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The M3 Muscarinic Acetylcholine Receptor Mediates p42mapk

Activation and c-fos mRNA Expression in Oligodendrocyte

Progenitors

By

FADIRAGHEB

Department of Pharmacology and TherapeuticsMcGill UniversityMontreal, Canada

March,1999

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillmentof the requirements for the degree of

Master of Science

Copyright © Fadi Ragheb) 1999

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TABLE OF CONTENTS

ABSTRACT•..•••...••.•.•.••.•••••.•••..••••.•••.•••.•••..••.•••••.•.•.••.•..••••••..••..•••.••...•.•..•••••••••••2ABSTRAIT•••••••••••••.•..•.•••...•••••••••.••.••••••••••••••••••••.•••.•••.••••••••.••.•••••••.•••.••..•..••••.••4ACKNOWLEDGMENTS.....•••••....•••..•.••.•••••••••••.•..•••.•••.•••••••••••••••.••••••...••....•••.••••••7ABBREVIATIONS••.••.••.•••.•••••..•.•••...•......•...••.•.••....••••••••••••••••••••••.•••.•••••.••••.••••••••9

CHAPTER 1: INTRODUCTION 12

BRJEF OVERVIEW OF HISTORICAL DEFINITIONS AND FUNCTION Of OL•..•...•.•..••.•13Introduction , _ 13

STAGES OF OLiGODENDROCYTE DEVELOPMENT 15Origin of Oligodendrocytes , , , ." ,. '" '" , 15Overview of O/igodendrocyte Developmental Lineage _ , 16

OLIGODENDROCYTE TYPE 2A A5TROCYTE PROGENITOR , , 19Developmenta/ Regulation , , 20Ofigodendrocyte Progenitor Differentiation and Maturation , , , 22

THE MUSCARINIC ACETYLCHOLINE RECEPTOR••.•.••....•.••••.........•••••..••.•.......•....••24Introduction , '" ., , , '" 24Molecular Bi%gy of the mAChR. , '" " _ 26Pharmacological Definitions of Subtypes , -28

MUSCARINIC COUPLING Ta G-PROTEINS AND EFFECTOR MOLECULES•.•••..•.•.•.••••31Introduction , ., , , '" ,_ , '" 31Composition and Structure ofHeterotrimeric G-proteins , , 31Adenylate Cyc/ase , 34Phospholipase C '" , , '" ., 36Phospho/ipase A2 _ , 37

MITOGEN-ACTIVATED PROTEIN KINASE•...••.••..•.........1••••••••••••••••••••••••••••••••••••••••••39Brief Overview: The MAPK Classes , , , , 39G-protein Coupled Receptors Signaling to MAPK. _ , , 40Novel Signaling Pathways Linking G-protein Coup/ed Receptor to MAPK Pathway.. .42Mapk Regulation of AP-1 Transcription Factor and the Induction of c-fos geneExpression , , , '" 45

CHAPTER TWO: STATEMENT OF PURPOSE..•.•...•••.••..•..••••..............•.49

CHAPTER THREE: MATERIALS AND METHODS••...•••••••.•••..................53Materia/s '" '" _.. , '" '" '" , , , '" , .54Cell Culture , '" .. , " , , , ,.. ,.54Immunocytochemistry , , , '" , , 56Western /mmunoblot Analysis _ '" .. ' , .. ' '" , ,.. ,..57Data Ana/ysis '" '" _ '" 58

CHAPTER FOUR: RESULTS....•.•.••••••••••••••...••.•••...•.••••.•••••...•.........•.•.60Resu/ts 61Figures 70

CHAPTER FIVE: DiSCUSSiON..•.•••.••••••••••••••••.••••••••••••••••••.••...•......••••82

CtiAf»1rE:Ft 51)(: FtE:f=E:FtE:..c:E:!) St6

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ABSTRACT

Oligodendrocytes, the ceUs that produce and maintain myelin in the central

nervous system CCNS), express several muscarinic acetylcholine (ACh) receptor

(mAChR) subtypes. Carbachol (CCh), a stable analogue of ACh, stimulates various

intracellular signais through mAChRs, including p42-mitogen-activated protein kinase

(p42mapk) activation and c-fos gene expression. The present study examined the mAChR

subtype(s) and the signaling pathway(s) involved in both responses using purified cultures

of oligodendrocyte progenitors from newbom rats.

The M3 mAChR selective antagonist 4-DAMP and its irreversible analogue 4­

DAMP-mustard, but not pirenzepine (M1) or methoctramine (M21M4), prevented p42mapk

activation and c-fos mRNA expression. Pretreatrnent with a phospholipase C (PLC)

inhibitor U73122 but not the inactive analogue U73343 blocked both responses, while the

phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect, suggesting the

involvement ofPLC but not PI3K.

In view of the fact that the M3 mAChR mediates p42mapk activation as weB as c­

fos mRNA expression, the MAPK Kinase (MEK) inhibitor PD 098059 was used to

establish a link between both responses. PD 098059 pretreatment caused no significant

attenuation in c-fos mRNA levels, demonstrating that this response may not be dependent

on p42mapk activation. However, the phospholipase Al (PLA2) inhibitors quinacrine and

AACOCf3 abolished c-fos mRNA expression, implicating arachidonic acid and/or its

bioactive eicosanoid metabolites.

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• ln summary, these results demonstrate that in oligodendrocyte progenitors, the M3

mAChR is mediating both p42D1apk activation and c-fos gene expression via PLC but not

PI3K, whereby c-fos gene expression is not regulated by p42mapk but by PLA2.

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RÉSUMÉ

Les oligodendrocytes produisent et maintiennent la myéline dans le système

nerveux central (SNC). Ces cellules possèdent plusieurs sous-types des récepteurs

acétylcholine (Ach) muscariniques (mAChR). Un analogue stable de l'acétylcholine,

carbachol (CCh) stimule de nombreux signaux intracellulaires à partir des mAChRs, dont

l'activation de la kinase p42mapk et l'expression du gène c-fos. La présente étude a

exanliné le(s) sous-type(s) des mAchRs et ces deux réponses intracellulaires, en utilisant

des cultures purifiées des prédécesseurs d'oligodendrocytes provenant de rats nouveaux

nés.

4-0AMP et son analogue irréversible 4-0A~IP-mustard sont des antagonistes

sélectifs du sous-type M3 des mAChRs et ont obvié l'activation de p42mapk et l'expression

de c-fos. à l'opposé de pirenzine (Ml) et methoctramine (M2/M4). Pré-traitement avec

U73122, qui inhibe la phospholipase C (PLC), a supprimé les deux réponses.

Wortmannin, un inhibiteur de la phosphoinositide 3-kinase (PI3K), et U73343, un

analogue inactif d'U73 122, n'ont eu aucuns effets sur ces deux événements. Ces résultats

suggèrent que PLC, mais pas PB-K, est impliqué dans la signalisation intracellulaire à

partir du récepteur M3 avec l'utilisation du CCh.

Étant donné que le récepteur M3 de cette famille cause à la fois l'activation de

p42mapk et l'expression de c-fos, PD 098059, un inhibiteur de la MAPK Kinase (MEK),

fut utilisé pour déterminer si un lien existe entre ces deux réponses. Pré-traitement avec

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PD 098059 n'a pas causé d'atténuation significative sur l'expression d'ARN messagères,

démontrant que cette réponse ne dépend pas de l'activation de p42mapk• D'autre p~ les

inhibiteurs de la phospholipase A2 (PLA2) quinacrine et AACOCf3 ont aboli

l'expression d'ARN messagères du gène c-fos. Ces résultats impliquent l'acide

arachidonique et/ou ses métabolites eicosanoïdes qui sont bioactifs.

En sommaire, ces résultats démontrent que dans les prédécesseurs

d'oligodendrocytes, le M3 mAChR est responsable pour l'activation de p42mapk et

l'expression du gène c-fos. PLC, mais pas PI3-K, est impliqué dans ces deux réponses.

Par contre, l'expression de c-fos dépend de PLA2 mais pas de p42mapk•

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Dedicated to my parents, for their love, confidence, and support that

they have given me during these years

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ACKNOWLEDGMENTS

1 would like to thank tirst and foremost my supervisor Dr. Guillermina Almazan

for developing a scientist out of me, committed, dedicated, and inspired. Her constant

support, help, and contribution in my work are of priceless value, and her leading

example as both a scientist and a compassionate individual is of lasting influence and

guidance.

1 would also like to thank Arnani Khorchid and H. N. (Shirley) Liu for their

valuable contribution and assistance in my project , and for the lasting friendship that has

been formed during these years. 1 would like to also acknowledge Ana-Katherine

Martinez for her appreciated input and advice.

My special thanks go to my adviser, Dr. Barbara Esplin for her continuous

support and interest throughout my years al the department, and to Dr. L.f. Congote and

his Ph.O. student Marco of the Endocrinology Unit of the Royal Victoria Hospital for

providing me with the opportunity to believe in research and to allow me to observe and

experience it at first hand.

My gratitude extends to my parents that have always provided me with the

comfort and support, especially during our hard times at our early years in Canada. Their

love and guidance solidified my determination and perseverance. 1 would like to express

my deep gratitude for offering me the opportunity to have a solid education that wi 11

guide me for the rest of my life.

My expressed gratitude is also extended to my sister Shourouk, for her love,

confidence in me, and her patience, for it couldn't have been the same without her. 1aJso

express my admiration to my brother Loay for his role model personality, determination,

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perseverance, and for bis brotherly guidance and support. 1 wish to express my love to

my aIder sister Loma, for her well appreciated support and guidance throughout these

years.

1 wouid like to also extend my deep gratitude to Barbara Milosevic, for her

greatly appreciated encouragement and for her continuous support. 1 also extend my

appreciation to her valuable contribution in the construction of this thesis and my ASN

poster. 1 am also very grateful to Anne Morinville for her assistance in translating my

abstract, to Martin Gagnon for his technical support for constructing my figures, and to

Alan Forster for the valuable work on the photographs.

Last but not least, 1 would like to thank the Department of Pharmacology and

Therapeutics for offering me the opportunity to complete my project in a stimulating

scientific environment.

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• ABBREVIATIONS

AA Arachidonic acid

ACh Acetylcholine

ATP Adenosine triphosphate

Atr Atropine

bFGF Basic fibroblast growth factor

CaMK Calciurn/calmodulin-dependent protein kinases

cAMP 3',5' -cyclic adenosine monophosphate

CBP CREB-binding protein

CCh Carbachol

CNP 2'-3'-Cyclic nucleotide 3'phosphohydroIase

CNS Central nervous system

cPLA2 Cytosolic phospholipase A2

• CRE cA.MP response element

CREB cAMP response element binding protein

DAO Diacy19lycerol

DMEM Dulbecco's modified eagle medium

EDTA Ethylenediamine tetraacetic acid

EGF Epidermal growth factor

E-NCAM Embryonic neural cell adhesion molecule

ERK Extracellular-signal regulated kinase

FAK Focal adhesion kinase

FCS Fetai calf serum

GC Galactocerebroside

GDP Guanosine diphosphate

GFAP Glial fibrillary acidic protein

G-protein Guanosine triphosphate-binding protein

GTP Guanosine triphosphate• HBSS Hanks balanced salt solution

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• HETE Hydroxyeicosatetraeinoic acid

HHSiD Hexahydro-sila-difenidol

ICso 500/0 inhibitory concentration

IGF I~II Insulin-like growth factor l, II

IP3 Inositol trisphosphate

JNK NHrterminal Jun kinase

mAb Monoclonal antibody

mAChR Muscarinic acetylcholine receptor

MAG Myelin-associated glycoprotein

MAPK Mitogen-activated protein kinase

MBP Myelin basic protein

MEK MAP kinase kinase

Meth Methoctramine

min minutes

MOG Myelin/oligodendrocyte glycoprtoein• nAChR nicotinic acetylcholine receptor

NT-3 Neurotrophin.3

0-2A Oligodendrocyte-type 2A astrocyte progenitors

aL a ligodendrocyte

aLP Oligodendrocyte progenitors

p42ffiapk p42-mitogen-activated protein kinase

PDGF Platelet-derived growth factor

PI Phosphatidylinosito1

PI3K PhosphatidyIinosito1-3-kinase

PIK Phosphatidylinositol kinase

PIP2 Phosphatidylinositol-4,5-bisphosphate

Pir Pirenzepine

PKA Protein kinase A

PKC Protein kinase C

PLA2 Phospholipase A2• PLC Phospholipase C

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• PLD Phospholipase D

PLP Proteolipid protein

PMA Phorbol-12,13 myristate acetate

PNS Peripheral nervous system

POA Proligodendroblast antigen

Pre-Gale Pre-Galactocerebroside

Pro-OL Pro-oligodendroblast

PTX Pertussis toxin

Quin Quinacrine

Raf MAP Kinase kinase kinase

SAPK Stress-activated protein kinase

SClP Suppressed cAMP-inducible POU

SFM Serum-free medium

SIE Sis-inducible element

SRf Serum response factor• TCf Temary complex factor

Thr Threonine

TM Transmembrane

TPA Phorbol-12, 13 myristate acetate

TRE TPA response clement

Tyr Tyrosine

Wort Wortmannin

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CHAPTERONEINTRODUCTION

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INTRODUCTION

(1) HISTORICAL DEFINITION AN}) FUNCTION OF

OLIGODENDROCYTES

(A) INTRODUCTION

Oligodendrocytes, which with astrocytes fonn the subclass of macroglia and

together with the microglia subclass fonn the family of neuroglial cells, are derived from

stem cell precursors arising from the subventricular zones of the developing central

nervous system (eNS). Astrocytes contribute to the structural support of the central

nervous system and function as a source of growth factors and cytokines, while microglia

are bone·marrow derived macrophages which serve as immunocompetent cells in the

central nervous system. Oligodendrocytes serve the specialist role of synthesizing and

maintaining the myelin sheath of neuronal axons. Myelin, a compact and well-organized

membranous structure. is an insulating sheath that wraps around electrically conductive

axons to maintain the electrical propagation down the axon and to prevent its dissipation

in both the CNS and the peripheral nervous system (PNS). The process of myelination

depends on glial-neuronal interaction, whereby a stable relationship between each

differentiated oligodendrocyte and short segments of several adjacent axons regulate this

process.

Rudolph Virchow (Virchow, 1858) was among the pioneers to describe neuroglia

and propose two functions for these cells: mechanical support of nerve cells and tissue

repaîr. He had also introduced the term myelin and described sheaths around nerve

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fibers. Discoveries achieved in the twentietb century have confirmed the assigned

functions by Virchow, and have separated the functional roles played by astrocytes,

oligodendrocytes, and microglia in the central nervous system. Oligodendrocytes were

not identified as a distinct neuroglial subpopulation until many years after by Robertson

(Robertson, 1899), Cajal (Cajal, 1913), and Hortega (Hortega, 1921; 1928). Through

silver nitrate impregnation techniques, Hortega described the existence ofthese ceUs and

explained their morphology as oligo-dendro-glia, which translates from the Greek

language as literally "few-tree-glue". Hortega's contribution ificluded the recognition of

oligodendrocytes as the ceUs responsible for myelin formation that enwrap axons in the

white matter of the CNS.

CNS myelination is crucially dependent on the interaction of oligodendrocytes

and axons. Myelination occurs when the membranous processes of oligodendrocytes

contact and ensheath axons, and compact to fonn the myelin lamellae needed for

saltatory axonal conduction. This process also relies on cell surface and extracellular

matrix molecules that promote interactions between myelin and axons (Bartsch et al.,

1993). ln the CNS, one oligodendrocyte can myelinate more than one axonal segment,

while Schwann ceUs, the myelinating ceUs of the PNS, cao myelinate only one axon

(Morrell et al., 1993).

Due to their critical role embodied in myelin production and thus nerve impulse

maintenance, oligodendrocytes' death, degeneration, or myelin integrity aberration

results in debilitating sensory or motor conditions or diseases in humans, namely multiple

sclerosis (Quarels et al., 1993). Therefore, understanding the cellular homeostasis of

oligodendrocytes and myelin production, in addition to the environmental cues that

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contribute to their development is of crucial importance. The development of severaJ in

vitro culture techniques for oligodendrocytes (McCarthy and de Vel1is, 1980; Szuchet

and Steffanson, 1980; Szuchet et al., 1980) simplified the study of their development,

metabolism, and myelin production, and facilitated the isolation and purification of

progenitor and mature oligodendrocytes from mammalian tissue.

(B) STAGES OF OLIGODENDROCYTE DEVELOPMENT

• Origin of Oligodendrocytes

Oligodendrocytes are generated over a short period of brain development during the

late gestational and early postnatal periods in mammals. In the forebrain and cerebellum,

oligodendrocyte progenitors (OLPs) commence their life as the immature

neuroectodermaI ceUs of the subventricular zone in the subependymal layer on the

surface of the lateral ventricles (Paterson et al., 1973; for review see Goldman, 1992;

Goldman, 1995). The subventricular zone is the germinal matrix area adjacent to

cerebral ventricles. Subsequently, these primordial glial ceUs migrate into gray and white

matter, whereby they differentiate and proliferate during migration. Their migration

occurs in a posto-anterior gradient (relative from the brainstem to the forebrain) to the

various areas of the brain, and also move radialy from the subventricular zone into thcir

terminal destination, the white matter nerve tracts. OLPs migrate along these nenTe

tracts, whereby they are responsive to environmental cues, leading them to proliferate,

differentiate, and synthesize myelin sheaths to enwrap adjacent axons.

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• Overview of the OUlodendrocyte Developmental Lineale

Oligodendrocytes undergo controlled migration, proliferation and differentiation.

They both secrete and respond to severai growth factors, largely influencing their cellular

architecture and morphology. From the subventricular zones~ oligodendrocytes originate

as neuroectodermal ceUs that migrate and mature into postmitotic myelin-producing cells.

Oligodendrocyte progenitors expand from approximately 500~OOO at birth to a terminal

population of 60 million oligodendrocytes in the adult rat brain (Pfeiffer et al.~ 1993).

Confocal microscopy and image reconstruction studies of oligodendrocytes both in

culture and in situ contributed to oligodendrocyte developmental lineage investigation

(Warrington and Pfeiffer~ 1992; Warrington et al., 1993; Morgan et aL, 1992). During

their development, oligodendrocytes express developmental markers that are identified

by cell-specific antibodies. The expression of developmental markers provides a tool to

divide the lineage into distinct phenotypic stages characterized by the capabilities of the

celis to divide, migrate, differentiate, and produce myelin.

The youngest precursor ceUs are the Pre-GD3 ceUs (see fig. 1). The Pre-GD3

precursor cells are proliferative, monopolar ceUs expressing the embryonic neural cell

adhesion molecule (E-NCAM) (Hardy and Reynolds, 1991). These precursor cells

differentiate into proliferative and migratory bipolar GD3+ precursors that express

gangliosides recognized by the monoclonal antibodies (mAb) anti-GD3 and A2B5. They

are referred to as the o/igodendrocyte-type 2A astrocytes (O-2A) progenitors, due to their

bipotential to differentiate into either mature oligodendrocytes or type 2A astrocytes

(identified by the expression of glial fibrillary acidic protein, an intermediate filament

protein) (Raff, 1989) via different culturing factors. Eventhough the identity and

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existence of the OLs in vivo is confirmed, there is no evideoce of the existence of the type

2A astrocytes in vivo (Skoff and Knapp, 1991; Norton and Farooq, 1993). A

developmental progression proceeds from the 0-2A stage to the Pro-o/igodendroblast

(Pro-DL) stage. Pro-OL cells possess multipolar, postmigratory, proliferative potentials,

and the phenotypic stage is characterized with the mAbs 04 and AOO? that recognize the

sulphated surface antigeo POA (Bansal et al., 1992). A transient developmental stage

follows the Pro-aL stage, named the Pre-Galactocerebroside (Pre-Gale). The Pre-GalC

ceUs stain positive to the mAb R but oot to the anti-GalC mAb 01. The GD3, Pro-DL,

and Pre-GaiC stages have often been labeled collectively as the "O-2A" progenitor;

however, they are functionally distinct in terms of electrical activities and responses to

neural-cell-derived mitogens and phorbol esters (Warrington et al., 1993; Gard and

Pfeiffer, 1993).

Yet to commence myelin synthesis, the onset of differentiation is necessary.

Terminal differentiation of oligodendrocyte progenitors is identified by the synthesis and

transport to the surface of GalC and sulphatides, and the synthesis of 2'-3' -Cyclic

nucleotide 3'phosphohydrolase (CNP), and later myelin basic protein (MBP), proteolipid

protein (PLP), myelin-associated glycoprotein (MAG), and myelin/oligodendrocyte

glycoprotein (MaG), hence the synthesis ofmyelin membranes (Pfeiffer et al., 1993).

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•THE OLIGODENDROCYTE LINEAGE

MATURE Dla.cSULCNPPLPM8PYcG

IUM.lTUSE CL

Gale (A.mAt>. 01)

SUL (04/A007. R~)CNP

Pfe·~aIC

R-Ag (~·mAb)

FOAG03"-G03­Yim+- '{im­SCIP+-- SCIP-

Pro-OL

POA ((j.4,'AOC7)A2SSGD3VimSCI?

POGF+oFG~ bF3F A·mA!)

IlJ<l~l~_~GQ3 rQ-~~

Ci03A2BSVlmSCIP

-,?re-GD3

E·NC':'MVIM•

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FIGURE 1: The Oligodendrocyte Lineage. The figure shows the stages of

development that an oligodendrocyte progenitor undergoes before becoming myelin­

producing mature oligodendrocyte. Commonly used stage-specifie markers (antibodies

identifying sorne markers are shown in parenthesis) help identify the different stages

during oligodendrocyte development. Figure is adapted from Pfeiffer et al. (1993>.

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(C) OLIGODENDROCYTE-TYPE 2A ASTROCYTE PROGENITOR

• Phenotype and Characteristics

Extensive in vi/ro studies have been completed on the OL lineage, including

studies on their migration, differentiation, and proliferation. Rat brain oligodendrocyte

precursor ceUs, also named 0-2A progenitors, were first isolated and characlerized in

vitro from the optic nerve by Raff and colleagues (Raff et al., 1983). Consequently, most

attention has been focused on this precursor cell. In culture~ the 0-2A progenitor features

a simple bipolar morphology, and contains a small soma (lO - 15 J,LM). ft is highly

proliferative. It binds the mAb A2B5, a mAb that reacts with a tetrasialoganglioside

(Eisenbarth et al., 1979; Raff et al., 1983). The 0-2A progenitor expresses the

ganglioside GD3 (Goldman et al., 1984) and the NG-2 chondroitin sulfate, in addition to

the intermediate filaments vimentin (Raff et al., 1984; Dubois-Dalcq~ 1987)~ nestin

(Almazan et al., 1993; Gallo and Annstrong, 1995), but does not express antigens of

mature astrocytes or mature oligodendrocytes. It also transiently expresses a seminolipid

and an unknown antigen called "proligodendroblast antigen" (POA) recognized by both

the A007 or 04 monoclonal antibodies (Bansal et al, 1992). 0-2A progenitors express

amply specific transcription factors characteristic of the phenotype: the transcription

factors SCIP/tst-l (Collarini et al., 1992), and myelin transcription factor 1 (MyTl)

(Annstrong et al., 1995). These transcription factors control myelin gene expression~ and

are downregulated as the ceUs develop into post-mitotic oligodendrocytes.

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• Developmental Regulation

The development of oligodendrocytes depends on intrinsic properties of each cell

and the availability of growth factors. The development of oligodendrocytes is Iargely

influenced by soluble factors released in their proximity by other components of the

CNS, such as neurons, astrocytes, and microglia. In addition, oligodendrocytes secrete

autocrine growth factors that can regulate their own development (Compston et al.,

1997). Studies performed in vitro have shown that platelet-derived growth factor (PDGF)

plays a major role in the control of 0-2A development. PDGF is a mitogenic factor for

0-2A progenitors, yet in the continued presence of PDGF, 0-2A progenitors eventually

stop proliferating and acquire mature differentiated oligodendrocyte characteristics

(Noble et al., 1988; Richardson et al., 1988). The source of PDGF has been linked to

neurons expressing PDGF in vivo (Yeh et al., 1991; Sasahara et al., 1991), while in vitro,

type 1 astrocytes have shown to secrete this growth factor (Richardson et al., 1988). In

addition, basic fibroblast growth factor (bFGF) is another growth factor that is involved

in regulating oligodendrocyte development. bFGF is also a mitogen for 0-2A

progenitors (Eccleston and Silberberg, 1985; Saneto and deVeL1is, 1985), whereby unlike

the effect of PDGF, progenitors' continuous exposure to bFGF prevents 0-2A

progenitor differentiation into mature oligodendrocytes (McKinnon et al., 1990), but

rnaintains ils proliferative state. The combined effect of bFGF and PDGF treatment of 0­

2A progenitors result in long periods of proliferation without differentiation (Bogler et

al., 1990). bFGF is produced by astrocytes, microglia, and neurons (Pettmann et al.,

1986; Ferrara et al., 1988; Shimojo et al., 1991).

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On the other hand~ neurotrophin-3 (NT-3) bas been shown to induce proliferative

effects on 0-2A progenitors (Barres et al.~ 1994). In addition, 0-2A progenitors are

influenced by epidermal growth factor (EGF), thyroxine and T3, retinoic acid, and

glucocorticoids, factors that promote progenitor differentiation and maturation in vitro

(Laeng et al., 1994). Other factors that drive progenitors into differentiation include

insulin-like growth factor 1 and Il (IGF-I~ IGF-II) (McMorris and Dubois-Dalcq~ 1988),

in addition ta IGF-I's role in promoting the survival ofprogenitors (Barres et al., 1992).

Moreover, studies performed in our laboratory have demonstrated a role for

glutamatergic, adrenergic, and muscarinic acetylcholine receptors (mAChRs) in

regulating proliferation of 0-2A progenitors. We have previously reported the block of

proliferation induced by stimulating the AMPA/Kainate receptors on 0-2A progenitors

(Liu and Almazan, 1995), while adrenergic stimulation caused no change in the

proliferative state of the progenitors (unpublished results). On the other hand,

pharmacological studies performed in our laboratory characterized the mAChR's

expressed on 0-2A progenitors and their developmental regulation, showing evidence for

the expression of several mAChR subtypes whereby the M3 subtype is the main subtype

expressed during development (Molina-Hoigado et al., unpublished). An interesting

finding was the extensive downregulation ofmAChRs during the differentiation of 0-2A

progenitors to mature differentiated oligodendrocytes, which May indicate a raie for these

receptors during their early development. Moreover, proliferation studies on 0-2A

progenitors showed that cholinergie stimulation of 0-2A progenitors increased the

proliferation of these cells, whereby the muscarinic antagonist atropine blocked this

21

•

•

•

response, demonstrating that muscarinic receptors are responsible for this cholinergie

response (Cohen et al., 1996).

The potentiaI of differentiating into type 2 astrocytes is a characteristic of 0-2A

progenitors. [t is an interesting developmentaI feature, whereby progenitors stop their

differentiation into oligodendrocytes and instead become astrocytes. ln vitro, the

maintenance of progenitors in serum can elicit this switch (Raff et al., 1983). In this

process, progenitors begin to express type 2 astrocyte characteristics~ such as stellate

shape morphology, binding of A2B5, expression of the astrocyte intermediate filament

protein, glial fibrillary acidic protein (GFAP), and failure to express gangliosides, lipids

and proteins characteristic of myelin (Raff et al., 1983). These ceUs have been labeled

type 2 astrocytes in order to he able to distinguish them from type 1 astrocytes, which are

A2B5 negative and display a flat shape.

• Oligodendrocyte Progenitor Differentiation and Maturation

Under certain culture conditions, oligodendrocyte progenitors enter the

maturation and differentiation stage. By withdra\\ing growth factors and maintaining

serum-free culture conditions, oligodendrocyte progenitors differentiate and lose their

proliferative state (Raff et al., 1983). ln vitro, this step is characterized by the loss of

nestin, vimentin, and the antigen A2B5, and the fonnation of multipolar processes, in

addition to the expression of a stage specifie modified lipid sulfatides recognized by the

04 (AOO?) antibody (Sommer and Schachner, 1981; Sarlieve et al., 1983; SansaI et al.,

1989; Knapp, 1991). The cells become reactive to the R-mAB, which reacts with not

only galactocerebrosides, but also to another epitope that is also recognized by the 01

22

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•

•

mAb (Raff et al., 1978; Ranseht, 1982; Bansal and Pfeiffer, 1992; Sommer and

Schachner, 1981). Consequently, myelin specifie proteins are expressed to regulate and

commence the synthesis ofmyelin. Myelin specifie proteins inelude CNP, MAG, MOG~

MBP, and PLP (Pfeiffer et aI., 1981; Raff et al., 1983; Zurbriggen et al., 1984; Dubois­

Dalcq et al., 1986; Dubois-Dalcq, 1987; Knapp et al., 1987, 1988; Amur-Umarjee et al.,

1990; Yim et al., 1995). These events signify the terminal differentiation of the

oligodendrocyte progenitor and preludes the post-mitotie mature oligodendrocyte. At

this stage, the morphology of the mature oligodendroeyte changes, whereby the cell body

darkens, and numerous processes form the stage-characteristic lace like lattice. This

··web" of processes supports the myelin membranous sheath. In the presence of neurons,

this membranous sheath forms a multilamellar~ spirally wrapped sheaths around the

axons, insulating their eiectrically conductivity, and ensuring fast electrical transmission.

23

• (II) THE MUSCARINIC ACETYLCHOLINE RECEPTOR

•

•

• Introduction

The classical neurotransmitter acetyIchoIine (ACh) transduces its signaIs to ceUs

via binding to either the ionotropic nicotinic receptor (nAChR) or the metabotropic

muscarinic receptor (mAChR). Nicotinic and muscarinic receptor subtypes are expressed

in both the PNS and the CNS. In the periphery, mAChRs regulate the synaptic

transmission in the ganglia of the autonomie nervous system, in addition to modulating

the functions of target organs of the parasympathetic nervous system (for review see

Cauifield and Birdsall, 1998). Such reguJation inciudes mediation of smooth muscle

contraction, glandular secretion, and modulation of cardiac rate and force. In the CNS,

mAChRs have been implicated in many important processes, including memory,

learning, temperature regulation, cardiovascular regulation, and control of movement (for

review see Nathanson, 1996). Therapeutically, the administration ofmuscarinic agonists

facilitate Iong-term potentiation and cause Parkinson's disease-like trernors, while

muscarinie antagonists interfere with leaming and memory and overcome the tremors in

Parkinson' s disease. Other therapeutic applications include treatments for asthma,

analgesia, and disorders of intestinal, cardiac, and urinary bladder function (for review

see Caulfield and Birdsall, 1998). In addition, several reports have indicated that

muscarinic agonists cao act as mitogens in cultured neural ceUs, whereby mAChRs may

play an important role in the development of the CNS (Ashkenazi et al., 1989; Gutkind et

al., 1991)

24

•

•

•

The mAChRs are members of the superfamily of hormones and neurotransmitter

receptors. The mAChR is a seven transmembrane domain receptor that is coupled to a

guanosine triphosphate (GTP) binding protein (G-protein). mAChRs regulate the

activities of intracellular second messenger pathways through ion channel and enzyme

activationldeactivation by the interaction with their coupied G-proteins. So far, there are

5 mAChR subtypes: the Ml, M2, M3, M4, and the MS subtypes. The MIIM31M5

mAChRs couple to pertussis toxin- (PTX) insensitive G-proteins (Gq/1t), while the

M2/M4 mAChRs bind PTX-sensitive G-proteins (GiJo)' The former group of subtypes

activate phospholipase C (PLC), phospholipase A2 (PLA2), phospholipase D (PLD),

tyrosine kinase, and calcium influx but do not inhibit adenylate cyclase, while the latter

group of receptors inhibits adenylate cyclase but do not stimulate PLC. However, this

coupling specificity of the mAChR subtypes is not absolute, for the M2 and the M4

subtypes can weakly activate PLC when expressed at high levels in certain cell types

(Ashkenazi et al. 1989a; Tietje et al., 1990; Tietje and Nathanson, 1991). The PTX­

insensitive coupling to PLC is mediated by GCXq, Gall, Ga14, and Ga16, while the PTX­

sensitive coupling to adenylate cyclase inhibition is mediated by Gai or Gao (Katz et al.,

1992; reviewed by Felder, 1995).

Historically, the initial studies indicating the existence of mAChR subtypes were

reported when gallamine showed cardioselective actions (Riker and Wescoe, 1951).

Eventually, Barlow et al. (1976) described significant distinctions in the pharmacological

properties of ileal and atrial muscarinic receptors. Pirenzepine was introduced as a

therapeutic agent for peptic ulcer disease, contributing to the appreciation of the existence

of mAChR subtypes. Pharmacological binding studies reported by Hammer et al. (1980)

25

•

•

•

and functional studies reported by Brown et al. (1980) and Hammer and Giachetti (1983)

provided an explanation for pirenzepine's in vivo selectivity, suggesting the existence of

more than one mAChR subtype

• Molecular Biology of the mAChR

Subsequent to the early pharmacologicaI findings, molecular cloning studies

identified 5 different that are products ofdistinct but homologous (Bonner, (989). Numa

and colleagues cloned the ml and the m2 genes (Kubo et al., 1986a,b), since the

pharmacologicaI profiles of the cloned receptors resembled the MI and the M2 subtypes

that have been pharmacologically characterized earlier. The m3, m4, and mS genes were

thereafter discovered (Bonner et al., 1987, 1988; Peralta et al., (987). The cloned

vertebrate receptor genes cIoned are intronless, and are similar across mammalian species

(Hall et al., 1993; Eglen et al., 1996). Each gene is approximately 460-532 amino acids

in length (m1=460; m2=466; m3=589-590; m4=478-490; m5=531-532). These five

genes encode mAChR glycoproteins that display the structural features of the seven

transmembrane helix G-protein-coupled receptor family. Based on the phannacological

binding studies ofeach receptor resembling the cloned genes, it is now recommended that

Ml, M2, M3, M4, and M5 be used to describe both the pharmacological subtypes and the

molecular subtypes encoded by the cloned genes.

Similar to most members of the G-protein-coupled receptor family whose ligand­

recognition site binds small molecules, there are several major features of muscarinic

receptor structure. First, the ligand recognition site can he located within the outer half of

the membrane-embedded part of the protein. To bind the neurotransmitter ACh, aIl

26

•

•

•

mAChRs have an Asp residue in the distal N-tenninal part of the third transmembrane

domain which is thought to interact with the polar head group of the neurotransmitter and

other amine ligands (Curtis et al., 1989; Spalding et al., 1994). Second, the

transmembrane segments are a-helical, whereby three helices are oriented approximately

perpendicular to the membrane, and four helices are oriented at a more acute angle

(Baldwin et al., 1997). Furthermore, there are two conserved cysteine residues that forrn

a disulfide bond bet\veen the first and third extracellular loops (Kurtenbach et al, 1990;

Savarese et al., 1992). There exists also a conserved triplet of amino acids (Asp Arg Tyr)

at the cytoplasmic interface of TMIII with the second intracellular loop that is crucial for

both the expression and function of the receptor (Zhu et al., 1994; Jones et al .. 1995; Lu

et al., 1997). Moreover, the carboxy-terminus of the mAChR is located on the

intracellular side of the membrane, while the amino-terminus is located on the

extracellular side of the membrane containing one or more glycosylation sites. On the

other hand, the fourth intracellular region plus regions near the transmembrane area in the

second and third internaI loops seem to be targeted for phosphorylation by ligand­

stinlulated negative feedback loop mediated by mAChR kinase (Haga et al., 1993). Yet

to distinguish between the different mAChR subtypes, a sequence divergence in the

postulated third internai (oops (i3) exists between the MIIM21M3 sequences compared

with the M21M4 sequences (Hulme et al., 1990; Wess, 1996; Wess et al., 1997). The

sequence divergence probably specifies the different coupling preferences of the two

categorized groups of subtypes (Wess, 1993). In addition, sequences \-vithin the i3 loop

differ sufficiently between each mAChR subtype that allows raising subtype specifie

antisera (see Levey, 1993 for explanation).

27

•

•

•

• Pharmacologieal Definitions of Subtypes

The task of pharmacologically characterizing the different mAChR subtypes has

been a difficult process, especially with the initial lack of agonists \vith any selectivity in

addition ta the lack of any antagonist with very high selectivity for any single receptor

subtype. Studies were focused on discovering natural agonists or antagonists plus

attempts ta synthesize selective agonists or antagonists that can bind selectively to each

subtype ta distinguish between each of the five mAChRs. Yet the binding sites for ACh

and other agonists were found to be similar among aIl of the mAChR subtypes.

However, the expression of more than one subtype in mammalian tissues emphasized the

concept of high selectivity for antagonists or agonists that are needed to be used in

clinical application for disorders resulting from muscarinic aberrations.

The definition of antagonist affinities for the five muscarinic receptors has been

aided amply by the use of radioligand binding techniques, with ligands such as

eH]pirenzepine and eH]N-methylscopolamine, in addition to the procedure of

membrane preparations from ceUs transfected with the gene for a specifie receptor,

achieving the expression of ooly one single subtype at a time. Binding studies using two

ne\V antagonists, hexahydro-sila-difenidol (HHSiD) and its para-fluoro analogue (p­

HHSiD), enabled researchers to distinguish between the M2 muscarinic binding sites in

the heart and in the glandular tissues (for review see Caulfield and Birdsall, 1998). The

heart M2 receptor had a 70-fold lower affinity for p-HHSiD than for the "'M2" glandular

tissue receptor, which was then found to he distinct from the M2 subtype, and renamed

the M3 mAChR. Further studies developed two highly selective M2 antagonists that

display high selectivity between the Ml and the M4 subtypes (for review, see Grimm et

28

•

•

•

al., (994). On one hand, tripitramine displayed a 2000 fold higher binding affinity to the

Ml than to the MI, M3, and M4 subtypes, and (S)-dimethindene is an M2 selective

antagonist used in functional and binding studies.

More selective antagonists have been recently synthesized (see Table 1),

including the M2 antagonist methoctramine, the M3 antagonist 4-DAMP and its

irreversible analogue 4-DAMP-mustard (Michel et al., 1989; Lazareno et al., 1990;

Caulfield 1993; Kondou et aL, 1994). In addition, n\'o muscarinic snake toxins have been

discovered, the MT3 and the MT7 toxins. They display very high affinity for muscarinic

receptors. MT3 and MT7 toxins belong to the many components of the venom of the

green (Dendroaspis angusticeps) and the black mamba (Dendroaspis polylepis) (Hulme

et al., 1990; Caulfield, 1993; Maggio et al., 1994; Eglen et al., 1996; Nunn et al., 1996;

Adem and Karlsson, 1997; Caulfield, 1997; Levine and Birdsall, 1997; Jolkkonen et al.,

1994). These two toxins show promising subtype selectivity (MT7 is highly selective to

the Ml subtype; MT3 is highly selective to the M4) and should be useful in muscarinic

receptor classification (for review see Caulfield and Birdsall, 1998). However, more

highly selective agonists are needed to activate preferentially each subtype in order to

implement the use of muscarinic agonists in clinical applications.

29

•RECEPTORSUBTVPE

MI Ml M3 M4 MS

Atropine 9.0-9.7 9.0-9.3 8.9-9.8 9.1-9.6 8.9-9.7

Pirenzepine 7.8-8.5 6.3-6.7 6.7-7.2 7.1-S.1 6.2-7.1

Methoctramine 7.1-7.8 7.8-S.3 6.3-6.9 7.4-S.1 6.9...7.2

4-DAMP 8.6...9.2 7.8...s.4 8.9-9.3 8.4-9.4 S.9-9.0

MT3 7.1 <6 <6 8.7 <6

• MT7 9.S <6 <6 <6 <6

TABLE 1: Antagonistic amnity constants (log affinity constants or pKB values) for

mammalian muscarinic receptors. Various selective mAChR antagonists are shown

including their pKB valueso Data are from a variet}° of mammalian species, including

human. Values are adapted from Caulfield and Birdsall (1998).

•30

•

•

•

111- MUSCARINIC COUPLING TO G-PROTEINS AND EFFECTOR

MOLECULES

• Introduction

Similar to most seven transmembrane receptors, the mAChRs are coupled to G­

proteins that can transduce the exterior signal, in this case the binding of ACh to the

mAChR and the receptor's subsequent activation, into an intracellular signal govemed by

specifie second messenger cascades that cause many cellular responses. The link between

receptors and effectors is in many cases mediated by heterotrimeric G- proteins. G­

protein transduced cellular responses include biochemical activities, metabolic changes,

enzyme activationldeactivation, downstream gene transcription, protein synthesis, cell

division, and celI motility (see Table 2).

• Composition and Structure of Heterotrimeric G-proteins

Heterotrimeric G-proteins consist of an a subunit (39-52kDa), a r3 subunit (35/36

kDa), and a y suhunit (6-8 kDa) (for review see Clapham and Neer, 1997; Muller and

Lohse, 1995). G-proteins can switch between an active foon and an inactive fonn

through binding to either a GTP or a GDP nucleotide. When bound ta the GTP

nucleotide. the G-protein is in its active stable state where it can activate directly diverse

effectors. G-proteins possess an intrinsic GTPase function in the a subunit which

hydrolyses GTP to GDP, and thus induce the GDP-binding inactive fonn. At this

inactive stage, the a. subunit associates with the f3y subunit. The ACh-bound fonn of the

mAChR induces the exchange of GDP to GTP al the a. subunit. The binding of GTP

31

•RECEPTOR SUBTYPE

Preferred G-proCein

Ml

q/ll

M2

iJo

M3

qlll

M4

i/o

MS

q/ll

Second messengers PLC IP3fDAG AC (-) PLC IP3IDAG AC(-) PLCCa2+/PKC Ca2+/PKC lP3/DAG

PLA2 PLA2 Ca2+IPKC

Locations Brain (conex.hippocampus) Heart.Hindbrain Smooth muscle Forebrain Substan.Glands, Sympathetic ganglia Smooth muscle Glands, Brain Striatum nigra

(f3y activates)Inhibits Ca2+ channelsDecrease hean rateDecrease heart forceDecrease neurotransm. release

• FunctionalResponses

M..current inhibition K+ channels Smooth musclecontraction

inhibitsCa2+ channels

•

TABLE 2: Muscarinic receptors: G-protein coupling, transduction pathways,

localization, and functional responses. rnAChRs couple selectively to different G-

proteins, triggering various intracellular responses. A variety of functional responses are

mediated by the different subtypes, and each subtype maintains a specifie anatomical

location. Refer to text for abbreviations. As adapted from Caulfield and Birdsall (1998).

32

•

•

•

results in the dissociation of the a subunit and the Py complex~ as a result both subunits

cao activate their own specific signaIs and events.

There bas been at least 21 different a subunits~ 6 13 subunits~ and 12 y subunits

c10ned (Hepler and Gilman, 1992; Rayet al., 1995; Simon et al., 1991; Watson et al.,

1996). A combination of ~y subunits reveal 72 potential f3y combinations that can bind

21 different a subunits. The large number of combinations raises questions about

specifie eoupling and function to different receptors. Since aU a subunits have

eharaeteristie functions, G-proteins have traditionally been defined by their cr subunits.

mAChR subtypes are eoupled to specifie G-proteins defined by their a subunits. The odd

numbered subtypes, Ml/M3/M5, are coupled to the PTX-insensitive GClq/ll protein, while

the even numbered subtypes~ M21M4, activated the PTX-sensitive Gailo protein.

Recently many (3y funetional attributes have been identified~ eliciting more

investigations in their structure and function. There are sorne restrictions to f3y

combinations. 132 does not fonn a stable complex with y}, and similarly for 133 and YI and

Y2 (Clapham and Neer, 1993; Pronin and Gautam, 1992; Schmidt et al.~ 1992), rendering

such combinations inactive (Iniguez-Lluhi et al., 1992). The region of Gy that defmes the

specificity of its interaction with GJ31 or G132, for example, is located in a 14 amino acid

segment close to the middle of the molecule (Spring and Neer, 1994). In addition, tissue­

specifie loealization is another characteristic of the diversity of G-protein subunits,

whereby the YI isoform is restricted to retinal rods while 133 occurs preferentially in retinal

cones (Iniguez-Lluhi et al., 1993; Wilcox et al.~ 1994; Peng et al., 1992).

The J3y subunits are membrane bound with the exception of transducin-(3y (J31YI)~

whieh cao be found as a soluble complex (for review see Muller and Lohse, 1995;

33

• Clapham and Neer, 1997). Ily complexes cannot he dissociated except with denaturants.

Isoprenylation of the y subunit at the C-tennÏnus is required for the complex' membrane

association. However, isoprenylation is not required for complex formation \Vith the ~

subWlit, but it is compulsory for ~y's interaction witll the a; subunit and the activation of

an effector molecule (Iniguez-Lluhi et al., 1992; Clarke et al., 1992; Higgins and Casey,

1994; Simonds et aL, 1991). On the other band, covalent modification of the a subunit

using fatty acids is required. \Vith the exception of ex l, all Cl subunits are palmitoylated

through a thioester bond to a cysteine residue in the third position of the N-terminus.

Palmitoylation appears to be necessary for membrane attachment of the a subunit, as

weil as for coupling to receptors (Linder et al., 1993; Veit et al., 1994; Wedegaertner et

al., 1993; Mumby et al., 1994; Parenti et al., 1993). Furthermore, a subunits are

• myristoylated at the N-terminal glycine via an amide linkage, and this modification

allows the a subunits to bind with an increasing affinity to the ~y complex (Buss et al.,

1987; Linder et al., 1991).

•

• Adenylate Cyclase

The decrease in adenylate cyclase activity induced by muscarinic activation has

been weil documented (for review see Felder, 1995). The expression of the M2 and the

rvl4 receptors in cell lines displayed coupling to adenylate cyclase inhibition (Buckley,

1990; Ashkenazi et al., 1989b; Hulme et al., 1991; Baumgold, 1992; Grimm et al., 1994;

Felder, 1995). In addition, G-protein reconstitution experiments showed that the G i

subtype coupled to the mAChRs is responsible for this response (Parker et al., 1991).

Adenylate cyclase catalyzes the breakdown of ATP into cAMP, which in tum cao

34

• activate cAMP-dependent protein kinases (pK.A). However, other studies have shown

that the expressed Ml subtype can weakly couple to adenylate cyclase inhibition through

a PTX·sensitive mechanism in RAT-l ceUs (Stein et al., 1988). On the other hand,

previous reports have shown that the endogenous M3 subtype can cause an accumulation

of cyclic-adenosine mono-phosphate (cAMP) in neuroblastoma ceUs (Baurngold and

Fishman, 1988), and also in other several ceUs such as the rat olfactory bulb (Olianas and

Onali, 1992)~ mouse parotid acinar ceUs (Watson et al., 1990), rat adrenal gland

(Regunathan et al. 1990), and rat sympathetic neurons (Suidan et al.~ 1991). Also, the f3y

subunits of the G-proteins coupled to the MI and the MS mAChRs can also weakly

stimulate adenylate cyclase types II and IV (Ashkenazi et al., 1989a; Pieroni et al., 1993).

The coupling of mAChRs to adenylate cyclase activation can be regulated through

• calcium and protein kinase mechanisms (Jansson et al., 1991; Baumgold et al., 1992).

cAMP production may occur through calciumlcalmodulin sensitive (types 1 and III) or

-insensitive (types II, IV, V, and VI) adenylate cyclases. The adenylate cyclase type

coupled to mAChRs is dependent on the cell line in which the mAChRs are expressed.

Nevertheless, the accumulation of cAMP may actually be the result of mAChR-mediated

phosphodiesterase inhibition in a variety of cell types including astrocytoma ceUs

(Meeker and Harden, 1982). In oligodendrocyte progenitors, previous studies from our

laboratory have reported the anenuation of J3-adrenergic-stimulated cAMP formation by

the M2 mAChR subtype, speculating a role for the Gai protein coupled to the M2

receptor in inhibiting adenylate cyclase (Cohen and Almazan, 1994).

• Phospholipase C (PLC)

•35

• The receptor-mediated activation of PLC leads to the hydrolysis of

phosphotidylinositol-4,5-bisphosphate (PIPz) to inositol trisphosphate (IP3) and

diacylglycerol (DAG). The family of PLC enzymes is grouped into three classes, J}, y,

and 8, with subtypes within each group (Rhee and Choi, 1992). Phosphoinositide

breakdown by ACh has been weil studied and linked to the M IIM31M5 mAChRs through

the coupling of Gqlll protein, while the M2 and M4 subtypes have been reported to

weakly activate PLC (Lambert et al., 1992). In the case of the Ml subtype,

phosphoinositide breakdown is mediated by PLC~1 through Gq/l l alpha subunits

(Berstein et al., 1992; Sawaki et al., 1993; Hildebrandt and Shuttleworth, 1993). On the

other hand, the M5 subtype have been linked to the activation of PLCf3 and

PLCy (44)PLCy activation is normally stimulated by tyrosine kinase-dependent

• phosphorylation, a mechanism induced by the M5-mediated calcium influx that activates

voltage-independent calcium channels and subsequent tyrosine kinase phosphorylation

(Gusovsky et al., 1993). However, the M2 subtype did not stimulate the phosphorylation

of PLCy or the mediation of calciwn influx. Later studies have shown, though, that the

M2 and M4 receptors cao also stimulate with lower efficiency phospholipase C through a

PTX-sensitive G-protein, namely through Gaz and Ga iJ (Ashkenazi et al., 1989;

Dell' Acqua et al., 1993). AIso, f3y subunits were shown to couple the M2 receptor and

phospholipase C-f32 (Katz et al., 1992). Studies in our laboratory have reported that in

oligodendrocyte progenitors, inositol trisphosphate accumulation was mediated by the

Ml and not by the M2 mAChR (Cohen and Almazan, 1994). Protein kinase C (PKC)

seemed to play a regulatory role in the mAChR-mediated accumulation of inositol

•36

•

•

•

trisphosphate, since an acute pretreatment with phorbol-12, 13 myristate acetate (PMA)

abolished the formation of inositol trisphosphates (Cohen and Almazan, 1994).

IP3 cao act 00 its respective receptor in the eodoplasmic reticulum. (an IP3­

sensitive calcium channel), releasing calcium from its intracellular stores, while DAG cao

activate, with the cooperation of calcium, certain isozymes of PKC. PKC consists of

three subgroups: the classical, which include a, fil, 1311, and y, isozymes that are activated

by DAG and calcium; the novel, which inc1ude Ô, E, e, l'l, J.L, and are activated by DAG

alone; the atypica/, which include ç, À, 1., and are independent of both calcium and DAG

(for review see Mellor and Parker, 1998). The atypical isozymes are activated by

phosphatidylserine and phosphatidylinositol (3,4,5)-trisphosphate. Note that certain

isozymes are involved in stimulating proliferation (Stephens et al., 1993), while others

are involved in negatively regulating mAChR activity by phosphorylating sites on the i3

loop (Haga et aL, 1993).

• Phospholipase A2 (PLA2)

The enzymatic function of PLA2 is to catalyze the hydrolysis of membrane

phospholipids, mainly phosphatidylcholine, to generate free arachidonic acid (AA) and

the corresponding lysophospholipid. AA is a Mediator of many cell"Ular and

physiological mechanisms including inflammation, pain, and intracellular signaling

through itself and its bioactive eicosanoids. Lysophospholipids that are generated from

the hydrolysis of membrane phospholipids are recycled to the membrane. AA can he

converted to bioactive eicosanoids including prostaglandins, thromboxanes, leukotrienes,

37

•

•

•

epoxides~ and hydroxyeicosatetraeinoic acids (HETEs). Reports published have

described the release of AA througb mAChR stimulation, in addition to the release of

eicosanoid metabolites in a variety of tissues including heart, brain, and muscle.

Pharmacological studies linked the odd numbered mAChRs MIIM31M5 to the activation

of PLA2 (Conklin et al., 1988; Felder et al., 1990; Liao et al., 1990), whereby CCh

stimulated the release of AA in a PTX-insensitive manner, ruling out the involvement of

G i or Go- On the other band, M2 and M4 subtypes expressed at physiological levels

showed no coupling to PLA2 (Conklin et al., 1988). The activation of PLA2 has been

shown to require an IP3-independent calcium influx and protein kinase C (Felder et al.,

1990; Brooks et al., 1989). The high molecular weigbt cytosolic PLA2 (cPLA2) seemed

to he the isozyme that is involved in the mAChR-mediated AA release (Sharp et al.~

1991; Clark et al., 1991). It is activated with nanomolar levels of calcium, and has a

selectivity for phospholipids with arachidonic acid in the sn2 position.

38

• IV- MITOGEN-ACTIVATED PROTEIN KINASES

•

•

• Brief Overview: The MAPK Classes

The mitogen-activated protein kinase (MAPK) pathway is involved in cell

differentiation and proliferation. Il is activated by bath tyrosine kinase receptors as weil

as G-protein coupied receptors. Receptor tyrosine kinases activate MAPKs through a

rnultistep process. The binding of the ligand to the receptors leads to tyrosine

phosphorylation of a docking site on the receptor for the adapter protein GRB2/SEM-5

(for review see Schlessinger~ 1993). Subsequently~ the recruitment of a guanine

nucleotide exchange protein Sos occurs~ which recruits Ras to the plasma membrane~ and

results in the exchange of GDP for GTP by Ras. GTP-bound Ras then activates a

cascade of protein kinases listed sequentially as MAP kinase kinase kinase (A-Raf~ B­

Raf, and Raf-I) and MAP kinase kinase (MEKI and MEK2). MEKs then phosphorylate

p44mapk and p42mapk MAPKs (ERK1 and ERK2) on both threonine and tyrosine residues.

As a result, MA.PKs phosphorylate and regulate the activity of key enzymes and nuclear

proteins, which can eventually regulate the expression of genes essential for proliferation.

However, the mechanism of G-protein coupled receptors· activation of MAPKs are less

understood, yet many groups have elucidated various signaling cascades.

In mammalian systems, II distinct MAPK and 7 MEK genes have been identified

to date. Members of the MAPK family include (i) extracellular signal-regulated kinases

(ERKI and ERK2) which are also known as p42mapk and p44mapk and are phosphorylated

by MEK 1 and MEK2; (ii) NH2-terminal Jun kinase/stress-activated protein kinases

(JNKlSAPK) a~ f} and~ y, which are phosphorylated by MEK4 and MEK7; and (iii) p38

39

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•

MAPKs a~ 13, y, and Ô, which are phosphorylated by MEK3 and MEK6 (for review see

Lewis et al., 1998). MAPKs are activated by MEKs through phosphorylation at two

regulatory phosphorylation sites with sequence T(P)-X-Y(P) located in the 'activation

Hp' between subdomains 7 and 8 of the conserved kinase core sequence. In addition to

the conserved kinase core~ p42mapk and p44mapk contain C-terminal extensions and a 25

amino acid insert bet\veen subdomains 9 and 10. Both enzymes are activated by

phosphorylation within their activation Hp at Thr183-Glu-TyrI85 (as in rat sequence), a

reaction that has Tyr phosphorylation preceeding Thr phosphorylation (for review see

Lewis et al., (998). Both events of phosphorylation are needed to fully activate both

isozymes.

• G-protein Coupied Receptors Signaling to MAPK

Activation of MAPKs mediated by G-protein coupled receptors has been extensively

studied, including investigations on bombesin, endothelin-l, adrenaline, somatostatin,

LHRH, TRH, thromboxane A2, and muscarinic receptors. These receptors have been

shown to couple to PTX- sensitive and insensitive G-proteins, thus signaling to MAPK

using both G-proteins (Gilo and Gq/ll ). To study the mechanism of activation of MAPK

by G-protein coupled receptors such as the mAChRs, transfected cell lines that express

p42mapk (labeled as MAPK in this text) together with mAChR subtypes \-vere used (for

review see Gutkind et al., 1997). In transfected cell lines such as COS-7, CCh

stimulation increased MAPK activity in a concentration dependent manner through the

Ml and the M2 subtypes (Crespo et al., 1994), whereby the Ml-mediated activation was

PTX-insensitive, while the M2-mediated activation displayed PTX-sensitivity. To

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elucidate the role of the a subunits of G-proteins, studies were performed in transfected

cells expressing wild type or activated G-protein a subunits, including Gaq and Gail

which couple MI and M2 subtypes respectively. However, these transfected ceUs did not

exhibit elevated MAPK phosphorylating activity (Crespo et al., 1994). As a result,

speculations of molecules in addition to a subunits of G-proteins play a role in the

activation of ~fl and M2-mediated MAPK activation.

Recently, available evidence has supported an active role for the GlJy complex in

signal transduction (Clapham and Neer, 1993). Since ex subunits did not stimulate the

activation of MAPK studies investigating the role of GlJy subunits were performed.

Indeed, transfecting ceUs with fll and y2 subunits and MAPK effectively elevated its

phosphorylating activity (Crespo et al., (994), demonstrating that the J3y-heterodimers

directly elicit biochemical pathways leading to MAPK activation. AIso, sequestration

studies performed on {3y complexes by overexpressing Gai proteins abolished the M2 and

~y-mediated activation of MAPK, and reduced this response when elicited by MI

stimulation. Therefore, these results indicate that Ml signaling to MAPK involves J3y­

dependent and independent pathways, while the M2-mediated activation of MAPK

appears to be strictly dependent on GIJ1'

Mutant-ras studies were performed to investigate whether signaling from MI and

M2 receptors or {3y dimers involve p21 ras (Ras). These studies have sho\\'Il that mutant

ras expression abolished the elevated MAPK activity in response to Ml, M2, and J3y

dimer overexpression (for review see Gutkind, 1998; Lewis et al., 1998). Similar studies

were done to elucidate the role of PKC, and the results suggested that signaling from Ml

receptors to MAPK involves a PKC-dependent and PKC-independent pathway, and both

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pathways converge at Ras. The PKC-independent pathway is mediated by the G13y

complex. On the other hand, the M2-mediated MAPK activation is PKC-independent,

and involves GitJy dimers, acting on a Ras-dependent pathway.

• Novel Signaling Pathways Linking G-protein Coupled Receptors to MAPK

Recent studies have shown that the activation of G-protein coupled receptors,

such as the mAChRs, induces the phosphorylation of the adapter protein Shc on tyrosine

residues and the formation of Shc-GRB2 complexes (see fig. 2) (Chen et al., 1996; van

Biesen et al., 1995). Other studies have demonstrated that Src, or a Src-like kinase,

mediates the ~y-induced phosphorylation of Shc, and eventually the recruitment of

GRB2 and Sos, consequently the activation of the Ras-MAPK pathway (Lutrell et al.,

1996). More studies have linked other non-receptor tyrosine kinases in signaling to the

MAPK pathway, including several Src-like kinases such as Fyn, Lyn. Yes, and Syk

(Ptasznik et al, 1995; Wan et al., 1996), in addition to the PKC-dependent Pyk2 (Della

Rocca et al., 1997; Dikic et al., 1996; Levet al., 1995). Pyk2 is related to the focal

adhesion kinase (FAK), a kinase involved in integrin signaling, where it contributes to

the formation of focal complexes in the celI.

Furthermore, reports have demonstrated that signaling from G-protein coupled receptors

to MAPK involves a wortmannin sensitive phosphatidylinositol-3-kinase (PI3K) (Hawes

et al., 1996; Lopez-Ilasaca et al., 1997). The PI3K family contains several species

including PI3Ka, PI3Kfi, and PI3Ky. The tyrosine kinase receptor-regulated

heterodimeric PI3Ka and PI3K(J consist of pllO catalytic subunits and different p85

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•

adapter molecules. On the other band, the novel PI3Ky cao he activated by Py subunits of

heterotrimeric G proteins (Stoyanov et al., 1995). Lopez-Ilasaca et al. (1997)

demonstrated the activation of MAPK by the expression of PB Ky in Cos-7 ceUs, while

PI3Ka did not affect the activity of MAPK. The expression of a mutated PI3Ky that

lacks lipid kinase activity abolished MAPK activation mediated by the M2 receptor or by

Gllr expression (Lopez-Ilasaka et al., 1997).

43

•mAChR

o CH,

JL ~CtiJHzN" ~~ CH:J

ACh agonist carbachol

+

•

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•

•

•

FIGURE 2: Divergent kinase cascades that Iink mAChRs to the MAPK pathways.

mAChRs couple to G-proteins that cao activate different signaling cascades upon

cholinergie stimulation of the receptor. Refer to text for discussion of pathways and for

abbreviations. Adapted and modified from Gutkind (1998>.

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• MAPK Regulation of AP-l Transcription Factor and the Induction of c-fos Gene

Expression

One of the most investigated targets of MAPK signaling in mammals is the AP-l

transcription factor. AP-I consists of members of the Joo and Fos transcription factor

families which belong to the bZIP group of DNA binding proteins. Fos and Jun family

combine in various ways with other transcription factor families, including cAMP

response element binding protein (CREB)/ATF, NFAT, ETS, NFkB, and nuclear

hormone receptors. It has been shown that several signaIs can induce AP-l activity,

including growth factors, cytokines, neurotransmitters, and cellular stress (Davis, 1994;

Karin, 1995; Angel et al., 1991; for review see Whitmarsh and Davis, 1996; Gutkind,

1998). AP-l is able to bind the TPA-response element (TRE) with the consensus

sequence TGACTCA (Angel and Karin, 1991) (see fig. 3). The binding affinity for a

particular TRE sequence is dependent on the composition of the AP-l transcription

factor. Many immediate early genes, including c-fos, posses TRE sequences in their

promoter regions. On one hand, MAPK signaling pathways influence AP-I activity by

bath increasing the aboodance and activity of AP-l components (Karin, 1995).

On the promoter of the c·fos gene, there are three major transcriptional control

elements present that are targeted by inducible signaling pathways: the cAMP-response

element (CRE), the serum response element (SRE), and the sis-inducible element (SIE)

(Janknecht et al., 1995). The efficient regulation of these elements is required for the

expression of c-fas. The CRE is bound by the dimeric CREB and related transcription

45

• Ap·1 TRANSCRIPTION FACTOR

•

p38

JNK jERK~

C

Protein kinases

/

AP-1

JNK

/ \ p38, /

c-fos promoter

•___---L. T_R_E I_

10...----... INDUCTION

•

•

•

FIGURE 3: Regulation of AP-l transcription factor by MAP kinases. The AP-l

transcription factor is highly regulated by different MAPK's including ERKs, JNKs,

p38s, in addition to other protein kinases. The AP-l transcription is formed by the

dimerization of c-fos and c-Jun transcription factors. Both c-fos and c-jun gene

expression is highly regulated by protein kinases. Upon stimulation of their expression,

c-Fos and c-Jun proteins dimerize and regulate the expression ofother genes, including c­

fos. Refer to text for abbreviations. Adapted and modified from Whitmarsh and Davis

(1996).

46

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•

factors. CREB is activated by protein kinase A (PKA)7 a sereine/threonine kinase that is

activated by cAMP. Phosphorylated CREB binds CREB-binding protein (CBP) which

can facilitate the transcriptional machinery on the c-fos promoter. CaIciumlcalmodulin­

dependent protein kinases (CalMK) have been impiicated in CRE-dependent c-fos

induction. by phosphoryiating CREB and therefore activating the transcription factor.

The c-fos SRE, on the other hand7 mediates serum induction of the c-fos

prornoter, hence the name. Yet SRE-mediated induction was shown to be also stimuIated

by growth factors, cytokines, and cellular stress (Cahill et ai., 1995; Cahill et al., 1996).

To start SRE-mediated transcription, the serum response factor (SRF) which recruits a

ternary complex factor (TCF) binds the SRE (Cahill et al., 1995). TCF proteins belong to

the ETS-domain family of DNA binding proteins that includes Elk-I, SAP l, and

SAP2INETIERP (Cahill et aI., 1995; Cahill et al., 1996; Price et al., 1995). In response

to several signais including growth factors and phorbol esters, the TCf Elk-I is

phosphorylated in its activation domain by members of ERK (Gille et aI., 1992; Marais

and Treisman, 1993; Janknecht et aI., 1993; Gille et al., 1995), JNK (Cavigelli et al.,

1995; Whitmarsh et al., 1995; Zinck et al., 1995; Gille et al., 1995), and p38 MAPKs

(Raingeaud et al., 1996), which increase its ability to form complexes with the SRf at the

SRE, therefore activating transcription (Gille et al., 1992; Gille et al., 1995). In

retrospect, the phosphorylation of the TCF Elk-l by ail three groups of MAPKs is crucial

for its transcriptional activity, whereby Elk-l acts to converge multiple MAPK signal

cascades at the SRE to increase c-fos transcription in response to a variety ofextracellular

signais, including the activation of G-protein coupled receptors such as mAChRs. Note

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that there are other TCF proteins that form temary complexes with the SRF at the SRE,

such as SAP-I. Its activity is also regulated by MAPKs including the ERK and the p38

MAPKs (Priee et al., 1995; Janknecht et al., 1995).

The formation of the AP-l transcription factor is limited by the induction of both

c-fos and c-jun genes, since AP-I dimers constitute the binding of c-fos and c-jun

transcriptional factors. The transcriptional response of the c-jun promoter is mediated by

two TREs, Jun 1 and Jun2 (Angel, 1995). These promoter elements are constitutively

bound where they preferentially bind c-Jun and ATF-2 (Angel, 1995). Both transcription

factors are phosphorylated on two sites by the JNK group of MAP kinases, therefore

enhancing the transcriptional activity and leading to the induction of the c-jun gene. As a

result, c-Jun protein synthesis is elevated. Subsequently, c-Jun transcription factors bind

to c-Fos transcription factors to fonn the AP-I complex, which in tum can regulate the

induction of c-fos and other immediate early genes at the AP-l response elements.

48

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CHAPTERTWO

STATEMENT OF PURPOSE

49

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STATEMENT OF PURPOSE

Earlier studies have demonstrated the ability of the oligodendrocyte lineage to

respond to cholinergie stimulation. Larocca et al. (1 987b) provided evidence for the

presence of the ~11 and the M2 mAChRs in pwified myelin isolated from adult rat brain~

eliciting the interest in investigating cholinergie responses and signaling in

oligodendrocytes. The purified myelin mAChRs couple to phosphoinositide hydrolysis

and inhibition of adenylate cyclase (Larocca et al.~ 1987a; Khan and Morell~ 1988).

Studies performed in our laboratory have pharmacologically characterized the mAChRs

present in 0-2A progenitors and in mature oligodendrocytes, displaying predominance of

the M3 subtype (see fig. 1) (Molina-Holgado et al., unpublished). However, the

pharmacological studies showed extensive downregulation of the mAChRs in mature

oligodendro'cytes during in vitro development, suggesting a role for mAChRs in their

progenitor stage (Molina-Holgado et al., submitted).

Cholinergie stimulation of 0-2A progenitors triggers various intracellular

responses that are mediated by mAChRs. Studies reported from our laboratory have

shawn that mAChR activation of progenitors causes an increase in their proliferation

(Cohen et al., 1996). Similarly, activation of mAChRs induees complex signal

transduction pathways in progenitors, including accumulation of 1P3 (Cohen and

Almazan~ 1994), increases in intracellular calcium levels (Cohen and Almazan~ 1994;

Kastritis et al., 1993; Takeda et al., 1995), production of calcium waves (Simpson and

Russell, 1996), inhibition of an inwardly rectifying potassium channel (Karschin et al.,

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1994), and inhibition of f}-adrenergic-stimulated cAMP production (Cohen and Almazan,

1994).

We have also recently demonstrated the cholinergie activation of p42mapk in 0-2A

progenitors, an event that is mediated by mAChRs in the presence of extracellular

calcium (Larocca and Almazan, 1997). In addition, Pende et al. (1997) demonstrated the

phosphorylation of CREB by CCh stimulation, an event that is dependent on p42mapk

activation. Moreover, we have shown that cholinergie stimulation of 0-2A progenitors

increased c-fos mRNA expression, an event that is mediated by mAChRs as weIl (Cohen

et al., 1996). CCh stimulated p42IMPk activation and c-fos mRNA expression in a time­

and concentration- dependent manner, involving DAG-independent PKC pathways

(Larocca and Almazan, 1997; Cohen et al., 1996). Yet the identity of the mAChR

subtype(s) that induce(s) the activation of p42IMPk and the expression of c-fos mRNA in

oligodendrocyte progenitors has not been determined. AIso, the signaling pathways that

connect mAChR activation to p42mapk activation and c-fos mRNA expression have not

been fully elucidated. Therefore, we decided to investigate the mAChR subtype(s)

involved in both responses, and to further define the intracellular effectors that link the

mAChR to the critical MAPK pathway and to the nucleus.

51

• AC) 120c:c.~ 100.cen~z-::r: 6

C")..-o~

"0(1)Coencft

i i-11 ..10

i·9

i-8

i..7

i·6

i-5

i-4

i-3

•• atropineo 4-DAMP• pirenzepine

o methoctramine

• tropicamide

B120

.,......cS 100Ü

:2 80E~

60~0'-

a. 40--J:C')

20-0

i i i i i i i , i• -11 -10 -9 -8 ..7 -6 -5 -4 ..3

•

•

•

FIGURE 1: Competition and PI hydrolysis studies OD mAChRs in oligodendrocyte

progenitors. (A) Competition binding experiments were performed on whole cells.

Progenitors were exposed to increasing concentrations of muscarinic antagonists

[atropine (non-selective); pirenzepine (Ml selective); (methoctramine (M2 selective); 4­

DAMP CM3 selective); tropicamide CM4 selective)] and 1 nM of the radiolabelled

acetylcholine analogue eH]NMS. (B) Inhibition of CCh-stimulated eHJIP

accumulation by increasing concentrations of muscarinic antagonists. Adapted and

modified from Molina-Holgado et al. {submittedl.

52

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CHAPTER THREE

MATE~SANDMŒTHODS

53

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MATERIALS AND METHOnS

Materials

The following reageots were obtained from the indicated supplier: carbachol~

atropine methyl bromide from Sigma-Aldrich Canada (Oakvil1e, Ontario); PD 098059,

methoctramine, 4-DAMP methiodide (4-DAMP), 4-DAMP-Mustard (4-DAMP-M),

pirenzepine, and wortmannin were purchased from RBI (Natick, MA); {1-[6-«(l7b-3­

methoxyestra-I,3,5(lO)-trien-17-yl)amino)hexyl]-IH-pyrrole-2,5-dione} (U73122), {I­

[6-«1 7b-3-methoxyestra-I,3,5(1O)-trien-17-yl)amino)hexyl]-2,5-pYrr0ldinedione}

(U73343) were from Calbiochem (La Jolla, CA). Phospho-specific p42mapk antibody

(Thrl83 and Tbr185) was obtained from Promega (Montreal, QC). The A2B5 mAb IgM

was from American Type Culture Collection CRL 1520. Second antibodies used for

immunofluorescence were purchased from Southern Biotechnology or Jackson

Immunoresearch Laboratories. The media Dulbecco's modified Eagle medium (DMEM),

Ham's FI2 medium and Hank's Balanced Salt Solution (HBSS) as weil as 7.5% bovine

serum albwnin (BSA) fraction V, fetal calf serum (FCS), penicillinlstreptomycin were

[rom Gibco BRL (Burlington, ON); PDGF AA, bFGF from UBI; poly-D-Iysine, poly-L­

omithine, Triton-X-I 00. AlI other reagents were obtained from standard suppliers.

• CeU Culture

Oligodendrocyte progenitor primary cultures were prepared as described by

Almazan et al. (1993), modified from McCarthy and de Vellis (1980). The cultures were

prepared from the brains of newbom Sprague-Dawley rats. The meninges and blood

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vessels were removed from the cerebral hemispheres in Ham's F12 medium. The tissue

suspension was passed through a 230 mm nylon mesh and collected by filtering through a

ISO mm and then a 100 mm nylon mesh. The resulting suspension was centrifuged for 10~

min at 1200 rpm and then resuspended in DMEM supplemented with 12 % FCS. Cells

were plated on poly-L-ornithine precoated 80 cm2 flasks and incubated at 37 oC with 5%

C02 in air. Culture medium was changed after 3 days and every two days thereafter. The

initial mixed glial cultures, gro\\-n for 9 to Il days, were placed on a rotary shaker at 225

rpm at 37°C for 3 br to remove loosely attached macrophages. Oligodendrocyte

progenitors were detached following shaking for 18 br at 260 rpm. Filtered through a 30

mm nylon mesh, the ceUs were plated on bacterial grade Petri dishes for 3 br. Under these

conditions, astrocytes and microglia become attached to the plastic surface and

oligodendrocyte progenitors remained in suspension. The final cell suspension was plated

on 6-well dishes (Falcon) pre-coated with poly-D-Iysine. Cultures were fed with serum

free medium (SFM) containing 2.5 ng/ml PDGF AA and 2.5 ng/ml bFGF to promote

self-renewal and prevent differentiation and changed every two days. SFM consisted ofa

DMEM-F 12 mixture (1: 1), 10 mM HEPES, 0.1 % bovine serum albumin (BSA), 25

mg/ml human transferrin, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM

progesterone, 10 nM biotin, 5 mg/ml insulin, 16 mg/ml putrescine, 30 nM selenium, 50

units/ml penicillin and 50 mg/ml streptomycin as previously reported. The cultures were

immunocytochemically characterized by specifie markers for different ceU types. More

than 95% of the cells reacted positively with mouse monoclonal antibody A2B5, a

marker for oligodendrocyte progenitors in culture and less than 5% were

galactocerebroside positive oligodendrocytes, glial fibrillary acidic protein positive

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•

astrocytes or complement type-3 positive microglia. These enriched oligodendrocyte

progenitor cell cultures were used in these studies.

• Immunocytochemistry

In order to determine the lineage composition of the cellular preparations,

immunocytochemistry was performed with A2B5 mAb that is specific to 0-2A

progenitors as shown by Cohen et al. (1996). Purified oligodendrocyte progenitors were

grO\\TI on poly-D-lysine-coated glass coverslips in 24-well tissue culture plates in SFM

plus growth factors for 3-5 days. For immunocytochemical detection "ith A2B5, cells

were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 min at

room temperature (RD. After fixation, the cells were washed severa! times with PBS

and blocked for 15 min in PBS containing 0.2% BSA, 5% goat serwn with 0.1% Triton

X-IOO for 45 min at RT. Monoclonal A2B5 mAb, applied as (1:500), was diluted in PBS

containing 10% FCS and 5% goat serwn with 0.1 % Triton X-IOO for 45 min at RT. The

second antibody SAM-IgM-FITC was applied (from Jackson Immunoresearch

Laboratories) and was diluted 1: 100. Coverslips were mounted in Immuno-mount and

were examined under a Leitz Diaplan epifluorescent microscope. On the day of the

experiment, 18 h after removal of the growth factors, more than 95% of the ceUs \vere

AlB5 positive oligodendrocyte progenitors (fig. 1)

• Western Immunoblot analysis

After preincubation with HBSS for 4 hr, cells were stimulated with

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•

pharmaeologieal agents for the indieated time periode CeUs were lyzed in SDS sample

buffer (62.5 mM Tris-HCI, pH 6.8,2% w/v sodium dodeeyl sulfate (SOS), 10% glyeeroI,

50 mM dithiothreitol, 0.1 % w/v bromophenol blue) and boiled for 5 min. 30 J.lg of eaeh

protein extract were electrophoresed on 10% polyaerylamide gels and transferred to

Immobilon-P membranes (Millipore). Blots were blocked with 5% milk in Tris-buffered

saline containing 0.01% Tween 20 and then incubated overnight at 4 oC with anti­

phospho-speeifie MAPK (1: 10000). Immunoreactivity was visualized by a

chemiluminescence deteetion system (Amersham) and quantified by densitometry using a

Master Scan Interpretative Densitometer.

• RNA extraction and Northem blot analysis

Total RNA was extracted from oligodendrocyte progenitors using a guanidium

thioeyanate method. The RJ."I'A pellets were resuspended in 50% formamide/2.2 M

formaldehyde/20 mM MOPS and denatured for 30 min at 65 oC. 10 J.lg of each extract

samples were eleetrophoresed on a 1.3% agarose-formaldehyde gel and transferred to

Hybond-N membranes. The c-fos probe was labeled with [a-32P]dCTP using a random

primer kit to a specifie aetivity of 108 epm/mg DNA. Following 2 hr of prehybridization,

membranes were ineubated at 42 oC for 48 hr with 106 epm/ml hybridization solution

(50% formamide, 25 mM sodium phosphate buffer (pH 6.5), 0.8 M NaCI, 0.5% SOS, 1

mM EDTA). Membranes were washed for 30 min at 50 oC in 0.1 X saline sodium citrate,

0.1 % SDS and exposed to X-ray film for 1-2 days. SignaIs were quantified using a

MaterSean Interpretive Oensitometer.

57

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• Data analysis

One way analysis of variance followed by the Tukey-Kramer test was used to

detennine statistical significance: P values less than 0.05 were considered significant.

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•

FIGURE 1: Purified oligodendrocyte progenitors (O-2A) were irnmunostained with the

A2B5 mAb, a marker for 0-2A progenitors in culture. Progenitor cultures showed 95%

purity.

59

1.

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CHAPTER FOUR

RESULTS

60

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•

RESULTS

• Activation of p42maPk by CCh is mediated through the M3 mAChR in

oligodendrocyte progenitors

The activation of p42maPk by cholinergie stimulation has been well documented in

other systems. Reports included studies on transfected cell lines and primary tissue

culture cells. We have shown that in cultured oligodendrocyte progenitors, CCh

stimulation activated p42nlapk via the rnAChRs (Larocca and Almazan, 1997). Larocca

and Almazan (1997) have demonstrated the expression of p42mapk by indirect

immunofluorescence using specifie anti_p42nlapk antibodies, whereby intense staining was

evident in the cytoplasm and the cellular processes but not in the nucleus. They have also

confirmed the expression ofp42mapk by Western blot analysis. In gel kinase activity using

MBP as a substrate demonstrated that CCh increased p42mapk, s kinase activity by two to

three above control levels. The activation of p42mapk by CCh was time- and

concentration-dependent, where maximallevels were reached at 2-5 min stimulation at an

optimal concentration of 10-100 J,lM. This response was blocked by atropine and EDTA

pretreatment, demonstrating that the activation of p42 mapL: by CCh is mediated by

mAChRs and is dependent on calcium.

Therefore, in view of these previous studies on p42mapk activation in addition to

the previous binding reports that pharmacologically characterized the mAChRs in

oligodendrocyte progenitors (Molina-Holgado et al., submitted), the present study

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•

investigated the mAChR subtype that mediates the activation of p42mapk• Progenitor

cultures were pretreated with relatively selective mAChR antagonists at a concentration

of 1 JlM, including pirenzepine (Pir), an MI antagonist, methoctramine (Meth), an

M2/M4 antagonist, and 4-DAMP, an M3 antagonist, and the non-selective muscarinie

antagonist atropine (Atr) for 20 min. The 1 f.lM concentration of each muscarinic

antagonist used is sufficient to block its respective subtype(s) (Michel et al.~ 1989;

Lazareno et al., 1990; Caulfield 1993; Kondou et al., 1994; Molina-Holgado et al.,

submitted results). The cultures were then stimulated with 100 flM CCh for a period of 5

min, which corresponds to the optimal concentration and time period for CCh-induced

p42mapk activation (Larocca and Almazan, 1997). The levels of phosphorylated p42mapk

were detected using Western blot analysis and were quantified densitometrically (fig. 1).

Stimulating with 100 f.lM CCh increased p42mapk activation by 2-fold, while atropine

(non-selective) and 4-DAMP (M3-selective) blocked the response (fig. 2). On the other

hand, pirenzepine (Ml) and methoctramine (M21M4) had no effect on the CCh­

stimulated activation of P42mapk•

To consolidate the results obtained with saturating concentrations of 4-DAMP,

cultures were pretreated with different concentrations of the antagonist, ranging from 1

nM to 1 JlM for 20 min. The cultures were then stimulated with 100 flM CCh for 5 min

(fig. 3). 4-DAMP reduced the activation of p42maPk dose dependently, with an

approximate ICso of 50 oM. Furthermore, an irreversible M3 selective antagonist, 4­

DAMP-mustard (4-DAMP-M) (Barlow et al., 1991), blocked the activation of p42mapk,

thus confirrning that the M3 mAChR is the subtype responsible for the cholinergie

activation of P42mapk (Table 1).

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• Carbaehol stimulates e-fos mRNA expression in oligodendro~yteprogenitors

through the M3 mACbR subtype

The activation of the M l/M3/M5 mAChRs activate PLC, leading to the activation

of PKC and the accumulation of intracellular calcium. Activated PKC cao potentially

stimulate a range ofcellular responses, including the induction of the proto-oncogenes c­

fos and c-jun (Boyle et al., 1991; Distel and Spiegelman, 1990). These genes are a1so

referred to as immediate early genes, and their expression is regulated by transcription

regulatory factors that are activated by a variety of neurotransmitters, and are involved in

celI growth, proliferation, and differentiation.

Previous studies have shown that cholinergic stimulation of oligodendrocyte

progenitors increases c-fos mRNA expression in a time- and a concentration-dependent

manner (Cohen et al., 1996). This response was abolished by pretreatment with the non­

selective muscarinic antagonist atropine. In addition, the expression of c-fos mRNA was

inhibited by the PKC inhibitor H7, but not by H-89, the PKA inhibitor. The induction of

c-fos mRNA was also dependent on the influx of extracellular calcium and the release of

calcium from intracellular stores, since both the extracellular calcium chelator EDTA and

the intracellular calcium chelator BAPTA blocked the response. However, Cohen et al.

(1996) did not address the identity of the mAChR mediating the induction of c-fos

rnRNA. Therefore, it was interesting to investigate whether the predominant mAChR

subtype, the M3, was also responsible for the induction ofc-fos.

Therefore, oIigodendrocyte progenitors were pretreated with selective mAChR

antagonists at 1 J.lM concentration for 20 min. The cultures were subsequently stimulated

with 100 J.lM CCh for 30 min, and the levels of c-fos mRNA levels expressed were

63

•

•

•

measured by Northern blot analysis (fig. 4). The autoradiograms were quantified

densitometrically (fig. 5). CCh alone increased the levels of c-fos mRNA by more than

six-fold above non-stimulated controls. Pretreatment of the cultures with either atropine

(non-selective) or 4-DAMP CM3 selective) abolished the CCh-stimulated c-fos mRNA

expression. On the other hand, 1 IJ.M of methoctramine (M21M4 selective) had no effect,

while pirenzepine reduced but did not entirely block c-fos expression. These resuIts

suggest that the M3 mAChR, which is also the predominant mAChR subtype found in

oligodendrocyte progenitors, is mediating the induction ofc-fos mRNA.

To define the ICso of 4-DAMP on c-fos mRNA expression, cultures were

pretreated with the antagonist (l DM to 1 J.l.M) for 20 min, followed by a 30 min

stimulation with 100 IJ.M CCh (fig. 6). 4-DAMP antagonized the effect of CCh on c-fos

rnRNA in a dose-dependent manner. A total block was obtained with 100 nM, and the

approximate ICso was calculated to be 10 nM. Furthermore, 1 J.LM 4-DAMP-mustard

(M3 irreversible) antagonized the CCh response (Table 1), confirming that the M3 is the

mAChR subtype responsible for the CCh-stimulated c-fos mRNA expression.

• The CCh-stimulated activation of p42mapk requires PLC

Previous studies have demonstrated the accumulation of inositol trisphosphates in

oligodendrocyte progenitors using [3H]-myo-inositol labeled young oligodendrocyte

cultures following stimulation with CCh (Cohen and Almazan, 1994). This response was

prevented by pretreatment with the non-selective mAChR antagonist atropine. The

accumulation of inositol trisphosphates was concentration- and time- dependent. In

64

•

•

•

addition, the same study reported a CCh-induced increase in the intracellular calcium

levels. The increase in intracellular calcium was dependent on bath the mobilization of

intracellular stores and the influx ofextracellular calcium.

Since mAChR stimulation increases the accumulation of calcium and inositol

trisphosphates, by the activation of PLC, and the mAChR-mediated activation of p42n13Pk

is calcium dependent and involves the M3 subtype which can couple to PLC via the Gqlll

G-protein (Larocca and Almazan, 1997), the present study investigated linking PLC

activation to the M3-mediated activation of p42mapk• To address this question, an

inhibitor of the enzyme PLC, U73122, and its close but inactive analogue U73343, were

used (Tatrai et al., 1994). Progenitor cultures were pretreated with 10 f.lM of either

U73122 or U73343 for 20 min prior to the 5 min treatment with 100 J.1M CCh (fig. 7).

Both inhibitors alone did not significantly reduce the levels of p42mapk activation.

However, pretreating the cultures with the active PLC inhibitor U73122 caused a

significant reduction in the CCh-stimulated leveIs of p42mapk activation, while the

inactive analogue U73343 had not effect. Therefore, these resuIts suggest that PLC

activation is implicated in the CCh-mediated activation of p42mapk

• The CCh-stimulated increase in c-fos mRNA expression requires PLC

The report of Cohen and Almazan (1994) demonstrated that cholinergie

stimulation of oligodendrocyte progenitors causes the accumulation of inositol

trisphosphates and intracellular calcium. Interestingly, the induction of the c-fos gene

was previously shown to be calcium dependent (Cohen et al., 1996). These response.r~

65

•

•

•

suggested a cholinergie activation of PLC via the odd numbered mAChRs, and the

activation of PLC may subsequently induce the expression of c-fos rnRNA through the

action of calcium.

To investigate the role of PLC in the cholinergie expression of c-fos m.RNA,

cultures were pretreated for 20 min with U73122 and its inactive analogue U73343,

followed by CCh stimulation for 30 min (fig. 8). The pretreatment with U73122 but not

U73343 caused a significant reduction in the levels of the CCh-stimuJated response,

displaying the involvement of PLC in the CCh-stimulated increase of c-fos mRNA

expression.

• Cholinergie p42..•pk activation and c-fos mRNA expression are Dot mediated by

PI3K

Phosphoinositide 3-kinase (PI3K) is a signaling Molecule that is regulated by

receptors with intrinsic or associated tyrosine kinase activities (Stoyanov et al., 1995; for

review, see Kapellar and Cantley, 1994). This enzyme phosphorylates the inositol ring of

phosphotidylinositol (PI) and phosphoinositides at the D3 position. There are several

species of PI3K that have been cloned and characterized including the heterodimeric

PI3K-a. and PI3K-f3, in addition to PI3K-y. PI3K-y can be activated in vitro by both Ct.

and f3y subunits of heterotrimeric G-proteins that are coupled to G-protein coupled

receptors (Stoyanov et al., 1995). In transfected cell lines, wortmannin, a PI3Ky

inhibitor, has been shown to inhibit p42maPk activation mediated by CCh. PI3Ky was

implicated in linking the f3y complex to the MAPK cascade (Lopez Ilasaca et al., 1997).

66

•

•

•

To determine this link, cultures were pretreated for 20 min with 100 nM wortmannin

(Wort) (Arcaro and Wymann, (993) followed by 5 min stimulation with 100 J,lM CCh

(fig. 9A). Pretreatment with wortmannin had no effect on the levels of CCh-stimulated

p42mapk activation, demonstrating no dependence on PI3Ky action.

In addition, PI3K has been shown to have various different downstream targets

including PKC isozymes from both the conventional and novel families. in addition to

other kinases including Alet, Blet, and Rac (for review see Fukui et al., 1998). Many of

these targets are involved in cell growth, development. and motility. responses that

involve signaling to the nucleus. Interestingly, the c-fos proto-oncogene is also involved

in protein synthesis and cell proliferation, whereby its product, the transcription factor

Fos, is important in regulating the expression of other proteins via the AP-I transcription

factor. In view ofthese findings, the present study investigated whether PI3K May play a

role in regulating the cholinergie expression of c-fos mRNA (fig. 9B). However,

pretreating progenitor cultures with wortmannin did not significantly reduce the CCh­

stimulated increase in c-fos mRNA expression, demonstrating that PI3K May also not he

implicated in this event.

• PD 098059, the MAPK Kinase (MEK) Inbibitor, reduces but does not abolish

tbe CCb-stimulated expression of c-fos mRNA in oligodendrocyte progenitors

Having established that both p42mapk activation and c-fos gene expression are

downstream from the M3 mAChR in oligodendrocyte progenitors, we attempted to

demonstrate a positive link between them. To address this issue, the MEK inhibitor PD

098059 (Alessi et al., 1995; Dudley et al., 1995) was added at different concentrations

67

•

•

•

ranging from 1 J..IM to 100 IJ.M for 20 min followed by a 30 min stimulation with 100 J,.lM

CCh (fig. 10). Pretreating the cultures with 10 J,.lM PD 098059, a concentration that is

sufficient to inhibit p42mapk activation (Larocca et Almazan, 1998), did not alter the CCh­

stimulated levels of c-fos mRNA. Furthermore, a significant reduction was ooly attained

at 50 J.LM and 100 J,.lM concentrations of PD 098059. The inability of PD 098059 to

completely block the stimulation of c-fos mRNA expression by CCh indicates that the

MAPK pathway May not be the only pathway linking muscarinic activation of

oligodendrocyte progenitors to c-fos gene expression. Altematively, PD 098059 at SO­

100 J.LM concentrations may be inhibiting other kinases since 10 J.lM concentration was

sufficient to completely block p42mapk activation (Larocca and Almazan, 1997).

• The CCh-mediated c-fos mRNA expression is abolished by PLA2 inhibitors

PLA2 catalyzes the hydrolysis of phosphatidylcholine to generate free

arachidonic acid (AA). An important domain of lipid research had focused on the

biochemistry of AA, and its cellular and physiological responses which include, but are

not limited to, inflammation, pain, and intracellular signaling. AA itself and its bioactive

eicosanoids are involved in many intracellular signaling pathways. The release of AA is

namely achieved in response to neurotransmitters, growth factors, or lipid stimulation of

cell systems. One such neurotransmitter is ACh.

Previous reports provided evidence for the release of AA via mAChR stimulation

in many systems, including the brain (for review see Felder, 1995), and phannacological

studies have demonstrated the coupling of the odd numbered mAChRs Ml /M3/M5 to the

activation of PLA2, namely to the cPLA2 (Conklin et al., 1988; Felder et al., 1990; Liao

68

•

•

•

et al., 1990; Sharp et al., 1991; Clark et al., 1989). Cholinergie stimulation of AA release

involved, as predicted, a PTX-insensitive G-protein, confirming the involvement of the

odd nwnbered mAChRs which couple to such G-proteins.

Many investigations have focused on the cell signaling function played by AA

and its metabolites in ceUs. Studies have reported the activation of members of the

MAPK family by AA and its Metabolites in several systems and have demonstrated the

co-compartmentalization of MAPKs with cPLA2 (Madamanchi et al., 1998; Hernandez

et al., 1998; Yu et al., 1998). In addition, recent reports provided evidence for PLA2 and

AA Metabolites in signaling to the c-fos serum response element (SRE) via Rac and Rho­

dependent mechanisms (Kim et al., 1998; Kim and Kim, 1998; Kim et al., 1997).

Interestingly, it has been recently reported that the activation of JNK by G-protein

coupled receptors involved Rac and Rho-dependent signaling cascades (Coso et al.,

1995b; Coso et al., 1996; Minden et al., 1995; for review see Gutkind, 1998). These

results suggested a raie for PLA2 in signaling downstream of the mAChRs, and linking

its activation to nuclear responses, including the expression of c-fos mRNA.

Therefore, to investigate the role of AA in mediating the cholinergie stimulation

of c-fos mRNA expression, two cPLA2 inhibitors were used, quinacrine and AACOCf3

(Sakuta and Yoneda, 1994; Wissing et al., 1997). Progenitor cultures were pretreated

with 50 JlM quinacrine or 100 J..l.M AACOCf3 for 20 min followed by 100 J.lM CCh

stimulation for 30 min (fig. Il). The pretreatment of progenitors by either inhibitor

abolished the expression of c-fos mRNA induced by Ceh. These results suggest a role

for the activation of cPLA2 in mediating the increased expression of c-fos mRNA by

cholinergie stimulation in oligodendrocyte progenitors.

69

•

Totalp42mapk ~

Phospho­p42mapk ~

·ta.......

11' 11' 11 ft Ir h

0II

~ ~

• ~ u < ~ a..ë a::: .r;

u êD ~0 + + «

ü ~ Q+

•

•

•

•

FIGURE 1: Western immunoblot autoradiogram demonstrating that p42mapk

activation is mediated by the M3 mACbR. An autoradiogram showing a Western

immunoblot analysis using phosphorylated p42mapk.recognizing antibody. The

membranes were also immunoblotted with non-phosphospecific p42mapk antibody that

recognizes total p42mapk, showing equal loading for each condition. AIl antagonists were

added for 10-20 min before CCh stimulation at a concentration of 1 J.1M (except for 4­

DAMP, 100 nM). p42mapk activation is abolished by atropine (+ Atr) and 4-DAMP (+ 4­

DAMP), but not by pirenzepine (+ Pir) or methoctramine (+ Meth). Note that the plus

sign (+) preceding the antagonists refers to pretreatment of progenitors with the

respective antagonists followed by the stimulation with 100 J.1M CCh.

70

• FIGURE 2

D.:icec+

....-c.+

.cooo~..Coo

-

T

- T'

T

-

-T

-.

T T

-

1 1- 1 1 T la

60

•

•

• FIGURE 2: p4Z··pk activation is mediated by the M3 mAChR. Immunoblot

autoradiograms were quantified densitometrically and expressed in arbitrary optical

density (0.0.) units as mean ± SEM of two independent experiments perfonned in

triplicates. Muscarinic antagonists were preadministered for 10-20 min [Atropine (+ Atr,

1 flM); Pirenzepine (+ Pir, 1 J,lM); Methoctramine (+ Meth, 1 flM); 4-0AMP (+ DAMP,

100 nM)] followed by 100 J,lM CCh stimulation for S min. Atropine and 4-DAMP

blocked the CCh-stimulated levels. Statistical differences are indicated: control versus

(vs) CCh (p<O.OOI); control vs Atr + CCh (p>0.05, Dot significant); control vs Pif + CCh

(p<O.OI); control vs Meth + CCh (p<O.OOl); control vs 4-0AMP + CCh (p>O.OS); CCh

vs Atr + CCh (p<O.OOO1); CCh vs 4-DAMP + CCh (p<O.OOO 1).

•

•71

• FIGURE 3

-5-6-7-8-9

o

-25 4--------r----r--------r-----r----,

-10

100

cS 750(1)~'"o c: 50cOoc..- '":S~ 25.-oCc:

•

CAMP [log(M)]

•

•

•

•

FIGURE 3: 4-DAMP blo~ks the activation of p42 IDapk in a concentration-dependent

manDer. Progenitors were pretreated with increasing concentrations of the M3 more

selective antagonist 4-DAMP (1 nM - 1 J,lM), followed by 100 J.lM CCh stimulation for S

min. p42mapk activation was determined by Western immunoblot analysis, and

autoradiograms were quantified densitometrically. Values are expressed in arbitrary

O.D. units as percentage of inhibition of the CCh-stimulated activation of p42mapk

(Response) as mean ± SEM of two independent experiments performed in triplicates.

Statistical differences are indicated: Stimulated levels (Response, 100 J.lM CCh) vs 1 fJ.M

4-DAMP (p<O.OOI); Stimulated levels vs 100 nM + 4-DAMP (p<O.OOI); Stirnulated

levels vs 50 nM + 4-DAMP (p>O.OS); Stimulated levels vs 10 nM 4-0AMP + CCh

(p>O.OS); Stimulated levels vs 1 nM 4-DAMP + CCh (p>O.OS).

72

•28S rRNA

c-fos mRNA

• 11 11 11' 1J 11 110 .c ~ a. ~ ~~ u - ~ - CL- <: CDc (J <: ~0 + +(J C)

++

•

•

•

•

FIGURE 4: Northern blot autoradiogram demonstrating that e-fos mRNA

expression is mediated by the M3 subtype. Progenitors were pretreated with

muscarinic antagonists at 1 JlM (except for 4-DAMP, 100 nM) for 10 - 20 min, followed

by 100 ~M CCh stimulation for 30 min. Levels ofc-fos mRNA expressed were detected

by a standard Northem blot procedure. Ooly atropine and 4-DAMP pretreatment blocked

the CCh-stimulated c-fos mRNA expression levels. The membranes were also stained

with methylene blue to detect rRJ.'IA bands, demonstrating equal RNA loading in each

different condition.

73

• FIGURE 5

.-D.+

.c...Cl)~

+

Q.:E<1:C+

~...<t+

.coo

-~...&:oo

- TT

-

,

-

T

r 1 r 1 r~

1T1 1T , 1~

o

1500.........-0Cl)..,>.-Cl)~

...J::I

<ci 1000~dECl)

>~; 500.... ~1 Cl)u~

"'-"

•

•

• FIGURE S: CCh..stimulated c..fos expression is mediated by the M3 mAChR. The

Northem blot autoradiogram was quantified densitometrically and ail values are

expressed in arbitrary 0.0. units as mean % SEM of two independent experiments

performed in triplicates. Only atropine and 4-DAMP blocked the CCh-stimulated c-fos

mR.~.A. levels. StatisticaJ differences are indicated: control vs CCh (p<O.OO1); control vs

Atr + CCh (p>O.OS); control vs 4-DAMP + CCh (P>O.OS); control vs Meth + CCh

(p<O.OOl); control vs Pir + CCh (p<O.OOI); CCh vs Atr + CCh (p<O.OOI); CCh vs 4­

DAMP + CCh (p<O.OO 1); CCh vs Melh + CCh (p>O.OS); CCh vs Pir + CCh (p<O.O 1).

•

•74

• FIGURE 6

•

150

.c:8 100

o~cC00; Cl. 50.- 0:eœ.ca:c-

cft. 0

-10 -9 -8 -7 -6 -5

•

DAMP [log (M)]

• FIGURE 6: 4-DAMP blocks the expression of c-fos mRNA in a concentration­

dependent manDer. Progenitors were pretreated with increasing concentrations of 4­

DA~1P (l nM - 1f.1M) for 10 - 20 min, followed by 100 f.1M CCh stimulation for 30 min.

e-fos mRNA levels were determined by Northem blot analysis, and were quantified

densitometrieally. Values are expressed in arbitrary 0.0. units of percentage inhibition

of CCh-stimulated c-fos mRNA expression levels (Response) as mean ± SEM of two

independent experiments performed in triplicates. Statistical differences are indicated:

CCh-stimulated levels (Response) vs 1 f.1M 4-DAMP + CCh (p<O.OO 1); ReSPOnse vs 100

nM 4-DAMP + CCh (p<O.OO1); Response vs 50 nM (p<O.OO1); Response vs 50 nM 4­

DAMP + CCh (p<O.OOl); Response vs 10 nM 4-DAMP + CCh (p<O.OI); Response vs 1

nM 4-0AMP + CCh (p>O.OS).

•

•75

•Control

CCh

4-DAMP-M

4-DAMP-M + CCh

p42111apk Activation(relative O.D. units)

51.3 ± 4.2

122.S ± 0.2

41.3 ± 0.1

35.4 ± 3.1

c-fos mRNA Level(relative O.D. units)

45.4 ± 19.1

662 ± 107.7

49.2 ± 7.3

74.6 ± 7.4

•

•

TABLE 1: 4-DAMP-mustard (4-DAMP-M) blocks the CCh-stimulated p42maPk

phosphorylation and c-fos mRNA expression. Progenitors were preincubated with 1

JlM 4-DAMP-M for 20 min followed by 100 fJ.M Ceh. p42mapk activation levels are

detected using Western blot analysis, and c-fos rnRNA levels are detected using Northern

blot analysis. 4-DAMP-M blocked both CCh-stimulated responses. Statistical

differences indicated: control vs CCh (p<O.OS, for both responses); control vs 4-DAMP-

M + CCh (p>O.OS); CCh vs 4-DAMP-M + CCh (p<O.OS).

76

• FIGURE 7: CCh-stimulated p42-aPk activation requires PLC. Progenitors were

pretreated with 10)lM U73122 or 10 J..lM U73343 for 20 min prior to 5 min stimulation

with 100 J..lM CCh (+ Antagonist). Levels ofp42mapk activation were detected using the

Western blot analysis and were quantified densitometrically. Levels are expressed as

arbitrary optical density units (O.D.) as mean :i: SEM of triplicates. U73122 but not

U73343 blocked the stimulated levels. Statistical differences are indicated: control vs

CCh (p<O.OOl); control vs U73343 (p>O.OS); control vs U73122 (p<O.OS); control vs

U73343 + CCh (p<O.OOI); control vs U73122 + CCh (p>O.OS); CCh vs U73343 + CCh

(p>O.OS); CCh vs U73122 + CCh (p<O.OO 1).

•

•77

• '. •c-fos mRNA Level

(relative 0.0. units)

o01oo

--L

aoo

-L

01oo

1\)ooo

."-C)C::DmGO

Control

CCh

U73122

U73343

+ U73122

+ U73343

1 1 1 1

i

---i

1-

· il-

i'-- ~.· -i

-

· ,

----i

.'.p42mapk Activation

(relative 0.0. units)-L

1\) 01 ~ 00010010

•."-Clc:::am~

CONTROL

CCh

U73122

U73343

+ U73122

+ U73343

1 J1 11

-

-1

-r-

-fJ

t----1

f-J

. . -;

-l""-

r--1

- J-I

- 1

--1--

• FIGURE 8: CCb-stimulated c-fos expression requires PLC. Progenitors were

pretreated with 10 J.lM U73122 or U73343 for 20 min prior ta 100 J.1M CCh (+

Antagonist) stimulation for 30 min. c-fos mRNA levels were detected using a Northem

blot procedure and autoradiograms were quantified deositometrically. Levels are

expressed as arbitrary optical density (0.0.) units as Mean ~ SEM oftriplicates. U73122

but not U73343 blocked the CCh-stimulated levels. Statistical differences are indicated:

control vs CCh (p<O.OOI); cootrol vs U73122 (p>O.OS); control vs U73343 (p>O.OS);

control vs U73122 + CCh (p>O.OS); control vs U73343 + CCh (p<O.OI); CCh vs U73122

+ CCh (p<O.OO 1); CCh vs U73343 + CCh (p>O.OS).

•

•78

f\) ~ al 0000000

•

Control

CCh

Wort

+Wort

c-fos mRNA Level(relative 0.0. units)

-'" -'" f\)U1 0010o 0 0 0o 0 0 0 0

1 1 1 1

1--

f

~

. --1

- -i ..

- -f

'•

m

Control

CCh

Wort

+Wort

p42mapk Activation(relative 0.0. units)

1 1 1 1

. of

-i

of

-;

•»

:!!Clc::0mco

•

•

•

FIGURE 9: p42mapk activation and c-fos mRNA expression are Dot mediated by

PI3K. Progenitor cultures were pretreated with 100 nM wortmannin (Wort) for 20 min

followed by 100 J.lM CCh stimulation. A: p42maPk levels were detected using Western

blot analysis, and were quantified densitometrically and expressed as arbitrary optical

density (0.0.) units as mean ± SEM for two experiments in triplicates. Wortmannin

pretreatrnent did not significantly reduce stimulated levels. Statistical differences

indicated: control vs CCh (p<O.OOl); control vs Wort (p>O.OS); control vs Wort + CCh

(p<O.OOI); CCh vs Wort + CCh (P>O.OS). B: c-fos mRNA levels were detected using

Northem blot analysis, and were quantified densitometrically and expressed as arbitrary

0.0. units as Mean ± SEM of triplicates. Wortmannin (Wort) pretreatment did not

significantly reduce stimulated levels. Statistical differences indicated: control vs CCh

(p<O.OOI); control vs Wort (p>O.OS); control vs Wort + CCh (p<O.OOI); CCh vs Wort +

CCh (p>O.OS).

79

• FIGURE 10

2000

.........-U)aJ.., 1500:>.-aJl:-J::J

• <.zq 10000: 0Ecu::-C/).-0 ..... 500.... .!E1(1)u'-

'-'"

0

CCh (~lM)

-

T

-T T

-

TT

- ,

1 11 1 1 1 1 Î

100 100 100 100 100

•

PD (.lL M) 1 10 50 100

• FIGURE 10: PD 098059 does Dot block CCh..stimulated e..fos gene expressiOD.

Progenitors were pretreated with different concentrations of the MAPK Kinase inhibitor

PD 098059 (1 - 100 J-lM) for 20 min followed by 30 min 100 J.&.M CCh stimulation. c-fos

rnRNA levels \Vere detected using Northem blot analysis, and were quantified

densitornetrically. Values are expressed in arbitrary optical density (0.0.) units as mean

± SEM of triplicates. Only at high concentrations (SO - 100 J-lM) did PD 098059 reduce

the CCh-stimulated levels. Statistical differences are indicated: control vs CCh

(p<O.OOI); control vs 1 J.&.M PD 098059 (p<O.OOI); control vs 10 J.A.M PD 098059

(p<O.OOI); control vs 50 IJ.M PD 098059 (p<0.0l); control vs 100 J.A.M PD 098059

(p<O.Ol).

•

•80

-. FIGURE 11

- ~ t:: M C M0 (.) .- LL -- LI.~ :::J :::J... (.) (J (Jc 0 a0 0 00 (J + (J

<t et<t et

+

-

- T

-

-

T " T-T -- T

1 11 1 r l-1 T 1 1 1 -1o

1250..--...

-0~~ 1000eue:..I::J

•<te 750

•Z·a: OE ~ 500(1);om.... -6 ~ 250

"'-'

•

•

•

•

FIGURE Il: PLA2 mediates the CCh-induced e-fos gene expression. Progenitors

were pretreated with 50 flM of quinacrine (Quin) or AACOCf3 for 20 min followed by

100 J.LM CCh stimulation for 30 min. mRNA levels were detected using Northem blot

analysis, and autoradiograms were quantified densitometrically and expressed as arbitrary

optical density (O.D.) units as mean % SEM of triplicates. Both antagonist pretreatments

bLocked the CCh-stimulated levels. Statistical differences are indicated: control vs CCh

(p<O.OOl); control vs Quin (p>0.05); control vs AACOCf3 (p>O.OS); control vs Quin +

CCh (p>O.OS); control vs AACOCfJ + CCh (p>O.OS); CCh vs Quin + CCh (p<O.OOI);

CCh vs AACOCfJ + CCh (p<O.OO 1).

81

•

•

•

CHAPTER FIVE

DISCUSSION

82

•

•

•

DISCUSSION

In summary~ the present study demonstrates that CCh~ a stable acetylcholine

analogue~ activates p42mapk via the M3 mAChR subtype in oligodendrocyte progenitors.

By pretreating oligodendrocyte progenitors with the M3 selective antagonist 4-DAMP

and its irreversible analogue 4-DAMP-mustard~ the CCh-stimulated p42mapk activation

was abolished. After establishing M3~s predominance in activating p42mapk~ the next step

was to use several pharmacological agents to unravel the pathway leading to its

activation. The present study demonstrated the involvement of PLC in the cholinergie

activation of p42mapk~ since a PLC inhibitor~ U73122, and not its close but inactive

analogue, U73343~ abolished this response. Moreover, pretreating with the PBK

inhibitor wortmannin did not reduce the levels of p42maPk activation~ indicating no role

for PBK in linking the M3 mAChR to p42mapk activation.

In addition, the present study has also focused on the pathways linking mAChR

stimulation to c-fos gene expression. We demonstrate that the cholinergie stimulation of

c-fos rnRNA expression is also mediated by the M3 mAChR subtype. This response was

blocked by 4-DAMP and 4-DAMP-mustard in a concentration-dependent manner.

Similar to p42mapkactivation~ PLC but not PI3K was implicated in the CCh-stimulated

response~ since the pretreatment with U73122 but not U73343 nor wortmannin blocked

the levels of c-fos mRNA expression. As both c-fos rnRNA expression and p42mapk

activation seem to be regulated by the same receptor and effector molecule~ it was

interesting to establish whether c-fos rnRNA expression was regulated by the activity of

p42mapk. Contrary to our predictions, pretreatment with PD 098059, a MAPK kinase

83

•

•

•

(MEK) inhibitor, did not decrease c-fos mRNA expression except at 50 and 100 flM

concentrations. At these high concentrations, the expression levels were reduced but

were not blocked. As a result, p42mapk activation may not be essential to the CCh­

induced changes in c-fos mRNA expression. However, by pretreating progenitors with

the PLA2 inhibitors quinacrine and AACOCf3, c-fos mRNA levels were abolished,

suggesting a role for PLA2 in linking mAChR stimulation to c-fos gene expression.

mAChRs are G-protein coupled receptors, and the different subtypes couple to

different classes of G-proteins. The MIIM31M5 subtypes couple to the PTX-insensitive

class of G-proteins, namely the Gq/l t, while the t\·12/M4 subtypes couple to the PTX­

sensitive class, mainly the Gilo (Offennanns, 1994). The stimulation of p42maPk is one of

the many signais mediated by G-protein coupled receptors such as the mAChRs. In

other systems, for example, the activation of p42mapk via the MI mAChR is mainly

mediated by the ex subunits of the Gq/11 protein (Hawes et al., 1995), whereby M IIM31M5

mediated p42mapk activation is PTX-insensitive (Wotta et al., 1998). On the other hand,

G iJo-coupled M2 mAChRs activate p42mapk primarily through the Gi/o rly subunit (Hawes

et al., 1995; Crespo et al., 1994).

Previous studies have shown that CCh activated p42mapk in oligodendrocyte

progenitors via the stimulation of mAChRs (Larocca and Almazan, 1997). Intense

immunostaining of oligodendrocyte progenitors demonstrated the presence of p42mapk in

the cytoplasm and in the cellular processes. However, Larocca and Almazan (1997) did

not account for the mAChR subtype that is involved in the activation of p42mapk. The

objective to identify the mAChR subtype stems from previous investigations that

characterized pharmacological1y and functional1y the mAChRs present on

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oligodendrocyte progenitors. Competition binding studies demonstrated the presence of

several mAChRs~ with the M3 being the predominant subtype expressed in

oligodendrocyte progenitors (Molina-Holgado et al.~ submitted). CCh-mediated PI

hydrolysis inhibition using mAChR antagonists also confirmed the predominance of the

M3 subtype. Therefore, we inferred a role for the M3 subtype in mediating the activation

of p42mapk• Confrrming our predictions, we indeed demonstrate that the M3 selective

antagonist 4-DAMP and its irreversible analogue 4-DAMP-mustard blocked the CCh­

stimulated activation of p42mapk• Both aotagonists were applied at low concentrations

(l00 nM for 4-DAMP, 1 J.lM for 4-DAMP-mustard) that yield more selectivity to the M3

subtype (Michel et al., 1989; Lazareno et al.~ 1990; Caulfield 1993; Kondou et al., 1994;

Molina-Holgado et al., submitted results). 4-DAMP inhibited P42milPk activation in a

concentration-dependent manner, showing that the ICso of 4-DAMP is at a relatively low

concentration of 50 oM. On the other hand, both 1 J.lM pirenzepine, an MI antagonist,

and 1 J.lM methoctramine, an M21M4 antagonist, did not affect the stimulated levels.

These results denote that the M3 is the mAChR responsible for the CCh-stimulated

p42ffiapk activation.

Subsequently~ we investigated the signaling pathway that links the M3 mAChR to

p42 mapk activation. The M3 mAChR has been shown to couple to PLe through the

interaction of the a subunit of the PTX-insensitive Gq/ 11 protein (for review see Exton,

1996; Lee and Rhee, 1995). The activation of PLC by CCh or other ACh analogues leads

to the generation of two second messengers, inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (DAG). It has been weil established that IP3 cao cause an increase in the

concentration of intracellular calcium as a result of intracellular storage mobilization and

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DAG can activate the cIassical TPA-sensitive PKCs (Berridge, 1993). Previous reports

provide evidence for the activation of PLC by CCh stimulation of oligodendrocyte

progenitors (Cohen and Almazan, 1994). CCh induces the generation of IP3 through PI

hydrolysis as weil as the accumulation of intracellular calcium through the mobilization

of intracellular storage sites and the extracelluar influx through calcium ion channels

(Cohen and Almazan, 1994). Furthermore, another report demonstrated that chelation of

extracellular calcium by EDTA blocked the activation of p42mapk in oligodendrocyte

progenitors (Larocca and Almazan, 1997). However, the requirement of intracellular

calcium may not he ruled out, since the intracellular calcium chelator BAPTA was not

used in the study by Larocca and Almazan (1997). Therefore, we speculated a role for

PLC and its generated second messengers in linking the stimulation of mAChRs to

dO\-Vllstream activation ofp42mapk•

1t is interesting to note that other studies have shown that MEKs and MAPKs can

be stimulated by the elevation of intracellular calcium through calcium ionophores,

thapsigargin, elevated extracellular calcium, or membrane depolarization (for review see

Lewis et aL, 1998). On the other hand, the question of PKC stimulation of the MAPK

cascade has been addressed, yet in Many cases the mechanism is cell type dependent.

Phorbol esters activate MAPKs in nearly every cell (for review see Lewis et al., 1998).

Larocca and Almazan (1997) demonstrated that CCh-induced p42mapk activation in

oligodendrocyte progenitors involved TPA-insensitive (or DAG-independent) PKC

isozyme(s), since the PKC inhibitors H7 and bisindolylmaleimide GFlü9203X, but not

chronic pretreatment with TPA, abolished the mAChR-mediated response.

To investigate the role ofPLC, the present study used the PLC inhibitior U73122

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and its inactive analogue U73343 (Tatrai et al.~ 1994). The active PLC inhibitor U73122,

but not the inactive analogue U73343, blocked the CCh-stimulated levels of p42mapk

activation. However, U73122 administration without CCh stimulation reduced the basal

levels significantly, in comparison to U73343 which had no effect on basal levels.

Previous studies have reported that in addition to PLC inhibition, U73122 may act on

other sites. Such mechanisms include the inhibiton of both intracellular calcium

channels, which are activated by fonnation of IP) by PLC, and plasma membrane bound

calcium channels (Pulcinelli et al., 1998). In the absence of CCh stimulation, muscarinic

receptor-coupled PLC is inactive, and thus Ïntracellular calcium channels activated by IP3

are closed. On the other hand, plasma membrane-bound calcium channels are

independent of PLC activation and may play a role in transient calcium currents.

Therefore, U73 122 may antagonize plasma membrane calcium channels and as a resuit

decrease the basal levels of p42maPk activation, while attenuating the CCh-stimulated

response by inhibiting both PLC activity and calcium channels.

Previously, Lopez-I1asaca et al. (1997) have demonstrated that p42mapk activation

by G-protein coupled receptors can be mediated by PI3K, a phosphatidylinositol kinase

(PIK), through a process that requires G-protein's f3y subunits. The wortmannin-sensitive

PI3K isozyme, PI3Ky, cao be activated by both the a and the f3y subunits of

heterotrimeric G-proteins (Stoyanov et al., 1995). Several studies have reported on the

regulation of differeot PIKs and PI pools by mAChRs. For example, wortmannin­

sensitive PIKs, such as PI4K (Sorensen et al., 1998), play a role in the endocytosis of

mAChRs (Sorensen et al., 1998), in addition to the regulation of PI pools by mAChRs, a

mechanism that involves the activation of different PIKs (Willars et al., 1998). The

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present study investigated whether a wortmannin sensitive PIK is required for stimulating

p42mapl.; by CCh. Pretreating progenitors with wortmannin showed that wortmannin was

not effective in reducing the levels of CCh-stimuIated p42mapk activation, and thus PI3K

or any other wortmannin-sensitive PIK May not be impIicated in the CCh-mediated

process. These results were similar to other recent studies that demonstrate wortmannin

insensitivity of the activation ofp42mapk by the M3 mAChR (Wotta et al., 1998).

Therefore~ in view of our present study, we conclude that the M3 subtype is

mediating the activation of p42mapk in oligodendrocyte progenitors via the action of PLC,

presumably by calcium mobilization, but not through PI3K. We specuIated that the

cascade linking the M3 mAChR to p42mapk activation does not require classical DAG­

sensitive PKC isozymes. The requirement of calcium for the activation of p42mapk has

been previously reported in oligodendrocyte progenitors (Larocca and Almazan, 1997).

However, contrary to previous reports indicating the involvement of a classical TPA­

sensitive PKC isozyme(s) (Schonwasser et al., 1998; Young et al., 1996), Larocca and

Almazan (1997) have shown that in oligodendrocyte progenitors, the activation of

p42mapk May require an atypical PKC isozyme that is TPA-insensitive. Ono et al. (1989)

demonstrated the expression of the atypical PKCÇ in oligodendrocyte progenitors.

Asotra and Macklin (1994) have aIso shown the expression of severaI phospholipid­

dependent PKC isozymes in oIigodendrocyte progenitors, including PKCÔ and PKCE. In

other cell lines, studies have shown that atypical PKC isozymes can activate p42mapk oruy

at the level of MEK but not Raf-l, suggesting a different signaling pathway linking

atypical PKCs to MEK activation (Schonwasser et al., 1998). Therefore, it is tempting to

speculate that the CCh-induced activation of p42mapk in oligodendrocyte progenitors

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involves an atypicaI PKC isozyme in addition to a PLC-dependent pathway that generates

calcium, and further studies are needed to elucidate the PKC isozyme(s) involved.

Moreover, the present study investigated nuclear responses downstream of the

mAChRs. Studies in cholinergie nuclear responses in oligodendrocyte progenitors have

been previously reported (Cohen et al., 1996). The investigation of nuclear responses

induced by cholinergie stimulation of oligodendrocyte progenitors can help unravel the

role of mAChRs in oligodendrocyte development. Previous pharmacological and

functional studies on progenitor and mature oligodendrocytes have displayed the

presence of several mAChRs subtypes in progenitors that undergo extensive

downregulation during the process of maturation (Molina-Holgado et al., submitted).

This interesting fmding poses the question of the developmental role pIayed by mAChRs

in the maturation of oligodendroeyte progenitors. Indeed, we have demonstrated that

cholinergie stimulation of oligodendroeyte progenitors induces several developmental

and signaling responses, including increased proliferation of progenitors, second

messengers accumulation, p42mapk activation, and c-fos gene expression (Cohen et al.,

1996; Cohen and Almazan, 1994; Larocca and Almazan, 1997; Cohen et al., 1996).

Expression of c-fos proto-oncogene has been implicated in cell growth, proliferation,

protein synthesis, and inhibition ofdifferentiation (for review see Janknecht et al., 1995).

The prevention of differentiation by c-fos gene expression is an interesting observation

because CCh can stimulate proliferation and not differentiation of progenitors in addition

to the expression of c-fos rnRJ.'lA (Cohen et al., 1996).

Therefore, we investigated the downstream signaling events linking mAChR

activation to nuclear responses such as c-fos gene expression. First, the mAChR subtype

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that is mediating the expression of e-fos mRNA was pharmaeologieally detennined.

Sinee the predominant subtype expressed in oligodendrocyte progenitors is the M3

subtype, and this subtype was also shown to mediate the activation of p42mapk, we

postulated that the M3 is also the subtype responsible for the cholinergie stimulation ofc­

fos mRNA expression. By using selective mAChR antagonists, we have provided

evidence in this study that the M3 mAChR does indeed mediate the CCh-stimulated

expression of c-fos mRNA. Both 4-DAMP and 4-DAMP-mustard bloeked this response.

Attenuation of c-fos gene expression by 4-DAMP demonstrated dose-dependency

whereby the ICso of 4-DAMP on c-fos mRNA expression is 10 nM. Both pirenzepine and

methoctramine had no effeet on the levels ofexpression.

Furthermore, since the M3 mAChR is coupled to a PTX-insensitive G-protein

which can activate PLC, we postulated a role for PLC in mediating the CCh-stimulated c­

fos nlRNA expression. As we have demonstrated for the stimulation of p42mapk, U73122

but not U73343 was effective in blocking the CCh-stimulated response. Since Cohen and

Almazan (1994) have provided evidenee for the activation of PLC by CCh, our fmdings

support a role for PLC in mediating the CCh-induced c-fos gene expression. The

present study also confmns earlier reports by Cohen et al. (1996) which have shown that

CCh-induced c-fos gene expression is calcium dependent, since PLC can generate an

increase in intracellular calcium through the action of IP3 on the endoplasmic reticulum.

However, Cohen et al. (1996) have shown that only the atypical TPA-insensitive PKC

isozymes may be involved in this response. Our results therefore indicate a potential role

for PLC in mediating the CCh-induced response only through calcium and not through

the classical TPA-sensitive PKC isozymes. Further investigations are needed to elucidate

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the different TPA-insensitive PKC isozyme(s) that may play a role in modulating the

CCh-stimulated expression ofc-fos mRNA.

Other signaling Molecules that cao potentially mediate c-fos mRNA expression

were investigated. Wortmannin, a PI3K inhibitor, was used to identify a possible role

for PI3K or any other wortmannin-sensitive PIK in mediating the CCh-stimulated c-fos

gene expression. Previous studies have shown that PI3K is a signaling molecule that is

regulated by receptors with intrinsic or associated tyrosine kinase activities, in addition to

its activation by both a and r3y subunits of heterotrimeric G-proteins (for review, see

Kapellar and Cantley, 1994). PI3K is involved in the regulation of protein synthesis and

cell proliferation by mitogens, whereby it can activate a variety of signaling Molecules

that include p70S6-kinase which activates the ribosomal 86 protein (Valius et al., 1993;

Seva et al., 1997), Many PKC isozymes from both conventional and novel families, plus

other kinases including Akt, Bkt, and Rac (for review see Fukui et al., 1998). The

different activation targets of PI3K lead us to speculate a role for PI3K in stimulating the

expression of proto-oncogenes such as c-fos. However, similar to the mode of p42mapk

activation by CCh, our present findings showed that wortmannin did not significantly

reduce the levels ofc-fos mRNA expression upon CCh stimulation.

The fact that both p42mapk activation and c-fos mRNA expression are mediated by

the same mAChR, the M3 subtype, suggests that this subtype cao play a major role in

cholinergie regulation of oligodendrocyte progenitors. It cao also he speculated that the

M3 mAChR cao also mediate the proliferative action of CCh on oligodendrocyte

progenitors (Cohen et al., 1996). Our findings denote that both responses are mediated

by similar pathways, involving PLC activation, calcium accumulation, and a TPA-

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insensitive PKC isozyme, but not wortmannin-sensitive PIKs. As a resul~ we questioned

whether both CCh-induced responses are linked. Our results demonstrate that

pretreatment with PD 098059, a MEKI inhibitor, at concentrations 1 to 10 flM, did not

significantly reduce the CCh-stimulated levels of c-fos mRNA. ft is important to note

that PD 098059 cao inhibit MEK1, the kinase that activates p42Rl3Pk, at 1-10 IJ.M, since

the ICso of this inhibitor on MEKI is 2-7 flM (Alessi et al., 1995; Dudley et al., 1995).

At concentrations of 50 and 100 flM, however, PD 098059 caused a significant reduction

but not a total block of the response. Sïnce Larocca and Almazan (1997) have previously

demonstrated that 10 flM concentration of PD 098059 is sufficient to completely block

the levels of p42mapk activation in oligodendrocyte progenitors (Larocca and Almazan,

1997), our current results suggest that the MAPK pathway rnay not be the major pathway

linking mAChR stimulation to c-fos rnRNA expression. The attenuation of c-fos mRNA

levels at 50 and 100 J.lM PD 098059 concentration May result from non-selective action

of this inhibitor at other MEKs such as MEK2 (ICso = 50 flM). These findings are

intriguing, since Many studies have linked the stimulation of the MAPK pathway to

downstream regulation of the c-fos promoter via the activation of transcription factors by

severa! MAPK species (for review see Janknecht et al., 1995; Gutk.ind, 1998).

Recent studies in oligodendrocyte progenitors showed a CCh-induced activation

of the transcription factor cyclic-adenosine monophosphate (cAMP) response element

binding protein (CREB) and the activation of the calcium-calmodulin kinase II (CaMK

II) (Pende et al., 1997; Sato-Bigbee et al., 1999). Both effector Molecules have been

shown to regulate the promoter of immediate early genes, such as c-fos. Pende et al.

(1997) have displayed that both responses are calcium dependent (Pende et al., 1997).

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Calcium cao trigger various intracellular events including both short term and long term

(Ghosh and Greenberg, 1995). It has also been postulated that calcium signaIs can

regulate the function of several transcription factors, including CREB (Sheng et al.,

1990), in addition to the modulation of several key regulatory enzymes such as PKC's

and CaMK's. Both of these families of kinases have been shown to be able to

phosphorylate CREB in vitro (Yamamoto et al., 1988; Sheng et al., 1991). Once

phosphorylated, CREB cao modulate the transcriptional activity of several genes by

binding to the cAMP response element (CRE) site at the promoter region. CREB can

modulate the expression of c-fos by binding to the CRE that is present on the c-fos

promoter (for review, see Karin.. 1995; Gutkind, 1998). Contrary to our speculations, the

study has aiso shown that pretreatment with the MEK inhibitor PD 098059 blocked the

activation of CREB by CCh, suggesting the involvement of the MAPK pathway in

stimulating CREB (Pende et al., 1997). Since our present study demonstrated no

significant decrease in the Ievels of c-fos mRNA expression upon PD 098059

pretreatment~ it can therefore be postuJated that while the CCh-stimulated activation of

both CaMKII and CREB are calcium dependent and may play a role in regulating

nuclear responses, the activation of CREB may not contribute to the CCh-stimulated

changes in c-fos gene expression.

The c-fos promoter contains several response elements such as SRE and SIE that

are regulated by transcription factors other than CREB, including AP-I, SRf, Elk-l, and

Sap-l (for review see Gutkind, 1998; Janknecht et al., 1995). Moreover, the c-Jun-NH2

terminal kinase (JNK) has been weB characterized and identified as a serine/threonine

kinase that cao phosphorylate transcription factors involved in c-fos gene regulation, such

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as c-Jun, c-Fos, and Elk-l. Upon stimulation, c-Fos and c-Jun dimerize to fonn the AP-l

transcription factor. Furthermore, the activation of EIk-1 facilitates binding to the SRF.

The formation of the AP-l transcription factor and the binding of SRf to Elk-l are

crucial regulatory events for c-fos gene expression. Recently, the p38 species ofMAPKs

has aIso been implicated in regulating the activity of certain transcription factors such as

MEF2 and ATF2 that can modulate the c-jun promoter (Han et al., 1997; Gutkind, 1998).

This field of research has been poody understood and it is currently being investigated.

As a result, further work is required to identify a role for other pathways or other ~1APK

species in the transduction cascade that links modulations in c-fos gene expression to

receptor activation.

Our investigations of the pathways downstream of the mAChRs that cao lead to

c-fos rnRNA expression explored the role of PLA2 in linking receptor activation to gene

expression. The product of PLA2 enzymatic activity, arachidonic acid (AA), can be

converted to prostaglandins, thromboxanes, leukotrienes, and other bioactive eicosanoids.

This major eicosanoid pathway has been shown to be activated by cholinergie stimulation

via the odd numbered mAChRs in many systems (for review see Felder, 1995). Recent

reports have described a linkage between AA and its metabolites to the stimulation of the

c-fos SRE through Rac and Rho-dependent mechanisms (Kim et al., 1998; Kim et al.,

1997). Via the activation of Rac and Rho-related proteins, G-protein coupled receptors,

including the mAChRs, cao stimulate MAPK species such as JNK (Coso et al., 1995b;

Caso et al., 1996; Minden et al., 1995), and subsequently the modulation of gene

expression such as c-jun and c-fos (for review see Gutkind, 1998). These findings

provided ample evidence that c-fos gene expression may be PLA2-dependent. In

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oligodendrocytes, studies have shown that AA cao Mediate severa! responses, including

MBP phosphorylation aod the inhibition of potassium conductances (Takeda and Soliven,

1997; Soliven and Wang, 1995). Our results confirmed our extrapolations, since the

pretreatment of progenitor cultures with either PLA2 inhibitors, quinacrine or AACOCF3,

abolished the stimulated levels of c-fos mRNA. However, further studies are needed in

order to determine whether AA itself or any of its Metabolites Mediate this response, and

therefore, more studies are needed to unravel the metabolite(s} leading to this nuclear

response.

In summary ~ our present study demonstrates that the M3 is the mAChR subtype

mediating the CCh-stimulated p42mapk activation and c-fos mRNA expression in

oligodendrocyte progenitors. The activation of p42mapk is dependent on PLC but not on a

wortmannin-sensitive PI3K. Similarly, the CCh-stimulated c-fos mRNA expression also

involves the activation of PLC and PLA2 but not a wortmannin-sensitive PI3K.

Although both p42rt141Pk activation and c-fos mRNA expression are mediated by the same

mAChR subtype, our study demonstrates that the activation of p42mapk is not necessarily

required for the CCh-mediated c-fos rnRNA expression. Further studies are needed to

identify the downstream signaling pathways that lead to the activation of p42mapk and the

increased expression ofc-fos mRNA, including the identification of the PKC isozyme(s)

that mediate(s) both responses, the investigation of other MAPK species that contribute

to the regulation of c-fos gene expression, in addition to the exploration of the role(s) of

AA and/or its Metabolites in mediating this nuclear response.

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CHAPTERSIX

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