Mitogen-activated protein kinase signalling in oligodendrocytes: a comparison of primary cultures and CG-4

Int. J. Devl Neuroscience 19 (2001) 427– 437 www.elsevier.nl/locate/ijdevneu

Mitogen-activated protein kinase signalling in oligodendrocytes: a comparison of primary cultures and CG-4 Rochelle L. Stariha, Seung U. Kim * Department of Medicine, Di6ision of Neurology, UBC Hospital, Uni6ersity of British Columbia, 2211 Wesbrook Mall, Vancou6er, BC, Canada V6T 2B5 Received 22 January 2001; accepted 13 February 2001

Abstract Oligodendrocytes play a significant role in the central nervous system, as these cells are responsible for myelinating axons and allowing for the efficient conduction of nerve impulses. Therefore, any understanding we can gain about the functional biology of oligodendrocytes will give us important insights into demyelinating diseases such as multiple sclerosis, where oligodendrocytes and myelin are damaged or destroyed. Currently, much attention has focussed on the role of a family of mitogen-activated protein kinases in OL. This kinase family includes the extracellular signal-regulated protein kinases (ERKs), the stress-activated c-Jun N-terminal kinase (JNK), and the 38 kDa high osmolarity glycerol response kinase (p38). The actions of mitogen-activated protein kinases in oligodendrocytes appear to range from proliferation and cell survival to differentiation and cell death. In the past, studies on oligodendrocytes have been hampered by the difficulties inherent in producing large enough quantities of these cells for experimentation. This problem arises in large part due to the post-mitotic nature of mature oligodendrocytes. Over the years, a cell line known as Central Glia-4 (CG-4) has become a popular oligodendrocyte model due to its potentially unlimited capacity for self-renewal. In this review, we will look at the suitability of the Central Glia-4 cell line as an oligodendrocyte model, specifically in respect to studies on mitogen-activated protein kinase signalling in oligodendrocytes. © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cell culture; CG-4 cell line; MAP kinases; Oligodendrocytes; Signal transduction

1. Overview of oligodendrocytes and cell signalling Oligodendrocytes (OL) play a very important role as the myelinating cells of the central nervous system (CNS). Since the myelin they create allows for the efficient conduction of nerve impulses, destruction or disruption of these cells can lead to severe pathological conditions. For instance, in the demyelinating disease multiple sclerosis (MS), loss of OL and myelin can lead to motor and sensory problems such as paralysis and loss of vision. Many studies, therefore, have been conducted on OL functional biology. By uncovering the relevant intracellular signalling pathways in these cells, scientists hope to find ways of enhancing OL survival and myelination capacity. Eventually, these findings * Corresponding author. Tel.: + 1-604-8227570; fax: +1-6048227897. E-mail address: [email protected] (S.U. Kim).

could lead to new treatments for demyelinating diseases. Studies on OL have been classically conducted on primary cultures isolated from mammalian brain. However, progress has been hindered by the fact that mature, myelinating OL are post-mitotic. Over the past years, a cell line isolated from rat brain known as Central Glia-4 (CG-4) has become a popular OL model (Louis et al., 1992). This cell line has facilitated many investigations on intracellular signalling in OL. Using a combination of primary OL and CG-4 cells, researchers have begun to piece together a role for mitogen-activated protein kinases (MAPKs) in OL signal transduction. The MAPKs are a family of kinases that includes the extracellular signal-regulated protein kinase (ERK), the stress-activated c-Jun N-terminal kinase (JNK), and the 38 kDa high osmolarity glycerol response kinase (p38) (Cano and Mahadevan, 1995; Kanashiro and Khalil, 1998; Wagey and Krieger, 1998). These mem-

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bers of the MAPK family each seem to play a unique role in OL function, including enhancement of OL differentiation, proliferation, cell survival or cell death. This review will compare primary OL and CG-4 culture models, outlining the advantages and disadvantages of each. Then, using information gained from both primary OL and CG-4, the potential role of MAPKs in OL proliferation, process extension, differentiation, cell survival and cell death will be discussed.

2. The primary oligodendrocyte culture model Primary OL have distinct immature and mature developmental stages. For instance, immature OL can proliferate but cannot myelinate, while mature OL can myelinate but cannot proliferate (Keirstead and Blakemore, 1997, 1999). These developmental stages are best categorized in the rat CNS, where the OL differentiation pathway can be tracked using cell surface marker expression (Bansal and Pfeiffer, 1992; Bansal et al., 1992). In the first stages of development, rat OL are bipolar and express the cell surface marker A2B5 (Raff, 1989). These progenitor cells are often referred to as O2A progenitor cells, as they have the capacity to differentiate into either mature OL or type-2 astrocytes. It should be noted that O2A progenitor cells are only characterized in rat, and are not characterized in mouse or in any other mammalian species. The concept of O2A, however, helps investigators to understand the early development of OL. Under serum-free conditions, O2A cells begin to lose expression of the A2B5 marker and acquire expression of the O4 oligodendrocyte marker. Gradually, as they mature into fully differentiated OL, these cells become multipolar and non-proliferative. They also begin to express galactocerebroside (GalC), and eventually make myelin proteins such as 2%,3%-cyclic nucleotide 3%-phosphohydrolase (CNP), myelin basic protein (MBP), and proteolipid protein (PLP) (Ranscht et al., 1982; Bansal and Pfeiffer, 1992; Bansal et al., 1992). OL can be cultured in the immature O2A stage from newborn rat brain (Noble et al., 1988; Raff and Lillien, 1988; Raff et al., 1988; Richardson et al., 1988; Grinspan et al., 1990). They can also be cultured in the mature GalC stage from adult mammalian tissue; for instance, purified or enriched populations of mature OL have been isolated from cow, pig, sheep and human tissue (Szuchet et al., 1980; Kim et al., 1983; Smyrnis et al., 1986; Kim, 1990; Althaus et al., 1991; Kim and Kim, 1991; Stariha et al., 1997). The obvious advantage of primary OL cultures is that the cultured cells have direct in vivo counterparts, unlike artificial cell lines. However, the post-mitotic nature of primary mature OL is a disadvantage, making it difficult to generate a sufficient quantity of mature OL for experimentation.

As well, O2A cells do not divide indefinitely, again creating a quantity problem. The CG-4 cell line, on the other hand, has a potentially unlimited capacity for self-renewal.

3. The CG-4 culture model The CG-4 cell line arose as a spontaneous mutation from rat O2A primary cultures (Louis et al., 1992). It is a bipotential cell line, able to differentiate into either OL or astrocytes, and thus it resembles cells of O2A lineage (Fig. 1). In its bipotential, or progenitor, form, it is bipolar and expresses A2B5. When cultured a serum-free medium containing B-104-conditioned media, these cells maintain their progenitor morphology and are highly proliferative. Upon removal of the B104-conditioning, however, CG-4 take on the morphological characteristics of mature OL within 2 days. Furthermore, these cells express GalC and lose much of their proliferative capacity. As mentioned above, the advantage of CG-4 is that they can provide unlimited numbers of OL-like cells. The disadvantage is that this cell has no real physiological counterpart. The question that must be addressed is, how significant are the differences between primary OL and CG-4? In other words, can data generated from CG-4 be applied to primary OL?

4. A comparison of primary OL and CG-4 CG-4 cells were first characterized by immunocytochemistry (Louis et al., 1992). In their progenitor state, 95% of CG-4 were found to express the immature OL marker A2B5. Furthermore, only 2–3% of the cells stained for GalC, an early marker of mature OL, and B 1% stained for glial fibrillary acidic protein (GFAP), a marker for astrocytes. In their multipolar, mature OL state, all CG-4 were found to express GalC and over 50% were also found to express MBP. Only 2% of CG-4 were found to retain a progenitor phenotype under these conditions, as assessed by A2B5 staining. Furthermore, these OL-like cells were no longer self-renewing, but rather degenerated after  8 days. In contrast, it was found that addition of 20% serum caused 50% of bipotential CG-4 to take on astrocyte morphology and stain with GFAP. These astrocyte-like cells remained highly proliferative. The first striking parallel between primary O2A cells and bipotential CG-4 is their similar differentiation pathways: Both cells differentiate into mature OL in serum-free media, and both cells differentiate into type2 astrocytes in serum-containing media. Furthermore, both O2A and bipotential CG-4 express the neural progenitor marker nestin, and both cells lose expression

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of this marker as they differentiate into mature OL (Gallo and Armstrong, 1995). Looking at the two differentiation pathways in more detail, PDGF and bFGF can be seen to enhance immature OL proliferation and delay differentiation to mature OL in both cell types. For instance, studies on primary OL have shown

Fig. 1. Central glia-4 cells. (A) CG-4 maintained in serum-free media plus B-104 mitogens; (B) CG-4 cultured in serum-free media for 1 day; (C) CG-4 cultured in 20% serum-containing media for 1 week.

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that PDGF can delay OL differentiation when injected into the cerebrospinal fluid of neonatal rats (Butt et al., 1997). As well, decreases in PDGF signalling have been shown to correspond to an exit of proliferative, immature OL from the cell cycle, and anti-PDGF antibodies have been shown block OL mitosis (Dutly and Schwab, 1991; Calver et al., 1998). Once immature OL have exited the mitotic cell cycle, they are able to spontaneously differentiate into mature OL (Dutly and Schwab, 1991). Basic FGF has also been shown to act as a mitogen for immature OL and to prevent differentiation of OL into GalC positive cells (Gard and Pfeiffer, 1993; Grinspan et al., 1993, 1996). Similarly, bFGF and PDGF are required to keep CG-4 in their bipotential form, while removal of these mitogens allows the cells to stop rapid proliferation and begin differentiation into mature OL-like cells (Louis et al., 1992). As well as their similar responses to PDGF and bFGF, primary OL and CGA also have similar responses to ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). Studies on primary cultures of O2A have shown that CNTF and LIF can induce conversion of O2A into astrocytes, as has been assessed by GFAP staining (Hughes et al., 1988; Lillien et al., 1990; Mayer et al., 1994; Gard et al., 1995a,b). Studies conducted on CG-4 also indicate a role for CNTF and LIF in the differentiation of progenitor cells towards astrocytes (Kahn and De Vellis, 1994; Kahn et al., 1997). Furthermore, CNTF and LIF are well known OL survival factors: CNTF has been shown to enhance OL survival after TNF-a treatment or removal of trophic factors, and LIF has also been shown to promote OL survival in vitro (Barres et al., 1993; Louis et al., 1993; Mayer et al., 1994; D. Souza et al., 1996; Vos et al., 1996; Jiang et al., 1999). In CG-4, CNTF and LIF have been shown to enhance the survival of trophically deprived OL-like cells (Kahn and De Vellis, 1994). The signal transduction pathways activated by CNTF in both primary OL and CG-4 also appear to be similar, as both lead to the activation of JAK/stat signalling cascades (Kahn et al., 1997; Dell’Albani et al., 1998). While there are many similarities between primary OL and CG-4, there are also significant differences between these two cell types. For instance, O2A cells drastically upregulate their expression of the ganglioside GM3 when they differentiate into mature OL. CG-4, on the other hand, express very small amounts of GM3, but larger amounts of GD1b (Yim et al., 1995; Schnaar et al., 1996). Furthermore, while CG-4 express myelin associated glycoprotein (MAG), their expression of this protein is much lower than in primary OL. The amounts of GalC and sulfatide are also lower in CG-4 than OL, but increased GalC synthesis does occur in CG-4 differentiating toward OL-like cells (Yim et al., 1995; Bichenkov and Ellingson, 1999). The

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general decrease in OL-related proteins in CG-4 as compared to primary OL suggest that CG-4 perhaps do not achieve as high a degree of differentiation as primary OL. However, the fact that they still express relevant OL proteins indicates that CG-4 could make a reasonable OL model. As well as expressing OL-related proteins, such as MAG, CG-4 also share expression of other proteins with primary OL. For instance, Western blotting and immunofluoresent staining studies have shown that CG-4 and primary OL both have similar expression of neural cell adhesion molecules, cadherins and betacatenin (Hughson et al., 1998). Both OL and CG-4 also express functional ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate sub-types, and both cell types express thyroid receptors (Baas et al., 1994; Pende et al., 1994; Yoshioka et al., 1995, 1996; Meucci et al., 1996; Pombo et al., 1999). As well, the developmental expression of the krox-24 differentiation regulator protein is similar in both CG-4 and primary OL (Sock et al., 1997). We have also determined, through a multi-blot array of  60 different kinases, including those of the MAPK family, that the protein expression pattern is very similar between primary OL and CG-4 (unpublished observations). However, if CG-4 cells are to be used as an OL model, they must share similar actions as well as similar expression patterns. One important action of O2A cells is their ability to migrate. Tests conducted on myelin and pleiotrophin coated dishes have found that CG-4 and O2A cells are, indeed, both able to migrate on a CNS substrate (Amberger et al., 1997; Rumsby et al., 1999). CG-4 have also been used in transplantation experiments to determine if they can mimic OL in vivo. It was found that, as for O2A cells, CG-4 can differentiate into both astrocytes and OL in vivo. Perhaps most significantly, they were also able to migrate and remyelinate in irradiated tissue (Olby and Blakemore, 1996). In conclusion, therefore, while primary OL and CG-4 have some different characteristics, the two cells are largely similar. Most importantly, they both express myelin proteins and they can both migrate and myelinate in the CNS. Therefore, since CG-4 can overcome the experimental restrictions cause by primary OL quantity problems, they should be a valuable model with which to study OL functional biology.

5. MAPK overview Using primary cultures and CG-4 cultures, many studies have been conducted on the role of MAPKs in OL. As mentioned above, the MAPKs are a family of structurally related proteins that includes ERK, JNK, and p38. While the functions of these MAPK family

members vary, all MAPKs require dual-phosphorylation on both tyrosine and threonine residues for full activation. (For complete reviews, see Cano and Mahadevan (1995) and Wagey and Krieger (1998)). Perhaps the most well-known and best-characterized MAPK is ERK. Two isoforms of this kinase are relevant to this review: the 42 kDa ERK2 and the 44 kDa ERK1. We will consider two pathways leading to ERK activation in OL (Fig. 2). First, in the classical activation pathway, ERK can be activated by a series of phosphorylation events stimulated by binding of an appropriate growth factor (i.e. EGF, PDGF) to its receptor. Receptors leading to the activation of ERK can also include the Trk neurotrophin receptors (Schlessinger and Ullrich, 1992). Binding of an appropriate factor to its receptor causes receptor dimerization and phosphorylation, which in turn triggers a series of phosphorylation events. While there are many adaptor and docking proteins involved in this signalling cascade, the main kinases of interest are the sequentially activated Ras, Raf, MEK, and ERK. The second ERK activation pathway of interest in this review is dependent on phorbol ester stimulation. In this case, a biologically active phorbol ester such as phorbol 12,13-myristate acetate (PMA) or phorbol dibutyrate (PDB) diffuses into the OL and activates protein kinase C (PKC) (Stariha et al., 1997) (Fig. 2). PKC is an important signalling molecule in most biological systems, and can also be activated by phospholipase C activation following growth factor receptor dimerization as described above (Kanashiro and Khalil, 1998; Wagey and Krieger, 1998). PKC consists of three subgroups: conventional PKC’s, novel PKC’s, and atypical PKC’s. In OL, the main sub-group that appears to mediate intracellular signalling events is the conventional PKC’s (Yong et al., 1994). This sub-group is calcium-dependent and is comprised of isoforms a, b1, b2, and g. Activation of conventional PKC’s by phorbol ester treatment can in turn lead to activation of Raf, thus feeding into the ERK activation cascade. Once ERK has been activated in OL, it has many potential substrates. Included in these are myelin basic protein (MBP), a major component of myelin, and transcription factors, such as c-fos (Vartanian et al., 1986; Bhat et al., 1992). The second member of the MAPK family is JNK. JNK is a stress-activated protein kinase, and thus is referred to as stress-activated protein kinase or SAPK in some systems (Fig. 2). The cellular stresses responsible for activating this kinase include anisomycin, okadaic acid, UV-irradiation, and ceramide. Also, as will be seen again when considering the p38 member of the MAPK family, JNK can be activated by receptors of the TNF-a receptor superfamily. This family of receptors includes the low affinity neurotrophin receptor, p75. Activators of these receptors, therefore, in-

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Fig. 2. A simplified version of potential MAPK signalling pathways in oligodendrocytes. ERK, JNK, p38: mitogen-activated protein kinases (MAPKs). PKC: protein kinase C. MBP: myelin basic protein. c-fos, c-jun, ATF-2: nuclear transcription factors. Cell stress: UV irradiation, anisomycin, okadaic acid.

clude neurotrophins and TNF-a, as well as IL-1 (Fig. 2). The classical JNK substrate is the N-terminus of c-Jun. The third member of the MAPK family, p38, is more similar to JNK than to ERK. For instance, it too can be activated by TNF-a, IL-1 and ceramide treatment. It is also classified as a stress-activated protein but, unlike JNK, osmotic stress is the main activator of p38 (Fig. 2). Thus, this kinase is referred to as high-osmolarity glycerol response protein, or HOG, in some systems (i.e. yeast). The main substrates for p38 are small heat shock proteins and the ATF-2 nuclear transcription factor. The ERK, JNK, and p38 members of the MAPK family are therefore structurally related proteins that share a requirement of dual phosphorylation for activation. However, the pathways leading to the activation of these kinases are different, as are their subsequent substrates. In this review, we will discuss the various roles of ERK, JNK, and p38 in OL process extension, proliferation and differentiation, cell survival and cell death.

6. The role of ERK in OL proliferation

6.1. Mature OL Both in vivo studies and in vitro observations have

allowed scientists to conclude that, under normal conditions, mature OL 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 OL can be induced to proliferate. For instance, Althaus et al. were able to induce proliferation of a sub-set of mature OL using nerve growth factor (NGF) treatment, and subsequently showed that NGF treatment can activate ERK1 in OL (Althaus et al., 1992, 1997). 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 an ERK kinase (MEK) inhibitor, thus strengthening the link between increased ERK activation and OL proliferation.

6.2. O2A The proliferation of O2A cells, as well as mature OL, has also been linked to ERK activation. For instance, treatment of progenitor OL with the neurotrophin NT-3 has been shown to increase both cell proliferation and ERK phosphorylation (Cohen et al., 1996). Since treatment of progenitor OL with NGF was shown to be less effective at inducing ERK phosphorylation than NT-3,

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and since NGF did not stimulate progenitor proliferation, it could be concluded that a certain level of ERK activation must be reached before O2A are induced to proliferate. Other studies have shown that the ERK2 isoform, rather than the ERK1 isoform, is preferentially activated by NT-3 (Kumar et al., 1998). Since the ERK1 isoform has been implicated in the proliferation of mature OL, and ERK2 has been implicated in the proliferation of O2A cells, it seems that ERK isoforms may be developmentally regulated in OL (Rus et al., 1997; Kumar et al., 1998).

6.3. CG-4 Studies on the role of ERKs in the proliferation of CG-4 have not yet been undertaken. Therefore, it is not possible to assess the contribution of CG-4 research to the elucidation of the ERK proliferation pathway in OL at this time.

lation of OL with PMA not only induced process extension, but also caused movement of ERK from the cytoplasm to the nucleus. Furthermore, this PMA stimulus caused activation of both ERK1 and ERK2 isoforms. We then went on to show that use of an ERK kinase (MEK) inhibitor not only blocked phorbolester-induced process extension, but also decreased ERK activation by  70%. Hence, we propose that a certain threshold of ERK activation must be reached before OL are induced to extend processes. This concept of a critical level of ERK activation has already been noted in the induction of O2A proliferation. As was mentioned above, increases in O2A proliferation were noted only after a high enough level of ERK activity was reached (Cohen et al., 1996).

7. The role of ERK in OL process extension

7.1. Mature OL Most of the current knowledge on process extension of signalling cascades in mature OL has come through experiments utilizing activation of protein kinase C (PKC). For instance, it has been well documented that treatment of mature OL with a biologically active phorbol ester, such as phorbol 12,13-myristate acetate (PMA) or phorbol dibutyrate (PDB), induces extension of processes (Fig. 3) (Yong et al., 1988; Althaus et al., 1991; Yong et al., 1991, 1994). The importance of PKC in this phorbol esterinduced extension has been confirmed by showing that inhibitors of PKC can block this event (Yong et al., 1988; Althaus et al., 1991; Yong et al., 1994). Investigators have since attempted to determine the signalling events following PKC activation in OL, and have found various downstream regulators of this kinase. Signal transduction events following from PKC activation to OL process extension include increased intracellular calcium levels, activation of metalloproteinase 9, and activation of ERK (Stariha et al., 1997; Uhm et al., 1998; Yoo et al., 1999). As mentioned above, the PKC pathway is thought to feed into the ERK activation pathway at the level of Raf (Fig. 2). A set of complementary experiments showing that NGF treatment of mature OL can cause both increased process extension and increased ERK activity initially outlined a potential role for ERKs in OL process extension (Althaus et al., 1992, 1997). Our lab has since corroborated and further defined this role (Stariha et al., 1997). First, we determined that stimu-

Fig. 3. Human oligodendrocytes. (A) Control OL (52 days in vitro); (B) OL treated with 100 nM PDB for 1 h (52 days in vitro) (Bar indicates 20 mM).

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While these results indicate a potential contradiction in the signalling pathways between primary OL and CG-4, they do not necessarily negate the use of CG-4 as an OL model. Rather, they stress the importance of careful controls and thorough comparisons between the two systems before any major conclusions are drawn.

8. The role of ERK in OL differentiation Fig. 4. ERK1 Activation in CG-4. The activation of ERK1 can be seen by the band-shift noted by (x). (a) CG-4 incubated in serum-free media plus B-104 mitogens for 30 min. (b) CG-4 incubated in serum-free media for 30 min.

7.2. CG-4 A problem inherent with the use of CG-4 to assess OL process extension is the rapid endogenous extension of processes from CG-4. In primary bovine OL cultures, for instance, processes are lost during the culture procedure and have a very slow endogenous regrowth rate. Therefore, any increases in process extension can be easily assessed since control cells show little or no process formation (Stariha et al., 1997). CG-4 cells, on the other hand, begin to extend processes within hours of removal of B-104 mitogens. It is nearly impossible, under these circumstances, to assess if any factors can enhance extension over and above the endogenous rate. It is, however, possible to assess if any factors can inhibit the extension of process from CG-4. Preliminary experiments in our lab have used this inhibition model to assess the role of ERKs in CG-4 process extension, with surprising results. After outlining a role for ERKs in the process extension of primary OL, we switched to a CG-4 culture model. Since ERK activation is required for process extension in primary OL, we attempted to inhibit the endogenous extension of process from CG-4 by using an ERK kinase (MEK) inhibitor. While we used various concentrations of two separate MEK inhibitors, PD 98059 and Uo-126, we found that no concentrations inhibited process extension. However, at higher concentrations the inhibitors were both toxic (unpublished observations). A Western blot comparison between CG-4 in the presence or abs6nce of B-104 mitogens showed that ERK was active in the bipotential cells, but not in the cells induced to differentiate and extend processes (Fig. 4). We then tested whether blocking ERK activation in the bipotential cells could induce differentiation and process extension. While a Western blot analysis confirmed that the MEK inhibitors could decrease ERK activity, the bipotential cells were not induced to extend processes (unpublished observations). A possible explanation is that the ERK activity still remaining after MEK inhibition is enough to inhibit process extension.

8.1. O2A Although there have been no definitive studies of ERKs during the differentiation of O2A cells to mature OL, various roles for these kinases can be suggested. For instance, it has been shown that PMA treatment of O2A cells leads to activation of both ERK1 and ERK2 (Bhat and Zhang, 1996). Furthermore, it has also been shown that PMA treatment of O2A cells leads to a decrease in the number of cells acquiring a mature phenotype, while the use of a PKC-inhibitor negates this PMA-induced decrease in differentiation (Baron et al., 1998, 1999; Heinrich et al., 1999). This makes it tempting to speculate that activation of ERKs in O2A cells prevents differentiation. However, since treatment of O2A with NT-3 has also been shown to increase differentiation, and since ERK can conceivably be activated downstream of NT-3, one could just as easily speculate that activation of ERKs in O2A cells induces differentiation (Heinrich et al., 1999). In order to definitively characterize the role of ERKs, experiments utilizing MEK inhibitors must be undertaken.

8.2. CG-4 Given the differences in ERK pathways already noted between mature OL and CG-4, it is perhaps not surprising that PKC-regulated differentiation pathways appear to be different in CG-4 than in primary OL. Although the role of ERK/PKC has not been conclusively studied in CG-4, our preliminary results show that use of the PKC inhibitor prevents differentiation in CG-4. Once again these results point to a potential contradiction with results seen in primary cultures, where activation of PKC has been shown to inhibit differentiation (Baron et al., 1998, 1999). These findings suggest that the CG-4 model may not adequately describe the role of PKC and ERKs in primary OL.

9. The role of ERK in OL survival and death

9.1. Mature OL Experiments done on primary, mature OL have shown a correlation between ERK activation and in-

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creased survival. For instance, NT-3 or NGF treatment of mature OL has been shown to cause both increased ERK phosphorylation and increased OL survival (Cohen et al., 1996). Further experiments were conducted in which the effect of ERK activity on cell survival was compared to the effect of JNK activity on cell survival (Casaccia-Bonnefil et al., 1996; Yoon et al., 1998). In a first set of experiments, NGF was used to selectively activate JNK in OL that expressed p75 but not TrkA (Casaccia-Bonnefil et al., 1996). In this case, cell death occurred. However, in OL expressing both p75 and TrkA, JNK and ERK were both activated and the cells survived (Yoon et al., 1998). These complimentary experiments suggest that JNK activation induces cell death, while ERK activation promotes cell survival.

9.2. CG-4 Studies on the effect ERK activation in CG-4 both compare and contrast to primary culture results. First, in comparison to primary culture results, increased ERK activation has been correlated to increased survival of CG-4 that are differentiated into mature OL-like cells. In these experiments, activation of ERK was carried out by treatment with prosaposin, a neurotrophic factor (Hiraiwa et al., 1999). In primary cultures, increased ERK activity and increased OL survival were also seen after neurotrophic factor treatment (Cohen et al., 1996; Yoon et al., 1998). In contrast, ERK activation has been implicated in H2O2-induced CG-4 cytotoxicity (Bhat and Zhang, 1999). In these experiments, CG-4 were differentiated into mature OL-like cells before exposure to H2O2. After treatment, the activities of all the MAPKs were increased. In other words, ERK, JNK and p38 were all activated. However, while both MEK and p38 inhibitors were tested, only the MEK inhibitor blocked the cytotoxicity. Therefore, ERK activation appears to be able to activate cell death in CG-4. There are two questions that must be asked, given the differences noted previously between ERK cascades in primary OL versus CG-4. First, is ERK an OL death-inducing agent in some circumstances and an OL survival-promoting agent in others? Or, second, is the ERK signalling pathway fundamentally different between primary OL and CG-4? The second scenario would make it possible for ERK to mediate cell survival in primary OL, while at the same time making it possible for ERK to mediate either cell survival or cell death in CG-4. Without parallel studies between primary OL and CG-4, it is dangerous to assume ERK can also have cell death-inducing characteristics in primary OL.

10. The roles of JNK and p38 in OL survival and death

10.1. Mature OL Both JNK and p38 have the distinction of being considered stress-related kinases, and are therefore often associated with cell death. In the case of JNK, studies on human OL have shown that TNF-a treatment can cause activation of both OL apoptosis and activation of JNK (Ladiwala et al., 1999). As well, studies on rat OL have used NGF to show activation of both OL apoptosis and JNK (Casaccia-Bonnefil et al., 1996). Other studies have confirmed the activation of JNK by TNF-a, as well as by ceramide, sphingosine and sphingomyelinase (Fig. 2). However, these studies did not assess the potential apoptotic effects of this JNK activation (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 5B203580 decreased this ceramide-induced apoptosis, while transfection of dominant negative p38 attenuated the apoptotic response. Interestingly, no activation of JNK was noted with ceramide treatment. Furthermore, cells transfected with dominant negative c-Jun did not have attenuated responses to the ceramide-induced apoptosis. These responses contradict a role for JNK in ceramide-induced apoptosis, while at the same time pointing to a role for p38. More experiments need to be done to define the function of these two kinases in the OL apoptotic response. One possibility is that JNK predominantly mediates TNF-a responses, while p38 predominantly mediates ceramide responses. Since the kinetics of JNK 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 a cell will be induced to undergo apoptosis (Zhang et al., 1996).

10.2. CG-4 Few studies have been done on p38 and JNK in CG-4. However, it has been shown that, as in primary OL, TNF-a and ceramide can increase JNK activity in CG-4 (Zhang et al., 1996, 1998). Perhaps this indicates that the JNK pathways may be similar between primary OL and CG-4, even though their ERK pathways differ. There has also been a set of CG-4 experiments corroborating a role for p38 in OL apoptosis. In these studies, p38 activity was linked to a cytotoxic response involving induction of inducible nitric oxide synthase in CG-4 (Bhat et al., 1999). Thus p38 appears to have

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cell-death related activities in both primary OL and CG-4.

11. Conclusion In conclusion, the roles of the various members of the MAPK family in oligodendrocyte functional biology are quite varied. While ERK activation appears to play a role in the proliferation, process extension and survival of OL, JNK and/or p38 activation appears to mediate OL cell death. Through our studies of the MAPK cascades in OL, we have noted differences in the ERK signal transduction cascades between CG-4 and primary OL. While these findings bring into question the suitability of CG-4 as an OL model, they do not necessarily negate the use of CG-4 in all OL experiments. For instance, CG-4 still appears to be a good model for OL glutamate responses and migration studies. We merely suggest that our findings underline the use of caution when interpreting the significance of CG-4 responses to primary OL responses.

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