and 10 mm when

Carbamathione was injected 30 min after occlusion. The sham-operated group showed no ischemic injury as determined by TTC staining.

Unlike mice with brain damage, when normal animals run in to a corner they naturally rear forward and upward, then turn back, in either direction, to face away from the corner and toward the open end of the setup (the corner test) [56]. This determines an animal’s asymmetric direction of turning when encounter corner is used as an indicator of brain injury. We used an experimental corner set up composed of two boards (30 x 20 x 1 cm3) arranged to form 30⁰ corner. We measured the rate of turns when normal mice faced a 30° corner (i.e., turning to either the left or right) to have a rate of 50 ± 8% (symmetric

53 and no bias) before BCAO-30 min surgery. Mice were tested before sacrifice at 4 days after the BCAO-30 procedure (Figure 25 and 25b). We observed a significant increase in asymmetric turning (~90% to one side) in BCAO-30 mice. when facing a 30° corner.

BCAO mice without treatment showed significant asymmetric turning (~90%) beginning one day after the procedure and persisting for at least four days. Mice with treatment

(BCAO + Carbamathione) exhibited behavior not significant differently from that seen in the sham-operated mice.

To test whether motor dysfunction might contribute to the learning deficits observed in corner test, the locomotor activity of mice was measured on a force-plate actometer [57, 58]. We observed similar difference in behavior between sham group and

BCAO with Carbamathione treated group. The travel distances and the number of Sham group, BCAO with Carbamathione group were statistically significantly different compare to BCAO with vehicle group. (Figures 26b and 26c, distance: Sham: 151.25 ± 8.39 m, n =

7; BCAO with vehicle: 100 ± 3.90 m; BCAO with Carbamathione 148.15 ± 7.45, n = 11, p < 0.05).

Figure 24: Morphological analysis of effect of Carbamathione on ischemia- induced brain injury in BCAO stroke model.

55 a) The TTC brain slices in 2, 4, 6, 8, and 10 mm from the frontal pole of the sham group, vehicle-treated group and Carbamathione treated group 30 min after occlusion at 4 days. 1 Sham group, 2 Vehicle-treated group, and 3 Carbamathione treated group 30 min after occlusion. b) The TTC results of the infarct size of the brain slices of the vehicle- treated group and Carbamathione treated group at 4 days after 30 min occlusion. Quantitative analysis revealed that both treated groups after 4 days produced a significant reduction in the infarction percent. Sham-operated group showed no infarct zone. c) The TTC results of the whole brain infarct size of the vehicle treated group and Carbamathione treated group at 4 days after 30 min occlusion. Data represent infarct volume as percent of both hemisphere volume and values are mean ± SEM of 4 experiments for BCAO plus vehicle and BCAO plus Carbamathione at 4 days. (n=4, * p<0.05, ** p<0.01).

Figure 25: Corner test results of BCAO mice.

Percentage asymmetry is observed as a mouse enters a 30⁰ corner and either turns left or right. In the above graph a), percent asymmetry is compared before and after BCAO for sham, BCAO/ Vehicle, and BCAO/Carbamathione (n=11). It is observed that mice treated with Carbamathione after BCAO had a significant better percent asymmetry than those of BCAO/Vehicle and like the percent asymmetry of Sham mice. b) Average results

57 (n=11) of Corner test densitometric scanning are presented. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups Sham and BCAO + Vehicle; *statistical significance between groups BCAO + Vehicle and BCAO + Carb).

Figure 26: Effect of Carbamathione on locomotor activity test after BCAO

59 a) Sample traces b) Locomotor summary. Locomotor activity of BCAO mice with and without Carbamathione treatment were done after 4 days. Results show increased amount of activity with administration of Carbamathione. The summary of locomotor data is provided in (b) expressed in mean ± SEM where n=11 for Sham, BCAO/Vehicle and BCAO/ Carb. Data shows animals treated with Carbamathione have activity consistent with sham animals. c) Average results (n=11) of Locomotor test densitometric scanning are presented. (All data are presented as mean+/-SEM, #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups Sham and BCAO + Vehicle; *statistical significance between groups BCAO + Vehicle and BCAO + Carb).

3.4.1 Carbamathione can modulate unfolded protein response and decrease

apoptosis by down-regulation of apoptotic markers

Our data showed that after BCAO, in the Carbamathione treated group GRP 78 dramatically decreased after 4 days compare to vehicle treated group. GRPs are commonly used as an indicator for the UPR. As we can see in Figure 27a and 27b, Carbamathione could decrease the expression of GRP 78 in comparison to the vehicle-treated group. ATF4,

XBP1, and ATF6 all unite on the promoter of the gene encoding the protein CHOP, which stimulates transcription of the mRNAs encoding Bim and BCL2 [10]. p38MAPK plays an important role in stimulating CHOP activity. JNK activates BIM, but inhibits BCL2. We measured the expression of CHOP by western blot analysis in the BCAO stroke model. As shown in Figure 27c and 27d, the expression of CHOP was up-regulated in the BCAO model by comparison to the sham-operated group. Western blot analyses showed that

Carbamathione can significantly decrease the levels of CHOP in the Carbamathione treated group (Figure 27d).

Figure 27: Effect of Carbamathione on expression of UPR (GRP 78) and CHOP protein in BCAO stroke model.

a) and b) Results from western blot analysis as described in material and methods. Graph a) Average results (n=9) of GRP 78 densitometric scanning are presented. Graph b) Average results (n=9) of CHOP densitometric scanning are presented. 1,4—Sham, 2,5 —BCAO with vehicle treated, 3,6— BCAO with Carbamathione treated. 1,2,3- right side of mouse brain and 4,5,6- left side of mouse brain. (All data are presented as mean+/-SEM, where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2, 4 and 5; *statistical significance between groups 2 and 3, 5 and 6).

Given that Bcl2 and Bax are downstream targets of cell death pathway. consistent with the previous studies, ischemia induced a significant increase of proapoptotic protein Bax and a decrease of antiapoptotic protein Bcl2. Compared with the vehicle treatment, dose of

Carbamathione significantly increased the expression of Bcl2 and decreased Bax at protein levels on day 4 (Figure 28b and 28d). our result indicates that measurement of the ratio of pro-survival Bcl2 to pro-death Bax on day 4 displayed a clear 2-fold up-regulation of Bcl2

/ Bax following Carbamathione treatment (Figure 29).

Figure 28: Effect of Carbamathione on expression of Bcl2 and Bax protein in BCAO stroke model.

a) and b) Results from western blot analysis as described in material and methods. Graph a) Average results (n=9) of Bcl2 densitometric scanning are presented. Graph b) Average results (n=9) of Bax densitometric scanning are presented. 1,4—Sham, 2,5 — BCAO with vehicle treated, 3,6— BCAO with Carbamathione treated. 1,2,3- right side of mouse brain and 4,5,6- left side of mouse brain. (All data are presented as mean+/-SEM, where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2, 4 and 5; *statistical significance between groups 2 and 3, 5 and 6). .

Figure 29: Bcl2/Bax ratio expression are in right and left part of BCAO brain.

The graph shows the ratio of Bcl2 to Bax in the BCAO brain in Carbamathione- treated group and vehicle-treated group. 1,4—Sham, 2,5 —BCAO with vehicle treated, 3,6— BCAO with Carbamathione treated. 1,2,3- right side of mouse brain and 4,5,6- left side of mouse brain. (All data are presented as mean+/-SEM, where #/*p<0.05, ##/**p<0.01, ###/***p<0.001. #statistical significance between groups 1 and 2, 4 and 5; *statistical significance between groups 2 and 3, 5 and 6). .

63 DISCUSSION

It is commonly accepted that a high concentration of the excitatory neurotransmitter glutamate causes excitotoxicity and it plays a key role in ischemia/reoxygenation-induced neuronal death [10, 77]. Despite extensive research to develop medicines for stroke based on the established methods either as glutamate receptor antagonists, Ca2+ channel blockers, enzyme inhibitors, inhibitors of apoptotic pathways, or ROS scavengers, etc., these attempts have been unsatisfactory [15]. Faiman et.al., 2013 suggested that

Carbamathione increased dopamine, decreased GABA, and had a biphasic effect on glutamate in both the Nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) concurrently, it has been reported for a Disulfiram (DSF) metabolite. The mechanism by which Carbamathione produced these simultaneous changes in dopamine, GABA, and glutamate in both the NAc and mPFC was unclear currently. Since Carbamathione affects glutamate binding [31], the present study was carried out to test the hypothesis. The neuroprotective effects of Carbamathione in an in vitro model of glutamate excitotoxicity, hypoxia/reoxygenation. PC-12 cell culture and in vivo model of BCAO stroke in mouse were investigated. First, we examined the neuroprotective effect of Carbamathione on the cell viability of PC-12 cell culture under glutamate excitotoxicity and hypoxia/reoxygenation condition. Carbamathione showed a significant increase in cell viability following glutamate excitotoxicity and hypoxic insult in PC-12 cell culture. These data led us to continue our experiment on in vivo model of BCAO stroke in mouse.

Glutamate release from pre-synaptic membrane is one of consequences of hypoxic or

64 ischemic cell injury, which has an essential effect on neuronal death [27, 51, 77]. The results show that Carbamathione is a very effective agent for protecting PC-12 cells from hypoxia/reoxygenation induced cell damage. This may be due to the inhibition of

Carbamathione on glutamate receptors. Glutamate release triggers activation of glutamate receptors and leads to the injury of surrounding neurons. Therefore, Carbamathione may play a dual role in preventing hypoxia-induced cell death, in which it could suppress the glutamate release from the presynaptic sites of neurons and the activation of glutamate receptors postsynaptically on neurons.

Carbamathione is an active metabolite of disulfiram, which has an antagonistic effect on brain glutamate receptors [44]. Disulfiram has been used in the treatment of alcoholism for decades, and it has been demonstrated that it exerts its anti-alcohol effect only after bio-activation to the active metabolite DETC-MeSO [78]. As shown earlier,

DETC-MeSO is a potent and selective carbamoylating agent for sulfhydryl groups in glutamate receptors [79]. The carbamoylation of glutathione by DETC-MeSO leads to the formation of S-(N, N-diethylcarbamoyl) glutathione (Carbamathione) (Figure 2). The percentage of Carbamathione formed from disulfiram is unknown but is presumed to be small since [80] estimated that 0.05% of DDTC is converted to DDTC-Me, a precursor to

Carbamathione (Figure 2). Carbamathione’s effect on NAc and mPFC dopamine, GABA, and glutamate is dose-dependent, Thus, the change in concentration of these neurotransmitters formed after disulfiram administration is smaller when compared to

Carbamathione [44]. The parent drug DETC-MeSO partially blocks glutamate binding to synaptic membrane preparation from the brain and prevents seizures in mice induced by glutamate analogs and hyperbaric oxygen [31].

65 It is important to note that the half-life of Carbamathione in the brain is only 5.55 min with most of the drug being cleared from the brain at 30-45 min after administration.

However, the changes in basal concentrations of GABA and glutamate upon

Carbamathione administration were observed to last for over 2 hour [49]. This suggests that Carbamathione may either affect the vesicular storage and release of these neurotransmitters over a prolonged period and recovery is slow while Carbamathione is rapidly removed, or that a metabolite of Carbamathione is responsible for the observed effect on these neurotransmitters. Studies in progress suggest the latter may be the case

[44]. Due to the advantage of partially blocking the glutamate receptors, it can reduce neuronal injury following glutamate over excitation and yet maintain normal glutamate neurotransmission. These data led us to select administration of Carbamathione 30 min after occlusion with the dose of 14 mg/kg to see its neuroprotective effect on ischemic stroke. Our post-BCAO/reoxygenation treatment group was done to mimic clinical treatment following the stroke. Our experiment demonstrated that Carbamathione can markedly reduce the volume of the lesion and improve the behavior in BCAO when it was administered post-BCAO/reoxygenation up to 4 days. we have further investigated various signal transduction pathways associated with cell injury and death using this above specific group.

A high concentration of glutamate has been related to several neurodegenerative diseases including Alzheimer’s disease [81] and Huntington’s disease [82] as well as to strokes [83]. One of the pathways involved in glutamate-induced cell death is calcium overload [84]. As The glutamate, excitatory effects are elicited through two kinds of glutamate receptors, ionotropic and metabotropic receptors [85]. Glutamate ionotropic

66 receptors are ligand-gated cation channels permeable to Ca2+. Although fundamentally all members of the glutamate receptor family are believed to be involved in mediating excitotoxicity, NMDA glutamate receptors are believed to be the crucial mediators of death during excitotoxic injury [10, 85]. During ischemia and through glutamate excitotoxicity,

Ca2+ influx through NMDA receptors promotes cell death more efficiently than through other types of Ca2+ channels [85].

It is believed that brain ischemia followed by glutamate excitotoxicity leads to intracellular calcium overload and initiates a series of intracellular events, such as the release of apoptotic proteins leading to apoptotic cell death [86]. As we believed that

Carbamathione can regulate intracellular calcium homeostasis through preventing Ca2+ influx, we continued testing mitochondrial function by measuring different pro- and anti- apoptotic markers. Our results demonstrated that Carbamathione can down-regulate the proapoptotic proteins Bax. On the other hand, measurement of the anti-apoptotic protein

BCL2 showed an increased ratio of BCL2/Bax in the brain of Carbamathione-treated versus vehicle-treated groups. We showed that 4 days after BCAO, Carbamathione could decrease Bax protein expression, while Bcl2 protein expression increased. A high ratio of

Bcl2 to Bax can prevent release cytochrome-c from mitochondria which results in decreased caspase-3 activity and helps in survival. Carbamathione can decrease caspase-3 activation in the glutamate and hypoxia/reoxygenation treated PC-12 cell.

Heat shock proteins (HSPs) help in the folding of newly synthesized proteins in normal cells. HSPs are also engaged in the creation and maintenance of multi-protein complexes, intra-organellar protein trafficking, and degradation of misfolded polypeptides.

Up-regulation of Hsp 27 and Hsp 70 mRNA and protein after many insults has been

67 discovered in neuronal cultures and brain tissue extracted from animals exposed to stress

[87]. In vitro prove of HSP-transfected neurons boosts the protective role of Hsp 27 and

Hsp 70. Trials in in vivo models of epilepsy and stroke indicate that transgenically overexpressed or virally supplied HSPs are not always able to lower lesion size, but can augment cell survival [88]. Since Hsp 27 and Hsp 70 are the main inducible HSPs in the central nervous system [89], we investigated the effect of Carbamathione on the model of stroke specially glutamate excitotoxicity and hypoxia/reoxygenation on these two HSPs by western blotting. Hsp 27 (the major inducible member of the Hsp 70 family) and Hsp 70 showed decreased expression in cells of vehicle-treated glutamate or hypoxic/reoxygenation groups in comparison to control group. Using Carbamathione could increase the expression of Hsp 27 in both glutamate and hypoxia/reoxygenation of

Carbamathione treated versus control group, while HSP70 also changed in the same manner. Hsp 27 can cause an important reduction in stroke lesion size [88], and our data confirm this report. It was stated although that Hsp 27 is naturally expressed in glial cells and not in neurons; however, additionally to the Hsp 27-expressing astrocytes, Hsp 27- positive neurons have also been identified [90]. Glial cells appear to be vital for structural and metabolic boost of neurons, maintaining synapse homeostasis and regulating the rate of neuronal repair [89, 90]. Thus, their ability to secrete Hsp 27 on stress might be a crucial mechanism for protecting neurons. Our finding that Carbamathione changed Hsp 70 expression, while we saw a major decrease in infarct size, adds strength to the plan that boosted Hsp 70 may signal special protective activities as a chaperone. In this post, increasing Hsp 27 expression might be more vivid due to its links with cytoskeletal stability and its ability to fix proteins in an ATP independent fashion in conjunction with its anti-

68 apoptotic functions. Hsp 27 has been reported to protect cells in vitro by affecting with both caspase-dependent and caspase-independent apoptotic pathways. Few tests display that this protein can block caspase-3 activation after ischemia [89, 90]. Surprisingly, substituting Hsp 27 in rat neurons with a caspase-blocking agent does not stop cytotoxicity, telling that, in addition to caspase-3 blockage, Hsp 27 must use chaperone functions that stop necrosis and caspase-independent events [91]. Other groups have shown that Hsp 27 overexpression can restore cytochrome-c after release from the mitochondria and inhibit its binding to the apoptosome [92]. Some research has linked cytoskeletal stability to mitochondria and apoptosis. It was found that Hsp 27 acts further upstream and stop cytochrome-c release via bid relocalization, which is in turn coupled with F-actin filaments modulated by this chaperone [93]. It has also been displayed that phosphorylated and unphosphorylated Hsp 27 attaches actin and tubulin filaments and can stimulate axonal growth and regeneration after injury [93]. Above data are consistent with that obtained from virally delivered HSPs in experimental stroke [88]. A kinase known to inhibit apoptosis is known as Protein Kinase B (AKT). Many studies have indicated that activated

AKT (P-AKT) augments neuroprotection during cerebral ischemia [94]. We documented that Carbamathione activates AKT, thereby eliciting protection against glutamate excitotoxicity and hypoxia/reoxygenation injury. Use of Carbamathione caused a markedly increased level of P-AKT expression in the cells of the Carbamathione-treated group. The cellular decision to suffer apoptosis is regulated by the integration of multiple survival and death signals. The AKT serine/threonine kinases are serious mediators of cell survival in response to Ca2+ influx [94]. A few pro-apoptotic proteins have been reported as direct

AKT substrates, including glycogen synthase kinase 3 (GSK-3), caspase-9, p53, Forkhead,

69 and BAD which are inhibited upon phosphorylation by AKT. p- AKT also has been revealed to prompt some anti-apoptotic markers, such as BCL2 and mTOR [95]. Our data on proapoptotic markers Bax and the anti-apoptotic marker BCL2 support the role of P-

AKT up-regulation in protecting cells in the cells of the Carbamathione-treated group.

Many pathological processes such as alterations in calcium homeostasis, glucose deprivation, and hypoxia control to the accumulation of unfolded proteins in the ER, resulting in ER stress. Primarily in reply to stressful stimuli, the ER will provoke the UPR which is a self-protective signal transduction pathway [96]. Sustained ER stress hints to cell death and is linked with the pathogenesis of some neurodegenerative disorders that appear misfolded proteins including Alzheimer's disease, Parkinson's disease, and ALS

[97]. GRP 78 is usually known as a main ER chaperone, and it is in the ER lumen but is also sensed as a transmembrane protein as well as found outside the ER. GRP 78 is an endoplasmic reticulum (ER)-resident molecular chaperone whose expression acts as a good marker of ER stress [98]. Though the ER is linked in neuronal degeneration in some situations, its function in delayed neuronal cell death after ischemia remains uncertain. As we reported in results, GRP 78 showed a significant decrease in BCAO 4 days after reoxygenation, also we detected changes in GRP 78 expression after treatment with

Carbamathione treated cells against hypoxia/reoxygenation injury. However, in the glutamate excitotoxicity, Carbamathione increased the expression of GRP 78 by comparison to the glutamate treated control group. Based on our previous studies on

Sulindac and DETC MeSO [10, 63], it was shown that ER stress is reduced by ischemic preconditioning and that ER stress reduction by preconditioning was the result of ER molecular chaperone induction [99]. GRP 78 serves as a molecular chaperon in glutamate

70 excitotoxicity or as a ER stress marker in hypoxia/reoxygenation injury and in BCAO, when Carbamathione drug was used. GRP 78 has a higher affinity for unfolded proteins, and therefore, following ER stress and accumulation of unfolded proteins, GRP 78 dissociate from each sensor (ATF6, PERK, and IRE-1 inside the ER lumen) and proceeded to cause the UPR. In this paper, our primary aim is to identify which ER stress-induced pathway can be affected by Carbamathione treatment during the process of hypoxia/reoxygenation in the PC-12 cell culture model. Following detachment of GRP 78,

PERK is stimulated and then phosphorylates a subunit of eIF2a [22].It stops general cap- dependent translation thus decreasing more collection of proteins within the ER lumen

[100]. PERK−/− mouse embryonic fibroblasts lack this translational block and thus are oversensitive to ER stress [100]. This translational block is not complete, and it does not apply to certain proteins such as ATF4. Following its translation, ATF4 translocate to the nucleus where it initiates transcription of ER genes and gene products (including GRP 78) involved in amino acid biosynthesis, redox reactions, and protein secretion as well as proapoptotic mechanisms including synthesis of the transcription factor CHOP [101]. As

ATF4 is down-stream proteins in the PERK pathway of ER stress, it is proper to calculate expression levels of this protein to decide the range of the PERK pathway response in the presence or in the absence of Carbamathione treatment. We found that there was a marked increase in ATF4 expression, indicating that the PERK pathway is activated in hypoxia/reoxygenation model of stroke. Our data showed that the PERK pathway is clearly inhibited in the cells following use of Carbamathione in hypoxia/reoxygenation. Upon sensing of ER stress and detachment of GRP 78, ATF6 translocates to the Golgi apparatus.

The ratio of cleaved ATF6 to full-length ATF6 proves that Carbamathione increases ATF6

71 cleavage in the PC-12 cells in hypoxia/reoxygenation using the in vitro model of stroke.

Certain previous studies have shown that protective effects with induced ATF6 cleavage signaling to UPR [102–104]. Knockdown of ATF6 in cardiac myocytes exposed to ischemia/reperfusion increased reactive oxygen species and necrotic cell death, both of which were diminished by ATF6 overexpression [103]. IRE1 through binding to GRP 78 is held in an inactivated state, and upon detachment of GRP 78, IRE1 is activated by dimerization and auto phosphorylation. Following its activation, IRE1 allows translation and generation of XBP1 [101]. XBP1 triggers the transcription of many proteins involved in the maintenance of ER homeostasis such as ER chaperones GRP 78, ER associated degradation (ERAD), as well as transcription factors such as CHOP and XBP1 [105]. XBP-

1 showed a significant decrease in the PC-12 cells of Carbamathione-treated in the hypoxia/reoxygenation. So, IRE1-mediated XBP 1 activation may represent a mechanism through which IRE1 can control absolute levels of CHOP, a pro-apoptotic transcription factor, is a convergence for all three arms of the UPR, with binding sites for ATF6, ATF4, and XBP1s present within its promoter, and CHOP facilitates ER stress-induced cell death through the regulation of Bcl2 family members. CHOP-mediated down- regulation of Bcl2 has also been reported as a means by which CHOP can tip the balance in favor of proapoptotic Bcl2 proteins and cell death. Simultaneously, these results indicate that the ER stress signals to the mitochondria via the regulation of Bcl2 proteins by CHOP. Our data revealed that CHOP and Bax that were up-regulated in the neurons of the BCAO model, show significantly decreased expression in the brain of the Carbamathione-treated group.

We demonstrated that Carbamathione can significantly reduce the expression of

CHOP/GADD153 in the PC-12 cells of the Carbamathione-treated group against glutamate

72 excitotoxicity and hypoxia/reoxygenation. These findings provide proof that activation of the PERK and some stage in the IRE-1 pathway can be inhibited by Carbamathione, and through these two pathways, PERK may inhibit ER-induced apoptosis. Furthermore, the results indicating suppression of both CHOP and Bax by Carbamathione treatment provide substantial evidence that Carbamathione can contribute to an effective inhibition of ER stress induced by stroke model. Based on past studies, our results shown that taurine significantly inhibit the activation of the ATF6 and the IRE1 pathways, but not the PERK pathway under chronic exposure to hypoxia/reoxygenation or glutamate [27] .

The ER pathway can activate caspase-12 [21], which plays an essential role in programmed cell death progression during the pro-apoptotic phase of the ER stress response [106]. Caspase-12, which was identified as the first ER associated member of the caspase family, is activated by ER stress, and this novel caspase is associated in the cell death-executing mechanisms of ER stress [29, 106]. Caspase-12, a specific ER membrane– associated caspase, is proteolyzed to cleaved caspase-12, which induces the caspase pathway cascade [30]. Both expression of CHOP and activation of caspase-12 initiate cell death. GRP 78 and caspase-12 are also upregulated after exposure to hypoxia/reoxygenation. IRE1s are also reported to activate the JNK-signaling pathway mediated by TRAF2 functions in response to the perturbation of protein folding in the ER

[107].Yoneda. et.al., 2010 showed that TRAF2 plays an essential role in the activation of caspase-12. In unstressed cells, TNF receptor-associated factor 2 (TRAF2) formed a stable complex with procaspase-12. The stimuli that induce ER stress led to the dissociation of procaspase-12 from TRAF2, and simultaneously dimerization (or oligomerization) of procaspase-12 was promoted. These findings raise the possible mechanisms that the

73 dissociation of TRAF2 from caspase-12 is a trigger for the activation of caspase-12 during

ER stress and that the resultant-free procaspase-12 is clustered to the ER [29]. We have

analyzed the expression of caspase-12 in the absence or presence of Carbamathione after

treatment with hypoxia/reoxygenation, and demonstrated that the caspase-12 or cleaved

caspase-12 expression was clearly reduced by Carbamathione following

hypoxia/reoxygenation. The results indicating suppression of both CHOP and caspase-12

by Carbamathione treatment provide substantial evidence that Carbamathione can

contribute to an effective inhibition of ER stress induced by hypoxia/reoxygenation.

Conclusion

The first phase of our In vitro study focused on the use of glutamate-induced toxicity in undifferentiated PC12 cells to simulate an in vitro model of stroke-like conditions. Using this model, we investigated the protective effect of Carbamathione

(Carb) against glutamate induced-toxicity; specifically, glutamate toxicity involving endoplasmic reticulum stress. Our study has provided evidence that (1) high extra cellular glutamate concentration induced toxicity resulting in cell death, (2) Carb was able to protect undifferentiated PC 12 cells from glutamate-induced cell death, (3) Carb will downregulate the ER stress apoptotic marker CHOP, cell death marker cleaved caspas-3 and Bax. Carb will increase the survival of PC12 cells against glutamate-induced apoptosis by upregulating Bcl2, P-AKT, GRP 78 and Hsp 27

The second phase of our in vitro study, we provided evidence that Carb protected against ER stress and apoptosis induced by hypoxia/reoxygenation injury via upregulating levels of anti-apoptotic Bcl2, molecular chaperones such as HSP 27, HSP 70, cell survival marker P-AKT and downregulating proapoptotic Bax. Carb reduced the expression level

74 of cleaved caspase 3, a cell death executioner caspase and Caspase-12. We show that Carb was protective against ER stress apoptosis by downregulating ER stress-induced apoptotic

CHOP and downregulating the expression levels of ATF4, XBP1 and ATF6, which are respective downstream targets in the ER stress sensor pathways: PERK, IRE1 and ATF6, respectively. The protective effect of Carb was also evident from the attenuation of stress markers such as GRP78 (ER intraluminal stress sensor). The effect of Carb on the above- mentioned proteins was significant compare to control and hypoxia/reoxygenation treated cells expect for its effect on cleaved ATF6. The explanation of effect of Carb on ATF6 pathway is debatable. Overexpression of the active form of ATF6 induces protective UPR and improves insulin signaling upon ER stress [103]. So, Carb significantly inhibited apoptosis by activation of the PERK and IRE1, but did not inhibit the ATF6 pathway.

In Vivo study we provided evidence that Carb reduced infarct volume, improved

behavioral score, protected against apoptosis by modulating the expression of Bcl2 family

members via upregulating levels of anti-apoptotic Bcl2 and downregulating proapoptotic

Bax expression level. In addition, we demonstrated that Carb was protective against ER

stress apoptosis by downregulating ER stress-induced apoptotic CHOP and

downregulating the expression levels of GRP 78 (ER stress marker).

In summary, many neurological disorders such as Alzheimer’s disease, stroke and

Parkinson’s disease have been related to the overactivation of glutamatergic transmission

and excitotoxicity as a known pathway of neuronal injury [108–110]. Past studies have

also shown that ER stress is induced in neurons by glutamate toxicity [111, 112]. Recently,

kainic acid (KA), a non-NMDA glutamate receptor agonist, was found to cause the

75 disintegration of the ER membrane in hippocampal neurons and to cause ER stress [51,

113].

Glutamate release from presynaptic membrane is one of consequences of hypoxic or ischemic cell injury, which has an essential effect on neuronal death. The results show that Carb is a very effective agent for protecting cortical neurons from hypoxia/reoxygenation induced cell damage. This may be due to the inhibition of Carb on glutamate receptors. Glutamate release triggers activation of glutamate receptors and leads to the injury of surrounding neurons. Therefore, Carb may play a dual role in preventing hypoxia-induced cell death, in which it could suppress the glutamate release from the presynaptic sites of neurons and the activation of glutamate receptors postsynaptically on neurons. We demonstrated glutamate induced ER stress associated with the up-regulation of the proteins Bax and cleaved Caspase-3. However, Carb has been not investigated or applied in treatment of many diseases, and therefore the protective mechanism is still not fully elaborated. Our results show that Carb reduces the ER stress induced by glutamate in

PC-12 cell cultures. The present study demonstrated that Carb may exert its protective effect on PC-12 cells through suppression of ER stress induced by hypoxia/reoxygenation or glutamate. Furthermore, the effect of Carb treatment on the three ER stress-induced signaling pathways was also investigated on PC-12 cells undergoing hypoxia/reoxygenation or glutamate exposure. As depicted in Figure 29, this study indicates that Carb may significantly inhibit the activation of the PERK and the IRE1 pathway, but did not inhibit the ATF6 pathway under chronic exposure to hypoxia/reoxygenation which is consistent with our previous study on DETC-MeSO treatment in the stroke on the three ER stress-induced signaling pathways showed that

76 DETC-MeSO significantly inhibited apoptosis by activation of the PERK and IRE1, but not the ATF6 pathway [10]. In the ER stress-induced apoptosis-signaling pathway, TRAF2 links between the stress sensor molecule IRE1 and the caspase-12 [29].

Figure 30: Scheme for protective effects of Carbamathione against activation of ER stress pathways.

After neurons were submitted with glutamate or hypoxia, the homeostasis in neuronal culture is disturbed, which initiates dimerization and auto phosphorylation of ER membrane proteins PERK and IRE1. ATF6 (P90) is activated by limited proteolysis after its translocation from the ER to the Golgi apparatus to form cleaved ATF6 (P50). Activated PERK phosphorylates eIF2a, which induces ATF4 expression. ATF4, being a transcription factor, translocate to the nucleus and induces the transcription of genes required to block the translational pathway. These three pathways will induce the up-regulation of CHOP. Caspase-12, a specific ER membrane-associated caspase, is proteolyzed to Cleaved Caspase-12, which induces the caspase pathway cascade. Both expression of CHOP and activation of Caspase-12 initiate cell death. Carbamathione treatment greatly inhibits PERK and IRE1 pathways but did not inhibit ATF6 pathway after hypoxia/reoxygenation.

Carbamathione can exert protective effects on neurons in the BCAO model through inhibition of ER stress. Also, the effect of Carbamathione treatment on the three ER stress-

77 induced signaling pathways indicated that Carbamathione significantly inhibited apoptosis by activation of the PERK and IRE1, but did not inhibit the ATF6 pathway. However, details of the important signaling pathways remain to be revealed and Carb can work through other pathways such as mitochondrial signaling or mTOR signaling but I did not analyze this aspect. Carbamathione can up-regulate AKT phosphorylation to prevent ischemia-induced apoptosis and to attenuate ER stress. Administration of Carbamathione showed that an increase in CHOP and Bax was prevented in BCAO stroke model, indicating that Carbamathione can decrease apoptosis both in mitochondrial Ca²⁺-induced apoptosis (up-regulation of BCL2/ Bax and down-regulation of caspase-3) and ER-induced apoptosis (down-regulation of CHOP). These data support the hypothesis that

Carbamathione, a partial antagonist of the NMDA receptor, can decrease apoptosis by decreasing ER stress in the mouse BCAO stroke model

The proposed mechanism of Carb against glutamate excitotoxicity or hypoxia/reoxygenation injury or mouse model of BCAO will be shown in Figure 29 and summarize below.

1) Reduced cellular energy metabolism during ischemia causes increased and deceased reuptake of glutamate, as well as increased extracellular K⁺ due to inhibition of

Na⁺-K⁺ ATPase [12] . This is happened in vitro model of stroke such as glutamate excitotoxicity or hypoxia/reoxygenation injury as well as in BCAO model.

2) Persistent glutamate activation of NMDA receptors with simultaneous membrane depolarization leads to prolong opening of NMDA receptor channels, permitting massive Ca²⁺ influx across membrane. Depolarization is also thought to cause additional Ca²⁺ entry in to cell through VOCC [114].

78 3) Increased intracellular Ca²⁺ leads to Neuronal toxicity and that affect ER

Mitochondria and Golgi. This calcium accumulation leads to severe damage to organelles.

Accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen triggers an adaptive stress response known as the UPR that is mediated by three types of ER stress sensors: IRE1a, PERK, and ATF6. Active IRE1a processes the mRNA encoding XBP1, a transcription factor that upregulates many essential UPR genes [27]. Upon the induction of

ER stress, ATF6 is processed at the Golgi apparatus, releasing its cytosolic domain which then translocates to the nucleus where it increases the expression of some ER chaperones and ER-associated degradation (ERAD)-related genes [19]. In addition, activation of

PERK decreases the general protein synthesis rate through phosphorylation of the initiation factor eIF2a. eIF2a phosphorylation increases translation of the ATF4 mRNA, which encodes a transcription factor that induces the expression of genes involved in amino acid metabolism, antioxidant responses, apoptosis, and autophagy [15].

Carbamathione is a partial NMDA glutamate antagonist and does not inhibit

ALDH2 [35, 115] .So, Carb affect glutamate bindings to NMDA receptors and decreased intracellular Ca²⁺ level and calcium induced neurotoxicity. Carb downregulated GRP 78, the ER stress marker and CHOP by modulating ER stress sensor pathway particularly

IRE1ɑ and PERK. Carb provides cell survival by inhibiting proapoptotic marker Bax and increasing antiapoptotic marker Bcl2.

However, since we used whole cell lysate in our western analysis it is difficult to make a definitive conclusion on the action of Bcl2 at either the ER or mitochondria.

Nevertheless, our study has provided new and valuable evidence on the anti-apoptotic mechanism of Carb against ER stress induced apoptosis in ischemia demonstrated by the

79 positive modulation of the PERK and IRE1α pathway and the downregulation of CHOP and GRP78.

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