Neurochemistry International 140 (2020) 104809

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Neurochemistry International

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Review Novel aspects of glutamine synthetase in ammonia homeostasis

Yun Zhou a,*, Tore Eid b, Bjørnar Hassel c, Niels Christian Danbolt a a Neurotransporter Group, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317, Oslo, Norway b Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT, USA c Department of Complex Neurology and Neurohabilitation, Oslo University Hospital, University of Oslo, N-0450, Oslo, Norway

ARTICLE INFO ABSTRACT

Keywords: Elevated blood ammonia (hyperammonemia) is believed to be a major contributor to the neurological sequelae Ammonia following severe liver disease. Ammonia is cleared via two main mechanisms, the urea cycle pathway and the Brain glutamine synthetase reaction. Recent studies of genetically modifiedanimals confirmthe importance of the urea Glutamine synthetase cycle, but also suggest that the glutamine synthetase reaction is more important than previously recognized. Glutamate transporter While the liver clears about two-thirds of the body’s ammonia via the combined action of the urea cycle and Liver Neurotransmitter glutamine synthetase, extrahepatic tissues do not express all the components required for performing a complete Urea cycle urea cycle and therefore depend on the glutamine synthetase reaction for ammonia clearance. The brain is particularly vulnerable to the effects of hyperammonemia, which include impaired extracellular potassium buffering and brain edema. Moreover, the glutamine synthetase reaction is intimately linked to the metabolism of the excitatory and inhibitory neurotransmitters glutamate and gamma aminobutyric acid (GABA), implicating a key role for this enzyme in neurotransmission. This review discusses the emerging roles of glutamine synthetase in brain pathophysiology, particularly aspects related to ammonia homeostasis and hepatic encephalopathy.

experimental portacaval shunting in dogs caused hyperammonemia and encephalopathy (Hahn et al., 1893). The link between hyper­ 1. Introduction ammonemia and encephalopathy has been solidified by a substantial body of evidence from patients (Clemmesen et al., 1999) and animal In mammals, ammonia is a biologically significant intermediate in models of liver disease (for reviews see: Butterworth et al., 1987; several biochemical processes and not merely a waste product from Clemmesen et al., 1999; Bernal et al., 2007; Aldridge et al., 2015). metabolism of nitrogenous compounds. Hyperammonemia (elevated Interestingly, other factors contribute to the encephalopathy because concentrations of ammonia in blood) may be caused by a number of the severity of the encephalopathy does not correlate with the blood conditions, such as liver failure, portocaval shunting, colonization of the ammonia concentration (Shawcross et al., 2011), and because there is gut with urease-producing bacteria, and inborn errors of metabolism not a consistent linear relationship between ammonia levels in the blood (Haberle, 2013). In addition, the brain may be exposed to high con­ and the brain (e.g. Cauli et al., 2008; Zwirner et al., 2010; Kanamori centrations of ammonia during bacterial encephalitis and brain abscess et al., 2002; Dahlberg et al., 2016; Shawcross et al., 2011). One possible formation (Dahlberg et al., 2016). factor is inflammation (Shawcross et al., 2007; for reviews see: Coltart Like several other biochemicals, e.g. glutamate (for reviews see e.g.: et al., 2013; Jayakumar et al., 2015; Tranah et al., 2013; Romero-Gomez Danbolt, 2001), ammonia is toxic if present at excessive concentrations. et al., 2015; Aldridge et al., 2015). Another complicating issue is that the The clinical features of hyperammonemia range in severity and include pathogenesis of ammonia-related encephalopathy is not exclusively personality changes, confusion, seizures, coma, and death (for reviews driven by the liver, but also involves other organs with high ammonia see: Cash et al., 2010; Prakash and Mullen, 2010). Some of the symptoms turnover, such as the skeletal muscle and the kidneys (Aldridge et al., are probably direct consequences of ammonia’s interference with 2015). cellular processes (Bosoi and Rose, 2009), while others are indirect, The scope of the present review is to provide an up to date account of likely reflecting tissue edema (Rovira et al., 2008; Alonso et al., 2014; the mechanisms underlying ammonia metabolism, with particular Larangeira et al., 2018). emphasis on the emerging importance of glutamine synthetase (Glul; EC The toxicity of ammonia was noted more than a century ago when

* Corresponding author. E-mail address: [email protected] (Y. Zhou). https://doi.org/10.1016/j.neuint.2020.104809 Received 29 March 2020; Received in revised form 8 July 2020; Accepted 9 July 2020 Available online 3 August 2020 0197-0186/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Y. Zhou et al. Neurochemistry International 140 (2020) 104809

Abbreviations EAAT2 Excitatory Amino Acid transporter (slc1a2; Pines et al., 1992) ALT alanine aminotransferase (EC 2.6.1.2) FTCD glutamate formiminotransferase (EC 2.1.2.5) AST aspartate aminotransferase (EC 2.6.1.1) GDH glutamate dehydrogenase ASS argininosuccinate synthase (EC 6.3.4.5) GLS2 liver glutaminase (EC 3.5.1.2) ASL argininosuccinate lyase (EC 4.3.2.1) HAL histidine ammonia lyase (EC 4.3.1.3) ARG1 arginase 1 (EC 3.5.3.1) HE hepatic encephalopathy CAS Chemical Abstracts Service registry number OAT ornithine aminotransferase (EC 2.6.1.13) CSF cerebrospinal fluid OCT ornithine transcarbamylase (EC 2.1.3.3) CPS1 carbamoyl-phosphate synthetase 1 (EC 6.3.4.16) MSO methionine sulfoximine (CAS 15985-39-4) CNS central nervous system MTLE mesial temporal lobe epilepsy Cre Cre recombinase (cyclization recombinase) NAGS N-acetylglutamate synthase (EC 2.3.1.1) Glul glutamate ammonia ligase = glutamine synthetase (EC RRID Research Resource Identifiers (https://scicrunch.org/ 6.3.1.2) resources)

6.3.1.2) in ammonia homeostasis and brain function. We will not discuss In contrast, glutamine synthetase and ornithine aminotransferase the pathophysiological sequelae of hyperammonemia, as those have (OAT; EC 2.6.1.13) are both localized exclusively in a rim of 2–3 layers been thoroughly reviewed by others (for review see: Visek, 1968; But­ of perivenous hepatocytes. Further, these hepatocytes also take up terworth et al., 1987; Braissant et al., 2013; Aldridge et al., 2015; Bosoi glutamate and ammonium by, respectively, the glutamate transporter and Rose, 2009)). subtype EAAT2 (slc1a2; see below) and the ammonium transporter Rh B glycoprotein (slc42a2; Haussinger, 1983; Gebhardt et al., 1988; Stoll 2. Zonation of ammonia and glutamate metabolism in the liver et al., 1991; Taylor and Rennie, 1987; Weiner et al., 2003). In the adult liver, glutamate dehydrogenase (GDH; EC 1.4.1.2) is present in high The recent availability of genetically modified animals has made it concentrations around the terminal portal and hepatic veins (thus in easier to uncover the interaction between ammonia metabolism and both periportal and perivenous hepatocytes), but is less abundant in the glutamate metabolism. For instance, urea cycle mutations (e.g. mice intermediate zone (Lamers et al., 1988). The pattern of zonation is lacking CPS1) are associated with severe increases in blood glutamine regulated through β-catenin-dependent and glucagon-dependent mech­ and ammonia levels (Khoja et al., 2019). Additionally, mice lacking anisms (Burke et al., 2009; Cheng et al., 2018). hepatic glutamate dehydrogenase display altered ammonia homeostasis Anterograde and retrograde perfusion experiments, as well as tar­ characterized by increased blood ammonia and reduced conversion to geted lesion experiments, have confirmed the metabolic zonation and urea (Karaca et al., 2018). Loss-of-function mutation in the glutamine suggest that the urea cycle pathway is a low affinity, high capacity transporter SNAT3 (slc38a3) causes several metabolic perturbations in ammonia clearance system that is preferentially localized to the peri­ the liver such as decreased glutamine levels, as well as increased urea portal hepatocytes. In contrast, the glutamine synthetase reaction in the levels (Chan et al., 2016). perivenous hepatocytes is believed to represent a high affinity, low ca­ Although indistinguishable from each other by hematoxylin & eosin pacity ammonia clearance system independent from the urea cycle staining, hepatocytes are functionally different depending on their pathway (Gebhardt and Mecke, 1983; Haussinger, 1983; Gebhardt et al., localization along the porto-central axis of the liver (for review see: 1988). Rappaport, 1958; Gumucio and Miller, 1981; Jungermann and Katz, 1989). For example, the metabolism of nitrogen and glutamate in he­ 3. The importance of the urea cycle in ammonia metabolism patocytes at the entrance of the sinusoids (periportal hepatocytes) is quite different from that of hepatocytes close to the central venules In 1932, Krebs and Henseleit used the surviving tissue slice technique of (perivenous/pericentral hepatocytes, summarized in Fig. 1). This Warburg to show that mammalian liver slices form urea from ammonia pattern first became apparent when the cellular localizations of and carbon dioxide (Krebs, 1942). Their pioneer study not only refuted ammonia-metabolizing enzymes and transporter proteins were deter­ the prevailing hypothesis that ammonium cyanate or related compounds mined by in situ hybridization (Moorman et al., 1994; Berger and were involved in urea formation, but also initiated a new era of Hediger, 2006), by immunohistochemistry (Gebhardt and Mecke, 1983; biochemistry related to urea biosynthesis by definingthe metabolic roles Gaasbeek Janzen et al., 1984; Gaasbeek Janzen et al., 1987; Gebhardt of ornithine, citrulline and arginine. With the efforts from numerous et al., 1988; Gascon-Barre et al., 1989; Kuo et al., 1991; Matsuzawa other biochemists, the chemical reactions involving the five key urea et al., 1997; O’sullivan et al., 1998; Najimi et al., 2014; Hu et al., 2018) cycle enzymes were clarified, i.e. carbamoyl-phosphate synthetase 1 and by microarray analysis (Braeuning et al., 2006). (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthase The following enzymes are predominantly present in periportal he­ (ASS), argininosuccinate lyase (ASL), and arginase 1 (ARG1). As shown patocytes: carbamoyl-phosphate synthetase 1 (CPS1; EC 6.3.4.16), in Fig. 2, the initial step in the urea cycle is the synthesis of carbamoyl ornithine transcarbamylase (OTC; EC 2.1.3.3), argininosuccinate syn­ phosphate from bicarbonate and ammonia via the mitochondrial thase (ASS; EC 6.3.4.5), argininosuccinate lyase (ASL; EC 4.3.2.1), enzyme carbamoyl phosphate synthetase 1 (CPS1; Krebs, 1942; Cohen arginase 1 (ARG1; EC 3.5.3.1), glutaminase (GLS2; EC 3.5.1.2), alanine and Hayano, 1948; Grisolia and Cohen, 1952). The next step is the aminotransferase (ALT; EC 2.6.1.2), aspartate aminotransferase (AST; formation of citrulline from carbamoyl phosphate and ornithine, cata­ EC 2.6.1.1), and three enzymes involved in histidine catabolism [histi­ lyzed by ornithine transcarbamylase (OTC) which is the only urea cycle dine ammonia lyase (HAL; EC 4.3.1.3), urocanate hydratase (UROC1; EC gene found on the X chromosome (KREBS et al., 1955; Marshall and 4.2.1.49) and glutamate formiminotransferase (FTCD; EC 2.1.2.5)]. Cohen, 1972). This is followed by the cytosolic components of the cycle, These hepatocytes also have the largest uptake capacity for glutamine beginning with the enzyme arginosuccinate synthase (ASS), which cat­ and alanine (Haussinger, 1983; Miyanaka et al., 1998; Gebhardt et al., alyzes the formation of arginosuccinate from citrulline and aspartate 1988; Stoll et al., 1991; Taylor and Rennie, 1987; Baird et al., 2004; (Saheki et al., 1975; Takada et al., 1979; Rochovansky et al., 1977; Botini et al., 2005). Ratner and Pappas, 1949). Arginosuccinate is cleaved via

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Fig. 1. Hepatic zonation of ammonia metabolism and glutamate metabolism. Simplified schematic illustration of ammonia removal via the urea cycle and via the glutamine synthetase pathway in the liver. The urea cycle pathway is a low affinity, high capacity ammonia clearance system that is preferen­ tially localized to the periportal hepatocytes, while the glutamine synthetase reaction in the perivenous hepatocytes represent a high affinity, low capacity system for escaping ammonia. Dysfunctions in either urea cycle or glutamine synthesis induces hyper­ ammonemia and causes encephalopathy (Qvarts­ khava et al., 2015; Hakvoort et al., 2017). These processes are intermingled with glutamate meta­ bolism via a differential involvement of enzymes and transporter proteins. Amino acids (AA, except gluta­ mate and aspartate) are taken up and catabolized inside periporal hepatocytes (Haussinger¨ et al., 1992). The two well studied examples are abundant plasma amino acids glutamine (Gln) and alanine, which possibly undergo transport inside the cells by slc38a5 (Taylor and Rennie, 1987). Liver glutaminase (GLS2) hydrolyses glutamine to glutamate and ammonia, and aminotransferases (ATs), e.g. alanine aminotransferase, transfer an amino group to alpha-ketogutarate (α-KG) to produce glutamate. The reaction catalyzed by glutamate dehydrogenase (GDH) is reversible. GDH produces ammonia that provides one nitrogen of the urea molecule. Also, it can furnish mitochondrial glutamate, which supports the formation of aspartate for the urea cycle (Nissim et al., 2003; Karaca et al., 2018). Glutamate partici­ pates in the urea cycle in several ways. First, it plays a regulatory role in the urea cycle because N-acetyl­ glutamate (NAG) is an obligatory activator of car­ bamyl phosphate synthetase 1 (CPS1) and is produced from glutamate and acetyl-coenzyme A. Second, glutamate donates its amino group to form aspartate, which then supplies the second nitrogen of the urea molecule. Third, as described above, the GDH reac­ tion glutamate is a donor of NH3. Perivenous hepa­ tocytes, on the other hand, take up glutamate (Glu) and aspartate (Asp) by the EAAT2 (slc1a2) isoform (Stoll et al 1991; Hu et al 2018). However, these cells apparently can produce glutamine in the absence of exogenous glutamate, and release glutamine probably via slc38a3. Besides alpha-ketoglutarate (α-KG) that is produced by Krebs cycle, ornithine is catabolized in ornithine aminotransferase (OAT)-containing hepa­ tocytes to provide a substrate for glutamine synthesis (Kuo et al 1991; O’sullivan et al 1998). The enzymes or transporter proteins that are predominantly pre­ sent in the periportal zone are indicated in red, while those in perivenous zone are indicated in green, those which are present in both zones are indicated in white and those with little information are indicated in gray.

arginosuccinate lyase (ASL) to arginine and fumarate which enters the transporters that carry ornithine and aspartate across the mitochondrial citric acid cycle (Ratner et al., 1953; Murakami-Murofushi and Ratner, membrane were eventually identified. Ornithine reenters the mito­ 1979). Finally, arginine is cleaved by arginase 1 to urea and ornithine chondria via ORNT1 (slc25a15) in exchange for citrulline (Camacho (Wu and Morris, 1998). Through extensive enzymological studies, it was et al., 1999; Kuno et al., 1990), while aspartate is released to cytosol via also revealed that acetylglutamate, catalyzed by N-acetylglutamate Citrin (slc25a13; Kobayashi et al., 1999). As expected, congenital or synthase (NAGS; EC 2.3.1.1), acts as allosteric activator of carbamoyl acquired deficiencies in any of the above listed enzymes and trans­ phosphate synthetase (Grisolia and Cohen, 1953; Shigesada and Tati­ porters can result in various degrees of hyperammonemia and enceph­ bana, 1971; Hall et al., 1958). Because the urea cycle is carried out in alopathy (summarized in Table 1). The history of the science related to two subcellular compartments (mitochondria and cytosol), mitochon­ the urea cycle and the metabolism of arginine and citrulline has been drial transporters had to be important, and in agreement, the two reviewed in detail elsewhere (Ratner, 1954; Holmes, 1980; Cohen, 1981;

3 Y. Zhou et al. Neurochemistry International 140 (2020) 104809

Fig. 2. The urea synthesis in the periportal hepatocytes. Cartoon depicting the formation of urea with the involvement of intermediate metabolites ornithine, citrulline and arginine. The chemical reactions and the involved enzymes/transporters (highlighted by blue) are detailed in the text.

Wu and Morris, 1998). 2019), as well as in enterocytes and immune cells (Stettner et al., 2018). The current non-surgical treatments of urea cycle disorders include Deficiency in these enzymes leads to reduced arginine levels and various approaches aimed at lowering ammonia levels (Tuchman et al., impaired synthesis of nitric oxide with multi-organ dysfunction as the 2008; Haberle et al., 2012a) such as ammonia scavenging agents (e.g. downstream consequence (Erez et al., 2011). sodium phenylacetate and sodium benzoate; Maestri et al., 1991; Enns et al., 2007), a low-protein diet with essential amino acid supplemen­ 4. The importance of liver glutamine synthetase in ammonia tation (Adam et al., 2013), hemodialysis, and arginine or citrulline metabolism supplementation (Brusilow, 1984; Donn and Thoene, 1985; Nagasaka et al., 2006). NAGS deficiencyin humans can be effectively treated with The urea cycle has long been considered to be the primary pathway N-carbamylglutamate (Caldovic et al., 2004; Heibel et al., 2011), and of nitrogen disposal. In the same year when Krebs described the Citrin deficiencyhas been successfully treated with a low-carbohydrate biosynthesis of urea from ammonia and carbon dioxide, he also noted diet with arginine supplementation (Saheki and Song, 1993). Even that the isolated rat liver removed more ammonia from the nutrient though these therapies have significantly improved the overall medium than was accounted for by the formation of urea (Krebs and morbidity and mortality, residual disease sequelae such as develop­ Henseleit, 1932). In 1935 he described the synthesis of glutamine from mental delay and language difficultiesoften persist (Maestri et al., 1991; ammonia and glutamate in the guinea pig kidney, brain, retina and other Kim et al., 2012). Intriguingly, the sequelae of urea cycle disorders tissues (Krebs, 1935). As shown in Table 1, the presence of hyper­ cannot be entirely explained by ammonia toxicity, because patients with glutaminemia in hyperammonemic patients with urea cycle disorders ASL and ORNT1 deficiencies may have neurological impairment in the has been known since the earliest case reports. Speck (1947) and Elliot absence of hyperammonemia (Baruteau et al., 2017; Kim et al., 2012). (1948 ab) independently discovered, respectively, in sheep brain and Moreover, some urea cycle enzymes and transporters are not exclusively pigeon liver, an enzyme system which catalyzes the synthesis of gluta­ expressed in the liver. For instance, ASS, CPS1 and ORNT1 show similar mine by combining glutamate with ammonia (Elliott, 1948; Elliott and patterns of developmental changes in the liver and small intestine Gale, 1948; Speck, 1947). The enzyme was later termed “glutamine (Begum et al., 2002). ARG1 is also found in the brain (Yu et al., 2001) synthetase (GS)" or “glutamate-ammonia ligase (Glul). Meister and and ASL is present in the brain stem (in locus coeruleus; Lerner et al., co-workers used a highly effective stereochemical approach to

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Table 1 Phenotypes of urea cycle disorders in human and mouse.

Gene Species Biochemical characteristics Phenotype Ref.

CPS1 Human severe hyperammonemia; elevated plasma glutamine; coma; death; protein intolerance; Gelehrter and Snodgrass (1974); reduced citrulline; absence of orotic aciduria developmental retardation; lethargy McReynolds et al. (1981); Wong et al. (1994) Mouse severe hyperammonemia; elevated plasma glutamine and die within 36 h of birth Schofield et al. (1999) (neonatal alanine; reduced citrulline and arginine Khoja et al. (2019) onset) Mouse (late hyperammonemia; encephalopathy; weight loss; death Khoja et al. (2018) onset) elevated plasma glutamine

NAGS Human neontal hyperammonia; elevated plasma glutamine; low can be treated with N-carbamylglutamate Bachmann et al. (1981); Al Kaabi citrulline level; absence of orotic aciduria treatment and El-Hattab (2016) Mouse hyperammonemia; elevated plasma glutamine, glutamate death within 48 h; can be rescued by carbamyl- Senkevitch et al. (2012) and lysine; reduced citrulline, arginine and ornithine l-glutamate and l-citrulline

OCT Human hyperammonemia; hyperglutaminemia coma; convulsions; metabolic acidosis Levin et al. (1969); Kang et al. (1973); Campbell et al. (1973) Mouse elevated plasma ammonia and glutamine; decreased a markedly decreased lifespan; retarded DeMars et al. (1976); citrulline and arginine; orotic aciduria growth; reduced fitness; hepatic fibrosis Batshaw et al. (1995); Wang et al. (2017)

ASS1 Human hyperammonemia; elevated plasma glutamine citrullinemia type I; progressive lethargic; Quinonez and Thoene (1993) premature death; feed poorly; spasticity; mental retardation; seizure; neurological deficits Mouse acute hyperammonemia; elevated plasma citrulline; early neonatal death; severe retardation; Patejunas et al. (1994) decreased arginine alopecia; lethargy; ataxia Perez et al. (2010)

Slc25a13 Human late onset of serious and recurring hyperammonia; often neonatal intrahepatic cholestasis; Saheki and Song (1993); Saheki citrullinemia (moderate compared with CTLN1 patients); neuropsychiatric symptoms; often leading to et al. (2002) decreased plasma glutamine; elevated arginine; an rapid death; reduced carbohydrate intakes increase in plasma threonine-to-serine ratio Mouse elevated ammonia, glutamate and glutamine; higher deficits in ureogenesis and gluconeogenesis; Moriyama et al. (2006) glycerol phosphate shuttle activity fatty liver (strain-dependent); no adult-onset Sinasac et al. (2004) type II citrullinemia

Slc25a13 Mouse hyperammonemia under fed conditions; citrullinemia; decreased survival; growth retardation; fatty Saheki et al. (2007); Saheki et al. and hypoglycemia liver; low-carbohydrate diets including (2012) mGPD increased protein content lead to metabolic correction

ASL Human episodic hyperammonemia; argininosuccinic aciduria; lethargy; poor feeding; tachypnea; seizures, Allan et al. (1958); elevated plasma citrulline, glutamine, alanine and coma; death; cognitive impairments; learning Nagamani et al. (2012) glycine disabilities; trichorrhexis nodosa Mouse hyperammonemia; elevated glutamine, arginosuccinic lethality, reduced body weight Reid Sutton et al. (2003) acid and citrulline; low plasma arginine

ARG1 Human hyperarginemia; hyperammonemia (less frequent) failure to thrive; cognitive dysfunction; Chandra et al. (2019); spasticity; seizures Crombez and Cederbaum (2005); Qureshi et al. (1983) Mouse late-onset hyperammonemia; moderate hyperarginemia; premature death; encephalopahy Iyer et al. (2002) reduced ornithine Deignan et al. (2006) Ballantyne et al. (2016) Kasten et al. (2013)

ORNT1 Human hyperammonaemia; hyperornithinaemia; intellectual disabilities; spastic paraparesis; Camacho et al. (1999); Salvi et al. homocitrullinuria hepatopathy; epilepsy (2001); Tezcan et al. (2012); Kimber et al. (1990); Fecarotta et al. (2006) understand key aspects of Glul function, such as its substrates, catalytic et al., 2010; Boissonnet et al., 2012), suggesting an important role of intermediates, and potential inhibitors (Meister, 1968a, 1968b; Rowe Glul in ammonia metabolism. This notion is further strengthened by the and Meister, 1970; Manning et al., 1969; Wellner and Meister, 1966; report of three human subjects (not related to each other) with partial Cooper et al., 1976). One such inhibitor was methionine sulfoximine congenital Glul deficiency (Haberle et al., 2005; Haberle et al., 2006; (MSO; CAS15985-39-4) which was produced during chemical bleaching Haberle et al., 2012b; reviewed in Spodenkiewicz et al., 2016). The (agenization) of flourwith agene (nitrogen trichloride; CAS 10025-85-1) subjects had increased blood ammonia levels, suffered from severe and causes acute and chronic seizures when ingested by or administered neonatal epileptic encephalopathy, and died young from multi-organ to animals (Campbell et al., 1951; Silver and Johnson, 1947). The use of failure. The severe and systemic manifestations of the Glul deficiency agene as bleaching agent was banned in the USA in 1949. are consistent with the widespread distribution and important roles of The significance of the Glul pathway for ammonia removal became the enzyme. evident when 15N-labeled ammonia was administered to laboratory Investigations of the physiological consequences of Glul have, until animals, resulting in more 15N-labeling of glutamine than of urea (Duda recently, relied heavily on the use of MSO. This approach, however, has and Handler, 1958; Jahoor et al., 1988). Further, systemic administra­ limitations. First, systemically administered MSO inhibits Glul in the tion of MSO reduces glutamine synthesis in multiple organs and leads to entire body, and thus it is unclear whether the neurological symptoms hyperammonemia (Warren and Schenker, 1964; Kant et al., 2014; Blin from systemic MSO are caused by inhibition of liver Glul, brain Glul, or et al., 2002; Rowe and Meister, 1970; Griffith and Meister, 1978; Cloix both. Second, MSO has several known effects other than inhibiting Glul,

5 Y. Zhou et al. Neurochemistry International 140 (2020) 104809

Table 2 Phenotypes of conditional glutamine synthetase knockout mice.

Organ/Cell Phenotypes Ref.

Global (all cells from conception) The blastocysts fail to implant and die at ED3.4. He et al. (2007) Brain/most CNS astrocytes (hGFAP-Cre) The mice die soon after birth. They fail to feed and have slightly increased cortical ammonia levels. He et al. (2010a) Cerebral cortex (glutamatergic neurons and The mice are viable, but they have reduced life span, develop spontaneous seizures from 6 weeks of age, Zhou et al. (2019a) glia; Emx1-IRES-Cre) and have decreased locomotive activities with episodes of wild running. Oligodendrocytes (MOGi-Cre) The mice are viable, but neuronal glutamatergic transmission is disrupted in a myelin-independent way. Xin et al. (2019) There are deficits in cocaine-induced locomotor sensitization. Macrophages (CSF1R-CreERT) The mice are viable. But they have an increased capacity to induce T-cell recruitment, a reduced T-cell Palmieri et al. (2017) suppressive potential and an impaired ability to foster endothelial cell branching or cancer cell motility. Endothelial cells (VE-cadherin(PAC)- The mice are viable, but have impaired retinal vessel sprouting during vascular development. Eelen et al. (2018) creERT2 and Pdgfb-creERT2) Liver (Alb-Cre and Alfp-Cre) The mice are viable, but they have systemic hyperammonia, and exhibit behavioral abnormalities Hakvoort et al. (2017); including increased locomotion, impaired fear memory, a slightly reduced life span, reduced exploratory Qvartskhava et al. activity, and delayed habituation to a novel environment. (2015); Chepkova et al. (2017) Muscle (MCK-Cre) The mice are healthy and fertile, but they have a 5-fold higher escaped ammonia when fasting. He et al. (2010b) Kidney (PEPCK-Cre) The mice are viable, but under basal conditions they have increased urinary ammonia excretion. During Lee et al. (2016); Lee metabolic acidosis or during dietary protein restriction, they have impaired adaptive responses in et al. (2017) ammonia excretion. Pancreas (Pdx1-Cre) Pancreatic ductal carcinoma development is supressed. Bott et al. (2019) such as impairing glutathione synthesis by inhibiting ɣ-glutamylcysteine 21Mgn/J, stock no. 003574; RRID: IMSR_JAX:003574)] becomes active synthetase (Bernard-Helary et al., 2002; Shaw and Bains, 2002; Kam and after birth and results in a gradual deletion which completes at the adult Nicoll, 2007). stage (Fig. 3A). To more accurately assess the roles of Glul in health and disease, several transgenic approaches have been undertaken. The firstapproach 5. The excitatory amino acid (glutamate) transporter subtype 2 was to completely delete the Glul gene throughout the body from (EAAT2) is co-expressed with glutamine synthetase in pericentral conception. However, such deletion leads to embryonic demise at em­ hepatocytes, but is not critical for ammonia detoxification bryonic day 3 (E3), suggesting that Glul is crucial for life (He et al., 2007). In a series of subsequent experiments that took advantage of the The pericentral hepatocytes also express EAAT2 (Berger and Cre-LoxP technology (Le and Sauer, 2000), several knockout mouse lines Hediger, 2006; Najimi et al., 2014; Zhou et al., 2014; Hu et al., 2018). with more restricted Glul deletions were independently created by three EAAT2 is the major glutamate transporter in the brain (Haugeto et al., research groups (He et al., 2010a: MGI:4462791, RRID: 1996; Tanaka et al., 1997; Holmseth et al., 2012a), and is the only IMSR_JAX:029827; Qvartskhava et al., 2015: MGI:5750936; Zhou et al., significantly expressed transporter for glutamate and aspartate in he­ 2019a: MGI:6285753) (Table 2). patocytes (Azimifar et al., 2014; Hu et al., 2018). Characterization of mice with liver-selective deletion of Glul The co-expression of Glul with EAAT2 in perivenous hepatocytes elegantly revealed the importance of the enzyme for ammonia homeo­ raises the question of how much blood-derived aspartate and glutamate stasis. The enzyme-deficient mice developed hyperammonemia and contribute to the synthesis of glutamine. Recent investigations using encephalopathy (Qvartskhava et al., 2015; Chepkova et al., 2017). liver-specific EAAT2 knockouts, suggest that EAAT2 cannot supply Further, by giving excess amounts of ammonia to the other enough glutamate to fully exploit the capacity of Glul (Hu et al., 2018). liver-selective Glul knockouts (RRID:IMSR_029827), it was discovered This assumption is based on the facts that the expression of hepatic Glul that Glul in the liver metabolizes approximately 35% of blood ammonia, is orders of magnitude higher than that of EAAT2 (Hu et al., 2018), and the urea cycle metabolizes another 35%, and the rest is degraded else­ that the transport catalyzed by EAAT2 is slow (Otis and Jahr, 1998; Otis where in the body (Hakvoort et al., 2017). While the data agree with and Kavanaugh, 2000; Bergles et al., 2002; Grewer and Rauen, 2005). In prior estimates that 80–87% of blood ammonia is metabolized by the line with this idea, mice lacking hepatic EAAT2 do not display loco­ liver (Aldrete, 1975), the equal contribution of Glul and the urea cycle to motor dysfunction (Hu et al., 2017) like mice lacking hepatic Glul the metabolism was unexpected, as most prior studies had suggested (Qvartskhava et al., 2015). The reason why EAAT2 may not be essential that the urea cycle was the most important. for liver function is that hepatocytes can produce glutamate via other Deletion of Glul from the liver was reported in one study to give pathways such as oxidation of ornithine by ornithine aminotransferase pericentral macrovesicular steatosis (Hakvoort et al., 2017). This would (OAT; O’sullivan et al., 1998) or from α-ketoglutarate (Braeuning et al., be in line with another study showing that hyperammonemia facilitates 2006) (Fig. 1). development of non-alcoholic steatohepatitis (De Chiara et al., 2020). It should be noted, however, that EAAT2 expression in perivenous However, when the liver selective deletion of Glul was replicated by us hepatocytes may be upregulated in disease, e.g. cholestasis (Najimi (Fig. 3D) and by Qvartskhava and coworkers (Qvartskhava et al., 2019), et al., 2014). Further, dietary supplementation with certain amino acids steatosis was not observed. We also did not observe postnatal growth (e.g. aspartate, glutamate, ornithine and arginine) attenuates retardation in our liver-specific glutamine synthetase knockouts carbohydrate-induced hyperammonemia in Citrin/mitochondrial (Fig. 3C) as Hakvoort and colleagues did. Hakvoort and Lamers glycerol-3-phosphate dehydrogenase double knockouts which have an concluded that the differences in steatosis were unlikely due to the impaired malate-aspartate shuttle (Saheki et al., 2019). Cre-line (Alfp-Cre) itself. Therefore, they speculated whether the dif­ ferences could be due to variations in the genetic background of the mice 6. Controversies concerning glutamine as a nitrogen donor for as they had used FVB/N mice while Qvartskhava and we had used urea synthesis C57BL6J mice (Hakvoort and Lamers, 2019). We, however, propose that the difference in steatosis development might be due to the age at which In addition to ammonia-derived nitrogen, other nitrogen donors for Glul is deleted from the liver: the Alfp-Cre construct initiates deletion of urea synthesis have been proposed. In periportal hepatocytes, Glul already at embryonic day E9.5 (Kellendonk et al., 2000; Parviz glutamate-nitrogen enters the urea cycle predominantly as aspartate et al., 2003), whereas the other Cre-line [Alb-Cre (B6.Cg-Tg (Alb-Cre) (Jahoor et al., 1988). But where does the glutamate-nitrogen come

6 Y. Zhou et al. Neurochemistry International 140 (2020) 104809

Fig. 3. Glutamine synthetase (GS) deletion that is controlled by the albumin promotor (Alb-Cre) occurs progressively in the liver during postnatal develop­ ment, being nearly complete in the young adult stage (about 7 weeks of old). Panel A. Immunocytochem­ istry analysis of the liver shows the incomplete dele­ tion of the glutamine synthetase protein (red) in the developing liver (P22) and almost complete deletion in mature liver (P55 and P89). Scale bar, 40 μm. Panel B. Immunoblotting analysis of liver-specific GS knockouts (cKO) and wild-type (WT) littermates (58–64 days old) confirm the absence of the gluta­ mine synthetase in the liver and the preservation in the brain. Panel C. The Alb-Cre driven glutamine synthetase knockouts (male, n = 11) have normal postnatal growth as their wildtype littermates do (n = 13) in C57BL6J mice. Panel D. Steatosis is absent in the liver-specific glutamine synthetase knockout fed with regular chows (3 months and 12 months of age; n = 3). The frozen tissue sections are stained for Oil Red O. Livers from high-fat fed mice were used as a positive control (data not shown). Scale bar, 100 μm. The data in the figure has only been published in abstract form (Hu et al., 2017).

et al., 2004). Considering that periportal hepatocytes can take up Table 3 glutamine and also express the liver isoform of glutaminase (which Localization of glutamine synthetase in rodents. catalyzes the conversion of glutamine to glutamate), it follows that the Organ Cellular localization Ref. amide-nitrogen of glutamine may represent a source of urea-nitrogen. Liver perivenous hepatocytes Gebhardt and Mecke (1983); This concept is experimentally supported by observations that addi­ Gaasbeek Janzen et al. tion of glutamine to liver slices results in considerable enhancement of (1987); Hu et al. (2018) urea production (e.g. Bach and Smith, 1956; Nissim et al., 1992). Brain astroglia Martinez-Hernandez et al. However, a major weakness of these studies is a lack of in vivo validation (1977) 15 oligodendrocytes Tansey et al. (1991); Saitoh (Kamin and Handler, 1957). By using N-glutamine, Handler concluded 15 and Araki (2010) that there is negligible formation of urea from N-glutamine in vivo Retina Müller cells Riepe and Norenburg (1977) (Duda and Handler, 1958), but this has been contradicted by another in endothelial cells Eelen et al. (2018) vivo study reporting that glutamine is more important than alanine as a Kidney proximal tubules, intercalated Burch et al. (1978), cells and distal convoluted Verlander et al. (2013) nitrogen donor for urea synthesis (Jahoor et al., 1988). Thus, further tubules investigations are required to fully resolve the role of glutamine in urea Skeletal myocytes Iqbal and Ottaway (1970); synthesis in vivo. muscle He et al. (2010b) Adipose tissue adipocytes van Straaten et al. (2006); 7. Glutamine synthetase in extrahepatic tissues Lie-Venema et al. (1997) Epididymis epithelial cells in the epididymis van Straaten et al. (2006); and testis and Leydig cells Lie-Venema et al. (1997) Glul is strongly expressed in extrahepatic tissues such as the brain, Stomach acid-producing parietal cells van Straaten et al. (2006) the kidneys, the skeletal muscle, the adipose tissue, the male repro­ Gastro- colon Lie-Venema et al. (1997) ductive organs, the pancreas, and the gastro-intestinal tract (see intestinal tract Table 3). These extrahepatic tissues are responsible for approximately Pancreas islets Zhou et al. (2014) one-third of the total ammonia detoxificationcapacity via the glutamine synthetase reaction (Hakvoort et al., 2017). Similar to the liver, the extrahepatic Glul-expressing cells often express glutamate transporters from? Alanine-nitrogen has been suggested because hepatic ammonia (Tables 3 and 4). Interestingly, the pancreas represents an exception, as uptake is accompanied by uptake of alanine in almost equimolar glutamate transporters are not expressed in the islets cells and are only quantities (see Fig. 1; Yang et al., 2000), and because alanine is a major present at very low levels in the exocrine pancreas (Zhou et al., 2014). amino acid extracted by the liver as a substrate for gluconeogenesis Skeletal muscle and kidneys have a particularly high capacity for (Felig et al., 1970). The in vitro work by Brosnan and coworkers confirms ammonia removal (Cruz et al., 2017). Selective deletion of Glul in the the contribution of alanine-nitrogen to urea and to glutamine synthesis, murine skeletal muscle suggests that the maximal capacity of ammonia although the capacity of alanine as a nitrogen source is limited (Brosnan absorption in skeletal muscle is approximately 10% (He et al., 2010b).

7 Y. Zhou et al. Neurochemistry International 140 (2020) 104809

Table 4 Localization of excitatory amino acid transporters (EAATs) in rodents.

Organ Subtype Cellular localization Ref.

Liver EAAT2 EAAT2 is the only EAAT-type of transporter in the liver and is selectively Berger and Hediger (2006); expressed in the perivenous hepatocytes Hu et al. (2018) Brain EAAT1 EAAT1 is selective for astroglia. Note that there are some reports claiming Lehre et al. (1995); Ginsberg et al. (1995); Berger and Hediger EAAT1 to be present in neurons, but this has not been supported by later (1998); Berger and Hediger (2000); Hanson et al. (2015) publications. EAAT2 EAAT2 protein is strongly expressed in astroglia Danbolt et al. (1992); Levy et al. (1993); Rothstein et al. (1994); Lehre et al. (1995); Berger and Hediger (1998); Schmitt et al. (1997); Berger and Hediger (2000) EAAT2 mRNA is present in astrocytes and in the majority of neurons in Torp et al. (1994); Schmitt et al. (1996); Torp et al. (1997); Berger multiple CNS regions and Hediger (1998); Berger and Hediger (2000); Berger and Hediger (2001) Torp et al. (1994); EAAT2 is expressed in brain neurons during development, in neurons in Northington et al. (1998); Northington et al. (1999); Martin et al. diseased brain tissue and in cultured neurons (1997); Mennerick et al. (1998); Plachez et al. (2000) Early electron microscopy suggested that nerve terminals, at least in cortex Beart (1976); McLennan (1976); Gundersen et al. (1993) and striatum, are able to take up glutamate though the responsible carrier(s) was not determined. Some of the EAAT2 protein (about 10%) is found in neurons, and is targeted to Chen et al. (2004); the axon-terminals where EAAT2 is the only glutamate transporter. Furness et al. (2008); Zhou et al. (2019b); Petr et al. (2015): Danbolt et al. (2016a) EAAT3 EAAT3 is selective for neurons and present in most if not all neurons, but is Kanai and Hediger (1992); Rothstein et al. (1994); targeted to the cell body and dendrites. Bjoras et al. (1996); Torp et al. (1997); Holmseth et al. (2012a) EAAT4 EAAT4 is expressed in cerebellar Purkinje cells with the highest concentrations Dehnes et al. (1998); Massie et al. (2008) where Zebrin II is, and is also present in scattered neurons in the fore- and midbrain. It is especially enriched in the parts of the dendritic and spine membranes facing astrocytes. Retina EAAT1 EAAT1 is the most abundant of the EAATs in the retina and is expressed by the Lehre et al. (1997); EAAT2 Müller cells. EAAT2 is found in cone photoreceptors and bipolar cells, while Rauen and Kanner (1994); Euler and Wassle (1995); Rauen et al. EAAT5 EAAT5 has been less studied, possibly expressed in Müller cells. (1996); Arriza et al. (1997); Rauen et al. (1999); Rauen (2000); Veruki et al. (2006) Kidney EAAT3 EAAT3 is the only EAAT-type of transporter expressed in the kidney, and is Kanai and Hediger (1992); Hu et al. (2018) found in the proximal tubules Heart EAAT1 Rat hearts contain EAAT3 and EAAT1, but at lower levels than in the brain. Martinov et al. (2014) EAAT3 EAAT2 and EAAT4 were not detected. Skeletal Not reported Muscle Gastro- EAAT3 EAAT3 is the only EAAT-type of transporters in enterocytes, and is found in the Kanai and Hediger (1992); intestinal small intestine and colon with the highest levels in the distal ileum. Hu et al. (2018); Berger and Hediger (2006) tract Exocrine EAAT2 EAAT2 is present at low levels in salivary glands. Berger and Hediger (2006) glands Adipose tissue EAAT1 Several EAATs have been reported in adipocytes, but EAAT1 is the only EAAT Berger and Hediger (2006); Adachi et al. (2007) confirmed by validated in situ hybridization and proteome analysis Pancreas In-depth proteome analysis and characterization of pancreas-specific EAAT2 Zhou et al. (2014) knockout mice show that EAAT2 is not expressed in pancreatic islets as some have reported, but may be expressed at low levels in the exocrine pancreas. In fact, the islets do not express any of the EAATs nor any of the VGLUTs

Although this estimate is much lower than what was proposed in the past (50%; Hod et al., 1982; Lockwood et al., 1979), it should be kept in Table 5 mind that skeletal muscle can upregulate Glul activity and therefore Phenotypes of local administration of MSO. increase ammonia metabolism during liver failure (Hod et al., 1982; Region Phenotype Ref. Desjardins et al., 1999; Clemmesen et al., 2000; Chatauret et al., 2006). Skeletal muscle Abolish the arterio- Hod et al. (1982) The kidneys also adapt to the consequences of a failing liver by reducing venous difference of ammonia release into the systemic circulation by slowing down ammonia + ammonia production and by increasing ammonium (NH ) excretion in Trigeminal subnucleus Altered mechanical Chiang et al. (2007); 4 caudalis activation threshold and Tsuboi et al. (2011) the urine (Dejong et al., 1993; Jalan and Kapoor, 2003). responses to graded A full discussion of ammonia metabolism in the kidneys and skeletal mechanical stimuli; muscle, however, is beyond the scope of this review, as these topics are attenuated nocifensive covered well elsewhere (e.g. Mutch and Banister, 1983; Graham and behavior Entorhinal-hippocampal Recurrent seizures Eid et al. (2008); MacLean, 1998; van de Poll et al., 2004; Weiner and Verlander, 2013; area/hippocampus/central Dhaher et al. (2015); Weiner et al., 2015; Olde Damink et al., 2002; Adeva et al., 2012). nucleus of the amygdala Gruenbaum et al. (2015); 8. Glutamine synthetase is essential for brain function Trigeminal motor nucleus Altered jaw-opening Mostafeezur et al. reflex (2014) Ventriculocisternal space Changes in ventilation Hoop et al. (1988) The importance of brain Glul was first uncovered by local adminis­ tration of MSO to the central nervous system (CNS). Depending on the injection site, MSO caused recurrent seizures, altered responses to graded mechanical stimuli, attenuated nocifensive behavior, or altered

8 Y. Zhou et al. Neurochemistry International 140 (2020) 104809 jaw-opening reflex (Table 5) (Eid et al., 2008; Dhaher et al., 2015; EAAT2/slc1a2; Danbolt et al., 1992; Lehre et al., 1995), converts Gruenbaum et al., 2015; Chiang et al., 2007; Tsuboi et al., 2011; Mos­ glutamate into glutamine via the Glul pathway (Martinez-Hernandez tafeezur et al., 2014). The MSO studies are, however, associated with et al., 1977) and releases glutamine into the extracellular space. Neurons some uncertainty because MSO not only inhibits glutamine synthetase, are able to take up glutamine (Schousboe et al., 1979; Dyste et al., 1989) but also decreases tissue glutathione (Shaw and Bains, 2002), increases via glutamine transporters. It is possible that several glutamine trans­ astrocyte glycogen (Bernard-Helary et al., 2002), and excites neurons porters are involved as there are at least 14 different solute carrier via a Glul-independent mechanism (Kam and Nicoll, 2007). Depletion of proteins with the ability to transport glutamine (for reviews see: neuronal glutathione is shown to increase cytosolic glutamate and af­ Mackenzie and Erickson, 2004; Bhutia and Ganapathy, 2016; Danbolt fects excitatory neurotransmission (Sedlak et al., 2019). et al., 2016a; Hellsten et al., 2017). Inside neurons, glutamine can be Several knockout approaches for Glul have recently become avail­ converted back to glutamate by phosphate-activated glutaminase able (as explained above), making highly accurate in vivo studies of the (PAG/GLS1; Kvamme et al., 2001). A full discussion of glutamine and enzyme possible. However, deletion of Glul in the entire CNS resulted in glutamate metabolism in the brain have been thoroughly reviewed by early neonatal death (He et al., 2010a). More restricted deletions would others (e.g. Yudkoff, 2017; Hertz and Zielke, 2004; McKenna and Fer­ thereby be necessary in order to obtain longer survival. Unfortunately, reira, 2016; Bak et al., 2018; Hertz and Chen, 2018; Hertz and Rothman, there are not that many astroglial Cre drivers, and that the commonly 2016). used ones (e.g. hGFAP-Cre) are active in most CNS astrocytes resulting in The glutamate-glutamine cycle model, however, probably over­ too extensive deletion. It was therefore a breakthrough when we (Zhou simplifies a complex reality, and not all available data fit with this et al., 2019a) were able to selectively delete Glul in the cerebral cortex model. (A) Although it is clear that neurons can take up glutamine, it is (i.e. neocortex and hippocampus) using the Emx1_IRES Cre line. These not known how and to what extent glutamine can enter the nerve ter­ mice survive well into adulthood and exhibit numerous pathological minals as no glutamine transporter protein has so far been shown features such as altered locomotive activity, progressive neuro­ immunocytochemically at the electron microscopic level to be present in degeneration, and spontaneous recurrent seizures (Zhou et al., 2019a). axon-terminals in brain tissue (for review see: Mackenzie and Erickson, In contrast, deletion of glutamine synthetase in the liver (Hakvoort et al., 2004; Conti and Melone, 2006; Grewal et al., 2009; Zhou and Danbolt, 2017), the kidneys (Lee et al., 2016), the skeletal muscle (He et al., 2014). Possible candidates comprise SNAT7 (slc38a7) and SNAT8 2010b), the pancreas (Bott et al., 2019) or the adipose tissue (Zhou Y and (slc38a8), but uncertainty remains as the corresponding knockout mice Danbolt NC, personal observations) barely affected mortality and had a are not yet available as negative controls and quantitative information mild phenotype with no evidence of behavioral seizures (Table 2). has yet to be obtained (Hagglund et al., 2011, 2015). Recently, it has been reported that the glutamine transporter SNAT1 (Slc38a1) partici­ 9. Glutamine production and roles of glutamine in pates in the regulation of the GABA levels in GABAergic terminals neurotransmission although the SNAT1 protein itself was not detected at the terminals (Qureshi et al., 2019; Qureshi et al., 2020). There is electrophysiological Glutamine is abundantly present in the central nervous system, and evidence for glutamine uptake at the Calyx of Held synapse (Billups the synthesis is largely catalyzed by astroglial glutamine synthetase et al., 2013). However, this synapse consists of a special giant nerve (Zhou et al., 2019a). The proposed major role is to be a precursor of the terminal which may not be representative for the majority of synapses in neurotransmitters glutamate (excitatory) and ɣ-amino butyric acid the mammalian CNS. The majority of mammalian synapses have bou­ (GABA; inhibitory). For more than half a century, compartmentation of tons that are very small and we know less about them (von Gersdorff and glutamate-glutamine metabolism has been central to how we envision Borst, 2002). (B) Another unexplored observation is that glutamine glutamine as a precursor for neuronal glutamate (Berl et al., 1962; Berl administered to the intact brain is mostly metabolized to CO2 (Zielke et al., 1968; Berl et al., 1970; Martinez-Hernandez et al., 1977; van den et al., 1998), in agreement with an important role for glutamine as a Berg and Garfinkel,1971 ; Bradford et al., 1978; Hamberger et al., 1979). neuronal energy substrate (Bradford et al., 1978; Hamberger et al., As outlined in Fig. 4, neurotransmitter glutamate is released by exocy­ 1979; Zielke et al., 2009). (C) Electrophysiological analyses of isolated tosis of synaptic vesicles from nerve terminals (for review: Sudhof, brain slices suggest that glutamatergic neurotransmission can be sus­ 2014). The surrounding astrocytes rapidly take up the released gluta­ tained in the absence of the cycle (Kam and Nicoll, 2007). (D) The ter­ mate via two astroglial glutamate transporters (EAAT1/slc1a3 and minals can obtain glutamate independently of glutamine as there is

Fig. 4. Overview of the glutamate–glutamine cycle. (1) Glutamate (Glu) is released from vesicles in the axon-terminals into the extracellular fluidfrom where it can (2) bind to glutamate receptors (GLU-R) before it is inactivated by uptake into astrocytes (3) via excitatory amino acid transporters (EAATs). (4) Inside astrocytes it is converted to glutamine (Gln) via the enzyme glutamine synthetase (Glul). (5) Gln is next shuttled from astrocytes to neurons via mechanisms that remain to be definedunequivocally. (6) Once inside the neurons, Gln is converted back to Glu by glutaminase, which is particularly enriched in mitochondria. (7) Finally, Glu is concentrated in synaptic vesicles via vesicular glutamate transporters (VGLUTs), thus completing the cycle. It should ne noted that (8) axonal terminal expressing EAAT2 will partly short circuit the cycle and that (9) there is an export of glutamine from the brain to the blood.

9 Y. Zhou et al. Neurochemistry International 140 (2020) 104809 some de novo synthesis (Hassel and Bråthe, 2000), and as they can take studies. Moreover, the use of Glul knockout tissue as a control for up glutamate directly via EAAT2 because a portion of EAAT2 (5–10%) is antibody specificityhas shown that Glul is expressed in a subpopulation present in axonal terminals (Furness et al., 2008; Danbolt et al., 2016a; of oligodendrocytes that emerges at P21 and is particularly densely Petr et al., 2015; Zhou et al., 2019b; McNair et al., 2019). (E) As stained in caudal regions of the CNS (Xin et al., 2019). The late onset and McKenna and Yudkoff pointed out earlier, the model ignores the facts regional heterogeneity in Glul expression, may explain earlier incon­ that the cycle is not stoichiometric and that the brain avidly exchanges sistent observations. Finally, selective removal of Glul in endothelial many metabolites with the blood (McKenna, 2007; Yudkoff, 2017). In cells impairs retinal vessel spouting during vascular development (Eelen line with this, the supply of glutamine to terminals may not always keep et al., 2018), and deletion of Glul in macrophages in tumor-bearing mice up with glutamate release (Waagepetersen et al., 2005; Kam and Nicoll, promotes tumor vessel pruning, vascular normalization, accumulation 2007; Marx et al., 2015), because there is a glutamine loss due to the of cytotoxic T-cells, and metastasis inhibition (Palmieri et al., 2017). export from the brain to the blood (Cangiano et al., 1983). This export The above findings challenge the concept that Glul in the CNS is process is mediated by several transporters including LAT1 (slc7a5), exclusively expressed in astrocytes and therefore the two-compartment LAT2 (slc7a8) and SNAT3 (slc38a3) which are robustly expressed in (neuron-glia) model of the glutamate-glutamine metabolic cycle, which brain microvessel endothelial cells (Duelli et al., 2000; Dolgodilina is thought to be critical for the synthesis of the major excitatory and et al., 2016; Dolgodilina et al., 2020). Thus, the relative contributions of inhibitory neurotransmitters glutamate and GABA (e.g. van den Berg the various mechanisms are still debated and a complicating factor is and Garfinkel,1971 ; Rothman et al., 1999). While knockout approaches that this may differ between brain regions and in disease. Marx and can be used to gain a more detailed and accurate understanding of the co-workers has recently tried to put available quantitative data together glutamate-glutamine cycle, they are associated with limitations. For (Marx et al., 2015). instance, the insertion of a promotor-Cre construct into a genome sometimes disturbs the expression of other genes (Forni et al., 2006; 10. Glutamine synthetase in the CNS may be expressed in cells Giusti et al., 2014). It is therefore important to assess whether the Cre other than astrocytes promotor interferes with the study. Likewise, when tamoxifen-inducible constructs have been used, possible side-effects of tamoxifen must be It is well established that Glul is expressed in astrocytes (Marti­ carefully controlled for. This is particularly relevant to cancer and nez-Hernandez et al., 1977); however, it is still debated whether the immunological research, as tamoxifen is an anti-cancer drug and ap­ enzyme is expressed in significant quantities in other CNS cells such as pears to have immunomodulatory effects which are independent of the oligodendrocytes, endothelial cells, microglia and neurons. The lack of a estrogen-receptor (Corriden et al., 2015; for review see: Behjati and consensus is partly due to how Glul is quantifiedin vitro and the fact that Frank, 2009). Another reason to be cautious is that gene expression the expression of the enzyme is regulated by cell to cell interactions and profilesof many cell types, including endothelial cells and macrophages, soluble factors that may be very different in vitro versus in vivo (e.g. are significantlyinfluenced by the microenvironment and may therefore Barakat-Walter and Droz, 1990; Cahoy et al., 2008). This point was well differ between locations and functional states (Gordon and Pluddemann, illustrated by Barakat-Walter and Droz (1990) who found that dorsal 2017; Garlanda and Dejana, 1997). Thus, the expression patterns of Glul root ganglion cells grown in vitro express Glul while those grown in vivo in endothelial cells of the brain and in microglia (a macrophage-like cell do not. Another factor contributing to the lack of consensus is the residing in the CNS) need further studies. It will be interesting to learn different results obtained with immunocytochemistry performed in more about the metabolic consequences of deleting glutamine synthe­ different laboratories with different antibodies and labeling protocols tase in endothelial and microglia in vivo. (Martinez-Hernandez et al., 1977; Norenberg and Martinez-Hernandez, 1979; Cammer, 1990; Tansey et al., 1991; D’Amelio et al., 1990; 11. Brain glutamine synthetase-deficiency and liver-induced Yamamoto et al., 1989; Takasaki et al., 2010), suggesting that differ­ hyperammonemia affect the brain in different ways ences in the specificityof immunohistochemical procedures play a role, as this is extensively discussed in prior publications (e.g. Danbolt et al., The classical hyperammonemia syndrome with increased blood 1998; Holmseth et al., 2006; Lorincz and Nusser, 2008; Saper, 2009; ammonia levels leads to astroglial swelling without significantneuronal Holmseth et al., 2012b; Danbolt et al., 2016b). death (Diemer and Laursen, 1977; Laursen and Diemer, 1979; Martin It is important to resolve the questions with regards the cellular et al., 1987; Desjardins et al., 2012; for reviews see: Butterworth et al., expression of Glul because if the enzyme is expressed in other cell types, 1987; Aldridge et al., 2015), and to glutamine accumulation in the brain then this will impact the interpretation of a number of studies of the (Cooper et al., 1979; Ratnakumari et al., 1992; Haussinger et al., 1994; ammonia metabolism. Fortunately, the recent development of knockout Bosman et al., 1990; Rao and Norenberg, 2001) despite models is expected to close key gaps in knowledge and open up for new ammonia-mediated downregulation of astroglial Glul (14–25%, Girard fields of research that may ultimately translate to novel treatments for et al., 1993; Desjardins et al., 1999; Haussinger et al., 2005). ammonia-related disorders. By knocking out Glul in a cell-specific Selective deletion of hepatic Glul causes a similar syndrome char­ manner, the functional consequences can be studied with a high level acterized by hyperammonemia with astroglial swelling, no visible of accuracy and the tissue can also serve as a specificity control for neurodegeneration, and a minor reduction in life span (Qvartskhava immunocytochemical staining of the enzyme. For example, one of the et al., 2015). The mice display altered behavior characterized by Glul knockout approaches (Emx1Cre/Glul-Flox) targets astrocytes, glu­ increased locomotion, reduced exploratory activities and delayed tamatergic neurons, and oligodendrocytes in the cerebral cortex, but habituation to a novel environment as well as impaired fear memory spares cortical GABAergic neurons and cells in other parts of the brain (Qvartskhava et al., 2015; Chepkova et al., 2017). (Zhou et al., 2019a; Gorski et al., 2002). Another approach selectively In contrast, selective deletion of Glul in the whole brain in mice re­ deletes Glul in oligodendrocytes (Xin et al., 2019), endothelial cells sults in a 14-fold decline in cortical glutamine and a 1.6-fold increase in (Eelen et al., 2018) and macrophages (Palmieri et al., 2017). cortical ammonia levels (He et al., 2010a). These mice die about three Surprisingly, mice lacking oligodendrocyte Glul have about 20% days after birth unless they are fed by hand. In contrast, when Glul is reduction in brain glutamate and glutamine levels and exhibit disturbed selectively deleted in the cerebral cortex (Zhou et al., 2019a), there is a glutamatergic neurotransmission, but unaffected myelination (Xin et al., 4-fold reduction in cortical glutamine and the majority of the animals 2019). The finding of normal myelination in vivo is in contrast to an in survive for several months. There is, however, a progressive gliosis and vitro study where glutamine synthetase deficiencyaffects differentiation impaired neurovascular coupling. These animals exhibit overall of oligodendrocytes (Saitoh and Araki, 2010). These results emphasize decreased locomotion with brief episodes of wild running, and many the importance of validating in vitro findings with subsequent in vivo develop spontaneous recurrent seizures and neurodegeneration that

10 Y. Zhou et al. Neurochemistry International 140 (2020) 104809 commence around 6 weeks of age. A portion of animals die suddenly and Al Kaabi, E.H., El-Hattab, A.W., 2016. N-acetylglutamate synthase deficiency: novel unexpectedly, leading to an overall reduced life-span (Zhou et al., mutation associated with neonatal presentation and literature review of molecular and phenotypic spectra. Mol. Genet. Metab. Rep. 8, 94–98. https://doi.org/10.1016/ 2019a). j.ymgmr.2016.08.004. The phenotype of the cortical Glul knockout mice are in good Aldrete, J.S., 1975. Quantification of the capacity of the liver to remove ammonia from agreement with the observations that patients with partial loss of the circulation of dogs with portacaval transposition. Surg. Gynecol. Obstet. 141, 399–404. function mutations in the Glul gene had brain atrophy and severe Aldridge, D.R., Tranah, E.J., Shawcross, D.L., 2015. Pathogenesis of hepatic neonatal epileptic encephalopathy (for review see: Spodenkiewicz et al., encephalopathy: role of ammonia and systemic inflammation. J. Clin. Exp. Hepatol. 2016). These humans had insufficient Glul activities, not only in the 5, S7–S20. https://doi.org/10.1016/j.jceh.2014.06.004. Allan, J.D., Cusworth, D.C., Dent, C.E., Wilson, V.K., 1958. A disease, probably brain, but in the entire body including liver, skeletal muscle, kidneys and hereditary characterised by severe mental deficiency and a constant gross skin. They died young from multiple organ failures. Further, the defects abnormality of aminoacid metabolism. Lancet 1, 182–187. https://doi.org/10.1016/ were present already at conception, and in this context it should be s0140-6736(58)90666-4. Alonso, J., Cordoba, J., Rovira, A., 2014. Brain magnetic resonance in hepatic noted that the deleterious effects of hyperammonemia may be more encephalopathy. Semin. 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