Cytoplasmic and nuclear localization of myelin basic proteins reveals heterogeneity among oligodendrocytes

Journal of Neuroscience Research 46:24&257 (1996)

Rapid Communication Cytoplasmic and Nuclear Localization of Myelin Basic Proteins Reveals Heterogeneity Among Oligodendrocytes Rebecca J. Hardy, Robert A. Lazzarini, David R. Colman, and Victor L. Friedrich, Jr. Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York

Myelin basic proteins (MBPs) are major proteins of central nervous system (CNS) myelin, where they fa- cilitate the apposition of cytoplasmic faces of myelin lamellae. Myelin-bearing oligodendrocytes transport MBP mRNA to myelin, where newly translated protein is directly inserted into the myelin sheath. An apparent absence of MBPs in oligodendrocyte perikarya has suggested that MBP localized to the soma is translationally inert. We now demonstrate by confocal immunofluorescence microscopy that not only are MBPs present in the majority of oligodendrocyte perikarya but oligodendrocytes are heterogeneous with respect to their localization of MBPs; MBPs are concentrated in some cells at the plasmalemma and distributed in others throughout the cytoplasm and, surprisingly, the nucleus. MBPs are present in the nuclei of over half of oligodendrocytes in the adult, but in almost all MBP+ oligodendrocytes during myelinogenesis. Transport of MBPs into nuclei appears to be a regulated process since some cells exhibit robust MBP accumulation in their cytoplasm but exclude MBPs from their nuclei. We show that oligodendrocyte nuclei contain all four major MBP isoforms, but that in transgenic mice, the epitope-tagged 14 kD MBP isoform preferentially segregates to the plasmalemma. Our data demonstrate that oligodendrocytes are not required to exclude MBPs from their perikarya and suggest that MBPs have a specific function in the oligodendrocyte perikarya and nucleus.

Key words: myelination, development, nucleus, oligodendrocytes, confocal laser scanning microscopy, immunocytochemistry

0 1996 Wiley-Liss, Inc.

INTRODUCTION Myelin basic proteins (MBPs) are major proteins of

myelin, where they mediate the compaction of myelin

lamellae to form the major dense line (Privat et al., 1979; Readhead et al., 1987). In the central nervous system (CNS), MBPs are expressed exclusively by oligodendrocytes and initially appear in newly differentiated oligodendrocyte perikarya 2-3 days after their final division (Reynolds and Wilkin, 199 1). Following myelinogenesis, however, MBPs are no longer detected in oligodendrocyte somas and appear to be restricted to myelin (Sternberger et a]., 1978; Roussel and Nussbaum, 1981; Reynolds and Wilkin, 1988).

In rodents, MBPs are encoded by a single 30 kb gene composed of 7 exons and alternative splicing of this gene results in the production of discrete mRNA species which encode the 4 major MBP isoforms of 21.5, 18.5, 17, and 14 kD (deFerra et al., 1985). Exons 1, 3, 4, 5, and 7 are common to all these forms, whereas only the 21.5 and 17 kD forms contain sequences encoded by exon 2, and only the 21.5 and 18.5 kD forms contain exon 6. The functions of the individual MBP isoforms are not clear, although the 14 and 18.5 kD isoforms greatly predominate in compact myelin (Barbarese et al., 1977) and the 14 kD isoform can induce major dense line formation by itself in transgenic MBP-deficient mutant shiverer mice (Kimura et al., 1989). Studies involving the expression by transfection of individual MBP isoforms in both HeLa cells and oligodendrocytes from shiverer mice showed that the 18.5 or 14 kD isoforms (i.e., those without exon 2) were associated with cell membranes, whereas the 21.5 or 17 kD isoforms were localized within the cytoplasm and nucleus (Staugaitis et al., 1990; Allinquant et al., 1991).

MBP is unique among myelin proteins in that its mRN A is translocated centrifugally from the oligoden-

Received April 15, 1996; revised May 8, 1996; accepted May 16, 1996.

Address reprint requests to Rebecca J . Hardy, Brookdale Center for Molecular Biology, Box 1126, Mount Sinai School of Medicine, New York, NY 10029.

0 1996 Wiley-Liss, h e .

MBP Localization in Oligodendrocyte Perikarya 247

(Birmingham, AL), Amersham (Amersham, UK), and Vector (Burlingame, CA).

Tissue Preparation Wild-type (C57BL6xDBA Fls) or MBP transgenic

mice (Gow et al., 1992) were perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. In some experiments, improved MBP immunolabeling was achieved using a modified Bouin’s fixative. However, this fixative did not yield optimal cell surface labeling using 0 4 or R-mAb mAbs. Adult mice were 3-12 months of age. Brains to be vibratome sectioned were postfixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C. Tissue was then embedded in 4% low melting point agarose and 75 p,m sections were cut on a vibratome. Brains to be cryostat sectioned were cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4”C, embedded in Tissue-Tek (Miles Scientific, Elkhardt, IN), and snap frozen. Sections (5-10 km) were thaw mounted onto gelatin-coated slides and refrozen for stor- age at -20°C.

Immunocytochemistry Vibratome sections from adult tissue were perme-

abilized either by snap freezing in liquid nitrogen or by incubating in 0.1% Triton X-100 for 10 min. Cryostat sections were incubated in methanol at - 20°C for 10 rnin and then incubated in 40 p,g/ml sodium borohydride in PBS for 10 rnin followed by 2 washes in PBS (10 rnin each). Both adult and developing tissue sections were blocked in 10% normal goat serum, 1% gelatin, 5% bo- vine serum albumin (BSA), and 0.05% sodium azide in PBS for 30 rnin at room temperature (RT). They were then incubated overnight with primary antibodies diluted in the same solution and then in fluorochrome- or biotin-conjugated species or subclass-specific secondary antibodies for 3 hr at RT followed by fluorochrome-conjugated streptavidin for 1 hr at RT. Each of these steps was followed by 3 washes in PBS (20 min each). Sections were then mounted on slides and coverslipped using 2.5% diazabicyclo-octane (Johnson et al., 1982) in glycerol/0.5 M Tris, pH 8.6 (9:1), or, where indicated, in 1.3% phenylenediamine in phosphate-buffered glycerol (Quinn and Weber, 1988). For immunolabeling in conjunction with BrdU labeling, sections were immunolabeled with MBP and 0 4 or R-mAb antibodies and corresponding fluorochrome-conjugated secondary antibodies as described above. Double stranded DNA was then denatured using 2 N HC1 treatment, as described previously (Hardy and Reynolds, 1991), after which sections were immunolabeled using anti-BrdU antibodies as described above. For triple labeling experiments, we used Cy 5@-conjugated secondary or streptavidin reagents (Molecular Probes,

drocyte soma into cellular processes and cytoplasmic compartments of the myelin sheath (Colman et al., 1982; Trapp et al., 1987; Ainger et al., 1993; Landry et al., 1994). Considering the high affinity of MBPs for membranes, it has been proposed that the translocation of MBP mRNA to the myelin sheath is a mechanism by which newly synthesized protein can be inserted directly into the myelin membrane (reviewed in Brophy et al., 1993). MBP mRNAs are detected in cell bodies, however (Trapp et al., 1987; Landry et al., 1994), but the apparent absence of MBP polypeptides from oligodendrocyte perikarya has suggested that in myelin-bearing oligodendrocytes, MBP mRNA localized in the cell body is translationally inert, and hence translation of mRNA occurs only at the myelin sheath (Colman et al., 1982; Allinquant et al., 1991; Brophy et al., 1993; Pfeiffer et al., 1993).

Here we report that not only are MBPs present in the cell bodies of the majority of mature, myelin-bearing oligodendrocytes, but individual oligodendrocytes are heterogeneous with respect to their intracellular localization of MBPs. Such heterogeneity is developmentally regulated and results from substantial variation among individual oligodendrocytes in their expression of MBP isoforms .

MATERIALS AND METHODS Antibodies

Rabbit anti-MBP antiserum, raised against the gel- purified 14 kD MBP isoform, recognizes all 4 MBP isoforms (Staugaitis et al., 1990). Additional rabbit anti- MBP antiserum raised against a peptide encoded by exon 1 of the MBP gene (Zeller et al., 1989) and mouse anti- MBP monoclonal antibodies (mAbs) raised against a peptide encoded by exon 5 (Boehringer, Indianapolis, IN) were also used. Rabbit anti-proteolipid protein (PLP) antiserum used in Western blotting has been described previously (Colman et al., 1982). 0 4 mAbs (Sommer and Schachner, 1981) react with sulfatide on the surface of differentiated oligodendrocytes and myelin, as well as an unidentified antigen (POA) on the surface of oligodendrocyte progenitors (Bansal et al., 1989, 1992). R-mAb (Ranchst et al., 1978) reacts with a variety of antigens on newly differentiated oligodendrocytes including galactocerebroside and sulfatide (Bansal et al., 1989). Mouse anti-histone mAbs were purchased from Chemicon (Temecula, CA), biotin-conjugated mouse anti-P-galactosidase (P-gal) mAbs from Sigma (St. Louis, MO) , and mouse anti-5’-bromo-2-deoxyuridine (BrdU) mAbs from DAKO (Carpinteria, CA). Fluoro- chrome- and biotin-conjugated secondary antibodies and fluorochrome-conjugated streptavidin were purchased from Jackson (West Point, IL), Southern Biotechnology

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Fig. 1. Conventional epifluorescence imaging of MBPs in oligodendrocyte cell bodies. a,b: Cerebral cortex of an adult MBP transgenic mouse, labeled by two-color immunofluorescence for MBP (a) and P-galactosidase (b). MBP+ cell bodies (a, arrows), visible among the immunofluorescent myelin sheaths, are positively identified as oligodendrocytes since

Eugene, OR), which emit at 647 nm, in addition to flu- orescein and Texas Red-conjugated reagents.

Confocal Microscopy Vibratome sections were analyzed with a Leica

TCS confocal microscope, equipped for three-color si- multaneous fluorescence detection (green, red, and long wavelength red), as well as a transmitted light detector yielding phase and brightfield images. Phase and brightfield images were collected simultaneously with the confocal fluorescence images and were in perfect register with them. Each fluorescence channel was digitized in 8 bit words (256 intensity levels) and scanned field sizes were adjusted to give 0.05-0.1 pdpixel . Single confocal optical sections had an effective thickness of approximately 0.5 pm. For the analysis of cell shape we ac- quired three-dimensional image data sets by collecting images spaced regularly along the vertical axis at around 0.2 pm intervals. In some cases, cells were scanned twice: once at low magnification and low resolution (pixel size 0.5 pm) to record information on cell shape and again at higher magnification and high resolution (pixel size 0.15 pm) to record the distribution of MBPs within cell bodies (e.g., see Fig. 4). Composite figures were assembled in Adobe Photoshop@ and printed on a dye sublimation photoprinter. For better intensity dis- crimination, blue and green are rendered in the figures with saturation decreasing as signal levels increase; high signal levels therefore appear bright and unsaturated.

Transgenic Mice Transgenic mice were generated using mouse

cDNA encoding the 14 kD MBP isoform (exons 1, 3,4, 5, and 7) with an 11 amino acid tag sequence (Ala-Ser- Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg) at the N-termi-

they contain the transgenic oligodendrocyte-specific marker protein P-galactosidase (b, arrows). c: MBP+ cell bodies in cerebral cortex of an adult wild-type mouse fluoresce with intensity comparable to nearby myelin sheaths. a,b: 5 pm thick cryostat section; c: 75 pm thick Vibratome section.

nus, cloned in the MP vector, which we have previously demonstrated to confer oligodendrocyte-specific expression (Gow et al., 1992).

Cell Fractionation and Western Blotting Brains from postnatal day (P) 14, P25, or adult

MPP transgenic mice were homogenized on ice in 0.25 M sucrose in 50 mM Tris, pH 7.4, using a Wheaton homogenizer. Protease inhibitors (aprotin 0.1 pg/ml, leu- peptin 1 pg/ml, pefabloc 0.1 mM, pepstatin 1 pg/ml) were present throughout. Brain homogenates were adjusted to 1.25 M sucrose and spun at 5,OOOg for 10 min through a 1.4 and 1.5 M sucrose cushion. The nuclear pellet was then resuspended in 0.25 M sucrose, read- justed to 1.25 M sucrose, and spun at 5,000g for 10 min. The resultant pellet was washed twice in 50 mM Tris, pH 7.4, and then resuspended in 100 p1 of the same buffer. In some experiments, pellets were washed in 0.1% NP- 40, followed by 50 mM Tris, pH 7.4, prior to resuspen- sion. Myelin fractions were collected from the same aged mice using sucrose density gradient centrifugation followed by hypotonic shock as previously described (Col- man et al., 1982). Myelin and nuclear fractions were diluted in loading buffer [final concentration: 0.25 M Tris , pH 6.8, 10% glycerol, 1 % sodium dodecyl sulfate (SDS), 1% P-mercaptoethanol, 0.05% bromophenol blue] and subjected to SDS discontinuous polyacrylamide gel electrophoresis (PAGE). Proteins were then transferred to Immobilon P membrane (for MBP) or ni- trocellulose (for PLP) and detected using MBP or PLP antibodies at 1 : 1,000 dilution, followed by alkaline phosphatase-conjugated donkey anti-rabbit secondary antibody (Jackson) diluted 1:5,000, using the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate detection system.

Fig. 2. Confocal microscopic imaging of oligodendrocyte cell bodies showing two distinct patterns of MBP localization. a x : Medulla of an adult wild-type mouse, imaged simultaneously for MBP immunofluorescence (a,c) and for transmitted light phase contrast (b,c). Nuclei are visible in the transmitted light image (b). MBP immunofluorescence is distributed in rings around three nuclei (b,c, asterisks); the overlay (c) clearly reveals that the perikaryal cytoplasm of one oligodendrocyte lacks MBP immunofluorescence (a,c, arrow) and that MBP immunoreactivity is restricted to the region of the plasma-

Fig. 3. Nuclear, cytoplasmic, and plasmalemmal MBP immunolabeling in oligodendrocytes of wild-type adult mouse cerebral cortex. a<: A single confocal optical section of tissue immunolabeled for MBP (a,c, green) and histones (b,c, red) shows MBP concentrated at the perikaryal plasmalemma (a, arrowheads), but clearly also present both in perikaryal cytoplasm and within the nucleus. By contrast, an immediately adjacent cell (presumably not an oligodendrocyte) lacks immu-

lemma. d-f: Caudoputamen of an adult wild-type mouse, imaged simultaneously for MBP immunofluorescence (d,f) and transmitted light phase contrast (e,f). MBP is distributed throughout the cytoplasm (a,e,f, arrowhead) while the nucleus shows no MBP signal (e,f, asterisk). The bright band running diagonally across the upper right is a bundle of myelinated axons. For best clarity in the overlays (c,f), MBP immunofluorescence is represented in the psuedocolor scale shown in the inset (d) .

noreactivity throughout its cell body, including the nucleus (asterisks). d-f: Another oligodendrocyte displays robust MBP labeling of the cytoplasm and of the nucleus. This tissue was counterstained with phenylenediamine, which preferentially labels heterochromatin (e,f). Such regions are devoid of MBP immunoreactivity (d-f, arrow). Again, MBP immunoreactivity is absent from the nucleus and cytoplasm of an adjacent, non- oligodendrocytic, cell (d-f, + ).

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Fig. 4. Nuclear and cytoplasmic MBP immunolabeling in im- mature oligodendrocytes both with and without connections to myelin. a-c: An oligodendrocyte in subcortical white matter of postnatal day 3 mouse forebrain immunolabeled with 0 4 mAb (a,c, green) and MBP antiserum (b,c, red) and imaged in two separate sets of serial optical sections. In a wide-field, low resolution reconstructed ‘‘thick section” image of the 0 4 labeling, this cell shows no attachment to myelin sheaths (a). A second image set obtained at higher resolution shows MBP

immunoreactivity in the cytoplasm and nucleus of this cell (b,c). d-f: An oligodendrocyte in postnatal day 5 mouse medulla, labeled with 0 4 (d,f, green) and MBP antiserum (e,f, red) and imaged in serial optical sections. The reconstructed “thick section” shows a process connecting the cell body with a myelin sheath (d, arrow). A single confocal optical section through the cell body reveals MBP immunoreactivity within both the cytoplasm and the nucleus (e,f).

RESULTS ~ P s Are Present in Oligodendrocyte Perikarya in Adult Mouse Brain

We found that MBP immunoreactivity was readily detected by immunofluorescence in the cell bodies of oligodendrocytes in adult mice. For positive identification of oligodendrocytes, we made use of the marker transgene MPP, which directs expression of the Escher-

ichia coli lac2 gene in oligodendrocytes in sufficient amounts to yield clear immunolabeling of oligodendrocyte cell bodies, even when surrounded by myelin (Gow et al., 1992). Following double immunolabeling of cryostat or vibratome sections of adult mice, we found P-gal+ oligodendrocyte cell bodies clearly immunore- active for MBP in brain and spinal cord regions which were sparsely or moderately myelinated (Fig. 1). In heavily myelinated regions, however, resolution of oligodendrocyte plasmalemma from surrounding MBP + myelin sheaths was not possible using regular epifluo-

Fig. 5. MBP immunolabeling at the plasmalemma of newly differentiated oligodendrocytes. a-c: Two cells in postnatal day 6 mouse medulla, immediately following 6 hr of BrdU administration, and mple immunolabeled as indicated, are imaged in a single confocal optical section. An 04+1MBP- oligodendrocyte progenitor has incorporated BrdU (a,c, arrow) but a more differentiated 04+/MBP+ oligodendrocyte has not ( a x , arrowhead). d-f: An oligodendrocyte in postnatal day 8 mouse medulla, 2 days following BrdU administration, and double immunolabeled with antibodies to MBP (d,f, red) and BrdU (e,f, blue), is imaged in a single confocal optical section. This oligodendrocyte is newly differentiated from a progenitor cell which incorporated BrdU 2 days previously (e,f). MBP immunoreactivity is present at the plasmalemma (d,f, arrowheads) but is virtually absent from the cytoplasm (d,f, arrow).


immunolabeling of tissue sections from the MBP-deficient mouse mutant shiverer (not shown).

We found that nuclear MBPs occurred only in conjunction with cytoplasmic MBPs; cells with MBP labeling at the plasmalemma, but not in the cytoplasm, did not contain MBPs in their nuclei (Fig. 2a-c). The intensity of nuclear MBP immunofluorescence varied widely but was almost always equivalent to or lower than the signal from the surrounding cytoplasm; rarely did MBPs appear concentrated in the nucleus. Some cells had substantial MBP immunofluorescence in perikaryal cytoplasm but no de- tectable signal in the nucleus; i.e., MBPs were excluded from the nucleus in these cells (Fig. 2d-f).

MBPs Are Present in the Cytoplasm and Nuclei of Oligodendrocytes of Developing Mice

We examined the distribution of MBPs within oligodendrocytes at several stages of development. MBP + cells were present in the medulla at birth and increased in number in this region and throughout the midbrain and then the forebrain over the next month. The vast majority of MBP + oligodendrocytes (>90%) in neonatal mouse brain showed some degree of MBP immunoreactivity within their cytoplasm and within their nuclei. From P1 to P14, MBP + oligodendrocytes without cytoplasmic and nuclear MBP labeling remained a minority of total oligodendrocytes, but such cells were seen with increas- ing frequency from P14 onward through to adulthood. However, as late as P20-P35, the majority of MBP+ oligodendrocytes (>go%) possessed both cytoplasmic and nuclear MBPs.

rescent microscopy. We therefore examined sections of adult mouse brain by confocal microscopy and were able to verify the presence of MBPs in oligodendrocyte cell bodies, even within white matter (e.g., Fig. 2a-c). Al- though the intensity of MBP labeling varied among cells, the majority of oligodendrocyte cell bodies throughout the brain, as identified by p-gal labeling, contained MBPs. Immunolabeling of oligodendrocyte perikarya was achieved with several different anti-MBP preparations. Wild-type adult mouse brain showed MBP immunolabeling patterns identical to those seen with the MPP transgenic mouse; indeed, the use of confocal microscopy precluded the need to locate oligodendrocyte cell bodies using MPP mice, as this enabled us to clearly identify individual MBP + oligodendrocyte perikarya, even in white matter.

Perikaryal MBPs Show Three Distinct Patterns of Distribution

The high resolution achieved with the confocal microscope enabled us to view the distribution of MBPs within wild-type adult oligodendrocyte perikarya and we observed three different patterns of MBP distribution. In some cells, MBP immunoreactivity was found only at the plasmalemma, where labeling was sometimes as strong as that in nearby myelin sheaths (Fig. 2a-c); the perikaryal cytoplasm appeared entirely unlabeled (Fig. 2a-c, arrow). In other cells, MBPs were distributed throughout the perikaryal cytoplasm (Fig. 2d-f). In this case, due to the strong labeling of the cytoplasm, it was not always clear whether MBPs were also associated with the plasmalemma, although in some cells a visible concentration of MBPs at the plasmalemma was main- tained (e.g., Fig. 3a). Finally, some strongly MBP+ oligodendrocytes showed high MBP levels throughout their cell bodies, apparently within both the perikaryon and the nucleus (Fig. 3). To confirm that MBPs were indeed contained within the nucleus of these cells, we counterstained sections using either immunolabeling of histones or the fluorescent stain phenylenediamine. Thin optical sections showed MBP immunoreactivity was clearly present in and broadly distributed throughout nuclei, although commonly excluded from presumptive nu- cleoli and from heterochromatin (Fig. 3).

MBPs were located in both the cytoplasm and nucleus of oligodendrocytes present throughout the brain, in both gray and white matter. Such cells represented approximately half of all MBP+ cells in the adult mouse. We detected nuclear immunof luorescence in oligodendrocytes with all MBP antibodies tested and under various fixation conditions. Non-oligodendrocyte nuclei in the same preparations were never labeled, indicating antibody binding specificity. Furthermore, no labeling of nuclei or of any other structures was observed following

Oligodendrocytes Contain Cytoplasmic and Nuclear MBPs Prior to the Initiation of Myelination

It has previously been suggested that cytoplasmic and nuclear MBPs may have a role in the initial stages of myelinogenesis in young oligodendrocytes (Allinquant et al., 1991). We therefore investigated whether there was a correlation between the three distinct patterns of perikaryal MBP expression and morphological charac- teristics of axon ensheathment, e.g., whether cells had connections to myelin sheaths. We found that developing oligodendrocytes contain cytoplasmic and nuclear MBPs both before and during myelinogenesis.

To visualize oligodendrocyte processes in developing tissue we used the 0 4 mAb, which labels differentiated oligodendrocytes in fixed tissue sections (Hardy and Friedrich, 1996). By double immunolabeling with 0 4 mAb and antibodies to MBP, we were able to visualize cells with large, elaborate process extensions. Dur- ing early development, many 04+/MBP+ cells were seen with multiple processes in regions which were yet to be myelinated. Figure 4a shows such a cell, which lies in the formative subcortical white matter at postnatal day 3,

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in which no myelin is present at this age. Although the cell has no apparent connections to myelin sheaths, MBPs are present within the perikaryal cytoplasm and nucleus (Fig. 4b,c). Oligodendrocytes with connecting processes to myelin sheaths can be seen in the medulla at this time (Fig. 4d), and high resolution scanning often revealed MBPs within the connecting processes of these cells and within both the cytoplasm and nucleus (Fig. 4e,f). In most cells observed during development, strong labeling of the cytoplasm obscured any concentration of immunoreactivity at the plasmalemma.

Presence of MBPs in the Cytoplasm and Nucleus Is Developmentally Regulated

Since MBPs appeared in the cytoplasm and nucleus of oligodendrocytes before they had commenced axon ensheathment, we were interested to examine the distribution of MBPs when they are first expressed in matur- ing oligodendrocytes. Oligodendrocyte progenitors are mitotic and can be labeled using the mAb 04 . Following differentiation, oligodendrocytes retain the ability to bind 0 4 and can additionally be recognized by another mAb, R-mAb (Gard and Pfeiffer, 1989; Warrington and Pfeiffer, 1992). We labeled dividing oligodendrocyte progenitors in P6 mice with the thymidine analog BrdU (3 i.p. injections over a 6 hr period). After 6 hr and 1 and 2 days, animals were sacrificed and triple immunolabeled for MBP, BrdU, and either 0 4 or R-mAb. Thus progenitor cells labeled during their last division could be followed during subsequent differentiation and could be assayed for the presence of MBPs in their cytoplasm and nucleus.

Immediately following BrdU administration, only 0 4 + /MBP- oligodendrocyte progenitors had incorporated the label, whereas differentiated oligodendrocytes, as defined by R-mAb labeling, and 0 4 + /MBP + oligodendrocytes had not (Fig. 5a-c). One day later, differentiated R-mAb + oligodendrocytes could be seen with BrdU in their nuclei. Thus, 0 4 + cells which had un- dergone their final division had differentiated into oligodendrocytes. However, no 0 4 + /MBP + or R-mAb + / MBP+ cells which also contained BrdU were seen at this time. The first 0 4 + lMBP + cells which contained BrdU appeared in the medulla, 2 days after BrdU injec- tion. On examination by confocal microscopy, we observed that 90% of MBP + /BrdU + oligodendrocytes (i.e., cells just beginning to synthesize MBP) invariably displayed strong immunoreactivity at the plasmalemma, with little cytoplasmic or nuclear labeling (Fig. 5d-f).

Our data on the distribution patterns of MBPs in oligodendrocyte perikarya during development and into maturity are shown schematically in Figure 6 . The youngest oligodendrocytes expressing MBPs, 2 days after their final division, contain protein only at the plas-

Fig. 6. Changes in MBP distribution during oligodendrocyte development. MBPs are absent from oligodendrocyte progenitors (A) and as they appear in newly differentiated oligodendrocytes are localized only at the plasmalemma (B). MBPs then quickly accumulate in the cytoplasm and nucleus of oligodendrocytes before they begin axon ensheathment (C) and remain there during myelinogenesis (D). In oligodendrocytes containing cytoplasmic MBPs, a concentration of MBPs at the plasmalemma cannot always be unequivocally determined. Mature, myelin-bearing oligodendrocytes fall into three cate- gories: those with MBPs in the nucleus and cytoplasm (E), those containing cytoplasmic MBPs but not nuclear MBPs (F), and those with MBPs at the plasmalemma but not in the cytoplasm or nucleus (G). These three phenotypes may represent individual subsets of oligodendrocytes or transient stages ex- perienced by all myelin-bearing oligodendrocytes.

malemma. MBPs evidently then accumulate rapidly in the cytoplasm and enter the nucleus prior to the initial axon ensheathment. The localization of MBPs to the cytoplasm and nucleus then persists in the vast majority of oligodendrocytes during myelinogenesis. Only in fully mature animals (older than 3 months) did an appreciable number of myelin-bearing oligodendrocytes lack diffuse cytoplasmic and nuclear MBPs.

14 kD MBP Isoform Preferentially Localizes to the Plasmalemma

Earlier work has shown that distinct MBP isoforms are differentially distributed when individually transfected into cultured cells: isoforms containing sequences encoded by exon 2 are targeted to the nucleus, whereas those lacking these sequences predominate in the plasmalemma (Staugaitis et al., 1990; Allinquant et al., 1991). Such patterns of MBP localization are strikingly similar to those we observed for oligodendrocytes in mature mouse brain. We therefore wanted to determine if the differential distribution of MBP isoforms accounted for the heterogeneity in MBP localization we observed in mature oligodendrocytes . We generated transgenic mice bearing epitope-tagged 14 kD MBP, which lacks exon 2, driven by 1.9 kb of the MBP regulatory region, previ-

MBP Localization in OIigodendrocyte Perikarya 253

godendrocyte nuclei of wild-type mice, we analyzed nuclear fractions from both developing (P14 and P25) and adult mouse brain by immunoblotting with antisera to MBP to PLP (Fig. 8a). While PLP could easily be detected in a purified myelin fraction, no PLP was detected in nuclear fractions, indicating that the fraction was free of contamination by myelin. Immunoblotting the same fractions with antisera to MBP not only confirmed the presence of the 14 kD MBP isoform in wild-type mice, but revealed that all 4 major MBP isoforms were present in the nuclear fraction (Fig. 8a). These data also confirm that MBP immunoreactivity found in oligodendrocyte nuclei in vivo is due to the presence of full-length MBP isoforms and not to degradation products of MBP isoforms entering the nucleus. The MBP antisera used did not react with nuclear proteins other than MBPs as no proteins were labeled with MBP antibodies on immunoblots of nuclear fractions prepared from shiverer mice (data not shown).

We wondered whether the MBPs in our nuclear fractions might have been released from compact myelin and then associated non-specifically with nuclei during homogenization. We have previously demonstrated im- munodetectable @gal in the cytoplasm of oligodendrocytes in MPP mice using conventional epifluorescence microscopy (Gow et al., 1992); however, during the present study utilizing the confocal microscope we found that P-gal is also present in the nucleus of oligodendrocytes. Hence in nuclear fractions from MPP transgenic mice we were able to identify oligodendrocyte nuclei using @gal antibodies. While we found immunofluorescence for MBPs in P-gal + (oligodendrocyte) nuclei, we found no MBP labeling in P-gal- (non-oligodendrocyte) nuclei (Fig. 8b-d). Furthermore, not all @-gal+ oligodendrocyte nuclei contained MBP immunoreactivity , in agreement with in vivo observations. It is therefore unlikely that MBPs bound non-specifically to nuclei during the fractionation process.

Fig. 7. Double immunolabeling of oligodendrocytes in transgenic mice bearing epitope-tagged 14 kD MBP isoform with antibodies to MBPs (wild-type and transgenic; a,c) and the epitope tag (b,d). a,b: This oligodendrocyte contains MBPs in its plasmalemma, cytoplasm, and nucleus (a, asterisk) but transgenic 14 kD MBP is found concentrated at the plasmalemma (b, asterisk indicates nucleus). c,d: The same images superimposed with a graph of relative intensities of immunofluorescence, sampled along the diagonal line shown. Trans- genic 14 kD MBPs are also present in the nucleus of this oligodendrocyte at a level significantly above background (d), aithough below that for total MBPs (c). Total MBPs are also concentrated in the cytoplasm (c, arrow).

ously shown to be sufficient to confer oligodendrocyte- specific expression (Gow et al., 1992). Double immunofluorescent analysis of transgenic mice using antibodies to the tagged transgenic 14 kD MBP and to all MBP isoforms (wild-type and transgenic) revealed that the transgenic 14 kD MBP isoform was expressed in all oligodendrocytes. Analysis of the distribution of transgenic and wild-type MBPs within individual oligodendrocytes revealed that transgenic 14 kD MBP preferentially segregated to the plasmalemma (Fig. 7). However, measurable levels of transgenic 14 kd MBP were found in the cytoplasm and nucleus of these cells (Fig. 7d).

Oligodendrocyte Nuclei Contain All 4 Major MBP Isoforms

As the presence of sequences encoded by exon 2 had been thought to be necessary for nuclear entry of MBPs (Staugaitis et al., 1990; Allinquant et al., 1991), the presence of the tagged 14 kD MBP in the nuclei of some oligodendrocytes was surprising. In order to determine whether the 14 kD MBP isoform is present in oli-

DISCUSSION We have demonstrated the presence of MBPs in the

majority of oligodendrocyte cell bodies in mature mouse brain at sufficient levels to allow detection by immunofluorescence. It has generally been believed that MBP polypeptides are absent from oligodendrocyte perikarya following myelinogenesis (Roussel and Nussbaum, 1981; Reynolds and Wilkin, 1988; Hardy and Reynolds, 1991), although one previous study has documented MBP immunoreactivity in adult rat oligodendrocyte perikarya using immunoperoxidase (Sternberger et al., 1985). Our results now demonstrate that not only do adult oligodendrocytes contain MBPs in their cell bodies, but that oligodendrocytes in mature tissue show re-

254 Hardy et al.

M N M N

a-- MBP PLP

Fig. 8. Analysis of nuclei isolated from P14 mouse brain. a: Immunoblots of nuclear fraction. Fractions of myelin (10 p.g protein) and isolated nuclei (25 p.g protein) were run on a 15% polyacrylamide gel, transferred to blotting membrane, and probed with antibodies to MBP or PLP. All 4 major isoforms of MBP (arrowheads) are present in the nuclear fraction (N) in similar proportions to those in the myelin fraction (M). Nuclear fractions contained no contamination from myelin as determined by the presence of PLP. b-d: Nuclei isolated from MPP

markable heterogeneity in the distribution of MBPs: around half of oligodendrocytes show labeling restricted to the plasmalemma, while the remainder have strong labeling distributed throughout perikaryal cytoplasm, the majority of which also display MBP immunoreactivity in their nucleus. This heterogeneity is most likely the result of differential distribution of distinct MBP isoforms; the 14 kD MBP isoform is predominantly located at the plasmalemma in transgenic mice. However, transgenic 14 kD MBPs are also found in some oligodendrocyte nuclei and all 4 major MBP isoforms are present in nuclear fractions of wild-type oligodendrocytes.

The MBP gene is part of a larger transcription unit of 105 kb named the Golli-mbp gene (Campagnoni et al., 1993). This gene is alternatively spliced to give a family of MBP-related mRNAs, some of which share sequences with the classical MBPs. It is therefore possible that MBP antisera might also recognize Golli-mbp proteins (Compagnoni et al., 1993). Golli-mbps are present in nuclei of oligodendrocytes in wild-type mice, and in some neuronal nuclei in both wild-type and shiverer mice (Campagnoni, personal communication). In the present study, no immunoreactivity was observed in tissue sections or Western blots of nuclear fractions from shiverer mouse using the MBP antibodies employed here, indicating that our reagents do not label Golli-mbps. Fur- thermore, in our material, nuclear MBP immunoreactivity was restricted to oligodendrocytes, whereas Golli-mbps are also present in neurons. The MBP immunoreactivity we observed in wild-type and transgenic oligodendrocytes is therefore due to the presence of classical MBPs and not to Golli-mbp polypeptides.

transgenic mouse brain, triple immunolabeled with antibodies to MBP (b), p-gal (c), and histones (d) and viewed by confocal microscopy. Oligodendrocyte nuclei, identified by their con- tent of transgenic @-gal (c), contain MBP, while the nuclei of other cells, which lack the P-gal marker, show no MBP labeling (asterisk). Histone labeling (d) defines the boundary of each nucleus; MBP immunoreactivity is always within these bounds, excluding adherent cytoplasm as a significant contrib- utor to the data in a.

MBPs Are Differentially Distributed in Oligodendrocyte Perikarya

In this paper, we demonstrate that the tagged 14 kD MBP isoform is enriched in the plasmalemma of oligodendrocytes in vivo in transgenic mice. We believe that the tag sequence itself is not responsible for the behavior of the transgenic protein, as transgenic mice bearing truncated MBP polypeptides, also attached to the tag sequence, show distinct patterns of distribution from those described here for tagged full-length 14 kD MBP (Rickman and Lazzarini, unpublished observations). Furthermore, in transgenic mice an identical tag sequence attached to the neurofilament coding sequence does not affect the localization or function of neurofila- ments, whether it is placed at the N-terminus of, or within, the protein (Elder et al., 1994).

The distinct patterns of MBP distribution in the perikaryon of normal adult oligodendrocytes shown here are intriguing in the light of transfection studies utilizing single isoforms of MBP, in which isoform-specific dis- tributions were seen: the 14 and 18.5 kD isoforms were restricted to the plasmalemma, whereas the 17 and 21.5 kD isoforms were localized to the cytoplasm and nucleus (Staugaitis et al., 1990; Allinquant et al., 1991). We now show that these two isoform-specific patterns of MBP distribution are also seen in oligodendrocytes in the in- tact animal. In view of the identical behavior of tagged 14 kD MBP in transgenic mice and of 14 kD MBP in transfected oligodendrocytes, it seems probable that na- tive 14 kD MBP also segregates to the plasmalemma in oligodendrocytes in vivo.


contain all 4 major MBP isoforms and that 14 kD MBP isoform can enter the nucleus in transgenic mice. While the 14 or 18.5 kD isoforms are excluded from nuclei when individually transfected into HeLa cells or cultured shiverer oligodendrocytes (Staugaitis et al., 1990; Al- linquant et al., 1991), it is now apparent that the 14 and 18.5 kD MBP isoforms are able to enter the nucleus in wild-type oligodendrocytes in vivo, when other MBPs are also present. Therefore, nuclear entry of the 14 and 18.5 kD isoforms in vivo may be facilitated by the presence of the exon 2 containing MBP isoforms. In transfected HeLa cells, IgG bound to the 21.5 kD MBP isoform can enter nuclei, indicating that proteins can utilize these MBPs to “piggy-back” into the nucleus (Pedraza et al., 1995). It is possible that in vivo MBPs may enter oligodendrocyte nuclei as a complex and that sequences present in exon 2 are obligate for nuclear entry. How- ever, distinct mechanisms for nuclear MBP entry may operate in other species. For example, in the chick, M A containing exon 2 is not detected (Zopf et al., 1989), but oligodendrocytes nevertheless contain MBPs in their nucleus (Hardy, unpublished observations).

Several lines of evidence suggest that the presence of MBPs in oligodendrocyte nuclei is functionally significant. First, nuclear import of MBPs is temperature dependent and requires the presence of ATP, indicating that it is an active process and is not merely the result of non-specific diffusion of MBPs into the nucleus (Pe- draza, personal communication). Second, some oligodendrocytes containing abundant cytoplasmic MBPs have no MBPs within their nuclei, i.e., MBPs are ex- ctuded from the nucleus in these cells, again indicating that the movement of MBPs between cytoplasm and nucleus is a regulated process. The existence of an energy- dependent, regulated mechanism for the nuclear import of MBPs suggests that they serve a specific function within the nucleus.

The regulation of the entry of MBPs into the nucleus may be related to the myelinating state of each cell: oligodendrocytes undergoing active myelinogenesis or myelin maintenance contain increased levels of cytoplasmic and/or nuclear MBPs, whereas those containing only plasmalemmal MBPs may be in a quiescent state. Thus nuclear MBPs may be involved in the regulation of gene expression or mRNA processing within oligodendrocytes during the different phases of myelination. The present observations of oligodendrocytes in adult tissue provide evidence that mature, myelin-bearing cells can be di- vided into at least two groups, based on their distribution of perikaryal MBP polypeptides. These groups may represent stable, functionally specialized populations, or re- flect transient changes in gene expression or protein processing in oligodendrocytes. Nevertheless, it is now evident that myelin-bearing oligodendrocytes are heter-

MBP mRNA Translocation Does Not Restrict MBP Polypeptides to the Myelin Sheath

MBP mRNA is vigorously translocated from oligodendrocyte perikarya to myelin sheaths (Colman et al., 1982; Trapp et al., 1987; Verity and Campagnoni, 1988; Ainger et al., 1993). Although MBP mRNA can be detected by in situ hybridization in oligodendrocyte cell bodies and processes, as well as at the myelin sheath (Trapp et al., 1988; Landry et al., 1994), the encoded peptides have eluded detection in the perikarya of oligodendrocytes in mature tissue. This had led to the speculation that MBP mRNA is translationally inert before its anchorage at the myelin sheath. This could en- sure that MBP does not accumulate in oligodendrocyte cell bodies, where it has been thought to have no function.

However, the present results show that MBP polypeptides do in fact accumulate in the soma and proximal processes of mature myelin-bearing oligodendrocytes . Given the avidity of MBP for membrane lipids (Smith, 1977; Boggs et al., 1982), it seems probable that MBP in the oligodendrocyte perikarya and proximal processes is locally translated. Therefore, not only is MBP mRNA translated in oligodendrocyte perikarya during development, when it is thought that cells lack the ability to transport MBP mRNA away from the soma (Brophy et al., 1993), but it is most likely that MBP mRNA is also translated in the cell bodies and processes of mature cells. The present data show that the oligodendrocyte is not required to restrict the presence of MBP polypeptides to myelin, as has been suggested (reviewed in Brophy et al., 1993). Furthermore, the presence of MBPs in mature oligodendrocyte perikarya is likely to be reflective of a regulatory role for these proteins in the myelination pro- gram.

MBPs Are Present in Oligodendrocyte Nuclei The present data show that MBPs are present in a

substantial fraction of oligodendrocyte nuclei in adult mice and in almost all oligodendrocyte nuclei during myelinogenesis. These observations extend previous work showing nuclear MBPs in occasional oligodendrocytes in situ at P9 (Allinquant et al., 1991). Furthermore, we confirm the presence of full-length MBPs in oligodendrocyte nuclei by immunoblotting isolated nuclei. It is possible that MBPs non-specifically enter oligodendrocyte, but not non-oligodendrocyte, nuclei during fractionation. However, we think this unlikely, as the proportion of oligodendrocyte nuclei which contained MBPs in the nuclear fraction was similar to the proportion of oligodendrocytes with nuclear MBPs in fixed tissue slices (i.e., around half, in adult animals).

We show here that oligodendrocyte nuclei in vivo

256 Hardy et al.

ogeneous, and myelin metabolism in the mature animals is subject to complex regulation.

ACKNOWLEDGMENTS We thank Ms. Sunghae Yoon for excellent techni-

cal assistance, David Rickman for transgenic constructs, and Ms. Jill Gregory for artwork. This work was sup- ported by National Institutes of Health (NIH) grant NS33165 to V.L.F. and R.A.L. and NIH grant NS31032 to D.R.C. This is manuscript 199 from the Brookdale Center for Molecular Biology.

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Cytoplasmic and nuclear localization of myelin basic proteins reveals heterogeneity among oligodendrocytes