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Correspondence to: Bernd Hamprecht, Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany.
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Summary |
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The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons. (J Histochem Cytochem 45:685-694, 1997)
Key Words: astroglial cells, energy metabolism, immunocytochemistry, nitrogen metabolism, oligodendroglial cells, O2A progenitor cells
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Introduction |
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The initial step in the catabolism of essential branched-chain amino acids (BCAA) is reversible transamination catalyzed by the branched-chain amino acid aminotransferase (BCAT). Early investigations of the distribution of BCAT in rat tissues revealed that most organs and tissues contained some BCAT activity and that activity was distributed between the mitochondrial and cytosolic compartments (
BCAA, along with other large neutral amino acids, readily pass the blood brain barrier in exchange with brain glutamine (
Understanding the involvement of different brain cell types in BCAA metabolism, and the metabolic function(s) of the two BCAT isoenzymes in neural tissue, requires knowledge of the location of BCATm and BCATc in different cells within the brain. As a first step, we have investigated the cellular distribution of BCAT isoenzymes in rat glial cells, using pure astroglial and astroglia-rich rat brain primary cultures. Quite surprisingly, BCATm was found to be the predominant isoenzyme in astroglial cells. BCATc was found in astroglia, but at lower levels and with a heterogeneous distribution. However, strong staining of BCATc was found in oligodendroglial cells and O2A progenitor cells. The possible physiological implications for brain metabolism of this unique distribution of BCAT isoenzymes are discussed.
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Materials And Methods |
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Materials
Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Gibco (Eggenstein, Germany). All cell culture plastic ware was from Nunc (Wiesbaden, Germany). Acrylamide, N,N'-methylene bisacrylamide, and sodium dodecyl sulfate (SDS) were from Bio-Rad Laboratories (München, Germany). Anti-glial fibrillary acidic protein (GFAP) monoclonal antibody, 5-bromo-4-chloro-3-indolyl-phosphate, 4-nitroblue tetrazolium chloride, and the molecular mass marker (Combothek) were from Boehringer Mann-heim (Mannheim, Germany). Triton X-100 and Tween 20 were from Serva (Heidelberg, Germany). Nitrocellulose filter sheets were purchased from Millipore (Eschborn, Germany). Alkaline phosphatase-conjugated goat anti-rabbit IgG, fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse IgG, and tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG were obtained from Sigma. The monoclonal antibody A2B5 was a gift from Dr. F. Walsh (London, UK). Monoclonal antibodies (MAbs) against galactocerebroside (GalC;
Cell Culture
Astroglia-rich primary cultures derived from the brains of newborn Wistar rats (bred in the animal facilities of the institute) were prepared and cultured as described previously (
Immunoblotting
The cultures were washed with PBS and scraped off the culture dish, homogenized in 100 mM Tris-HCl, (pH 7.4; 0.25 ml/mg protein) by 20 up-and-down strokes at 1400 rpm in a Potter-Elvehjem-type glass/teflon homogenizer. Blots were performed on proteins separated by SDS-PAGE electro-phoresis (
Immunocytochemistry
Cells grown to confluency on glass coverslips (22 x 22 mm) for 6-8 days after seeding were prepared for immunocytochemistry essentially as reported previously (
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Results |
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The presence of BCAT isoenzymes in homogenates of cultured rat brain cells was demonstrated by Western blot analysis (Figure 1), using an antiserum generated against BCATm from rat heart mitochondria (
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Western blot analysis established the presence of the two BCAT isoenzymes in the cultures. The next step was the cellular allocation of either isoenzyme in astroglia-rich primary cultures and pure astroglial cultures by examining the co-localization of the BCAT isoenzymes and cell type-specific markers. This was performed by using, in an indirect immunofluorescence double-labeling technique, antibodies against GFAP (
In the double-labeling experiments shown in Figure 2, antisera against the BCAT isoenzymes were applied in combination with an antibody against the astroglial marker GFAP, using pure astroglial primary cultures. Staining for BCATm revealed co-localization of BCATm and GFAP in flat cells of irregular morphology (Figure 2A and Figure 2B). Fluorescence corresponding to BCATm appeared in a distinctive punctate pattern in the cytoplasm of the cell somata, whereas the nuclei were clearly unstained. This is compatible with a mitochondrial localization for the antigen. Comparison of the intracellular distribution of the BCATm staining (Figure 2A) with the corresponding cell shapes (Figure 2A and Figure 2B) demonstrated that the stained mitochondria appeared to be concentrated unevenly in the region surrounding the nucleus. Because of the thickness of the cells, some of the fluorescent grains representing mitochondria were out of focus. Figure 2D and Figure 2E show the results of the simultaneous application of antibodies against BCATc and GFAP on the same cultures. The fluorescence signal for BCATc was distributed throughout the entire cell. In the area of the nuclei, the staining showed a higher intensity. Furthermore, not all GFAP-positive cells displayed the same BCATc immunoreactivity.
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In the experiments shown in Figure 3B and Figure 3E, an anti-GalC MAb was used for characterization of oligodendroglial cells in astroglia-rich primary cultures in combination with antisera against each BCAT isoenzyme (Figure 3). The cells that stained intensively with anti-GalC MAb had small, round somata, from which extended several thin and long processes characteristic of oligodendroglia. This morphology can also be seen in the corresponding phase-contrast micrograph, which shows cells sitting on a confluent layer of flat astroglial cells (Figure 3C and Figure 3F). With BCATm antiserum, virtually no staining was detected in the GalC-positive cells (Figure 3A). In contrast, the oligodendroglial cells were intensively and evenly stained for BCATc throughout the cell soma and the thin extended processes (Figure 3D).
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O2A lineage cells also were present in the mixed glial cultures. A tetrasialoganglioside specific for O2A progenitors, which could be stained with the antibody A2B5, allowed identification of the cells. Figure 4B and Figure 4E show the stellate shape of typical O2A lineage cells, with spike-like projections extending from the small cell bodies. O2A lineage cells were only slightly immunoreactive for BCATm (Figure 4A), but their cytoplasm stained intensively for BCATc (Figure 4D-F). On the other hand, not all BCATc-positive cells were apparently stained by the A2B5 antibodies (Figure 4D-F, long arrows).
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Discussion |
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The results of the present study suggest cellular localization of the BCAT isoenzymes in two types of glial cells in brain, astrocytes, and oligodendrocytes. The data show that in pure astroglial primary cultures the mitochondrial isoenzyme BCATm is located in astroglial cells. The mitochondrial location of the enzyme is confirmed by the punctate staining pattern in these cells. BCATm is the predominant BCAT isoenzyme outside of the CNS. It is a bifunctional protein that catalyzes the transamination of BCAA and the transport of the -keto acid products (
-keto acids (BCKA) generated by BCATm can be translocated into the cytosol and therefore are not necessarily available for oxidation by the mitochondrial branched-chain
-keto acid dehydrogenase. A second possible function of BCATm is transport of BCAK (
The fact that all GFAP-positive astroglial cells in culture appeared to stain strongly for BCATm may point to a constitutive expression of the gene of this isoenzyme, at least in cultured astroglial cells. In contrast, the staining for BCATc varied considerably, suggesting the existence of at least two populations of astroglial cells, one with low or moderate staining intensity and another population that did not stain for BCATc. This may indicate that expression of BCATc is a regulated astroglial function. The elevated staining by the BCATc antiserum of the nuclear area may be due to a thick layer of cytosol around the nucleus rather than a reflection of nuclear location of the isoenzyme. Oligodendroglial cells and O2A progenitor cells were also present in astroglia-rich cultures. In contrast to astroglial cells, oligodendroglial cells, identified by their specific surface marker GalC, contained BCATc as the only BCAT isoenzyme (Figure 3). The lack of tetrasialoganglioside (Figure 4) points to an advanced degree of maturity (
The pattern of expression of BCAT isoenzymes raises some questions about the physiological significance of these enzymes in brain metabolism. The BCAA are believed to play an important role in brain nitrogen metabolism. It is documented that BCAA are readily taken up into brain via the blood-brain barrier (-ketoisocaproate from leucine-fed astroglial cells into the culture medium (
-ketoisocaproate, and (d) on the observation that
-ketoisocaproate is transaminated to leucine in synaptosomes (
-ketoacid in the neuron could then cycle back to the astrocyte, where it would donate its amino group for the formation of glutamate, and subsequently of glutamine via glutamine synthetase. If glutamate dehydrogenase is active, the BCAA can also provide free ammonia for glutamine synthesis. This results in a counter-traffic of glutamine and
-keto-isocaproate from astrocytes to neurons and of glutamate and leucine in the opposite direction, assuming that BCAT activity is present not only in astroglial cells but also in neurons (Figure 5). Preliminary evidence for the presence of BCAT in neurons in culture (
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The pattern of expression of the BCAT isoenzymes in cultured astroglial and oligodendroglial cells, i.e., the variable co-existence of both isoenzymes in the former and the exclusive presence of BCATc in the latter cells, may not necessarily reflect the expression pattern in brain. In culture the cells are taken out of their normal cellular context, and cellular interactions occurring in brain by cell surface molecules or humoral factors that would be necessary for an expression to be induced or suppressed may be missing in culture. Because such factors may change during development, the expression of the BCAT isoenzymes in different brain cell types could vary with the stage of brain development and the region of the brain. Therefore, the cellular distribution of these isoenzymes must be studied in various brain areas as a function of the development from embryonic to adult animal life. Discrepancies between in vitro and in vivo functions may provide access to the factors regulating the expression of the BCAT isoenzymes.
The equilibrium constants for most aminotransferase reactions, including BCAT (-ketoglutarate in the cell, as well as on the rate of product removal (
-keto acids by the branched chain
-keto acid dehydrogenase enzyme complex. In the rat, skeletal muscle is believed to be the primary source of BCKA for oxidation by liver (
In addition to shuttling nitrogen among cells in the brain, oxidation of the BCKA may play a role in brain energy metabolism. Release of the BCKA by astrocytes (
The role of BCATc in oligodendroglial cells is not known at this time. If the catabolic pathways are present in these cells, then BCAA or BCKA can be degraded, thus serving as an energy source. The main synthetic function of oligodendrocytes is to produce myelin. Because leucine and isoleucine are eventually degraded to acetyl-CoA, they might also serve as lipid precursors in these cells.
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Acknowledgments |
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Supported by the University of Tübingen, by NATO Collaborative Research Grants Program CRG 950864, and by grant DK 34738 from the National Institutes of Health.
We thank Drs Barbara Ranscht and Frank Walsh for the anti-galactocerebroside and A2B5 antibodies, respectively. We also thank Dr Brigitte Pfeiffer for helpful suggestions concerning the immunocytochemistry and Dr Heinrich Wiesinger for critically reading the manuscript.
Received for publication May 21, 1996; accepted December 5, 1996.
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