Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical...

Journal of Neurochemistry Raven pres$ Ltd., New York 0 1991 International Society for Neurochemistry

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

Francine A. Tansey, Muhammad Farooq, and Wendy Cammer

Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, U.S.A.

Abstract: The results of recent immunocytochemical exper- iments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost

~~

fivefold higher than those in white matter homogenates. Im- munocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections. Key Words: Glutamine synthetase-Oligodendrocytes-Astrocytes-Glial cells- Bovine brain-Rat brain. Tansey F. A. et al. Glutamine synthetase in oligodendrocytes and astrocytes: New biochemical and immunocytochemical evidence. J. Neurochem. 56, 266-272 (1991).

The concept that the glial cells provide the com- partment in which glutamate, y-aminobutyric acid (GABA), and aspartate are converted to glutamine was derived initially from metabolic evidence (see, e.g., Balazs et al., 1973; Pate1 et al., 1982). Later, immunocytochemical methods were used to show that glutamine synthetase (glutamate-ammonia ligase; EC 6.3.1.2; GS), an enzyme on which those processes de- pend, was found exclusively in glial cells (Martinez- Hernandez et al., 1977). Ultrastructural and further light microscopic examination of GS immunostaining in the corpus striatum, cerebral cortex, cerebellar cortex, and hippocampus of the rat suggested that GS was confined to astrocytes (Norenberg and Martinez-Her- nandez, 1979; Norenberg, 1983). More recently, however, a method for fixation of tissue was used that op- timizes the preservation of cytoplasmic antigens in glial cells before paraffin embedding (Cammer et al., 1985), and GS was localized in CNS regions, such as rat spinal cord and penventricular white matter, in paraffin sections from rat brain, in which immunostaining for this antigen had not previously been described. The sur- prising result was the observation of GS immunoreac- tivity in oligodendrocytes, as well as astrocytes, in par-

affin sections from those regions in that species (Cam- mer, 1990).

The present study was designed to use biochemical methods for the estimation of GS activity in cells from rat CNS and to examine an additional species, additional regions of rat forebrain, and a vaned protocol for tissue fixation. Thus, the major cell types-astrocytes, neurons, and oligodendrocytes-have been isolated from rat forebrains and assayed for GS. Vibra- tome sections, in addition to paraffin sections, of tissue from various regions of forebrain have now been immunostained, and the biochemical and immunocytochemical data from rat and bovine forebrains have consistently shown GS in oligodendrocytes as well as in astrocytes.

MATERIALS AND METHODS

Materials Reagents for the enzyme assays were obtained from Sigma

Chemical Co. (St. Louis, MO, U.S.A.), chemicals forge1 elec- trophoresis from Bio-Rad (Richmond, CA, U.S.A.), fluores- cent second antibodies from Kirkegaard and Perry (Gaith- ersburg, MD, U.S.A.), and other immunochemical reagents from Vector (Burlingame, CA, U.S.A.). Antibodies against

Received March 27, 1990 revised manuscript received June 26, 1990 accepted June 28, 1990.

Address correspondence and reprint requests to Dr. W. Cammer at Department of Neurology, F-140, Albert Einstein College of Med- kine, 1300 Moms Park Avenue, Bronx, NY 1046 1, U.S.A.

Abbreviations used: CNP, 2’,3’-cyclic nucleotide Y-phosphohydrolase; FEA, formalin/ethanol/acetic acid/water; GABA, y-aminobutyric acid; GFAP, dial fibrillary acidic protein; GS, glutamine synthetase.

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GLUTAMINE SYNTHETASE IN GLIAL CELLS 26 7

TABLE 1. Specific activities of GS in cells isolutedfrom rat brains and in rut bruin homogenutes

Homogenate or SA (nmol/min/ Cell SA/ cell type mg of protein) homogenate SA

Homogenates 189 * 12 (5) 1 .o Oligodendrocytes 229 f 26 (3) 1.2 Astrocytes 910+.7 (3) 4.8 Neurons 75 k 8 (3) 0.40

Data are mean k SE values (no. of independent samples assayed). SA, specific activity.

sheep GS were raised in rabbits and characterized as described (Cammer, 1990), and the monoclonal mouse anti-human 2‘,3’-cyclic nucleotide 3’-phosphohydrolase (CNP), which cross-reacts with the rat enzyme, was a gift from Dr. T. J. Sprinkle (Sprinkle et al., 1987). The monoclonal mouse anti- human glial fibrillary acidic protein (GFAP) was purchased from Boehringer-Mannheim (Indianapolis, IN, U.S.A.).

Preparation of cells Cells were prepared from the forebrains of 3-4-week-old

rats as described (Farooq and Norton, 1978; Snyder et al., 1980). Oligodendrocytes were prepared from bovine brains as described (Farooq et al., 1981). Because rat forebrain ho-

mogenates were trypsinized briefly during the cell isolation procedures, trypsinized homogenates were also assayed for GS and were found to have specific activities similar to those in untrypsinized homogenates. The latter are shown in Ta- ble 1.

Enzyme assay To determine GS activities, the rate of reaction of hy-

droxylamine with glutamine was measured (Meister, 1985). This reaction has been shown to be catalyzed by GS in brain cells (Juurlink, 1982). The high reaction rates make possible precise assays of small samples within a wide range of specific activities, eg., in neurons and astrocytes. In brief, samples were incubated at 37°C for 0-15 min with ~-glutarnine, hy- droxylamine, and ATP in the medium described (Meister, 1985). The reaction was stopped by addition of a ferric chlo- ride reagent containing HCl and trichloracetic acid, the pre- cipitated protein was removed by centrifugation, and the op- tical densities of the supernatants were determined at 535 nm. Zero-time blanks, separate reagent blanks for each sub- strate, and a y-glutamyl hydroxamate standard curve were measured routinely. Protein concentrations in the samples were measured by the method of Lowry et al. ( 1 95 1).

Immunocytochemistry Paraffin sections and Vibratome sections from rat and bo-

vine forebrains were used for the immunocytochemical studies, Paraffin sections (see Figs. 1 and 2) were prepared as

FIG. 1. lmmunostaining of GS in rat brain: (a) cortical gray matter and (b) white matter (corpus callosum). Paraffin sections were prepared and immunofluorescence staining was performed as described in Materials and Methods. Anti-GS was used at a dilution of 1:150 with fluorescein-conjugated goat anti-rabbit IgG. The long arrows point to rows of stained cells that resemble interfascicular oligodendrocytes, and the short arrow points to two cells that resemble astrocytes. Bar = 50 pm.

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268 F. A . TANSEY ET AL.

described (Cammer et al., 1985), after fixation of the tissue in formalin/ethanol/acetic acid/water (FEA; 4:69:5:22 by volume). Bovine tissue was transported on ice from the slaughterhouse, blocks 5- 10 mm thick were fixed overnight in FEA, and Vibratome sections (- 50 pm thick) were prepared. Some blocks from rat forebrain (see Fig. 3) were prepared after perfusion with periodate/lysine/parformaldehyde (McLean and Nakane, 1974) and postfixation overnight in the same solution, followed by cutting and staining of Vi- bratome sections. Immunofluorescence staining and avidin-

biotin immunostaining were performed as described by Cammer and Tansey (1988) and Tansey et al. (1988), respectively.

RESULTS Among the cells isolated from rat brains (Table l),

astrocytes had the highest specific activities of GS. Oli- godendrocytes, but not neurons, also had specific activities slightly higher than those in the crude homog-

FIG. 2. Double immunofluorescence staining of GS and GFAP in rat brain: (a) mostly periventricular white matter, rhodamine; (b) same field, fluorescein; (c) optic tract (white matter), rhodamine; (d) same field, fluorescein; (e) hippocarnpus (gray matter), rhodamine; (f) same field, fluorescein; (9) optic tract (white matter), normal rabbit serum at 1:150 instead of anti-GS; and (h) same field, fluorescein. Rabbit anti- GS was used at 1:150 and mouse anti-GFAP at 1:lOO. Paraffin sections were prepared and irnmunostained as described in Materials and Methods. Second antibodies were rhodamine-conjugated goat anti-rabbit IgG (for GS) and fluorescein-conjugated goat anti-mouse IgG (for GFAP). Bar = 50 pm.

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enates. If contamination with astrocytes were respon- sible for the GS activity in oligodendrocytes from rat brain, the level of contamination would have to be almost 25% (Table 1). This clearly is not the case for the product of the cell isolation method used here (see Snyder et al., 1980).

In rat brain, anti-GS immunostained astrocytes in the gray matter (Fig. la), but in the white matter of corpus callosum, rows of cells that appeared to be interfascicular oligodendrocytes were stained (Fig. 1 b, long arrows), along with some astrocytes (short arrow). The GS-positive cells in the white matter adjacent to the ventricles and in the optic tracts, for example, were not GFAP positive (Fig. 2a-d), whereas those in gray matter did contain GFAP (Fig. 2e and f).

For identification of the GS-positive cells in white matter, Vibratome sections from rat brain were double- immunostained with rabbit anti-GS and a mouse monoclonal antibody against CNP, which is a marker for myelin and oligodendrocytes (Sprinkle, 1989) (Fig. 3). The GS-positive cells in white matter also contained CNP (arrows in Fig. 3a and b), and when the rabbit prebleed was substituted for anti-GS, the cells stained only for CNP (Fig. 3c and arrows in d). In gray matter, the GS-positive structures, which resembled protoplasmic astrocytes (arrowheads in Fig. 3e) and astrocytic end-feet on blood vessels (arrow in Fig. 3e), were not stained by the anti-CNP (Fig. 3f).

Oligodendrocytes were also isolated from bovine brain white matter, and the GS specific activities were compared with those in homogenates of white matter and gray matter (Table 2). The specific activity in oligodendrocytes was almost five times that in the starting material, i.e., white matter. Homogenates of gray matter also had very high specific activities.

In Vibratome sections from bovine brains (Fig. 4), the most intensely GS-positive cells in the white matter were the small, ovoid oligodendrocytes (Fig. 4a-c, arrowheads), and staining was also observed in astrocytes (arrow in Fig. 4d). Immunofluorescence (Fig. 5a and b) also demonstrated GS in both oligodendrocytes (short arrows) and astrocytes (long arrows) in white matter. In gray matter (shown at higher magnification in Fig. 5c), astrocytes were GS positive, and the neuropil was filled with GS-positive astrocytic processes, which probably account for the high specific activities in gray-matter homogenates (Table 2). GS-positive staining in the neuropil was also observed in some gray- matter regions of rat brain (see, e.g., Fig. 2a, lower right corner).

DISCUSSION

The new biochemical and immunocytochemical data add to recent evidence that there is GS in oligodendrocytes as well as astrocytes in the rat CNS (Cam- mer, 1990). Oligodendrocytes in tissue cultures have also been shown to contain GS (Wamnga et al., 1988). In the present study, the data obtained when GS was

FIG. 3. Double immunofluorescence staining of GS and CNP in rat forebrain: (a) white matter, rhodamine; (b) same field, fluorescein; (c) same region, normal rabbit serum in place of anti-GS, rhodamine; (d) same field, fluorescein; (e) gray matter, rhodamine; and (f) same field, fluorescein. Vibratome sections were prepared and immunostained as described in Materials and Methods. Rabbit anti-GS was used at 1:lOO and mouse anti-CNP at 1:2. The second antibodies were rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated goat anti-mouse IgG. The thin arrows in (a), (b), and (d) point to oligodendrocytes, the thick arrow in (e) to astrocytic end-feet on the walls of a blood vessel, and the arrowheads in (e) to astrocytes. Bar = 25 pm.

assayed in bulk-isolated cells showed significant specific activities in oligodendrocytes from bovine and rat brains.

Values for GS specific activities found in the liter- ature and obtained using the more common assay

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TABLE 2. GS activity in bovine brain homogenates and oligodendrocytes

Fraction

~~

SA (nmol/min/ Cell SA/white- mg of protein) matter SA

White-matter homogenate 95.5" Gray-matter homogenate 429, 350 Oligodendrocytes 421, 501

- 4.8

~

SA, specific activity. Four independent samples were assayed. The SE was 7.3.

method, with glutamate plus ammonia and ATP as substrates, vary widely, ranging from 12 to 50 nmol/ min/mg of protein, all in the brains of rats 2 1 8 days old or in astrocytes (Wu, 1964; Nicklas and Browning, 1978; Hertz, 1979; Nicklas et al., 1979; Dennis et al.,

1980; Juurlink, 1982; Patel et al., 1983; Subbalakshmi and Murthy, 1985; Marcus et al., 1986). The transferase reaction, which is much more rapid and was measured in the present study, has been validated for general use (Meister, 1985), with appropriate controls, as were performed here, and also for use with cells from brain, in particular (Juurlink, 1982).

Patel et al. (1982) have measured GS specific activities in astrocytes and neurons isolated from the cere- bella of much younger (8-day-old) rats and have found, in the astrocytes, specific activities four- to eightfold higher than those in the neurons. In the present study, the enrichment in astrocytes, compared with neurons, was - 12-fold. The differences between these ratios do not represent a severe discrepancy, in view of the differences between the ages of animals, the cell isolation methods, and the regions of the brains that were used. GS in synaptosomal fractions has been attributed to

FIG. 4. lmmunostaining of GS in bovine brain: (a-c) white matter, where arrowheads point to some examples of oligodendrocytes; (d) gray matter, where the arrow points to a glial cell that is probably an astrocyte; and (e) white matter, normal rabbit serum at 1:500 in place of anti-GS. Pieces of white matter and gray matter from corpus callosum and cortex, respectively, were fixed, and Vibratome sections were cut and immunostained with anti-GS, at 1:500. by the avidin-biotin method (see Materials and Methods and Tansey et al., 1988). Slides were photographed with Nomarski optics. Panels (c) and (d) are shown at twice the magnification of (a), (b), and (e). Bar = 50 pm.

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GLUTAMINE SYNTHETASE IN GLIAL CELLS 2 71

FIG. 5. lmmunofluorescence staining of GS in bovine forebrain: (a and b) white matter, where short arrows point to some examples of oligodendrocytes and long arrows to astrocytes; (c) gray matter at twice the magnification of the other panels (note the appearance of the cells as protoplasmic astrocytes and the staining of the neuropil, which probably is due to astrocytic cell processes); and (d) gray matter stained with the prebleed at 1 :500. Pieces of white matter and gray matter from corpus callosurn and cortex, respectively, were fixed, and Vibratome sections were cut and immunostained with anti-GS at 1:500 and rhodamine-conjugated goat anti-rabbit IgG. Bar = 50 pm.

contamination with glial cell fragments or to an actual neuronal locus for some of the GS in brain (Ward and Bradford, 1979; Dennis et al., 1980). The present data suggest that there is little, if any, GS activity in neuronal cell bodies and that if there is GS in nerve endings, the levels are too low to be detected by the present immunocytochemical methods. An early study of fractions from rabbit brain showed substantial specific activities of GS in synaptosomes and neurons, as well as in glial cells (Weiler et al., 1979). However, the authors believed that many of the cytoplasmic constituents might have been lost from the isolated cells. Because of this technical uncertainty and additional problems of interpretation, the authors attached relatively little importance to their data on GS. In addition, the species difference would add difficulty in comparing those data with the present data. Thus, the possibility that there is some GS in neurons has not been ruled out, but all

the other studies suggest that, if present, it would be at levels much lower than those in glial cells.

Immunocytochemical methods showed GS in cells resembling oligodendrocytes in white matter of bovine brains and, in rat brain, in the three white-matter regions that were examined, i.e., corpus callosum, periventricular white matter, and optic tract, and with two methods of preparing the tissue. Double immunofluorescence staining showed that the GS-positive cells in rat brain that resembled oligodendrocytes were GFAP negative and CNP positive (Figs. 2 and 3) and could therefore be designated as oligodendrocytes.

The functions proposed for GS in glial cells have included roles in the metabolism of glutamate and GABA and in detoxification of ammonia (Balms et al., 1973; Benjamin, 1983; Norenberg, 1983). The recognition that GS and carbonic anhydrase coexist in astrocytes in the gray matter and in oligodendrocytes in

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the white matter suggested that these enzymes may be required together for certain additional functions. Van Gelder (1983) has suggested a transport role for these enzymes in astrocytes, and it is also possible that these enzymes may function together to generate bicarbonate and glutamine for the synthesis of pyrimidines. The potential role of glutamine in dial cells as a precursor and/or nitrogen source for biosyntheses of purines, pyrimidines, and some amino acids is widely recognized (see, e.g., Cooper et al., 1983; Kvamme, 1983).

Acknowledgment: We thank Dr. T. J. Sprinkle for the monoclonal antibody against CNP, Mariann Downing and Ying Ren for technical assistance, and ReneC Sass0 for typing this manuscript. This work was supported by U.S. Public Health Service grants NS12890 and NS23705 and by grant 1089 from the National Multiple Sclerosis Society.

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