Expression of QKI Proteins and MAP1B Identifies Actively Myelinating Oligodendrocytes in Adult Rat Brain

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Molecular and Cellular Neuroscience 17, 292–302 (2001) doi:10.1006/mcne.2000.0941, available online at http://www.idealibrary.com on

Expression of QKI Proteins and MAP1B Identifies Actively Myelinating Oligodendrocytes in Adult Rat Brain Hoi Yee Wu, Mary R. L. Dawson, Richard Reynolds, 1 and Rebecca J. Hardy Department of Neuroinflammation, Division of Neuroscience, Imperial College School of Medicine, Charing Cross Campus, London W6 8RF, United Kingdom

We have studied developing oligodendrocytes in tissue sections as they initiate myelination and have found that the transition from premyelinating oligodendrocytes into myelin-bearing cells is accompanied by a dramatic upregulation in expression of the RNA binding QKI proteins. We show that in mature oligodendrocytes in culture, the localization of cytoplasmic QKI isoforms requires an intact cytoskeleton. Together with previous observations, this indicates that cytoplasmic QKI proteins facilitate movement of mRNAs to myelin via the cytoskeleton. In the adult rat brain, we found that a subset of oligodendrocytes displays characteristics of actively myelinating cells seen during development, i.e., connections to myelin sheaths and elevated levels of QKI proteins and also MAP1B. These observations suggest that instead of merely maintaining myelin, oligodendrocytes in the normal adult CNS are capable of responding to demands for new myelin sheaths. This has important implications for the prospect of repair of myelin in demyelinating conditions such as multiple sclerosis.

INTRODUCTION Myelination in the rodent brain begins during late embryonic development when postmitotic, differentiated oligodendrocytes and the initial stages of myelin formation are first seen in the hindbrain (Hardy and Friedrich, 1996a). The maturation of oligodendrocytes and production of myelin then continue rostrally to-

1 To whom correspondence should be addressed at the Department of Neuroinflammation, Division of Neuroscience, Imperial College School of Medicine, Charing Cross Campus, Fulham Palace Road, London W6 8RF, United Kingdom. Fax: 44 (0)20 8846 7025. E-mail: [email protected].

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ward the rostral forebrain during the first few postnatal weeks. Prior to their initiation of myelination, oligodendrocytes differentiate from their progenitor cells and begin to synthesize myelin proteins such as CNP and MBP (reviewed in Rogister et al., 1999). These steps in the development of oligodendrocytes are well characterized both in vivo and in vitro. Later stages of myelinogenesis, such as oligodendrocyte process contact with axons, initiation of the myelination program, and the process of active myelination, are less easy to study in two-dimensional dissociated cell culture and are consequently more poorly understood. Recent studies of the morphology of oligodendrocytes in intact tissue sections have identified several stages in the progression of oligodendrocytes from the newly differentiated cell to the myelin-forming cell (Hardy and Friedrich, 1996b; Trapp et al., 1997, Butt et al., 1997). First, newly differentiated cells elaborate multiple branching processes with no apparent contacts with target axons; these cells have been termed premyelinating cells (Hardy and Friedrich, 1996b), express the myelin proteins CNP, MBP, and DM20 (Hardy and Friedrich, 1996b; Trapp et al., 1997), and are apparently poised to begin myelination. Premyelinating oligodendrocytes then progress to the transitional stage whereby one of their processes contacts an axon to be myelinated and begins to elaborate a membrane “tube,” a precursor of myelin (Hardy and Friedrich, 1996b). This is repeated by additional processes and is accompanied by pruning of oligodendrocyte branches such that only those supporting myelin sheaths remain. Once this process is complete, transitional cells become mature myelinating cells. These morphological studies give us a good basic understanding of the initial stages of myelination, but 1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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as yet the molecular mechanisms which lead to these changes have not been characterized. A cellular process that certainly accompanies the transformation of premyelinating oligodendrocytes into myelinating cells is remodeling of the cytoskeleton as membrane elaboration and pruning of unattached processes occur. Most of the studies on cytoskeletal function in oligodendrocytes have been carried out on cultured cells (reviewed in Richter-Landsberg, 2000). For example, oligodendrocytes in culture have been shown to express the microtubule-associated protein MAP1B (Vouyiouklis and Brophy, 1993). In neurons, MAP1B is associated with growth cones and axonogenesis (reviewed in Gordon Weeks and Fischer, 2000), indicating that its role in oligodendrocytes is in process modeling during myelinogenesis. As well as playing a role in altering the shape of oligodendrocytes ready for myelination, the cytoskeleton is also used to traffic mRNA of certain myelin proteins along processes to the myelin sheath (Brophy et al., 1993; Carson et al., 1998). These mRNAs are then locally translated to facilitate direct insertion of certain proteins into myelin. It has been demonstrated that translocation of MBP mRNA requires an intact microtubule network and is dependent on the microtubule motor kinesin (Carson et al., 1997). It remains unclear, however, exactly how MBP and other transported myelin mRNAs use the cytoskeleton to move from perikaryal cytoplasm to the myelin sheath prior to their translation. One candidate molecule involved in myelin mRNA localization to myelin is the QKI family of RNA binding proteins. We know that these proteins have a critical function in myelinogenesis as their absence in myelinforming cells is responsible for the phenotype of the dysmyelinating mouse mutant quakingviable (Hardy et al., 1996). Three QKI isoforms have been characterized to date, QKI5, QKI6, and QKI7. These differ only in short carboxy-terminal tails (Ebersole et al., 1996), which are thought to mediate intracellular localization, at least in the case of the nuclear-specific isoform, QKI5 (Wu et al., 1999). All isoforms contain a KH RNA binding motif, are normally expressed in myelin-forming cells (Hardy et al., 1996; Vernet and Artzt, 1997), and can bind mRNA (e.g., Chen and Richards, 1998; Saccomanno et al., 1999). Indeed, a recent study has demonstrated binding of the QKI7 isoform to MBP mRNA (Li et al., 2000). This information, together with the observation that MBP mRNAs are improperly localized to the myelin sheath in quakingviable mice (Barbarese, 1991; Li et al., 2000), predicts a role for QKI proteins in MBP mRNA localization in oligodendrocytes. In this study we have investigated the morphological

and molecular characteristics of oligodendrocytes during the early stages of myelination and have found that as oligodendrocytes develop from premyelinating cells to myelinating cells they dramatically upregulate their expression of QKI proteins. We show that in cultured oligodendrocytes, cytoplasmic QKI proteins are associated with the cytoskeleton; QKI6 and QKI7 both associate with microtubules, whereas QKI6, but not QKI7, associates with actin filaments. Taken with previously published data, this strongly suggests that the role of cytoplasmic QKIs in myelinating oligodendrocytes is to transport MBP and other myelin mRNAs along the cytoskeleton to myelin prior to its translation. While myelinogenesis is primarily a developmental event, there is evidence that myelin continues to be produced well into adulthood (Sturrock, 1980, 1987). In this study we show that normal adult rat forebrain contains a significant population of oligodendrocytes which display the phenotypic characteristics of actively myelinating oligodendrocytes seen during development. These cells are most notable for their elevated levels of QKI proteins and by expression of the cytoskeletal protein MAP1B. These observations suggest that oligodendrocytes present in normal adult brain do not merely maintain existing myelin, but can respond to demands for new myelin sheaths. This has important implications for the reensheathment of demyelinated axons in multiple sclerosis (MS).

RESULTS QKI Levels Are Dramatically Increased in Myelinating Cells First we studied the expression of QKI isoforms during the early stages of myelination in developing rat forebrain. Previously, we and others have shown that oligodendrocytes of the corpus callosum and subcortical white matter begin to express myelin proteins at around 1 week after birth (Levine and Goldman, 1988; Hardy and Reynolds, 1991). At this time, the predominant type of oligodendrocyte present is the premyelinating cell, i.e., that which is yet to begin to show features of myelination. These cells have multiple branching processes, but no apparent contacts to axons (Figs. 1A and 1B; Hardy and Friedrich, 1996b). Also present at this age are oligodendrocytes which have already begun the myelination program and have processes which extend to nascent myelin sheaths (Figs. 1A and 1C). Their cell bodies are often somewhat larger than their predecessors, with abundant cytoplasm, and

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drocytes contain low levels of QKI proteins, whereas cells which have initiated myelination contain dramatically elevated levels of QKI6 and QKI7 (Figs. 1D and 1E). Observation at high magnification confirmed that oligodendrocytes with elevated levels of QKI proteins invariably had observable connections to myelin sheaths (Figs. 1F and 1G) and while the majority of QKI6 and QKI7 was found in perikaryal cytoplasm, some was also found in these connecting processes (Figs. 1F and 1G, arrowheads). Therefore, as oligodendrocytes mature from premyelinating cells, make contact with axons, and begin to elaborate myelin, they dramatically upregulate expression of QKI proteins. Cytoplasmic QKI Proteins Associate with the Cytoskeleton in Cultured Oligodendrocytes

FIG. 1. QKI levels are dramatically upregulated in myelinating oligodendrocytes. Double immunofluorescent labeling of 7-␮m coronal sections of postnatal day 7 rat forebrain with antibodies against MBP (A–D, F) and antisera against QKI7 (E, G). (A) MBP⫹ premyelinating oligodendrocytes (arrowheads) are seen in subcortical white matter, adjacent to newly formed myelinating oligodendrocytes (arrows). (B) A premyelinating oligodendrocyte with multiple branching processes yet no apparent connections to myelin sheaths. (C) A myelinating oligodendrocyte, elaborating fewer processes, some of which connect to myelin sheaths. (D, E) While all MBP⫹ oligodendrocytes express QKI7, myelinating oligodendrocytes (arrows) contain elevated QKI7 levels compared to premyelinating oligodendrocytes (arrowhead). (F, G) At higher magnification, MBP⫹ oligodendrocytes which contain high levels of QKI7 can be clearly seen to have connections to myelin sheaths, confirming their identity as myelinating cells. QKI7 is found in the perikaryal cytoplasm and also in proximal processes (small arrowheads).

they display upregulated expression of myelin proteins (Fig. 1D, cf. arrowheads and arrows). Both types of oligodendrocyte express the myelin proteins MBP and CNP and also QKI proteins (Figs. 1D and 1E). As we have previously shown in mice, QKI5 was found in the nucleus of oligodendrocytes, whereas QKI6 and QKI7 were found predominantly in the cytoplasm of perikarya and proximal processes (data not shown). However, not all CNP⫹ and MBP⫹ cells express similar levels of QKI proteins; premyelinating oligoden-

In order to shed light on the role of QKI proteins in the initial stages of myelination, we proceeded to monitor the expression of QKI proteins in mature oligodendrocytes in culture. Here they terminally differentiate, express myelin proteins, and elaborate membrane sheets, thought to be the in vitro equivalent of the initial stages of the myelin sheath (Reynolds et al., 1989; Rogister et al., 1999). First we determined the localization of QKI proteins within mature oligodendrocytes. We found that, as in vivo, the QKI5 isoform was restricted to the nucleus in these cells (Fig. 2A) and in contrast, QKI6 and QKI7 were found in the cytoplasm of the cell body and process network (Figs. 2C and 2E). Interestingly, QKI6 and QKI7 appeared to be colocalized with cytoskeletal elements. Double immunofluorescent labeling of mature oligodendrocytes revealed that both of these QKI isoforms were indeed colocalized with ␤-tubulin and MAP1B (Fig. 2). In order to determine whether the localization of QKI6 and QKI7 requires an intact microtubule network, we treated mature cultured oligodendrocytes with the microtubule-disrupting drug nocodazole (10 ␮g/ml). After 2 h of treatment, cells were fixed and immunolabeled with antibodies to ␤-tubulin and QKI proteins. Nocodazole treatment significantly disrupted the microtubule network during this period, and this resulted in similar significant disruption of both QKI6 and QKI7 distribution (Fig. 3). QKI5 nuclear localization was not affected by nocodazole treatment (not shown). As well as having an obvious colocalization with microtubule proteins in mature oligodendrocytes, QKI6 was also found in regions of oligodendrocyte membrane sheets rich in actin microfilaments, as determined using fluorescent conjugated phalloidin which binds specifically to actin (Figs. 4A and 4C,

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295 the nucleus was also not disrupted by cytochalasin B treatment (not shown). Cells with the Phenotype of Actively Myelinating Oligodendrocytes Are Present in Adult Rat Brain

FIG. 2. Cytoplasmic QKI proteins colocalize with the cytoskeleton in cultured oligodendrocytes. Double immunofluorescent labeling of cultured mature oligodendrocytes with antisera against QKI5 (A), QKI6 (C), or QKI7 (E) and either ␤-tubulin (B, F) or MAP1B (D). (A, B) QKI5 is restricted to the nucleus of mature, branching oligodendrocytes. (C, D) QKI6 is found in the perikaryal cytoplasm as well as in both major and minor branching processes and shows a distribution similar to that of MAP1B. (E, F) QKI7 appears to be localized in a manner similar to QKI6 and completely colocalizes with ␤-tubulin expression.

arrowheads). However, the proportion of QKI6 that localized to actin was small in relation to that which localized to microtubules (Figs. 2– 4) and was sometimes not apparent in immunofluorescently labeled cells. Nevertheless, to determine if, at least in part, QKI protein localization requires intact actin filaments, we treated mature oligodendrocytes with the actin-disrupting drug cytochalasin B (10 ␮g/ml). After 30 min treatment, phalloidin staining revealed disruption and beading of actin filaments in processes and membrane sheets (Figs. 4E and 4H). QKI6 localization was also partially disrupted such that most actin-containing “beads” also contained QKI6 (Figs. 4D and 4F, arrows), although the majority of QKI6 remained undisturbed, presumably because it was associated with microtubules undisturbed by cytochalasin treatment. In contrast, QKI7 localization appeared to be unaffected by cytochalasin B treatment (Figs. 4G and 4I, arrows). QKI5 localization to

In rodents, myelination proceeds throughout the first few months of postnatal life. After this time, levels of mRNA encoding myelin proteins fall precipitously (reviewed in Campagnoni and Macklin, 1988) and immunoreactivity for the structural myelin proteins is no longer observed in the cell bodies of oligodendrocytes. This has been taken to mean that once the major period of myelination is complete, the function of the oligodendrocyte is merely to maintain existing myelin. However, there is some evidence that new myelin is generated well into adulthood (Sturrock, 1980, 1987), indicating that at least a proportion of oligodendrocytes in the adult CNS elaborate new myelin. Having identified several molecular and morphological features of actively myelinating cells found during development, we decided to look at adult rat brain to see if any cells of this phenotype were present. Oligodendrocytes in mature brain generally have characteristics distinct from their developmental counterparts. They have small cell bodies and scarce cytoplasm, probably because once myelination is complete, their metabolic burden is significantly decreased (Figs. 5A and 5C, arrowheads). Oligodendrocytes continue to express the myelin enzyme CNP, but MBP levels are considerably reduced in the cell body, presumably because the majority of MBP is now synthesized at the myelin sheath (Colman et al., 1982; Carson et al., 1998). In addition, we found that levels of cytoplasmic QKI isoforms (QKI6 and QKI7) were dramatically reduced in mature oligodendrocytes, to levels similar to those found in astrocytes (Fig. 5B, small arrows). While the majority of oligodendrocytes in adult rat brain displayed this phenotype, we nevertheless observed a small but significant population of oligodendrocytes that instead had the phenotype of actively myelinating oligodendrocytes seen during development. Although these cells often had larger cell bodies, had increased cytoplasm, and expressed more CNP than their neighbors, these properties alone are often not sufficient to distinguish them from more matureappearing cells. However, other features definitively distinguish them from the majority of oligodendrocytes in mature brain. For example, they contained significant levels of MBP in their cell body, a characteristic of immature myelinating cells (Fig. 5G). Most dramatically, however, these cells contained significantly

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FIG. 3. Localization of cytoplasmic QKI proteins requires an intact microtubule network. Double immunofluorescent labeling of cultured oligodendrocytes with antisera against QKI6 (A, D) or QKI7 (G) and ␤-tubulin (B, E, H). Cultures were either untreated controls (A–C) or treated with 10 ␮g/ml nocodazole (D–I). (A–C) In control cultures, QKI6 colocalizes with ␤-tubulin in major processes (as does QKI7, see Figs. 2E and 2F), but is also found in ␤-tubulin-free sites at the periphery of the cell (A–C; arrowheads). (D–F) On treatment with the microtubule-disrupting drug nocodazole, both ␤-tubulin and QKI6 localization are similarly disrupted. (G–I) Similarly, QKI7 localization is disrupted in a manner identical to that of ␤-tubulin following nocodazole treatment.

higher levels of QKI proteins than their more matureappearing counterparts (Figs. 5B and 5D, large arrows). Oligodendrocytes with this phenotype were found scattered throughout the cerebral cortex and accounted for up to 12% of the oligodendrocyte population (total CNP⫹ cells per 7-␮m section, 450 ⫾ 25; CNP⫹/QKI7⫹ cells per 7-␮m section, 55 ⫾ 10; total cells counted, 2702). Myelin is less abundant in the cortical gray matter and we were able to determine that cells with elevated levels of QKI proteins invariably had observable connections to myelin sheaths (Figs. 5E to 5H). This fact, and the phenotypic similarity of these cells to myelinating oligodendrocytes seen during development, suggests that these cells were actively myelinating oligodendrocytes. They were seen in both gray matter and white matter regions such as the corpus callosum, striatum, hypothalamus, thalamus, cerebellum, and brain stem, where their frequency did not appear to differ significantly from that in the cerebral cortex. Because QKI expression correlates with expansion of the microtubule network in oligodendrocytes in cul-

ture, and because QKI protein localization is dependent upon an intact microtubule network, we looked at the expression of cytoskeletal proteins in cells with the phenotype of actively myelinating oligodendrocytes in mature rat brain. Although it has previously been shown that oligodendrocytes in culture express MAP1B (Vouyiouklis and Brophy, 1993), it has not been possible to confirm these findings in vivo, mainly due to the abundance of nonoligodendrocyte MAP1B expression found during development of the rodent brain. However, as MAP1B expression levels are dramatically reduced in mature tissue (Gordon Weeks and Fischer, 2000), we were able to demonstrate unequivocal expression of MAP1B in a proportion of oligodendrocytes (Figs. 6A and 6B, arrow). As might be expected, we found that oligodendrocytes containing MAP1B were those with an immature phenotype, i.e., those with high levels of QKI proteins (Figs. 6C and 6D). MAP1B and cytoplasmic QKI proteins were found colocalized in the cell bodies and processes of these cells (Figs. 6C and 6D, inset, arrowheads). We also found that oligodendro-

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FIG. 4. Localization of QKI6, but not QKI7, requires an intact actin filament network. Immunofluorescent labeling of cultured oligodendrocytes with antisera against QKI6 (A, D) or QKI7 (G), costained with fluorescent-conjugated phalloidin (B, E, H). Cultures were either untreated controls (A–C) or treated with 10 ␮g/ml cytochalasin B. (A–C) In control cultures, QKI6 colocalizes with actin at the periphery of mature oligodendrocytes (arrowheads). (D–F) On treatment with the actin-disrupting drug cytochalasin B, actin filaments begin to “bead.” QKI6 localization is similarly disrupted and it localizes within actin beads (D, E, F; small arrows). In contrast, QKI7 does not localize to actin beads (G, H, I; arrows) and is apparently undisrupted by cytochalasin B treatment.

cytes with elevated levels of QKI proteins expressed abundant ␤-tubulin (Figs. 6E and 6F). Therefore, apparently immature myelinating oligodendrocytes found in mature rat brain express the cytoskeletal protein MAP1B, whereas their more mature counterparts do not. New Oligodendrocytes Are Generated from Cycling Cells in the Adult Rat Cortex The adult rat brain contains a widely distributed and numerous population of oligodendrocyte precursor cells that can be labeled with antibodies to the NG2 chondroitin sulfate proteoglycan (Dawson et al., 2000). Therefore, we next investigated whether the actively myelinating oligodendrocytes that we observed in the intact mature rat brain were recently generated from adult progenitor cells. To address this question, we labeled cycling cells by giving adult rats repeated injections of BrdU (three injections, 2 h apart) and followed their fate in the mature cerebral

cortex. After a 6-h pulse of BrdU, 70 –75% of cells incorporating the label also expressed NG2 (Fig. 7A), consistent with earlier studies (Horner et al., 2000; Dawson et al., 2000). No CNP⫹ oligodendrocytes were seen to have incorporated the label at this time, similar to previous studies carried out on developing animals (Reynolds and Wilkin, 1991). Since the halflife of BrdU in injected rats is of the order of a few hours, any cells subsequently seen to contain BrdU will have incorporated it into their DNA within this 6-h period. Six days after BrdU administration, the vast majority of BrdU⫹ cells were still NG2⫹. We did, however, observe some CNP⫹ cells that were double labeled with BrdU antibodies (Fig. 7B). That is, cycling CNP⫺ cells that had incorporated BrdU 6 days previously had by now differentiated into CNP⫹ oligodendrocytes. These cells had the multibranching morphology of premyelinating oligodendrocytes and were found mainly in layer I of the cerebral cortex (Fig. 7B). By 9 days after BrdU injection, CNP⫹/BrdU⫹

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DISCUSSION We have demonstrated that a subset of oligodendrocytes found in the mature CNS have the distinct phenotype of actively myelinating oligodendrocytes seen in abundance during development. These cells have dramatically increased levels of the RNA binding proteins QKI6 and QKI7, and they express MAP1B, indicative of process remodeling. This suggests that a population of oligodendrocytes present in normal, mature rat brain actively generates myelin and that expression of high levels of QKIs and MAP1B are molecular markers for these cells.

FIG. 5. A subset of oligodendrocytes in adult rat brain contains elevated levels of QKI7. Double immunofluorescent labeling of adult rat cortex with antibodies to CNP (A, C, E) or MBP (G) and antisera against QKI7. (A, B) CNP⫹ oligodendrocyte cell bodies are easily detected in adult rat cortex. While the majority of these cells contain low levels of QKI7 (arrowheads), similar to those found in astrocytes (B, small arrows), a subset of oligodendrocytes expresses significantly higher levels of QKI proteins (arrows). (C, D) CNP⫹ oligodendrocytes with high levels of QKI7 (arrow) are found among CNP⫹ cells with low levels of QKI7 (arrowheads). (E, F) When viewed at higher magnification, oligodendrocytes with elevated QKI7 levels are seen to have observable connections to myelin sheaths; QKI7 can be seen within processes to myelin (arrowheads). (G, H) Oligodendrocytes with elevated levels of QKI7 also express MBP in their cell body and proximal processes often connecting to myelin sheaths (arrowheads).

cells were more frequently observed and were seen throughout the dorsal layers of the cerebral cortex (Fig. 7C).

FIG. 6. Adult oligodendrocytes with elevated levels of QKI proteins also express MAP1B. Double immunofluorescent labeling of adult rat cortex with antibodies to CNP (A) and MAP1B (B), QKI7 (C) and MAP1B (D), and QKI7 (E) and ␤-tubulin (F). (A, B) Unlike most CNP⫹ oligodendrocytes in adult rat cortex (arrowheads), a subset of CNP⫹ cells expresses MAP1B (arrows). (C, D) These oligodendrocytes also contain elevated levels of QKI7 (arrow). In these cells, both QKI7 and MAP1B are found in perikaryal cytoplasm and also in processes leading to myelin (C, D, inset, arrowheads). (E, F) This same subset of oligodendrocytes also contains elevated levels of ␤-tubulin (arrow).

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A Role for QKI Proteins in mRNA Transport in Oligodendrocytes We have previously demonstrated that the KH domain-containing RNA binding proteins of the QKI family are expressed in glial cells in the developing rodent nervous system and at high levels in myelin-forming cells (Hardy et al., 1996). Absence of the QKI6 and QKI7 isoforms in myelin-forming cells in the mouse mutant quakingviable leads to chronic dysmyelination, demonstrating that correct regulation of QKI protein expression is critical for proper formation of myelin (Ebersole et al., 1996; Hardy et al., 1996; Hardy, 1998). In this study, we show that the expression levels of QKI proteins are regulated in close association with the initial stages of myelination, further emphasizing an important role for these proteins in myelin formation. We also show for the first time that cytoplasmic QKI proteins require an intact cytoskeleton for their proper intracellular localization. QKI6 associates with microtubules and actin filaments, whereas QKI7 associates with microtubules only. It is not known whether cytoplasmic QKI proteins bind directly to cytoskeletal elements or associate with the cytoskeleton through intermediary proteins. In either case, it seems likely that the ability of QKI6 to interact with microtubules or actin filaments is mediated through different domains of the protein. As QKI6 and QKI7 differ only in their short carboxy-terminal tails (8 and 14 amino acids for QKI6 and QKI7, respectively; Ebersole et al., 1996), the QKI6-specific tail is most likely responsible for conferring the ability to associate with actin filaments. In contrast, ability to associate with microtubules would be conferred by sequences common to both QKI6 and QKI7. Of course these sequences are also found in QKI5, but this isoform is excluded from cytoplasmic cytoskeletal interactions as it is targeted to the nucleus by its own specific carboxy-terminal tail (Wu et al., 1999). Several lines of evidence suggest a role for QKI proteins in the translocation of MBP and other myelin mRNAs to the myelin sheath. First, MBP mRNA is not effectively transported to myelin in the absence of QKI proteins in quakingviable mutants (Barbarese, 1991; Li et al., 2000). Second, it has been demonstrated that QKI7 binds to MBP mRNA and that the MBP mRNA 3⬘UTR, known to contain sequences important for localization, is important for this binding (Li et al., 2000). Our data now suggest that cytoplasmic QKI proteins mediate mRNA interactions with the cytoskeleton during transport of mRNAs in oligodendrocytes. Furthermore, distinct QKI isoforms may facilitate the traffic of mRNAs on different cytoskeletal highways. The major route used by MBP mRNAs leaving

the oligodendrocyte cell body is the microtubule network (Carson et al., 1997), which could be mediated by either QKI6 or QKI7. However, local delivery of mRNAs to the periphery of the cell may be mediated by QKI6 association with actin filaments. The notion that a single protein can transport mRNA on both actin filaments and microtubules is not new. For example, the KH domain-containing RNA binding protein ZBP1/Vg1 is involved in both actinmediated transport of actin mRNA in fibroblasts and microtubule-mediated transport of Vg1 mRNA in Xenopus oocytes (reviewed in Oleynikov and Singer, 1998). Adult Brain Contains Actively Myelinating Oligodendrocytes We have demonstrated that in the normal adult rat brain, a small but significant proportion of oligodendrocytes displays the phenotype of myelinating cells seen during development. Most notably these cells have elevated levels of QKI proteins and, unlike their more mature counterparts, express the cytoskeletal protein MAP1B. Due to the elevated expression of myelin proteins in these cells and in view of the putative role of QKI proteins in the movement of MBP and other mRNAs to newly formed myelin, we consider it likely that adult oligodendrocytes with this phenotype are actively myelinating cells. However, our data cannot distinguish between cells that are carrying out myelination for the first time and preexisting myelinating cells that have reverted to a more immature phenotype. Previous studies have shown the presence of MAP1B in cultured oligodendrocytes (Vouyiouklis and Brophy, 1993) but this has not been possible to demonstrate in vivo, due to the ubiquitous nature of MAP1B expression in the developing brain. Here we show that MAP1B expression is normally downregulated during oligodendrocyte maturation, but is present in oligodendrocytes containing high levels of cytoplasmic QKI proteins. This observation reinforces the notion that this subset of oligodendrocytes is actively producing new myelin. MAP1B is thought to play a role in growth cone modeling and axonogenesis during development and is found in areas of axon growth in mature brain (reviewed in Gordon Weeks and Fischer, 2000). Thus, the presence of MAP1B indicates that these oligodendrocytes are undergoing cytoskeletal reorganization and process remodeling, consistent with the formation of new myelin. This suggests that the actively myelinating cells we have observed in mature tissue are not merely maintaining existing myelin, but are generating new connections to axons and producing new myelin sheaths.

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FIG. 7. Oligodendrocytes are generated from cycling cells in the adult rat cortex. Double immunolabeling of adult rat cortex following administration of BrdU using antibodies to BrdU (red) and NG2 (A, green) or CNP (B, C, green). BrdU was administered to adult rats in three injections, 2 h apart. (A) Immediately following the 6-h BrdU pulse, most cells that have incorporated BrdU are also NG2⫹. These cells are sometimes seen in pairs, indicating that cell division has already taken place by this time. (B) By 6 days after BrdU administration, some BrdU⫹ cells express CNP and these cells have the multiprocessed morphology of premyelinating cells; this example lies in layer I of the cortex. (C) BrdU⫹/CNP⫹ cells were more abundant by 9 days after BrdU administration and were found in deeper cortical layers.

Mature Rat Brain Is Capable of Generating New Oligodendrocytes and Myelin Despite the evidence showing that the vast bulk of myelination occurs during the first few months of postnatal life, there are some data which show that myelin is generated well into adulthood. For example, the number of myelinated axons increases in the anterior commissure and corpus callosum of adult rats (Sturrock, 1980, 1987; Nunez et al., 2000), and myelin content of the brain increases in a linear fashion with age (Norton and Poduslo, 1973). We have now identified a subset of oligodendrocytes in both adult gray and adult white matter that is likely to be producing new myelin. We have also shown that new oligodendrocytes are generated during adulthood from cycling cells. This finding complements recent retroviral lineage tracing experiments which show that oligodendroglia generated from cycling cells continue to accumulate in the cerebral cortex of 8-month-old rats (Levison et al., 1999). These results suggest that either adult oligodendrocytes or their progenitors are capable of responding to signals demanding the production of myelin. Whether this is a response to plasticity of adult brain or a general requirement for additional myelin in older animals remains to be determined. For example, studies in primates have shown that oligodendrocyte degeneration is a common feature of the aging brain (Peters, 1996). Therefore, it is feasible that oligodendrocytes and myelin are generated as part of an ongoing repair process to replenish degenerating cells. Whatever the impetus for oligodendrocyte and myelin generation in mature brain, the capacity to stimulate active myelination has important implications for repair processes following pathological damage to myelin and oligodendrocytes in diseases such as MS. Recent studies have suggested that a lack of oligodendrocytes,

or their progenitors, is not necessarily responsible for the failure of remyelination in chronic MS lesions (Lucchinetti et al., 1999; Wolswijk, 2000), and so stimulation of active myelination by surviving oligodendrocytes may provide the key to promoting repair. The unique expression of elevated QKI levels and of MAP1B in actively myelinating cells in the adult may prove a useful marker for remyelination in studies assessing putative repair strategies for MS.

EXPERIMENTAL METHODS Antibodies and Reagents Anti-QKI5, -QKI6, and -QKI7 rabbit polyclonal antisera were a gift from Karen Artzt (University of Texas, Austin) and have been previously characterized (Hardy et al., 1996b). Rabbit polyclonal NG 2 antibodies were obtained from Joel Levine (SUNY Stony Brook). Monoclonal antibodies were purchased as follows: CNP from Chemicon (Harrow, UK), MAP1B and ␤-tubulin from Sigma (Poole, UK), MBP from Boehringer Mannheim (Roche Diagnostics, Lewes, UK), and BrdU from DAKO (Cambridge, UK). Anti-rabbit or subclass-specific antimouse fluorescence- and biotin-conjugated secondary antibodies were purchased from Amersham Pharmacia (Amersham, UK), Southern Biotechnology (Cambridge Bioscience Ltd., Cambridge, UK), or Sigma. Alexa-conjugated streptavidin (Alexa 488 and Alexa 546) was purchased from Molecular Probes (Cambridge Bioscience Ltd.). Tissue Processing and Immunostaining Sprague–Dawley rats age postnatal day 7 or adult (250 g, 3 months) were perfused through the left ven-

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tricle with 4% paraformaldehyde in PBS. Brains were removed and postfixed for 4 h at 4°C in the same fixative. Following cryoprotection overnight at 4°C in 30% sucrose in PBS, brains were frozen in OCT, and 7-␮m coronal (forebrain) or sagittal (cerebellum and hindbrain) sections were cut and thaw mounted onto coated slides. Sections were stored at ⫺20°C until required. Sections were immunolabeled as previously described (Hardy et al., 1996). Briefly, following pretreatments (10 min in ⫺20°C MeOH for CNP and MBP labeling; 10 min in 0.1% Triton in PBS for MAP1B labeling), sections were washed thoroughly in PBS and incubated overnight in primary antibodies diluted in 10% normal goat serum in PGBA (PGBA is 0.1% gelatin, 1% BSA, and 0.1% sodium azide in PBS). Sections were washed (3 ⫻ 10 min in PBS) and incubated in fluorescence- or biotin-conjugated secondary antibodies diluted in PGBA for 1 h. Following washes, sections were incubated in fluorescence-conjugated streptavidin diluted in PGBA, if necessary. Sections to be processed for double immunolabeling were then incubated with primary antibodies and secondary and tertiary reagents for 3, 1, and 1 h, respectively. For all immunolabeling procedures which included QKI protein antisera, 0.5 M Tris HCl, pH 7.4, was substituted for PBS, as previously described (Hardy et al., 1996). Cell Culture Oligodendrocyte progenitors were isolated from P4 to P5 rat cerebral hemispheres using immunoselection with O4 antibodies. Briefly, cerebral hemispheres were enzymatically dissociated with trypsin (0.5 g/L), EDTA (0.2 g/L) solution (Sigma T-3924) and then mechanically dissociated to a single-cell suspension. Adherent cells were removed from the population by incubation of the cell suspension in a 75-cm tissue culture flask for 20 to 30 min at 37°C. Unbound cells were resuspended in Earle’s balanced salt solution ⫹ 5% FCS and incubated on an O4-coated panning dish. After 1 h at 4°C, unbound cells were washed from the dish (five washes in DMEM) and bound cells removed from the surface by vigorous pipetting. Cells were plated at a density of 2 ⫻ 10 4/cm 2 on poly-l-lysine-coated glass coverslips in 24-well plates in OLP medium (DMEM with 10 ␮g/ml insulin, 50 ␮g/ml transferrin, 0.05% BSA, 3 nM sodium selenite, 2 mM N-acetyl-l-cysteine, 2 mM glutamine, 1 mM sodium pyruvate, 20 nM progesterone, 30 ng/ml thyroid hormone, 10 nM hydrocortisone). Cells were processed for immunolabeling or drug treatment on day 4 to 6 of culture.

Immunolabeling of Cell Cultures Cells grown on coverslips were fixed for 20 min in 4% paraformaldehyde in PBS at room temperature. Cells were then permeabilized in 0.1% Triton X-100 for 5 min prior to incubation with primary antisera diluted in 10% NGS in PGBA for 45 min. Following three washes in PBS, cells were incubated in fluorescence- or biotinconjugated secondary antibodies for 30 min, followed by incubation in fluorescence-conjugated streptavidin for a further 30 min if necessary. BrdU Incorporation and Immunolabeling Adult Sprague–Dawley rats (250 g, approximately 3 months) were given three injections of bromodeoxyuridine (50 ␮g/g body weight, intraperitoneal) at 2-h intervals and were sacrificed at 2 h, 6 days, or 9 days after the last injection. Tissue was prepared and cryostat sections were cut as described above. Sections to be immunolabeled using BrdU antibodies were incubated in 70% ethanol at ⫺20°C for 10 min, followed by 2 ⫻ 10 min washes in PBS ⫹ 0.1% Triton X-100. They were then incubated for 20 min in 1 N HCl at 45°C, followed by 2 ⫻ 10 min washes in 0.1 M sodium borohydride, pH 8.6, and 2 ⫻ 10 min washes in PBS ⫹ 0.1% Triton X-100. Anti-BrdU antibodies and either anti-NG2 or anti-CNP antisera, diluted in PBS ⫹ 0.1% Triton X-100, were added to sections and incubated for 48 h at 4°C. Following 3 ⫻ 10 min washes in PBS ⫹ 0.1% Triton X-100, secondary antibodies were added to sections for 45 min at room temperature.

ACKNOWLEDGMENTS We thank Karen Artzt for gifts of QKI antisera, Joel Levine for NG 2 antibodies, and Sue Ansell and Helen Payne for technical assistance. This work was funded by the Wellcome Trust and the Multiple Sclerosis Society of Great Britain and Northern Ireland. R.J.H. was a Wellcome Research Career Development Fellow.

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