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The expression of protein kinases and the role of extracellular signal-regulated protein kinases 1 and… Heisel, Rochelle 2001

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THE EXPRESSION OF PROTEIN KINASES AND THE ROLE OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASES 1 AND 2 IN OLIGODENDROCYTES  by  ROCHELLE HEISEL  B . S c , The University of British Columbia, 1994  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES Faculty of Medicine  Experimenta.l Medicine Program . We accept this thesis as confirming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 2001  copyright Rochelle H e i s e l , 2001  UBC  Special Collections - Thesis Authorisation Form  Page 1 of 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department o f  f N2dLv^.rsff^ r  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada  Date  ftc^-^H  (cOl  http://www.library.ubc.ca/spcoll/thesauth.html  8/24/01  ABSTRACT  Oligodendrocytes (OL), the myelinating  cells of the central  nervous  system, extend processes to contact axons and wrap them in an insulative layer of myelin.  This series of studies was undertaken to examine the role of  extracellular signal-regulated protein kinases (ERKs) 1 and 2 in O L process extension. First, it was determined that stimulation of mature primary bovine O L with the phorbol ester P M A could induce both process extension and ERK1/2 activation. Furthermore, application of the MEK1 inhibitor P D 98059 was able to both  block  PMA-induced  phosphotransferase activity.  process  extension  and  reduce  ERK1/2  Thus it appears that a threshold of  ERK1/2  phosphotransferase activity is required for primary O L process extension. To continue to elucidate the signalling cascades involved in O L process extension, the Central-Glial 4 (CG-4) cell line was assessed for suitability as an OL model.  CG-4 are bipotential cells capable of differentiating  astrocytic or oligodendrocytic (CG-4 OL) cells.  into either  A multi-kinase Western blot  profile was conducted to compare the kinase expression patterns of primary rat OL to CG-4 OL. Overall, the expression of a wide variety of kinases, including conventional protein kinase C (PKC) isoforms, mitogen-activated protein kinases, protein kinase A and protein kinase B were very similar between the two cell types.  However,  some differences  in kinase expression were  detected.  Increased expression of focal adhesion kinase, P K C - s and cyclin-dependent kinase (CDK) 7 in CG-4 cells could be a function of the self-renewal capacity of this cell line.  Increased expression of Pak-a, P K C - 5 and C D K 5 in primary OL  ii  could explain why these primary cells can achieve a greater degree of differentiation than C G - 4 OL. After verifying the suitability of the C G - 4 cell line as an O L model, further process extension studies were undertaken. It was found that transient ERK1/2 activation is required to prevent bipolar C G - 4 cells from acquiring a multipolar phenotype. This transient ERK1/2 activation was provided by addition of medium containing B-104 mitogens.  In B-104 mitogen-free medium, ERK1/2 was not  activated and the C G - 4 cells acquired a multipolar phenotype.  Furthermore,  P M A was able to activate ERK1/2 in lieu of B-104 mitogens, while at the same time inhibiting the formation of a multipolar phenotype.  To verify a role for  ERK1/2 activation in the inhibition of a multipolar phenotype, C G - 4 cells were exposed to B-104 mitogens in the presence of the MEK1/2 inhibitors P D 998059 or UO-126. Surprisingly, even pretreatment of the cells with either M E K inhibitor could not induce the formation of multipolar processes. Western blots, however, indicated that neither inhibitor was able to completely abolish ERK1/2 activity. Therefore, it is possible that the MEK1/2 inhibitors were unable to reduce ERK1/2 activity below the level necessary to inhibit multipolar process formation. In summary, ERK1/2 activation can both induce process extensions from primary O L and inhibit the formation of a multipolar phenotype in C G - 4 OL. This could indicate that the C G - 4 cell line does not make a suitable model for O L signal transduction studies. Therefore, caution must be taken when applying the results of signal transduction experiments conducted on C G - 4 cells to primary OL.  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  ix  List of Figures  x  List of Abbreviations  xiii  Acknowledgements  xvi  Dedication  xvii  C H A P T E R 1: G E N E R A L INTRODUCTION  1.1 An Introduction to Oligodendrocytes (OL) and Central-Glial 4 (CG-4) Cells 1.1.1 The Biology of Primary Oligodendrocytes:  1 2  O L Progenitor Cells 1.1.2 The Biology of Primary Oligodendrocytes:  4  Mature O L 1.1.3 Discovery and Characterization of the C G - 4 Cell Line  6  1.1.4 A Comparison of the Properties of Primary O L  8  and C G - 4 Cells 1.2 Signal Transduction 1.2.1 General Protein Kinase C Signalling  12 12  IV  1.2.2 P K C Signalling Cascades in Primary O L  17  1.2.3 General M A P K Signalling  23  i)  ERKs  23  ii) J N K  26  iii) p38  27  1.2.4 ERK1/2 Signalling in Primary O L and C G - 4  28  1.2.5 J N K and p38 Signalling in Primary OL and C G - 4  30  1.3 Rationale and Objectives  32  C H A P T E R 2: M A T E R I A L S A N D M E T H O D S  2.1 General Materials  35  2.1.1 Chemical Reagents  35  2.1.2 Laboratory Supplies  38  2.1.3 Antibody Reagents  38  2.2 General Methods  39  2.2.1 Primary Bovine O L Cultures  39  2.2.2 Primary Rat OL Cultures  41  2.2.3 Bipotential C G - 4 Cultures  41  i) B-104 Conditioning  42  2.2.4 Differentiation of C G - 4  42  2.2.5 Treatment Protocols  43  i) Phorbol Ester Treatments  43  ii) Ro-32 Treatments  44  iii) UO-126 and PD 98059 Treatments  44  2.2.6 Cell Lysis  45  i) Cytosolic Lysates  45  ii) Membrane Lysates  46  2.2.7 Determination of Protein Concentration  47  2.2.8 Mono Q Fractionation  47  2.2.9 ERK1/2 Kinase Assays  48  2.2.10 SDS-polyacrylamide Gel Electrophoresis  48  2.2.11 Western Blotting  50  i ) Oligodendrocytes  50  ii) C G - 4  51  2.2.12 Immunoprecipitations  51  2.2.13 Immunocytochemistry  52  C H A P T E R 3: T H E R O L E O F E R K 1 A N D 2 IN P R I M A R Y OL P R O C E S S E X T E N S I O N  3.1 Introduction  53  3.2 Results  54  3.3 Discussion  61  vi  C H A P T E R 4: A C O M P A R I S O N O F T H E KINASE E X P R E S S I O N P R O F I L E B E T W E E N P R I M A R Y R A T OL A N D C G - 4  4.1 Introduction  71  4.2 Results  72  4.2.1 Comparison of Primary O L to C G - 4 OL  77  4.2.2 A Comparison of the Three Differentiation States  87  of C G - 4  4.3 Discussion  88  C H A P T E R 5: T H E R O L E O F E R K IN C G - 4 P R O C E S S E X T E N S I O N 5.1 Introduction  96  5.2 Results  97  5.2.1 P K C Inhibitor Studies  93  5.2.2 M E K Inhibitor Studies  106  5.2.3. Phorbol Ester Studies  106  5.3 Discussion  114  5.3.1 P K C Inhibitor Studies  114  5.3.2 M E K Inhibitor Studies  117  5.3.3 Phorbol Ester Studies  118  vii  C H A P T E R 6: S U M M A R Y A N D F U T U R E DIRECTIONS  6.1 The Role of E R K 1 and 2 in Primary OL Process Extension  119  6.2 A Comparison of the Kinase Expression Profile Between Primary  122  Rat OL and C G - 4 6.3 Process Extension in C G - 4  124  6.4 Conclusions  126  REFERENCES  131  A P P E N D I X A: Kinetworks™ Analysis  168  viii  List of Tables  Table 1.  The Shared Properties of Primary O L and the  9  CG-4 Cell Line  Table 2.  Kinetworks™ Protein Kinase Screen  76  Table 3.  Protein Kinases Undetected by the Kinetworks™ Analysis  78  Table 4.  Kinetworks™ Phosphoprotein Analysis  105  ix  List of Figures  Figure 1.  The Conserved Domains of P K C Isoforms  14  Figure 2.  General Schematic of ERK1/2 Signalling via  25  Growth Factor Activation  Figure 3.  The Effects of P M A and P D 98059 on Primary  55  Bovine OL  Figure 4.  P D 98059 Dose Response  56  Figure 5.  E R K 1 / 2 Phosphotransferase Activity  58  Figure 6.  Anti-ERK Western Blot  59  Figure 7.  Anti-Phosphotyrosine Western Blot  60  Figure 8.  Raf Immunoprecipitations  62  Figure 9.  The Effects of Wortmanin, Forskolin, and KN-62  69  on Primary Bovine OL  Figure 10.  The Effects of Wortmanin, Forskolin, and KN-62 on PMA-induced Process Extension in Primary Bovine OL  70  Figure 11.  C N P Staining of C G - 4 OL and Primary Rat OL  Figure 12.  C G - 4 OL and Primary Rat OL  74  Figure 13.  The Three Differentiation States of the C G - 4 Cell Line  75  Figure 14.  M A P K Expression Profile  79  Figure 15.  Western Blot Kinase Expression Profile  80  Figure 16.  Western Blot Kinase Expression Profile  81  Figure 17.  P K B - a and P K A Expression Profile  84  Figure 18.  P K C Expression Profile  85  Figure 19.  C D K 7 and C D K 5 Expression Profile  86  Figure 20.  The Inhibitory Effects of Ro-32 on the Development  98  of a Multipolar Phenotype in C G - 4 OL  Figure 21.  The Effects of 70/30, N1, and Ro-32 on ERK1/2  99  Activation in C G - 4 Cells  Figure 22.  Morphological Changes of C G - 4 Cells  101  xi  Figure 23  The Effects of Ro-32 on ERK1/2 Activation of C G - 4 Cells  102  Figure 24  The Effects of 70/30 and N1 on P K C - a Activity  104  in C G - 4 Cells  Figure 25.  The Effects of P D 98059 and UO-126 on C G - 4  107  Cell Morphology  Figure 26.  The Effects of P D 98059 and UO-126 on ERK1/2  108  Activation in C G - 4 Cells  Figure 27.  The Effects of P D 98059 on ERK1/2 Activation  109  in C G - 4 Cells  Figure 28.  Transient Inhibition of a Multipolar/ Phenotype  111  in C G - 4 Cells by P M A Treatment  Figure 29.  The Activation of P K C and ERK1/2 in C G - 4 Cells  112  Treated with P M A  Figure 30.  P M A Dose Response  113  xii  List of Abbreviations  5% HS  Dulbecco's Modified Eagle Mediim plus 5% horse serum  70/30  70% N1 + 30 % B-104-conditioned medium  ADB  assay dilution buffer  AEC  3-amino-9-ethyl-carbazole  ATP  adenosine triphosphate  BSA  bovine serum albumin  bFGF  basic fibroblast growth factor  CG-4  Central-Glial 4 cell line  CG-4 OL  oligodendrocytic C G - 4  cm  centimeter  CNP  2,3-Cyclic nucleotide 3-phosphohydrolase  CNS  central nervous system  CNTF  ciliary neurotrophic factor  DAG  diacylglycerol  DMEM  Dulbecco's Modified Eagle Medium  DMSO  dimethylsulfoxide  DTT  dithiothreitol  ECL  enhanced chemiluminescence  EDTA  ethylenediaminotetraacetic acid  ERK  extracellular signal-regulated protein kinase  GalC  galactocerebroside  GDP  guanosine diphosphate  GFAP  glial fibrillary acidic protein  Grb-2  Growth factor receptor binding-2  GTP  guanosine triphosphate  HCI  hydrochloric acid  HOG  high osmolarity glycerol response kinase  hr  hour  Xlll  IPs  inositol (1,4,5) tris-phosphate  IL-1  interleukin-1  JNK  c-Jun N-terminal kinase  LIF  leukemia inhibitory factor  M  molar  mA  milliampere  MAP-2  microtubule-associated protein 2  MAPK  mitogen-activated protein kinase  MAPKAP-2  MAPK-activated protein kinase-2  MARCKS  myristoylated alanine-rich C-kinase substrates  MBP  myelin basic protein  MEK  M A P K / E R K kinase  MGDG  monogalactosyl diglyceride  p-g  microgram  ul  microlitre  u.m  micromillimeter  uM  micromolar  min  minute  ml  milliliter  mm  millimeter  MMP-9  metalloproteinase 9  MOPS  3-[N-morpholino]ethanesulfonic acid  MS  multiple sclerosis  N1  defined C G - 4 feeding medium  NaCI  sodium chloride  NGF  nerve growth factor  NGS  normal goat serum  nM  Nanomolar  NT-3  neutrophin-3  02A  oligodendrocyte type 2-astrocyte  OL  Oligodendroctye  xiv  Pak  p21-associated protein kinase  PBS  phosphate buffered saline  PDGF  platelet-derived growth factor  Penstrep  penicillin and streptomycin solution  PI 3-K  phosphatidylinositol 3-kinase  PICK'S  protein's that interact with C-kinase  PIP  phosphatidylinositol 4,5-bisphosphate  2  PKA  protein kinase A  PKB  protein kinase B  PKC  protein kinase C  PKI  protein kinase inhibitor  PL  poly-L-lysine  PLC  phospholipase C  PLP  Proteolipid  PMA  phorbol 12,13-myristate acetate  PMSF  phenyl methylsulphonyl fluoride  PS  Phosphatidylserine  RACK'S  receptors for activated-C kinase  R-mAb  Ranscht monoclonal antibody  rpm  revolutions per minute  RT  room temperature  SAPK  stress-activated protein kinase  SDS-PAGE  sodium dodecylsulphate-polyacrylamide gel electrophoresis  SH  src-homology  SOS  son-of-sevenless  STATs  signal transducers and activators of transcription  TBS  Tris buffered saline  TBST  Tris buffered saline + Tween-20  TNF  tumour necrosis factor  Tris  tris (hydroxylmethyl)  Tween 20  polyoxyethylene-20-sorbitan monolaurate  methylamine  XV  Acknowledgements  I would like to acknowledge Dr. Seung Kim, and to thank him for the invaluable opportunity to conduct my research under his supervision. I would also like to thank the members of my committee, Dr. Seung Kim, Dr. Roger Brownsey, Dr. Charles Krieger, and Dr. Steven Pelech, for sharing their time and expertise. So many people have helped me during the course of my research. I would like to thank all the Fellows and students of Dr. Kim's lab, especially Dr. Coral Sanfeliu, Dr. Kozo Hatori, Dr. Atsushi Nagai, Dr. Akihiko Ozaki, Hyun Beom Choi and Jae Kyu Ryu for their technical and moral support. I would also like to thank our technician, Mrs. Margaret Kim, for all her assistance. Thank you to Dr. Steven Pelech for giving me the opportunity to learn molecular biology research in his laboratory. For their guidance in the early stages of my research, I would like to thank Mohammed Hasham, Dr. Bill Sahl, and Dr. Jasbinder Sanghera. Hong Zhang, Jane Shi and Harry Paddon - thank you for your patience and advice! Venska Wagey and Maggie Hampong - thank you for your help, and most of all for your friendship! Finally, a big "thank you" to all my family and friends. You stood by me through the ups and downs, and your belief in me has made this possible. A special thank you to my husband, Olaf - you not only put up with the many stressful days and sleepless nights, but you also made me smile and helped me through them all!  xvi  . my/vawnfoj &<my wnd' Qf$&w4iMa ^hz^iAaj wAaae wn  . my Au^lundj  (B/a^ (£%pei6e/ 9  J  wAa maAe$ me 60 Aa/ifey/  CHAPTER 1: GENERAL INTRODUCTION  1.1 A n Introduction to Oligodendrocytes and Central-Glial 4 Cells  In the developing central nervous system (CNS), pluripotent stem cells have the capability of differentiating into cells of either neuronal or glial lineage. Cells of neuronal lineage develop into nerve cells, while cells of glial lineage can develop into astrocytes or oligodendrocytes (OL). While glial cells were originally thought to be merely supporting cells for neurons, they are now known to be highly specialized cell types.  For example, astrocytes provide framework and  trophic support for neurons, and can also buffer excess potassium. A s the myelinating cells of the C N S , OL also play a very important physiological role. These cells are responsible for wrapping nerve cell axons in an insulative coating of myelin, thus allowing for the efficient conduction of nerve impulses. Disruption of O L and the myelin they create can lead to severe pathological consequences, such as the paralysis, numbness, and loss of vision associated with the disease multiple sclerosis (MS). The significance of O L is reinforced by studies on M S , which have shown that spontaneous remyelination by surviving O L can alleviate symptoms  (Rodriguez,  1992; Prineas et  spontaneous remyelination is incomplete.  al., 1993).  Unfortunately,  this  Thus, in order to treat diseases  associated with O L dysfunction, it is crucial to understand the functional biology of these cells.  1  Studies of O L functional biology have historically relied on the use of primary O L cultures.  However, due the post-mitotic nature of mature OL,  scientists have searched for an OL cell line capable of self-renewal. Relatively recently, a cell line known as Central-Glial 4 (CG-4) has emerged as an OL model (Louis et al., 1992).  C G - 4 cells have become a popular choice for a  number of reasons. First, this cell line initially arose as a spontaneous mutation from a primary culture of rat OL progenitor cells. A s such, it is not considered to be a transformed cell line, and it shows a normal karyotype after 25 passages. Second, phenotypic marker analysis has shown that C G - 4 cells can display OLspecific markers, such as myelin basic protein (MBP).  Third, transplantation  studies have shown that C G - 4 cells can myelinate demyelinated axons (Duncan et al., 1996). Therefore, using both C G - 4 cultures and primary O L cultures, the knowledge  base on OL functional  biology has steadily  increased.  The  characteristics of primary O L and C G - 4 cell models will be reviewed below.  1.1.1 The Biology of Primary Oligodendrocytes: OL Progenitor Cells  The classical pathway of OL maturation is best characterized for the developing rat C N S . However, this developmental pathway may not adequately define the steps of OL maturation in the C N S of humans and other mammalian species.  Despite this potential species variation, the well-defined rat OL  developmental pathway provides a useful framework with which to characterize the various stages of OL maturation.  2  For neonatal rat brain cells committed to the glial lineage, the first main step of OL development begins with a bipotential progenitor known as an oligodendrocyte type 2-astrocyte (02A). The 0 2 A cell is a proliferative, bipolar and motile progenitor that has the capacity to differentiate into either OL or type 2-astrocytes (Raff et al., 1983; Small et al., 1987).  It can be identified with a  monoclonal antibody, A2B5, which recognizes the gangliosides GQ1b, G D 3 , GT1, and G D 2 (Kundu et al., 1983).  In vitro, addition of serum to the culture  medium favours the differentiation of 0 2 A cells into astrocytes. Conversely, the use of serum-free medium induces 0 2 A cells to develop into mature OL (Raff et al., 1983). Addition of mitogens such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), however, act to keep 0 2 A cells in their proliferative, bipotential progenitor form and to prevent differentiation (Gard and Pfeiffer, 1993; Grinspan et al., 1993; 1996). For instance, injection of P D G F into the cerebrospinal fluid of neonatal rats has been shown to delay  02A  differentiation, and anti-PDGF antibodies have been shown to block 0 2 A mitosis (Dutly and Schwab, 1991; Butt et al., 1997). Furthermore, decreases in P D G F signalling have been shown to correspond to an exit of 0 2 A cells from the proliferative cell cycle (Calver et al., 1998). Once these cells exit the cell cycle, they are able to spontaneously differentiate into mature OL (Dutly and Schwab, 1991).  While 0 2 A cells are commonly isolated from neonatal rat tissue,  accounts of isolation of similar cells from human fetal tissue indicate that there is at least some species similarity in O L development (Kennedy and Fok-Seang, 1986; Weidenheim et al., 1994; Rivkin et al., 1995).  3  A s well as the discovery of 0 2 A cells in neonatal rat brain, 0 2 A cells have also been discovered in the adult rat brain (Ffrench-Constant and Raff, 1986; Wolswijk and Noble, 1989). The function of the adult rat 0 2 A is most likely to maintain a pool of cells capable of both self-renewal and generation of new mature OL by asymmetrical division (Noble et al., 1992; Wren et al., 1992). This is in contrast to neonatal rat 0 2 A cells, which divide symmetrically and are therefore not capable of self-renewal once differentiation has occurred. To date there is only one account of isolation of a proliferative human oligodendrocyte progenitor from adult tissue. This cell was shown to be a uni-, bi- or tri- polar cell that was positive for A 2 B 5 staining. It was able to differentiate into astrocytes in serum-rich medium, and into O L in serum-deprived medium (Scolding etal., 1995; 1999).  1.1.2 The Biology of Primary Oligodendrocytes: Mature OL  0 2 A cells committed to the OL lineage next mature into pro-OL that are still proliferative and bipotential but no longer motile (Levi et al., 1987; Gard and Pfeiffer, 1993; Warrington et al., 1993).  These cells also lose their A 2 B 5  reactivity, and can instead be identified by an 0 4 antibody that recognizes sulfatide (Trotter and Schachner, 1989; Gard and Pfeiffer, 1990). In human fetal tissue, a sub-set of precursor O L that do not react with 0 4 but that do react with the Ranscht monoclonal antibody (R-mAb) have been identified (Satoh and Kim, 1994). The R-mAb recognizes galactocerebroside (GalC) as well as sulfatides  4  (Ranscht et al., 1982). These 04-/R-mAb+ precursors have not been identified in neonatal rat tissue. Furthermore, 04+ cells isolated from the adult human C N S lack the  bipotential  and proliferative  capacities of  rat-derived  04+  cells  (Armstrong et al., 1992). These differences underline the existence of species variation between rat and human OL. Finally, pro-OL derived from neonatal rat brain differentiate into postmitotic, multipolar OL that express cell surface markers which are recognized by 04,  anti-GalC, and  antibodies.  anti-2,3-cyclic  nucleotide  3-phosphohydrolase (CNP)  Fully mature O L will then produce the rest of the myelin proteins,  such as myelin basic protein (MBP) and proteolipid protein (PLP) (Ranscht et al., 1982; Bansal and Pfeiffer, 1992; Bansal et al., 1992). Mature OL are commonly isolated not only from rat, but also from cow, pig, sheep, and human tissue (Szuchet et al., 1980; Kim et al., 1983; Smyrnis et al., 1986; Kim, 1990; Stariha et al., 1997). A s mentioned above, mature O L function as the myelinating cells of the C N S . The first step in myelination involves the extension of processes from OL, as this extension allows OL to contact unmyelinated axons. OL myelinate axons of 1 um or more, and one OL can myelinate as many as 50 axons (Compston et al., 1997). Signals such as substratum adhesions and cell-cell contact between specific cell adhesion molecules on O L and neurons probably stimulate myelin sheath production once contact between OL and axons has been made (Yim et al., 1986; Poltorak et al., 1987). Myelin itself is not merely a lipid-rich insulative coating, but it also possesses specific enzymes, such as C N P , and a variety of  5  ion channels. Once an OL has created a functional myelin sheath, it continues to support the metabolic activities of the sheath (Compston et al., 1997). There appears to be a direct correlation between O L cultured in vitro and OL found in vivo, making primary OL cultures a useful model with which to study OL function.  For instance, both in vitro and in vivo OL show extension of  processes. They also both express the major myelin lipids and proteins, such as M B P and P L P (Yim et al., 1986; Vartanian et al., 1992). A s well, myelin lipids and proteins are produced in vitro along a similar time course to those produced in vivo (Zeller et al., 1985; Dubois-Dalcq et al., 1986).  In vitro studies on OL  functional biology, however, are hampered by the post-mitotic nature of mature cells. This lack of proliferation makes it inherently difficult to produce sufficient quantities for experimentation. Although proliferating 0 2 A cells can be cultured and induced to differentiate into mature OL, these progenitor cells do not divide indefinitely and can be cultured only in relatively small amounts from rat brain. Therefore, there is also a cell quantity problem associated with primary 0 2 A cultures.  To overcome this problem, scientists have begun to use a self-  renewing cell line known as the C G - 4 cell line to conduct studies on OL.  1.1.3 Discovery and Characterization of the Central Glial-4 (CG-4) Cell Line  The C G - 4 cell line arose as a spontaneous mutation from rat 0 2 A primary cultures (Louis et al., 1992). These 0 2 A primary cells were initially cultured in the usual fashion, and most cells showed the classical differentiation to mature  6  OL after approximately 5-6 weeks in culture. However, it was noted that a subset of cells did not differentiate but rather continued to proliferate. This sub-set of continuously self-renewing cells was subsequently found to be a bipotential cell line capable of differentiating into either OL-like cells or astrocyte-like cells. This cell line is named C G - 4 , and has been shown to resemble cells of 0 2 A lineage. C G - 4 cells were first characterized by immunocytochemisty (Louis et al., 1992). In their progenitor, bipolar state, 95% of C G - 4 cells were found to stain with A2B5. Furthermore, only 2-3% of the cells stained for GalC, and less than 1% stained for glial fibrillary acidic protein (GFAP). A s GalC is an O L marker, and G F A P is an astrocyte marker, these immunocytochemical results confirmed that the cells had not yet differentiated into either OL or astrocytes. To maintain C G - 4 cells in their proliferative, bipotential state, serum-free medium containing mitogens provided by the B-104 neuroblastoma cell line was used.  Further  analysis determined that b F G F and P D G F could replace this B-104 conditioning. The bipotential C G - 4 cells were shown to differentiate into OL-like cells upon removal of the B-104 conditioning (Louis et al., 1992). After 48 hr in serumfree  medium with  no  B-104  conditioning, the  morphological characteristics of mature OL.  C G - 4 cells took  on  the  These OL-like cells were multi-  polar, and lost their capacity for proliferation. A n immunocytochemical analysis showed that all of these differentiated C G - 4 cells expressed GalC, over 50% expressed M B P , and only approximately 2 % still stained with A2B5. To differentiate the bipotential C G - 4 cells into astrocyte-like cells, medium containing 20% fetal calf serum (FCS) was used (Louis et al., 1992).  After  7  approximately 1 week of culturing in this serum-containing medium, the C G - 4 cells took on the morphological characteristics of astrocytes. Furthermore, they remained highly proliferative and 50% stained for the astrocyte marker G F A P .  1.1.4  A Comparison of the Properties of Primary O L and C G - 4 Cells  Striking parallels can be found between primary cultures and C G - 4 cultures when comparing 0 2 A cells to bipotential C G - 4 cells (Table 1).  First,  both 0 2 A and bipotential C G - 4 cells stain with the A 2 B 5 antibody. Second, both cell types can differentiate either into mature OL in serum-free medium or into astrocytes in serum-containing medium.  0 2 A and bipotential C G - 4 cells also  both contain nestin, which is a progenitor cell marker that is lost upon differentiation (Gallo and Armstrong, 1995). A s noted above, b F G F and P D G F are instrumental in keeping 0 2 A cells in their proliferative state.  Removal of  these mitogens in both 0 2 A and C G - 4 cultures allows the cells to exit the cell cycle and to begin differentiation towards OL. Furthermore, 0 2 A and C G - 4 cells can both differentiate into astrocytes in the presence of mitogens such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) (Raff et al., 1983; Hughes et al., 1988; Lillien et al., 1990; Kahn and De Vellis, 1994; Mayer et al., 1994; Vos et al., 1996). The signal transduction pathways activated by C N T F also appear to be similar in both cell types, as they both involve signal transducers and activators of transcription (STATs) (Kahn et al., 1997; Dell'Albani et al., 1998).  8  Property  Primary 02A C e l l s and Bipotential C G - 4 C e l l s  Primary Mature O L and CG-4 OL  Phenotypic marker expression  A2B5, nestin  Gale, M B P , C N P  Effects of s e r u m containing medium  Differentiation to astrocytes  N/A  Effects of serum-free medium  Differentiation to oligodendrocytes  N/A  Effects of C N T F and LIF  Increased differentiation to astrocytes  Increased survival with removal of trophic factors  Effects of b F G F  Maintenance of cells in a bipotential, proliferative state  Primary OL: stimulation of process extension in concert with astrocyte extracellular matrix C G - 4 0 L : N/T  Thyroid Receptor Expression  Thyroid receptor-a  Thyroid receptor-a and -p  Predominant glutamate receptor expression  A M P A and Kainate  A M P A and Kainate  Krox-24 e x p r e s s i o n  High expression  Low expression  Migration ability  Good migration on pleiotrophin and myelin substrates  Generally considered to be non-migratory cells  Myelination potential  Must differentiate into mature OL prior to affecting myelination  Can myelinate  Tabl e  1: The Shared Properties of Primary O L and the C G - 4 Cell Line  N/A = not applicable N/T = not tested  9  As well as possessing similar differentiation  pathways, Q2A and  bipotential CG-4 cells also possess similar functions (Table 1). For instance, one important function of 02A cells is their ability to migrate, as this ability gives them the potential to move into areas of demyelination and affect repair. In vitro tests conducted on myelin and pleiotrophin coated dishes have found that 02A and bipotential CG-4 cells can migrate on these CNS substrates (Amberger et al., 1997; Rumsby et al., 1999). Perhaps most significantly, 02A and CG-4 cells are both able to remyelinate demyelinated axons in irradiated tissue (Groves et al., 1993; Franklin etal., 1995). Similarities between the primary OL culture model and CG-4 culture model do not only exist between 02A cells and bipotential CG-4 cells. In fact, there are also many parallels between mature primary OL and CG-4-derived OL (CG-4 OL). For instance, CNTF and LIF have been shown to increase the survival of both primary OL and CG-4 OL in response to removal of trophic factors (Barres et al., 1993; Kahn and De Vellis, 1994; D'Souza et al., 1996; Vos et al., 1996; Jiang et al., 1999). Both similarities and differences, however, were discovered between the two cell types in an extensive study comparing the expression of glycolipids and myelin-associated glycoprotein in primary OL and CG-4 cells (Yim et al., 1995; Schnaar et al., 1996). These studies found that while the levels of GalC and sulfatide increased during differentiation of bipotential CG-4 cells towards CG-4 OL, the final levels of these proteins were still lower than the levels of GalC and sulfatide found in mature, primary OL. As well, although both cell types expressed a high amount of the ganglioside GD3, CG-4 OL expressed  10  much less G M 3 and more GD1b and GT1b than primary OL. Finally, C G - 4 O L also expressed lower levels of myelin-related glycolipids and myelin-associated glycoprotein than primary O L . This decrease in OL-related proteins in C G - 4 O L as compared to primary O L indicates that C G - 4 O L perhaps do not achieve as high a degree of differentiation as primary OL. However, the fact that they still express relevant O L proteins indicates that the C G - 4 cell line could be a reasonable O L model. C G - 4 cells also share the expression of various non-OL related proteins with primary O L .  These proteins are often found in both the mature and  immature C G - 4 and primary O L differentiation stages. For instance, both C G - 4 cells and primary O L show developmental expression of thyroid receptors, which is significant as thyroid hormones play a role in the regulation of myelination (Baas et al., 1994; Pombo et al., 1999). The developmental expression of the krox-24 differentiation regulator protein is also similar in both C G - 4 cells and in primary O L , indicating that O L differentiation can be studied using C G - 4 cell cultures (Sock et al., 1997). Both cell types also express functional ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate sub-types (Pende et al., 1994; Yoshioka et al., 1995; Meucci et al., 1996; Yoshioka et al., 1996). This glutamate receptor expression makes C G - 4 cells useful for studies on calcium regulation and excitotoxicity in OL.  Finally, neural cell adhesion molecules, cadherins, and beta catenin are  similarily expressed in both cell types (Hughson et al., 1998). Therefore, while  11  there are differences between primary O L and C G - 4 cells, the two systems appear to be similar enough to both be used as O L model systems.  1.2 Signal Transduction  Signal transduction involves the transmission of signals from the plasma membrane of a cell to intracellular protein intermediates.  Protein intermediates  called protein kinases phosphorylate substrates and thereby influence cellular functions such as proliferation and differentiation.  Protein phosphatases reverse  these kinase-mediated phosphorylation events. To understand which signalling pathways  govern the  survival, death, proliferation,  differentiation,  process  extension and myelination capacities of OL, scientists have employed both the primary O L culture and C G - 4 culture models.  Currently, many studies of OL  intracellular signalling events have focused on intracellular signalling enzymes known as protein  kinase C (PKC) and mitogen-activated  protein kinases  (MAPKs). These kinases will be reviewed below.  1.2.1 General Protein Kinase C Signalling  The term P K C encompasses a family of widely expressed proteinserine/threonine kinases. In the C N S , P K C has been linked to events such as modulation of ion channels, neurotransmitter release, long-term potentiation, and differentiation (Spinelli and Ishii, 1983; Baraban et al., 1985; Madison et al.,  12  1986; Malenka et al., 1986; 1987).  P K C signalling in O L has been linked to  proliferation, process extension, and myelination capacity (Yong et al., 1988; Bhat, 1989; Asotra and Macklin, 1993; Yong et al., 1994). Direct substrates for P K C isoforms can include the cytosolic myristoylated alanine-rich C kinase substrates (MARCKs), the cytoskeletal proteins vinculin and talin, and the kinase Raf (Morrison et al., 1988; Stumpo et al., 1989; Simons and Elias, 1993; PerezMoreno et al., 1998).  Activation of Raf can link P K C signalling to M A P K  signalling, as will be discussed later under M A P K s . There  are three  main sub-groups  of  P K C isoforms;  namely,  the  conventional, novel, and atypical sub-groups [reviewed in: (Newton, 1995; 1997; Kanashiro and Khalil, 1998; Ron and Kazanietz, 1999)]. All three sub-groups are composed of a regulatory N-terminal region and a catalytic C-terminal region. They also share similar conserved domains known as the C 1 , C 2 , C 3 , and C4 domains (Fig. 1). The C1 conserved domain is characterized by two cysteine-rich zinc finger repeats.  These repeats represent a binding site for diacylglycerol (DAG) and  phorbol esters. An autoinhibitory pseudosubstrate domain can be found just Nterminal  to  C1.  The  C2  domain  recognizes  acidic  lipids,  phosphatidylserine (PS), and can bind calcium in some isoforms.  such  as  The C 3  domain contains the A T P binding site, and the C 4 domain contains the substrate binding site. Thus, C1 and C 2 are part of the regulatory region, while C 3 and C4 are part of the catalytic region (Fig. 1).  13  Regulatory Region C1 NH-  3  C3  t t t t Zn  PseudoSubstrate  NH  C2  Catalytic Region C4  t  Zn  DAG/ phorbol esters  C a & acidic phospholipids + 2  C2  C1  ATP  Acidic phospholipids  PseudoSubstrate  t  C3  DAG/ phorbol esters  ATP  C1  C3  Zn  t t  ATP  cPKC's  LCOOH  nPKC's  C4  Zn  PseudoSubstrate  COOH  Substrate  t t t t t Zn  L  Substrate  C4  L  COOH  aPKC's  Substrate  Figure 1: The Conserved Domains of PKC Isoforms The conventional P K C ' s (cPKC's) have four conserved domains. The first conserved domain (C1) has two zinc finger repeats in a cystein-rich motif. This domain binds DAG/phorbol esters, and can also be found in the novel P K C ' s (nPKC's). domain in the atypical P K C ' s  The C1  (aPKC's), however, has only one zinc-finger repeat,  and therefore does not bind D A G /phorbol esters. The second conserved domain (C2) binds calcium and acidic phospholipids in c P K C ' s , but does not bind calcium in nPKC's and does not exist in a P K C ' s .  The C 3 and C 4 domains bind A T P and  substrate, respectively, and can be found in all P K C isoforms. 14  In O L signalling pathways, the conventional P K C (cPKC) isoforms have been proven to play an important role. The conventional P K C ' s include the a, pi, pll, and y isoforms. These isoforms are regulated by phospholipids, calcium, and DAG/phorbol esters (Ogawa et al., 1981; Castagna et al., 1982; Kikkawa et al., 1983; Nishizuka, 1983; Blumberg et al., 1984).  The proposed mechanism of  c P K C activation has been previously reviewed by Newton (Newton, 1997). This mechanism involves the binding of D A G to C1 and P S to C 2 , localizing P K C to the cell membrane and causing a conformational change that removes the autoinhibitory sequence from the active site. Although binding of either D A G or P S is sufficient for weak PKC-membrane interactions, binding of both is required for high affinity interactions and removal of the autoinhibitory sequence. Binding of calcium to C 2 is thought to increase the affinity of P K C for P S , thus decreasing the amount of D A G required for full activation. Phorbol esters can compete with D A G for binding to C 1 , and are thus also able to target P K C to the membrane for activation. Since D A G is a pivotal molecule in the activation of the conventional P K C ' s , signalling pathways which lead to the production of D A G can result in the activation of P K C [reviewed in: (Nishizuka, 1992; Haeffner, 1993)]. For instance, activation of receptor tyrosine kinases or G-protein associated receptors can lead to the activation of phospholipase C (PLC). Activation of P L C in turn leads to the break down phosphatidylinositol 4,5-bisphosphate  (PIP2)  into inositol  1,4,5-  trisphosphate (IP3) and D A G . Phospholipase D can also contribute to the production of D A G by orchestrating the breakdown of phosphatidylcholine, which  15  is subsequently dephosphorylated. Once DAG is produced, as mentioned, it is instrumental in targeting PKC to the membrane for activation. PKC binding proteins are also thought to play a role in PKC signalling [reviewed in (Faux and Scott, 1996; Ron and Kazanietz, 1999)].  Briefly, it is  thought that PKC binding proteins help to compartmentalize the different PKC isoforms so that they can be brought into close proximity with their target substrates.  PKC binding proteins include receptors for activated C-kinase  (RACK'S), proteins that interact with C-kinase (PICK's), and substrates that interact with C-kinase (STICK's). Finally, PKC can also be regulated by phosphorylation events. A protein known as PDK-1 has been identified as a kinase able to phosphorylate various PKC isoforms, including a and pll (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998). This phosphorylation event is followed by autophosphorylation of PKC on two C-terminal sites.  It is proposed that these phosphorylation and  autophosphorylation events are required to create and maintain a catalytically competent conformation of PKC. They may also play a role in PKC localization (Bornancin and Parker, 1996; 1997; Newton, 1997). In brief, the other two remaining sub-groups of PKC isoforms are the novel and atypical sub-groups. The novel sub-group (nPKC) consists of isoforms 8, s, cp, and r|.  These nPKCs, unlike the conventional PKCs, are calcium  independent. Like the conventional PKC's, however, they can be activated by PS in the presence of DAG or phorbol esters. The atypical PKC sub-group includes the  i , and X isoforms. These atypical isoforms have only one cysteine  16  rich repeat in the C1 domain and appear to be DAG/phorbol ester independent. They also appear to be calcium independent, but can be at least partially activated by P S .  1.2.2 P K C Signalling Cascades in Primary O L  The effects of P K C activation have been studied in both immature and mature OL primary cultures.  In general, P K C activation appears to promote  proliferation in progenitor OL and to promote dedifferentiation and process extension in mature OL. However, studies of P K C in O L are often contradictory. Progenitor O L have been shown to increase proliferation in response to b F G F , while use of the H-7 P K C inhibitor has been shown to abolish this b F G F induced proliferation (Radhakrishna and Almazan, 1994).  Other studies have  shown that short-term treatment of 04+ OL with phorbol 12,13-myristate acetate (PMA), a biologically active phorbol ester, can also increase proliferation. Once again, this increase in proliferation was blocked by the use of the H-7 P K C inhibitor (Bhat, 1992). One mediator of PKC-induced proliferation appears to be c-fos.  Induction of c-fos in OL occurs along with induction of bFGF-stimulated  proliferation, while the use of H-7 blocks both bFGF-induced proliferation and cfos gene induction (Bhat et al., 1992; Radhakrishna and Almazan, 1994). One possible explanation for these results is that b F G F engages growth factor receptors that activate P L C , which in turn could lead to activation of P K C and eventual activation of extracellular-signal regulated protein kinases (ERKs) 1 and  17  2.  These E R K s could then translocate to the nucleus, inducing c-fos gene  expression and promoting the transcriptional events required for cell proliferation. Another potential mediator of PKC-induced proliferation is the M A R C K S protein, as PMA-treatment of progenitor O L has been shown to cause M A R C K S phosphorylation.  In OL, M A R C K S can be found in both the cytoplasm and  process extensions (Bhat, 1991; 1995). These experiments indicate a role for P K C activation in the proliferation of immature OL. However, other studies have shown that long-term exposure of progenitor O L to P K C activators causes either no increase or a decrease in proliferation (Deloulme et al., 1992; Liu and Almazan, 1995).  A possible  explanation for these results is the phenomenon of P K C down-regulation. Downregulation of P K C activity can occur either with the use of P K C inhibitors or with prolonged exposure (upwards of 1 hr) to P K C activators.  In the latter scenario,  prolonged phorbol ester treatments have the potential to down-regulate P K C activity, despite the fact that short-term phorbol ester treatments normally upregulate P K C activity. Although Western blot studies have shown that progenitor O L do not down-regulate P K C upon long-term exposure to phorbol esters, they have also shown that O L changing from A 2 B 2 expression to 0 4 expression do begin to down-regulate P K C (Asotra and Macklin, 1993; 1994).  Asotra and  Macklin speculate that perhaps this differential modulation of P K C activity between immature and differentiating O L could be attributed to differences in calpain-like activity.  Since calpain has been shown to mediate phorbol ester-  induced P K C degradation, they hypothesize that a lack of calpain-like activity in  18  immature O L may protect these cells from phorbol ester-induced P K C downregulation. Asotra and Macklin also show that the change from A2B5+ immature OL to 04+ differentiating O L occurs over two culture days, thus making the change to O L which are susceptible to P K C down-regulation very rapid. Under experimental conditions, growth factors are often removed from the culture medium prior to treatment of progenitor OL with P K C activators. A s mentioned above, removal of growth factors has been shown to induce O L differentiation. Therefore, it is possible that the removal of growth factors prior to exposure of progenitor OL to P K C activators is enough to make these cells susceptible to P K C down-regulation. In contrast to its stimulation of immature O L proliferation, P K C appears to inhibit  immature  progenitors  O L differentiation.  have reported  Two separate studies on A2B5+ OL  a decrease in the  percentage of A2B5+ OL  differentiating into mature O L after activation of P K C (Baron et al., 1998; Heinrich et al., 1999). First, A2B5+ OL progenitors were assessed for the development of a CNP+ phenotype in the presence or absence of PMA. The results showed that OL were most likely to differentiate from A2B5+ bipolar cells to CNP+ multipolar cells in the absence of P M A . Furthermore, the P K C inhibitor BIM negated the effects of P M A treatment on these cells (Baron et al., 1998; 1999). Subsequent studies used the neurotrophic factor NT-3 to induce differentiation of bipolar progenitors toward multipolar GalC+ OL.  In these studies, it was shown that  P K C inhibitors (staurosporine and chelerythrine chloride) could increase NT-3induced morphological differentiation (Heinrich et al., 1999).  It has also been  19  demonstrated that P M A treatment can cause transient reversion of O L from an A2B5+/04+  phenotype to an even more immature A2B5+/04-  (Avossa and Pfeiffer, 1993).  phenotype  Finally, the presence of P M A in mixed rat brain  cultures has been shown to prevent the developmental expression of M B P and P L P in O L (Baron et al., 2000a). From  the  results  of  these  experiments,  it  seems  reasonable  to  hypothesize that P K C exerts its mitogenic effects on A2B5+ O L progenitors at least partially by preventing proliferative O L progenitors from differentiating into post-mitotic, mature OL. Thus, there appears to be a dual and complementary role for P K C activation in these cells. While P K C prevents the differentiation  of immature OL, studies have  shown that P K C activation can be linked to differentiation events in mature OL. For instance, most studies conducted on mature OL corroborate a role for P K C activation in enhancement of process extensions.  From early studies, it was  noted that treatment of mature OL with P M A induced process extension over basal levels. A s well, the use of a variety of P K C inhibitors was shown to block this PMA-induced extension (Yong et al., 1988; Althaus et al., 1991; Yong et al., 1994). Further analysis of the phorbol ester-induced process extension response used Ca -dependence and specific pharmacological agonists to determine that +2  the oc-isoform of P K C contributes most to these process extension signalling pathways (Yong et al. 1994).  Soon, P K C activators other than phorbol esters  were found to induce O L process extension. For instance, it was discovered that monogalactosyl diglyceride (MGDG) could enhance O L process re-growth in  20  adult porcine OL, while at the same time stimulating P K C - a activity (SchmidtSchultz and Althaus, 1994). However, a definitive link between MGDG-induced process extension and P K C activity has yet to be established. It has also been reported that a combination of b F G F and astrocyte extracellular matrix can enhance O L process re-growth in adult human OL. Furthermore, selective P K C inhibitors (calphostin C and C G P 41251) have been shown to abolish this bFGF/astrocyte extracellular matrix-induced enhancement of processes (Oh et al., 1997).  Finally, metalloproteinase 9 (MMP-9) has been presented as a  downstream effector of the PKC-induced process extension response. In these experiments, phorbol ester treatment extension and M M P - 9 activity.  was shown to induce both process  The use of a P K C inhibitor, calphostin C, was  able to both block process extension and decrease M M P - 9 activity.  Perhaps  most convincingly, inactivation of M M P - 9 was shown to inhibit the phorbol esterinduced extension of processes (Uhm et al., 1998). Since most studies on mature O L process extension involve exposure of OL to phorbol esters for upwards of 24 hr, the question of P K C down-regulation must be addressed. It has been shown that P K C activity actually increases 400500%  in mature O L treated with phorbol dibutyrate (PDB) for 48 hr, thus  indicating a lack of P K C down-regulation in mature OL.  Furthermore, P K C  enzyme activity assays have shown that P K C activity does not decrease even after 12 days of P D B treatment (Yong et al., 1994). It should be noted, however, that contrary  results have been obtained.  Through  immunocytochemical  analysis, down-regulation of P K C - a and other P K C isoforms in both GalC+ and  21  MBP+ O L has been observed (Asotra and Macklin, 1993; 1994). One possible explanation for these varied results is species specificity; the experiments indicating that P K C is not downregulated involved human OL, whereas the experiments indicating that P K C is downregulated involved rat OL. Not only does P K C activation play a role in the process extension of mature OL, it also plays a role in their myelination capacity. On the one hand, studies have shown that phorbol ester-induced P K C activation in mature O L can lead to increased phosphorylation of M B P , as well as increased M B P synthesis (Vartanian et al., 1986; Yong et al., 1994). On the other hand, a contradictory study points to a role for P K C in demyelination and O L dedifferentiation (Pouly et al., 1997). The main differences between these studies are the varied culture methods employed. The studies pointing to a role for P K C in myelination were conducted on primary cultures enriched for mature OL, while the study pointing to a role for P K C in demyelination was conducted on aggregating brain cultures. While aggregating brain cultures have the advantage of allowing neural cell interactions  that  more  closely  approximate  in  heterogeneous nature makes interpretation difficult.  vivo conditions,  their  For instance, it is not  possible to determine if the noted demyelination was a result of phorbol esters acting directly on OL. It is possible that other neural cells were also affected by the phorbol ester, and that these cells then mediated the demyelination. A s well, without an analysis of cell marker expression, it is not possible to ascertain if the decreased O L differentiation seen in the aggregating cultures was truly a result of mature OL dedifferentiation.  It is also possible that a lack of immature OL  22  differentiation led to the decreased expression of myelin proteins observed in these phorbol ester-treated cultures.  1.2.3 General M A P K Signalling  The mitogen-activated protein kinases (MAPKs) are a family of structurally related protein-serine/threonine kinases that are characterized by a requirement for dual-phosphorylation on both tyrosine and threonine  residues for full  activation. The M A P K family includes the extracellular-signal regulated protein kinases (ERKs), the stress-activated c-Jun N-terminal kinases (JNKs), and the high osmolarity glycerol response kinase (p38)  [reviewed  in: (Cano and  Mahadevan, 1995; Pelech and Charest, 1995; Widmann et al., 1999)]. Studies of M A P K s in O L have determined unique roles for the varied members of this family.  These roles will be discussed in the next section, and range from  proliferation and differentiation to survival and apoptosis.  i) ERK1/2 The best-characterized members of the M A P K family are the 44 kDa ERK1 and the 42 kDa E R K 2 . Although they will not be discussed here, E R K s 3,4,5 and 6 also exist. Classical activation of E R K s 1 and 2 involves binding of a growth factor to its tyrosine-kinase receptor. Growth factor receptors involved in ERK1/2 activation include the P D G F receptor and the Trk A nerve growth factor receptor.  Dimerization of the receptor follows ligand binding, leading to  23  autophosphorylation and receptor activation (Bishayee et al., 1986; 1989; Heldin et al., 1989). Receptor activation ultimately leads to the sequential activation of the following kinases: Ras, Raf-1, MEK1/2, ERK1/2 (Fig. 2).  This sequential  cascade utilizes interactions among many adapter proteins. First, the activated receptor recruits an adapter protein such as growth factor receptor binding protein-2 (Grb-2).  Grb-2 is recruited by virtue of its src homology-2 (SH2)  domain, which is a recognition domain containing a specific conserved protein pattern that helps mediate protein-protein interactions (Matuoka et al., 1993; Schlessinger, 1994). Next, Grb-2 interacts with a guanine nucleotide exchanger known as Son-of-sevenless (SOS), also via its S H domain. These interactions draw S O S to the membrane, where it can interact with Ras and activate it by catalyzing the exchange of G D P for G T P (Chardin et al., 1993). Activated Ras can then contribute to activation of Raf-1 by localizing Raf-1 to the plasma membrane and exposing the Raf-1 kinase domain.  Although the exact  mechanism of Raf-1 activation remains unclear, P K C is one of the proteins that is thought to play a role (Morrison and Cutler, 1997). Activation of Raf-1 in turn leads to activation of M A P K / E R K kinase (MEK) via phosphorylation (Morrison et al., 1988; Van Aelst et al., 1993; Williams and Roberts, 1994). MEK1 and M E K 2 are dual-specificity kinases that activate ERK1 and E R K 2 by phosphorylating both tyrosine and threonine residues in a T E Y motif (Zhang et al., 1994). It is thought that tyrosine phosphorylation of ERK1/2 allows for substrate binding, while threonine phosphorylation allows for the correct alignment of catalytic residues (Marshall, 1994). Once ERK1 and E R K 2  24  \  (  Growth Factor  c-fos Figure 2: General Schematic of ERK1/2 Signalling via Growth Factor Activation  Growth factor receptor dimerization and autophosphorylation (P) follows ligand binding. This in turn leads to recruitment of an adapter molecule (Grb-2) to the receptor, which subsequently leads to the recruitment a guanine nucleotide exchange factor (SOS). Sos then converts R a s - G D P to R a s - G T P , and a sequential cascade of kinase phoshorylations leads from Raf-1 to MEK1/2 to ERK1/2 activation.  Cytoplasmic  substrates of ERK1/2 can include M B P , MAP-2, and Rsk, while translocation of ERK1/2 to the nucleus can cause c-fos gene induction via Elk-1 phosphorylation. 25  have been activated, they can phosphorylate a variety of substrates. Cytosolic substrates of ERK1/2 include M B P , microtubule-associated protein-2 (MAP-2), and kinases of the Rsk family (Sanghera etal., 1990a; 1990b; G o t o h e t a l . , 1991; Blenis, 1993).  Translocation of activated ERK1/2 to the nucleus can lead to  phosphorylation of the transcription factor Elk-1, which in turn can lead to the induction of the c-fos gene (Gille et al., 1995; Hodge et al., 1998).  Cellular  responses to ERK1/2 activation include increased survival and proliferation. Since there are no commercially available inhibitors of ERK1 and E R K 2 , experiments on these kinases often rely on inhibitors of MEK1 and M E K 2 . Two such M E K inhibitors are P D 98059 and UO-126 (Dudley et al., 1995; Favata et al.,  1998).  Both inhibitors  appear to act on MEK1/2 through  allosteric  mechanisms, and do not compete with A T P or ERK1/2 for binding.  ii) J N K The J N K members of the M A P K family are kinases that become activated in response to cellular stresses.  In fact, J N K s are sometimes referred to as  stress-activated protein kinases, or S A P K s . The stresses that can activate J N K s include exposure to anisomycin, UV-irradiation, and ceramide (Westwick et al., 1995; Meier et al., 1996).  A s well, tumor necrosis factor alpha (TNF-cc) and  interleukin-1 (IL-1), which engage receptors of the TNF-cc superfamily, have been shown to activate J N K (Westwick et al., 1994; Rosette and Karin, 1996). Another member of this receptor superfamily that can lead to J N K activation is the p75 low affinity neurotrophin receptor. The kinases directly upstream of J N K , known  26  as M E K 4 and M E K 7 , activate J N K by dual phosphorylation of tyrosine and threonine in a T P Y motif (Derijard et al., 1995). Unlike activation of M E K 1/2 in the ERK1/2 cascade, the Ras-Raf-1 signalling cascade does not lead to activation of M E K 4 or M E K 7 in the J N K cascade. Rather, a M A P K / E R K kinase kinase (MEKK) is thought to be upstream of M E K 4 and M E K 7 , and p21-activated kinase (Pak) has also been shown to act upstream of M E K 4 (Bagrodia et al., 1995; Frost et al., 1996; Fanger et al., 1997; Tibbies and Woodgett, 1999). The classical substrate of J N K is the N-terminus of c-Jun, and cellular responses to J N K activation include apoptosis and cessation of cell growth.  iii) p38 A third member of the mammalian M A P K family, p38, has a counter-part in yeast known as high osmolarity glycerol response kinase (HOG).  Not  surprisingly, therefore, p38 is classically activated by osmotic stress. There is also evidence of overlap between p38 and J N K cascades, as ceramide, T N F - a , and IL-1 have been shown to activate both J N K and p38 (Raingeaud et al., 1995). The kinases directly upstream of p38, M E K 3/6, activate p38 via dual phosphorylation of tyrosine and threonine in a T G Y motif (Derijard et al., 1995; Han et al., 1995; Stein et al., 1996).  Signals downstream of p38 are often  transduced through a kinase known as MAPK-activated protein kinase-2 (MAPKAPK-2), which in turn phosphorylates substrates such as the small heatshock proteins (Guay et al., 1997; Larsen et al., 1997). Activation of p38 can also lead to induction of the ATF-2 nuclear transcription factor (Raingeaud et al.,  27  1996). Cellular responses to p38 activation can include apoptosis or proliferation (Craxton et al., 1998; Hida et al., 1999).  1.2.4 ERK1/2 Signalling in Primary O L and C G - 4  Studies on the role of ERK1/2 in O L have predominantly primary cultures.  These studies show a general trend whereby  activation leads to increased OL proliferation  and survival.  employed ERK1/2  For instance,  circumstantial evidence linking progenitor proliferation to activation of ERK1/2 has shown that treatment of cells with NT-3 increases both cell proliferation and ERK1/2 phosphorylation (Cohen et al., 1996). In parallel experiments, however, it was noted that N G F treatment could also phosphorylate ERK1/2 but could not induce O L proliferation.  Since treatment of progenitor OL with N G F was less  effective at sustaining ERK1/2 phosphorylation than NT-3, and since N G F did not stimulate progenitor proliferation, it could be concluded that a certain level of ERK1/2 activation must be reached before O L are induced to proliferate.  Other  studies have led to speculation that the E R K 2 isoform, rather than the ERK1 isoform, is preferentially activated by NT-3 in progenitor OL (Kumar et al., 1998). It is widely accepted, that, under normal in vitro and in vivo conditions, mature O L do not proliferate (Althaus et al., 1984; Kim and Yong, 1990; Althaus et al., 1991; Keirstead and Blakemore, 1997; 1999).  However, there is some  evidence that a select sub-population of mature O L can be induced to proliferate. Althaus et al. (1992; 1997) were able to induce proliferation of a sub-set of  28  mature OL using nerve growth factor (NGF) treatment, and subsequently showed that such N G F treatment also activates ERK1 in OL.  Furthermore, the  complement complex C5b-9 has also been shown to increase both mature OL proliferation and ERK1 activation (Rus et al., 1997). This complement-induced increase in proliferation was blocked with the use of the P D 98059 MEK1 inhibitor, thus strengthening the link between increased E R K activation and OL proliferation. Since ERK1 has been implicated in the proliferation of mature OL, while E R K 2 has been implicated in the proliferation of 0 2 A cells, it seems that ERK1/2 isoforms may be developmentally regulated in OL (Althaus et al., 1992; Kumar etal., 1998). Experiments done on primary, mature OL have shown a correlation between ERK1/2 activation and increased survival. For instance, NT-3 or N G F treatment  of  mature  OL  has  been  shown  to  increase  both  ERK1/2  phosphorylation and O L survival (Cohen et al., 1996). Further experiments were conducted in which the effect of ERK1/2 activity on cell survival was compared to the effect of J N K activity on cell survival.  In a first set of experiments, N G F was  used to selectively activate J N K in OL that expressed p75 but not TrkA. In this case, cell death occurred (Casaccia-Bonnefil et al., 1996).  However, in O L  expressing both p75 and TrkA, E R K s 1 and 2 were activated to a much greater extent than J N K .  In this experiment, the cells survived (Yoon et al., 1998).  These complimentary studies indicate that J N K activation induces cell death, while E R K activation promotes cell survival.  29  A s of yet, few studies of E R K s in C G - 4 cells have been undertaken. However, ERK1/2 expression in C G - 4 has been noted, and C G - 4 cells cultured in serum-free medium for 24 hr show an upregulation of ERK1/2 phosphorylation in response to phorbol ester treatment (Bhat and Zhang, 1996). There have also been studies undertaken on the relationship of ERK1/2 activation to cell survival in C G - 4 cells.  First, increased ERK1/2 activation has been correlated to  increased survival of C G - 4 O L after treatment with prosaposin, a neurotrophic factor (Hiraiwa et al., 1997). While these results compliment primary culture data that also show increased OL survival after  neurotrophin-induced  ERK1/2  activation, other C G - 4 culture experiments appear to contradict primary culture results.  For instance, C G - 4 O L exposed to H 0 2  2  have been shown to  demonstrate increased M A P K activity and increased cytotoxicity.  Use of the PD  98059 MEK1 inhibitor was able to block this cytotoxicity, implicating ERK1/2 in the cytotoxic response (Bhat and Zhang, 1999). However, as mentioned above, ERK1/2 activation appears to promote cell survival in primary OL.  It is still  unclear whether ERK1/2 responses differ between primary O L and C G - 4 OL, or whether ERK1/2 activation mediates H 0 cytotoxicity in primary O L as well as in 2  2  C G - 4 OL.  1.2.5 J N K and p38 Signalling in Primary O L and C G - 4  Both J N K and p38 have the distinction of being considered stress-related kinases, and are therefore often associated with cell death. In the case of J N K ,  30  studies on human OL have shown that TNF-a treatment can cause activation of both OL apoptosis and activation of J N K (Ladiwala et al., 1998; 1999). A s well, studies on rat OL have used N G F to show activation of both OL apoptosis and J N K (Casaccia-Bonnefil et al., 1996). activation  Other OL studies have confirmed the  of J N K by TNF-a, as well as by ceramide, sphingosine and  sphingomyelinase. However, these studies did not assess the potential apoptotic effects of this J N K activation on OL (Zhang et al., 1996; 1998). A case can also be made for a role for p38 in OL apoptosis.  In  experiments that used ceramide to induce OL apoptosis, p38 was activated (Hida et al., 1999).  Furthermore, use of the p38 inhibitor SB203580 decreased this  ceramide-induced apoptosis, while transfection of cells with dominant negative p38 attenuated the apoptotic response. Interestingly, no activation of J N K was noted with ceramide treatment.  Furthermore, transfection of cells with dominant  negative c-Jun did not lead to attenuation of ceramide-induced apoptosis. These responses contradict a role for J N K in ceramide-induced apoptosis, while at the same time indicating a role for p38. More experiments are needed to define the function of these two kinases in the OL apoptotic response. One possibility is that J N K predominantly mediates TNF-a responses, while p38 predominantly mediates ceramide responses. Since the kinetics of J N K activation have been shown to be different in response to TNF-a than in response to ceramide, it is conceivable that the kinetics of activation and not just activation itself play a role in determining whether or not OL will undergo apoptosis (Zhang et al., 1996).  31  While studies of J N K in CG-4 cells have not yet been undertaken, studies of p38 in CG-4 cells tend to corroborate an apoptotic role for this kinase in OL. For instance, it has been shown that p38 activity is linked to CG-4 cytotoxicity via induction of nitric oxide synthase (Bhat et al., 1999). The similarities noted in the M A P K signalling pathways between primary OL and CG-4 cells, as well as similarities in their differentiation pathways and protein expression patterns, indicate that both primary O L and CG-4 cells can potentially be used to further elucidate the signal transduction pathways involved in O L functional biology.  1.3 Rationale and Objectives  Studies indicate that, after the onset of pathological demyelination in diseases such as M S , surviving mature OL do not, in fact remyelinate. Rather, it seems that a surviving pool of progenitor OL contribute to the remyelination process by migrating into the area of demyelination and proliferating (Keirstead and Blakemore, 1997; 1999).  Presumably, they then differentiate into new  mature OL, which in turn attempt to remyelinate the axons.  Since the  remyelination effected by the progenitor O L population in M S is incomplete, it would be beneficial if the surviving mature O L could somehow also be induced to contribute to the remyelination process. To determine how to stimulate remyelination from mature OL, experiments have been undertaken to elucidate the signal transduction pathways involved in  32  OL process extension.  The experimental endpoint of process extension is  relevant because axons must be contacted by O L before they can be myelinated. A s well, this endpoint is easily monitored, since O L lose their processes during the culture procedure and must re-grow them in vitro. Since the use of primary culture often results in a limited number of OL, the C G - 4 culture model has also been used to facilitate research on O L signal transduction. Signal transduction studies on OL functional biology have thus far included the P K C and M A P K signalling cascades. P K C has been shown to play a role in immature O L proliferation, as well as in mature OL process extension. Specifically, treatment of mature primary OL with phorbol esters has been shown to both activate P K C and induce O L process extension (Yong et al., 1988; Althaus et al., 1991; Yong et al., 1994). M A P K s have been shown to contribute to survival and death signals in OL, and may potentially also be downstream of P K C in process extension signalling cascades. The objectives of the experiments in this thesis were therefore three-fold: 1 - The first objective was to use primary O L cultures to uncover any potential role for the ERK1/2 members of the M A P K family in O L process extension. This involved examination of the specific phosphotransferase activity levels of ERK1/2 in the presence or absence of phorbol ester induction of O L process extension. A s well, the effects of the MEK1 inhibitor P D 98059 on both OL process extension and ERK1/2 activity were assessed. 2 - The second objective was to identify a suitable cell line model for use in OL signal transduction studies.  In particular, the utility of the C G - 4 cell line was  33  examined through a series of multi-kinase Western blots. These blots were used to compare and contrast the expression profile of various kinases in primary OL and in C G - 4 cells.  Since the C G - 4 cell line initially arose as a spontaneous  mutation from primary rat 0 2 A cultures, primary rat O L were employed in these experiments. 3 - The third objective was to use the C G - 4 cell line to further investigate the involvement of ERK1/2 in O L process extension. Specifically, the effects of the phorbol ester P M A , the MEK1/2 inhibitors P D 98059 and UO-126, and the P K C inhibitor Ro-32 on the development of a multipolar phenotype were assessed. In conclusion, the objectives for this thesis were to use both the primary OL culture model and the C G - 4 culture model to outline the role of E R K s 1 and 2 in O L process extension.  34  CHAPTER 2: MATERIAL AND METHODS  2.1 General Materials  2.1.1 Chemical Reagents  Acrylamide  Fisher  [y- P] adenosine triphosphate  Amersham/Pharmacia  Albumin, bovine  Sigma  3-amino-9-ethyl-carbazole (AEC) tablets  Sigma  Avidin-Biotin Complex kit  Vector  Bio-Rad Protein Assay Dye Reagent  Bio-Rad  Biotin  Sigma  Bis-acrylamide  Fisher  3-bromo-4-chloro-3-indoyl phosphate  Sigma  Bromophenol blue  ICN  B S A protein standard  Biorad  Dimethylsulfoxide  Sigma  Dithiothreitol (DTT)  BDH  DNase  Sigma  Dulbecco's Modified Eagle Medium  Sigma  Enhanced chemiluminescence kit (ECL)  Amersham  32  Ethylene bis (oxyethylenenitrilo) tetraacetic acid (EGTA)  Fisher/ICN  Ethylene diamine tetraacetate disodium salt (EDTA)  Fisher/ICN  Fetal calf serum  Hyclone  Fibronectin  Sigma  Gentamicin  Gibco-BRL  Glycerol  Anachemia  p-glycerophosphate  ICN/Fisher  Glycine  ICN/Fisher  Horse serum  Hyclone  Insulin  Sigma  Leupeptin  Roche  M B P substrate  Kinetek/Sigma  M B P synthetic peptide  Kinetek  P-mercaptoethanol  Fisher  Methanol  Fisher  p-methyl aspartic acid  Sigma  Mono Q  Pharmacia  3-[N-mopholino]ethanesulfonic acid (MOPS)  Sigma/ICN  Nitro blue terazolium  Sigma  Noniodet P-40  BDH  Normal goat serum  Sigma  PD 98059  Calbiochem  Penstrep (10 units/ml penicillin, 10 mg/ml streptomycin)  Stemcell  Percoll  Pharmacia  Phenyl methylsulphonyl fluoride (PMSF)  Sigma  Phorbol 12,13-myristate acetate  Sigma  Phosphate Buffered Saline  Oxoid  Phosphoric acid  Fisher  Poly-L-lysine  Sigma  Ponceau S  Sigma  Progesterone  Sigma  Protein kinase inhibitor peptide  Sigma  Putrescine  Sigma  Ro-32  Calbiochem  Scintillation fluid  Fisher  Skim milk  Safeway  Sodium azide  Fisher  Sodium chloride (NaCI)  Fisher  Sodium dodecylsulphate (SDS)  Fisher  Sodium orthovanadate ( N a V 0 )  Fisher  Sodium selenite  Sigma  Transferrin (human)  Sigma  Tris hydroxylmethyl aminomethane hydrochloride (Tris-HCl)  Fisher  4  3  4  Tris (hydroxylmethyl) methylamine (Tris)  Fisher  Triton X-100  Pharmacia  Trypsin  Sigma  Tween-20 (polyoxyethylene-20sorbitan monolaurate)  Fisher  UO-126  Calbiochem  2.1.2 Laboratory Supplies  3MM filter paper  VWR  Bottle top filters, 0.22 um pore size  Nalgene  Culture Dishes: Cell+  Sarstedt  Disposable Cell Scrapers  Sarstedt  M A P K assay kit  Upstate Biotechnology  Nitrocellulose membrane  VWR  Plastic coverslips, Aclar  Allied Signal  Syringe filters, 0.22 urn pore size  Gelman  Syringes  BD  2.1.3 Antibody Reagents  Anti-CNP  Sigma  Anti-ERK (ERK1-CT)  Upstate Biotechnology  Anti-phosphotyrosine (4G10)  Upstate Biotechnology  Anti-PKC-a  Transduction Labs  Anti-PKC-p (C16)  Santa Cruz  Biotinylated anti-mouse IgG  Vector  Goat anti-mouse IgG alkaline phosphatase conjugate  BioRad  Goat anti-mouse IgG-horse radish peroxidase conjugate  BioRad  Goat anti-rabbit IgG alkaline phosphatase conjugate  BioRad  Goat anti-rabbit IgG-horse radish peroxidase conjugate  Calbiochem  Multiblot primary antibodies  Provided by Kinexus  2.2 General Methods  2.2.1 Primary Bovine O L Cultures  Bovine O L cultures were prepared following the procedures described previously (Kim et al., 1983; Kim, 1990).  Fresh adult bovine brains were  obtained from an abattoir and transported on ice in a phosphate buffered saline solution (PBS).  The P B S also contained 10 units/ml penicillin and 10 mg/ml 4  streptomycin in a 1:100 dilution (Penstrep). After cleaning off the blood vessels and meninges, the brains were manually chopped into pieces of approximately ~3x3x3 mm. The pieces were then suspended in a P B S + penicillin/streptomycin  39  solution containing 0.25% trypsin and 20 ng/ml DNase. They were placed on a cell shaker in a 37 °C incubator and incubated for approximately 1 hr. Following trypsinization, the dissociated cells were passed through a nylon filter with a pore size of 100 urn and centrifuged at 1400 rpm for 10 min.  The cell pellets were  then resuspended in P B S , and 20 ml of cell suspension was layered over 9 ml of Percoll plus 1 ml of 10x P B S . After centrifugation for 30 min at 15 000 rpm, the cell and Percoll mixture was separated into five distinct layers. The top layer consisted of mainly debris, the second layer of myelin, the third layer of neural cells, the fourth layer of blood cells, and the fifth layer of remaining Percoll. The debris and myelin layers were removed by aspiration, and the third layer of cells was collected.  These cells were diluted with P B S and centrifuged for 10 min at  1400 rpm. The pellets were then resuspended in P B S and centrifuged for 10 min at 1200 rpm. Finally, the pellets were resuspended in feeding medium consisting of Dulbecco's Modified Eagle Medium plus 5% horse serum, 0.04 mg/ml gentamicin, and penicillin/streptomycin. After a final centrifugation for 10 min at 1200 rpm, the cells were resuspended in feeding medium (5% HS) and plated on 10-cm-diameter plastic Petri dishes. Twenty-four hours later, most O L had not attached to the plastic dishes, while most other cells (i.e. astrocytes and microglia) had attached. The floating O L were therefore collected and replated. This replating procedure was carried out at least four times to obtain an enriched culture of OL.  O L were  maintained in a 37 °C incubator with a 5% C O 2 content.  40  2.2.2 Primary Rat O L Cultures  Rat brains were removed from 21-25 day old Sprague-Dawley or Wistar rats that were sacrificed by exposure to carbon dioxide. The brains were then processed in a similar fashion to bovine brains, except that the trypsinization time for rat material was 30 min as opposed to a trypsinization time of 1 hr for bovine material.  2.2.3 Bipotential CG-4 Cultures  The CG-4 cell line was originally provided by Dr. J . C . Lewis of Amgen. CG-4 cells were maintained as bipotential cultures, then differentiated to either OL-like or astrocyte-like cells as needed. To maintain CG-4 cells in a bipotential state, the cells were fed with 70% defined medium supplemented with 30% B104 conditioned medium (70/30). The defined medium (N1) was made by adding 50 ug/ml transferrin, 5 ug/ml insulin, 100 uM putrescine, 20 nM progesterone, 30 nM selenium, and 10 ng/ml biotin to D M E M .  Gentamicin and penicillin-  streptomycin were also added to the medium. The cells were maintained in 10cm-diameter round culture dishes coated with poly-L-lysine (PL) and overlayed with fibronectin. For P L coating, dishes were incubated in P L for 1 hr at 37 °C, washed three times with distilled water, and left to air dry. After drying, the dishes were overlayed with 50 ug of human fibronectin/dish and incubated for 24  41  hr at 37 °C. The dishes were rinsed once with D M E M before being used for C G 4 plating.  i) B-104 Conditioning Prior to the creation of conditioned medium, B-104 cultures were maintained in D M E M containing 10% heat inactivated fetal calf serum (FCS). Once the cells were confluent, they were rinsed three times with P B S and incubated in N1 medium plus 2 mM glutamine for 72 hr.  Subsequently, the  conditioned medium was collected and filtered (0.22 um).  The medium was  either used immediately for C G - 4 cultures or stored at -20 °C.  2.2.4 Differentiation of C G - 4  Bipotential C G - 4 cells were differentiated into OL-like cells (CG-4 OL) by removal of B-104 conditioning from the medium. medium differentiated in C G - 4 OL after 24-48 hr.  Cells fed with 100% N1 Bipotential C G - 4 cells were  induced to differentiate into astrocyte-like cells by incubation in N1 medium supplemented with 20% F C S .  The differentiation  into astrocyte-like cells  occurred over a one week period.  42  2.2.5 Treatment Protocols  Prior to treatments  and morphological monitoring,  bovine OL were  adhered to 12-mm-diameter round Aclar coverslips coated with PL. Coverslips were coated by incubation for 1 hr in PL, then washed twice in distilled water and left to air-dry.  O L suspended in 5% H S medium were then dropped onto the  coverslips and allowed to settle for 24 hr. CG-4 cells were maintained in 70/30 medium prior to treatments.  They  were adhered to PL+ fibronectin coated dishes as described above. All treatment reactions were stopped by flooding the O L or C G - 4 cells with ice cold P B S prior to cell lysis.  i) Phorbol Ester Treatments Phorbol 12,13-myristate acetate (PMA) stocks of 1 mM were made by dissolving P M A powder in D M S O . The 1 mM stocks were then stored at -20 °C until just prior to use, when they were further diluted to 100 u.M in D M S O . After a final dilution in feeding medium, the solutions were filtered through a 0.22 um pore size filter. OL were exposed to 100 nM P M A for 24 hr. They were then monitored morphologically using a phase contrast microscope. Parallel O L cultures were exposed to 100 nM P M A for 15 min prior to lysis. CG-4  in N1  medium were exposed to  1, 5,  10, 20,  or 40  nM  concentrations of P M A and observed under a phase contrast microscope over  43  the next 24-72 hr. Parallel C G - 4 cultures were exposed to 1, 5, 10, 20, or 40 nM concentrations of P M A in N1 medium for 15 min prior to lysis.  ii) Ro-32 Treatments Ro-32 was dissolved in D M S O and filtered through a 0.22 pm pore size filter to make 1 mM stock solutions. Solutions were stored at -20 °C prior to use. C G - 4 cells to be lysed were exposed to 1, 2.5, 5, 10 or 20 pM concentrations of Ro-32 in N1 medium for 30 min, 1 hr, or 2 hr. Parallel cultures were exposed to 1, 2.5, 5, 10 or 20 pM concentrations of Ro-32 in N1 medium and observed under a phase contrast microscope over the next 24-72 hr. C G - 4 cells in 70/30 medium were also treated with 5 pM Ro-32, and either observed over the next 24-72 hr or lysed after 30 min. In some cases, the cells were pretreated with Ro-32. First, the 70/30 medium was collected after 48 hr. Second, 5 uM Ro-32 was added to the collected medium, and the medium was then replaced. Twenty minutes later, the medium was aspirated off and replaced with fresh 70/30 medium containing fresh 5 uM Ro-32.  iii) UO-126 and P D 98059 Treatments Both UO-126 and P D 98059 were dissolved in D M S O to make 1 mM stock solutions.  Stock solutions were stored at -20 °C prior to use.  After further  dilution in feeding medium to reach the appropriate concentration, the solutions were filtered through a 0.22 um pore size filter.  44  OL were incubated in 12.5, 25, 50, or 100 uM concentrations of P D 98059 for 15 min prior to exposure to P M A .  The optimal dose of P D 98059 was  assessed morphologically by determining the lowest dose to completely inhibit PMA-induced process extension without causing cytotoxicity.  To do this, OL  were exposed to P D 98059 for 15 min prior to addition of 100 nM P M A . The extent of process inhibition as compared to cells treated with 100 nM P M A alone was monitored using a phase-contrast microscope. C G - 4 cells to be lysed were incubated in 12.5, 25, 50, or 100 uM concentrations of UO-126 or P D 98059 in 70/30 medium for 30 min.  Also,  selected cultures were pretreated with the inhibitors for 20 min prior to the 30 min treatment just described. To pretreat cells, 70/30 medium was collected from the cultures after 48 hr. UO-126 or PD 98059 was then added, and the medium was replaced. After 20 min, the medium was aspirated off and replaced with fresh 70/30 medium containing the appropriate inhibitor. Once again, parallel cultures were used for morphological monitoring  2.2.6 Cell Lysis  i) Cytosolic Lysates OL in suspension were flooded with ice cold P B S , collected, and pelleted by centrifugation at 1200 rpm for 10 min. The supernatant was aspirated off and the pellet resuspended in a homogenization buffer consisting of 50 mM p-glycerol phosphate, 20 mM M O P S , 5 uM E G T A , 5 uM p-methyl aspartic, 2 mM EDTA,  45  and 2 mM Na3V0 . This buffer was stored at 4 °C, while 1 u.g/ml leupeptin and 5 4  mM P M S F were added directly before use. The resuspended pellets were then sonicated on ice and centrifuged at 13 000 rpm for 30 min at 4 °C. The cytosolic proteins were  collected with the  supernatant,  the  protein  concentrations  determined, and the proteins loaded onto a Mono Q column. Adherent C G - 4 cells were prepared for lysis by washing in ice cold P B S . The cells were then collected by scrapping in a lysis buffer (75 mM (3glycerolphosphate, 20 mM M O P S , 15 mM E G T A , 2 mM EDTA, 1 mM N a V 0 , 1 3  4  ug/ml leupeptin and 5 mM P M S F ) . After collection, the cells were sonicated and centrifuged as described for OL. The lysates were stored at -70 °C until use.  ii) Membrane Lysates To obtain membrane proteins, C G - 4 cells were initially processed as described for cytosolic proteins.  After sonication and centrifugation, cytosolic  proteins were collected with the supernatant and kept on ice. In the meantime, the remaining pellets were resuspended in lysis buffer containing 1% Triton X 100. The resuspended pellets were then centrifuged at 100 000 rpm for 10 min in an ultracentrifuge. After centrifugation, the membrane proteins were collected with the supernatant.  The cytosolic and membrane fractions were kept on ice  while protein determinations were performed. The samples were then equalized for protein content (0.5-1 mg/ml) and boiled in 5x concentrated sample buffer. Samples were stored at -70 °C until use.  46  2.2.7 Determination of Protein Concentration  Protein concentrations were determined using the method of Bradford (1976).  First, a 1.42 mg/ml B S A standard was diluted with d H 0 to a total 2  volume of 100 uJ/tube in order to create a series of standard tubes ranging from 0 to 30 (ig BSA/tube. Second, 5 uJ of each unknown sample was diluted with d H 0 2  to a total volume of 100 uJ/tube. To each tube was added 2.5 ml of Bio-Rad Protein Assay Dye Reagent. After gently vortexing to mix and allowing to stand for approximately 5 min, the samples absorbances were read at a wavelength of 595 nM using a spectrophotometer.  The concentrations of the samples were  then determined using a linear regression plot.  2.2.8 Mono Q Fractionation  Cytosolic extracts from O L were fractionated  by fast protein  liquid  chromatography ( F P L C ; Pharmacia) on a Resource Q column (Pelech et al., 1991). The column was equilibrated with Mono Q buffer (10 mM M O P S , pH 7.2; 25 mM p-glycerol phosphate, 5 mM E G T A , 2 mM EDTA, 1 mM dithiothreitol (DTT), 2 mM N a V 0 ) prior to loading. Samples containing approximately 1 mg 3  4  of protein were loaded onto the column, which was eluted at a flow rate of 0.8 ml/min with a 15-ml linear 0-0.8 M NaCI gradient.  Fractions of 0.25 ml were  collected and further analyzed by kinase phosphotransferase assays  and  Western blotting.  47  2.2.9 ERK1/2 Kinase Assays  The ERK1/2 activity in the Mono Q fractions of O L cytosolic lysates was assayed for phosphotransferase activity  using a M A P K  assay  kit.  The  phosphotransferase activity was assessed using either 1 mg/ml M B P or 1 mg/ml synthetic M B P peptide.  The synthetic peptide is an E R K substrate designed  around the Thr-97 substrate recognition sequence for ERK1 in M B P (Clark-Lewis et al., 1991). Five times concentrated M B P substrates were made up in a pH 7.2 assay dilution buffer (ADB) containing 1 M p-glycerophosphate, 1 M M O P S , 0.25 M E G T A , 0.1 M EDTA, 1 M magnesium chloride, 0.25 M DTT, and 5 mM pmethyl aspartic acid. The 30 pi total reaction volume contained 10 pi of sample. The reaction mix also contained 0.5 uM protein kinase inhibitor peptide (PKI) and 50 uM [y- P] adenosine triphosphate (ATP). The reactions were carried out for 32  10 min and stopped by spotting 10 ul into wells containing filter paper. The wells were washed in 1 %  H3PO4  (v/v) to remove excess A T P . The filter papers were  subsequently punched out of the wells and into scintillation vials containing scintillation fluid. The vials were then quantitated for radioactivity in a scintillation counter.  2.2.10 SDS-polyacrylamide Gel Electrophoresis  Mono Q fractions of OL lysates were subjected to protein separation via sodium  dodecylsulphate-polyacrylamide  gel  electrophoresis  (SDS-PAGE)  48  according to the general methods of Laemmli (1970). Each lane was loaded with 90 u.l containing the Mono Q fraction sample diluted with 5x concentrated S D S sample buffer (250 mM Tris-HCl (pH 6.8), 10% S D S (w/v), 2 5 % glycerol (v/v), 10% p-mercaptoethanol, 0.02% bromophenol blue (w/v)). were boiled for 5 min prior to loading onto gels.  The sample mixes  The samples were then  electrophoresed through 4% stacking and 11% separating gels made with an acrylamide to bis-acrylamide ratio of 37.5:1. Electrophoresis was conducted at 8 mA for 15 hr in a running buffer consisting of 25 mM Tris, 192 mM glycine, and 3.4 mM S D S . In general, the S D S - P A G E methods for C G - 4 cells were the same as just described for OL.  However, for better bandshift resolution, samples were  electrophoresed through 13% low-bis separating gels as opposed to the regular 11% separating gels.  These low-bis gels consisted of an acrylamide to bis-  acrylamide ratio of 150:1. Protein samples of 30-70 ug were loaded onto large gels of 1.5 mm thickness. They were then electrophoresed through the stacking gel at 30 mA and the separating gel at 45 mA for approximately 3 hr. Once the dye front reached the bottom of the gel, electrophoresis was continued for another 50 min at 45 mA. Protein samples of 20-30 ng were loaded onto minigels of 1.5 mm thickness. Electrophoresis was performed at 30 mA through the stacking gel and at 35 mA through the separating gel for approximately 90 min. Once the dye front reached the bottom of the gel, electrophoresis was continued for another 40 min at 35 mA. Both large and mini-gels were cooled during the gel running procedure.  49  2.2.11 Western Blotting  i) Oligodendrocytes Following S D S - P A G E , the separating gels were soaked in transfer buffer (20 mM Tris, 120 mM glycine, 20% methanol (v/v), pH 8.6) prior to layering on a nitrocellulose membrane. The gel and membrane were sandwiched between 3 MM filter papers and sponges, which were also soaked in transfer buffer prior to layering. The proteins were then electrophoretically transferred from the gel to the membrane for 3 hr at 300 mA on ice using a Hoeffer transfer cell system. To assess the transfer of proteins from gel to membrane, the membranes were incubated in a Ponceau S protein stain for 2-5 min following transfer.  The  membranes were then destained by rinsing in distilled water followed by rinsing in T B S (50 nM Tris-HCl, 150 mM NaCI, pH 7.5). Membranes were blocked for 30 min using 5% skim milk (w/v) in T B S . Following blocking, the membranes were rinsed briefly in T B S T (0.05% Tween-20 (v/v) in T B S ) and then incubated in an anti-ERK1/2 primary antibody overnight at 4 °C. In general, primary antibodies were diluted to an optimal concentration in T B S T containing 0.05% sodium azide (w/v).  Blots were rinsed 4x in T B S T after primary antibody incubation.  rinse lasted 5-10 min, with agitation. alkaline  Each  The blots were then incubated in an  phosphatase-conjugated secondary antibody  for  2  hr  at  room  temperature. After rinsing 4x in T B S T , the blots were developed with nitro blue terazolium and 5-bromo-4-chloro-3-indoyl phosphate in an alkaline phosphatase buffer (0.1 M Tris, 0.1 M NaCI, 5 mM MgCI 6 H 0 , pH 9.5) 2  2  50  For probing with the 4G10 anti-phosphotyrosine antibody, membranes were blocked for at least 6 hr using 3% B S A (w/v) in low-salt T B S (20 mM Tris, pH 7.5, and 50 mM NaCl). 0.05% Nonidet P-40.  All rinses were done in low-salt T B S containing  Development of the blots was otherwise as described  above.  ii) C G - 4 The general procedures for C G - 4 Western blots were the same as described above for primary OL, with the following alterations. Large gels were transferred overnight at 100 mA at 4 °C, while mini gels were transferred at 300 mA for 45 min on ice. Membranes were blocked for 45 min in a T B S solution containing 2.5% skim milk and 1.5% B S A . After incubations in primary and secondary antibodies, the membranes were developed using an enhanced chemi-luminescence kit. The results were acquired with the use of a Fluoro S Max Multi-imaging system.  2.2.12 Immunoprecipitations  OL lysates containing 500 pg of protein were incubated in an equal volume of 3% N E T F (3% NP-40 (v/v) in 100 m NaCl, 5 mM EDTA, 50 mM TrisHCl (pH 7.4), 50 mM NaF) plus 10 ul antibody and 30 pi Protein A Sepharose slurry. They were rotated for 3 hr at 4 °C prior to washing twice in 3% N E T F and twice in KM buffer (12.5 mM sodium-p-D-glycerophosphate, 12.5 mM M O P S (pH  51  7.2), 5 mM E G T A , 20 mM MgCI , 50 mM NaF and 0.25 mM DTT (pH 7.2)). The 2  samples were then boiled for 5 min in 5x sample buffer and stored at -70 °C until used for Western blotting.  2.2.13 Immunocytochemistry  CG-4  O L and primary OL adhered to coverslips were fixed and  permeabilized in methanol for 10 min at -20 °C. The cells were then washed in P B S and incubated in P B S + 5% normal goat serum (NGS) for 45 min at room temperature (RT). Next, the coverslips were incubated overnight in a monoclonal anti-CNP antibody (1:100) at 4 °C. After washing 3x in P B S , the coverslips were then incubated for 1 hr at RT in a biotinylated anti-mouse IgG secondary antibody (1:100). They were then washed 3x in P B S prior to incubation in an avidin-biotin complex solution for 30 min at RT. Colour development was done using 3-amino-9-ethyl-carbazole (AEC) plus hydrogen peroxide. Coverslips were inverted and mounted onto slides using gelvatol.  52  CHAPTER 3:  THE ROLE OF ERKS 1 AND 2 IN PRIMARY OL PROCESS  EXTENSION 3.1 Introduction  Based on findings that treatment of OL with phorbol esters can induce OL process extension via P K C activation, and that P K C can be found upstream of ERK1/2 in many cell systems, it is possible that E R K s 1 and 2 are involved in mediating O L process extension. This potential role for ERK1/2 also takes into account that process extension precedes myelination, and that M B P is a wellknown ERK1/2 substrate as well as a major component of myelin. Furthermore, activation of ERK1/2 has already been shown to play a role in the extension of neurites from neurons, P C 1 2 cells, and neuroblastoma cells (Kim et al., 1997; Encinas et al., 1999; Perron and Bixby, 1999; Schmid et al., 1999; Walowitz and Roth, 1999). The following experiments were conducted to assess the activation state of E R K s 1 and 2 in O L induced to extend processes as compared to control OL. Mature bovine O L were used in these experiments in order to generate a sufficient quantity of cells. Bovine O L were also chosen for their slow basal rate of process re-growth, allowing for easily observable differences between treated and untreated cultures. Process extension is therefore defined in this thesis as the appearance of thin branches extending from the cell bodies of OL, as opposed to a complete lack of branches.  53  3.2 Results  To assess the potential role of ERK1/2 in phorbol ester-induced process extension, three parallel O L cultures were set-up. The first set of O L were left untreated, the second set were treated with 100 nM P M A or P D B , and the third set were pre-treated with 50 |uM P D 98059 for 15 min prior to the addition of 100 nM P M A or P D B . Observation of the cultures over the next 24-72 hr showed that untreated cells did not extend processes, while cells treated with 100 nM phorbol ester showed intricate'process extensions (Fig. 3).  It was found that 100 nM  PDB (data not shown) or 100 nM P M A (Fig. 3) could both produce this effect. Furthermore, O L pre-treated with 50 uM P D 98059 prior to addition of phorbol ester did not extend processes (Fig. 3). A dose-response was conducted using P D 98059 concentrations of 12.5, 25, 50, and 100 uM. Cells were pre-treated with P D 98059 prior to addition of P M A , then observed over the next week.  It was determined that 50 uM of the  M E K inhibitor was the lowest concentration sufficient to abolish the induction of processes (Fig. 4). Furthermore, as these inhibitor-treated cells were still phase bright under a phase-contrast microscope even one week after treatment, 50 uM was deemed to be a non-toxic inhibitor dose (Fig. 4).  OL treated with 50 uJvl  PD98059 alone also showed no process extensions or toxic effects. To verify that activation of ERK1/2 is required for phorbol ester-induced process extension, OL were lysed and the cytosolic lysates were subjected to Mono Q fractionation. Eluted protein fractions were then subjected to  54  Figure 3: The Effects of PMA and PD 98059 on Primary Bovine Oligodendrocytes Photographs were taken 24 hr after treatment using a phase contrast microscope. A = untreated O L B = O L treated with 100 nM P M A for 24 hr C = O L pretreated with 50 uM P D 98059 for 15 min prior to treatment with 50 uM P D 98059 + 100 nM P M A for 24 hr. Note the extensions visible after P M A treatment, but not after P D 98059 + P M A treatment. This data is representative of five experiments. Bar = 20 urn 55  Figure 4: PD 98059 Dose Response Primary bovine O L were observed under a phase contrast microscope 1 week after treatment. A = untreated, control O L B = O L treated with 100 nM P M A C = O L treated with 25 uM PD 98059 for 30 min prior to treatment with  25 uM PD 98059 + 100 nM P M A D = O L treated with 50 uM PD 98059 for 30 min prior to treatment with  50 uM PD 98059 + 100 nM P M A Note that OL are still viable even one week after treatment. Also note that 25 pM PD 98059 was unable to completely abolish OL process extension. The ability of 50 uM PD 98059 to cause non-toxic inhibition of OL process extension was verified 5 times. Bar = 20 pm  56  phosphotransferase kinase assays using either M B P or a synthetic peptide substrate. It was found that ERK1/2 phosphotransferase activity was increased by the phorbol ester treatment, and that this increase was reduced by pretreatment of the cells with P D 98059 prior to addition of P D 98059 + P M A . By measuring the area under the peaks created by graphing the phosphotransferase activity values against the Mono Q fraction numbers, it was determined that P M A could increase E R K phosphotransferase up to 12-fold. Furthermore, P D 98059 was able to decrease this activity by approximately 7 0 % (Fig. 5). To ensure that the eluted and assayed fractions represented ERK1/2, samples of each Mono Q fraction were analyzed with both anti-ERK and antiphosphotyrosine (4G10) Western blots. Prior to primary antibody incubation, the membranes were first subjected to a Ponceau S protein stain in an effort to account for the shift in peak phosphotransferase values between control or P M A treated lysates and P D 98059 + P M A treated lysates. The stain indicated a total shift in protein elution between the two samples, rather than a specific shift in ERK1/2 elution. After destaining, the membranes were probed with either antiE R K or anti-phosphotyrosine primary antibodies.  The anti-ERK blots clearly  showed the presence of both ERK1 and E R K 2 in the eluted fractions (Fig. 6). Furthermore, E R K 1 and 2 bandshifts to slower migrating forms were noted. These bandshifted fractions corresponded to fractions that showed increased activity in the phosphotransferase assays. The 4G10 Western blots also verified ERK1 and E R K 2 activity in these same fractions (Fig. 7).  A s well, both blots  confirmed that the ERK1/2 activity was not abolished, but only decreased, in PD  57  1800  250  Mono Q Fraction #  Figure 5: ERK1/2 Phosphotransferase Activity Control = OL left untreated P M A = O L treated with 100 nM P M A for 15 min PD 98059 + P M A = OL treated with 50 uM P D 98059 for 15 min prior to treatment with 50 uM P D 98059 + 100 nM P M A for 15 min more. After cell lysis, 1 mg of protein from each sample was subjected to Mono Q fractionation. Fractions containing 250 uJ were collected and 10 uJ of each fraction were assayed for ERK1/2 phosphotransferase activity. using either an M B P  Assays were conducted  or a peptide """ substrate. Note the increase in ERK1/2  activity in P M A treated samples.  Also note that the MEK1 inhibitor did not  completely abolish PMA-induced ERK1/2 activity.  This trend was observed in 5  experiments.  58  59  CO  CO  CO  c  <  -t—•  c  CO O  CD  E  "CD CO  o o  1—  CD CL X CD  g  C/>  L CL  C  o c o <  CD LO O  CO  0  CO  00  CD  o  Q CL  CO  O  C L  E CO  CO TD CD CD  o c o  O LO  JZ  c  CD  -t—'  CD  o  E  CD 2 2,  XI 13  CO  CD  C/>  CO CO  CO CD  CD  JZ  c •a c o  O  i—  CO  CQ c  c E  CD W CD  LO i—  <D  c  < o_  '53 o  o o  CL V) o  CO  co  c  < 2!  3 O)  ii  c E  CL  E co  LO  JZ o CO CD  CD LO  E o i= c  o  00  CD  Q CL  CD  •4—* O  i_  o sz  Q  -CD  C L  o  .c -*—' O  o •_  + — *  o  a  "D CD  CT3  CD  o II CD  C L  o  o  LO  CO  E "D CD  -4—'  CO CD  o II  O  CD  i— O  E c E LO  CO  CN  a: LU "D CD  _co cr o  JZ CO C L  O  o >, -*—' c o nT E CD  JZ •4—'  -*—'  CD  o  1—  CD  CO  JZ -t—'  CD  -f—'  o  •a CD CO  o C L T S  60  98059 treated cells. A densitometry comparison of the two most intense 4G10 bands in PMA-treated lanes to the two most intense 4G10 bands in P D 98059 + PMA-treated lanes determined that the PMA-bands were approximately 30% more intense than the P D 98059 + PMA-bands. Finally, immunoprecipitations and Western blots were undertaken to determine if Raf is potentially upstream of ERK1/2 in OL. All three known Raf isoforms, Raf-1, Raf A, and Raf B, were detected (Fig. 8).  3.3 Discussion  These experiments were conducted in order to confirm or deny a role for E R K s 1 and 2 in O L process extension.  Through a series of morphological  studies coupled with phosphotransferase assays and Western blots, ERK1/2 activation was shown to be a necessary component of phorbol ester-induced process extension.  Other studies have demonstrated that E R K 1 / 2 activation  may also play a role in NGF-induced process extension, as N G F has been shown to induce both process extension and ERK1/2 activation in mature O L (Althaus et al., 1992; 1997). This indicates that ERK1/2 activation could be a general requirement of OL process extension, rather than simply a component of phorbol ester-induced O L process extension. The potential substrates of ERK1/2 in OL also support a general role for this kinase in process extension. One such substrate, M B P , is a major myelin protein that comprises up to 25-30% of the total protein in myelin (Deber and  61  B  Raf B  89 kDa « _ Raf-1 Raf A 4  66 kDa  Figure 8: Raf Immunoprecipitations A = Raf-1 immunoprecipitation B = Raf A immunoprecipitation C = Raf B immunoprecipitation OL were lysed and 500 ug of cytosolic lysate were subjected to Raf immunoprecipitations. Note the evidence of all three Raf isoforms in O L . These blots are representative of 3 experiments.  62  Reynolds, 1991). M B P has been shown to associate with the O L cytoskeleton, specifically in the thick processes of mature cells, and radiolabelling studies have shown that phosphorylation of M B P occurs with the onset of myelin formation (Ulmer and Braun, 1983; Wilson and Brophy, 1989). Thus it is conceivable that the ERK1/2 activation observed in these studies could lead to phosphorylation of M B P in preparation for myelination following process extension. E R K 1 / 2 also has the potential to directly affect cytoskeletal dynamics via influencing the cytoskeletal protein, actin. Studies have linked ERK1/2 activation to actin reorganization in embryonic avian corneal cells, actin-mysoin assembly in immune cells, and redistribution of the actin cytoskeleton in progenitor O L (Baron et al., 1999; Chu et al., 2000; Stupack et al., 2000). Since O L processes themselves are not comprised simply of actin filaments, but also of microtubules, microtubule modulation is another possible target of activated ERK1/2.  One  possible scenario involves ERK1/2 phosphorylation of stathmin, a microtubule depolymerizing factor. Phosphorylation of stathmin by ERK1/2 has been shown to decrease its disruptive effects on microtubule polymerization (Moreno and Avila, 1998). This, in turn, has been shown to lead to microtubule rearrangement resulting in the formation of microtubule bundles that extend to the cell rim (Lovricetal., 1998). Another potential role for ERK1/2 is induction of the c-fos gene, since activation of ERK1/2 can lead to c-fos gene induction via phosphorylation of the Elk-1 transcription factor. A s well, c-fos induction in response to P K C activation has already been documented in progenitor OL, and is therefore also likely to  63  occur in mature O L (Bhat et al., 1992).  Induction of c-fos has been linked to  differentiation in osteoclasts, as well as to neurite extension in rat cerebellar granule cells and neuroblastoma cells (Rossino et al., 1995; Reddy and Roodman, 1998; Vaudry et al., 1998).  Therefore, it is not unreasonable to  hypothesize a similar role for c-fos induction in OL. It is conceivable that the actions of ERK1/2 during process extension involve both the cytoplasmic/cytoskeletal and nuclear substrates mentioned above.  This  hypothesis  is  supported  by  immunocytochemical  studies  demonstrating the presence of ERK1/2 in the cytoplasm, processes, and nucleus of O L induced to extend processes (Stariha et al., 1997). Therefore, contact with M B P , actin and stathmin in the cytoplasm and processes is viable, as is induction of c-fos in the nucleus. The ability of the MEK1 inhibitor P D 98059 to abolish phorbol esterinduced process extension provides tangible evidence of a role for ERK1/2 in process extension.  However, MEK1 is upstream of ERK1/2, and there are no  commercially available E R K inhibitors.  Therefore, studies were undertaken to  verify the inhibition of ERK1/2 by the MEK1 inhibitor. The ERK1/2 activity profile was measured in four separate ways. The first two measurements involved assessment of ERK1/2 phosphotransferase activity, and the second two measurements involved Western blotting.  The two  phosphotransferase assays were conducted using either M B P or a synthetic peptide substrate (Fig. 5).  The synthetic substrate was used to reduce the  chances of artificially inflating the ERK1/2 activity levels via the presence of other  64  MBP-kinases in the eluted fractions. A s might be expected, therefore, the activity counts were lower when using the synthetic peptide than when using M B P . However, both substrates showed the same trend  of substantial  ERK1/2  activation in lysates from PMA-treated OL, and a substantial reduction of ERK1/2 activation in lysates from OL pre-treated with P D 98059 prior to addition of P M A . Although it is possible that the level of ERK1/2 activity in the PMA-treated fractions could still be inflated by the presence of other MBP-kinases, M B P kinases other than ERK1/2 would not likely be affected by P D 98059. Therefore, some of the activity seen in the MEK1-inhibited fractions could be due to other MBP-kinases, and the drop in ERK1/2 activity could actually be greater than 70%. Examples of such MBP-kinases are Raf-1 and protein kinases B and C. Oddly, the activity elution profiles consistently showed that ERK1/2 in P D 98059 treated samples eluted later than ERK1/2 in control or PMA-treated samples.  This anomaly was examined further via Western blotting.  Western  blots using an anti-ERK antibody confirmed the presence of E R K in the fractions that showed activity in the kinase assays (Fig. 6).  They also confirmed that  ERK1/2 in control and PMA-treated samples eluted earlier than those seen in P D 98059 + PMA-treated samples. To visualize the general protein elution profile for the various treatments, the membranes were subjected to a Ponceau S protein stain. It was observed that the entire protein elution profile appeared to shift in the P D 98059 + PMA-treated samples. Since the samples had always been applied to the column in the same order, namely control samples followed by PMA-treated samples followed by P D 98059 + PMA-treated samples, it was  65  speculated that the P D 98059 was affecting the elution profile.  A s well, it is  possible that the column was becoming progressively clogged over time. In the above-mentioned anti-ERK1/2 Western blots, eluted fractions were probed with an antibody that recognizes both ERK1 and E R K 2 .  ERK1/2  bandshifts were used as an indication of ERK1/2 activity, and were noted predominantly in PMA-treated samples. While these bandshifts are difficult to distinguish due to the large amount of ERK1/2 present in the samples, parallel anti-phosphotyrosine blots clearly showed a predominance of phosphorylated ERK1/2 in PMA-treated samples (Fig. 7).  Furthermore, both blots confirm that  ERK1/2 activity is not abolished, but merely reduced, in P D 98059 + PMA-treated OL. This lack of complete abolition of ERK1/2 activity indicates that ERK1/2 is required to reach a certain level of activity before O L will commit to process extension.  In O L treated with P D 98059 + P M A , therefore, the less robust  ERK1/2 activation may not be enough to cross the necessary activity threshold. It is plausible that a major cell function, such as the differentiation  and  myelination associated with process extension, would not be turned on or off with only minor changes in kinase activity.  Having a threshold of activity could be  akin to having a safe-guard against premature differentiation of O L in response to small E R K 1 / 2 activity spikes. Another factor that must be taken in consideration, but which cannot be answered by these experiments, is whether or not compartmentalization of ERK1/2 also affects process extension. In such a case, it would not simply be activation of ERK1/2 but also translocation of activated ERK1/2 to the nucleus and/or processes that would mediate process extension.  66  This possibility is supported but not verified by the noted translocation of ERK1/2 to the nucleus and processes of PMA-treated OL (Stariha et al., 1997). Finally, the anti-ERK Western blot results indicate that E R K 1 , rather than E R K 2 , is the dominant isoform in mature bovine OL. While the 4G10 Western blots indicate similar phosphorylation levels of ERK1 and E R K 2 in these studies, another set of studies using in gel assays have shown that NGF-induced O L process extension leads to phosphorylation of predominantly ERK1 over E R K 2 (Althaus et al., 1997).  Thus, it appears that ERK1 may play a larger role in  process extension than E R K 2 .  However, isoform specific immunoprecipitations  and kinase assays would be required to make this distinction. Preliminary studies were also undertaken to determine other potential components of the signalling cascade necessary for O L process extension. A s expected, a phosphotransferase assay utilizing a c-Jun substrate for J N K and an ATF-2 substrate for p38 showed no significant phosphotransferase activity upon PMA-treatment of O L (data not shown). A s a control, MBP-phosphotransferase activity was still observed.  These experiments were done to verify that  specifically E R K s , and not M A P K s in general, are responsible for process extension. extension  Furthermore, the potential contribution signalling cascade was  assessed  by  of Raf to the process Raf  immunoprecipitations  followed by Western blotting and MBP-phosphotransferase assays.  Three Raf  isoforms, Raf-1, Raf A, and Raf B were present in these cells, as seen via Western blotting (Fig. 8). However, Raf-1 was the only isoform that showed an approximately 2-fold increase in M B P phosphotransferase activity (data not  67  shown).  Raf-1 activation has also been linked to phorbol ester stimulation in  bovine luteal cells and NIH3T3 fibroblasts (Reuter et al., 1995; Chen et al., 1998) . Preliminary morphological studies were undertaken to determine if a variety of protein kinase inhibitors and/or activators could either induce process extension or inhibit phorbol ester-induced process extension. The PI 3-kinase inhibitor, wortmanin, was employed as there is evidence linking PI 3-kinase to the E R K pathway and to neurite extension (Kim et al., 1998; Pandey et al., 1999). The calcium-calmodulin dependent kinase II inhibitor, KN-62, was employed as studies have linked calcium modulation to process extension in O L (Yoo et al., 1999) . A s well, the protein kinase A (PKA) activator, forskolin, was employed, since P K A is another fairly ubiquitous kinase that has been implicated in O L differentiation and proliferation (Baron et al., 1998; Shi et al., 1998; Baron et al., 2000b). Wortmanin was shown to have no effect on control O L at concentrations up to 200 nM (Fig. 9).  A s well, pretreatment of cells for 24 hr with 200 nM  wortmanin followed by 24 hr of wortmanin + PMA-treatment showed no change in the formation of PMA-induced process extensions (Fig. 10).  Similar results  were found with KN-62 at concentrations of 5 -10 u.M, although 20 u.M of KN-62 was cytotoxic (Fig. 9, 10).  Finally, forskolin did not appear to induce process  extensions at a dose of 100 nM, nor did it inhibit PMA-induced process extensions (Fig. 9, 10).  Therefore, the only kinases definitively linked to OL  process extension at this time are P K C and ERK1/2.  68  c  Figure 9: The Effects of Wortmanin, Forskolin, and KN-62 on Primary Bovine OL Primary bovine O L were treated with various compounds for 24 hr. Photographs were taken using a phase contrast. A - control O L B - OL treated with 200 nM wortmanin C - O L treated with 100 nM forskolin D - OL treated with 5 uM KN-62 Note that none of the treatments induced noticeable process extensions from the OL. (For PMA-induced process extensions, see Fig. 10.) This indicates that PI 3-kinase (panel B), P K A (panel C), and calcium/calmodulin-dependent kinase II (panel D) do not play a role in OL process extension. This data is representative of 3 experiments. Bar = 20 um 69  Figure 10: The Effects of Wortmanin, Forskolin, and KN-62 on PMA-induced Process Extension in Primary Bovine OL Primary bovine O L were pretreated with various compounds for 24 hr prior addition of PMA. Photographs were taken using a phase contrast microscope. A - O L treated with 100 nM P M A for 24 hr. B - O L treated with 200 nM wortmanin for 24 hr followed by 200 nM wortmanin + 100 nM P M A for 24 hr more. C - O L treated with 100 nM forskolin for 24 hr followed by 100 nM forskolin + 100 nM P M A for 24 hr more. D - O L treated with 5 uM KN-62 for 24 hr followed by 5 uM KN-62 + 100 nM P M A for 24 hr more. None of the treatments visibly affected PMA-induced process extension in three experimental trials. (For untreated control OL, see Fig. 9).  Bar = 20 u.m 70  CHAPTER 4:  A COMPARISON OF THE KINASE EXPRESSION PROFILE  BETWEEN PRIMARY RAT OL AND CG-4  4.1 Introduction  After conducting process extension studies on primary cultures of mature OL as described in Chapter 3, the quantity and variability problems associated with primary O L cultures led to a search for a better model.  To generate  sufficient quantities of mature primary O L for experimentation, bovine brains had been employed. However, although it was possible to produce a large quantity of cells from these brains, there was no control over the age or genetics of the animals.  It was therefore logical to switch to a model that would allow for the  generation of a large number of cells, while at the same time allowing for a certain amount of control over culture variability. The search for an O L model brought to attention the C G - 4 cell line. Based on immunocytochemical data, C G - 4 cells are similar to primary OL. A s well, C G 4 cells and primary O L share the expression of many proteins and share many similar actions, as described in the Introduction.  However, since there are also  reports of differences between C G - 4 cells and primary OL, it was necessary to verify the suitability of the C G - 4 model for signal transduction studies.  An  exhaustive analysis of the kinase expression patterns between C G - 4 cells and primary O L was undertaken to determine this suitability.  Since the C G - 4 line  71  initially arose from a spontaneous mutation of rat 0 2 A cells, the comparison was made using primary rat OL.  4.2 Results  In an initial comparison of primary rat O L to C G - 4 OL, it was found that both cell types stained with an anti-CNP antibody (Fig. 11). Both cell types also developed a multipolar morphology, although the processes on primary O L had the potential to become slightly more intricate than the processes on C G - 4 OL (Fig. 12). For a further comparison of primary rat O L to CG-4-derived OL, cell lysates were sent to Kinexus Bioinformatics Corporation for a multi-kinase immunoblotting  expression profile.  C G - 4 cells in bipolar and  astrocytic  differentiation states were also sent for analysis. Prior to lysis, C G - 4 cells were allowed to differentiate over a one week period (Fig. 13). The Kinetworks™ analysis is a proprietory method used to quantitate the level of expression of at least 75 known protein kinases. The screen employs the use of independently validated commercial antibodies for the protein kinases shown in Table 2. After the Kinetworks™ screen, selected protein kinase expression levels were confirmed at least twice on verification Western blots using fresh cell lysates. Any changes in kinase expression were quantitated by averaging the results of densitometric measurements taken from two verification Western blots.  72  Figure 11: CNP Staining of CG-4 OL and Primary Rat OL The panels on the left represent C G - 4 cells differentiated into C G - 4 OL by incubation in N1 medium for 48 hr.  The panels on the right  represent primary rat O L maintained in 5% HS medium. Note that both cell types stain for the OL marker, C N P . Bar = 10 um  73  Figure 12: CG-4  OL and Primary Rat OL  The upper panel represents CG-4 cells differentiated into OLlike cells in N 1 medium. The lower panel represents primary rat OL maintained in 5 % H S medium.  Photographs were taken  using a phase contrast microscope. Note that both cell types are multipolar, although the processes on the primary O L appear to be slightly more intricate than those on the CG-4 OL. Bar = 1 0 urn  75  Bmx Btk Calmodulin-dependent kinases kinase Calmodulin-dependent kinase 1 Calmodulin-dependent kinase 4 Cyclin-dependent kinases 1 (Cdc2) Cyclin-dependent kinase 2 Cyclin-dependent kinase 4 Cyclin-dependent kinase 5 Cyclin-dependent kinase 6 Cyclin-dependent kinase 7 Cyclin-dependent kinase 9 Casein kinase 1 5 Casein kinase 1 s Casein kinase 2 a/a'/a" Cot (Tpl2) Csk DAPK DNAPK Extracellular regulated kinase Extracellular regulated kinase Extracellular regulated kinase Extracellular regulated kinase Focal adhesion kinase Fyn GCK G R K 2 (BARK) G S K 3 a/p Hpk1 nhibitor NF K B kinase a JAK1 JAK2 Ksr1 Lck yn Mek1 Mek2 Mek4  1 2 3 6  MEK6 Mek7 Mnk2 Mos Mst1 Nek2 p38 Hog M A P K P a k a ( P a k 1) Pak p (Pak 3) PDK1 (PKB kinase) Pim1 P K A (cAMP-dep. protein kinase) PKBa(Aktl) P K G 1 (cGMP-dep. protein kinase) PKR Protein kinase C a Protein kinase C p i Protein kinase C y Protein kinase C 5 Protein kinase C X Protein kinase C E Protein kinase C C, Protein kinase C 9 Protein kinase C LI Pyk2 Raf1 RafB ROK a Rsk1 Rsk2 S 6 K p70 S A P K (JNK2) Src Syk Yes Z A P 7 0 kinase ZIP kinase  Table 2: Kinetworks™ Protein Kinase Screen The expression levels of the 75 protein kinases listed above were assessed by the Kinetworks™ analysis performed by the Kinexus Bioinformatics Corporation.  76  4.2.1 Comparison of Primary OL to CG-4 OL  A number of kinases were not detected by the Kinetworks™ analysis in either primary OL or in the CG-4 line. These kinases include ERK6 from the MAPK family, as well as PKC's y, A,, and 6. For a complete list of kinases not detected in primary OL or in CG-4 cells, see Table 3. Among the kinases that were detected by the Kinetworks™ analysis were members of the MAPK family.  In fact, most members of the MAPK family  showed similar expression profiles in primary OL and in CG-4 OL. For a change in kinase expression to be considered relevant, the averaged densitometric values of two blots must have shown a minimum of 1.5-fold increase or decrease in expression.  Furthermore, any trends of increased or decreased protein  expression must have been supported by the original Kinetworks™ analysis. Based on these criteria, there were no major differences detected in ERK1, ERK2, p38, or SAPK expression (Fig 14). As well, upstream regulators of the MAPK family, such as MEK, Raf-1 and Raf B, and downstream substrates, such as Rsk-1, also showed similar expression levels (Fig. 15). There was, however, a noticeable change in Pak-a expression levels, and Pak-a has been shown to act upstream of the JNK and HOG members of the MAPK family.  Pak-a  expression was approximately 5-fold higher in primary OL than in CG-4 OL, and also at least 2-fold higher in primary OL than in astrocytic or bipotential CG-4 cells (Fig 16).  77  Btk Calmodulin-dependent kinase 4 Casein kinase 1 s CSK DAPK DNAPK Extracellular regulated kinase 6 GCK HPK1 Ksr1 Lck Lyn Mnk2  Mst1 Nek2 Pak p Pim1 PKG1 (cGMP-dep. protein kinase) PKR Protein kinase C y Protein kinase C X Protein kinase C 0 Pyk2 ROK a Syk Yes  Table 3: Protein Kinases Undetected by the Kinetworks  im  Analysis  Antibodies that targeted the protein kinases listed above were present in the Kinetworks™ screen performed on extracts from primary rat OL, C G - 4 OL, bipotential C G - 4 cells and astrocytic C G - 4 cells. However, the expression of these kinases was not detected in any of their cell lysates.  78  ERK1 ERK2  45 kDa  p38 36 kDa  p54 SAPK p46 SAPK  45 kDa B  O  ROL  Figure 14: MAPK Expression Profile Twenty jxg of cytosolic protein lysate were loaded into each lane. A = astrocytic CG-4 cells, cultured for 1 week in N1 medium + 20% F B S B = bipotential CG-4 cells, cultured for 1 week in 70/30 medium O = oligodendrocytic CG-4 cells, cultured for 1 week in N1 medium R O L = mature rat oligodendrocytes, primary culture Note that the ERK1/2 and p38 expression levels are similar across all lanes. Based on averaged densitometric values from two blots, there is a  2-fold  upregulation of p54 S A P K  in bipotential  CG-4  cells in  comparison to astrocytic CG-4 cells.  79  66 kDa  Raf-1  97 kDa  Raf-B  m~  45 kDa  Mek-2  97 kDa Rsk-1 B  O  ROL  Figure 15: Western Blot Kinase Expression Profile  Twenty jxg of cytosolic protein lysate were loaded into each lane. A = astrocytic CG-4 cells, cultured for 1 week in N1 medium + 20%FBS B = bipotential CG-4 cells, cultured for 1 week in 70/30 medium O = oligodendrocytic CG-4 cells, cultured for 1 week in N1 medium ROL = mature rat oligodendrocytes, primary culture Note that the expression levels Raf-1, Raf-B, Mek-2 and Rsk-1 are similar across all lanes.  80  66 kDa  97 kDa A  B  O  ROL  Figure 16: Western Blot Kinase Expression Profile Twenty ug of cytosolic protein lysate were loaded into each lane. A = astrocytic CG-4 cells, cultured for 1 week in N1 medium + 20%FBS B = bipotential CG-4 cells, cultured for 1 week in 70/30 medium O = oligodendrocytic CG-4 cells, cultured for 1 week in N1 medium R O L = mature rat oligodendrocytes, primary culture Based on averaged densitometric values from two blots, R O L has at least a 2-fold higher expression of Pak-a and a 2-fold lower expression of FAK as compared to A, B, or 0.  81  Other kinases assessed included P K A , P K B , and P K C family members. While P K A and P K B expression was similar between primary O L and C G - 4 OL, there were differences among some P K C isoforms (Fig. 17,18). In terms of P K C a, the Kinetworks™ analysis detected kinase expression in C G - 4 OL but not in primary OL. Verification blots, however, did detect P K C - a expression in both cell types.  These verification blots showed a slight increase (1.3-fold) of P K C - a  expression in C G - 4 OL as compared to primary O L (Fig. 18). The Kinetworks™ analysis and verification blots both indicated increased expression of PKC-p1 in C G - 4 O L as compared to primary OL. Again, however, this increase was still only approximately 1.3-fold (Fig. 18). The expression of PKC-y was undetectable in either cell type. Two novel P K C isoforms, s and 8, were also studied. It was found that primary O L expressed levels of P K C - s that were at least 2-fold lower than in any of the C G - 4 cells, and levels of PKC-8 that were at least 2-fold higher than in any of the C G - 4 cells (Fig. 18). The expression of the atypical PKC-c; isoform also appeared significantly higher in C G - 4 O L than in primary O L in the Kinetworks™ analysis, but this difference was not confirmed on the verification blots (Fig. 18). Outside of the M A P K and P K C pathways, interesting results were noted in two other kinases: cyclin-dependent kinase (CDK) and focal adhesion kinase (FAK). The expression of C D K 5 was visible in primary OL, but not in C G - 4 cells, on the Kinetworks™ analysis. Although verification blots did detect C D K 5 in the C G - 4 cell line, they also indicated that C D K 5 expression was approximately 2fold higher in primary O L than in any of the C G - 4 cells (Fig. 19). In contrast to  82  C D K 5 , the expression of C D K 7 was approximately 1.6-fold higher in C G - 4 OL and bipotential C G - 4 cells than in primary O L (Fig. 19). Finally, F A K expression was approximately 2-fold higher in all C G - 4 cells as compared to primary O L (Fig. 16).  83  «— PKB-a  45 kDa PKA  A  Figure 17:  B  O  ROL  P K B - a and PKA Expression Profile  Twenty \ig of cytosolic protein lysate were loaded into each lane. A = astrocytic C G - 4 cells, cultured for 1 week in N1 medium + 20% F B S B = bipotential C G - 4 cells, cultured for 1 week in 70/30 medium O = oligodendrocytic C G - 4 cells, cultured for 1 week in N1 medium R O L = mature rat oligodendrocytes, primary culture Note that the expression of P K B - a and P K A is similar in all lanes.  84  97 kDa  PKC-a  66 kDa  97 kDa PKC-P1  66 kDa  97 kDa 66 kDa  97 kDa  «— PKC- C  B  O  ROL  Figure 18: PKC Expression Profile Twenty ug of cytosolic protein lysate were loaded into each lane. A = astrocytic C G - 4 cells, cultured for 1 week in N1 medium + 2 0 % F B S B = bipotential C G - 4 cells, cultured for 1 week in 70/30 medium O = oligodendrocytic C G - 4 cells, cultured for 1 week in N1 medium R O L = mature rat oligodendrocytes, primary culture Based on averaged densitometric values from two blots, B has at least a 2-fold lower expression of  PKC-a and PKC-P1 than  O. A s well, R O L has an approximately 1.3-  fold lower expression of these isoforms than O. Finally, the expression of  PKC-e is at  least 2-fold lower and the expression of P K C - 5 is at least 2-fold higher in R O L as compared to A, B, or O.  85  36 kDa _  CDK7  36 kDa _  CDK5  B  ROL  Figure 19: CDK7 and CDK5 Expression Profile Twenty ug of cytosolic protein lysate were loaded into each lane. A = astrocytic CG-4 cells, cultured for 1 week in N1 medium + 20% F B S B = bipotential CG-4 cells, cultured for 1 week in 70/30 medium 0 = oligodendrocytic CG-4 cells, cultured for 1 week in N1 medium R O L = mature rat oligodendrocytes, primary culture Based on averaged densitometric values from two blots, the expression of C D K 7 is approximately 1.6-fold higher in B and 0 than in R O L . A s well, the expression of C D K 5 is approximately 2-fold higher in R O L than in A, B, or O. Also note the apparent bandshift in C D K 5 in B and O.  86  4.2.2 A Comparison of the Three Differentiation States of C G - 4  The Kinetworks™ multi-kinase immunoblot was also used to compare the bipotential, astrocytic, and oligodendrocytic forms of the C G - 4 cell line.  It was  determined that most members of the M A P K family did not change expression drastically during the differentiation of bipotential C G - 4 cells to astrocytic or oligodendrocytic C G - 4 cells (Fig. 14). The largest M A P K change was a 2-fold increase of p54 S A P K expression in bipotential C G - 4 cells as compared to astrocytic C G - 4 cells. A s well, Pak-a expression in oligodendrocytic C G - 4 cells was at least 2.5-fold  lower than  in astrocytic or bipotential  C G - 4 cells.  Furthermore, the Pak-a levels in all three C G - 4 differentiation states were at least 2-fold lower than those seen in primary O L (Fig. 16). The levels of P K A and P K B were also similar among all three C G - 4 differentiation states (Fig. 17). P K C isoforms.  However, there were some differences among  For instance, P K C - a expression was at least 1.5-fold higher in  oligodendrocytic and astrocytic C G - 4 cells than in bipotential C G - 4 cells (Fig. 18). There was also a 1.5-fold higher expression of P K C - p i in oligodendrocytic C G - 4 cells as compared to bipotential C G - 4 cells, although an apparently similar increase of PKC-(31 expression in astrocytic C G - 4 cells could not be confirmed (Fig. 18).  While verification blots indicated an apparent increase of P K C - 8  expression in astrocytic C G - 4 cells as compared to bipotential C G - 4 cells, this trend was not observed in the Kinetworks™ analysis (Fig. 18).  87  Finally, there was little difference in the expression levels of C D K 7 and C D K 5 among the three C G - 4 differentiation states. The largest difference was an approximately  1.4-fold higher expression of C D K 7  in bipotential  and  oligodendrocytic C G - 4 cells as compared to astrocytic C G - 4 cells (Fig 19).  4.3 Discussion  The main purpose of the Kinetworks™ analysis was to determine if the C G - 4 O L model is suitable to use for signal transduction studies on O L process extension.  Since process extension studies conducted on primary OL have  already outlined a role for the M A P K and P K C families, the similarities in M A P K and P K C expression patterns between primary OL and C G - 4 O L indicate that the C G - 4 cell line may be a suitable O L model. To begin with, all three branches of the M A P K cascade are present in both primary O L and C G - 4 OL.  Furthermore, no major changes in the expression  levels of these M A P K s were noted between primary O L and C G - 4 OL.  Most  upstream and downstream regulators of the M A P K cascades, such as various M E K , Raf, and Rsk isoforms, were also similarly expressed (Fig. 14,15). However, there was a large increase in the expression of one upstream regulator of the M A P K cascade, Pak-a, in comparison to all three C G - 4 differentiation states (Fig. 16). Pak-a is known to be enriched in the brain. The human homologue of Pak-a, Pak-1, has been implicated in the activation of various M A P K s (Knaus  88  and Bokoch, 1998). For instance, activation of J N K has been demonstrated by over-expression of Pak-1 in 293 cells, and has also been noted after addition of Pak-1 to cell free Xenopus  oocyte extracts (Polverino et al., 1995; Frost et al.,  1996) . A s well, other studies have demonstrated mediation of p38 activity by Pak-1 (Frost et al., 1996).  Expression of dominant negative Pak-1 has been  shown to suppress induction of p38 activity, while Pak-1 has been shown to be an upstream component of p38 activation in NK cells (Zhang et al., 1995; Mainiero et al., 2000).  Although the consequences of JNK/p38 activation in  many of these studies were not assessed, both J N K and p38 have been linked to apoptosis in O L (Casaccia-Bonnefil et al., 1996; Hida et al., 1999; Ladiwala et al., 1998).  Therefore, the elevated expression of P a k - a in primary cultures might  indicate that these cells are more susceptible to the induction of apoptotic cell death than the CG-4 cell line. However, since Pak-1 has also been shown to act in an anti-apoptotic manner via phosphorylation of the pro-apoptotic protein Bad, further studies would be needed to assess this hypothesis (Schurmann et al., 2000). There is also evidence for Pak-a influence on the E R K members of the M A P K family. While studies have indicated that Pak isoforms do not directly lead to activation of ERK1/2, over-expression of the Pak-1 regulatory domain has been shown to inhibit ERK1/2 activation (Polverino et al., 1995; Frost et al., 1996; 1997) . It is unclear from these ERK1/2 results why Pak-a should be upregulated in primary  O L as opposed to CG-4  cells.  However, the  morphological  consequences of P a k - a activation may provide some clues.  89  Pak-a has been shown to play a role in the morphological signalling pathways of various cell types. For instance, targeting of Pak-1 constructs to the plasma membrane in P C 1 2 cells has been shown induce neurite extension independently  of  kinase activity  (Daniels et al., 1998).  Therefore,  the  upregulation of Pak-a expression in primary OL may explain why these cells are able to form an overall more intricate network of processes than C G - 4 OL (Fig. 12). However, other reports have indicated that activation of Pak isoforms, rather than simply their expression, can cause retraction of the cell periphery (Manser et al., 1997). The role of Pak isoforms in cellular process extension, therefore, appears to depend on regulation of its activation state. Interestingly, C D K 5 has been shown to stimulate neurite outgrowth by inactivation of Pak-1, and C D K 5 appears to be highly expressed in primary O L (Nikolic et al., 1998) (Fig. 19). A s was mentioned above, the expression of P K A and P K B was also similar among primary OL and C G - 4 cells (Fig. 17). The P K A pathway has been implicated in OL differentiation and proliferation, while P K B has been linked to PI 3-kinase signalling. PI-3 kinase, in turn, has been linked to OL survival (Vemuri and McMorris, 1996).  Therefore, it seems that kinases from a variety of  important signalling cascades are expressed in both the cell line and in primary cells.  This again speaks to the suitability of the C G - 4 model for OL signal  transduction studies. The expression of conventional P K C isoforms was also largely similar between primary O L and C G - 4 OL, with a few significant differences. First, the noted lack of P K C - y expression in primary OL and C G - 4 OL is corroborated by  90  some studies and contradicted by other studies (Asotra and Macklin, 1993; Schmidt-Schultz and Althaus, 1994; Yong et al:, 1994). This lack of consensus on the expression of PKC-y in O L could be due to species variation.  For  instance, PKC-y has been detected in cultured porcine OL, but not in human OL. Furthermore, PKC-y has been found to be poorly expressed in rat OL, even though expression of this isozyme was detected in high levels in purified myelin. Since there is also evidence of developmental-dependent expression of various P K C isozymes in O L , the level of maturation of the O L used in the abovementioned studies could also have affected the amount of PKC-y detection.  In  contrast to PKC-y, primary O L and C G - 4 O L were both found to express P K C - a and pi (Fig. 18).  Previous studies have also demonstrated the expression of  these isoforms in OL, as well as an upregulation of P K C - a and pi in mature OL as compared to immature OL (Asotra and Macklin, 1993). This upregulation can again be seen when comparing bipotential C G - 4 cells to C G - 4 OL, indicating that upregulation of P K C - a and p i may be required for O L cell differentiation (Fig. 18). The slight upregulation of P K C - a in astrocytic C G - 4 cells as compared to bipotential C G - 4 cells also speaks to a possible role for this isozyme in differentiation. In terms of the functions of P K C - a and P K C - p in OL, studies have indicated that P K C - a rather than P K C - p is the important mediator of OL process extension. In one such study, the P K C - a and p agonist thymeleatoxin was found to stimulate OL process extension. However, the P K C - p agonist, resiniferatoxin, was found to have no effect (Yong et al., 1994). In another study, M G D G was shown to increase both O L process extension and P K C - a activity (Schmidt-  91  Schultz and Althaus, 1994). Given that P K C - a may play a role in O L process extension, it is interesting that C G - 4 OL appear to express slightly higher levels of P K C - a than primary OL. Since C G - 4 O L extend processes within hours of attachment to a substratum, while primary O L can take days or weeks to send processes, the higher expression of P K C - a in C G - 4 O L may partially explain the rapid ability of these cells to extend processes. Perhaps the most significant differences in P K C isoform expression were in the 5 and s isoforms (Fig. 18). P K C - 5 was expressed to a greater degree in primary O L than in any C G - 4 differentiation state. A putative function of P K C - 8 is inhibition of cell proliferation, as over-expression of P K C - 5 has been shown to inhibit proliferation of NIH 3T3 cells (Mischak et al., 1993). Since the astrocytic and bipotential forms of the C G - 4 cell line are highly proliferative, while primary OL are not, it seems reasonable that PKC-8 may play a role in the prevention of primary O L proliferation. This argument does not apply to C G - 4 OL, however, as these cells also exhibit diminished proliferative capacity. function of P K C - 8 is differentiation.  Another putative  Differentiation of murine erythroleukemia  cells has shown to be blocked by decreases in PKC-8 expression, and inhibition of PKC-8 has been shown to block neurite outgrowth in P C - 1 2 and H19-7 cells (Leng et al., 1993; Corbit et al., 1999).  In this case, the lower level of PKC-8  expression in C G - 4 O L could account for the generally lower level of differentiation attained by these cells in comparison to primary O L (Yim et al., 1995).  92  P K C - s , by contrast, shows lower expression in primary O L than in any of the C G - 4 differentiation states. This isoform could play on oncogenic role, as overexpression of  PKC-E  in rat colonic epithelial cells has been shown to cause  cell transformation (Perletti et al., 1996). Thus, the higher expression of P K C - s in CG-4 cell as compared to primary O L indicates that this isoform may contribute to the mutation of primary 0 2 A cells into the C G - 4 cell line. However, conflicting studies also indicate that  PKC-E  may play a role in differentiation, rather than  proliferation, as over-expression of this isoform has been shown to increase EGF-induced neurite outgrowth (Brodie et al., 1999). A s well as the M A P K and P K C families, the cyclin-dependent kinase (CDK) family demonstrated interesting kinase expression patterns. On the one hand, the expression of C D K 5 was higher in primary O L than in any of the C G - 4 differentiation states. On the other hand, expression of C D K 7 , especially in the bipotential and oligodendrocytic C G - 4 cells, was higher in the C G - 4 cell line than in primary O L (Fig. 19). The  C D K family of kinases is involved in cell cycle dynamics by  modulation of cell cycle regulatory protein known as cyclins. Specifically, C D K 7 has been implicated in the activation of cyclin A and cyclin B complexes, leading to cell division (Larochelle et al., 1998). With respect to its proliferative function, inhibitors of C D K 7 have been shown to inhibit tumour cell proliferation (Hajduch et al., 1999). C D K 7 has also been found to be moderately upregulated in tumour cells as compared to normal control cells (Bartkova et al., 1996). Although the CG-4 cell line is purported to be non-tumorigenic, the moderate upregulation of  93  C D K 7 in C G - 4 cells as compared to primary O L could be a function of the selfrenewal capacity of this cell line. C D K 5 , on the other hand, although it has been shown to associate with cyclins D1 and E, has not yet been shown to function as a kinase in these complexes (Xiong et al., 1992; Miyajima et al., 1995; Lee et al., 1997).  It has, however, been shown to phosphorylate neurofilaments and to  colocalize with actin and tubulin (Veeranna et al., 2000). These results imply that C D K 5 plays a role in cytoskeletal dynamics. A s primary O L are eventually able to produce more elaborate and intricate process extensions than C G - 4 , perhaps the upregulation of C D K 5 in these cells speaks to its role as a cytoskeletal modulator.  There also appears to be a C D K 5 bandshift in the bipotential and  oligodendrocytic C G - 4 cells (Fig. 19). This could represent phosphorylation/ activation of this kinase, and activation of C D K 5 has been shown to be involved in the outgrowth of neurites from neuronal ceils (Nikolic et al., 1998; Li et al., 2000; Zukerberg et al., 2000). Therefore, the seemingly endogenous activation of the C D K 5 in bipotential and oligodendrocytic C G - 4 could be one reason why these cells are able to extend processes within hours of attachment to a substratum. Finally,  there  was  higher  expression  of  F A K in  all three  CG-4  differentiation states than in primary O L (Fig. 16). Functions associated with F A K include cell adhesion and motility, as well as cell survival (Schlaepfer et al., 1999).  In terms of cell adhesion, plating of B A L B / c 3T3 fibroblasts onto a  fibronectin substrate has been shown to activate F A K (Hanks et al., 1992). Since both C G - 4 and primary O L can adhere to a poly-L-lysine/fibronectin substratum,  94  this perhaps explains why F A K expression is noted in both cell types. Another well-documented role for F A K is cell migration.  Over-expression of F A K in  Chinese hamster ovary cells has been shown to stimulate migration, and F A K deficient fibroblasts have been shown to demonstrate reduced migration (llic et al., 1995; Cary et al., 1996). Since in vivo studies have shown that bipotential C G - 4 cells have the ability to migrate, while mature primary O L do not, this could explain why F A K expression is higher in bipotential C G - 4 than in primary O L (Duncan, 1996). It does not, however, explain why the expression of this kinase should also be higher in astrocytic and oligodendrocytic C G - 4 cells. A possible explanation for the noted upregulation of F A K in all three C G - 4 differentiation states could be its tumorigenic effects.  Studies have shown that blocking F A K  expression in tumour cells can induce apoptosis, and elevated F A K levels have been found in a variety of tumours (Weiner et al., 1993; Owens et al., 1995; X u et al., 1996; Kornberg, 1998).  It is possible that the general increase in F A K  expression seen in the C G - 4 cell line is characteristic of its immortalization potential rather than its cell adhesion/motility functions.  95  CHAPTER 5: PROCESS EXTENSION IN CG-4  5.1 Introduction  After surveying the kinase expression profile of the CG-4 cell line as described in Chapter 4, experiments using CG-4 cells were undertaken to further the process extension experiments begun on primary OL. CG-4 cells begin to extend processes almost immediately after adherence to a substrate in serumfree medium, and thus do not require a phorbol ester stimulus.  A s well,  bipotential CG-4 cells maintained in 70/30 medium already demonstrate long, bipolar processes.  However, since switching CG-4 cells to an N1 medium  induces the formation of multipolar processes, the endpoint of these experiments was the expression of a multipolar phenotype. A multipolar phenotype is defined in this thesis as one in which the CG-4 cells exhibit upwards of four extensions that form a network of branches encircling the cell body. Since inhibition of P K C can lead to an inhibition of phorbol ester-induced process extension in primary OL, the Ro-32 P K C inhibitor was initially applied to CG-4 cells in an attempt to block multipolar process extensions. Subsequent experiments were undertaken to determine the effects of MEK1/2 inhibition and P K C activation on the formation of multipolar processes in the CG-4 cell line.  96  5.2 Results  5.2.1 P K C Inhibitor Studies  CG-4 cells were maintained in 70/30 medium, then switched to N1 medium containing the P K C inhibitor Ro-32.  Concentrations of Ro-32 ranging  from 1 to 20 u.M were applied, and concentrations of 5-10 u.M were able to prevent the formation of multipolar processes (Fig. 20). A concentration of 20 u.M Ro-32 was found to be cytotoxic. Western blots were performed to determine if CG-4 cells cultured in N1 medium  express  an  endogenous  level  of  phosphorylated  ERK1/2.  Phosphorylation of ERK1/2 was used as an indication of increased ERK1/2 activation.  Western blots were also used to determine if inhibition of any  endogenous E R K 1 / 2 activity by Ro-32 is responsible for the inhibition of process formation observed in Figure 20. The Western blots indicated that CG-4 cells in N1 medium with or without Ro-32 did not express the phosphorylated and more active forms of E R K 1 / 2 .  CG-4 cells in 70/30 medium, however, did express  marked levels of the phosphorylated and bandshifted forms of ERK1/2 (Fig 21). The bandshift in ERK1 was particularity evident, whereas the phosphorylated E R K 2 co-migrated very close to the dephosphorylated E R K 1 . These blots also indicated that the phosphorylation of ERK1/2 seen in bipotential CG-4 cells is transient, as the phosphorylated ERK1/2 isoforms were nearly undetectable 1-2 hr after the application of fresh 70/30 medium. To assess if the transient ERK1/2  97  98  99  activation provided by the addition of fresh 70/30 medium every 48 hr prevents CG-4 cells from developing a multipolar morphology, CG-4 cells were cultured in 70/30 medium for 4 days with no medium change. With a lack of fresh 70/30 medium, these cells did indeed begin to form multipolar process extensions (Fig. 22). The next experiments were undertaken in order to clarify the effects of Ro32 and ERK1/2 activation in CG-4 cells.  First, bipotential CG-4 cells were  switched to fresh 70/30 medium containing 5 u.M Ro-32 in an attempt to block ERK1/2 activation. Second, the cells were lysed after 30 min of Ro-32 treatment and subjected to Western blotting. It was found that Ro-32 had no effect on the transient ERK1/2 activation noted in bipotential CG-4 cells, even when these cells were pre-incubated with the inhibitor prior to the medium change (Fig. 23). Not surprisingly, therefore, a parallel experiment showed that Ro-32 had no morphological effects on CG-4 cells in 70/30 medium. Since Ro-32 is a P K C inhibitor, CG-4 cells in both 70/30 and N1 media were next assessed for any endogenous P K C - a activity.  CG-4 cells were  switched to either fresh 70/30 or N1 medium for 15 min, then lysed. The lysates were separated into cytosolic and membrane fractions and subjected to Western blotting.  The activity of P K C - a was assessed by observing any translocation  from cytosolic to membrane fractions. Although it is possible that some of the P K C - a in the cytosolic fraction could be active, translocation of P K C - a to the membrane fraction commonly signifies activation of this kinase. P K C - a was not detected in the membrane fractions of CG-4 cells in either 70/30 or N1 medium.  100  Figure 22: Morphological Changes of CG-4 Cells CG-4 cells in the left panel had 70/30 medium replaced every 48 hr for 4 days.  C G - 4 cells in the right panel remained in the same 70/30  medium for 4 days. Note that, in CG-4 cells which did not receive fresh 70/30 medium every 48 hr, multipolar processes began to form. Photographs were taken using a phase contrast microscope and are representative of 3 experiments. Bar = 20 pm  101  pERK1 45  ERK1 pERK2  kDa  ERK2  36 kDa  Figure 23: The Effects of Ro-32 on ERK1/2 Activation in CG-4 Cells CG-4 cells were lysed 30 min after various treatments. 1 = CG-4 cells exposed to 70/30 medium 2 = CG-4 cells exposed to 70/30 medium + 5 u M Ro-32 3 = CG-4 cells exposed to N1 medium 4 = CG-4 cells pretreated with 5 uM Ro-32 for 20 min prior to exposure to 70/30 medium + 5 uM Ro-32 Note the ERK1/2 bandshift in all 70/30-treated samples, regardless of the addition of Ro-32.  The inability of Ro-32 to inhibit E R K 1 / 2 activation in  duplicate experiments indicates that the E R K 1 / 2 activation seen in 70/30 treated cells is not necessarily linked to P K C activation.  102  In confirmation of the multi-kinase blots conducted in Chapter 4, however, P K C - a was detected in the cytosolic fractions (Fig. 24). A parallel blot also confirmed that phosphorylation of ERK1/2 can occur independently of P K C - a activity in these cells (Fig. 24). To  determine  phosphorylated Kinetworks™  if  between  kinases  other  than  C G - 4 cells in either  ERK1/2 70/30  or  are N1  differentially medium,  a  phosphokinase profile was performed 20 min after medium  change. Since it is unclear why Ro-32 prevented the expression of a multipolar phenotype in C G - 4 fed with N1 medium + 5 u.M Ro-32, these Ro-32 treated cells were also profiled.  The multi-kinase blot did not reveal significant differences  between C G - 4 cells in either N1 or 70/30 medium, other than the increased ERK1/2 phosphorylation previously noted in Figure 21 (Table 4). In terms of Ro32 treatment, preliminary results from the Kinetworks™ phosphoprotein screen indicated a decrease of STAT3 phosphorylation in C G - 4 cells treated with N1 medium + 5 u.M Ro-32 as compared to C G - 4 cells in either N1 or 70/30 medium alone (Table 4).  103  N1  70/30  97 kDa 66 kDa  45 kDa  PKC-a  r pERK1 ERK1 pERK2 ERK2  Figure 24: The Effects of 70/30 and N1 on PKC-a Activity in CG-4 Cells C G - 4 cells were maintained in 70/30 medium for at least 48 hr prior to medium change. Cells were lysed 15 min after medium change. The blots on the left correspond to cells fed with N1 medium, while the blots on the right correspond to cells fed with 70/30 medium.  P K C - a activation was  assessed by separating the lysates into cytosolic (C) and membrane (M) fractions. ERK1/2 activation was assessed by observing bandshifts. Note that P K C - a did not appear to be activated by either N1 or 70/30 medium, as the kinase is not detected in the membrane fractions. Furthermore, the ERK1/2 bandshift noted in cells fed with 70/30 medium occurred independently of P K C - a translocation. This data is representative of three experiments. 104  Phosphoprotein  N1 + Ro-32  NR1 Adducin ERK1 PKB/Akt GSK3 a/p Protein kinase C s Protein kinase C a Protein kinase C a / p Src STAT3  8 14 24 6 18 10 21 17 12 14  N1  70/30  9 13 19 6 14 8 20 21 13 24  11 16 57 9 14 11 18 22 10 22  Table 4: Kinetworks™ Phosphoprotein Analysis The phosphoproteins listed above are part of the phosphoprotein screen conducted by Kinexus. The numerical values represent the relative quantity (%) of the E C L signals from individual bands within each lane.  C G - 4 cells were  maintained in 70/30 medium for at least 48 hr prior to treatment, and then lysed for analysis 20 min after treatment. N1 + Ro-32 = C G - 4 treated with N1 medium containing 5 uJv1 Ro-32 N1 = C G - 4 treated with N1 medium 70/30 = C G - 4 treated with fresh 70/30 medium Note the approximately 3-fold increase in the relative quantity of phosphorylated ERK1 in 70/30 samples as compared to N1 samples.  These was also an  approximately 1.7-fold increase in the relative quantity of phosphorylated STAT3 in N1 samples as compared to N1 + Ro-32 samples.  105  5.2.2. M E K Inhibitor Studies  Since all the results described above implicate E R K 1 / 2 activation in the bipolar nature of CG-4, these cells were next treated with MEK1/2 inhibitors. PD 98059 and UO-126 were employed in an attempt to inhibit ERK1/2 and to induce the expression of a multipolar phenotype.  However, morphological monitoring  showed that inhibitor concentrations ranging from 12.5 to 50 ulvl did not affect the bipolar CG-4 cell phenotype, while 100 uM was cytotoxic (Fig. 25).  Even  preincubation of the cells in either inhibitor for 20 min prior to treatment had no morphological effect. Next, Western blots were performed to determine the level of phosphorylated and active ERK1/2 in these cells.  The blots showed that,  while both 50 uM P D 98059 and 50 uM UO-126 were able to reduce ERK1/2 phosphorylation, neither inhibitor could completely abolish their phosphorylation (Fig. 26).  To assess if a higher M E K inhibitor concentration could abolish  ERK1/2 phosphorylation in CG-4, Western blots using 100 uM PD 98059 were conducted. These blots confirmed that ERK1/2 inhibition via MEK1 inhibition is possible in these cells (Fig. 27).  5.2.3. Phorbol Ester Studies  To test the hypothesis that activation of ERK1/2 appears to prevent the formation of multipolar processes in CG-4 cells, the phorbol ester P M A was employed in an attempt to phosphorylate ERK1/2 via activation of P K C - a . CG-4  106  Figure 25: The Effects of PD 98059 and UO-126 on CG-4 Cell Morphology C G - 4 cells were maintained in 70/30 medium for at least 48 hr prior to treatment.  Photographs were taken using a phase contrast microscope  24 hr after treatment. A = C G - 4 in 70/30 medium B = C G - 4 in N1 medium C = C G - 4 in 70/30 medium + 50 u M PD 98059 D = C G - 4 in 70/30 medium + 50 uM UO-126 Note that neither MEK1/2 inhibitor induced C G - 4 cells to adopt the oligodendrocytic, multipolar phenotype observed in panel B. Results are representative of 4 experiments. Bar = 20 urn 107  45 kDa  * = L  r  45 kDa  pERK1 ERK1 ERK2  pERK1 ERK1 pERK2 ERK2  1  Figure 26: The Effects of PD 98059 and UO-126 on ERK1/2 Activation in CG-4 Cells CG-4 cells were exposed to the MEK1/2 inhibitors P D 98059 and UO-126 during the change to fresh 70/30 medium. ERK1/2 bandshifts were observed in order to assess if the inhibitors could block the ERK1/2 activation associated with the addition of fresh 70/30 medium. Treatments were 30 min in length. 1 = 70/30 medium  i  6 = N1 medium  2 = 70/30 medium + 25 u M UO-126  7 = CG-4 pretreated with 50 uM P D 98059  3 = 70/30 medium + 50 uM UO-126  for 20 min prior to treatment with  4 = 70/30 medium + 25 uM P D 98059  70/30 medium + 50 \iM P D 98059  5 = 70/30 medium + 50 uM P D 98059 In the upper panel, note that there was no marked reduction of phosphorylated ERK1 in MEK1/2 inhibitor-treated samples.  Phosphorylated E R K 2 comigrated near the  dephosphorylated E R K 1 . In the lower panel, note that even pretreatment of the cells with P D 98059 did not decrease ERK1/2 phosphorylation to the level seen in N1 treated cells. This data is representative of duplicate experiments. 108  p E R K 1  45 k D a _  t< —  E R K 1 p E R K 2 E R K 2  1  2  3  Figure 27: The Effects of PD 98059 on ERK1/2 Activation in CG-4 Cells CG-4 cells were treated with a high dose of the M E K 1 inhibitor P D 98059 to determine if the inhibitor has the ability to inhibit ERK1/2 phosphorylation. 1 = CG-4 cells in fresh 70/30 medium for 30 min 2 = CG-4 cells pretreated with 100 uM PD 98059 for 20 min prior to treatment with 100 uM P D 98059 in fresh 70/30 medium for 30 min more. 3 = CG-4 cells in N1 medium for 30 min This high dose of the P D 98059 abolished the E R K 1 / 2  phosphorylation  associated with the addition of fresh 70/30 medium in duplicate experiments.  109  cells in N1 medium were exposed to P M A concentrations ranging from 1 to 40 nM P M A , and cell analysis was carried out 24-48 hr later. A P M A concentration of 20 nM was the lowest concentration sufficient to completely abolish the formation of a multipolar phenotype over a 24 hr period. After 48 hr, multipolar processes began to form even in PMA-treated cells (Fig. 28). Next, cells were treated with 20 nM P M A in N1 medium for 15 min prior to lysis and Western blotting. The lysates were separated into cytosolic and membrane fractions. The Western blots indicated a significant translocation of PKC-a, a very small translocation of P K C - p , and the appearance of activated ERK1/2 (Fig. 29). To further monitor the relationship between PKC-a translocation and E R K activation, a dose response using a concentration range of 1 nM to 20 nM P M A was conducted.  After exposure to P M A for 15 min, C G - 4 cells were lysed,  fractionated, and Western blotted.  The Western blots indicated that P K C  translocation is dose dependent, and that noticeable ERK1/2 phosphorylation only occurs with the PKC-a translocation seen at the 20 nM P M A dose (Fig. 30). Therefore, 20 nM P M A was the lowest dose able to both completely block the formation  of  multipolar  processes  and  to  markedly  increase  ERK1/2  phosphorylation.  110  111  PKC-a  PKC-P  97 kDa 66 kDa  45 kDa  pERK1 ERK1 pERK2 ERK2  Figure 29: The Activation of PKC and ERK1/2 in CG-4 Cells Treated with PMA C G - 4 cells were maintained in 70/30 medium for at least 48 hr prior to feeding with N1 medium alone or N1 medium containing 20 nM P M A . Cells were lysed 15 min after treatment. 1 = Cytosolic fraction, N1 treatment 2 = Membrane fraction, N1 treatment 3 = Cytosolic fraction, N1 + P M A treatment 4 = Membrane fraction, N1 + P M A treatment Note that the translocation of P K C - a was much greater than the translocation of PKC-P after P M A treatment. Furthermore, this translocation corresponded to an activation of ERK1/2. These results are representative of 3 experiments.  112  113  5.3 Discussion  5.3.1 P K C Inhibitor Studies  These C G - 4 experiments were undertaken to first confirm the role of ERK1/2 in OL process extension, and to then expand the knowledge base on signal transduction events involved in O L process extension.  Since P K C  inhibitors have been shown to inhibit phorbol ester-induced process extension in primary OL, the P K C inhibitor Ro-32 was applied to C G - 4 cells in an attempt to inhibit the formation of multipolar processes. Switching C G - 4 cells from 70/30 medium, which contains B-104 mitogens, to N1 medium, which does not contain these mitogens, induces the formation of multipolar processes in C G - 4 (Fig. 13). Therefore, studies were undertaken to determine whether including Ro-32 in the N1 medium could inhibit this process formation. A s can be seen in Figure 20, C G - 4 treated with Ro-32 in N1 medium did indeed demonstrate inhibition of multipolar process formation. Working with the hypothesis that ERK1/2 activation is a crucial component in the induction of O L process extension, it was hypothesized that C G - 4 cells in N1 medium may express an endogenous level of ERK1/2 activation. In such a scenario, the treatment of these cells with Ro-32 might have inhibited this activation and thereby inhibited process formation.  However, the Western blot  depicted in Figure 21 shows that C G - 4 cells in N1 medium do not contain endogenously active ERK1/2, and therefore Ro-32 does not affect the level of  114  ERK1/2 activity in these cells. Instead, C G - 4 cells in 70/30 medium were found to show a transient activation of E R K 1 / 2 up to 1 hr after the application of fresh medium. ERK1/2 activation was subsequently hypothesized to inhibit multipolar process formation rather than to induce process formation in C G - 4 cells. To test this hypothesis, C G - 4 cells were left in 70/30 medium for 4 days with no medium change. The medium is normally replaced every 48 hr in order to maintain C G - 4 cells in their bipotential state, and thus bipotential C G - 4 cells are subject to transient ERK1/2 activation every 48 hr. A s can be seen in Figure 22, without the application of fresh 70/30 medium, C G - 4 cells began to develop multipolar processes. This result implies that transient but repetitive ERK1/2 activation is required in order to prevent multipolar process formation in the C G - 4 cell line. The  lack of ERK1/2 activation in C G - 4 cells switched to N1 medium  negates the possibility of ERK1/2 inhibition by Ro-32 in these cells, but it does not rule out the possibility of P K C inhibition.  Therefore, C G - 4 cells were  assessed for P K C - a translocation 15 min after change to either N1 or 70/30 medium.  P K C - a was the isoform chosen for assessment as it is one of the  conventional P K C isoforms, and Ro-32 is a conventional P K C inhibitor (Wilkinson et al., 1993). Furthermore, P K C - a rather than P K C - p is the isoform implicated in primary O L process extension, while P K C - y is virtually undetectable in these cells.  The blots indicate, however, that P K C - a does not translocate to the  membrane fraction in these cells, and is therefore not likely a potential candidate for Ro-32 inhibition.  115  The Western blots in Figure 23 also show that ERK1/2 phosphorylation is independent of P K C - a activation in C G - 4 cells fed with 70/30 medium. Since 70/30 medium, by definition, contains 30% B-104 mitogens, and since N1 medium does not contain these mitogens, the B-104 conditioning must be responsible for this observed ERK1/2 phosphorylation.  The most credible  scenario involves phosphorylation and activation of E R K 1 / 2 via activation of protein tyrosine kinase receptors, leading to the sequential activation of Ras, Raf1, MEK1/2, and E R K 1 / 2 .  It has been previously determined that the active  components of B-104 conditioned medium are b F G F and P D G F (Louis et al., 1992). Therefore, these two growth factors are the best candidates for activation of protein tyrosine kinase receptors leading to ERK1/2 activation in bipotential C G - 4 cells. Speculating that the ERK1/2 activation cascade in C G - 4 cells involves b F G F and P D G F activation of tyrosine kinase receptors, as opposed to activation of P K C - a , it becomes clear why Ro-32 had no effect on ERK1/2 phosphorylation in these cells.  However, the fact remains that Ro-32 was able to inhibit the  formation of processes of C G - 4 cells in N1 medium.  Therefore, a multi-  phosphoprotein immunoblot conducted by Kinexus Bioinformatics Corporation was used to survey potential candidates for Ro-32 inhibition in these cells. Since the I C  50  of Ro-32 is 9 nM for P K C - a and 28 nM for P K C - p , while the minimum  concentration required for abolition of process formation in C G - 4 cells is 5 uJM, it is possible that this inhibitor was demonstrating non-specific effects in the C G - 4 cell line. The phosphorylated proteins surveyed included P K C - a , p, and e, but  116  these kinases showed no decrease in phosphorylation with the  inhibitor.  However, the survey did indicate that S T A T 3 may be non-specifically inhibited by Ro-32. Since S T A T 3 has been shown to play a role in the differentiation of P C 12 cells, inhibition of this kinase by Ro-32 could explain why CG-4 cells in N1 medium were prevented from differentiating into a multipolar phenotype (Wu and Bradshaw, 2000). Further experiments are necessary to confirm any significant inhibition of S T A T 3 by Ro-32.  5.3.2 M E K inhibitor studies  Once it became evident that the transient ERK1/2 activation associated with the application of fresh 70/30 medium could be involved in the inhibition of process formation in CG-4 cells, M E K inhibitors were applied in an attempt to block this activation and to induce process formation.  Surprisingly, neither PD  98059 nor UO-126 was able to induce process formation at non-toxic doses. Since Western blots indicate, however, that these inhibitors were unable to abolish the phosphorylation of ERK1/2 at these doses, it is still possible that ERK1/2 activation inhibits process formation in CG-4 cells. The concept of a threshold of activation has already been seen with respect to ERK1/2 in primary OL, and may again play a role in the CG-4 cell line.  In such a scenario, it is  possible that the MEK1/2 inhibitors were unable to drop the level of activated ERK1/2 below the threshold necessary to remove its inhibitory effects on process formation. It is unclear why the MEK1/2 inhibitors were unable to abolish ERK1/2  117  activity in these cells. One possibility is that the inhibitor dose was simply not high enough, while another possibility is that ERK1/2 is downstream of a kinase other than MEK1/2 in C G - 4 cells. To address these possibilities, a higher dose of P D 98059 was applied to the cells. Since 100 LIM P D 98059 was able to abolish  ERK1/2  phosphorylation,  this  indicates  that  ERK1/2  is  indeed  downstream of MEK1/2 in the C G - 4 cell line. However, it does not rule out the possibility that other pathways could also lead to ERK1/2 activation in these cells.  5.3.3 Phorbol Ester Studies  After  attempting  to  induce process formation  by  blocking  ERK1/2  activation in C G - 4 cells fed with 70/30 medium, P M A was used in an attempt to block process formation by inducing E R K activation in C G - 4 cells fed with N1 medium.  Since 20 nM P M A in N1 medium could inhibit the formation of a  multipolar phenotype over a 24 hr period, while at the same time leading to ERK1/2 phosphorylation, these results support a role for E R K 1 / 2 activation in the inhibition of process formation.  Furthermore, the ability of C G - 4 cells in N1  medium + 20 nM P M A to form multipolar processes after 48 hr strengthens the argument that repetitive transient activation of ERK1/2 is required to inhibit multipolar extensions.  118  CHAPTER 6: SUMMARY AND FUTURE DIRECTIONS  6.1 The Role of E R K s 1 and 2 in Primary O L Process Extension  The first objective of this thesis was to use a primary O L model to determine whether or not E R K s 1 and 2 play a role in process extension.  In  these experiments, bovine brains were used as a source of OL. Bovine brains were chosen as they are able to provide a large quantity of cells, and the basal rate of bovine O L process extension is very slow. From previous studies it has already been determined that phorbol esters can  induce  primary  OL  process extension.  Experiments  were  therefore  undertaken to assess if the MEK1 inhibitor P D 98059 could prevent this phorbol ester-induced response.  It was found that P D 98059 could inhibit process  extension, and could also inhibit the increase in ERK1/2 activity observed after P M A treatment.  The inhibitor did not, however, completely abolish ERK1/2  activity, leading to speculation that a threshold of ERK1/2 activity is required for OL to commit to process extension. There are other instances where the concept of a threshold of ERK1/2 activity may affect the functional biology of OL. First, in studies using NT-3 and N G F to induce progenitor O L proliferation, it was found that both compounds could lead to ERK1/2 phosphorylation.  However, only NT-3 could stimulate  progenitor O L proliferation (Cohen et al., 1996). Upon closer examination, it was shown that ERK1/2 was phosphorylated to a greater extent upon treatment of  119  cells with NT-3 than with N G F . It is therefore possible that N G F did not cause ERK1/2 activation to cross the threshold necessary to induce proliferation. Second, in the C G - 4 studies described in Chapter 5, inhibition of ERK1/2 phosphorylation was unable to induce morphological changes.  Once again,  however, this inhibition was shown to be minimal, allowing for the possibility that ERK1/2  activation  was  not  dropped  below  the  threshold  required  for  morphological change. From these results it may be concluded that OL do not commit to major proliferative or morphological responses unless significant kinase activity changes are affected. A s well as causing activation of ERK1/2, stimulation of primary O L with P M A has been shown to cause movement of ERK1/2 into the nucleus and processes of O L (Stariha et al., 1997).  The potential substrates of ERK1/2  activation during O L process extension are numerous; in the cytoplasm and processes, the most likely candidates are M B P and stathmin.  In the nucleus,  activated ERK1/2 most likely leads to induction of the c-fos gene. Perhaps the most probable ERK1/2 substrate is M B P , as there is already circumstantial evidence linking ERK1/2 activation to M B P phosphorylation.  First, our studies  have shown that PMA-stimulation of O L causes ERK1/2 activation and process extension (Stariha et al., 1997). Second, other studies have demonstrated that PMA-stimulation of O L causes M B P phosphorylation (Vartanian et al., 1986). Since M B P is a commonly used in vitro substrate of E R K , there is a potential in vivo link between ERK1/2 phosphorylation, M B P phosphorylation, and process extension in OL. Phosphorylation of M B P during O L process extension could be  120  a preparatory step for myelination. However, it is possible that one or more other ERK1/2 substrates are also phosphorylated during O L process extension. Other such substrates, including stathmin, may play a more integral role than M B P in the formation of the processes themselves. There  is also evidence that the  ERK1 and  ERK2  isoforms  are  developmentally regulated in OL. For instance, E R K 2 has been shown to play a role in the proliferation of progenitor O L (Kumar, 1998). Conversely, ERK1 has been shown to play a role in the proliferation of a subset of mature O L (Althaus, 1997). While our studies indicate that ERK1 is the dominant isoform in primary OL, the separate contributions of ERK1 and E R K 2 to process extension were not determined.  To examine this question, studies could be undertaken  to  individually immunoprecipitate ERK1 and E R K 2 from O L and compare their activities before and after stimulation and inhibition of process extension. Finally, our preliminary results indicate that p38, J N K , P K A , PI-3-kinase and calcium-calmodulin-dependent kinase II do not play a role in OL process extension.  However, future studies are needed to verify and expand these  results, as it is likely that kinases other than ERK1/2 and P K C - a are involved in the process extension phenomenon.  121  6.2 A Comparison of the Kinase Expression Profile Between Primary Rat OL and CG-4  The second objective of this thesis was to evaluate the usefulness of the CG-4 cell line as a model for O L signal transduction studies. A self-renewing O L model would greatly advance the knowledge of OL signal transduction, since primary mature O L are post-mitotic and therefore of limited quantity. Also, a cell line could overcome the inherent variability associated with primary cultures. To evaluate the CG-4 cell line for use in OL signal transduction studies, a multi-kinase  expression profile  Corporation.  Any interesting results were then further confirmed via Western  blotting.  was  conducted  by  Kinexus Bioinformatics  In general, there were few differences between the kinase expression  profiles of primary O L and CG-4 OL. The main areas of difference were in the expression of Pak-a, which was higher in primary O L than in CG-4 cells, and the expression of FAK, which was lower in primary O L than in CG-4 cells. There were also differences in the expression of PKC-5 and -s, as well as in the expression of C D K s 5 and 7. The putative functions of P a k - a vary widely. For instance, this kinase has been shown to activate J N K and p38 pathways, potentially leading to apoptosis. However, it has also been shown to act in an anti-apoptotic manner via phosphorylation of Bad (Schurmann et al., 2 0 0 0 ) . Pak isoforms have also been shown to both induce neurite extension and to cause retraction of the cell periphery (Manser et al., 1997; Daniels et al., 1998). Therefore, the significance  122  of increased Pak-a expression in primary O L is unclear. One possible scenario evolves after examining the expression of Pak-a separately from the activation of Pak-a.  Expression of Pak1 has been implicated  in the  above-mentioned  induction of neurite extension, while activation of Pak-a has been implicated in the retraction of the cell periphery.  It is therefore possible that the expression of  Pak-a in primary OL, as opposed to the activation of Pak-a in these cells, may explain why primary O L can form more intricate networks of processes than C G -  4 0L. In contrast to Pak-a, F A K expression is higher in CG-4 cells than in primary OL.  The most likely explanation for this increased expression is that  F A K is involved in the self-renewal capacity of the CG-4 cell line.  FAK  expression has been found to upregulated in a variety of tumours, and blocking F A K expression in tumour cells can induce apoptosis (Owens et al., 1995; X u et al., 1996). While the CG-4 cell line is not considered to be a tumour cell line, it is possible that this kinase contributes to the spontaneous mutation of primary progenitor  O L into immortalized  self-renewing  CG-4 cells.  The increased  expression of P K C - s in CG-cells as compared to primary O L may also play a similar role, since overexpression of P K C - s has been shown to be involved in cell immortalization (Perletti et al., 1996). A s opposed to PKC-s, PKC-8 expression is higher in primary O L than in CG-4 cells. PKC-8 has been implicated in both proliferation and differentiation, and it is unclear which of these roles it assumes in OL. A role for PKC-8 in O L differentiation could explain why this kinase is highly expressed in primary cells,  123  as primary O L are able to achieve a higher degree of differentiation than C G - 4 O L ( Y i m etal., 1995). Finally, the expression of C D K 5 was higher in primary O L than in C G - 4 cells. Conversely, the expression of C D K 7 was lower in primary O L than in C G - 4 cells.  Since C D K 7 has been found to be upregulated in tumour cells, it could  share a role with F A K and P K C - s in the mutation of primary O L into the selfrenewing C G - 4 cell line. A s opposed to C D K 7 , C D K 5 has been shown to play a role in cytoskeletal dynamics. Along with Pak-a, increased expression of this kinase in primary O L could explain why these cells form more intricate processes than C G - 4 OL. Overall, the differences in kinase expression between primary O L and C G 4 OL were few. Many major kinases, such as conventional P K C s , members of the M A P K family, P K A , and P K B , were all similarly expressed. Therefore, the C G - 4 cell line appears to make a suitable model for O L signal transduction studies.  6.3 Process Extension in C G - 4  After noting the similarities in the kinase expression patterns of C G - 4 cells and primary OL, particularly with respect to the M A P K pathway, it was surprising to discover that E R K s 1 and 2 do not appear to play a role in the induction of mulitpolar process extensions from C G - 4 cells. Rather, in contrast to their role in  124  primary OL, E R K s 1 and 2 appear to inhibit multipolar process formation in C G - 4 cells. First, transient ERK1/2 activation was noted in C G - 4 cells fed with 70/30 medium, but not in C G - 4 cells fed with N1 medium.  However, C G - 4 cells fed  with 70/30 medium maintain a relatively bipolar phenotype, while C G - 4 cells fed with N1 medium acquire a multipolar phenotype.  Therefore, the  ERK1/2  activation associated with 70/30 medium appears to prevent the expression of a multipolar phenotype. Further evidence of this was provided when C G - 4 cells in 70/30 medium did not have their medium renewed for 4 days. These cells were able to develop a multipolar phenotype, presumably due a lack of fresh 70/30 medium leading to a lack of transient ERK1/2 activation.  It is thought that the  active components in 70/30 are the b F G F and P D G F mitogens contained in the 30% B-104 conditioning. These two growth factors, therefore, are likely leading to ERK1/2 phosphorylation in C G - 4 cells fed with 70/30 medium. Second, C G - 4 cells fed with 70/30 medium were exposed to two different MEK1/2 inhibitors in an attempt to inhibit transient ERK1/2 activation and induce the formation of a multipolar phenotype. However, although the inhibitors were able to reduce ERK1/2 activation at non-toxic doses, they were unable to induce a multipolar phenotype.  These results do not necessarily negate a role for  ERK1/2 activation in the inhibition of multipolar process extensions, since the inhibitors were able to reduce but not to abolish ERK1/2 activity. A s mentioned above, the ERK1/2 activity after inhibitor treatment could still be above the threshold required to keep the C G - 4 cells in their bipolar state.  125  Finally, since N1 medium does not contain b F G F and P D G F , N1 medium alone does not cause ERK1/2 activation. Therefore, C G - 4 cells in N1 medium are normally able to form a multipolar phenotype. PMA-induced activation of ERK1/2 in C G - 4 fed with N1, however, was able to prevent the expression of a multipolar phenotype. This result supports a role for ERK1/2 activation in the inhibition of multipolar process extensions in C G - 4 cells.  6.4 Conclusions  The results presented in Chapter 3 of this thesis provide evidence for a role for ERK1/2 activation in the induction of process extensions from primary OL.  The results presented in Chapter 4 of this thesis then illustrate that the  kinase expression profiles of primary O L and C G - 4 OL are similar, implying that the C G - 4 cell line is a useful model for O L signal transduction studies. Then, in Chapter 5, the signal transduction results on the role of ERK1/2 in C G - 4 OL process extension were found to potentially contradict the results obtained from primary OL. While at first glance this seems to negate the usefulness of the C G 4 model for O L signal transduction studies, this is not necessarily the case. Due to the nature of primary O L as compared to C G - 4 OL, there were differences in the experimental protocols used for these two cell types.  Therefore, direct  comparisons should not be drawn without careful consideration. For instance, the primary bovine O L used in the experiments shown in Chapter 3 were mature cells that had lost their processes during the primary  126  culture  procedure.  These cells take weeks to  re-grow their processes  endogenously, and therefore phorbol esters were used to stimulate process extension and ERK1/2 activation. The endpoint was an observed extension of processes from phorbol ester-stimulated mature OL, as compared to a complete lack of processes in control OL. Such an endpoint is not possible with C G - 4 , as these cells endogenously extend processes within hours of  differentiation  towards mature OL-like cells. By the time C G - 4 cells have fully differentiated into mature C G - 4 OL, they already demonstrate a multipolar phenotype. Therefore, a distinction has to be made between "process extension" in primary O L and a "multipolar phenotype" in C G - 4 OL. "Process extension" in primary O L is defined as the appearance of processes extending from the cell body, as opposed to a complete lack of processes. A "multipolar phenotype" in C G - 4 cells is defined as a network of branched processes surrounding the cell body, as opposed to 2-4 unbranched processes that do not completely encircle the cell body. One way to mimic the primary O L experiments as closely as possible would be to initially differentiate the C G - 4 cells into C G - 4 O L by the application of N1 medium. These C G - 4 O L could then be replated, as most processes are lost during the replating procedure, and the effects of various kinase stimulators and inhibitors on the re-growth of processes could be monitored over the next few hours.  This type of experiment was attempted; however, the survival and  general well-being of the C G - 4 O L were adversely affected by the replating procedure. Since bipotential C G - 4 cells can be easily replated, a second type of experiment was devised.  127  In this second type of experiment, C G - 4 cells were maintained as bipotential cells in 70/30 medium prior to any treatments. This is a significant departure from primary OL, which were maintained as mature cells prior to any treatments. However, the advantage of such an experimental set-up is that the change from the bipolar phenotype of C G - 4 cells in 70/30 medium to the multipolar phenotype of C G - 4 cells in N1 medium can be easily monitored. Various kinase stimulators and inhibitors were therefore employed in an attempt to either stimulate or inhibit the induction of a multipolar phenotype. In this thesis, results obtained from the C G - 4 cell line provide strong evidence to support a role for ERK1/2 activation in the inhibition of a multipolar phenotype in C G - 4 cells.  In addition, results obtained from primary OL  experiments evidence a role for ERK1/2 activation in the formation of process extensions from mature OL. Two potential conclusions can be drawn from the combined results of these C G - 4 cell line and primary O L culture experiments: 1 - The first conclusion is that the C G - 4 cell line is not a useful model for O L signal transduction studies.  This conclusion is supported by the induction of  process formation via ERK1/2 activation in primary OL, and the inhibition of process formation via ERK1/2 activation in C G - 4 cells. 2 - The second conclusion is that E R K s 1 and 2 play a role in the induction of OL process extension, but only if the OL have fully matured.  This conclusion is  supported by studies of P K C in OL, since P K C activation has been shown to both prevent differentiation in immature O L and to induce process extensions in  128  mature OL. These P K C results demonstrate that the effects of kinase activation in O L can be dependent on the differentiation level of the cell.  In this second  conclusion, therefore, the CG-4 cell line is not necessarily negated as an appropriate model for O L signal transduction studies. Future experiments using primary 0 2 A cultures to outline the role of ERK1/2 in  process extension during  differentiation  would  help to  elucidate  any  differentiation-dependent effects of ERK1/2 in OL. For instance, the effects of ERK1/2 activation on the differentiation of primary 0 2 A cells into mature OL, as well as the effects of ERK1/2 activation on the differentiation of bipotential CG-4 cells into CG-4 OL, could be assessed using developmental marker expression. If ERK1/2 activation is shown to prevent the appearance of mature O L markers in both the primary model and CG-4 model, then the CG-4 cell line may still be a useful model for O L signal transduction studies.  In such a scenario, the  previously noted inhibition of a multipolar phenotype in CG-4 cells via ERK1/2 activation could be attributed to an inhibition of differentiation rather than a direct inhibition of process formation. It is of paramount importance that the usefulness of the CG-4 cell line as an OL model be addressed. Due to the large number of OL-like cells that can be obtained from this cell line, it has rapidly become the model of choice for studies on OL functional biology. Since the results of this thesis raise the possibility that some protein kinase signalling cascades may function differently in CG-4 OL than in primary OL, care should be taken before applying any results obtained from the CG-4 cell line directly to primary OL. In terms of signal transduction  129  studies, the functional effects of any signalling pathways should first be determined in both primary O L and CG-4 O L cultures.  If the functional results  are the same in both models, then the unlimited supply of cells afforded by the CG-4 model could be employed to further define such signalling pathways. Once the pathways have been well-defined in CG-4 OL, the results should again be verified in primary OL. In conclusion, therefore, this thesis underlines the importance of exercising caution when using the CG-4 cell line for O L studies.  To truly eludicate the  signal transduction pathways involved in O L functional biology, an approach that combines both the CG-4 and the primary O L culture models is the best option.  130  REFERENCES  1.  Althaus, H. H., Hempel, R., Kloppner, S., Engel, J . , Schmidt-Schultz, T., Kruska, L., and Heumann, R. Nerve growth factor signal transduction in mature pig oligodendrocytes. Journal of Neuroscience  Research;  1997:  50(5):729-42 2.  Althaus, H. H., Kloppner, S., Schmidt-Schultz, T., and Schwartz, P. 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Neuron; 2000: 26(3):633-46  167  I  168  Appendix A: Densitometric Analysis of Rat O L Blot, Part 1  ane  Band  1  1 2 3 4 5 1 5 1 2 3 4 1 2 6 7 2 5 2 2 2 3 3 7 1 3 2 3 2 5 2 3 3  1 1 1 1 2 2 3 3 3 4 5 6 6 6 8 8 9 10 11 11 13 13 14 14 15 16 18 18 19 19 20  RF 0.183 0.304 0.496 0.739 0.917 0.239 0.522 0.391 0.552 0.63 0.63 0.204 0.3 0.648 0.822 0.261 0.661 0.639 0.313 0.509 0.557 0.33 0.583 0.387 0.565 0.609 0.691 0.4 0.522 0.313 0.452 0.613  MOLWT 112 81 49.9 36 29.9 96.792 48.276 65.361 46.426 41.822 41.904 107.718 83.116 41.105 33.317 93.282 40.56 41.817 81.033 49.796 46.772 78.142 45.358 67.992 46.472 43.995 39.597 66.489 49.47 83.207 58.534 44.127  BandName standard standard standard standard standard Raf B ERK1-III PKB-CT ERK1-CT ERK1-CT ERK 2 PKC-mu PKC-beta 1 p38 CDK 5 nPKC-epsilon CDK 7 CDK 9 PKC-delta SAPK-beta SAPK-beta PKC-zeta MEK 1 PAK-alpha MEK2 MEK4 MEK 6 S6K GSK 3 a/b RSK1 COT CK2 alpha-Ill  PeakQty AverageQty TraceQty 3462 1140.636 4970 1381.182 5063 1611.818 2781 1010.857 3667 1019.9 7002 4374.765 19583 7593.708 7682 3875.929 60765 24627.727 24960 10813.429 23097 7719.143 16268 6667.75 47782 19599.455 28906 14316.2 22616 9118.4 5940 3232.714 15687 7107.708 5719 2475.368 30463 9844.55 4012 3057.909 8126 5184.167 37856 17068.077 5325 2625 23379 8073.875 6427 3364.714 30579 10854.533 10707 3732.688 38065 15949.375 13895 7252.111 63986 27906.947 11567 6057.933 54360 15539.056  1254.7 1519.3 1773 707.6 1019.9 7437.1 18224.9 5426.3 54181 22708.2 16210.2 10668.4 21559.4 28632.4 13677.6 4525.8 17058.5 4703.2 19689.1 3363.7 6221 22188.5 3412.5 12918.2 4710.6 16281.8 5972.3 25519 6526.9 53023.2 9086.9 27970.3  RelativeQty 14.566 17.638 20.584 8.215 11.841 12.62 30.926 5.154 51.458 21.567 33.387 21.648 19.407 25.774 12.312 9.137 34.439 23.544 38.613 5.964 11.031 32.221 4.955 25.633 9.347 21.29 17.132 25.611 6.55 46.482 7.966 39.679  169  ^ *_  I—  CN  f  CO  Q_ co  cr  o co co _>« CO  CN  c <  m  CO I—  o t?  CN  CD  cn  c  :  < c a> Q. CL <  Y" J i  CN  1|  n u  'r  •ii  LT-J  k-  170  Appendix A: Densitometric Analysis of Rat O L Blot, Part 2  Lane  Band  1 1 1 1 1 2 2 3 4 6 8 9 14 16 17 18 19  1 2 3 4 5 1 2 2 3 4 3 1 1 1 1 2 1  RF  MOLWT  0.204 112 0.332 81 0.506 49.9 0.783 36 0.949 29.9 0.579 45.867 0.639 42.698 0.343 78.393 0.352 76.578 0.459 57.584 0.665 41.533 0.339 79.128 0.382 70.931 0.253 100.651 0.352 76.453 0.524 50.29 0.236 106.29  BandName standard standard standard standard standard ERK1-CT ERK1-CT IKK alpha BMX Fyn CK1 delta Zap70 Raf-1 FAK GRK2 Zipk JAK1  PeakQty AverageQty TraceQty RelativeQty 2899 8974 4808 7336 6772 50678 22571 5350 6877 20521 3889 8473 12589 7441 8866 7435 7014  1254.625 1003.7 2218.636 2440.5 1324.727 1457.2 1939.692 2521.6 1952.818 2148.1 18220.81 38263.701 8801.188 14081.9 2680.923 3485.2 2942.182 3236.4 9489.688 15183.5 2068 3308.8 2619.529 4453.2 4934.095 10361.6 3714.067 5571.1 3013.875 4822.2 3811.6 3811.6 3438.571 4814  8.996 21.874 13.061 22.601 19.253 56.697 20.866 10.077 9.415 13.448 10.652 24.994 21.338 10.234 17.412 3.938 7.36  171  -i I  n  CN  cn 1-  mi  Vr  ro D_ O  I—t v  IN  6 o  m  o  4—  n-4 f -  01  I  m  w co >«  ro c < ,st..  1 I"  CO  CD  f 4  c  I  < X c  CD  f - II  Q. Q. <  i -  - 4  172  Appendix A: Densitometric Analysis of CG-4 OL Blot, Part 1  Lane  Band  1  1 2 3 4 5 1 2 4 1 2 3 2 3 2 3 2 2 3 3 2 1 1 5 5 2 6 7 8 2 3 5 3 4  1 1 1 1 2 2 2 3 3 3 4 5 6 6 7 8 8 9 10 12 13 13 14 15 18 18 18 19 19 19 20 20  RF 0.203 0.312 0.416 0.667 0.883 0.422 0.626 0.774 0.591 0.787 0.843 0.848 0.365 0.839 0.913 0.909 0.439 0.878 0.896 0.535 0.917 0.5 0.791 0.774 0.861 0.617 0.748 0.861 0.53 0.548 0.665 0.874 0.904  MOLWT 201 130 94 48.6 36.4 92.797 54.328 42.231 59.877 41.658 38.637 38.554 112.574 39.301 35.623 35.965 91.058 37.596 36.884 72.026 36.251 79.827 42.952 44.104 39.489 61.13 46.273 39.927 76.155 72.963 54.65 39.547 38.012  BandName standard standard standard standard standard Raf B ERK1-III ERK14II PKB-CT ERK1-CT ERK1-CT ERK 2 PKC-u p38 Cdk5 Cdk6 nPKC-epsilon Cdk7 Cdk9 PKC-delta Mos III PKC-zeta MEK 1 MEK 2 MEK 4 S6K GSK 3 a/b GSK 3 a/b RSK1 RSK1 COT CK2 alpha III CK2 alpha III  PeakQty AverageQty TraceQty RelativeQty 3637 3660 7544 5742 6256 11091 13687 16778 20110 64441 52405 40526 14667 64012 7335 43643 15160 60626 15920 20535 8885 64441 8657 9117 61707 58299 61298 18538 64441 64441 18630 60274 63338  1127.364 1333.444 2228.182 2419 1710.357 5097.357 6318.857 7054.538 6296.611 29718.348 16264.056 11461.813 8248.667 25781.111 5367.111 9761.905 6635.4 23132.895 6016.611 8752.929 3803.571 29415.588 4572.765 4617.176 25570.25 23130.235 20055.938 12996.444 33880.611 30276.833 9194.091 19534.824 35589.154  1240.1 1200.1 2451 1935.2 2394.5 7136.3 8846.4 9170.9 11333.9 68352.202 29275.301 18338.901 7423.8 46406.001 4830.4 20500.001 13270.8 43952.501 10829.9 12254.1 5325 50006.502 7773.7 7849.2 40912.401 39321.401 32089.501 11696.8 60985.102 36332.201 10113.5 33209.201 46265.901  7.224 6.991 14.277 11.273 13.948 8.232 10.205 10.579 8.528 51.429 22.027 34.631 10.07 32.892 3.424 47.512 15.785 52.279 39.668 27.318 14.848 44.065 6.85 13.718 36.638 23.12 18.868 6.878 36.636 21.826 6.076 24.667 34.365  173  -~  1 1  CN  •1  [  I  1•  T  T CN  1  i  JBB.  1  |  r  cn  CN  1  |  pr  — i — i  CN  -  m D_  1  i  j|  1  (NJ  ~ •  1  »  11  4  —  XL  i  1  O  1 1  6 T  y  i  o  ?  1  o  r  co CO  N  1  cc c  1  < F  CN  T  CO  1  O  i I  F  1  '  J  4  H r  1 1  T  1  I  1 1  m r~  I  4  i—i  <N  1  4  1  1  1  cn i i  CD  c  < X c CD  Q. Q. <  1  wm — + — i  1  174  Appendix A: Densitometric Analysis of CG-4 OL Blot, Part 2  Lane Band 1 1 1 1 1 2 2 8 14 16 17 19 20  1 2 3 4 5 1 2 1 2 1 1 1 1  RF  MOLWT  0.196 201 0.308 130 0.425 94 0.633 48.6 0.842 36.4 0.705 43.834 0.763 40.443 0.776 39.066 0.481 70.009 0.299 120.249 0.432 79.009 0.282 123.928 0.274 126.376  BandName standard standard standard standard standard ERK1-CT ERK1-CT Zap70 Raf1 FAK GRK2 JAK1 JAK2  PeakQty AverageQty 8268 5859 6701 6267 6222 53403 21009 3486 9758 14424 8355 9524 4398  TraceQty RelativeQty  2792 3071.2 1860.182 2046.2 2025.091 2227.6 2234.778 2011.3 1995.923 2594.7 24098.667 57836.802 10846.588 18439.201 2323.6 3485.4 5868.333 8802.5 5619.722 10115.5 3279.133 4918.7 4779.188 7646.7 1991.75 2390.1  15.115 10.07 10.963 9.899 12.77 61.782 19.697 8.393 15.661 12.988 15.047 9.925 11.882  175  m M — h -  1  T-CN  I l 1  •  1 *—  l  CN  iro l  I TI I ! 1  i I  _r  ._  .  1  1  T  1  1  1  |  <  _  T  i  :  IX- — Of) |  •1 A f  f  II 1 1 1 fl  CN  CO CO  LO  CN  i  ,  CL  CO  j  1  c  ro c  1 1  T  m  -4—'  in o  CN  I 1  1  1  I  CN  1I  i i  1i  o  1  1  1  IllT  O  1  1  1  6  _  cn  |  1  i  cn  CN  .  ro 0_  t  -r-rs  i  m  T  T !  1  1  1  iwm  J a  I 1  CO  LT)  —1—H  CN  [ 1 1  ft 9  i1  M  i  9  1  j  f  i  o  ccu  <  li  #  X '•xS  c  1  0 CL CL < 1  1  -it  176  Appendix A: Densitometric Analysis of Bipotential CG-4 Blot, Part 1  me 1 1 1 1 1 1 2 2 3 3 3 4 5 5 6 6 6 6 6 7 9 11 17 18 18 18 18 18 18  Band 1 2 3 4 5 6 2 4 2 3 4 2 3 4 2 4 5 3 2 3 2 1 5 6 7 2 3 1 2  RF 0.207 0.304 0.397 0.578 0.755 0.886 0.395 0.634 0.521 0.664 0.71 0.71 0.336 0.424 0.429 0.714 0.782 0.777 0.382 0.748 0.462 0.433 0.513 0.618 0.689 0.437 0.521 0.718 0.744  MOLWT 201 130 94 48.6 36.4 29.8 94.402 44.267 59.627 42.138 39.05 38.998 116.357 84.57 83.204 38.625 34.751 34.927 98.294 36.407 73.31 81.348 60.478 44.93 39.678 79.612 58.586 37.582 36.062  BandName standard standard standard standard standard standard Raf B ERK1 III PKB-CT ERK1-CT ERK1-CT ERK2 PKC-mu PKC-alpha PKC-beta p38 CDK 5 CDK 6 nPKC-epsilon CDK 7 PKC-delta PKC-zeta S6K GSK 3 a/b GSK 3 a/b RSK1 COT CK2 alpha-Ill CK2 alpha-Ill  PeakQty AverageQty TraceQty RelativeQt} 3038 3890 4088 5655 2863 7224 13931 20940 13060 46000 43073 42702 22874 6370 43558 39635 5030 13073 17536 38011 21977 50161 40349 23144 6210 60411 10460 20059 30380  1746.778 1209.818 1170.364 1472.231 1006.778 1652.083 6839.143 7194.923 4363.526 17297.9 13222.412 13684.385 12594.556 4318.071 14359.864 13462 3836 4442.733 8303.143 11451.6 8581.636 17111.95 14553.391 7514.813 4426.7 24692.621 6537.231 7774.462 16440.889  1572.1 1330.8 1287.4 1913.9 906.1 1982.5 9574.8 9353.4 8290.7 34595.801 22478.101 17789.701 11335.1 6045.3 31591.701 26924.001 4603.2 6664.1 17436.601 22903.201 18879.601 34223.901 33472.801 12023.7 4426.7 71608.602 8498.4 10106.8 14796.8  13.922 11.785 11.4 16.948 8.024 17.556 10.918 10.666 8.469 35.341 22.962 31.116 13.744 7.33 21.653 18.454 3.155 18.124 22.379 29.395 36.925 49.092 27.668 9.939 3.659 52.252 6.201 17.653 25.844  177  r  •=1—1—h-tol  II II  I  l!  N |  m  • f e l1 l 1  M H i CN  1 HH  ML  t f f t  U  j •  1  —1  1  m  I  ml  -+-  F  |  t  -J"  4  F1 — i F  •  Q*  .i  CO  i  i  ,  n  Q_  6 o  1  '  *•  -r 1  I^HR T—  1  "I•1 ,i  1 1  rsj  1  T  i 1  1  1 j  CN  .]  ^ L •f  I  D  r  •• •  1  fp  1 1  1 1 ll  j  i i  1 i  F  — |P  T  \  CN  i i  1 I  t  r  i 11 11  f  1  1  1  f 1  II  II  f-  CO  "(0  cc c  CO  E_ O CD  c  < 1  1  1  ^ c a> a.  Q. <  J  VH Pi t H 4 -  :  CP  r 1  tf  Wfc  iiiiiViiii'iiililBtftffli  CQ 4— O  H  CN  T  O Q.  <  CN  I, i.  ,1  •+-I  1  -q- ix)  »  (D  cn  1  1  c  1  1  4  1  178  Appendix A: Lane Band 1 1 1 1 1 2 2 6 9 11 12 14 16 17 18 19 20  1 2 3 4 5 3 4 5 1 1 1 2 1 2 9 2 1  RF  Densitometric Analysis of Bipotential CG-4 Blot, Part 2  MOLWT  BandName  0.093 201 0.227 130 0.361 94 0.611 48.6 0.838 36.4 0.72 42.313 0.771 39.615 0.603 49.801 0.383 85.954 0.818 37.142 0.579 52.832 0.453 71.521 0.252 118.748 0.421 76.808 0.607 49.474 0.234 124.473 0.22 129.397  standard standard standard standard standard ERK1 ERK1 Fyn Zap70 CaMK1 CaMKK-CT Raf1 Fak GRK2 Zipk JAK1 JAK2  PeakQty AverageQty TraceQty RelativeQty 2993 3164 6306 3234 6741 39665 32867 20811 7314 3183 1871 16743 46527 21498 8404 15005 5285  1268.636 1173.5 2038 1631.375 1993.1 15261.158 10866 8490.706 2547.889 2042.636 894.5 9231.471 14469.65 5773.056 6351.4 5966.389 2115.333  1395.5 1408.2 2038 1305.1 1993.1 28996.2 18472.2 14434.2 4586.2 2246.9 1073.4 15693.5 28939.3 10391.5 6351.4 10739.5 2538.4  12.866 12.983 18.789 12.032 18.376 49.543 31.562 13.038 27.873 8.287 9.331 29.55 30.096 31.578 5.72 13.329 18.378  179  1—(—- f -H  1—H  r—  I  i  i  CN  r 1  cn  s  '  Uj  p  i ^  f  CN  |  T  7  ,  cn  '  i  i 1 1 1 1 I 11 11 1 If 1 •  1  1  CD  i  D_  6  II  "  CNtjp  l1  l  i  i  1  i  1  1 1  l1  1  i  r -  CN  r  CO  <  »  1  CN  " T  o o l_  1  .. 1  o  !  -  |  Iff  i  i in  cn  1  '-+-i -I—»  1  cn  CN  1  g  W  CM  1 ' 1L 1  o  i  CO CO CO  c: < CO  r-  y ' y I  cn  CN  1  CN  4  1  1i  T fl  j  j  1  o  i  II ff  ll  ll  CN  T-  1  || f f l cn  •*  ' J St S 11 CN  I  4  rn  -h  1  1  i  1  | | 1 1 1  1 1  I  1  1 1  _ CN  I  1 1  1  11  CD  c  < _><  c a a <  CW  1|  1 1  1  180  Appendix A: Densitometric Analysis of Astrocytic CG-4 Blot, Part 1  Band  e 1  1 1 1 1 1 2 2 2 2 3 3 3 4 5 5 6 6 8 8 9 10 13 13 14 14 15 18 18 18 19 19 19 20 20  1 2 3 4 5 6 1 3 4 5 1 2 3 4 3 10 1 2 2 3 3 2 1 4 1 2 2 10 12 13 3 4 6 2 3  RF 0.124 0.253 0.343 0.601 0.803 0.936 0.358 0.56 0.681 0.72 0.53 0.72 0.772 0.776 0.302 0.802 0.427 0.772 0.362 0.806 0.819 0.461 0.427 0.728 0.504 0.707 0.784 0.53 0.668 0.784 0.409 0.422 0.573 0.767 0.789  MOLWT 201 130 94 48.6 36.4 29.8 90.953 54.103 43.405 41.057 58.688 41.141 38.2 38.037 110.626 36.725 77.505 38.428 92.341 36.714 36.082 72.092 79.826 41.459 65.535 42.85 38.396 62.264 45.684 38.622 85.658 82.816 55.87 39.757 38.538  BandName standard standard standard standard standard standard Raf B ERK3 ERK1-III ERK1-III PKB-CT ERK1-CT ERK1-CT ERK2 PKC-mu CDK 4 PKC-beta 1 p38 nPKC-epsilon CDK 7 CDK 9 PKC-delta PKC-zeta MEK 1 PAK alpha MEK 2 MEK 4 S6K GSK 3 a/b GSK 3 a/b RSK1 RSK1 COT CK2 alpha-Ill CK2 alpha-Ill  PeakQty 3249 2157 5246 2785 4542 9337 7174 9090 23449 8047 13647 35857 25798 17641 11733 7661 23500 29626 4164 27429 4192 7067 33653 4206 4054 5608 34739 29765 38478 11700 34795 40187 11201 34181 35899  AverageQty 1435.625 928.273 1737 1058.222 1455.222 2900.6 4493.059 5772.688 9670.105 4873.455 3895.526 15613.81 9310.235 5686.235 5468.2 4362.1 10562.471 11150.25 2008.385 8429.826 1943.556 2718.737 13245.389 2144.909 2530.6 2738.579 13398.474 13728.538 12669.5 8039.222 14273.308 29553 5935.7 14242.4 22899.909  TraceQty  RelativeQty 7.246 6.442 8.767 6.009 8.263 18.3 8.594 10.393 20.673 6.032 10.055 44.544 21.502 30.478 10.899 8.694 23.783 23.63 7.109 52.794 28.666 20.735 39.345 3.894 9.775 13.399 36.285 16.959 19.262 6.875 20.155 22.47 6.447 29.778 35.111  1148.5 1021.1 1389.6 952.4 1309.7 2900.6 7638.2 9236.3 18373.201 5360.8 7401.5 32789.001 15827.4 9666.6 5468.2 4362.1 17956.201 17840.401 2610.9 19388.601 3498.4 5165.6 23841.701 2359.4 3795.9 5203.3 25457.101 17847.101 20271.201 7235.3 18555.301 20687.101 5935.7 21363.601 25189.901  181  182  Appendix A: Densitometric Analysis of Astrocytic CG-4 Blot, Part 2  Lane 1 1 1 1 1 1 2 2 3 4 6 9 14 16 17 19 20  Band 1 2 3 4 5 6 1 2 1 2 4 1 2 1 3 1 1  RF  MOLWT  0.111 201 0.209 130 0.302 94 0.515 48.6 0.681 36.4 0.843 29.8 0.609 41.217 0.651 38.28 0.374 75.062 0.387 72.087 0.519 47.938 0.353 80.255 0.417 65.116 0.264 110.413 0.379 73.754 0.247 118.821 0.234 125.336  BandName standard standard standard standard standard standard ERK1-CT ERK1-CT IKK-alpha BMX Fyn Zap70 Raf-1 FAK GRK2 JAK1 JAK2  PeakQty AverageQty TraceQty RelativeQty 6243 3571 4359 3923 6475 6889 58849 32975 4034 7371 22279 7200 21891 49911 15877 16917 13131  1669.5 2337.3 1374.636 1512.1 1382.818 1521.1 1593.636 1753 1840.727 2024.8 1786 2143.2 17063.6 34127.201 8639.5 13823.2 2015.6 3023.4 3018.5 4829.6 9230.25 14768.4 2835.067 4252.6 11262.5 15767.5 15459.056 27826.301 5010.083 6012.1 7242.588 12312.4 4102.533 6153.8  13.126 8.492 8.542 9.844 11.371 12.036 51.375 20.809 9.694 10.321 12.968 14.532 23.426 26.616 16.57 16.147 21.636  183  

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