Distribution and role of calcium independent phospholipase a2 in the brain

SUMMARY One class of phospholipase A2 PLA2 that does not require calcium for its activity is the cytosolic calcium-independent PLA2 iPLA2.. In brain tissues, the basal expression and act

DISTRIBUTION AND ROLE OF PHOSPHOLIPASE A2 IN

ACKNOWLEDGEMENTS

I wish to express my deepest appreciation and heartfelt thanks to my

supervisor, Associate Professor Ong Wei Yi, Department of Anatomy, National

University of Singapore, for suggesting this study topic and his constant and patient guidance and encouragement throughout the course of the study During

my postgraduate study at National University of Singapore, he has not only introduced me to a new research field but has also been a role model for hardwork and commitment to research His deep and sustained interest and stimulating discussion have been most invaluable in the accomplishment of this thesis

I am very grateful to Professor Ling Eng Ang, former Head of

Department of Anatomy, National University of Singapore, for giving me the opportunity to do my postgraduate study at National University of Singapore I

am grateful to Professor Bay Boon Huat, Head of Department of Anatomy, for

his full support in using the excellent research facilities My deep indebtedness

goes to Associate Professor Gavin Stewart Dawe, Department of

Pharmacology, National University of Singapore, who gives me guidance and comment on my study and kindly offered help in teaching me the use of acoustic

startle reflex apparatus I am greatly indebted to Associate Professor Go Mei Lin, Department of Pharmacy, National University of Singapore, for their valuable

suggestions and friendly help during this study

I am also acknowledging my gratitude to Mrs Ng Geok Lan and Mrs Yong Eng Siang for their excellent technical assistance; Miss Chan Yee Gek, and Mdm Yu Ya Jun for Electron Microscopy work; Mr Yick Tuck Yong for his constant assistance in computer work; Mdm Ang Lye Gek Carolyne and Mdm Teo Li Ching Violet for their secretarial assistance

I would like to thank all other staff members and my fellow postgraduate students and vital friends in Histology Lab, Neurobiology Programme, Centre for

Life Science, National University of Singapore: Pan Ning, Lim Seok Wei, Jinatta Jittiwat, Tang Ning, Lee Hui Wen Lynette, Kim Ji Hyun, Ma May Thu, Poh Kay Wee, and Chia Wan Jie, for their help and support in many ways It

was a joyful experience working with all of them

I would like to take this opportunity to express my heartfelt thanks to my parents and sister for their full and endless support for my study I am very

grateful to Mr Chow Kum Seng Bernard and Mdm Cheong Hoon Moy Jean for

providing generous financial support Finally, I am greatly indebted to my

husband, Mr Xu Congyuan for his understanding and encouragement during

this study

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ……… …… ……… I TABLE OF CONTENTS……….……… ………… III SUMMARY……….……… … VIII LIST OF TABLES……… XI LIST OF FIGURES……… … XII ABBREVIATIONS……… … ………….XIII PUBLICATIONS……….……… ……….…… …….XVI

Chapter I INTRODUCTION 1

1.General introduction 2

1.1 sPLA2 isoforms 5

1.2 cPLA2 isoforms 6

1.3 iPLA2 isoforms 8

2.cPLA2 in normal brain 11

2.1 Molecular genotype 11

2.2 Localization and distribution of cPLA2 13

2.3 cPLA2 functions 13

2.4 Fatty acid released by cPLA2 14

2.5 cPLA2 inhibitors 16

3.iPLA2 in normal brain 17

3.1 Molecular genotype 18

3.3 iPLA2 functions 20

3.4 Fatty acid released by iPLA2 22

3.5 iPLA2 inhibitors 23

4.cPLA2 in pathological diseases 25

5.iPLA2 in pathological diseases 26

6 Roles of PLA2 in neurodegeneration 27

6.1 Cerebral ischemia 28

6.2 Alzheimer’s disease 28

6.3 Parkinson’s disease 30

7 Roles of iPLA2 in neuropsychiatric disorders 31

7.1 Schizophrenia 31

7.2 Depression 32

8 Animal models of neuropsychiatric disorders 33

8.1 Acoustic startle reflex 33

8.2 Vacuous chewing movement 36

8.2.1 Dopamine receptors and antipsychotic side effects 38

8.2.2 Dopamine D2 receptor occupancy 39

8.2.3 Other factors affecting VCM 40

9 Roles of PLA2 in mitochondrial diseases 41

Chapter II AIM OF STUDY 43

Chapter III EXPERIMENTAL STUDIES 46

Chapter 3.1 Distribution of Calcium-Independent Phospholipase A2 in the Rat Brain 47

1 Introduction 48

2 Materials and methods 49

2.1 Animals 49

2.2 Immunocytochemistry 49

2.3 Electro microscopy 51

3 Results 52

3.1 Light microscopy 52

3.1.1 Distribution of iPLA2 in the normal forebrain of Wistar rat 52

3.1.2 Distribution of iPLA2 in the basal ganglia and cerebellum of Wistar rat brain 54

3.2 Electron microscopy 56

4 Discussion 58

Chapter 3.2 Role of Calcium-Independent Phospholipase A2 in Cortex Striatum Thalamus Cortex Circuitry-Enzyme Inhibition Causes Vacuous Chewing Movements in Rats 1 Introduction 62

2 Materials and methods 64

2.1 Chemicals 64

2.2 Antisense oligonucleotides 64

2.3 Rats and treatment 68

2.4 Stereotaxic injections 70

2.5 Behavioral assessment 73

2.6 Western blot analysis 73

3 Results 74

3.1 Rats treated with BEL 74

3.3 Rats treated with iPLA2 inhibitors plus benztropine 79

3.4 Rats treated with iPLA2 antisense oligonucleotides 81

4 Discussion 83

Chapter 3.2 Role of Calcium-Independent Phospholipase A2 in Cortex Striatum Thalamus Cortex Circuitry-Enzyme Inhibition Causes Vacuous Chewing Movements in Rats 61

Chapter 3.3 Role of Phospholipase A2 in Prepulse Inhibition of the Auditory Startle Reflex in Rats 89

1 Introduction 90

2 Materials and methods 90

2.1 Rats and treatment 90

2.2 Antisense oligonucleotides 91

2.3 Acoustic startle reflex recordings 92

2.4 Statistical analysis 95

3 Results 96

3.1 Acoustic startle response 96

3.2 Prepulse intensity 97

4 Discussion 99

Chapter 3.4 Effects of Calcium-Independent Phospholipase A2 on Exocytosis in Rat PC12 Cells 103

1 Introduction 104

2 Materials and methods 105

2.1 Cell culture 105

2.2 Patch clamp and capacitance measurements 105

2.3 PC12 cell treatment 107

2.4 Mitochondrial membrane potential assay 108

3 Results 109

3.1 Effects of BEL and factors affecting BEL-induced exocytosis on capacitance measurements in PC12 cells 110

3.2 Effects of BEL on mitochondrial membrane potential 111

4 Discussion 112

Chapter IV CONCLUSION 116

Chapter V REFERENCE 121

SUMMARY

One class of phospholipase A2 (PLA2) that does not require calcium for its activity is the cytosolic calcium-independent PLA2 (iPLA2) In brain tissues, the basal expression and activity of iPLA2 is higher than either cytosolic calcium-dependent PLA2 (cPLA2) or secretory calcium-dependent PLA2 (sPLA2) (Molloy

et al 1998; Farooqui et al 1999), and its protein expression decreases during aging (Aid and Bosetti, 2007) iPLA2 is not only responsible for regulation of membrane phospholipid homeostasis (“housekeeping”) in cells (Balsinde et al

1995), but also plays important roles in intracellular signal transduction

The present study aims to elucidate the role and distribution of iPLA2 in the brain iPLA2 immunoreactivity was observed in structures derived from the telencephalon, whereas structures derived from the diencephalon were lightly labeled The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled Immunoreactivity was observed on the nuclear envelope of neurons, dendrites and axon terminals using electron microscopy Drug-induced tremulous oral movements have been linked to human Parkinsonian tremor (Salamore et al 1998) Inhibitors of iPLA2 could induce vacuous chewing movements (VCM) in rats Striatal injections of iPLA2 inhibitor, bromoenol lactone (BEL), resulted in significantly increased VCM in Wistar rats from 2 to 5 days after injection Significantly increased VCM was also observed after intrathalamic or intracortical injections of BEL In contrast, no significant effect was observed after BEL injection into the cerebellum The effects of BEL

were replicable using another PLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP) These findings suggest that increased VCM after MAFP injection was because of inhibition of iPLA2 The observations with BEL and MAFP point to a role for inhibition of PLA2 enzymatic activity in VCM

Prepulse inhibition (PPI) of the acoustic startle reflex has been widely used as a model of sensory information processing and sensorimotor gating (Graham, 1975; Kumari and Sharma, 2002) Our results demonstrate that systemic administration of the non-specific PLA2 inhibitor, quinacrine, resulted in significantly decreased PPI of the auditory startle reflex at 76, 80, and 84 decibel (dB), compared to saline injected controls Rats that received intrastriatal injection of antisense oligonucleotide to iPLA2 also showed significant reduction

in PPI at prepulse intensities of 76 and 84 dB compared to scrambled sense injected controls iPLA2 inhibition apparently has the same effect as increased dopamine receptor stimulation, in terms of its effects on PPI These findings appear consistent with the previous findings that iPLA2 inhibition causes VCM

We have shown that mitochondrial permeability transition pore (MPTP) is essential in meditating the effect of BEL on exocytosis in PC12 cells Blocking of MPTP with the inhibitors bongkrekic acid (BKA) and cyclosporine A (CsA) resulted in reduced exocytosis after intracellular addition of BEL p-trifluromethoxy carbonyl cyanide phenyl hydrazone (FCCP) which depolarizes mitochondria transition potential and deplete mitochondrial calcium did not have any effects on exocytosis after addition of BEL The above results are consistent

with previous studies that overexpression of iPLA2 has a protective effect on the mitochondria It is possible that inhibition of iPLA2 causes damage to the mitochondria and release of calcium resulting in exocytosis The results highlight the importance of normal “housekeeping” phospholipase A2 in maintaining the normal function of neurons

LIST OF TABLES

Table 1 Treatment group of Wistar rats……… 72

Table 2 Two treatment protocols of Wistar rats……….92

Table 3 Summary of four different types of trials……… 96

Table 4 Acoustic startle in baseline and treatment groups……… 97

LIST OF FIGURES

Figure 3.1 Distribution of iPLA2 in the normal forebrain of Wistar rat………53

Figure 3.2 Distribution of iPLA2 in the basal ganglia and cerebellum of Wistar rat brain……… 55

Figure 3.3 Electron microscopy………57

Figure 3.4 Degradation of mRNA by RNase H……… 65

Figure 3.5 Structure of phosphorothioate oligonucleotide……… 66

Figure 3.6 Effects of intracerebral BEL injections on VCM……… 76

Figure 3.7 Effects of intrastriatal MAFP injection on VCM……… 78

Figure 3.8 Effects of benztropine on BEL and MAFP induced VCM……… 80

Figure 3.9 Effects of antisense and sense injection of oligonucleotides to the striatum……….82

Figure 3.10 Prepulse inhibition (PPI) experimental setup in rats………94

Figure 3.11 Prepulse inhibition of the auditory startle at four prepulse intensities after quinacrine administration (5 mg/ml, i.p.) compared to saline controls……….98

Figure 3.12 Prepulse inhibition of the auditory startle response in rats treated with iPLA2 antisense oligonucleotide injections and sense controls……… 99

Figure 3.13 Effects of BEL and factors affecting BEL-induced exocytosis on capacitance measurements in PC12 cells……… 111

Figure 3.14 Effects of BEL on mitochondrial membrane potential……… 112

ABBREVIATIONS

AACOCF3, arachidonyl trifluoromethyl ketone

ABC, avidin-biotin complex

AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ASR, acoustic startle reflex

ATP, adenosine triphosphate

BEL, bromoenol lactone

CA1, hippocampal CA1 area

CA3, hippocampal CA3 area

DHA, docosahexaenoic acid

DNA, deoxyribonucleic acid

EBSS, Earl’s Balanced Salt Solution

EPS, extrapyramidal symptoms

EPSCs, excitatory postsynaptic currents

FCCP, p-trifluromethoxy carbonyl cyanide phenyl hydrazone

FFA, free fatty acid

FKGK11, 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one

IL-1β, interleukin 1 beta

IL-6, interleukin 6

iPLA2, calcium-independent phospholipase A2

MAFP, methyl arachidonyl fluorophosphonate

MPT, mitochondrial permeability transition

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

NMDA, N-methyl-D-aspartate

NPD1, neuroprotectin D1

OsO4, osmium tetroxide

PAF, platelet-activating factor

PAP-1, phosphatidate phosphohydrolase

RCM, renal cortex mitochondria

ROS, reactive oxygen species

sPLA2, secretory phospholipase A2

TBS, tris-buffered saline

TNF-α, Tumor necrosis factor-alpha

UCH-L1, ubiquitin C-terminal hydrolases L1

VCM, vacuous chewing movement

PUBLICATIONS

Various portions of the present thesis have been published:

International Refereed Journals

Lee LY, Ong WY, Farooqui AA, Burgunder JM (2007) Role of

calcium-independent phospholipase A2 in cortex striatum thalamus cortex enzyme inhibition causes vacuous chewing movements in rats Psychopharmacology 195:387-395

circuitry-Lee LY, Farooqui AA, Dawe GS, Burgunder JM, Ong WY (2009) Role of

phospholipase A2 in prepulse inhibition of the auditory startle reflex in rats Neurosci Lett 453:6-8

Chapter I INTRODUCTION

1 General introduction

A phospholipid molecule consists of a diglyceride, a phosphate group, which may itself be bound to one of a variety of polar head groups such as choline A diglyceride is a glyceride consisting of two fatty acyl chains, which are hydrophobic in nature, covalently bonded to a glycerol molecule through ester linkages One exception to this is sphingomyelin, which is derived from sphingosine instead of glycerol, and contains only one fatty acid linked to an amino group With their hydrophilic polar phosphate groups and long hydrophobic hydrocarbon “tails”, phospholipids readily form membrane-like

structures in water They are a major component of all cell membranes

All four ester moieties in a phospholipid are susceptible to enzymatic hydrolysis Phospholipases are a family of enzymes that catalyze the hydrolysis

of phospholipids They are classified into four groups based on their catalytic activity, namely phospholipase A1, A2, B, C and phospholipase D Phospholipase

A1 cleaves the carboxy ester linkages between the glycerol backbone of the phospholipid and the acyl chains attached at sn-1 position Phospholipase A2

hydrolyzes the acyl ester bond at the sn-2 position of glycerol in membrane phospholipids to produce free fatty acids and lysophospholipids Phospholipase

B cleaves both sn-1 and sn-2 acyl chains, they are also known as lysophospholipase Phospholipase C cleaves the ester linkage between sn-2 position of the glycerol backbone and the inorganic phosphate moiety of the polar

head group, whereas phospholipase D cleaves the bond between inorganic phosphate and the polar head group (Boyer, 1983)

Phospholipase A2 (PLA2) represents a large superfamily of enzymes that catalyze the hydrolysis of the fatty acid or alkyl bond of phospholipids at the sn-2 position, liberating free fatty acids and lysophospholipids Phospholipids are the major lipid constituents of neural membranes, and their metabolism is therefore important to maintain the integrity of the cell membrane There are many types of PLA2 expressed in mammalian tissues The human genome contains over 25 genes encoding PLA2s that have been broadly classified into types based on their substrate preference, cofactor requirements, and dependence on calcium for activity, size, and their catalytic mechanism (Cedars et al 2009) They are broadly defined into five different types based on functional criteria: secretory

Ca2+-dependent PLA2 (sPLA2), cytosolic Ca2+-dependent PLA2 (cPLA2), Ca2+independent PLA2 (iPLA2), platelet-activating factor hydrolases (PAF-AH) and lysosomal PLA2 (Cummings et al 2000; Balsinde et al 2002; Schaloske and Dennis, 2006) PLA2 enzymes also function in the digestion of dietary lipid and microbial degradation Their most widely-known function is the regulation of phospholipid acyl turnover as a housekeeping role for membrane repair or production of proinflammatory mediators They provide neural membranes with stability, fluidity, and permeability Neural membrane phospholipids are a reservoir for bioactive lipid metabolites and second messengers These enzymes have been purified from many different sources such as mammalian pancreas,

-1995) They are not only involved in neural cell proliferation, differentiation, and apoptosis, but also in the modulation of activities of transporters, ion channels, and membrane-bound enzymes (Farooqui et al 2000a) It is well known that phospholipids-mediated signaling involves the generation of bioactive lipid metabolites in response to agonist-receptor interactions at the plasma membrane level (Farooqui et al 2000a)

These bioactive lipid metabolites modulate certain genes in the nucleus The occurrence of phospholipids in nuclei has been documented by biochemical and ultracytochemical procedures (Fraschini et al 1995; Albi et al 1996) The total phospholipids content of nuclei is reported as 3% by weight compared with 75% for protein and 22% for DNA Major nuclear phospholipids include PtdIns,

PtdCho, and CerPCho This is in contrast to the plasma membrane that contains

a considerable amount of PtdCho, PtdEtn, PtdSer, and PtdIns (Hunt et al 2001; Irvine et al 2003) The phospholipids contents (per mg protein) of the nuclear membrane are approximately nine times that of whole nuclei Collective evidence suggests that the nucleus is the main site involved in active and autonomous phospholipids metabolism, with nuclear phospholipids having a composition and turnover rate different from phospholipids present in plasma membranes, microsomes, and mitochondria (Fraschini et al 1999; Tamiya-Koizumi et al 2002; Martelli et al 2004; Ledeen et al 2004)

The nuclear fraction contains many enzymes responsible for the metabolism of phospholipids, and at the same time, generates and regulates the

levels of second messengers These enzymes include PLA2, phospholipase C (PLC), phospholipase D (PLD), diacylglycerol lipase (DAG-lipase), phosphatidylinositol 4-kinase, Mg2+-dependent sphingomyelinase, and CTP:phosphocholine cytidylyltransferase (CCT) The second messengers generated by these enzymes modulate neural cell proliferation, differentiation, and apoptosis During these processes the quantitative ratios among various phospholipids undergo significant changes depending upon the functional state

of neurons, astrocytes, and oligodendrocytes It is interesting to note that some extracellular stimuli, such as retinoic acid, produce bioactive lipid metabolites only in the nucleus and not in the plasma membrane The enzymic properties of phospholipid metabolizing enzymes in the nuclear fraction are different from those found in plasma membrane, microsomes, and cytoplasm (Maraldi et al 1999)

1.1 sPLA 2 isoforms

The first type of PLA2 enzymes discovered were the secreted PLA2

(sPLA2) found in snake venoms, mammalian pancreas, and synovial fluid They are characterized by a low molecular weight (13 – 15 kDa), a catalytic site which contains histidine, Ca2+ bound in the active site, and six-conserved disulfide bonds with one or two variable disulfide bonds (Burke and Dennis, 2009) Millimolar concentrations of Ca2+ are needed for maximal enzymatic activity (Boyer, 1983; Wong and Dennis, 1990) It has been implicated as an important agent involved in both local and systemic inflammatory conditions (reviewed in

Vadas et al 1993) sPLA2 gene expression has also been shown to be induced

by various inflammatory stimuli, such as endotoxin, IL-1β, TNF-α, and IL-6 (Vadas et al 1993) The mechanism of governing the proinflammatory activity of sPLA2 is not known sPLA2 uses histidine to catalyze phospholipids at the sn-2 position, releasing non-esterified fatty acids and produce lysophospholipids (Balsinde et al 2002) However, sPLA2 has little preference for the type of fatty acid in the sn-2 position

Ten members of the sPLA2 family have been identified in mammals, which are numbered and grouped in the order of their discovery: group IB (GIB), IIA, IIC, IID, IIE, IIF, III, V, X and XII (Schaloske and Dennis, 2006) Based on the position of disulfide bonds and sequence alignment, the mammalian sPLA2 can

be subdivided into three structural classes (Murakami et al 2002) Natural gene mutations of sPLA2 have been found in a number of inbred mouse strains (Kennedy et al 1995) The sPLA2 gene was disrupted by a frameshift mutation in exon 3 This mutation terminates out of frame in exon 4, resulting in a disruption

of the calcium binding domain and loss of both activity domains encoded by exons 4 and 5 The identification of this mutation has important implications in mouse inflammatory models, which serve to define the physiological role of sPLA2 and ascertain its contribution in the inflammatory process

1.2 cPLA 2 isoforms

The cPLA2 family consists of six intracellular enzymes, cPLA2α, cPLA2β, cPLA2γ, cPLA2δ, cPLA2ε and cPLA2ζ They are intracellular PLA2 of a high

molecular weight (85 – 110 kDa), and play “housekeeping” roles by facilitating phospholipid remodeling and maintaining phosphatidylcholines (Balsinde et al

1996, 1997; Ramanadham et al 1999; Ma et al 2001) Much research has focused on cPLA2α because of its central and functional role through its ability to trigger release of arachidonic acid and eicosanoids (Leslie et al 1997) cPLA2β is found mainly in the cerebellum and shares more similarities with cPLA2α than with cPLA2γ (Farooqui and Horrocks, 2007) It has been reported that cPLA2γ is predominantly expressed in the brain, heart, and skeletal muscle in humans

(Pickard et al 1999; Underwood et al 1998) and this enzyme is activated in vivo

by serum (Stewart et al 2002) cPLA2δ was identified from psoriatic skin (Chiba

et al 2004) cPLA2δ, together with cPLA2ε and cPLA2ζ, were cloned and characterized from novel murine cPLA2, which forms a gene cluster with cPLA2β (Ohto et al 2005) The role of these paralogs of cPLA2 in brain tissues remains speculative

The PLA2 activity of cPLA2α is characterized by Ca2+-dependence for its activity and substrate preference for arachidonoyl phospholipids (Leslie 1997; Sharp et al 1991; Clark et al 1991; Hirabayashi et al 2004) Submicromolar concentrations of calcium binding is required for translocation of the enzyme from the cytosol to the endoplasmic reticulum, Golgi apparatus and perinuclear membranes, and it is subsequently activated by phosphorylation via the MAPK pathway (Balsinde et al 1999; Murakami et al 1998, 2000) With the exception

of cPLA2γ, all known cPLA2s require micromolar concentrations of calcium for

cPLA2 has a 20-fold preference for arachidonic acid over other unsaturated fatty

acids in phospholipid substrates (Clark et al 1991)

of 39 kDa and is markedly inhibited by glycosaminoglycans Heparan sulfate is most potent inhibitor, followed by hyaluronic acid, chondroitin sulfate, and heparin (Yang et al 1994a) N-acetylneuraminic acid, gangliosides, and sialoglycoproteins also inhibit the plasmalogen-selective PLA2 in a dose-dependent manner (Yang et al 1994b)

At present, seven distinct isoforms of iPLA2 have been identified at the genetic level and they are designated as iPLA2α (Andrews et al 1988), iPLA2β (Tang et al 1997), iPLA2γ (Mancuso et al 2000), iPLA2δ (neuropathy target esterase) (van Tienhoven et al 2002), iPLA2ε (adiponutrin), iPLA2ζ (TTS-2.2), and iPLA2η (GS2) (Jenkins et al 2004) Four of the iPLA2 isoforms (iPLA2α,

iPLA2β, iPLA2γ and iPLA2δ) are high molecular weight intracellular phospholipases (84 – 146 kDa), but the remaining three iPLA2 isoforms are between 28 – 57 kDa The most extensively studied enzyme for this group is iPLA2β, also known as Group VIA-2 PLA2, using the newer classification A structural feature of Group VIA PLA2 is the presence of several ankyrin repeats suggesting protein-protein interactions, and it is proposed that the enzyme activity requires oligomerization of four iPLA2 monomers (Winstead et al 2000) Group VIA iPLA2 contains a binding domain for calmodulin which negatively regulates its activity (Wang et al 2005; Jenkins et al 2006) Although calcium is not required for iPLA2 activity, their activity may be regulated by calcium binding proteins Group VIA PLA2 activity can be stimulated by reactive oxygen species (ROS) (Martinez and Moreno 2001) and it has been reported to be involved in eicosanoids synthesis in granulocytes (Larson Forsell et al 1998), phospholipids remodeling (Balsinde et al 1995), endothelium-dependent relaxation (Seegers et

al 2002), and a role in apoptosis (Atsumi et al 1998, 2000; Perez et al 2006)

The commonly known iPLA2, iPLA2β and iPLA2γ are referred to as Group VIA-1, Group VIA-2 and Group VIB respectively, using the newer Group numbering system for PLA2 (Dennis 1997; Six and Dennis, 2000; Balsinde et al 2002; Schaloske and Dennis, 2006) Group VIA-1 and A-2 are splice variants of the same gene and are expressed in the cytosol (Ma et al 1999) iPLA2γ is a distinct gene product localized to the endoplasmic, peroxisomal, and mitochondrial membranes (Mancuso et al 2000; Cummings et al 2002), and

iPLA2γ is involved in arachidonic acid release that ultimately leads to eicosanoids formation (Murakami et al 2005) iPLA2γ is widely expressed in human tissues but is particularly enriched in the heart, placenta, and skeletal muscles (Leslie, 2004) iPLA2γ showed different sensitivities towards enantiomers of bromoenol lactone, a specific suicide-inhibitor for iPLA2, as compared to iPLA2β (Jenkins et

al 2002)

iPLA2δ is a membrane protein (146 kDa) expressed in the neurons of

human and mice There is a homologue in Drosophila which plays a crucial role

in brain development (Schaloske and Dennis, 2006) iPLA2ε, iPLA2ζ, and iPLA2η were shown to hydrolyze both linoleic acid and arachidonic acid at the sn-2 position with no requirement for free Ca2+ ions requirement (Jenkins et al 2004) All three enzymes were inhibited by bromoenol lactone at submicromolar levels These enzymes also possess high triacylglycerol lipase and acylglycerol transacylase activities It was suggested that the three enzymes might play a role

in the regulation of triacylglycerol homeostasis (Jenkins et al 2004)

The generation of lysophospholipid acceptors by iPLA2 and regulation of fatty acyl-turnover resulted in them being labeled as “housekeeping” proteins (Hooks and Cummings, 2008) iPLA2 complete these tasks while releasing relatively low level of arachidonic acid or lysophosphatidic acid, which limits inflammation and cell death The functions of iPLA2 also appear cell dependent,

as their roles as housekeeping enzymes might not apply to all cells iPLA2 has been implicated in playing a fundamental role in mediating electrophysiologic

alterations during ischemia (Mancuso et al 2003), in modulating the function of mitochondria during the onset of diabetes (Han et al 2007), and as important effectors of calcium signaling via its role in capacitative Ca2+ entry (Smani et al 2008; Wolf et al 1997)

2 cPLA 2 in normal brain

The first report on the occurrence of phospholipases in brain appeared in

1962 (Gallai-Hatchard et al 1962), when it was shown that human postmortem brain tissue hydrolyzed labeled phosphatidylcholine (PtdCho) into free fatty acid and lysophosphatidylcholine Subsequently, Webster and Cooper (1968) reported the presence of PLA1 and PLA2 activities in rat brain slices and homogenates PLA2 activity was then determined in rat brain synaptosomes and

in neuronal and glial cell preparations obtained from rabbit brain (Woelk et al 1978)

2.1 Molecular genotype

cPLA2 was first characterized in platelets and macrophage cells (Clark et

al 1995) The inferred sequence of murine cPLA2 is more than 90% homologous

to the human cPLA2 sequences, indicating great structural similarity between cPLA2 from different species (Kramer and Sharp, 1997) The cDNA of cPLA2 was cloned from macrophages’ libraries (Clark et al 1995; Sharp et al 1991) and the promoter of the cPLA2 gene was isolated (Tay et al 1994; Miyashita et al 1995; Morri et al 1994) Together, the data showed that the cDNA of cPLA2 consisted

of a total of 2880 nucleotides, in which about 200 nucleotides comprised the 5’ untranslated region and about 500 nucleotides comprised the 3’ untranslated region The 3’ untranslated region of the cPLA2 mRNA contains regions with increased AU content and multiple conserved AUUUA sequences that appear to regulate mRNA stability enhancing expression in mitogen-stimulated mesangial cells (Hack et al 1995) Even though the complete chromosomal DNA for cPLA2

has not yet been sequenced, there appear to be at least 7 introns, and some features of the genetic control mechanisms of the cPLA2 gene were revealed by analysis of 5’-flanking sequence (Tay et al 1994; Miyashita et al 1995; Morri et

al 1994) Additional regulation appears to be mediated post-transcriptionally

The genomic location of cPLA2 and associated variable markers has been identified (Hack et al 1995) The genes for human cPLA2-α, -β, -γ map to chromosomes 1, 15, and 19, respectively (Farooqui and Horrocks, 2007) A polymorphic CA repeat appears 160 bases upstream from the transcriptional start site in both the rat and human 5’-flanking regions (Miyashita et al 1995; Morri et al 1994; Tay et al 1995) Even though the complete chromosomal DNA for cPLA2 has not yet been sequenced, there appears to be at least 7 introns, and some features of the genetic control mechanisms of the cPLA2 gene were revealed by analysis of 5’-flanking sequence (Tay et al 1994; Miyashita et al 1995; Morri et al 1994) Additional regulation appears to be mediated post-transcriptionally

2.2 Localization and distribution of cPLA 2

The basal expression of cPLA2α mRNA under normal conditions is very low in neuronal and glial cells of brain tissue (Owada et al 1994) In human cerebral cortex, cPLA2 is present in astrocytes of gray matter (Stephenson et al 1994) cPLA2 activity present in cytosolic fractions of the hindbrain displays the highest specificity activity, followed by spinal cord, midbrain, and forebrain (Ong

et al 1999) In cortical cultures, cPLA2α is expressed by both astrocytes and neurons (Luo et al 1998) In the rat, forebrain and midbrain are very lightly stained with cPLA2α antibody; the hindbrain in contrast, contains many densely labeled nuclei The dorsal and ventral cochlear nuclei, the initial portions of the ascending auditory pathway, and the superior olivary nucleus, showed dense staining as well (Farooqui et al 2000b) Purkinje neurons of the cerebellar cortex itself were labeled, and deep cerebellar nuclei, which receive afferents from the Purkinje neurons, were also labeled (Farooqui et al 2000b) Recent immunolabeling and in situ hybridization studies indicate that cPLA2α is localized

in somata and dendrites of Purkinje cells, whereas cPLA2β is present in granule cells of rat brain (Shirai and Ito, 2004)

2.3 cPLA 2 functions

Depending on the type of tissues or cells, cPLA2 has been implicated in various cellular responses such as differentiation, inflammation, mitogenesis, and cytotoxicity Intracellular PLA2 are the main mediators in agonist-regulated

specific for arachidonic acid and generates a major pool of arachidonic acid in response to pro-inflammatory factors (Serhan et al 1996; Balsinde et al 1999) Free arachidonic acid is further metabolized into prostaglandin H2 (PGH2) via prostaglandin G2 (PGG2) by two cyclooxygenase (COX) isoforms (Vane et al 1998) Degradation of neural membrane phospholipids by cPLA2 is the rate-limiting step for the generation of a variety of proinflammatory mediators including prostanglandins, leukotrienes, thromboxanes, and platelet activating factor Translocation of cPLA2 paralogs is needed for hydrolysis of membrane phospholipids Owing to the presence of Ca2+-dependent phospholipid-binding domain at the N-terminal region, cPLA2 is translocated in a Ca2+-dependent manner from cytosol to the nuclear or other cellular membranes (Clark et al 1995), where other downstream enzymes, including the cyclooxygenases and lipoxygenases responsible for the metabolism of arachidonic acid to eicosanoids, are located This gives cPLA2 access to its membrane-associated phospholipid substrate In neural membranes, cPLA2 activity and arachidonic acid release are linked to dopamine, glutamate, serotonin, P2-purinergic, cytokine, and growth factor receptors through different coupling mechanisms Some receptors involve G-proteins and others do not Collectively, these mechanisms modulate the release of arachidonic acid and levels of second messengers in brain tissue (Farooqui et al 2000b)

2.4 Fatty acid released by cPLA 2

Arachidonic acid (AA) is distributed rather evenly in the gray and white matter and among the different cell types in the brain In resting cells, AA is stored within the cell membrane, esterified to glycerol in phospholipids (Bloom et

al 2002) AA release from phospholipids in cells is stimulated by several neurotransmitters, including glutamate and its analogs, dopamine, serotonin, endothelin, and ATP, which in turn activate the multiple forms of cPLA2 (Sanfeliu

et al 1990; Kim et al 1995; Bruner and Murphy, 1993; Stella et al 1997; Dumuis

et al 1990; Lazarewicz et al 1988) The release of free AA within the brain tissue

is of considerable physiological importance, and it has three possible fates: reincorporation into neural membranes by reacylation reactions, diffusion outside the cells, and metabolism of AA in the brain AA is oxidized by three distinct enzyme pathways expressed in neural cells to form various oxygenated metabolites, namely cyclooxygenases, lipoxygenases, and cytochrome P450 epoxygenases (Funk, 2001) The metabolites generated are prostaglandins and thromboxanes, leukotrienes, and epoxyeicosatrienoic acid respectively (Farooqui and Horrocks, 2007), they are known collectively as eicosanoids They play important roles in regulating signal transduction, gene transcription processes and also in inducing and maintaining acute inflammatory responses (Wolfe and Horrocks, 1994; Phillis et al 2006)

Inhibition of cPLA2 was found to be favorable for neuronal survival after ischemia (Arai et al 2001) It was shown that the onset of hypoxia leads to a sustained increase in the concentration of intracellular Ca2+ (Grondahl et al

both neurons and glial cells In addition, AA release is also activated by ROS (Tournier et al 1997; Xu et al 2003) following ischemia (Phillis and O’Regan, 2003) The products of this oxidation, isoprostanes (Basu, 2004), and 4-hydroxynonenal (peroxidation product of AA) (Varadarajan et al 2000; Horrocks and Farooqui, 2004), are known to retain their toxic properties for longer time periods than free radicals

2.5 cPLA 2 inhibitors

The pharmacological inhibitor of choice for cPLA2 is arachidonoyl trifluoromethyl ketone (AACOCF3) The problem with the available inhibitors of PLA2 isoforms is their specificity AACOCF3 inhibits not only cPLA2 activity, but also inhibits cyclooxygenase and acyltransferase activities in non-neural cells (Cummings et al 2000; Fuentes et al 2003) Methyl Arachidonoyl fluorophosphonate (MAFP) is a selective, active-site directed, irreversible inhibitor of bovine brain cPLA2 (IC50, 0.5 µM) and iPLA2 (IC50, 0.75 µM), but has

no effect on sPLA2 It inhibits enzymic activity by reacting with a serine residue at the active site In cortical neuronal cultures, MAFP inhibits Aβ-mediated stimulation of cPLA2 activity (Kriem et al 2005) It is important to note that MAFP and AACOCF3 both inhibit cPLA2 at concentrations around 5-10 µM (Cummings

et al 2007; Ackermann et al 1994)

All isoforms of brain PLA2 are strongly inhibited by antimalarial drugs in a dose-dependent manner (Farooqui and Horrocks, 2007) One of the earliest, and still commonly used antimalarial drug is quinacrine (mepacrine) It is also well

documented that quinacrine might exert certain anti-inflammatory properties by acting as a phospholipase inhibitor (Horrobin et al 1977; Al Moutaery and Tariq, 1997) Quinacrine is one of the non-specific inhibitors of PLA2, inhibiting cPLA2

and iPLA2 activities from several sources (Ginsburg et al 1993; Hope et al 1993) Quinacrine appears in monkey brain 24 h after intrauterine administration (Dubin et al 1982), indicating that this inhibitor can cross the blood brain barrier

It has also been administered (5 mg/kg) to rats that underwent 2 h of middle cerebral artery occlusion (Estevez and Phillis, 1997) The administration of quinacrine resulted in a marked reduction in neurological deficits after 24 h of reperfusion (Estevez and Phillis, 1997) It could also suppress oxygen radical release from human macrophages (Struhar et al 1992)

3 iPLA 2 in normal brain

One class of PLA2 that does not require Ca2+ for its activity is the cytosolic calcium-independent PLA2 (iPLA2) Like cPLA2, iPLA2 catalyzes phospholipids via an active site serine nucleophile in concert with an essential active site aspartate to form a similar catalytic dyad (Tanaka et al 2004; Rydel et al 2003; Tang et al 1997; Wolf and Gross, 1996) iPLA2 activity was first recognized in studies carried out with lysosomal acidic hydrolases This enzyme has been purified, cloned, and sequenced (Ackermann et al 1994; Ma et al 1997) iPLA2

is a group of enzymes that are phylogenetically related to patatin lipases (Tanaka

et al 2004) Brain iPLA2 hydrolyzes the sn-2 fatty acid from phosphatidylcholine (PtdCho) with preference for and in order of linoleoyl > palmitoyl > oleoyl >

arachidonyl groups All iPLA2 splice variants contain conserved binding (GXGXXG) and lipase (GXSXG) sequence motif, with the catalytic serine and aspartate

of 88 kDa, along with an additional 54 amino acid proline-rich insertion in the last

of the eight ankyrin repeats (residues 395 – 449) This additional 54 amino acid insertion accounts for the major difference between the human enzyme and other species Overall, human and rodent iPLA2 shared 90% sequence identity

The gene coding for human iPLA2 enzyme was identified and localized on chromosome 22q13.1 (Larsson Forsell et al 1999) It consists of at least 17 exons span more than 69 kb By computational identification and analysis,

transcript of human iPLA2 gene generated multiple transcripts encoding iPLA2

enzyme with distinct tissue distributions and functions, due to alternative splicing (Larsson Forsell et al 1999) The first exon was found in the 5’-UTR and this region was suggested to be involved in negative regulation of translation initiation (Sachs, 1993) Additional sequences in the 5’-UTR suggests the presence of alternative splice sites in the first intron or the presence of an alternative promoter The putative promoter for the iPLA2 gene has been partially characterized and shown to contain a CpG island and several potential Sp-1-binding sites but lack a TATA-box, suggesting that iPLA2 is a housekeeping gene (Larsson Forsell et al 1999) The 5’-flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence Northern blot analysis detected four iPLA2 transcripts with distinct tissue distribution, and Western blot analysis of subcellular fractions showed association of iPLA2 with membranes

3.2 Localization and distribution of iPLA 2

The cytosolic fraction from brain tissues showed iPLA2 activity This enzyme was purified from rat brain to homogeneity using multiple column chromatographic procedures, which produced a very low yield The purified enzyme had a specific activity of 4.3 µmol/min/mg Regional distribution studies have indicated that iPLA2 is present in all brain regions with the highest activity in the striatum, hypothalamus, and hippocampus (Ong et al 2005)

High levels of expression of iPLA2 are present in most regions of the forebrain under basal conditions, consistent with an important “housekeeping” function of the enzyme In the study of normal monkey brain by Ong et al 2005, densely labeled iPLA2 immunoreactivity was observed in structures derived from the telencephalon, including the cerebral cortex, septum, amygdala, and striatum The neuropil of the cerebellar cortex is moderately densely stained Lightly labeled immunoreactivity was observed in structures derived from the diencephalon, including the thalamus, hypothalamus, and subthalamic nucleus The brainstem was also lightly labeled, with the exception of the central gray and the locus ceruleus At electron microscopy level, iPLA2 immunoreactivity was observed in the nuclear envelope of neurons, dendrites and axon terminals (Shirai and Ito, 2004; Ong et al 2005) Glial cells and mural cells in the walls of blood vessels do not show iPLA2 immunoreactivity In situ hybridization and immunolabeling study of the rat’s cerebellum found iPLA2 to be present in granule cells, stellate cells and also in the nucleus of Purkinje cells (Shirai and Ito, 2004) Strong signals of iPLA2 were observed in the olfactory bulb, hippocampus CA 1-3, and dentate gyrus

3.3 iPLA 2 functions

In brain tissues, the basal expression and activity of iPLA2 is higher than either cPLA2 or sPLA2 (Molloy et al 1998; Farooqui et al 1999), and its protein expression decreases during aging (Aid and Bosetti, 2007) iPLA2s are not only responsible for regulation of membrane phospholipid homeostasis

(“housekeeping”) in cells (Balsinde et al 1995) and play important roles in intracellular signal transduction, but also ensures smooth functioning of the electron transport chain by maintaining the integrity of mitochondrial membrane cardiolipin and the mitochondria permeability transition pore (Gadd et al 2006)

The mammalian brain is particularly enriched in long-chain polyunsaturated fatty acids (PUFA), namely the arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) Both in vivo and in vitro evidences support the release of DHA by iPLA2 (Strokin et al 2003; Strokin et al 2007; Green et al 2008) Bromoenol lactone (BEL) (Strokin et al 2003) and more specifically, the inhibition of group VIB iPLA2 by small interfering RNA (Strokin et

al 2007), inhibited DHA but not AA release from phospholipids of astrocytes stimulated with ATP In rat hippocampal slices, cAMP/PKA pathway is shown to

be involved in the stimulus-induced activation of iPLA2 and release of DHA (Strokin et al 2006) BEL also inhibited oxygen/glucose deprivation-induced DHA release from hippocampal phospholipids (Strokin et al 2006)

iPLA2 can also modulate calcium homeostasis by promoting replenishment of intracellular calcium stores (Wilkins and Barbour, 2008) iPLA2

was shown to be involved in the store-operated calcium entry in rat cerebellar astrocytes (Singaravelu et al 2006) iPLA2 plays an important role in synaptic plasticity by regulation of hippocampal α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors (Menard et al 2005b) iPLA2 also plays a role in memory This was evidenced by hippocampal injection of BEL which resulted in

the attenuation of short-term and long-term memory in inhibitory avoidance learning (Schaeffer and Gattaz, 2005) At the same time, BEL also impairs

spatial memory of mice (Fujita et al 2000)

3.4 Fatty acid released by iPLA 2

Docosahexaenoic acid (DHA) belongs to the n-3 (ω3) PUFA family with six double bonds Brain tissue is particularly rich in DHA In human brain gray matter, DHA accounts for approximately 24% of total acyl groups in PtdEtn and 37% of total acyl groups in PtdSer (Farooqui and Horrocks, 2007) It is highly enriched in excitable neural membranes of the cerebral cortex and retina (Lauritzen et al 2001) DHA, supplied with diets, was often found to be favorable for decreasing lipid peroxidation in the brain (Glozman et al 1998; Hossain et al 1998; Kubo et al 1998) In astrocytes, DHA is the major fatty acid released from iPLA2 (Strokin et al 2003) Inhibition of DHA release from hippocampal slices and enrichment the cultures with DHA provide neuroprotection after onset of oxygen-glucose deprivation (Strokin et al 2006)

The most important phospholipid subtype for DHA is plasmalogens (Pls) The turnover of DHA involves a deacylation/reacylation cycle (Farooqui et al 2000c) Newly taken up DHA incorporates predominantly into the Pls species (Farooqui and Horrocks, 2001; Nagan and Zoeller, 2001) The differences between DHA-containing bilayers and AA-containing bilayers are that, DHA-containing bilayers have extremely high water permeability, minimal interaction with cholesterol, and loose acyl chain packing (Mitchell et al 1998; Stillwell et al

2005) It is suggested that DHA acyl groups provide lipid microdomains that serve as platforms to interact with proteins in neural membranes for compartmentalization, modulation, and integration of signaling (Ma et al 2004)

An important property of Pls is that they contain a vinyl-ether bound side chain at the sn-1 position, in which the double bond of the vinyl-ether group is highly reactive for oxidation, especially by reactive oxygen species (ROS) An antioxidative effect of DHA observed in vivo stems from the fact that, Pls serve

as natural scavengers for ROS and can provide neuroprotection during the oxidative stress (Strokin et al 2006)

DHA is also the substrate for synthesis of neuroprotectin D1 (NPD1) and resolvin Both molecules exhibit strong neuroprotective and neurotrophic properties after ischemia-reperfusion (Marcheselli et al 2003), in cell survival and resolution of inflammation (Lukiw and Bazan, 2006, 2008; Schwab et al 2007) Several studies demonstrated that DHA-enriched diets provide protection from lipid peroxidation The neuroprotective role of DHA derivatives such as NPD1 and resolvin, suggests that iPLA2 would serve as a possible mediator in for the prevention of ischemic damage (Strokin et al 2006)

3.5 iPLA 2 inhibitors

The functions of iPLA2 in cell physiology were derived from studies using pharmacological and molecular inhibitors The pharmacological inhibitor of choice for iPLA2 is bromoenol lactone (BEL or (E)-6-(1-bromoethyl) tetrahydro-3-