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The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc-labelled neurons were either GABA-ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.
Studies using in vivo rat brain and ex vivo rat retina models demonstrate that the branched chain aminotransferases (BCATs) [E.C. 18.104.22.168] are key metabolic proteins, responsible for the production of 30% of de novo brain glutamate (LaNoue et al. 2001; Lieth et al. 2001). They catalyse the reversible transamination of the branched chain amino acids (BCAAs) leucine, isoleucine and valine to their respective α-ketoacids (BCKAs) and glutamate (Scheme 1) (Ichihara and Koyama 1966). These amino acids can cross the blood-brain barrier, where the influx of leucine is considerably higher than that of other amino acids (Oldendorf 1973; Smith et al. 1987). Studies using [15N]leucine have demonstrated that the BCAAs are nitrogen donors for the synthesis of glutamate and glutamine in brain explants and in primary neuronal cultures (Yudkoff et al. 1983, 1996a,b; LaNoue et al. 2001). De novo synthesis of glutamate also involves other enzymes, including pyruvate carboxylase and the TCA cycle enzymes, to produce α-ketoglutarate (LaNoue et al. 2001). The second and rate limiting step in the metabolism of the BCAAs is catalysed by the branched-chain α-keto acid dehydrogenase complex (BCKDC) composed of three subunits (E1–E3) (Harris et al., 1990). A metabolon between hBCATm and the E1 subunit has been reported that facilitates the channelling of branched-chain alpha-keto acids from BCATm to E1 (Islam et al. 2007), the kinetics of which are influenced by glutamate dehydrogenase (Islam et al. 2010).
There are two main human isoforms of BCAT, cytosolic BCAT (hBCATc) and mitochondrial BCAT (hBCATm), these enzymes sharing 58% homology in their primary amino acid sequence (Hutson 1988; Hutson et al. 1992; Davoodi et al. 1998). The mitochondrial isoform, hBCATm is expressed in most tissues and plays a significant role in skeletal muscle glutamine and alanine synthesis (Suryawan et al. 1998). These proteins are unique among the aminotransferase family where they are reversibly regulated through a redox sensitive CXXC motif, which is reversibly inhibited through S-nitrosation (Conway et al. 2004, 2008; Coles et al. 2009). The cytosolic isoform, hBCATc, is found predominantly in nervous tissue and accounts for 70% of rat brain BCAT activity (Sweatt et al. 2004a, b; Garcia-Espinosa et al. 2007). Two other variants have also been described that are homologous to hBCATm: a novel alternatively spliced variant found in placental tissue and a splice variant that is reported to act as a co-repressor of thyroid hormone nuclear receptors (Lin et al. 2001; Than et al. 2001).
As a result of the ease with which the BCAAs pass the blood–brain barrier and their role in glutamate metabolism, the subcellular localization of the BCAT proteins, particularly BCATc, in brain has been extensively investigated in rat brain and cell models (Bixel et al. 1997, 2001; Sweatt et al. 2004a, b; Garcia-Espinosa et al. 2007). In rat brain, the expression of BCATc was found to be solely in neurons, its subcellular distribution depending on neuronal type (Sweatt et al. 2004a, b). In glutamatergic neurons, such as granule cells of the cerebellar cortex and dentate gyrus, BCATc was localized to axons and nerve terminals, whereas in GABAergic neurons, such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc was concentrated in the cell body. BCATc was also found in autonomic nerve fibres of the digestive tract, as well as in axons of the sciatic nerve (Sweatt et al. 2004a, b). Although the mitochondrial isoform was not mapped in these studies, recent work has reported uniform staining of BCATm in the cell bodies of astrocytes throughout rat brain, supporting the hypothesis that BCATm contributes nitrogen to the glutamate-glutamine cycle via astrocytes in rat (Cole et al. 2012). However, in primary cell cultures of rat brain, BCATm was detected in astrocytes and microglia, and BCATc was found not only in neurons but also in developing oligodendrocytes (Bixel et al. 1997, 2001). In the developing rat brain, BCATc expression was up-regulated in neurons and glial cells as they responded to changes in connectivity and growth factors (Garcia-Espinosa et al. 2007).
The cell-specific compartmentalization of BCATc to neurons and BCATm to astrocytes, together with several key metabolic studies, suggested that in astrocytes transamination was in the direction of glutamate and α-keto acid synthesis, the reverse holding true for BCATc transamination in neurons (Chaplin et al. 1976; Brand 1981; Brookes 1993; Yudkoff et al. 1994). These findings led to the development of the BCAA-BCKA shuttle hypothesis, in which the BCAT proteins function as an anaplerotic pathway that interfaces with the glutamate-glutamine cycle to replenish ‘lost’ glutamate (Fig. 2) (Hutson et al. 1998, 2001). This proposed pathway allows for nitrogen transfer for glutamate synthesis in neurons and to buffer neuronal glutamate (Yudkoff 1997). However, in a rat cell culture model, BCATc was also detected at low levels in some astrocyte populations and it has therefore been unclear the extent to which these models are directly applicable to man (Bixel et al. 1997, 2001).
Figure 2. The glutamate-glutamine cycle and the branched chain amino acid (BCAA)-branched chain α-ketoacid (BCKA) shuttle. Glutamate is released from neuronal cells during excitatory neurotransmission. Excess glutamate not utilized is taken up via specific receptors in astrocytes. In astrocytes, much of the glutamate undergoes amidation to glutamine by glutamine synthetase. Glutamine is then released for uptake by neuronal cells, to replenish the glutamate pool. However, as glutamate can also be lost through oxidation or generation of glutathione/purines in astrocytes the glutamate-glutamine cycle must work with other anaplerotic pathways to regenerate the neuronal pool (McKenna 2007). BCAA metabolism is thought to participate in nitrogen shuttling in the de novo synthesis of glutamate. In brief, it was proposed that mitochondrial branched chain aminotransferase (BCAT) catalyses the transamination of the BCAA in astrocytes generating glutamate, which enters the glutamate-glutamine cycle with BCKA shuttled to neuronal cells for transamination with glutamate back to BCAA, by BCATc which exit the neuron and return to the astrocyte, with α-ketoglutarate entering the TCA cycle (Yudkoff 1997; Hutson et al. 2001).
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Our aim in this study was to examine the distribution of the hBCAT proteins in the human brain, to ascertain whether the proposed BCAA-BCKA shuttle might contribute to the regulation of glutamate in the human brain. The regulation of glutamate is of particular importance for the prevention of excitotoxicity, an important contributor to neuronal cell death in brain ischaemia and several neurodegenerative diseases. For the first time, this study maps the hBCAT proteins in the human brain. In contrast to reported findings in the cell models, our findings highlight the presence of hBCATm in endothelial cells throughout the brain vasculature and an absence of detectable labelling in astrocytes. As in the rat, we report that in man hBCATc is restricted to neurons, although widely distributed throughout the brain. We discuss the impact of these findings with respect to the current BCAA-BCKA shuttle and the potential impact on glutamate regulation in the human brain.
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- Materials and methods
Given the key role that BCAT enzymes play in brain glutamate metabolism, it is important to establish the distribution of these isoforms in the human brain relative to rat and cell culture models. Consistent with the rat and cell culture models, hBCATc was found predominantly in neuronal cells, supporting its proposed role in the synthesis of the neurotransmitter glutamate and as a precursor for GABA production. Distribution of hBCATc expression in hormone secreting cells offers the potential for additional roles for hBCATc. Moreover, for the first time we show that unlike the rodent model, which showed expression of hBCATm in astrocytes, there is extensive immunopositive staining for hBCATm in the vasculature of the brain with no evidence of staining within astrocytes, suggesting a different role for hBCATm in glutamate regulation in the human brain. This and other findings are discussed in further detail with respect to published models.
The role of BCAAs in glutamate production in neurons is based on studies that show BCAAs can label a significant proportion of the glutamate pool in neuronal cell culture, which has also been shown in vivo and ex vivo rat retina models (Yudkoff et al. 1983, 1994; Kanamori et al. 1998; LaNoue et al. 2001). Similar to the rat studies by Garcia-Espinosa et al. (2007) the majority of hBCATc-labelled neurons in the human brain were either GABAergic or glutamatergic, with GABAergic neurons shown to be more strongly immunopositive than glutamatergic neurons. However, many neurons that were neither glutamatergic nor GABAergic also labelled for hBCATc. The distribution and the varied intensities of expression in the hippocampus, where staining of the CA3 region was more intense than the CA1 region, mirrored the findings of Castellano et al. (2007) who investigated mRNA expression of hBCATc in post-natal and adult brains of mice. The cell bodies of neuronal cells within the temporal and hippocampus showed intense staining relative to the dendrite regions reflecting the possibility that their primary role would be to contribute to the glutamate metabolic pool used to generate glutamate rather than the glutamate pool used during excitation. Conversely, in the areas of the supraoptic tract intense staining along axons was noted indicating an additional role of hBCATc transamination in glutamate release in this region. Our findings are consistent with the rat and rat metabolic studies suggesting that hBCATc transamination could potentially contribute to the pool of neurotransmitters and the glutamate required for release during excitation.
Magnocellular neuroendocrine cells were shown to express high levels of hBCATc, in the supraoptic nucleus and the paraventricular nucleus of the hypothalamus. In response to osmoreceptors in the brain these cells which are rich in AMPA and NMDA receptors, respond to glutamate stimulation, which act together with intrinsic membrane conductance that control the bursting pulsatile release of oxytocin and vasopressin (reviewed in Pak and Currás-Collazo 2002). Precedence for BCAT metabolites in hormone secretion has been reported, where leucine has been shown to be a potent insulin secretagogue (Xu et al. 2001; Zhou et al. 2010). As intense hBCATc expression in the magnocellular neurons and also in the substantia nigra was reported it will be important to further investigate if these proteins play a role in hormone release in these cells. The presence of hBCATc in the neurons of the nucleus basalis of Meynert and the raphe nuclei raises a similar curiosity about hBCATc in acetylcholine and serotonin release or in intermediate production. Moderate staining of hBCATc in pigmented cells, such as the neurons of the locus ceruleus, that strongly express melanin concentrating hormone (MCH), may be related to the role of BCAAs in melanogenesis, where it has been demonstrated that incubation of melanoma cells with BCAA inhibited the production of melanin (Cha et al. 2012). The mechanism underpinning this inhibition was not determined. Therefore, given the role of leucine in insulin release, their contribution in these other endocrine cells warrants further investigation.
Previous studies using astroglial primary cultures derived from the brains of newborn Wistar rats showed that BCATm was largely located in astrocyte populations, the basis of which formed the hypothetical BCAA-BCKA nitrogen shuttle, where the BCAT enzymes facilitate the de novo synthesis of glutamate (Fig. 2) (Hutson et al. 1998, 2001; LaNoue et al. 2001). In rat, this allows for nitrogen transfer to regenerate glutamate in neurons (Yudkoff et al. 1996a, b; Yudkoff 1997). In contrast, here, no astrocyte staining was detected in the human brain, indicating that hBCAT transamination cannot occur in human astrocytes. Although, the explanation remains unclear, one possibility is that the expression of hBCATm is induced under conditions of cell proliferation, as demonstrated in studies by Perez-Villasenor et al. (Perez-Villasenor et al. 2005), or that the pattern of expression may be altered by placing cells in culture outside the normal milieu. Alternatively, these studies more likely highlight the difference in BCAT expression between species, especially in light of recent studies which have reported positive astrocyte staining for BCATm in rat brain tissue (Cole et al. 2012). Moreover, one must also consider the age of the brains used in this study and appreciate that expression of hBCATm may differ in younger brains of humans.
In these studies, the expression of hBCATm was pronounced throughout the endothelial layer examined in all areas of the human brain in all samples analysed indicating that transamination can occur in these cells, where the direction of metabolism will be directed by substrate concentration and the expression of other key metabolic systems. Cerebral endothelial cells have a high density of mitochondria (Oldendorf et al. 1977), giving greater capacity for energy production than other capillary endothelial cells and explaining the punctuate staining of mitochondrial hBCAT in these cells. The endothelial barrier has membrane-specific transporters, the L-system facilitative transporters (L1), on both the abluminal and luminal side which allow transport of BCAAs, particularly leucine (Oldendorf 1973; Smith et al. 1987; O'Kane and Hawkins 2003; O'Kane et al. 2004; Abbott et al. 2006). Conversely, only minimal glutamate entry is allowed on the luminal side, which is thought to protect the brain from peripheral glutamate plasma changes that could be potentially toxic to brain cells. Although controversy exists regarding the exact EAAT subtype and its expression on the abluminal side (Chaudhry et al. 1995; O'Kane et al. 1999) evidence in support of brain glutamate efflux is gaining impetus (Hosoya et al. 1999; Gottlieb et al. 2003; Uchida et al. 2011), the rate of which is considered to be influenced by blood glutamate (Gottlieb et al. 2003; Zlotnik et al. 2008, 2012; Teichberg et al. 2009; Campos et al. 2012). Currently, the proposed mechanism in animal models by which the endothelial cells remove excess glutamate include direct uptake of glutamate through Na+-dependent transporters, creating a rise in the glutamate gradient in endothelial cells, which has been proposed to subsequently exit into the blood through facilitated transport (O'Kane et al. 1999). Until the study by Helms et al. 2012, the potential for further metabolism of glutamate within endothelial cells was not considered. If glutamate dehydrogenase (GDH) is expressed in endothelial cells glutamate oxidation can occur, dependent on the concentration and redox state. However, our novel finding of the hBCATm transaminase in these cells raises the possibility of an additional mechanism to support glutamate synthesis or metabolism, in the human brain.
For glutamate oxidation to occur in endothelial cells driven by hBCATm, branched chain keto acids are required, although the source remains unclear. Oxidation of glutamate would release α-keto glutatarate that could enter the TCA cycle and the respective BCAA, which could be further oxidized for energy or be taken up by neuronal cells for further metabolism. Metabolism of brain glutamate by hBCATm could serve as an auxillary mechanism to the glutamate-glutamine cycle, the predominant system that regulates brain glutamate between neurons and astrocytes (Danbolt 2001). For this to be possible, levels of glutamate would need to exceed the Km of the glutamate transporter (approximated at 138 μM in rat models), which could only occur, albeit for ≤ 10 ms during excitation (Dzubay and Jahr 1999) or more importantly during periods of excitotoxicity, a feature in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease (Moussawi et al. 2011). Although the kinetics of glutamate efflux may differ in humans, glutamate uptake and metabolism by hBCATm would be of benefit to glutamate homeostasis, in particular when glutamate levels are high, indicating a possible role for this protein as an auxiliary neuroprotection mechanism. Transamination in the opposite direction of BCAA metabolism would require α-ketoglutarate, which would affect TCA cycle activity if it were not replenished. Notably, pyruvate carboxylase could fill this role through carboxylation of pyruvate, but this enzyme has not been mapped to this area and is considered specific to astrocytes. Other potential candidates include malic enzyme or phosphoenolpyruvate carboxylase kinase, but these are thought to generally operate in the direction of decarboxylation. Alternatively, another transamination reaction (e.g. ALT) could operate to transfer nitrogen from glutamate to pyruvate and regenerate α-keto glutarate and alanine. Evidence for nitrogen shuttling has been established in muscle, where ALT transaminases play a pivotal role in mediating the passage of intermediates. Indeed, if GDH is found to be present and BCATm/GDH association occurs, then the end product is ammonia and α-keto glutarate is regenerated (Hutson et al. 2011). Evidently, the metabolic reason for hBCATm expression in the endothelium is open to discussion, clarity of which will only evolve with improved models of human brain metabolism and also mapping of key metabolic proteins to this area.
In summary, we provide the first direct evidence of transaminases in the endothelial layer that can potentially operate as a support network to astrocytes mediating the fine tuning of glutamate homeostatsis. We propose that under normal physiological conditions the BCAAs are taken up by neuronal cells, where hBCATc controlled transamination acts as a pathway to replenish the glutamate pool. These findings may have further implications with respect to our understanding of glutamate toxicity, an important contributor to a range of neurodegenerative and vascular neurological diseases. Further research is needed into the role of BCAT in glutamate regulation and toxicity in the brain.