ARTICLE |
Correspondence to: Susan M. Hutson, Wake Forest U. School of Medicine, Department of Biochemistry, Winston-Salem, NC 27157. E-mail: shutson@wfubmc.edu
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Summary |
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Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the -keto acid products of BCAT catalyzed by the branched-chain
-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization. (J Histochem Cytochem 49:407418, 2001)
Key Words: brain cells, branched-chain amino acid, cell culture, energy metabolism, immunocytochemistry, nitrogen metabolism
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Introduction |
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THE ESSENTIAL BRANCHED-CHAIN AMINO ACIDS (BCAAs) leucine, isoleucine, and valine readily cross the bloodbrain barrier (
The second reaction in BCAA degradation is the oxidative decarboxylation of the branched chain -keto acids (BCKAs) catalyzed by the mitochondrial branched chain
-keto acid dehydrogenase (BCKD) enzyme complex. Because this step is essentially irreversible, passage of the
-keto acids corresponding to the BCAAs through this step commits the carbon skeleton to degradation. The multienzyme complex consists of three different subunits, E1, E2, and E3, and is structurally related to the pyruvate dehydrogenase enzyme complex (
BCAAs are believed to be involved in brain glutamate metabolism, especially because
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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 plasticware was from Nunc (Wiesbaden, Germany). Acrylamide and N,N'-methylene bisacrylamide were obtained from Bio-Rad Laboratories (München, Germany). 5-Bromo-4-chloro-3-indolyl-phosphate and 4-nitroblue tetrazolium chloride, premixed protein molecular weight markers (cat. no. 1495984), sodium dodecyl sulfate (SDS), and Tris were from Boehringer (Mannheim, Germany). N,N-N',N'-tetramethyl-ethylendiamine, Triton X-100, and Tween-20 were from Serva (Heidelberg, Germany). Nitrocellulose filter sheets were purchased from Millipore (Eschborn, Germany). Bovine serum albumin (BSA), poly-D-lysine, fluorescein isothiocyanate (FITC)-labeled anti-rabbit immunoglobulin G (IgG) (host animal, goat; affinity isolated antibody; cat. no. F-0382), FITC-labeled anti-mouse IgG (host animal, goat; affinity isolated antibody; cat. no. F-1010), tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-rabbit IgG (host animal, goat; affinity isolated antibody; cat. no. T-6778), anti-vimentin (clone V9; cat. no. V-6630), and anti-growth-associated protein 43 (GAP-43; clone GAP-7B10; cat. no. G-9264) monoclonal antibodies and alkaline phosphatase-conjugated anti-rabbit IgG (host animal, goat; affinity isolated antibody; cat. no. A-3687) were obtained from Sigma (Deisenhofen, Germany). Anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (clone G-A-5; cat. no. IF03) and Cy3-labeled anti-mouse IgG (host animal, goat; affinity isolated antibody; cat. no. 115-165-003) were purchased from Dianova (Hamburg, Germany). Anti-CR3 monoclonal antibody (clone OX42; cat. no. MCA-275R) was from Serotec (Wiesbaden, Germany). All other chemicals were of highest purity available and were obtained from Merck (Darmstadt, Germany).
The generation of the antisera against BCATc purified from rat brain cytosol and against BCATm isolated from rat heart mitochondria has been described previously (
Cell Culture
Neuron-rich primary cultures were prepared from the brains of 16-day-old rat embryos as described (
Immunocytochemistry
For BCAT/vimentin, BCAT/OX42, BCKD/GFAP, BCKD/vimentin, and BCKD/OX42 double staining, the cells were fixed at room temperature (RT) using 4% (w/v) paraformaldehyde in PBS, pH 7.4, for 10 min. The cells were washed twice in PBS for 5 min, then once with 0.1% (w/v) glycine in PBS, pH 7.4, for 5 min. Next, the cells were permeabilized with 0.3% (w/v) Triton X-100 in PBS for 10 min. The antisera against BCAT isoenzymes and the antivimentin monoclonal antibodies were diluted in PBS containing 0.3% (w/v) Triton X-100 and 10% (v/v) normal goat serum (Protocol A). This staining method proved to be advantageous for detection of the BCAT isoenzymes and BCKD. Because the anti-GAP-43 antibodies were unable to recognize their antigen when the cells were treated according to this procedure, for BCAT/GAP-43 and BCKD/GAP-43 double staining the cells were fixed with 4% (w/v) paraformaldehyde in PBS at pH 7.4 for 15 min. The cells were permeabilized by ice-cold methanol for 5 min using a modification of the method described by
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For immunostaining of cells on the coverslips, they were placed in a humidified chamber and then exposed to a mixture of the two primary antibodies for 2 hr and separately to the mixture of the two secondary antibodies for 1 hr. The figure legends indicate the corresponding antibody solutions used for the individual double-labeling experiments. The staining of the cultured cells was the same whether they were stained with individual antibodies or with both antibodies in the same cocktail. After each incubation, the cells were washed twice either with 0.1% (w/v) Triton X-100 in PBS for BCAT/vimentin staining or with 0.3% (w/v) BSA in PBS for BCAT/GAP-43 staining. The coverslips were mounted, cells down, using 50% (v/v) glycerol in PBS. The preparations were viewed by glycerol immersion optics using a Zeiss fluorescence microscope (IM35) with a Plan-Neofluar x25 objective. The specificity of the BCATc, BCATm, and BCKD antisera was evaluated by preabsorbing the antisera with their corresponding antigens before using them for immunocytochemistry. The suitable antigen concentration for preincubation of the antiserum was determined using ELISAs. ELISAs were performed by coating 96-well plates overnight at 4C with 100 ng BCAT. BCATm was purified from rat heart mitochondria (
Before use in immunocytochemistry, the BCATm (BCATc) antiserum (diluted 1:200) was preincubated with 8 µg BCATm (5 µg BCATc) in a total volume of 80 µl at 4C for 12 hr. We have shown previously that BCATm is abundant in astroglial cultures (
Immunoblotting
The cell culture homogenates were prepared as described previously (
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Results |
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To determine the distribution of BCAT isoenzymes in neurons, we have immunocytochemically examined neuron-rich primary cultures prepared from embryonic rat brains using antisera against rat BCATc and rat BCATm. Staining of the cultures for BCATc revealed strong immunoreactivity in cells with small and round somata bearing several thin and extended processes (Fig 2A and Fig 3A). These cells were identified as neurons by double staining with antibodies against the neuronal marker GAP-43 (Fig 2B;
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The distribution of both BCAT isoenzymes was also investigated in microglial cells. Astroglia-rich primary cultures, in which about 10% of the cell population are microglial cells, were used as the source to obtain almost pure microglial secondary cultures (
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The second step in degradation of BCAA is oxidative decarboxylation of the transamination products, the BCKAs, which is catalyzed by the mitochondrial BCKD enzyme complex. This step commits the BCAA carbon skeleton to the degradation pathway. Therefore, the cellular distribution of this enzyme was investigated in cultured brain cells. Double staining of neuron-rich primary cultures with BCKD antiserum, which recognizes the E2 subunit of the BCKD, and with monoclonal antibodies against the neuronal marker GAP-43, revealed co-localization of the two proteins, indicating that neurons express BCKD (Fig 6A6C). The fluorescence signal for BCKD was observed in the cell somata and also in the cell processes (Fig 6A). Because cultured neurons grow preferentially in cell clusters and the portion of cytoplasm around their cell nuclei is rather small, the characteristic punctate staining for the mitochondrial BCKD is difficult to see in neurons. In the experiment shown in Fig 6D6E, an astroglia-rich primary culture was used to examine the presence of BCKD in astroglial cells. BCKD antiserum was applied in combination with an antibody against the astroglial cell marker GFAP. Staining for BCKD revealed co-localization of BCKD and GFAP in flat and extended cells of irregular shape. In contrast to the staining of neurons, in astroglial cells the punctate fluorescence signal for mitochondrial BCKD could be seen clearly in the extended cytoplasm of these cells (Fig 6D). The cell nuclei were not stained. In addition to GFAP-positive astroglial cells, other GFAP-negative cell types stained positive for BCKD (Fig 6D6F). Most of these cells had small round somata carrying several thin and elongated processes characteristic of oligodendroglial cells and O2A lineage cells. In Fig 6G6I, a microglial secondary culture was stained simultaneously for BCKD and for the microglial marker CR3. CR3-positive cells displayed intense fluorescence staining of punctate and tube-like structures, which were located in the cytosol. Again, the nuclei were not labeled. The cell periphery of the microglial processes was less intensely stained, most likely due to the lower occurrence of mitochondria. The results suggest that, in contrast to each of the BCAT isoenzymes, BCKD is expressed ubiquitously in rat brain cell cultures.
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Discussion |
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This study shows that BCATc is the sole BCAT isoenzyme expressed in rat cortical neurons in culture, whereas BCATm is the isoenzyme form found in microglial cells in culture. In a separate immunocytochemical study, we found previously that BCATm is the predominant isoenzyme in cultured astroglial cells, while BCATc, but not BCATm, is found in high concentration in cultured oligodendroglial cells and O2A progenitors (
Yudkoff and co-workers (1996a,b; -ketoisocaproate, from the astrocytes (
-ketoisocaproate in astrocytes, synaptosomes, and cultured neurons (
The BCAA nitrogen shuttle has been expanded to include an obligatory role for these amino acids for optimal rates of de novo glutamate synthesis (-ketoglutarate and subsequently metabolized to lactate (
-amino nitrogen of glutamate (
The immunocytochemistry results presented here (Fig 2 and Fig 3) and by
The BCAA nitrogen shuttle hypothesis makes no prediction about the location of the BCKD enzyme complex, but changes in activity of this enzyme could also affect operation of the cycle. Becausee the BCKD irreversibly decarboxylates the BCAA transamination products, the BCKAs, it is the committed step in BCAA oxidation and results in a net transfer of nitrogen to the cell in which this reaction occurs. The ubiquitous expression of BCKDs in rat brain cells in culture suggests that all types of brain cells can oxidize the BCAAs. Recently, we immunocytochemically localized -methylcrotonyl-CoA carboxylase (
-MCC), an enzyme unique to the leucine catabolic pathway, in cultured astroglial and neuronal cells (
-MCC, and the majority of neurons also contained
-MCC. The expression of BCKD (present work) and
-MCC (
BCAT isoenzymes also exhibit cell-specific expression in cultured rat brain cells other than astrocytes and neurons (Fig 4;
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Footnotes |
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1 Present address: Freie Universität Berlin, Institut für Biochemie, Berlin, Germany.
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Acknowledgments |
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Supported by the NATO Collaborative Research Grants Program CRG 950864 and by grant DK 34738 (SMH) from the National Institutes of Health.
We thank Dr Brigitte Pfeiffer, Dr Heinrich Wiesinger, and Mr Oliver Kranich for kindly providing neuron-rich primary cultures, and Agus Suryawan for testing the specificity of the BCKD antiserum.
Received for publication May 31, 2000; accepted October 12, 2000.
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