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6 Branched Chain Amino Acids (BCAAs) in Brain

S. M. Hutson . A. J. Sweatt . K. F. LaNoue

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Introduction

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Branched

Chain

Amino

Acid Metabolism

 

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Branched

Chain

Amino

Acids

Are Nitrogen Donors in Peripheral Tissues

 

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Branched

Chain

Amino

Acids

as Nitrogen Donors in the Central Nervous System

 

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4.1

Intercellular Nitrogen Transfer

 

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Role of BCAA in de novo Glutamate Synthesis and the Branched Chain Aminotransferase (BCAT) Cycle Hypothesis

 

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Localization of BCAA Catabolic Enzymes in Rodent Brain

 

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6.1

The

Cytosolic BCATc Isozyme

 

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6.2

The Mitochondrial Enzymes BCATm and Branched Chain a-Keto Acid

 

Dehydrogenase (BCKD)

 

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6.3

The BCAT Cycle and GABA Metabolism

 

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Conclusions, Implications for Disorders of BCAA Metabolism, and Future Directions

 

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# Springer-Verlag Berlin Heidelberg 2007

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Branched chain amino acids (BCAAs) in brain

Abstract: The branched chain amino acids (BCAAs) play a role in glutamate neurotransmitter synthesis by providing a ferry system to move nitrogen between astrocytes and neurons for the synthesis of glutamate. This hypothetical nitrogen cycle, the branched chain aminotransferase (BCAT) cycle, is tied to the glutamate/glutamine cycle and is an arm of the glutamate/pyruvate cycle. Immunolocalization of the BCAT isozymes in rodent brain shows that the cytosolic BCATc is localized in selected glutamatergic and GABAergic neurons whereas the mitochondrial isozyme, BCATm, is found in astroglia. These results provide support for the cycle hypothesis and suggest BCAA also play a role in gaminobutyric acid (GABA) metabolism. When the BCAA catabolic disposal system is genetically impaired in humans, serum BCAA and BCKA levels rise and severe neurological dysfunction occurs. In the hereditary metabolic disease that results from mutations in the branched chain aketo acid dehydrogenase complex (BCKD), maple syrup urine disease, the most obvious symptoms are neurological. Useful animal models for the human condition are not yet available.

List of Abbreviations: BCAA, branched chain amino acid; BCAT, branched chain aminotransferase; BCATc, cytosolic branched chain aminotransferase; BCATm, mitochondrial branched chain aminotrans- ferase; BCKA, branched chain a-keto acid; BCKD, branched chain a-keto acid dehydrogenase; E1, branched chain a-keto acid decarboxylase; E2, dihydrolipoly transacylase; E3, dihydrolipoyl dehydrogenase; GABA, gaminobutyric acid; GABA-T, GABA, aminotransferase; GAD, glutamate decarboxylase; a KG, a-ketoglu- tarate; KIC, aketoisocaproate; KIV, a -ketoisovalerate; KMV, a -keto- b -methylvalerate; MSUD, Maple Syrup Urine Disease; PC, pyruvate carboxylase; TCA, tricarboxylic acid

1 Introduction

The branched chain amino acids (BCAAs), leucine, isoleucine, and valine, are classified as indispensable amino acids because they cannot be synthesized de novo in mammals. Therefore, they must be obtained from dietary sources and adequate intake of BCAAs is required for normal growth and development.

In addition to their role as protein building blocks, the BCAAs are involved in interorgan nitrogen transfer

between peripheral tissues. In the central nervous system (CNS), they are thought to provide nitrogen for neurotransmitter glutamate synthesis. Inborn errors of BCAA metabolism that increase BCAAs and/or their metabolites are toxic to the brain. Thus, regulation of BCAA catabolism is required to limit their breakdown when dietary intake is restricted and to efficiently clear BCAAs and their metabolites when dietary intake exceeds the body’s needs.

2 Branched Chain Amino Acid Metabolism

A schematic diagram of the BCAA catabolic pathways is shown in > Figure 6-1 . The first step in BCAA

catabolism is reversible transamination catalyzed by the branched chain aminotransferase (BCAT) iso- zymes. In this step, the aamino group of an individual BCAA is transferred to aketoglutarate to form glutamate and the respective branched chain a keto acids (BCKA: a ketoisocaproate [KIC], aketobmethylvalerate [KMV] or aketoisovalerate [KIV]). Glutamate, in addition to its role as the major excitatory neurotransmitter in the CNS, is also a key metabolic substrate for a variety of nitrogen transfer reactions and it is the entry point for BCAA nitrogen into other metabolic pools. Given the large size of the brain

glutamate pool, in short term studies BCAA nitrogen tends to accumulate in glutamate (Hutson et al., 2005). Nevertheless, BCAA nitrogen can flow from glutamate via other aminotransferase reactions to amino acids such as alanine and aspartate. In glia where glutamine synthetase is located, BCAA nitrogen flows from glutamate to the aamino group of glutamine (Minchin and Beart, 1975; Martinez Hernandez

et al., 1977; Norenberg, 1979). In GABAergic neurons, BCAA nitrogen enters into the gaminobutyric acid

pool (GABA) by decarboxylation of glutamate catalyzed by the vitamin B 6 dependent glutamate decarbox-

ylase (GAD) isozymes. Free ammonia can be formed by oxidative deamination of glutamate catalyzed by mitochondrial glutamate dehydrogenase.

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. Figure 6-1 BCAA catabolic pathways. The first step is transamination of the BCAAs (Leu, Ile, Val) catalyzed by the branched chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) to produce the branched chain a keto acids (BCKAs), aketoisocaproate (KIC), a ketob methylvalerate (KMV), and aketoiso- valerate (KIV). aKetoglutarate is the a keto acid acceptor of the BCAA aamino group. Glutamate (Glu) is the product. Glutamate, via the action of other aminotransferases, can donate its nitrogen group to other amino acids, such as Asp and Ala, or reform aketoglutarate via oxidative deamination catalyzed by mitochondrial glutamate dehydrogenase forming free NH 3 . Glutamate is decarboxylated by glutamate decarboxylase to form gaminobutyric acid (GABA) in GABAergic neurons. In astroglia, glutamate is also converted to glutamine by glutamine synthetase as part of the glutamate/glutamine cycle. The second catabolic step is oxidative decar- boxylation, catalyzed by the branched chain aketo acid dehydrogenase (BCKD) enzyme complex

chain a ‐ keto acid dehydrogenase (BCKD) enzyme complex In mammals, there are two BCATs—a mitochondrial

In mammals, there are two BCATs—a mitochondrial (BCATm) and a cytosolic (BCATc) isozyme that are encoded by two separate genes (Ichihara, 1985; Hutson et al., 1988; Bledsoe et al., 1997). Not only are the BCAT isozymes expressed in different cell compartments but they also show different patterns of expression in body tissues ( > Figure 6-2 ). With the exception of rodent liver, the mitochondrial isozyme is expressed ubiquitously, and outside the CNS it is often localized in secretory epithelial cells (Hutson et al., 1992; Sweatt et al., 2004a). On the other hand, the cytosolic isozyme is found almost exclusively in nervous tissue (Ichihara, 1985; Hutson et al., 1988; Sweatt et al., 2004a). BCATc is the predominant BCAT activity in rat brain accounting for 60–70% of total brain BCAT activity (Hutson, 1988; Hall et al., 1993). In the rat, BCATc is also expressed outside the CNS, localized in peripheral nerves, but neither BCATm nor BCKD are expressed in these nerves (Sweatt et al., 2004a). Coexpression of BCATm and BCATc within the same cell type has not been observed in the CNS (Hutson et al., 2001; Sweatt et al., 2004a; Cole et al., 2005). Therefore, in the brain and spinal cord, the BCAT isozyme localization is entirely cell specific. The second step in BCAA catabolism is irreversible oxidative decarboxylation of the BCKAs, producing branched chain acyl CoA derivatives. This reaction is catalyzed by the mitochondrial branched chain a keto acid dehydrogenase (BCKD) enzyme complex (Harris et al., 1986) ( > Figure 6-1 ). Because this step is

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Branched chain amino acids (BCAAs) in brain

. Figure 6-2 BCAA catabolic enzymes are found throughout the body. The localization of BCAA catabolic enzymes facilitates nitrogen cycling and net nitrogen transfer between and within different tissues and cell types. In the rat, BCATm is found in all tissues except liver, whereas in primates it is present in Kupffer cells of the liver but not in hepatocytes where the BCKD enzyme complex is localized (Suryawan et al., 1998; Sweatt et al., 2004a). BCATc is found primarily in the central nervous system in neurons but is also expressed in peripheral nerves (not shown) (Sweatt et al., 2004a)

in peripheral nerves (not shown) (Sweatt et al., 2004a) irreversible, it commits the BCAA carbon skeleton

irreversible, it commits the BCAA carbon skeleton to the degradative pathway and results in irreversible transfer (net transfer) of the aamino nitrogen from BCAA to glutamate and other dispensable amino acids. The mammalian BCKD complex contains multiple copies of three enzymes (Harris et al., 1990)—a branched chain aketo acid decarboxylase (E1); a dihydrolipoyl transacylase (E2) and a dihydrolipoyl dehydrogenase (E3). In this respect, the BCKD complex resembles the mammalian a ketoglutarate dehy- drogenase and pyruvate dehydrogenase enzyme complexes. The activity of the BCKD complex within a tissue is regulated by its phosphorylation state. Phosphorylation by BCKD kinase inactivates the complex, and dephosphorylation by a specific phosphatase activates the complex (Harris et al., 1990, 2004). Muta- tions in one or more proteins in this complex result in the inborn error of BCAA metabolism known as maple syrup urine disease (MSUD) (Chuang and Shih, 2001). The next step in the oxidation of BCAAs is dehydrogenation of the branched chain acyl CoA BCKD reaction products catalyzed by two different dehydrogenases (Chuang and Shih, 2001). After this step, the catabolic pathways of the three BCAAs diverge. The remaining enzymatic reactions resemble boxidation of fatty acids. Leucine is unique in that its catabolic pathway contains an ATPand biotin dependent carboxylation step (b methylcrotonylCoA carboxylase) and an intermediate that is a precursor of choles- terol. Leucine is ketogenic, forming acetylCoA and acetoacetate whereas valine is glucogenic entering the tricarboxylic acid cycle (TCA cycle) as succinylCoA. Both isoleucine and valine are metabolized to succinate via methylmalonyl CoA, catalyzed by propionylCoA carboxylase, and subsequent rearrangement to succinylCoA, catalyzed by the vitamin B 12 dependent enzyme methylmalonylCoA mutase. The other product of isoleucine metabolism is acetoacetate. Neurologic consequences are observed in most inborn errors of the enzymes in BCAA metabolism (Chuang and Shih, 2001).

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3 Branched Chain Amino Acids Are Nitrogen Donors in Peripheral Tissues

Early research on the metabolism of BCAAs in body organs and tissues led to the hypothesis that BCAAs play an important role in body nitrogen metabolism. They are nitrogen donors for the major nitrogen carriers, glutamine and alanine, that carry nitrogen from amino acid catabolism in peripheral tissues such as skeletal muscle to the liver and intestine (Odessey et al., 1974; Garber et al., 1976; Harper, 1989). In contrast to the degradative pathways of other indispensable amino acids, the enzymes for degradation of the BCAAs are expressed in tissues throughout the body rather than being confined to the liver ( > Figure 6-2 ) (Ichihara, 1985; Hutson, 1988; Hutson et al., 1988; Sweatt et al., 2004a). The initial step, transamination, is reversible. BCAT activity is low in liver and high in other organs so that the initial step in BCAA degradation is located in tissues other than liver (Harper, 1989) ( > Figure 6-2 ). BCAAs represent about 50% of skeletal muscle amino acid uptake, and most of the other plasma amino acids do not undergo catabolism in muscle. The BCAAs are used for maintenance of muscle nitrogen pools of glutamate and glutamine and provide part of the nitrogen for glutamine release from muscle and subsequent uptake by the intestines and kidney. In rat and human skeletal muscle, BCAT activity is high compared to BCKD activity (ratio of BCAT/actual BCKD activities is 90 and for total dephosphorylated BCKD activity 25) (Suryawan et al., 1998). The high ratio of BCAT/BCKD activities favors release of BCKAs rather than their oxidation (Hutson et al., 1978, 1980; Harper et al., 1984), and there is efficient transfer of BCAA nitrogen to glutamate and glutamine in skeletal muscle preparations (Hutson and Zapalowski, 1981). In addition, kinetic data indicate that in situ BCATs operate at substrate concentrations below their K m values so tissues respond rapidly to changes in BCAA and BCKA concentrations. In rat liver, the absence of the BCATm isozyme and high activity of BCKD favor liver oxidation of circulating BCKAs. This complex metabolic scheme also permits leucine to act as an effective nutrient signal both in liver and in peripheral tissues. Leucine stimulates, via an as yet incompletely characterized signaling cascade(s), protein synthesis in skeletal muscle, adipose tissue, and liver (Anthony et al., 2000; Lynch, 2001; Lynch et al., 2002). Whether or not leucine acts specifically as a nutrient signal in the CNS is not known.

4 Branched Chain Amino Acids as Nitrogen Donors in the Central Nervous System

There are parallels between the function of BCAAs in skeletal muscle and their role in the CNS. In both organs, BCAAs are nitrogen donors to glutamate (glutamine). As described above, in the CNS the glutamate that is formed from BCAA transamination is an excitatory neurotransmitter, substrate for glutamine synthesis in the glutamate/glutamine cycle, and substrate for synthesis of the inhibitory neuro- transmitter GABA. There is substantial uptake of BCAAs into the CNS in rats (Oldendorf, 1971), and in humans the cerebral arteriovenous difference for leucine exceeds that of other amino acids (Oldendorf, 1971; Grill et al., 1992). Uptake of BCAAs at the blood–brain barrier appears to be mediated by the facilitative large neutral amino acid transporter 1 system (Killian and Chikhale, 2001). Cerebral glutamine release has also been observed during BCAA uptake (Grill et al., 1992). Although release of BCKAs from peripheral tissues occurs in rodents and humans (Hutson et al., 1978; Hutson et al., 1980; Matthews et al., 1980), BCKA release from brain may be quite slow. Michaelis constants for uptake of BCKAs into rat brain (Conn and Steele, 1982) suggest there is limited transport of BCKAs across the blood–brain barrier at physiological concentrations, but it may occur under some circumstances (Matsuo et al., 1993). The low concentrations of BCKAs observed in brain (Hutson and Harper, 1981) are consistent with efficient BCKA oxidation or perhaps some release of BCKAs to the blood stream. Rat liver, which is a major site of BCKA oxidation, has BCKA concentrations that are as low as those measured in brain (Hutson and Harper,

1981).

Branched chain amino acid transport into the brain provides the major source of brain nitrogen. The exchange of aamino nitrogen between BCAAs and other brain amino acids is very active and catalyzed by BCATc and BCATm. In rodent and human brain, maximal BCAT activity exceeds total BCKD activity (BCAT/total BCKD activity 50) (Brosnan et al., 1985; Suryawan et al., 1998). Isotope studies

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demonstrated the rapid transfer of nitrogen from BCAA to glutamate (glutamine) both in vitro and in vivo in animal models (Yudkoff et al., 1983; Yudkoff et al., 1993; Yudkoff et al., 1994; Yudkoff et al., 1996a; Kanamori et al., 1998; Yudkoff et al., 2003; Sakai et al., 2004). Transfer of BCAA nitrogen into glutamate and glutamine occurs in primary rat astroglia cultures and synaptosome preparations (Yudkoff et al., 1983; Yudkoff et al., 1994; Yudkoff et al., 1996a, b; Hutson et al., 1998; McKenna et al., 1998a). Incorporation of 15 N from leucine into glutamate, glutamine, and GABA takes place in mice in vivo (Yudkoff et al., 2003). Using NMR, Kanamori et al. (1998) estimated that leucine provides 25% of glutamate nitrogen in rat brain, and during 9 h of continuous intragastric feeding in rats, Sakai et al. (2004) estimated that at least 50% of brain glutamate nitrogen was derived from leucine. The authors also concluded that there was significant reamination of the leucine aketo acid, KIC (Sakai et al., 2004). Although the relative rates of brain KIC reamination (nitrogen cycling) versus oxidation or BCKA release (net nitrogen transfer) in brain in situ remain to be determined, human brain aminotransferase and actual BCKD capacities are second only to skeletal muscle. These results suggest that the brain may be a quantitatively more important site of BCAA metabolism in humans where brain represents a higher percent of total body weight than in rats (Suryawan et al., 1998).

4.1 Intercellular Nitrogen Transfer

The first evidence for metabolic compartmentalization, intercellular transfer of BCAAs and their metabolites, and transfer of BCAA nitrogen to glutamate and glutamine within the brain came from studies of BCAA metabolism in primary rat brain cell cultures and synaptosome preparations. Early studies showed that fetal and adult rat brain preparations can transaminate and oxidize BCAAs and that the rate of BCAA transamina- tion is greater than the rate of incorporation into new protein (Chaplin et al., 1976; Shank and Aprison, 1981; Murthy and Hertz, 1987). Astrocyte cultures exhibit a high capacity for leucine uptake (Su et al., 1995), accumulate BCAAs against a concentration gradient (Su et al., 1995), readily transaminate BCAAs (Hutson et al., 1998), and transfer their nitrogen to glutamate and glutamine (Yudkoff et al., 1994). BCAAs also stimulate glutamine efflux (Hutson et al., 1998). On the other hand, cultured astrocytes oxidize BCAAs slowly (Bixel and Hamprecht, 1995; Hutson et al., 1998), releasing KIC and other leucine metabolites (Bixel et al., 1995). Although primary neuronal cultures also readily transaminate BCAAs, reamination of KIC is favored in neuronal compared to astrocyte cultures (Hutson et al., 1998). Yudkoff et al. (1996b) also concluded that in nerve endings the BCAT reaction proceeds largely in the direction of reamination. A model that took into account the most salient features of brain BCAA metabolism was proposed by Yudkoff (Yudkoff et al., 1996a; Yudkoff, 1997) and was originally called the ‘‘leucine/glutamine cycle.’’ In this model, leucine enters the brain across the capillary/glial interface, and then transaminates with aketoglutarate to form glutamate, which is then converted in the glia to glutamine. Glutamine and KIC, the aketo acid of leucine, are released from the glia. Glutamine and KIC are then taken up by neurons. KIC is reaminated in the neuron, consuming glutamate in the process. Leucine is released into the extracellular fluid where it can again be utilized by astrocytes, completing the cycle. As originally envisioned, this cycle provides a mechanism whereby BCAA nitrogen can be used to support brain nitrogen homeostasis. In the original thesis, this cycle buffers glutamate levels in neurons, thereby preventing a buildup of glutamate in neurons. Reamination of BCKAs serves to conserve BCAA for protein synthesis. Free ammonia is produced in the glutaminase step in neurons. In this cycle, glutamate dehydrogenase uses the ammonia from the glutaminase step for resynthesis of glutamate.

5 Role of BCAA in de novo Glutamate Synthesis and the Branched Chain Aminotransferase (BCAT) Cycle Hypothesis

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. Figure 6-3 The BCAT cycle and de novo Glu synthesis. BCAAs donate nitrogen via BCATm in glia for de novo glutamate synthesis with reamination of BCKA in neurons by BCATc and shuttling of nitrogen to glia via BCAAs. Ammonia (NH þ ) from Gln conversion to Glu in neurons is used to reaminate a ketoglutarate to reform glutamate used in BCATc step. Other key enzymes of the BCAT cycle are in astrocytes (1) pyruvate carboxylase and (2) glutamine synthetase, and in neurons (3) glutaminase and (4) glutamate dehydrogenase

4

in neurons (3) glutaminase and (4) glutamate dehydrogenase 4 operate under near equilibrium conditions in most

operate under near equilibrium conditions in most cells, buffering can be provided by a single isozyme in a single compartment such as the neuron. In the BCAT cycle hypothesis, emphasis is on the role of BCAAs in de novo synthesis of glutamate as opposed to a purely buffering role. De novo synthesis of glutamate is needed in the brain because some glutamate used in neurotransmission is lost in catabolic reactions. In addition, the neurotransmitter glutamate/glutamine cycle does not operate stoichiometrically. Results from studies of cultured neonatal cortical astrocytes (Sonnewald et al., 1993; Westergaard et al., 1995) have shown that a significant fraction of the glutamine returned to the neuron is not derived from the glutamate that was originally taken up by astrocytes. > Figure 6-4 highlights the pathways for glutamate oxidation to pyruvate in the glia and illustrates why some glutamine derived from glia must come from de novo synthesis of glutamate. In the degradation pathway, glutamate is first converted to a ketoglutarate (a KG). This step is catalyzed by transamination and/or under special circumstances by glutamate dehydrogenase (Rao and Murthy, 1993; McKenna et al., 1996a, b). As long as cycle intermediates are not removed and used to synthesize other metabolites, the citric acid cycle intermediates, including aketoglutarate, are not consumed. Nevertheless, studies show that a significant fraction of the glutamate taken up by astrocytes disappears because the glutamate is metabolized further to pyruvate and lactate and subsequently released by astrocytes (Sonnewald et al., 1993; Hassel and Sonnewald, 1995; Sonnewald et al., 1996) to be oxidized by neurons. This lactate is an effective substrate for maintaining energy metabolism in synaptic terminals (McKenna et al., 1998b). In fact, Magistretti and Pellerin have proposed that lactate released by astrocytes is the principal source of energy for neurons (Pellerin and Magistretti, 1994; Magistretti et al., 1999). Lactate is the normal product of glycolysis but is also produced from the decarboxylation of the citric acid cycle intermediates, oxaloace- tate (OAA), and malate, by reactions diagrammed in > Figure 6-4 (Yu et al., 1983; Shank et al., 1985). Pyruvate is reduced to lactate by lactate dehydrogenase. Alternatively, pyruvate can be transformed to oxaloacetate by pyruvate carboxylase (PC). We (Gamberino et al., 1997) and others (Schmoll and Hamprecht, 1994; Hassel and Sonnewald, 1995; McKenna et al., 1995) have identified these reactions in cultured astrocytes, and the cycle involving conversion of glutamate derived pyruvate back to oxaloacetate has been named the glutamate/pyruvate cycle. These studies show that the cycle is active in excised retinas (Lieth et al., 2001; Xu et al., 2004). Thus, the glutamate/pyruvate cycle provides a mechanism to convert excess glutamate, which is toxic, to useful nutrients such as pyruvate and lactate. The active conversion of glutamate to pyruvate in astrocytes would gradually lower brain glutamate concentrations unless a pathway were present in astrocytes to replenish the glutamate carbon by a process

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. Figure 6-4 Glutamate/pyruvate cycle. Glu taken up into astrocytes forms a ketoglutarate (aKG) in a transamination (TA) reaction. a Ketoglutarate (a KG) is converted to malate (Mal) and oxaloacetate (OAA) in the TCA cycle. Malic enzyme (ME) or the combined action of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) produce pyruvate (Pyr). Pyruvate forms lactate, catalyzed by lactate dehydrogenase, or pyruvate carboxylase (PC) resynthesizes OAA from pyruvate

or pyruvate carboxylase (PC) resynthesizes OAA from pyruvate called anaplerosis. Anaplerosis is the process by which

called anaplerosis. Anaplerosis is the process by which citric acid cycle intermediates are replenished by carboxylation to form oxaloacetate. The major anaplerotic enzyme in brain is PC. This biotin dependent enzyme catalyzes an ATPdependent reaction and uses HCO 3 as the source of CO 2 for carboxylation of pyruvate ( > Figure 6-4 ). The operation of PC produces oxaloacetate, which is rate limiting for de novo malate and aketoglutarate synthesis (Gamberino et al., 1997). aKetoglutarate provides the carbon skeleton for glutamate. There are high levels of PC in brain, and it is expressed exclusively in glia (Yu et al., 1983; Shank et al., 1985; Patel, 1989). Conversion of aketoglutarate to glutamate requires nitrogen from amino acids or ammonia. In the leucine/glutamine cycle, BCAA supply the nitrogen. The need for nitrogen for de novo glutamate synthesis and the observations that in primary rat brain cell cultures, the mitochondrial isozyme BCATm is expressed in astroglia whereas the cytosolic isozyme BCATc is expressed in neurons (Bixel et al., 1997; Bixel et al., 2001; Hutson et al., 2001) led to refinement of the leucine cycle to include a role for BCAA in de novo glutamate synthesis where the different BCAT isozymes catalyze nitrogen transfer in separate cell compartments. Hutson and coworkers (Hutson et al., 1998; Goto et al., 2005) discovered in the course of their studies on the BCAT isozymes that the antiepileptic drug gabapentin (neurontin) is a specific competitive inhibitor of cytosolic BCATc, but it does not inhibit mitochondrial BCATm. The structural basis for the specificity of gabapentin for the BCATc isozyme is now understood (Goto et al., 2005). Gabapentin was synthesized originally as a GABA analog but is actually structurally similar to the BCAA leucine (Su et al., 1995). Gabapentin is used in combination with other drugs for seizure control and is now used widely to treat neuropathic and other types of pain (Rogawski and Loscher, 2004). Gabapentin was used in the ex vivo rat retina preparation to test whether the BCAT isozymes operate as a cycle that provides nitrogen for de novo synthesis of glutamate (Hutson et al., 2001; LaNoue et al., 2001; Lieth et al., 2001). The experiments provided quantitative evidence that BCAA are necessary for optimal rates of de novo glutamate synthesis in

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the retina. Finally, they showed that transamination is the major source of nitrogen for de novo glutamate synthesis in the ex vivo rat retina, with 50% of this nitrogen coming from BCAA (LaNoue et al., 2001). Rates of transfer of leucine nitrogen to glutamate were comparable to rates of de novo glutamate synthesis in whole brain ( 1.3 nmol/mg protein/min) (Sakai et al., 2004; Xu et al., 2004). This BCATdependent nitrogen cycle is tied to the glutamate/glutamine cycle ( > Figure 6-3 ). In astroglia, BCAA nitrogen is used to form glutamate and BCKAs. The BCKAs and glutamine produced are transferred to neurons. In the neuron, glutamine deamination regenerates glutamate and ammonia is released (glutamate/glutamine cycle). The high levels of neuronal glutamate accelerate transamination of BCKAs to BCAAs in the neuronal compartment. The BCAAs are then free to diffuse back to the astroglia. As originally formulated, this hypothetical cycle serves to provide nitrogen for glutamate synthesis in astroglia and serves to buffer glutamate levels in neurons. Thus, the BCAT nitrogen shuttle is an accessory part of the forward arm of the glutamate/pyruvate cycle and is required as a means to regulate the size of the glutamate neurotransmitter pool.

6 Localization of BCAA Catabolic Enzymes in Rodent Brain

The BCAT cycle hypothesis predicts that in brain BCATc is localized in glutamatergic neurons and BCATm is expressed in astroglia surrounding the glutamatergic neurons. Because glutamate is the precursor of GABA, it is also likely that the BCAAs may provide nitrogen for GABA synthesis and BCATs might be expressed in GABAergic neurons. Published and unpublished results from our recent immunohistochem- istry studies of the localization of BCAT isozymes and BCKD in rat brain and spinal cord show expression of the BCATc isozyme in glutamatergic and GABAergic neurons (Sweatt et al., 2004a, b).

6.1 The Cytosolic BCATc Isozyme

In rodent brain and spinal cord, BCATc is found only in neurons (Sweatt et al., 2004b; Cole et al., 2005). Immunolabeling of BCATc in adult rat cerebellum and hippocampus shows that the enzyme is expressed by both glutamatergic and GABAergic neurons (Sweatt et al., 2004b). In several brain regions, we observed striking differences in the intracellular localization of BCATc between glutamatergic and GABAergic neurons. For example, the cell bodies of the glutamatergic granule cells of the cerebellum and of the dentate gyrus (hippocampal formation) are not labeled for BCATc, but their axonal processes, including the cerebellar parallel fibers and the mossy fiber projection in the hippocampus, are labeled for BCATc (Sweatt et al., 2004b). Furthermore, in the hippocampus, BCATc is concentrated in the synaptic varicosities formed between mossy fibers and their target neurons in field CA3. From these observations, it appears that the BCATc synthesized in glutamatergic neurons is transported into the axon and toward the synaptic terminal. In contrast, in GABAergic neurons, such as the Purkinje cells of the cerebellum and the pyramidal basket cells of the dentate gyrus, immunoreactivity for BCATc is concentrated in the cell body. In the remaining brain regions and spinal cord, the pattern of BCATc is similar to what has been observed in the hippocampus and cerebellum (unpublished observations).

6.2 The Mitochondrial Enzymes BCATm and Branched Chain a-Keto Acid Dehydrogenase (BCKD)

The expression of the two mitochondrial enzymes, BCATm and BCKD is different from that of BCATc. In the adult rat brain, BCATm is found in astroglia (Hutson et al., 2001; Cole et al., 2005) whereas BCKD is found only in neurons (Cole et al., 2005). The partitioning of BCATm to astroglia is striking in the hippocampus, where a field of BCATm expressing astroglia surrounds the BCATcrich mossy fiber projection (Cole et al., 2005) (unpublished observations). In brain regions containing white matter tracts, BCATm is prominent in astrocytes (Cole et al., 2005). This distribution indicates that astrocytes possess

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BCAA transamination capability, as predicted by the BCAT cycle hypothesis. In preliminary studies, the second enzyme in the BCAA catabolic pathway, BCKD, has been localized to neurons and, in the hippocampus, BCKD does not appear to colocalize with BCATc, i.e., BCKD is expressed in a different population of neurons than BCATc (Cole et al., 2005). Astroglia are not immunoreactive for BCKD. Thus, localization of BCATm in astroglia and BCATc in glutamatergic neurons positions these enzymes to catalyze the cyclic transfer of BCAA nitrogen as predicted by the BCAT cycle hypothesis. BCATm is positioned to provide nitrogen for de novo glutamate synthesis in the glia and BCATc in glutamatergic neurons reaminates BCKAs to form leucine which returns to the astrocytes. Irreversible nitrogen transfer, i.e., oxidation of branched chain aketo acids occurs only in neurons. In areas such as the hippocampus, oxidation is confined to a separate cellular region where BCATc is not located.

6.3 The BCAT Cycle and GABA Metabolism

The expression of BCATc in GABAergic neurons suggests that BCAAs and/or the BCAT cycle play a role in GABA metabolism. GABA is the major inhibitory transmitter in the brain. The BCAT isozymes, through their involvement in the metabolism of glutamate, can influence GABA pools. Neuronal GABA is synthe- sized through the decarboxylation of glutamate, catalyzed by the cytosolic enzyme GAD, of which there are two isozymes. GAD67 is found in the cytosol and GAD65 is associated with synaptic vesicles (Christgau et al., 1992; Condie et al., 1997). Most of the GABA released from neurons is taken up again by the neurons, while about 20% of released GABA is taken up by astrocytes. After release at the synapse, GABA, taken up by neurons, is repackaged in synaptic vesicles. GABA catabolism occurs in the mitochondrial compartment of neurons and astrocytes. Therefore, GABA is transported into mitochondria, where it undergoes transamination with aketoglutarate, a TCA cycle intermediate, catalyzed by GABA aminotransferase (GABA T). This reaction produces glutamate and succinic semialdehyde. Reduction of succinic semialde- hyde produces succinate, which is also a TCA cycle intermediate. This pathway is called the GABA shunt because it shunts the mitochondrial aketoglutarate directly to succinate bypassing succinyl CoA thioki- nase, i.e., there is no loss or gain of TCA cycle intermediates. Depending on the cell type (astrocyte or neuron), glutamate, which is the other product of mitochon- drial GABA T activity, can follow one of several metabolic pathways. In astrocytes, if glutamate exits the mitochondria on the glutamate/hydroxyl carrier, it can be used directly for the synthesis of glutamine (glutamate/glutamine cycle). BCATm is localized in astrocyte mitochondria. So another pathway is BCATmcatalyzed transamination of glutamate with a BCKA, which would regenerate aketoglutarate and BCAA. This pathway essentially preserves the TCA cycle intermediate aketoglutarate and recycles BCAAs within the astrocyte (LaNoue and Hutson, 2005). Alternatively, glutamate can be oxidatively deaminated by mitochondrial glutamate dehydrogenase to produce a ketoglutarate, but in the astrocyte this does not appear to be a major pathway of glutamate catabolism under physiological conditions (McKenna et al., 1996a; Gamberino et al., 1997). In GABAergic neurons, the only BCAT isozyme that is expressed is cytosolic BCATc. If the BCAT cycle operates as proposed for glutamate, then the cycle will function to provide glutamate for subsequent GABA synthesis in GABAergic neurons. For GABA that is metabolized in neurons, glutamate is formed in the mitochondria, requiring export on the glutamate/hydroxyl carrier before it can be a substrate for BCATc. In this case BCATc, depending on the availability of BCKAs, can serve as a buffer for excess glutamate. Alternatively, deamination by glutamate dehydrogenase would generate aketoglutarate, thus providing substrate for BCATccatalyzed glutamate synthesis. BCATc is found primarily in the cell body of GABAergic neurons (Sweatt et al., 2004b), therefore, it is positioned to interact with the cytosolic pool of glutamate as opposed to the neurotransmitter pool of glutamate that is located near the synapse. Because glutamate transporters are located on GABAergic cell bodies, glutamate released from excitatory nerve terminals that synapse on GABAergic cell bodies can enter the cytoplasmic pool (Conti et al., 1998). Other studies have shown that there is a connection between

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glutamate and GABA metabolism. Inhibition of GABA T resulted in an increase in GABA in the cortex and subsequent decline in glutamate and glutamine (Pie´ rard et al., 1999). Since glutamine is the precursor of glutamate, for the BCAT cycle to contribute directly to GABA synthesis glutaminase should be expressed in GABA neurons. To date, expression of glutaminase in GABAergic neurons has not been reported whereas published studies do support expression of glutaminase in glutamatergic neurons (Laake et al., 1999; Daikhin and Yudkoff, 2000). Thus, the BCAT cycle can only support GABA synthesis indirectly by its contribution to glutamate neurons. The role of BCATc in GABAergic neurons depends then on the supply of substrate, i.e., BCAA or BCKA. If the astrocyte arm of the cycle is active (glutamate neurotransmission), BCKAs will be available for BCATc in GABAergic neurons. Then BCATc catalyzed transamination of BCKA reduces the supply of glutamate for GABA synthesis. On the other hand, if there is active GABA neurotransmission and astrocytes are not producing BCKAs (see above and LaNoue and Hutson, 2005), then BCATc can contribute directly to GABA synthesis by transamination of BCAAs producing glutamate in the cytosol where it is available for GAD. Thus, the BCAT cycle may provide a mechanism for communication between glutamate neurotransmission and the metabolic pool of GABA.

7 Conclusions, Implications for Disorders of BCAA Metabolism, and Future Directions

Current theories of the function of BCAAs in brain and the localization of BCAA catabolic enzymes in brain and spinal cord support the involvement of the glutamatergic and/or GABAergic systems in the etiology of disorders of BCAA metabolism. The current consensus is that BCAAs donate nitrogen for synthesis of neurotransmitter glutamate and are involved in maintenance of neurotransmitter glutamate at a relatively constant level. The localization of BCAT isozymes in GABAergic neurons and surrounding astrocytes suggests a role for BCAAs in GABA metabolism, but whether or not BCAAs and BCATs operate as hypothesized for glutamate or serve a different function remains to be determined. In disorders of BCAA metabolism, such as MSUD, a disturbance and/or imbalance in the glutamatergic and GABAergic systems in brain may cause the neurological symptoms. Experiments conducted on post mortem brain tissue from MSUD calves showed changes in glutamatergic and GABAergic receptor densities compared to normal calf brains (Harper et al., 1989; Dodd et al., 1992), and the authors hypothesized that these changes might lead to an alteration in the excitation–inhibition balance and be proconvulsive (Dodd et al., 1992). Recent studies using in situ or in vitro systems incubated with concentrations of individual BCKAs or BCAAs that are equal to or exceed concentrations found in MSUD patients have generated a number of theories for the underlying cause(s) of the neurological damage observed in untreated MSUD. These theories range from metabolic imbalances to structural changes and include impaired glutamate transport, BCKAinduced apoptotic cell death, leucine induced oxidative damage, and altered phosphorylation of intermediate filament proteins (Jouvet et al., 2000; Bridi et al., 2003; Funchal et al., 2004; Bridi et al., 2005). Cell culture systems and in vitro preparations cannot substitute for the complex system represented by the intact brain. Progress in testing the BCAT cycle hypothesis, determining the quantitative importance of BCAAs as sources of nitrogen in glutamate and GABA biosynthesis and neurotransmission, as well as understanding the physiological changes that occur in patients with inborn errors of metabolism, such as MSUD, would be facilitated by development of transgenic animal models.

Acknowledgments

The work reported here was supported by Grants NS 38641 and DK34738 from the US National Institutes of Health.

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