Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

Abstract

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.

Keywords: MSUD; branched chain amino acid; branched chain aminotransferase; gamma-amino butyric acid; glutamate; traumatic brain injury.

Figure 1

Western blot analysis demonstrates the specificity of the BCKD antibody. Using a molecular weight marker in lane A, a single band was observed in lane B indicating that the antibody generated to identify BCKD does recognize a band of the appropriate size and is specific to this protein and does not recognize other antigens.

Figure 2

Immunolocalization of BCATm in the cerebellar cortex. (A) Peroxidase immunolabel for BCATm. Label is detected in the molecular (Mol) and granular layers (GL), but not in the Purkinje cell layer (PC). Dotted line indicates the pial surface. (B) Higher resolution image of a portion of the cerebellar white matter, showing label in cells that appear to be astrocytes. (C) Rhodamine-immunolabel of BCATm in the cerebellar white matter. (D) FITC-immunolabel of GFAP in the same section as in panel C. (E) Merged image of panels C and D, demonstrating label of BCATm in several astrocytes. Scale bars: (A) 100 μm, (B–E) 25 μm.

Figure 3

Immunolocalization of BCATm in the hippocampal formation. (A) Nissl-stained coronal section showing portions of the dentate gyrus (DG) and field CA1. Regions similar to those enclosed by boxes B and C are shown at higher resolution in panels B and C. (B) Peroxidase-visualized immunolabel of BCATm in the dentate gyrus. Approximate boundaries of the granule cell layer (GCL) are shown by the dotted lines. (C) Higher resolution image of BCATm-immunolabeled astrocytes of the molecular layer (MOL) of the dentate gyrus. (D) Rhodamine-immunofluorescent labeling for BCATm in the molecular layer of the dentate gyrus. (E) FITC-immunofluorescent labeling for GFAP in the same section as shown in panel D, showing GFAP in the proximal processes and cell bodies of two astrocytes. (F) Merged image of panels D and E demonstrating co-localization of BCATm and GFAP. Scale bars: (A) 200 μm; (B) 25 μm; (C) 10 μm; (D–F) 20 μm.

Figure 4

Immunolocalization of BCATm in the spinal cord. (A) Anti-BCATm strongly labels elongate, radially oriented elements in the white matter of the spinal cord. (B) Labeling for GFAP in the same section as shown in panel A shows that the radial elements are glial processes. (C) The merged image of panels A and B shows nearly complete colocalization of BCATm with GFAP. (D) (Inset) Cell bodies of glial cells are labeled only for BCATm. Scale bar: (A–C) 100 μm. (D) 20 μm.

Figure 5

Immunolocalization of BCKDC-E1α in the cerebellar cortex. Co-localization of BCKDC-E1α (red) and GFAP (green) in the cerebellar cortex. Labeling for BCKDC is very prominent in structures located along the plane of Purkinje cell bodies (PC, cells marked by ^*; BCKDC label indicated by arrows). Label for BCKDC is also seen in some cells of the molecular layer (Mol). Immunolabel for GFAP is associated with the distal process of the Bergmann glial cells (BG, arrows) and with astrocytes in the granule cell layer (GC). (Inset) Merged image of co-labeling for BCKDC (red) and glutamate dehyrogenase (GAD), a marker for GABAergic cells. Purkinje cell bodies are clearly demarcated by anti-GAD. Strongest labeling for BCKDC is either at the far periphery of the Purkinje cells, in a distinct structure associated with surfaces of Purkinje cell bodies. Scale bars: (A) 50 μm. (B–E) 25 μm.

Figure 6

Immunolocalization of BCKDC in deep cerebellar nuclei and white matter. (A) Labeling for BCKDC is strong in the cell bodies of neurons of the deep cerebellar nuclei. Dashed white line marks the border between the nucleus and an unlabeled white matter tract. (B) Immunolabeling for myelin basic protein (MBP), a marker for myelin and oligodendrocytes, in the same section as in panel A. (C) Merged image of panels A and B. There is virtually no co-labeling of cells for BCKDC and MBP. (D) Co-label of the Purkinje cell layer for BCKDC and MBP. Structures labeled for BCKDC are not labeled for MBP, indicating that they are not oligodendrocytes. (E) Control section showing the Purkinje cell layer of a section incubated with secondary antibodies only. The image was obtained using the same microscope settings, and processed in the same way as the image in panel A. Scale bars: (A–C) 100 μm. (D,E) 50 μm.

Figure 7

Immunolocalization of BCKDC in dentate gyrus. (A) BCKDC immunolabel is present in neurons of the granule cell layer (GCL). Arrows indicate specific labeling of large neurons of the polymorphic layer (PoM). (B) Immunolabel for the astrocyte marker GFAP in the same section as in panel A. Astrocyte processes are labeled in the granule cell polymorphic layers. Astrocyte cell bodies are also labeled in the polymorphic layer. (C) Merged image of panels A and B. There is no co-labeling of cells for BCKDC and GFAP. (D) Labeling of the dentate gyrus for BCATc, one of the enzymes that generates branched-chain α-keto-acids that are the substrates for BCKDC. As shown previously (Sweatt et al., 2004a), BCATc is present only in cells at the inner margin of the granule cell layer. Scale bars: (A–C) 25 μm. (D) 20 μm.

Figure 8

Immunolocalization of BCKDC in the cerebral cortex. BCKDC is clearly expressed in the perinuclear regions of neurons in the pyramidal cell bodies. GFAP immunoreactivity is observed in astrocytic processes primarily in the apical surface. Scale bars: 100 μm.

Figure 9

Immunolocalization of BCKDC in the hypothalamus. (A) Labeling for BCKDC is present in cells of hypothalamic nuclei, as well as in ependymal cells lining the third ventricle (III). (B) Immunolabel for S100 in the same section as in panel A. (C) Merged image of panels A and B. BCKDC-positive cells are not co-labeled for S100. Labeling for BCKDC in the ependymal cells clearly lies above a layer of cells that express S100. (D) Higher resolution image of the arcuate nucleus (Arc), near the transition to the median eminence (ME). (E) Immunolabel for S100 in the same section as in panel D. (F) Merged image of panels D and E. Neurons of the arcuate nucleus are labeled for BCKDC, with no overlap of labeling for S100. S100 labeling appears in occasional cells of the nucleus, as well as in processes of cells lying in the transition zone to the median eminence. Scale bars: (A–C) 100 μm. (D–F) 50 μm.

Figure 10

Immunolocalization of BCKDC in the spinal cord. (A) Labeling for BCKDC in the white matter is found on cell bodies and fine, radially oriented processes. (B) Labeling for an astrocyte marker, S100, is present over some cell bodies, but is mostly associated with serpentine structures that resemble blood vessels. (C) The merged image of panels A and B shows that BCKDC-positive cell bodies are often associated with S100-positive processes. Scale bars: (A–C) 50 μm.