Role of phospholipase a2 in orofacial pain and synaptic transmission 1

Although emerging evidences have shown the roles of PLA2 isoforms in nociception, direct evidences which indicate altered brain PLA2 activity and expression during allodynia or hyperalge

SYNAPTIC TRANSMISSION

MA MAY THU

(B.Sc (Hons), NUS)

SUPERVISOR: ASSOCIATE PROFESSOR YEO JIN FEI

A THESIS SUBMITTED FOR THE DEGREE OF

ACKNOWLEDGEMENTS

With thanks to my supervisor, Associate Professor Yeo Jin Fei, Head,

Department of Oral and Maxillofacial, National University of Singapore, who

extended utmost support to my entire project; to my co-supervisor, Associate

Professor Ong Wei Yi, Department of Anatomy, National University of

Singapore, who proposed the topic of my study, provided relentless guidance throughout my entire candidature and most importantly, influenced me greatly with his knowledge and passion for research

Numerous people contributed to the realization of this project: Tang Ning, for her indefatigable teachings and guidance in my study; Pan Ning, for her brilliant technical support; Jinatta Jittiwat, Nuntiya Sompran, Chan Yee Gek and Wu Ya Jun, for their assistance in Electron Microscopy; Chew Wee Siong,

Chia Wan Jie, Ee Sze Min, Guo Jing, Ho Mei Xuan, Kazuhiro Tanaka, Kim Ji Hyun, Lynette Lee Hui Wen, Lee Li Yen, Amy Lim Seok Wei, Loke Sau Yeen, Mary Ng Pei Ern, Poh Kay Wee, Tan Yan, Wong Li Ming, Wong Sin Kei, Yang Hui and Alicia Yap Mei Yi, for their selfless support of my interest in this

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ……… …… ….………II TABLE OF CONTENTS……… ……… ……… III SUMMARY……….……… ……VIII LIST OF TABLES……… XI LIST OF FIGURES……… ……… XII ABBREVIATIONS……….……… ……… ….XIV PUBLICATIONS……….……… ……….….… … XVIII

SECTION I INTRODUCTION……… … 1

1 Phospholipase A2……… …….… 2

1.1 Cytosolic phospholipase A2 (cPLA2)……… ………… … ……… 5

1.2 Ca2+-independent phospholipase A2 (iPLA2)………… …… ….….7

1.3 Secretory phospholipase A2 (sPLA2)……… ……… 8

1.3.1 sPLA2 isozymes………9

1.3.1.1 sPLA2-IB……… … 9

1.3.1.2 sPLA2-IIA……… ……… 10

1.3.1.3 sPLA2-IIC……… ……… ……… ………… …11

1.3.1.4 sPLA2-IID……… ……… … … 12

1.3.1.5 sPLA2-IIE……… ……….…….………… … ………12

1.3.1.6 sPLA2-IIF……….… …… 13

1.3.1.7 sPLA2-III………….……….….……….… 13

1.3.1.8 sPLA2-V……….… …….………14

1.3.1.9 sPLA2-X……… ……….…………15

1.3.2 sPLA2-XIIA……… ………16

1.4 Arachidonic acid……… ………18

1.5 Phospholipids and lysophospholipids……… …… ………20

1.6 Exocytosis……… ……… ……….23

1.6.1 PLA2 and neurotransmission……….……… … … 25

1.6.2 Phospholipids and neurotransmission……… … 27

1.6.3 Lysophospholipids and neurotransmission……… 28

1.6.4 Factors affecting exocytosis-lipid rafts and Ca2+….… … 30

2 Pain……… ………… …… 32

2.1 Orofacial pain……… ….….33

2.2 Nociception and nociceptors……….……… …… … ….33

2.3 Pain models……….……… …34

2.4 Pain pathways……… …….36

3 PLA2 and inflammatory pain……….……… ……39

3.1 PLA2 and receptors……….……… 45

SECTION II Experimental studies……….…… 48

CHAPTER 1 Changes in brain lipids contents after carrageenan-induced orofacial pain……….……… 49

1.1 Introduction……… ………50

1.2 Materials and methods……….……….52

1.2.1 Time course study of pain responses after facial CA injection……… 52

1.2.2 Assessment of responses to mechanical stimulation………53

1.2.3 Lipidomics analyses………54

1.2.3.1 Internal standard……… 55

1.2.3.2 Lipid extraction……….55

1.2.3.3 Analysis of lipids using liquid chromatography/mass spectrometry………56

1.3 Results……… ……… ……57

1.3.1 Time course study of pain responses after facial CA injection………57

1.3.2 Lipidomics analyses……… 58

1.4 Discussion……… ……….………64

CHAPTER 2 Differential expression pattern of PLA2 isoforms in CNS after orofacial pain ………….……… ………… 67

2.1 Introduction……… ……68

2.2 Materials and methods……… 70

2.2.1 Real-time RT-PCR……… 70

2.2.2 Western blot analysis……… 71

2.2.3 Immunohistochemistry……… ……… 72

2.3 Results……… ……… ……74

2.3.1 mRNA expression of PLA2 isoforms in the medulla oblongata……….74

2.3.2 sPLA2-III protein expression and localization in the CM… 76

2.4 Discussion……… ……….…78

CHAPTER 3 Role of group III sPLA2 in nociception and synaptic transmission in

the CNS………81

3.1 Introduction……… ……82

3.2 Materials and methods……… 85

3.2.1 Real-time RT-PCR……… 85

3.2.2 Western blot analysis……… 86

3.2.3 Immunohistochemistry……….……… 87

3.2.4 Electron microscopy………88

3.2.5 Capacitance measurement………88

3.2.6 Intracellular Ca2+ imaging……… ……….90

3.3 Results……… ……… …………92

3.3.1 Differential expression of sPLA2-III in rat CNS…….……… 92

3.3.2 Western blot analysis of sPLA2-III……….………93

3.3.3 Immunohistochemistry………95

3.3.4 Electron microscopy………97

3.3.5 Capacitance measurements……… …98

3.3.6 Intracellular Ca2+ imaging……….……… 99

3.4 Discussion……….……101

CHAPTER 4 Role of group IIA sPLA2 in nociception……… …….…105

4.1 Introduction………106

4.2 Materials and methods………109

4.2.1 Real-time RT-PCR………109

4.2.2 Western blot analysis………110

4.2.3 Immunohistochemistry……….111

4.2.4 Electron microscopy……….112

4.3 Results……… ………113

4.3.1 Real-time RT PCR………113

4.3.2 Western blot analysis………117

4.3.3 Immunohistochemistry……….118

4.3.4 Electron microscopy……….121

4.4 Discussion……….123

CHAPTER 5 Role of lysophospholipids in synaptic transmission…… ……….127

5.1 Introduction………128

5.2 Materials and methods………131

5.2.1 TIRFM……….131

5.2.2 Capacitance measurements………132

5.2.3 Amperometry measurements……… 133

5.2.4 Intracellular Ca2+ imaging……….………… 134

5.3 Results……… ………136

5.3.1 TIRFM……….136

5.3.2 Capacitance measurements………138

5.3.3 Amperometry measurements……… 141

5.3.4 Intracellular Ca2+ imaging……….………… 143

5.4 Discussion……….144

SECTION IV CONCLUSION……… 149

SECTION V REFERENCES……… 154

SUMMARY

Phospholipase A2 (PLA2, EC 3.1.1.4) are enzymes which hydrolyze the

acyl ester bond at the sn-2 position to generate free fatty acids such as

glycerophospholipids PLA2 isoforms include secretory phospholipase A2 (sPLA2), cytosolic phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2) Although emerging evidences have shown the roles of PLA2 isoforms in nociception, direct evidences which indicate altered brain PLA2 activity and expression during allodynia or hyperalgesia are lacking The function

of PLA2 during nociceptive transmission also needs to be explored

The present study elucidated changes in brain lipids in medulla oblongata after orofacial pain induced by facial carrageenan (CA) injection The caudal medulla oblongata (CM) showed decreases in phospholipids including phosphatidylethanolamine and phosphatidylinositol (PI) and increases in their corresponding lysophospholipids, lysophosphatidylethanolamine and lysophosphatidylinositol (lysoPI) These results indicated an enhanced PLA2 activity in the CM and release of AA after peripheral inflammation of the face This study further examined changes in expression level of PLA2 isoforms after nociception mRNA expression of sPLA2-III was highly expressed in the CM and this expression was significantly increased in the CA-injected rats However, no corresponding increase in sPLA2-III protein expression was detected These changes possibly take place in spinal trigeminal nucleus which communicates

nociceptive input from orofacial region, indicating the nociceptive function of sPLA2-III

The expression profile of sPLA2-III in CNS and its effects on exocytosis in rat PC-12 cells further illustrated the role of sPLA2-III in pain transmission Both sPLA2-III mRNA and protein expression were expressed at the highest levels in the brainstem and spinal segments The enzyme was localized to dendrites in spinal trigeminal nucleus, supporting its role in ascending pain pathway External application of sPLA2-III to PC-12 cells augmented capacitance measurement, indicating exocytosis and this was dependent on lipid rafts and external Ca2+ Moreover, sPLA2-III caused an increased in intracellular Ca2+ ([Ca2+]i), indicating that it could be a trigger for exocytosis Moreover, sPLA2-IIA with a strong secretory signal, showed high levels of mRNA and protein in the brainstem and spinal segments sPLA2-IIA was also localized to the dendrites in spinal trigeminal nucleus and dorsal horn of spinal cord The expressions of sPLA2-IIA were supported by previous studies which also illustrated a significant function of CNS sPLA2 in nociceptive transmission

PLA2 participates in the synaptic transmission through its secretion, i.e in sPLA2-III and -IIA, and also via its enzymatic product, lysophospholipids When the effects of lysophospholipids on exocytosis in PC-12 cells were elucidated, external infusion of lysoPI augmented vesicle fusion, indicating exocytosis Similarly, significant increase in capacitance measurement, or number of spikes detected at amperometry, indicating exocytosis was observed after external

application of lysoPI This process was affected by the lipid rafts and [Ca ]i LysoPI also caused an elevated [Ca2+]i, implying its effect on exocytosis

In conclusion, this study demonstrated significantly increased PLA2 activity and expression upon orofacial pain Moreover, due to their localization and roles

in synaptic transmission, both sPLA2-III and sPLA2-IIA are found to be important isozymes in the ascending pain pathway

LIST OF TABLES

SECTION I

Table.1.1 Summary of differential mRNA and protein expression of PLA2 isoforms in the CNS and peripheral organs based on various previous studies 17 SECTION II

Table.2.1.1 Changes in selected lipids in the RM and CM after facial CA

Table.2.4.1 Comparison of sPLA2 isoforms mRNA and protein expression in rat CNS between previous reports and current findings ……… … 116

LIST OF FIGURES

SECTION I

Figure.1.1 Site of action of phospholipase A1, A2, C and D on the phospholipid molecule……….3 Figure.1.4.1 Chemical structure of AA ……….19 Figure.1.5.1 Schematic diagrams of structures of lysophospholipids …….……22 Figure.1.6.1 Schematic diagram of neurotransmission ……….………24 Figure.2.3.1 Schematic diagram of structure of carrageenan…… ……… …35 Figure 2.4.1 Schematic diagram of pain pathway ……… 38 SECTION II

Figure.2.1.1 Responses to von Frey hair stimulation of the face after tissue inflammation induced by CA injection ………57

Figure.2.1.2 Lipidomic analysis of changes in selected lipids in right half of CM sacrificed at 3 days post-CA injection ……… ……… …………61

Figure.2.1.3 Lipidomic analysis of changes in selected lipids in right half of CM sacrificed at 3 days post-CA injection……… … ….63

Figure.2.2.1 Real-time RT PCR analysis of differentially expressed PLA2 subgroups in the RM and CM……… ……… …….75

Figure.2.2.2 Western blot analyses of sPLA2-III protein expression in different parts of the rat CM and light micrographs of sPLA2-III immunolabeled sections from normal and CA-injected rat CM.……… ……… … 77 Figure.2.3.1 Protein sequence of the human group III PLA2………….…………83 Figure.2.3.2 Real-time RT-PCR analysis of sPLA2-III in the various parts of CNS 92 Figure.2.3.3 Western blot analyses of sPLA2-III protein expression in different areas of the rat CNS ……….…………94

Figure.2.3.4 Light micrographs of sPLA2-III immunolabeled sections from a normal rat CNS ……… ………… …….……95

Figure.2.3.5 Light micrographs of sPLA2-III immunolabeled sections from a normal rat CNS ……… 96

Figure.2.3.6 Electron micrographs of sPLA2-III immunolabeled sections from the spinal cord of a normal rat ……….……… 97

Figure.2.3.7 Increase in membrane capacitance in a PC-12 cell indicating exocytosis, after addition of sPLA2-III ……… …… 99 Figure.2.3.8 Intracellular calcium imaging……… …… …100 Figure.2.4.1 Real-time RT-PCR analysis of differentially expressed sPLA2 subgroups in the CNS ………114 Figure.2.4.2 Western blot analyses of sPLA2-IIA protein expression in different parts of the rat CNS ……… ……117 Figure.2.4.3 Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS … ……….………118

Figure.2.4.4 Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS … ……….………120

Figure.2.4.5 Electron micrographs of sPLA2-IIA immunolabeled sections from the dorsal horn of the spinal cord of a normal rat ………….……….………122

Figure.2.5.1 TIRFM imaging of vesicles footprints and fusion events happened

at subplasmalemmal region ……… ……… …………137

lysophospholipids……….140 Figure.2.5.3 Capacitance measurements after various treatments ……… …141 Figure.2.5.4 Amperometry measurements ……… ……….…142 Figure.2.5.5 Intracellular calcium imaging ……….………143

AA Arachidonic acid

AACOCF3 Arachidonyl trifluoromethyl ketone

AMPA 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4yl)propanoic acid

[Ca2+]i Intracellular calcium

CICR Calcium- induced calcium release

CMC Critical micelle concentration

cPLA2 Calcium-dependent cytosolic phospholipase A2

DAB 3,3-diaminobenzidine tetrahydrochloride

DRG Dorsal root ganglion

EAAs Excitatory neurotransmitter amino acids

EDTA Ethylenediaminetetraacetic acid

EGFR Epidermal growth factor receptors

HPLC High performance liquid chromatography

I.C.V Intracerebroventricular

iNOS Nitric oxide synthase

iPLA2 Calcium-independent phospholipase A2

L-VSCCs L-type voltage sensitive calcium channels

MAPK Mitogen activated protein kinases

MBCD Methyl-beta-cyclodextrin

NADPH Nicotinamide adenine dinucleotide phosphate

PI-4,5-P2 Phosphatidylinositol 4,5-bisphosphate

SNAP-25 Synaptosomal-associated protein of 25 kDa

SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein

receptor SPANs Snake presynaptic phospholipase A2 neurotoxins

TBS Tris-buffered saline

TIRFM Total internal reflection microscopy

TNF-α Tumor necrosis factor-alpha

TTXR Tetrodotoxin-resistant

PUBLICATIONS

Several parts of this study have been published in international refereed journals

International Refereed Journals

1 Ma MT, Yeo JF, Farooqui AA, Zhang J, Chen P, Ong WY (2010) Differential

effects of lysophospholipids on exocytosis in rat PC-12 cells J Neural

Transm 117:301-308

2 Ma MT, Nevalainen TJ, Yeo JF, Ong WY (2010) Expression profile of multiple

secretory phospholipase A(2) isoforms in the rat CNS: enriched expression of sPLA(2)-IIA in brainstem and spinal cord J Chem Neuroanat 39:242-247

3 Ma MT, Zhang J, Farooqui AA, Chen P, Ong WY (2010) Effects of cholesterol

oxidation products on exocytosis Neurosci Lett 476:36-41

4 MaMT, YeoJF, Farooqui AA, OngWY (2011) Role of Calcium Independent

Phospholipase A2 in Maintaining Mitochondrial Membrane Potential and Preventing Excessive Exocytosis in PC12 Cells Neurochem Res 36:347-

354

5 MaMT, ShuiG, WenkMR, YeoJF, OngWY (2011) Systems wide analyses of

lipids in the brainstem during inflammatory orofacial pain – evidence for increased phospholipase A2 activity Eur J Pain 16:38-48

6 Yang H, Ma MT, Siddiqi NJ, Alhomida AS, Ong WY (2011) Enriched

expression profile of sPLA2-III in rat CNS and its effects on exocytosis in PC12 cells Neuroscience (submitted)

SECTION I INTRODUCTION

1 Phospholipase A 2

Phospholipase A2 (PLA2, EC 3.1.1.4) consists of a superfamily of enzymes

which specifically hydrolyze the acyl ester bond at the sn-2 position to liberate

free fatty acids, such as arachidonic acid (AA) and lysophospholipids, from glycerol in membrane phospholipids (Fig 1.1.1.) Different isoforms of PLA2 enzymes exists, including secretory phospholipase A2 (sPLA2), calcium (Ca2+)-dependent cytosolic phospholipase A2 (cPLA2), and Ca2+-independent phospholipase A2 (iPLA2) (Balsinde and Dennis 1996; Akiba and Sato 2004) These isoforms are differentiated according to their cellular localization, and activation pathways (Zhu et al 1996) and they play different roles in the central nervous system (CNS) Studies have related the role of cPLA2 to modulating neuronal excitatory functions while sPLA2 to inflammatory responses, and iPLA2

is more widely studied in neurologic disorders associated with brain iron accumulation which normally occur during childhood and has been associated with Schizophrenia (Ong et al 2010; Sun et al 2010) cPLA2 and sPLA2 are more commonly associated with many inflammatory diseases (Mayer and Marshall 1993)

Fig.1.1.Site of action of phospholipases A1, A2, C and D on the phospholipid molecule to produce

lysophospholipids and arachidonic acid (Farooqui and Horrocks 2007)

Secreted sPLA2 acts on the cells extracellularly to liberate AA, which is

then taken up by cells almost immediately Activation of this enzyme stimulates

cPLA2 which acts intracellularly, on the nuclear membrane and/or endoplasmic

reticulum to release AA inside the cell (Balsinde et al 1994) iPLA2 is involved in

phospholipid remodeling as it hydrolyzes membrane phospholipids in a Ca2+

-independent manner Lysophospholipids, product from enzymatic action of

iPLA2, are substrates for acyl transferases necessary for incorporation of

unsaturated fatty acids, AA, to produce phospholipids These are required for

cyclooxygenas

e

Prostaglandins Thromboxane

s Lipoxins Leukotriene

s Epoxides Fatty acid Alcohols

Lysophospholipid

s

Lysoplatelet –activating

factor (Lyso-PAF)

stimulation for sPLA2 and cPLA2, suggesting the significance of iPLA2 physiological function in basal cell metabolism (Dennis 1997)

Both membrane phospholipids and their hydrolytic products are essential for signal transduction pathway in the cells Phospholipids are precursors of diacylglycerol (DAG) which triggers protein kinase C (PKC) production after its translocation to the membranes (Farooqui et al 1988) DAG induces addition of regulatory domain of PKC into the hydrophobic core of membranes (Orr and Newton 1992) likely through altering the properties of membrane lipid bilayer (Senisterra and Epand 1993) Moreover, DAG is involved in neurotransmission

as it induces membrane fusion (Nieva et al 1989) associated with neurotransmitter release

phosphatidylinositol (PI) are hydrolyzed according to specific PLA2 activities to their respective lysophospholipids which reside on the neural membrane (Farooqui et al 1997b; Farooqui et al 2000b) Different phospholipids exert a variety of influences on the cell membranes, such as inducing neural transmission and cell excitotoxicity For instance, PS regulates binding ability of glutamate receptors necessary for maintaining the long-term potentiation (LTP) in both neonatal and adult rat brain (Baudry et al 1991; Gagne et al 1996) However, the production of PS in cerebellar slices is blocked by metabotropic glutamate receptor agonist (Buratta et al 2004), suggesting that the metabotropic glutamate receptor activation reduces the insertion of serine into

PS and influences the production of excitatory postsynaptic currents in rat cerebellar slices (Farooqui and Horrocks 2007)

Lysophospholipids are generated either through hydrolysis action of lysophospholipase or by recycling of phospholipids in remodeling pathway (Farooqui et al 2000a) The main function of these lipids is to alter membrane fluidity and permeability and to exert phospholipid remodeling and membrane perturbation (Farooqui and Horrocks 2006) AA, another enzymatic product due

to action of PLA2 on phospholipids, has a small portion of it converted to inflammatory mediators such as prostaglandin E2 (PGE2), leukotrienes and thromboxanes while most of it is reincorporated into brain glycerophospholipids during physiological conditions (Leslie 2004) AA is a precursor for pro-inflammatory mediators and regulates neural cell function directly by changing the fluidity and polarization state of membranes through stimulating PKC and triggering the release of Ca2+ (Molloy et al 1998)

1.1 cPLA 2

Another PLA2 isoform, cPLA2, has a molecular weight of 85 kDa and is highly distributed throughout rat CNS (Ong et al 1999b) In contrast to sPLA2,

this enzyme preferred AA in the sn-2 position of phospholipid substrates (Diez et

al 1992; Dennis 1997) precursors for various pro-inflammatory lipid mediators including prostaglandins and leukotrienes (Roshak et al 1994; Naraba et al 1998; Farooqui et al 2004) cPLA2 is transcribed in almost all peripheral tissues, although the pancreas, liver, heart and kidney exhibit very low levels of cPLA2

expression (Molloy et al 1998) cPLA2 mRNA is expressed at the highest levels

in most regions of the brain including the brainstem, hippocampus, striatum, spinal cord, midbrain and cerebellum using quantitative PCR using Western blot and cPLA2 activity assay (Shirai and Ito 2004; Lucas et al 2005; Ong et al 2010) cPLA2 immunoreactivity is detected in the dorsal and ventral horns of the spinal cord (Ong et al 1999a), in which the enzyme protein is localized in dendrites or dendritic spines that are postsynaptic to unlabeled axon terminals (Sandhya et al 1998; Ong et al 1999a)

In addition, this enzyme is localized in facial motor nucleus of the brainstem and initial parts in the ascending auditory pathway including cochlear nuclei (Sandhya et al 1998; Kishimoto et al 1999; Ong et al 1999b; Farooqui 2000; Shirai and Ito 2004) suggesting that cPLA2 is also important in pain pathway cPLA2 is involved in spinal nociceptive processing as cPLA2 activity in the spinal homogenates is significantly reduced after intrathecal (IT) injection of cPLA2 inhibitors (Lucas et al 2005) Although cPLA2 gene expression is low in the hippocampus, olfactory bulb and cerebellar granular cells, its activity is upregulated in the dentate granule gyrus after brain ischemia (Koike et al 1997) This upregulation contributes to degradation of neural membrane phospholipids which results in production of AA-derived lipid metabolites that are implicated in nociception, neuroinflammation, oxidative stress and neurodegeneration (Ong et

al 2010)

iPLA2 has an important “housekeeping” role during physiological conditions, in which iPLA2 activity is essential for prevention of vacuous chewing movements which is a rodent model for tardive dyskinesia Deficits in prepulse

inhibition of the auditory startle reflex has been detected in patients with schizophrenia and it could also occur in rodents (Lee et al 2007; Lee et al 2009; Ong et al 2010) iPLA2 maintains the structure of mitochondrial membrane lipid component, cardiolipin, to allow for smooth functioning of the electron transport chain and protects mitochondria from oxidant-mediated lipid peroxidation and dysfunction through regulation of the decylation/reacylation cycle (Seleznev et al 2006; Kinsey et al 2007; Kinsey et al 2008) Following pre-treatment with specific iPLA2 inhibitor, bromoenol lactone (BEL), it accelerated the oxidant-mediated mitochondrial lipid peroxidation and swelling (Morgan et al 2006; Gregory et al 2008)

1.3 Secretory phospholipase A 2 (sPLA 2 )

sPLA2 is produced intracellularly but is secreted and acts extracellularly Mammalian sPLA2 comprises of a family of lipolytic enzymes that are structurally related, a His-Asp catalytic dyad and disulfide-rich with low molecular masses (14-19 kDa) (Yang et al 2009) Their activities require Ca2+ in the millimolar (mM) range (Valentin and Lambeau 2000; Farooqui and Horrocks 2004; Farooqui et al 2004) There are 10 catalytically active sPLA2 isozymes in mammals (sPLA2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X and -XIIA) (Gelb et al 2000; Six and Dennis 2000; Ho et al 2001) Each sPLA2 has well-defined enzymatic properties, and displays distinctive tissue expression patterns Therefore it suggests that each sPLA2 isozyme acts on specific phospholipid membrane moieties in vivo (Murakami and Kudo 2004)

sPLA2 is expressed in all regions of a mammalian brain Brain sPLA2 contains a secretion peptide and requires Ca2+ for enzymic activity However, it is not selective of any particular fatty acyl chains in the phospholipids (Farooqui and Horrocks 2007) Total sPLA2 activity is shown to be of the highest in medulla oblongata, pons, and hippocampus, moderate in the hypothalamus, thalamus, and cerebral cortex while low level of sPLA2 activity is detected in the cerebellum and olfactory bulb (Thwin et al 2003) In addition, sPLA2 is expressed in rat brain synaptic vesicles, thus suggesting the presence of sPLA2 activity in the neurons and in differentiated pheochromocytoma-12 (PC-12) cells (Matsuzawa et al 1996)

1.3.1 sPLA 2 isozymes

1.3.1.1 sPLA 2 -IB

sPLA2-IB has a distinct 5 amino acid extension known as pancreatic loop

in the middle of the molecule with specific disulfide bond between residues 11 and 77, which defines group I (Six and Dennis 2000; Murakami and Kudo 2004)

As the expression of this isozyme is high in the pancreas, it is proposed to have

a crucial role in digesting the dietary phospholipids (de Haas et al 1968; Snitko

et al 1999) Mature form of sPLA2-IB binds to the M-type PLA2 receptor (present

on smooth muscle) with high affinity (Lambeau and Lazdunski 1999) M-type sPLA2 receptor knockout mice show features which allow resistance to endotoxin shock (Hanasaki et al 1997) sPLA2-IB is expressed abundantly in the CNS, particularly high in cerebral cortex and hippocampus and cerebellum (Kolko et al

2005; Lucas et al 2005) sPLA2-IB mRNA is also detected in rat cerebral neurons and cells of neurodermal origin (Kolko et al 2005; Kolko et al 2007) This isozyme modulates glutamatergic synaptic function due to its presence in areas which are rich in glutamatergic neurons (Kolko et al 2006), thus indicating its role in synaptic transmission

1.3.1.2 sPLA 2 -IIA

Group II sPLA2, however, has a distinct structure and a widely known role

in inflammatory pathway sPLA2-IIA is made up of disulfide linking residues 50 with residues at the C-terminus and contains a C-terminal extension of 7 amino acids in length which defines group II distinctively (Kramer et al 1989) sPLA2-IIA potently hydrolyzes phospholipids such as phosphatidylglycerol (PG) (Ono et al 1988) which is the main component on the exterior of the mammalian cells plasma membrane In addition, sPLA2-IIA causes the release of AA from intact cell membranes in various cell systems to potentiate its effect on the inflammatory pathway (Hanasaki et al 1999)

sPLA2-IIA is expressed in vitro, for instance cultured astrocytes (Svensson

et al 2005), suggesting its origin from the brain Moreover, this isozyme is ubiquitously expressed in the rat brain and also in the spinal cord (Molloy et al 1998; Lucas et al 2005) sPLA2-IIA is also expressed in peripheral organs such

as rat testis, hind paw skin, liver, heart, spleen and pancreas (Molloy et al 1998) sPLA2-IIA is highly involved in inflammatory processes occurring in the peripheral system High catalytic sPLA2-IIA activity is detected in synovial fluids from

patients with rheumatoid arthritis (RA) and osteoarthritis (OA) (Vadas and Pruzanski 1984; Pruzanski et al 1991; Pruzanski et al 1995) The sPLA2-IIA level in sera or exudative fluids is associated with the severity of inflammatory diseases (Nakano et al 1990; Crowl et al 1991; Oka and Arita 1991) sPLA2-IIA

is also synthesized and secreted from human synovial cells in response to a inflammatory cytokine, interleukin-1 (IL-1) (Koike et al 1997) Both sPLA2-IIA mRNA expression and activity are upregulated by cytokines including tumor necrosis factor-α (TNF-α) and IL-1 α/β and endotoxins (Oka and Arita 1991; Svensson et al 2005; Adibhatla and Hatcher 2007) Injection of this isozyme to the hind paw of rats with adjuvant arthritis (Murakami et al 1990) exacerbated inflammatory responses, indicating the role of sPLA2 in inflammatory pain (Koike

pro-et al 1997) Increased sPLA2-IIA mRNA expression and immunoreactivity are observed in the rat cerebral ischemic brain and in Alzheimer’s disease (AD) whose prominent pathological feature is inflammation (Lauritzen et al 1994; Lin

et al 2004; Adibhatla and Hatcher 2007)

1.3.1.3 sPLA 2 -IIC

Unlike sPLA2-IIA, sPLA2-IIC has an extra disulfide between residues 87 and 93 in an extended loop region Due to the lack of a portion of one exon in the human genome, this isozyme of sPLA2 is a pseudogene Nevertheless, sPLA2-IIC is shown to be expressed at high levels in rodent testis and pancreas (Chen

et al 1994a; Valentin et al 1999a) sPLA2-IIC mRNA expression level is low in peripheral tissues even though it is distributed throughout all regions of the CNS

(Molloy et al 1998; Lucas et al 2005) It has low level of expression in the thalamus, cerebellum and brainstem compared to the rest of the brain regions (Molloy et al 1998) Unlike sPLA2-IIA, sPLA2-IIC is less commonly known in inflammatory pathway

1.3.1.4 sPLA 2 -IID

The structure of sPLA2-IID most resembles to that of sPLA2-IIA Recombinant mouse and human of this sPLA2 isozyme are active against vesicles of PG, PE and PC with high substrate affinity for the active site that is similar for all these phospholipids (Valentin et al 1999b) sPLA2-IID is expressed

at high levels in immune and digestive organs, such as the pancreas and spleen,

in which its expression is upregulated by pro-inflammatory stimuli in some tissues (Ishizaki et al 1999; Valentin et al 1999b)

1.3.1.5 sPLA 2 -IIE

sPLA2-IIE contains histidine PLA2 catalytic residues, and 7 disulfide bonds

of which define group II members (Valentin et al 1999a) Human recombinant sPLA2-IIE prefers hydrolyzing PG substrate to PE and to a smaller extent PC (Suzuki et al 2000) Using Northern blot, the sPLA2-IIE mRNA is localized in mouse testis and liver (Valentin et al 1999a), and detected at high levels in brain, heart, lung and placenta using RT-PCR (Suzuki et al 2000) Furthermore, sPLA2-IIE mRNA expression is high in both the rat brain and primary neuronal cultures sPLA2-IIE expression is most abundant in hippocampus and cerebral

cortex of the rat brain (Kolko et al 2006) In mammalian cells, sPLA2-IIE is induced in response to pro-inflammatory stimuli, and is activated to promote AA release and eicosanoid generation (Sun et al 2010) sPLA2-IIE expression levels are also elevated in sPLA2-IIA-deficient mice upon endotoxin challenge, further suggesting its function in inflammatory response in vivo (Suzuki et al 2000)

1.3.1.6 sPLA 2 -IIF

sPLA2-IIF contains a unique 30-amino acid C-terminal extension with an additional cysteine residue, leading to the formation of a homodimer or a heterodimer with a second protein (Valentin et al 1999a) Purified form of sPLA2-IIF hydrolyzes PG better than PC vesicles (Valentin et al 1999a) sPLA2-IIF is abundant in testis of adult mice and in mouse embryos, implying that its expression is regulated during development (Murakami and Kudo 2004)

1.3.1.7 sPLA 2 -III

Among the sPLA2 isozymes of small molecular weight, sPLA2-III is an unusually large protein (55 kDa) It is made up of three domains, sPLA2 domain (19 kDa) surrounded by large and unique N- and C- terminal regions The sPLA2 domains exhibit distinctive characteristics of group III sPLA2 purified from the bee venom, including 10 cysteines, the key residues of the Ca2+ loop and catalytic site (Valentin et al 2000) The sPLA2 domain of human sPLA2-III is 31% homologous to the bee venom sPLA2 and demonstrates similar features of group III sPLA2s (Valentin et al 2000) Although the residues surrounding the sPLA2

domain suggests the presence of protease cleavage sites (Valentin et al 2000), the mechanism of processing sPLA2-III in the cells is still not known (Murakami and Kudo 2004) A partially purified human sPLA2-III hydrolyzes PG vesicles more than PC vesicles (Six and Dennis 2000) sPLA2-III also shows a homologous His/Asp dyad in the active site, suggesting the similar hydrolase mechanism as described previously for the other Histidine PLA2’s (Kuchler et al 1989; Davidson and Dennis 1990; Scott et al 1991)

sPLA2-III is expressed in mammals including humans where it is transcribed in different tissues (Valentin et al 2000) It is expressed at a high level in the kidney, heart, liver, and skeletal muscle In addition, sPLA2-III is also expressed in both the peripheral and the CNS, vascular endothelium of various tissues, and macrophages (Murakami et al 2005; Masuda et al 2008; Mounier et

al 2008) Similar to sPLA2-IIA, sPLA2-III is also recognized to play a role in

inflammation Transgenic (Tg) mice overexpressing human sPLA2-III developed

skin inflammation (Sato et al 2009)

1.3.1.8 sPLA 2 -V

sPLA2-V has a significant activity against PE and PC vesicles when expressed in vitro This is in contrast to the characteristics of the sPLA2-IIA (Chen

et al 1994b; Chen et al 1994c; Chen and Dennis 1998; Han et al 1998; Han et

al 1999) Although sPLA2-V mRNA has been detected in high abundance in the heart (Chen et al 1994b) and skin tissues, it is expressed at low levels in peripheral organs such as in spleen, pancreas and testis and is not detectable in

the kidney or liver (Tischfield 1997; Molloy et al 1998) sPLA2-V is transcribed at high levels in rat and mouse brain (Chen et al 1994c) In CNS, high level of sPLA2-V mRNA is expressed in neurons of the cerebral cortex and hippocampus (Molloy et al 1998; Kolko et al 2006) It is also detected in the rat cerebellum by immunostaining and in situ hybridization (Shirai and Ito 2004) Using quantitative PCR and Western blot analysis, sPLA2-V is identified in the rat spinal cord (Lucas

et al 2005; Svensson et al 2005) sPLA2-V could also be detected at high levels

in various tissues and immune cells (Sawada et al 1999), especially in response

to inflammatory stimuli (Sawada et al 1999; Valentin et al 1999b; Murakami and Kudo 2001) Therefore, sPLA2-V is suggested to be involved in inflammatory conditions and signal transduction in vivo (Balboa et al 1996; Murakami et al 1998; Balsinde et al 1999; Murakami et al 1999; Balsinde et al 2000)

1.3.1.9 sPLA 2 -X

Recombinant sPLA2-X is active against PE and PC vesicles (Cupillard et

al 1997; Hanasaki et al 1999) Human sPLA2-X mRNA expression is detected in spleen, thymus, and blood leukocytes (Cupillard et al 1997), while its protein expression is detected in lung alveolar endothelial cells (Hanasaki et al 1999) sPLA2-X is also transcribed in the peripheral neurons at low but significant levels, and exhibit neuritogenic effect, dependent on lysophospholipids such as lysophosphatidylcholine (lysoPC) (Ikeno et al 2005) However, low level of sPLA2-X expression is present in neurons of the rat brain, in primary neuronal cell cultures and in the cerebral cortex (Kolko et al 2006) In contrast to human

sPLA2-X, mouse sPLA2-X transcript is expressed in the digestive organs and testis (Cupillard et al 1997; Valentin et al 1999a) sPLA2-X has been shown to

be play a role in inflammatory pathway or signal transduction through production

of AA and its pro-inflammatory mediators, in human monocytic human acute monocytic leukemia cells study when added exogenously (Hanasaki et al 1999) The mature enzyme of sPLA2-X is able to interact with the M-type sPLA2 receptor with high affinity (Morioka et al 2000b) to mediate signal transduction during inflammation

1.3.2 sPLA 2 -XIIA

sPLA2-XIIA comprises of a central catalytic domain with a His/Asp catalytic dyad and cysteines are on the exterior of catalytic domain (Gelb et al 2000) sPLA2-XIIA also has a small molecular weight of 19 kDa and is expressed at high levels in the heart, skeletal muscle, kidney, and pancreas (Ho et al 2001) Its function in inflammation or synaptic transmission is still not yet widely studied

Table 1.1 Summary of differential mRNA and protein expression of PLA 2 isoforms in the CNS and peripheral organs based on various studies done so far

Differential levels of expression in

various organs

Molecular weight (kDa)

Peripheral organs

1.4 Arachidonic acid

Arachidonic acid (AA) (Fig 1.4.1.), all-cis-5, 8, 11, 14-eicosatetraenoic acid (20:4n-6, a ω-6 fatty acid), is an essential fatty acid released by the action of PLA2 (both cytosolic and secretory forms) on membrane phospholipids AA is involved in both physiological and pathological processes (Dennis et al 1991; Farooqui et al 1997a; Farooqui et al 1997b) AA is a precursor for the production of prostaglandins and leukotrienes involved in the development of a range of neurological disorders in mammals (Bosetti 2007) AA is metabolized to eicosanoids (prostaglandins, leukotrienes and thromboxanes) (Wolfe and Horrocks 1994) which are implicated in neural and immune functions (Katsuki and Okuda 1995) This fatty acid and its metabolites regulate a variety of processes in mammals including neuropsychiatric disorders, inflammation, and diseases such as AD and Parkinson’s disease (PD) (McGahon et al 1997; Smalheiser and Swanson 1998; Hudson et al 1999; Patrick et al 2000; Calder 2008; Meves 2008) Release of AA from the phospholipids is affected by cytokines, nitric oxide, and glutamate which regulate Ca2+ mobilization in the tissue and affect PLA2 activation Consequently, neuroinflammation due to upregulation of AA is observed in different neurological diseases (Lehtonen et al 1996; Mattson and Chan 2003; Rao et al 2007; Sun et al 2007; Orr and Bazinet 2008)

Fig 1.4.1 Chemical structure of arachidonic acid

During physiological conditions, AA level in brain tissue is very low (<10 μmol/kg) At this concentration, it acts as a second messenger molecule in the nervous system and is involved in a variety of functions AA activates PKC, which phosphorylates various substrates to modulate cytoskeletal dynamics (Leu and Schmidt 2008) Low levels of AA regulates ion channels, neurotransmitter release, LTP induction, neural cell differentiation, maintains neural membranes structural integrity and fluidity to and modulate neural inflammation and synaptic transmission (Farooqui et al 1997a; Farroqui et al 2002; Farooqui and Horrocks 2006)

AA generated due to N-methyl-D-aspartic-acid (NMDA) receptor stimulation at postsynaptic level may cross the synaptic cleft to presynaptic compartment to function as a retrograde messenger for LTP (Katsuki and Okuda 1995) In addition, AA facilitates retrograde neuromodulator in glutamatergic synapses after released upon activation of glutamate receptors (Katsuki and Okuda 1995) AA blocks uptake of glutamate, mediated by excitatory amino acid (EAA) transporters in normal cell, neuronal and glia cultures (Volterra et al 1994) AA also modulates acetylcholine production in the rat hippocampus (Almeida et al 1999) Therefore, high concentration of AA leads to adverse effects on neural membrane structures such as uncoupling oxidative

COOH CH3

phosphorylation and inducing efflux Ca2+ and mitochondrial K+ (Katsuki and Okuda 1995; Farooqui et al 1997a)

1.5 Phospholipids and lysophospholipids

The phospholipid bilayer and associated lipids provides not only a permeability barrier but also a structured environment that is necessary for a proper functioning of membrane-bound proteins (Maxfield and Tabas 2005) The neutral lipids PC, resides predominantly on the outer membrane, whereas anionic phospholipids PS, PE, and PI are located in the inner membrane (Adibhatla and Hatcher 2008) The transbilayer distribution of cholesterol between the leaflets determines membrane fluidity and modulates the membrane function (Adibhatla and Hatcher 2008)

In addition to their participation in structural components of the cell membrane, phospholipids serve as precursors for various second messengers such as AA, DHA, ceramide, 1,2-diacylglycerol, phosphatidic acid (PA), and lysophosphatidic acid (lysoPA) Lipid metabolism may be particularly important to the CNS, as this organ has the highest concentration of lipids next to adipose tissue (Adibhatla and Hatcher 2008) Phospholipids are hydrolyzed by PLA2 and generate lysophospholipids which are rapidly re-esterified by another fatty acid (Farooqui and Horrocks 2007) This deacylation-reacylation cycle is a vital mechanism to regulate the composition of saturated and polyunsaturated phospholipid acyl group in neural membranes (Yamashita et al 1997) which are

susceptible to oxidative damage as the membrane phospholipids are rich in polyunsaturated fatty acids (Farooqui and Horrocks 2007)

Lysophospholipids such as lysoPC, lysophosphatidylethanolamine (lysoPE), lysophosphatidylinositol (lysoPI), lysophosphatidylserine (lysoPS) and lysoPA (Fig 1.5.1.), are products of PLA2-catalyzed reactions and are precursors

of platelet activating factors (PAFs) (Rossi et al 2006) They undergo rapid decylation-recylation due to action of PLA2 acyltransferases introducing polyunsaturated fatty acids into phospholipids (Sun and MacQuarrie 1989; Farooqui et al 2000a) Lysophospholipid transacylase modulates lysophospholipid levels in neural membranes These reactions maintain lysophospholipids at very low levels in the brain tissue (Farooqui et al 2000a) Acetylation of lysoPC produces PAF, a potent pro-inflammatory lipid mediator involved in many pathological processes (Farooqui and Horrocks 2006) Snake presynaptic PLA2 neurotoxins (SPANs) hydrolyze phospholipids of cultured neurons to generate lysoPC and fatty acids (Rigoni et al 2005) This leads to a massive release of synaptic vesicles, with their incorporation into the presynaptic plasma membrane and resulting in surface exposure of synaptic vesicle luminal epitopes (Rigoni et al 2005)

H LysoPI