Prostaglandin E2 in -induced neuronal inflammation and injury

A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of

Master of Science

in the Division of Pharmaceutical Sciences of the James L. Winkle College of Pharmacy April 2017

by

Xu Kang B.S. Shenyang Pharmaceutical University June 2014

Committee Chair: Jianxiong Jiang, Ph.D.

ABSTRACT

Cyclooxygenase-2 (COX-2) is upregulated in many neurological conditions including strokes, epilepsies, neurodegenerative diseases, tumors, etc., and plays a pivot role in promoting inflammatory processes within the brain that might facilitate neuronal degeneration and functional impairments. Mounting evidence from preclinical and clinical studies suggests that the pro-inflammatory actions of COX-2 are largely attributed to its major prostanoid product

– prostaglandin E2 (PGE2). PGE2 is involved in a variety of physiological and pathological proceedings in the central nervous system (CNS) via activating four G-protein coupled receptors

(GPCRs) – EP1, EP2, EP3 and EP4. However, which EP receptor is the culprit of PGE2- mediated neuroinflammation and neurodegeneration remains largely unclear and is presumably dependent on the brain insult types and the responding molecular and cellular components. Here, we show evidence that the COX-2, PGE2 and pro-inflammatory cytokine interlukine-1β (IL-1β) are substantially induced in neuronal cells – Neuro-2a (mouse) and SH-SY5Y (human) – that are treated with 6-hydroxydopamine (6-OHDA). Taking advantage of our recently developed novel selective EP2 antagonists – TG4-155 and TG6-10-1, we further demonstrate that EP2 receptor is the major Gαs-coupled receptor that mediates PGE2-initiated cAMP- dependent signaling pathway in Neuro-2a and SH-SY5Y cells, and largely contributes to 6-

OHDA-induced neurotoxicity. Furthermore, microinjection of 6-OHDA into the striatum also causes COX-2 induction in the brain followed by the upregulation of many inflammation and gliosis-associated genes such as IL-1β, TNF-α, Iba-1 and GFAP in Sprague Dawley rats. Our results suggest that pharmacological inhibition of EP2 receptor might represent a novel strategy

III to prevent brain inflammation and injury in neurodegenerative diseases such as Parkinson’s disease.

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V

ACKNOWLEDGEMENT

I would like to take this opportunity to thank all the people here who made this thesis possible.

Most importantly, I would like to thank my advisor Dr. Jianxiong Jiang for his guidance, advice, patience, and encouragement during the past two years. Without his support, this work would not have been possible. I am thankful to my supervisory committee members Dr. Hao and

Dr. Gudelsky for their guidance and suggestions.

I would like to thank my lab friends Yifeng Du, Jiange Qiu, Avijit Dey and Qianqian Li for their help and support. I would also like to thank Dr. Kim Seroogy, Ann Hemmerl and Tara

Kyser for their help with the animal surgery.

I would like to show my appreciation to the University of Cincinnati and James L.

Winkle College of Pharmacy for academic and financial support.

I would like to give special thanks to my parents. They are the most wonderful parents I could ever have. I was the kind of child that is hard to discipline, but they have never given up on me. I am glad that I have such great parents who have devoted all they have to me, who are willing to grow together with me and who always trust me, comfort me and listen to me. I would also like to thank my host family here, Bill and Maureen, who have given me a family here.

Finally, I would like to show my appreciation to all my friends who have helped and loved me unconditionally when my life gets difficult, and with their support I’ve become more and more mature and brave.

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TABLE OF CONTENTS

CHAPTER 1 INTRODUCTION ...... 1

1.1 Background ...... 2

1.2 COX, PGE2 and EP receptors ...... 3

1.3 Parkinson’s disease cell model ...... 4

1.4 Parkinson’s disease animal model ...... 4

1.5 6-OHDA induces Parkinson’s disease as a neurotoxin . . . .5

CHAPTER 2 AIMS OF THE STUDY ...... 6

CHAPTER 3 MATERIALS AND METHODS ...... 8

3.1 Cell culture ...... 9

3.2 Chemicals and reagents ...... 9

3.3 Immunocytochemistry ...... 9

3.4 Cell viability assay ...... 10

3.5 Quantitative PCR ...... 10

3.6 PGE2 measurement ...... 11

3.7 Cell-based TR-FRET cAMP assay ...... 11

3.8 6-OHDA rat model surgery ...... 12

3.9 Statistical analysis ...... 13

CHAPTER 4 RESULTS ...... 14

VII

4.1 Neuro-2a and SH-SY5Y cells are TH positive and susceptible to 6-OHDA-mediated cytotoxicity ...... 15

4.2 6-OHDA induces COX-2 expression and nuclear translocation . . .15

4.3 6-OHDA leads to increased production of PGE2 and IL-1β . . .16

4.4 PGE2/EP2/cAMP signaling in Neuro-2a and SH-SY5Y cells . . .16

4.5 EP2 receptor inhibition is neuroprotective in 6-OHDA-induced cell injury .17

4.6 6-OHDA causes inflammatory induction in a rat model of 6-OHDA-induced neuronal injury ...... 18

CHAPTER 5 DISCUSSION ...... 31

5.1 6-OHDA provokes inflammatory processes . . . . .32

5.2 EP2 receptor is the dominant EP receptor in COX-2-initiated cAMP-dependent signaling pathway ...... 32

5.3 Roles of COX-2 in neurodegenerative process . . . . .33

5.4 Gαs coupled EP2 and EP4 receptors ...... 34

5.5 Conclusion ...... 35

REFERENCES ...... 36

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LIST OF FIGURES

Figure 1: COX-2/PGE2/EP2 signaling pathway after a variety of stimuli. 20

Figure 2: Mouse Neuro-2a and human SH-SY5Y cells are NeuN positive and 21

express tyrosine hydroxylase (TH).

Figure 3: 6-hydroxydopamine (6-OHDA) induces neurotoxicity. 22

Figure 4: 6-OHDA induces cyclooxygenase-2 (COX-2) expression. 23

Figure 5: 6-OHDA stimulation causes COX-2 translocation to the nuclei. 24

Figure 6: Effect of 6-OHDA on the syntheses of prostaglandin E2 (PGE2) and 25

interleukine-1β (IL-1β).

Figure 7: PGE2-mediated cAMP signaling in mouse Neuro-2a cells. 26

Figure 8: PGE2-mediated cAMP signaling in human SH-SY5Y cells. 27

Figure 9: EP2 receptor inhibition reduces 6-OHDA-triggered neurotoxicity. 28

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Figure 10: Induction of inflammatory genes COX-2, IL-1β, TNF-α, GFAP, and 29

Iba-1 in the brain following 6-OHDA microinjection into the rat

striatum.

X

LIST OF TABLES

Table 1: Sequences of the primers for qPCR. 30

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LIST OF ABBREVIATIONS

6-OHDA 6-hydroxydopamine

COX-2 Cyclooxygenase-2

ELISA Enzyme-linked immunosorbent assay

GFAP Glial fibrillary acidic protein

GPCRs G protein-coupled receptors

Iba-1 Ionized calcium binding adaptor molecule 1

IL-1β Interleukine-1β

L-DOPA L-dihydroxyphenylalanine

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PD Parkinson’s disease

PGE2 Prostaglandin E2 qPCR Quantitative polymerase chain reaction

ROS Reactive oxygen species

SNpc Substantial nigra

TH Tyrosine hydroxylase

TNF-α Tumor necrosis factor-alpha

TR-FRET Time-resolved fluorescence resonance energy transfer

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CHAPTER 1: INTRODUCTION

1

1.1 Background

Parkinson’s disease (PD) is a neurodegenerative disease that mostly effects the motor system of patients, with a median age-of-onset of approximately 55 years (Brundin et al., 2008).

The motor symptoms of PD result from progressive death of in the substantial nigra pars compacta (SNpc), a region of the midbrain, leading to irreversible destruction of the (Tieu, 2011). Many patientswith PD will have non-motor problems too, such as apathy, pain, sexual difficulties, bowel incontinence, and sleep disorders, which can happen during all stages of disease. It is very difficult for doctors to diagnose because patients may not link these symptoms to PD or they are too embarrassed to tell others (Chaudhuri, et al., 2009).

The causes of PD are poorly understood; both genetics and environment can trigger the onset of disease, and researchers believe that motor symptoms of PD result from the death of -generating cells in the , a region of the midbrain (Brundin et al., 2008).

The molecular mechanisms leading to loss of SNpc neurons are not fully understood, but it has been shown that brain inflammation, reactive oxygen species (ROS), oxidative stress, mitochondrial impairment, and dysfunction in proteasomal or autophagic protein degradation could be involved (Duty et al., 2011; Tieu, 2011). Dopamine is an organic chemical that plays important roles in the brain and body, and the precursor chemical L-dihydroxyphenylalanine (L-

DOPA) is synthesized in the brain and kidney. Initial pharmacological treatment with L-DOPA can attenuate the symptoms of PD, but the efficacy of this treatment gradually decreases over time. (Yasushi Takagi et al., 2005).

Other current drugs for PD treatment are dopamine agonists (directly activate dopamine receptors), anticholinergics (inhibit cholinergic receptors) and MAO-B inhibitors (prevent MAO-

2

B from breaking down dopamine) (http://www.webmd.com/parkinsons-disease/guide/drug- treatments). However, these drugs have debilitating side effects, e.g., abnormal thinking, hallucinations, respiratory depression, weakness, blurred vision, etc. (https://www.drugs.com/), and are not curative. Thus, novel drug targets for the treatment of PD are highly in demand.

1.2 COX, PGE2, and EP receptors

Cyclooxygenase (COX) is an enzyme responsible for the rate-determining step in the syntheses of prostanoids, including prostaglandin D2 (PGD2), PGE2, PGF2α, prostacyclin PGI2 and thromboxane TXA2, and has two isoforms – COX-1 and COX-2 (Hirata et al., 2011; Jiang et al., 2013a). COX-1 is constitutively expressed in a wide range of tissues to maintain homeostatic prostanoid levels that are essential for many biological functions such as angiogenesis, vasodilatation, platelet function, tissue maintenance, etc. COX-2 is normally expressed at low levels in tissues, but under conditions like infection, injury, and pain, it can be rapidly induced to initiate pro-inflammatory processes that facilitate and maintain disease states (Dubois et al., 1998;

Chen, 2010). PGE2 is a major COX-2 product within the brain and has four G protein-coupled receptors (GPCRs): EP1, EP2, EP3, and EP4 (Figure 1). Activation of these GPCRs has been proposed to promote neuronal inflammation and degeneration in many neurological diseases such as ischemic stroke (Kawano et al., 2006; Andreasson, 2010a; Ikeda-Matsuo et al., 2010), epilepsy (Varvel et al., 2015; Dey et al., 2016), neurodegenerative diseases (Liang et al., 2008;

Andreasson, 2010b; Johansson et al., 2015), brain tumors (Qiu et al., 2016, Jiang et al., 2017), inflammatory pain (Kawabata, 2011), etc. However, which downstream EP receptors are directly responsible for COX-2/PGE2-mediated brain inflammation and injury remains unclear and is

3 presumably dependent on the type of brain insults and the responding tissues and cells

(Andreasson, 2010b).

1.3 Parkinson’s disease cell models

Neuroblastoma cell lines, mouse-derived Neuro-2a and human-originated SH-SY5Y, have been used extensively in experiments to detect neurotoxic properties and effects of compounds because they preserve many features of SNpc neurons (Alberio et al., 2012; Mukai et al., 2012). Furthermore, their sensitivity to drugs and their proliferative ability make them ideal cell lines to study signaling pathways involved in inflammation, oxidative stress, and apoptosis in dopaminergic neurons.

In this study, we investigated COX-2-associated inflammatory processes in Neuro-2a and

SH-SY5Y cells after 6-OHDA insult. We also investigated the involvement of PGE2 and the corresponding EP receptors in 6-OHDA-induced neurotoxicity by using our recently developed novel selective small-molecule antagonists.

1.4 Parkinson’s disease animal models

Animal models for PD are needed in research in order to study the mechanisms of signaling pathways and the efficacy of compounds, which is important for both development of new drugs and testing of compounds. (Ronald Deumensa et al, 2002). The most commonly used animal models for PD are the 6-OHDA rat model and the MPTP mouse model. Recent studies in animal models suggest that inflammatory PGE2 signaling is involved in the pathogenesis of PD

(Carrasco et al., 2007; Jin et al., 2007; Johansson et al., 2013; Pradhan et al., 2016).

4

1.5 6-OHDA induces Parkinson’s disease as a neurotoxin

2,4,5-trihydroxyphenethylamine – also known as 6-hydroxydopamine (6-OHDA) – is a neurotoxic synthetic organic compound that is widely used to induce PD symptoms in animal models because it can selectively destroy dopaminergic neurons (Miller et al., 2009; Duty et al.,

2011). As an analog of dopamine, 6-OHDA enters the neurons via dopamine specific reuptake transporters where it generates ROS, leading to mitochondrial dysfunction and neuronal apoptosis (Tieu, 2011). However, the molecular mechanisms as to how 6-OHDA induce production of ROS and oxidative stress are unclear.

5

CHAPTER 2: AIMS OF THE STUDY

6

The main goal of this study is to understand the role of PGE2/EP2 signaling in the development of PD and to gain new insight into the prevention and treatment of Parkinson’s disease. Based on previous studies and our preliminary results, we hypothesized that inflammatory PGE2 signaling via EP2 receptor is involved in 6-OHDA-induced neuroinflammation and neuronal degeneration. The aims of this study are described below:

Aim 1: To examine the PGE2/EP2 signaling pathway in neuronal cell lines treated with 6-OHDA in vitro

Aim 2: To determine the effect of EP2 antagonists in 6-OHDA-induced toxicity in neuronal cells

Aim 3: To investigate the inflammatory responses in rat model of 6-OHDA-induced nigrostriatal injury

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CHAPTER 3: MATERIAL AND METHOD

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3.1 Cell culture

The mouse neuroblastoma cell line – Neuro-2a and the human neuroblastoma cell line –

SH-SY5Y were purchased from the American Type Culture Collection (ATCC®: #CCL-131™ and #CRL-2266™, respectively) (Manassas, VA, USA). The Neuro-2a cells were cultured in

Dulbecco's Modified Eagle Medium (DMEM) supplemental with 10% fetal bovine serum (FBS),

1% MEM non-essential amino acids solution, 100 U·mL-1 penicillin and 100 µg·mL-1 streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere consisting of

5% CO2/95% air. The SH-SY5Y cells were maintained in 50% DMEM + 50% Ham's F-12 medium supplemental with 10% FBS, 100 U·mL-1 penicillin and 100 µg·mL-1 streptomycin.

3.2 Chemicals and reagents

6-hydroxydopamine (6-OHDA), forskolin and rolipram were purchased from Sigma-

Aldrich (St. Louis, MO, USA). PGE2, butaprost, CAY10598 and GW627368X were purchased from Cayman Chemical (Ann Arbor, MI, USA). Compounds TG4-155 and TG6-10-1 were obtained from MedChem Express (Monmouth Junction, NJ, USA. Jiang et al., 2012).

3.3 Immunocytochemistry

Cells were cultured onto Poly-D-Lysine coated microscope cover glass in 24-well plates.

After each treatment, cells were fixed with 4% paraformaldehyde (PFA) in PBS, followed by permeation with 0.2% Triton X-100 in PBS. After incubation in blocking solution – 10% horse serum (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 2 h, the cells were incubated with primary antibodies: NeuN (EMD Millipore, #MAB377), tyrosine hydroxylase (TH) (Cell

Signaling Technology, #2792), or COX-2 (abcam, #ab15191) overnight. Cells were then stained

9 with Alexa Fluor® 488 or 568-conjugated secondary antibodies (Invitrogen) for 2 h and DAPI

(10 µg·mL-1 in PBS) for 10 min. Slides were mounted using DPX mountant (Electron

Microscopy Sciences, Hatfield, PA, USA). Images were obtained using EVOS FL Auto Cell

Imaging System (Invitrogen). The fluorescence intensity was quantified using ImageJ software developed at the National Institutes of Health (NIH).

3.4 Cell viability assay

Cells were cultured in 96-well plates with 100 µL medium. Cell viability was measured using the Vybrant MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After treatment, MTT was added into each well at a working concentration of 0.5 mg·mL-1. Incubate the cells at 37°C for 4 h. Living cells can convert MTT into formazan, which is insoluble but can be dissolved in

DMSO. Absorbance of the formazan was measured by a microplate reader (Molecular Devices,

Sunnyvale, CA, USA) at 540 nm with a reference wavelength at 630 nm. The dose-response curves were generated using OriginPro software (OriginLab, Northampton, MA).

3.5 Quantitative PCR

Quantitative PCR (qPCR) was used to quantify mRNA levels of interested genes. Total

RNA was isolated using Trizol (Invitrogen) with the PureLink RNA mini kit (Qiagen, Hilden,

Germany) from cultured cells and brain tissues. RNA concentration and purity were measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific). First-strand complementary DNA

(cDNA) synthesis was performed with 1.5 µg of total RNA, using SuperScript III One-Step RT-

PCR System (Invitrogen) according to manufacturer’s guidelines. 1.5 µL of cDNA was used to

10 set up each 20µL qPCR reaction mixture following SYBR Green PCR Master Mix manual

(Thermo Fisher Scientific). Real-time PCR was performed using the StepOnePlus Real-Time

PCR System (Applied Biosystems, Foster City, CA, USA). Cycling conditions were as follows:

95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and then 60 °C for 1 min. Melting curve analysis was used to verify single-species PCR product. Fluorescent data were acquired at the

60 °C step. The cycle thresholds for GAPDH was used as an internal control for relative quantification. Samples without cDNA template served as the negative controls. Primers used for qPCR were listed in Table 1. The mRNA level of an interested gene was normalized to the control in each experiment to limit unwanted sources of variation.

3.6 PGE2 measurement

ELISA kit from Arbor Assays is used to measure the PGE2 levels in culture medium

(Ann Arbor, MI, USA). Cells were cultured in 24-well plates. After each treatment, 50μL medium was taken for PGE2 measurement according to the manufacturer’s protocol. The optical density generated from each well was measured in a microplate reader (Molecular Devices) at

450 nm. Run standard curves for PGE2.

3.7 Cell-based TR-FRET cAMP assay

Cytosol cAMP was measured with a cell-based homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) method (Cisbio Bioassays, Codolet, France). The assay is based on generation of a strong FRET signal upon the interaction of two molecules: an anti- cAMP antibody coupled to a FRET donor (cryptate) and cAMP coupled to a FRET acceptor (d2).

Endogenous cAMP produced by cells competes with labeled cAMP for binding to the cAMP antibody and thus reduces the FRET signal. Cells were seeded into 384-well plates in 30 µL

11 complete medium (4,000 cells per well) and were grown overnight. The medium was carefully withdrawn and 10 µL Hank’s Buffered Salt Solution (HBSS) (Hyclone, Logan, Utah, USA) plus

20 µM rolipram was added into the wells to block phosphodiesterase. The cells were incubated at room temperature for 0.5 h, and then treated with vehicle or tested compound for 5-10 min before addition of EP agonists. The cells were further incubated at room temperature for 40 min, then lysed in 10 µL lysis buffer containing the FRET acceptor cAMP-d2 and 1 min later another

10 µL lysis buffer with anti-cAMP-cryptate was added. After 60-90 min incubation at room temperature, the FRET signal was measured by an Envision 2103 Multilabel Plate Reader

(PerkinElmer, Waltham, MA, USA) with excitation at 340 nm and dual emissions at 665 nm and

590 nm for d2 and cryptate (100 µs delay), respectively. The FRET signal was expressed as:

F665/F590 × 104 and normalized to the controls to indicate the cAMP levels.

3.8 6-OHDA rat model surgery

Adult male Sprague-Dawley rats weighing 225-250 g were used for this model. Animals were divided into 5 groups (3 in each group) and were anesthetized with Ketamine

[87mg/kg]/Xylazine [13mg/kg] (IsoThesia; Butler Animal Health Supply, Dublin, OH). Then the animals were placed in a stereotaxic apparatus (StereoDrive, Stoelting Co., Wood Dale, IL), and were given unilateral injections of 6-OHDA (Sigma, St. Louis, MO) using a 5 μl Hamilton

Gastight syringe (Reno, NV). For the unilateral partial lesion, a volume of 2 μl (2.5 μg/μl 6-

OHDA in 0.9% NaCl, 0.2% ascorbic acid saline solution) was injected over 10 min (from bregma: Site 1. AP: + 1.6 mm, ML: -2.4 mm, DV: -4.2 mm, Site 2. AP: + 0.2 mm, ML: -2.6 mm,

DV: -7.0 mm), with the needle left in place for 5 min before and after injection. Following the surgery, animals were given 0.1 ml buprenorphine hydrochloride to minimize pain. Animals

12 were put back into the cages until fully recovered. After 4 h, 1 day, 2 day, and 7 days, animals were perfused with ice cold PBS and the following tissues were collected: hippocampus, nigra, striatum, and cortex (ipsilateral and contralateral). qPCR was used to detect mRNA expression of

COX-2, IL-1β, TNF-α, GFAP, and Iba-1.

3.9 Statistical analysis

Statistical analyses were performed using Prism (GraphPad Software, La Jolla, CA, USA) by one-way ANOVA with post-hoc Bonferroni or Dunnett’s test. P < 0.05 was considered to be statistically significant. All data are presented as mean + or ± SEM.

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CHAPTER 4: RESULTS

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4.1 Neuro-2a and SH-SY5Y cells are TH positive and susceptible to 6-OHDA-mediated cytotoxicity

Neuro-2a is a mouse neuroblastoma cell line derived from the neural crest with many features of neurons, including neurofilaments (Olmsted et al., 1970). SH-SY5Y is a human cell line that was initially isolated from a bone marrow biopsy removed from a four-year-old girl with neuroblastoma (Biedler et al., 1973). Owing to their neuronal background and properties, these two cell lines are widely used as in vitro models to study neuronal function and differentiation, axonal growth, neuronal signaling, neurotoxicity, and neurodegeneration, particularly for investigating Parkinson’s disease (PD) (Tiong et al., 2010; Tremblay et al., 2010).

Both cell lines were purchased directly from the American Type Culture Collection (ATCC), and we first examined their neuronal background by immunochemistry. As shown in Figure 2, both cultured Neuro-2a and SH-SY5Y cells express NeuN (a canonical neuronal biomarker) and tyrosine hydroxylase (TH; the enzyme responsible for the first step in dopamine synthesis in

SNpc neurons). In fact, the vast majority of cells – 82.1% Neuro-2a and 73.1% SH-SY5Y– are both NeuN and TH positive.

As a synthetic oxidopamine, 6-OHDA is a widely-used neurotoxin to induce degeneration of dopaminergic neurons and to destroy the nigrostriatal pathway in rodent PD models (Tieu, 2011). Similar to previous findings (Tiong et al., 2010; Mukai et al., 2012), 6-

OHDA decreased viability of both Neuro-2a and SH-SY5Y cells in a concentration-dependent manner with similar EC50s: ~ 100 µM, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reduction assays (Figure 3).

4.2 6-OHDA induces COX-2 expression and nuclear translocation

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The mechanisms underlying 6-OHDA-mediated destruction of neurons are unclear, but it has been proposed that the neurotoxin enters dopaminergic cells via dopamine transporters to generate ROS such as superoxide (Lehmensiek et al., 2006; Tieu, 2011; Kwon et al., 2014).

COX-2, a primary pro-inflammatory mediator in both the periphery and brain, is known to generate oxidative stress in many inflammation-associated conditions. We found that at both low concentration (75 µM, ~ EC25) and high concentration (150 µM, ~ EC75), COX-2 was quickly and robustly induced by 6-OHDA treatment in Neuro-2a and SH-SY5Y cells, as measured by mRNA levels using qPCR (Figure 4). COX-2 is mainly present in the cytosol at basal levels

(Figure 5), where homeostatic prostanoids are synthesized to maintain normal physiological functions. However, nuclear translocation of COX-2 was observed using fluorescence microscopy after 6-OHDA stimulation in the majority of Neuro-2a and SH-SY5Y cells (Figure

5). These results all demonstrate that 6-OHDA can induced COX-2 activation in these cells lines.

4.3 6-OHDA leads to increased production of PGE2 and IL-1β In order to further test the effects of COX-2 in 6-OHDA-mediated neurotoxicity, we quantified the prostaglandins secreted to the culture medium by these cells using an ELISA kit, and found that 6-OHDA stimulation significantly increased PGE2 in the culture medium nearly

5-fold in Neuro-2a cells (P < 0.01; 75 µM 6-OHDA for 24 h) and 3-fold in SH-SY5Y cells (P <

0.01; 150 µM 6-OHDA for 24 h) (Figure 6A). By measuring mRNA levels by qPCR, we found that 6-OHDA treatment also significantly increased the pro-inflammatory cytokine interleukin-

1β (IL-1β) in these two cell lines (Figure 6B). These data suggest that 6-OHDA can promote pro- inflammatory processes in neuronal cells.

4.4 PGE2/EP2/cAMP signaling in Neuro-2a and SH-SY5Y cells

16

PGE2 has four receptors EP1, EP2, EP3, and EP4. EP2 and EP4 are Gαs-coupled receptors and mediate cAMP-dependent pathways that are involved in PGE2-mediated chronic inflammation and neurodegeneration (Andreasson, 2010b; Hirata et al., 2011; Jiang et al.,

2013a). In order to study PGE2/cAMP signaling in Neuro-2a cells, increasing concentrations of

PGE2 was used to treat cells and cytosolic cAMP levels were measured using time-resolved fluorescence energy transfer (TR-FRET) assay. As shown in the Figure 7, PGE2 (1 or 10 µM) induced cAMP accumulation in Neuro-2a cells in a concentration-dependent manner. Butaprost

(10 µM; selective EP2 agonist) and forskolin (100 µM; a direct activator of the adenylyl cyclase) induced maximal cAMP levels in these cells. By contrast, the EP4 selective agonist CAY10598 caused a moderate increase in cAMP levels in these cells at 10 µM, a concentration that should induce maximal effect on the EP4 receptor (Figure 7). Similarly, PGE2 (0.1, 1 or 10 µM) and butaprost (0.1, 1, or 10 µM), but not CAY10598 (up to 10 µM), recapitulated forskolin-induced

(100 µM) cAMP production in SH-SY5Y cells in a concentration-dependent manner (Figure 8A).

In order to study the role of the EP2 receptor in chronic inflammation and injury, we used a series of novel small molecules that are among the first-generation selective EP2 antagonists

(Jiang et al., 2010a; af Forselles et al., 2011; Ganesh et al., 2013), TG6-10-1 and compound

TG4-155. Interestingly, PGE2-induced (10 µM) cAMP accumulation in SH-SY5Y cells was completely blocked by EP2 antagonists TG4-155 and TG6-10-1 in a concentration-dependent manner, but not by the EP4 antagonist GW627368X. The TR-FRET cAMP assay results suggest that EP2 is the dominant Gαs-coupled PGE2 receptor in both Neuro-2a and SH-SY5Y cells.

4.5 EP2 receptor inhibition is neuroprotective in 6-OHDA-induced cell injury

Next, we studied whether PGE2/EP2 signaling is involved in 6-OHDA-induced neuronal injury. As shown in Figure 9A, both EP2 antagonists TG4-155 and TG6-10-1 (10 or 20 µM)

17 significantly reduced 6-OHDA-induced (75 µM) cytotoxicity in Neuro-2a cells in a concentration-dependent manner, as measured by MTT reduction assay 24 h after 6-OHDA incubation. This observation was recapitulated by incubation with the selective COX-2 inhibitor celecoxib (10 or 20 µM), but not by the EP4 selective antagonist GW627368X (20 µM).

Furthermore, 6-OHDA-induced (150 µM) cytotoxicity in SH-SY5Y cells was also blocked by pretreatment with EP2 antagonists TG4-155 and TG6-10-1 (20 µM) (Figure 9B). Together, these data suggest that 6-OHDA-induced cytotoxicity in Neuro-2a and SH-SY5Y cells can be attributed in part to PGE2 signaling via the EP2 receptor.

4.6 6-OHDA causes inflammatory induction in a rat model of 6-OHDA-induced neuronal injury

The neurotoxin 6-OHDA is widely used to induce depletion of dopaminergic neurons in animal models of PD. In the 6-OHDA rat model, we studied the expression of COX-2, IL-1β,

TNF-α, GFAP, and Iba-1 after unilateral lesion. COX-2 mRNA levels in the striatum were increased 4 h after administration of 6-OHDA. After one day, the levels of COX-2 returned to normal, whereas there was no observable change in COX-2 mRNA levels in the other three tissues (Figure 10). IL-1B mRNA levels in the striatum were increased at 4 h, 1 day, 2 day, 4 day, and 7 day after administration of 6-OHDA (Figure 10). TNF-α mRNA levels in the striatum and the cortex were increased at 4 h and 1 day post-administration of 6-OHDA, whereas there was no observable change at the other three time points (Figure 10). GFAP and Iba-1 mRNA levels in the striatum were increased at 1 day, 2 day, 4 day, and 7 day after 6-OHDA microinjection, whereas the 4 h time point showed no observable changes. In addition, there were no observable changes in the levels of GFAP and Iba-1 mRNA at the other three sites (Figure 10). These data

18 suggest that 6-OHDA causes overall induction of pro-inflammatory genes within the rat brain following 6-OHDA treatment.

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Figure 1. COX-2/PGE2/EP2 signaling pathway after a variety of stimuli.

20

Figure 2. Mouse Neuro-2a and human SH-SY5Y cells are NeuN positive and express tyrosine hydroxylase (TH). Immunostaining was performed to illustrate the expression of NeuN (red) and TH (green) in Neuro-2a cells (A) and SH-SY5Y cells (B). The cell nuclei were visualized by DAPI staining (blue) and cells were counted in 5 random fields at a magnification of 200×. Data were reported as the percentage of cells/field. Note that the majority of cells are both NeuN and TH positive. Scale bar = 100 µm.

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Figure 3. 6-hydroxydopamine (6-OHDA) induces neurotoxicity. 6-OHDA caused cytotoxicity in both Neuro-2a cells (A) and SH-SY5Y cells (B) in a concentration dependent manner, shown by the dose-response curves. Cells were treated with 6-OHDA at different concentrations for 24 or

48 h, and the cell viability was measured by MTT assay. 6-OHDA EC50 = 111 µM for 24 h incubation and EC50 = 109 µM for 48 h incubation in the Neuro-2a cells; 6-OHDA EC50 = 118 µM for 24 h incubation in the SH-SY5Y cells. Data are shown as mean ± SEM (n = 4-6).

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Figure 4. 6-OHDA induces cyclooxygenase-2 (COX-2) expression. Neuro-2a cells (A) and SH- SY5Y cells (B) were treated with 6-OHDA at different concentrations (75 or 150 µM) for 0, 6 or 24 h. COX-2 mRNA levels in these cells were measured by qPCR (*P < 0.05; **P < 0.01 compared to control, one-way ANOVA with Dunnett’s Multiple Comparison Test). Data are presented as mean + SEM (n = 3-5).

23

Figure 5. 6-OHDA stimulation causes COX-2 translocation to the nuclei. Neuro-2a cells (A) and SH-SY5Y cells (B) were treated with 6-OHDA (75 or 150 µM) for 24 h. Immunostaining was performed to show the expression of COX-2 protein (green) in these cells and the cell nuclei were visualized by DAPI staining (blue). The subcellular distribution of COX-2 protein was quantified by measuring the fluorescence intensities along a line across the cell body using NIH ImageJ software. Scale bar = 20 µm.

24

Figure 6. Effect of 6-OHDA on the syntheses of prostaglandin E2 (PGE2) and interleukine-1β (IL-1β). Neuro-2a cells (left) and SH-SY5Y cells (right) were treated with 6-OHDA at different concentrations (75 or 150 µM) for 0, 6 or 24 h. (A) PGE2 levels in the culture medium of these cells were measured by ELISA (*P < 0.05; **P < 0.01 compared to control, one-way ANOVA with Dunnett’s Multiple Comparison Test). (B) IL-1β mRNA levels in these cells were measured by qPCR (*P < 0.05; ***P < 0.001 compared to control, one-way ANOVA with Dunnett’s Multiple Comparison Test). Data are presented as mean + SEM (n = 3-4).

25

Figure 7. PGE2-mediated cAMP signaling in mouse Neuro-2a cells. Neuro-2a cells were treated with PGE2 (1 or 10 µM), EP2 selective agonist butaprost (10 µM), EP4 selective agonist CAY10598 (10 µM) or direct activator for adenylyl cyclase forskolin (100 µM) for 40 min. The cAMP concentrations in the cells were measured by time-resolved fluorescence energy transfer (TR-FRET) assay (**P < 0.01 compared to control, one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test). Data are shown as mean + SEM (n = 4).

26

Figure 8. PGE2-mediated cAMP signaling in human SH-SY5Y cells. (A) SH-SY5Y cells were treated with PGE2 (0.1, 1 or 10 µM), butaprost (0.1, 1 or 10 µM), CAY10598 (0.1, 1 or 10 µM) or forskolin (100 µM) for 40 min. The cAMP concentrations in the cells were measured by TR- FRET assay (*P < 0.05; **P < 0.01 compared to control, one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test). (B) SH-SY5Y cells were treated with EP2 antagonists TG4-155 (0.1, 1 or 10 µM), TG6-10-1 (0.1, 1 or 10 µM) or EP4 antagonist GW627368X (0.1, 1 or 10 µM), followed by incubation with PGE2 (10 µM) for 40 min. The cAMP concentrations in the cells were measured by TR-FRET assay (*P < 0.05; **P < 0.01 compared to control, one- way ANOVA with post-hoc Dunnett’s Multiple Comparison Test). Data are shown as mean + SEM (n = 6).

27

Figure 9. EP2 receptor inhibition reduces 6-OHDA-triggered neurotoxicity. (A) Neuro-2a cells were pretreated with TG4-155 (10 or 20 µM), TG6-10-1 (10 or 20 µM), COX-2 selective inhibitor celecoxib (10 or 20 µM) or GW627368X (20 µM), followed by treatment with 6- OHDA (75 µM) for 24 h. The cell viability was measured by MTT assay (*P < 0.05; ***P < 0.001, one-way ANOVA with post-hoc Bonferroni’s Multiple Comparison Test for selected pairs as indicated). (B) SH-SY5Y cells were pretreated with TG4-155 (20 µM) or TG6-10-1 (20 µM), followed by treatment with 6-OHDA (150 µM) for 24 h. The cell viability was measured by MTT assay (*P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA with post-hoc Bonferroni’s Multiple Comparison Test for selected pairs as indicated). Data are shown as mean + SEM (n = 4-6).

28

Figure 10. Induction of inflammatory genes COX-2, IL-1β, TNF-α, GFAP, and Iba-1 in the brain following 6-OHDA microinjection into the rat striatum. Male adult SD rats were injected with 6-OHDA at 2 sites in the striatum (AP: + 1.6 mm, ML: -2.4 mm, DV: -4.2 mm and AP: + 0.2 mm, ML: -2.6 mm, DV: -7.0 mm). The expression of inflammation-associated genes in the ipsilateral striatum, hippocampus, cortex and substantia nigra was measured by qPCR 4h, 1d, 2d, 4d or 7d after 6-OHDA injection (*P < 0.05, **P < 0.01 compared to the contralateral sites, one- way ANOVA with post-hoc Dunnett’s Multiple Comparison Test). Data are presented as mean + SEM (n = 3).

29

Gene Forward primer Reverse primer

Mouse

GAPDH 5'-TGTCCGTCGTGGATCTGAC-3' 5'-CCTGCTTCACCACCTTCTTG-3'

COX-2 5’-CTCCACCGCCACCACTAC-3’ 5’-TTGATTGGAACAGCAAGGAT-3’

IL-1b 5'-TGAGCACCTTCTTTTCCTTCA-3’ 5'-TTGTCTAATGGGAACGTCACAC-3’

Human

GAPDH 5'-GTCAAGGCTGAGAACGGGAA-3' 5'-AAATGAGCCCCAGCCTTCTC-3'

COX-2 5’-GGTCTGGTGCCTGGTCTGAT-3’ 5’-TCCTGTTTAAGCACATCGCATACT-3’

IL-1β 5'-TACCTGTCCTGCGTGTTGAA-3’ 5'-TCTTTGGGTAATT TTTGGGATCT-3’

Rat

COX-2 5’-ACCAACGCTGCCACAACT-3’ 5’-GGTTGGAACAGCAAGGATTT-3’

TNF-α 5’-CGTAGCCCACGTCCGTAGC-3’ 5’-GGTTGTCTTTGAGATCCATGC-3’

IL-1β 5’-CAGGAAGGCAGTGTCACTCA-3’ 5’-AACCGCATCACCATTCCTG-3’

GFAP 5’-CATCTCCACCGTCTTTACCAC-3’ 5’-TCCCACGAGTCACAGAGGA-3’

Iba-1 5’-TCGATATCTCCATTGCCATTCAG-3’ 5’-GATGGGATCAAACAAGCACTTC-3’

Table 1. Sequences of the primers for qPCR.

30

CHAPTER 5: DISCUSSION

31

5.1 6-OHDA provokes inflammatory processes

In this study, we found that 6-OHDA stimulation at both low and high concentrations caused rapid COX-2 activation and nuclear translocation in both mouse Neuro-2a and human

SH-SY5Y cells, leading to pro-inflammatory processes characterized by PGE2 synthesis and cytokine (IL-1β) induction.

6-OHDA is widely used in animal PD models to induce dopaminergic neuronal loss in the SNpc, presumably through generation of ROS that leads to oxidative stress and mitochondrial impairment (Duty et al., 2011; Tieu, 2011). ROS production could result from autoxidation of 6-OHDA and possibly via direct inhibition of the mitochondrial respiratory chain by the neurotoxin (Rodriguez-Pallares et al., 2007). In our study, we found that COX-2 is induced by 6-OHDA and causes the syntheses of PGE2 and IL-1β production in both Neuro-2a

(mouse) and SH-SY5Y (human) neuronal cell lines that express TH, suggesting that 6-OHDA induces inflammatory processes in these cells – at least partially – through COX-2 and the prostaglandin cascade.

5.2 The EP2 receptor is the dominant EP receptor in COX-2-initiated cAMP-dependent signaling pathway

PGE2 has four G-protein-coupled receptors: EP1, EP2, EP3, and EP4. EP2 and EP4 receptors are Gs-coupled GPCRs and can increase cAMP levels, whereas EP3 receptor is a Gi- coupled GPCR and can decrease cAMP levels. In this study, we found that PGE2 initiates cAMP-dependent pathways in mouse Neuro-2a and human SH-SY5Y cells mainly through the

EP2 receptor. 6-OHDA-induced cytotoxicity in these NeuN- and TH-positive cells is largely blocked by pharmacological inhibition of the EP2 receptor, but not the EP4 receptor. These

32 findings give us insight into the COX-2 downstream signaling pathway – COX-2/PGE2/EP2 pathway, which is involved in neurotoxin-mediated neuronal inflammation and injury.

5.3 Roles of COX-2 in neurodegenerative process

There is much evidence to show that COX-2 is increased in many neurological disorders including stroke (Andreasson, 2010a), epilepsy (Jiang et al., 2013c; Du et al., 2016), and neurodegenerative diseases (Trepanier et al., 2010; Teismann, 2012). In addition, COX-2 has been found to contribute to neuronal injury through initiation of pro-inflammatory processes and by imposing oxidative stress on the neurons (Hsieh et al., 2011; Chen et al., 2016). COX-2 is selectively induced in SNpc dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-administered mice (Teismann et al., 2003), and in 6-OHDA-treated rats (Lee et al., 2012); the pharmacological inhibition of COX-2 is protective for the TH neurons and can relieve the oxidative stress in these animals (Teismann et al., 2003; Sanchez-Pernaute et al., 2004), suggesting that COX-2 is essential in the neurodegenerative process of PD. However, because there is growing recognition of adverse effects of COX-2 inhibitor drugs on the cardiovascular and cerebrovascular systems, the use of COX-2 as a therapeutic target has decreased over the past decade (Grosser et al., 2010), which has given us insight into the downstream prostaglandin signaling pathways that might provide alternative targets for therapeutics with more specificity.

When there is external stimuli such as injury, infection, and inflammation, COX-2 is rapidly induced and converts arachidonic acids, which are released by the cell membrane, to

PGH2 in the cytoplasm where the unstable intermediate prostaglandins are further catalyzed to prostanoids by tissue-specific isomerases (Hirata et al., 2011). Unexpectedly, we observed

33 translocation of COX-2 from the cytoplasm to nucleus in 6-OHDA-treated Neuro-2a and SH-

SY5Y cells. The translocation of COX-2 between the nucleus and the cytosol has been reported in IL-1β-treated vascular endothelial cells (Parfenova et al., 2001), retinal Müller cells after hypoxia (Barnett et al., 2005), human breast carcinoma (Maroni et al., 2011), and bladder cancer cells (Thanan et al., 2012), but not in brain neurons prior to this study. COX-2 activity has been linked to several nuclear receptors and transcription factors such as peroxisome proliferator- activated receptors (PPARs), octamer-binding transcription factor 4 (Oct-4), and nuclear factor-

κB (NF-κB) (Carta et al., 2011; Du et al., 2011; Thanan et al., 2012; Lecca et al., 2015). Further study is needed to investigate if nuclear translocation of COX-2 in these two neuroblastoma cell lines indicates a novel function of COX-2 in the transcriptional regulation of genes that are associated with 6-OHDA-promoted oxidative stress and apoptosis.

5.4 Gαs coupled EP2 and EP4 receptors

EP2 and EP4 are PGE2 receptor subtypes that are coupled to Gαs and mediate cAMP- dependent signaling pathways in both brain neurons and (Andreasson, 2010b; Jiang et al.,

2013a). PGE2 signaling via the EP2 receptor leads to α-synuclein aggregate-mediated neurotoxicity in a mouse MPTP model of PD (Jin et al., 2007). Moreover, EP2 receptor activation facilitates microglial and astrocytic inflammatory responses to MPTP and causes loss of dopaminergic neurons in the SNpc (Johansson et al., 2013), suggesting that the EP2 receptor plays an important role in the pathogenesis of PD. Conversely, pharmacological activation of the

EP4 receptor has been found to prevent MPTP-induced SNpc neuronal loss, whereas genetic ablation of the receptor aggravates MPTP-associated pro-inflammatory processes (Pradhan et al.,

2016). Together, these previous findings suggest that EP2 and EP4 might have opposing

34 functions during neurodegenerative progression in PD. Consistently, we found that inhibition of the EP2 receptor – but not EP4 – significantly reduced neuronal death triggered by 6-OHDA.

Furthermore, neuroprotection of EP2 antagonists was observed at a similar level as the COX-2 selective inhibitor celecoxib, indicating that the COX-2-mediated neurotoxicity in these cells could mostly be attributed to EP2 receptor activation by PGE2. Nonetheless, whether our selective EP2 antagonists can provide sufficient neuroprotection for SNpc neurons following 6-

OHDA treatment in vivo is an important topic to explore in the future.

5.5 Conclusion

In summary, our results show that the PGE2 receptor EP2 dominantly mediates COX-2- initiated cAMP-dependent signaling in Neuro-2a and SH-SY5Y cells following 6-OHDA treatment, and largely contributes to 6-OHDA-induced neurotoxicity. Furthermore, microinjection of 6-OHDA into the striatum causes COX-2 induction followed by the upregulation of many inflammation and gliosis-associated genes incuding IL-1β, TNF-α, Iba-1 and GFAP in Sprague Dawley rats.. Whether selective inhibition on EP2 receptor can provide sufficient neuroprotection for SNpc neurons following 6-OHDA treatment in vivo is an important topic for the future study.

35

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