Differential Ultrastructural Localization of Myelin Basic

Joiirnal OJ/ Neirroclirmr~lry Raven Press, Ltd., New York 0 1989 International Society for Neurochemistry

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2’, 3’Cyclic Nucleotide 3’-Phosphodiesterase in the CNS of Adult Rats

Christof Brunner, Hans Lassmann, “Thomas V. Waehneldt, j-Jean-Mane Matthieu, and $Christopher Linington

Institute for Brain Research, Austrian Academy of Sciences and Neurological Institute, University of Vienna, Vienna, Austria; *Forschungsstelle Neurochernie, Max-Planck-Institut fur experimentelle Medizin, Gottingen, F.R.G.: ?Centre Hospitalier Universitaire Vaudois, Laboratoire de Neurochimie, Service de Pkdiatrie, Lausanne, Switzerland; and #Department of Medicine, University of Wales, Heath Park. Card& Wales

Abstract: In a light and electron microscopic immunocyto- elin. MOG was preferentially detected at the extracellular chemical study we have examined the distribution of myelin surface of myelin sheaths and oligodendrocytes and in only basic protein (MBP), 2’,3‘-cyclic nucleotide 3’-phosphodies- low amounts in the lamellae of compacted myelin and the terase (CNP), and myelin/oligodendroglial glycoprotein myelin/axon border zone. Our studies, thus, indicate further (MOG) within CNS myelin sheaths and oligodendrocytes of the presence of different molecular domains in compact my- adult Sprague-Dawley rats. Ultrastructural immunocyto- elin, which may be functionally relevant for the integrity and chemistry allowed quantitative analysis of antigen density in maintenance of the myelin sheath. Key Words: Myelin an- different myelin and oligodendrocyte zones: MBP was de- tigens-Myelin basic protein-Myelin/oligodendrogliaJ glyco- tectable in high density over the whole myelin sheath, but protein-2’,3‘-Cyclic nucleotide 3‘-phosphodiesterase-Quan- not in regions of loops, somata, or the oligodendrocyteplasma titative ultrastructural immunocytochemistry. Brunner C. et membrane. CNP reactivity was highest at the myelin/axon al. Differential ultrastructural localization of myelin basic interface, and found in lower concentration over the outer protein, myelin/oligodendroglial glycoprotein, and 2’,3’-cyclic lamellae of myelin sheaths, at the cytoplasmic face of oli- nucleotide 3’-phosphodiesterase in the CNS of adult rats. J. godendrocyte membranes, and throughout the compact my- Neurochem. 52,296-304 (1989).

The ultrastructural localization of antigens in the (Drummond and Dean, 1980; Sprinkle et al., 1980), myelin sheath by immunocytochemistry may help was localized in oligodendrocyte cytoplasmic com- elucidate the function of the various myelin compo- partments and in loose myelin lamellae of young rats nents. Myelin basic protein (MBP), a major myelin (Roussel et al., 1978). Its exact physiological role re- protein, was demonstrated in the dense line region of mains unclear at present. A recently described minor the sheath (Omlin et al., 1982) in newborn and young glycoprotein specific for central myelin (Linington et rats. This study strongly supported the concept that al., 1984) was detected immunohistochemically on the MBP is central to the formation and maintenance of external surface of oligodendrocytesand myelin of de- the compact lamellar myelin structure, acting to main- veloping rats (Linington et al., 1988). This distribution tain the close apposition of the cytoplasmic surfaces. is a crucial requirement for an antigen to be target of 2’,3’-Cyclic nucleotide 3’-phosphodiesterase (CNP), the antibody-mediated autoimmune demyelination (Lin- main component of the Wolfgram protein fraction ington and Lassmann, 1987; Lassmann et al., 1988;

~~ ~ Received April 25, 1988; revised manuscript received July 14, 1988; Ahhveviations used: CNP, 2’,3’-cyclic nucleotide 3’-phosphodies- accepted July 25, 1988. terase; GA, glutaraldehyde; LRW, LR-White (London Resin); MAG, Address correspondence and reprint requests to Dr. C. Brunner at myelin-associated glycoprotein; MBP, myelin basic protein; MOG, Neurological Institute, University of Vienna, Schwaaspanierstrasse myelin/oligodendrogJial glycoprotein; NF, neurofilament; PFA, 17, A-I090 Vienna, Austria. paraformaldehyde.

296 ULTRASTR UCTURAL DISTRIBUTION OF MYELIN ANTIGENS 297

FIG. 1. lmmunocytochemistry of CNP and MOG on paraffinsections. a: Cerebral hemisphere of an adult Sprague-Dawley rat immunostained for MOG. Myelin in white and gray matter is positive. X8. b Serial section adjacent to a, immunostained for CNP. Numerous digodendrocytes are labelled (arrows) in addition to myelin. X250. c: Cerebral cortex, immunostained for MOG. Shown are two oligodendrocytes (arrows) with membrane-associatedstaining. X630. d Cerebral cortex, immunostained for CNP. In addition to myelin two oligodendrocytes(arrows) show reaction product associated with their membrane. X630.

Linington et al., 1988). All these localization studies (5070,70%, 80%, 96%, 2 X loo%>,followed by mixtures of were performed with a preembedding peroxidase tech- methanol/LRW (2: 1, 1: 1, 1:2) and pure LRW at -2OOC for nique, which is, however, limited by the poor penetra- 30 min, each step. Pure LRW was changed three additional tion of sera and reagents into the tightly compacted times at 4°C; the resin was allowed to penetrate for at least adult myelin. In the present study we have obtained 12 h each time. The tissue blocks were placed into gelatine capsules, which were then filled with resin and polymerized further, more detailed information on the ultrastruc- at 60°C overnight. Semithin sections were selected from re- tural localization of these myelin antigens using a post- gions with good myelin preservation, sectioned for electron embedding immunogold technique on electron micro- microscopy, and mounted on gold grids for ultrastructural scopic sections of rat CNS embedded in the hydrophilic immunocytochemistry . resin LR-White (LRW). Sera and reagents Antibodies to the following determinants were employed MATERIALS AND METHODS in the present study: MBP (MCA 70, monoclonal, Serotec, Tissue processing 1:3,000); myelin/oligodendroglial glycoprotein (MOG, Adult Sprague-Dawley rats were narcotized with barbi- monoclonal, Linington et al., 1984; 1: 100); CNP (polyclonal; turate and perfused via the aorta with 4% freshly prepared Karin and Waehneldt, 1985; 1:200); and an epitope on 210- phosphate-buffered paraformaldehyde (PFA), or, alterna- kilodalton phosphorylated neurofilament protein (SM I 3 1, tively, with a mixture of 3.5% PFA and 0.5% glutaraldehyde monoclonal, Sternberger-Meyer, 1 :20,000). (GA). The brains were dissected and immersed in the same fixatives for 3 h. Thin razor blade tissue slices from PFA-fixed cerebral cor- TABLE 1. Irnrnunoreactivity of oligodendrocytes with tex and the subcortical white matter were prepared, subjected antisera to CNP, MOG, and MBP to preembedding immunocytochemistry with various antisera (see below), routinely osmicated, embedded in Epon, and CNP MOG MBP sectioned for light and electron microscopic analysis. Re- maining material was embedded in paraffin. ++/+ 11.1124.9 5.5/17.7 010 Tissue blocks from PFA/GA-fixed cortex and subcortical The values indicate numbers of immunostained oligodendrocytes white matter were embedded in the hydrophilic resin LRW per mm2 of cerebral cortex. An area of at least 1 mm2 was evaluated (London Resin, U.K.) according to the following procedure: per marker on consecutive serial sections. ++, distinct, continuous dehydration was performed with a graded methanol series membrane-associated labelling; +, weakly positive cells.

J. Neurochem., Vol. 52, No. 1. 1989 298 C. BRUNNER ET AL. ULTRASTRUCTURAL DISTRIBUTION OF MYELIN ANTIGENS 299

Secondary antisera and immunohistochemical reagents target antigen. Thus gold grains observed within 35 nm of included biotinylated, species-specific anti-mouse and anti- the myelin surface, myelin/axon interface, and the oligoden- rabbit immunoglobulins from sheep (Amersham, U.K., 1: drocyte membrane were regarded as associated with these ZOO), rabbit anti-mouse immunoglobulins (Dakopatts, Den- structures. Counts of gold grains in defined regions were nor- mark, 1 :500), avidin-horseradish peroxidase complex (Sigma, malized to background values, obtained from the signals over 1:70), and diaminobenzidine (Huka). Ten-nanometer col- nuclei and nonmyelin tissue. Thus, the values in Fig. 4 rep- loidal gold-coated swine anti-rabbit immunoglobulins were resent signal/background ratios. The total area analyzed provided by Dako, Denmark; 10-nm gold/streptavidin was quantitatively was on the average 280 pm*/marker. by Amersham (U.K.), and 15-nm colloidal gold-labelled Protein A (1 50) was prepared according to Slot and Geuze RESULTS (1 985). Immunocytochemistry on paraffin sections Immunocytochemistry Antibodies to MBP, MOG, and CNP uniformly The immunostaining procedure employed on paraffin sec- stained myelin sheaths in white and gray matter of adult tions has already been described in detail earlier by Lassmann rats (as documented for MOG and CNP in Fig. 1). et al. (1986). A similar technique was used for preembedding immunocytochemistry, but with serum incubation times in- Oligodendrocyte staining could best be evaluated in creased to 6 h and with extended washing steps (Lassmann the cortex, where myelin sheaths are sparse and do not et al., 1986). cover cytoplasmic or membrane-associated immuno- Immunogold electron microscopy was performed on LRW reactivity (Roussel and Nussbaum, 1981). Most oli- sections incubated for 30 min in 0.5% bovine serum albumin godendrocytes were found to be positive for CNP, but (BSA), the medium in which all sera were diluted. Sections fewer stained for MOG and none were positive for were then incubated with primary antisera (overnight at 4"C), MBP (Table 1; Fig. lb-d). followed by the Protein A-gold complex (1 h at room tem- perature). In the case of monoclonal primary antibodies, rab- Preembedding immunocytochemistry bit anti-mouse serum was used as a linker between the mouse On semithin sections from Epon-embedded material IgG and the Protein A. In addition, in some sections im- previously immunostained with anti-CNP or anti- munoreactivity was visualized with colloidal gold-coated MOG antibodies, reactivity for both markers was as- swine anti-rabbit immunoglobulins, or by biotinylated anti- sociated with myelin sheaths (Fig. 2b and h). In addi- mouse or anti-rabbit Ig followed by gold labelled streptavidin (Bancher et al., 1987). The sections were stained with uranyl tion, oligodendrocytes were positive for CNP (Fig. 2b), acetate and lead citrate and analyzed with a Jeol 100 S electron but only the occasional cell stained weakly for MOG. microscope. In the electron microscope, CNP reactivity showed For control, primary antibodies were either omitted or ir- a patchy distribution in the submembranous region of relevant antibodies from the same immunoglobulin class or oligodendrocyte cytoplasm (Fig. 2a and c). Myelin-as- normal rabbit serum were used. sociated staining was due to diffuse cytoplasmic reac- Quantitative evaluation of antigen distribution tivity in inner, outer, and paranodal loops (Fig. 2d-f). Representative micrographs were taken with a standardized In contrast, MOG was found to stain the surface of magnification of X49,OOO from well-preserved immuno- both myelin sheaths and oligodendrocyte processes stained myelin sheaths and oligodendrocytes in the gray mat- (Fig. 2g, i, and j), although the oligodendrocyte plas- ter of adult rats. Myelinated fibers were "dissected" on the malemma was only occasionally and weakly stained photographs into zones associated with the outer myelin sur- (Fig. 2j). face, the myelin/axon interface, compact myelin, and the axonal compartment, as shown in Fig. 4. Furthermore, an Immunogold electron microscopy oligodendrocyte membrane-associated zone, oligodendrocyte Quantitative evaluation of antigen distribution, as cytoplasm, and nucleus were defined. The size of Protein A/ visualized by the "on grid" immunogold method, re- gold-antibody complexes (Hainfeld, 1987) may result in gold vealed a high density of MBP reactivity over the whole grains appearing up to 35 nm from the actual site of the myelin sheath (Figs. 3a and f and 4). As the regions

4 FIG. 2. Preembedding immunoelectron microscopy of CNP and MOG. a: Oligodendrocyte with CNP-related reaction product in a patchy distribution close to the cytoplasmic side of the cell membrane (large arrows) and within the perinuclear cytoplasm (small arrows). Cerebral cortex, X9,500. b (inset): The same cell as shown in a in an adjacent semithin section, demonstrating the block edge free of unspecific staining; reaction product can be seen associated with some myelin sheaths (arrows) and the oligodendrocyte. XI,000. c: Detail from a; patchy submembranous reaction product (arrows), well-preserved cell organellae. 0, oligodendrocyte nucleus. X28,OOO. d: Myelinated axon in cerebral cortex of an adult rat; immunostain for CNP. lmmunoreactivityin the cytoplasm of an inner oligodendrocyte loop (arrow). X35,OOO. e: Same block and region as in d showing CNP reactivity in the paranodal zone oligodendrocyte cytoplasm (arrows). X34,OOO. f: Same block and region as in d and e showing CNP reactivity in an outer (large arrow) and inner (small arrow) oligodendrocyte loop. X28,OOO. g: Perivascular group of myelinated axons in the periventricular white matter of an adult rat; immunostain for MOG. Reactivity on the surface of myelin sheaths. E, ependymal cell with typical cilia (arrows c). X10,850. h (inset): Same region as shown in g (arrows g) in an adjacent semithin section. Peroxidase reaction product associated with myelinated fibers, no unspecific reactivity at the block edge. E, ependymal cell. X1,OOO. i: Detail from g showing MOG reactivity on the surface of myelin sheaths (large arrows), extending to the surface of adjacent cell processes (small arrows), possibly from oligodendrocytes.XI 8,450. j: Myelin surface reactivity for MOG (large arrows), less at the surface (small arrows) of an adjacent oligodendrocyte (0).X17,500.

J. Neurochern , I'd. 52, XO. 1. 1989 300 C. BRUNNER ET AL.

J Neirrochem , Vd. 52, No 1. 1989 ULTRASTRUCTURAL DISTRIBUTION OF MYELIN ANTIGENS 301 denominated “myelin surface associated” and “my- lamellae of adult myelin. Thus, antigenic sites within elin/axon interface” also included adjacent zones of these compartments may be missed by this method. extramyelin space and axoplasma, respectively, the fi- Postembedding immunocytochemistry with im- nal values in these compartments are lower than the munogold techniques on ultrathin sections of meth- corresponding counts over myelin only. This has to be acrylate-embeddedtissue, on the other hand, does allow considered likewise in the cases of CNP and MOG. detection of antigens in compacted tissue elements, In comparison to MBP, CNP and MOG reactivity such as mature myelin. Furthermore, the immune re- was lower and not evenly distributed over the entire action can be quantified, giving information on anti- myelin sheath: CNP was highest at the myelin/axon genic density in various structural elements. This interface (Figs. 3e and h and 4), and MOG at the myelin method, however, is handicapped by its relatively low surface (Figs. 3g and 4); the signal for both was relatively sensitivity due to the limited exposure of antigens on low over compact myelin. the section surface (Ellinger and Pavelka, 1985) and Significant oligodendrocyte-associated immuno- by its poor spatial resolution, which is determined by reactivity was found only for CNP: similarly by the the size of the antibody-gold complexes (20-30 nm; block staining method, clusters of gold grains were Hainfeld, 1987). concentrated in the submembranous region (Fig. 2b- With regard to myelin antigens, thus, preembedding d). In addition, oligodendrocyte loops were frequently immunoelectron microscopy provides information on decorated with gold grains due to CNP reactivity (Fig. the following aspects: first, the presence of the antigen 2e and h), but this compartment was not included in (or epitope) on the extracellular face of the cell mem- the quantitative analysis, as the total area was too small brane of myelin supporting cells and the myelin sheath, to give reliable results. and second, the subcellular localization of the antigen The same technique using an anti-neurofilament (or epitope) in the cytoplasm of myelin supporting cells antibody produced a highly specific staining pattern, and their processes. in which only the axoplasm was decorated with gold However, information on the distribution within the grains (Figs. 3i and 4). compacted myelin sheath, although obtained for major Additional control experiments performed either myelin proteins such as MBP (Omlin et al., 1982), is with the primary antibody omitted or with irrelevant limited with this technique, especially when antigens, antibodies of the same immunoglobulin class or normal which are present in the myelin sheath in minor con- rabbit serum excluded nonspecific reactions in the centrations, are studied. course of immunostaining procedures. Postembedding immunocytochemistry together with quantitative evaluation of the immunogold precipitates DISCUSSION allows conclusions on the distribution of the antigen within compacted myelin, but, because of poor spatial In the present study we show the differential ultra- resolution, is insufficient to determine the orientation structural distribution of three myelin proteins (MBP, of the antigen (or epitope) within the membrane (e.g., CNP, and MOG), indicating different molecular do- location on major or minor dense line). mains within the myelin sheath. We applied two dif- Thus, both techniques (pre- and postembedding ul- ferent immunoelectron microscopic techniques, both trastructural immunocytochemistry) give comple- of them having advantages and limitations. mentary information and should therefore be used si- Preembedding immunocytochemistry using im- multaneously to achieve optimal results. mune peroxidase techniques is a highly sensitive Our present results further extend the concept of method, especially suitable for the detection of extra- different molecular domains in the compact myelin. cellular antigens or epitopes located on the extracellular This implies that structural antigens, such as MBP surface of cells. However, penetration of sera and re- (Omlin et al., 1982; Braun, 1984; Lees and Brostoff, agents into some tissue compartments may be severely 1984) or possibly proteolipid protein (Laursen et al., inhibited, especially in the case of the tightly compacted 1984; Stoffel et al., 1983; Potter and Lees, 1988), which * FIG. 3. lmmunogold electron microscopy of MBP, MOG, CNP, and neurofilament (NF). Fifteen-nanometer gold-conjugated Protein A was used in all cases except for g (1 0-nm gold-conjugatedswine anti-rabbit immunoglobulins). a: Oligodendrocyte(0) and adjacent myelinated axons in the cerebral cortex; postembeddingstain for MBP. Reactivity was mainly over compact myelin (arrow); no significant signal over oligodendrocyte cytoplasm. X15,600. b: Oligodendrocyte (0) in the cerebral cortex of an adult rat; postembedding stain for CNP. Gold grains are mainly located at the cytoplasmic face of the cell surface (see also details c. d). LRW section, X19,600. c and d: Details from b, showing immunoreactivity associated with the periphery of the oligodendrocyte. X51.800. e: Same block and region as in b showing CNP reactivity over an outer oligodendrocyteloop (borders indicated by arrowheads) and inner- and outermost myelin lamellae. X47,200. f: MBP reactivity evenly and exclusively distributed over compact myelin; no reactivity in oligodendrocyte loop (arrowheads). X65,OOO. g: MOG reactivity preponderantly over the myelin surface associated zone, less in compact myelin and the myelin/axon interface. Gold grain size, 10 nm. X51,OOO. h: CNP reactivity in an inner oligodendrocyte loop (arrowheads) and over inner- and outermost myelin lamellae. X49,OOO. i: NF reactivity associated only with filamentous structures in the axoplasm. X56,OOO.

J. Neirrochem , Vol. 52, No. 1. 1989 302 C. BRUNNER ET AL.

FIG. 4. Quantitative evaluation of antigen distribution in different zones of myelin sheaths and oligodendrocytes. Myelin sheaths and oligodendrocytes were divided into: (1) perinuclear oligodendrocyte cytoplasm, (2) oligodendrocyte mernbrane-associatedzone, (3) axoptasma, (4) rnyelin/axon interface, (5) compact myelin, and (6)myelin surface-associatedzone. The bars represent the ratios between gold grain counts over the respective zones and background counts (see Materials and Methods). The fine-dotted basis zone indicates background counts + three standard deviations. are responsible for the formation and the maintenance Schachner, 1986). We have now found evidence that of the compact structure of mature myelin, are evenly CNP and MOG are also located in specific domains distributed throughout the entire compact myelin. On of the adult myelin sheath. the other hand, myelin antigens with other functions CNP is an enzyme involved in the hydrolysis of 2’,3’- may be located at specific sites of the myelin sheath. cyclic nucleotide phosphates (Drummond et al., 1962) As an example, myelin-associated glycoprotein (MAG), and is found by biochemical and immunocytochemical a cell adhesion molecule (Quarles, 1983/84; Martini methods in highest activities in central myelin and oli- and Schachner, 1986), is located mainly at the periax- godendrocytes (Kurihara and Tsukada, 1967; Roussel onal lamellae of the myelin sheath (Martini and et al., 1978; Roussel and Nussbaum, 1981; Sheedlo et

J. Neiirochem., Vol. 52, No I. I989 ULTRASTRUCTURAL DISTRIBUTION OF MYELIN ANTIGENS 303 al., 1983; Sprinkle et al., 1983; McMoms et al., 1984). ent compared to the only other characterized major Work by Braun et al. (1988) in normal and myelin- myelin glycoprotein MAG (Martini and Schachner, deficient (mld)mice also shows, that immunoreactive 1986). In the present study we were able to show that CNP is particularly concentrated at the periphery of the epitope of MOG recognized by the monoclonal the oligodendrocyte, generally adjacent to the Plasma antibody used is present on the extracellular surface of membrane but not especially associated with mem- adult rat myelin and oligodendrocytes. By means of branes. The identity between CNP and the Wolfgram postembedding immunocytochemistry, the highest protein has been demonstrated; they both show on antigen density was revealed in the outermost lamellae electrophoresis a doublet of 45-50 kilodaltons (Drum- of myelin sheaths, but was also found in lower con- mond and Dean, 1980; Sprinkle et al., 1980). A possible centrations in the center of compacted myelin and even role of the enzyme in myelin formation is assumed less at the myelin/axonal interface. from its immunocytochemical detection in rat oligo- Recently it has been shown that MOG may be an dendrocytes in the earliest stages of myelination, even important molecular target in antibody-mediated de- preceding MBP by 1-3 days (Roussel and Nussbaum, myelination. Demyelinating activity of sera from 1981). This role is assumed further from the close cor- guinea pigs with chronic relapsing allergic encephalo- relation of enzyme activity with basic protein levels myelitis correlates well with antibody titer to MOG during early development in rat brain (Sprinkle et al., (Linington and Lassmann, 1987); moreover, acute 1978) and from the simultaneous in vitro inhibition MBP specific T-cell line-mediated experimental allergic of guinea pig brain CNP and myelin protein synthesis encephalomyelitis can be modulated into a demyelin- by 2’-adenosinemonophosphate (Starich and Dreiling, ating disease by intravenous injection of monoclonal 1980). The physiological substrate(s), however, remain antibodies to MOG (Schliisener et al., 1987; Linington unclear at present. Recently, Newman et al. (1987) et al., 1988; Lassmann et al., 1988). speculated that mRNAs from the MBP gene might be In conclusion, our study shows, by ultrastructural modified also by posttranscriptional mRNA splicing, immunocytochemistry, that some myelin antigens are giving rise to MBP molecules with various molecular distributed unevenly in different domains of the myelin weights. Furthermore, an additional 5’-polynucleotide sheaths. This indicates that the myelin sheath, despite kinase activity was demonstrated for CNP (Sprinkle et of its uniform ultrastructure, is composed of regions al., 1987), also suggesting a role in ribosomal RNA with different antigenicity and function. turnover, possibly in connection with the peripheral synthesis of MBP and other myelin proteins. Immu- Acknowledgment: The authors wish to thank Helene nocytochemical data showing the localization of CNP Breitschopf, Angela Cervenka, Susanne Katzensteiner, and in oligodendrocyte processes and submembranous cy- Marianne Leiszer for excellent technical assistance and pho- toplasm support this concept. However, our finding of tographic work. This study was supported by Science Re- CNP as a component of compact myelin, preferentially, search Fund, Austria, Project P6438M, and the Swiss Na- but not exclusively, located in inner- and outermost tional Science Foundation, grant 3.129.88. myelin zones (see Fig. 4), might suggest an additional REFERENCES function within the mature sheath. Bancher C., Lassmann H., Budka H., Grundke-Iqbal I., Iqbal K., At present, no data are available on the function of Wiche G., Seitelberger F., and Wisniewski H. M. (1987) Neu- MOG in oligodendrocytes and myelin sheaths. 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