The multifunctional properties of astrocytes signify their importance in brain physiology and neurological function. In addition to defining the brain architecture, astrocytes are primary elements of brain ion, pH and neurotransmitter homoeostasis. GS (glutamine synthetase), which catalyses the ATP-dependent condensation of ammonia and glutamate to form glutamine, is an enzyme particularly found in astrocytes. GS plays a pivotal role in glutamate and glutamine homoeostasis, orchestrating astrocyte glutamate uptake/release and the glutamate–glutamine cycle. Furthermore, astrocytes bear the brunt of clearing ammonia in the brain, preventing neurotoxicity. The present review depicts the central function of astrocytes, concentrating on the importance of GS in glutamate/glutamine metabolism and ammonia detoxification in health and disease.

Astrocytes as homoeostatic cells in the CNS (central nervous system)

Of the four main classes of neuroglial cells of the central nervous system (astroglia, oligodendroglia, NG2 glia and microglia), astrocytes are arguably the most diverse. Indeed, astroglial cells include a huge variety of phenotypes with very different morphology and physiological properties. The largest and most characterized types of astroglia are represented by the protoplasmic astrocytes of the grey matter and fibrous astrocytes of the white matter. The morphological appearance of these cells, as well as their physiology, differs substantially between various brain regions. Another main group of astroglial cells covers the radial glia, which are bipolar cells with a small cell body and main processes, one of which forms endfeet on the ventricular wall and the other at the pial surface. Radial glia are the main glial type of the embryonic brain and are generally absent from the mature brain with the exception of the retina (Müller glia) and cerebellum (Bergmann glia). Astrocytes are further classified into the velate astrocytes of the cerebellum, the interlaminar and polarized astrocytes of the primate cortex, tanycytes (found in the periventricular organs, the hypophysis and the raphe part of the spinal cord), pituicytes in the neuro-hypophysis, and perivascular and marginal astrocytes. Astroglia also include several types of cells that line the ventricles or the subretinal space, namely ependymocytes, choroid plexus cells and retinal pigment epithelial cells (see [1,2] for reviews and references therein).

All of these highly diverse cells are united by their function which lies in homoeostasis of the nervous system [1]. To cater for this, astrocytes perform many different functions, which embrace every known housekeeping and homoeostatic task in the CNS (for detailed reviews see [312]). Astroglial cells are fundamental for brain development being, for example, chief promoters and supporters of synaptogenesis, synaptic maturation and maintenance [13,14]. In the adult brain, it is the astrocytes which act as stem cells in the neurogenic niches [15]. Astrocytes define overall brain architecture by forming glial barriers at the pia mater and between the brain parenchyma and vasculature; they divide the grey matter into neurovascular units and contribute to regulation of local blood flow; they are primary elements of brain ion homoeostasis; they control extracellular pH; they provide neurons with metabolic substrates and release scavengers of reactive oxygen species (for detailed reviews, see [312]). Astrocytes are also critical for numerous systemic homoeostatic functions such as central chemoception [1618] and regulation of sleep [19]. In the present review, we concentrate on a single homoeostatic function of astroglia, the function which relates to their ability to maintain turnover of two principal neurotransmitters in the brain, glutamate and GABA (γ-aminobutyric acid), this function being accomplished by glutamate, GABA and glutamine transporters, and by an astroglia-specific enzyme: GS (glutamine synthetase).

Astrocytes in glutamatergic neurotransmission: glutamate uptake and release

Fundamentally, astrocytes contribute to the homoeostasis and regulation of the extracellular level of three key neurotransmitters in the CNS, represented by glutamate, GABA and adenosine [8,20,21]. The astroglial role is multifaceted: (i) it includes an uptake of these neurotransmitters from the extracellular cleft, which defines the time course of neurotransmission and (at least in case of glutamate) toxicity; (ii) astrocytes are also capable of releasing neurotransmitters through vesicular and non-vesicular pathways; and (iii) astrocytes catabolize neurotransmitters into intermediates, which are then sent back to neurons to be transformed into active molecules, thus maintaining synaptic transmission.

Astroglial uptake of glutamate

Glutamic acid, or glutamate, is an amino acid, which, in the CNS, acts as the main excitatory neurotransmitter released from presynaptic terminals through vesicular exocytotic mechanism. To make this mechanism function properly, both extra- and intra-cellular glutamate dynamics should be tightly controlled. Glutamate should be removed from the synaptic cleft, and, at the same time, the synaptic cleft should be guarded from extrasynaptic glutamate or glutamate spillover from neighbouring synapses. Simultaneously, the glutamate inside the presynaptic terminal should be rapidly replenished. It is important to note that neurons are incapable of de novo synthesis of glutamate; the latter is synthesized in astrocytes [22] (see below). Astrocytes also provide a principal pathway for glutamate uptake: approximately 80% of all glutamate released during synaptic transmission is taken up by astroglial cells, only ~20% of this transmitter is being accumulated into postsynaptic neurons; presynaptic neurons do not take up much glutamate [20,23].

Glutamate uptake by astroglial cells is mediated through plasmalemmal glutamate transporters of the SLC1 (solute carrier 1) family [24] represented by five main types of EAATs (excitatory amino acid transporters), i.e. EAAT1/SLC1A3, EAAT2/SLC1A2, EAAT3/SLC1A1, EAAT4/SLC1A6 and EAAT5/SLC1A7, of which astrocytes specifically express EAAT1 and EAAT2; these in rodents are known as GLAST (glutamate/aspartate transporter) and GLT-1 (glutamate transporter-1). Transport of glutamate by EAATs is powered by transmembrane gradient of Na+ ions; translocation of a single molecule of glutamate (which is a monovalent anion at physiological pH) is linked to an influx of three Na+ and one H+, and efflux of one K+; all of these movements are downhill along respective concentration gradients. The glutamate transport cycle is therefore associated with net cation influx which defines the electrogeneity of glutamate transporters that could be measured as an inward current. Uptake of physiologically relevant concentrations of glutamate causes substantial Na+ influx, which may increase intracellular Na+ concentration by tens of millimolar [25,26].

Astroglial release of glutamate

Astrocytes can release glutamate by several different mechanisms: (i) reversal of uptake by plasma membrane glutamate transporters [27], (ii) opening of anion channels, be that volume-regulated anion channels [28] or Ca2+-activated anion channels [29], (iii) Ca2+-dependent exocytosis [30], (iv) glutamate exchange via the cystine/glutamate antiporter [31], (v) release through ionotropic purinergic receptors [32], and (vi) functional unpaired connexons, ‘hemichannels’, on the cell surface ([33] and see [34] for a review). These mechanisms can be classified further into the release through plasmalemmal molecular entities, channels and transporters, and by exocytosis; the latter characterized by the formation of an aqueous channel, the fusion pore, upon the merger of vesicular and plasma membranes. All of the mechanisms depend on the glutamate concentration in various cellular compartments and the extracellular space. Conceptually, its anionic concentration gradient between the cytosol and the extracellular space dictates the release directly via plasmalemmal channels/transporters. However, owing to coupling transport of other ions, EAATs have their reversal potential at ~+50 mV [25,35], which means that, under normal physiological conditions, EAATs could not revert to release glutamate [36]. With respect to exocytosis, cytosolic glutamate is sequestered into secretory vesicles via VGLUTs (vesicular glutamate transporters) before it is released through the fusion pore.

The cytoplasmic glutamate concentrations in astrocytes are maintained differently from those in neurons (for a review, see [20]). The glutamate concentration in the cytosol of synaptic terminals reaches 10–15 mM [37]. This permits VGLUTs (Km ~1–2 mM) to operate at nearly their maximal rates to concentrate glutamate into synaptic vesicles. Consequently, the intravesicular glutamate concentration reaches 60 mM [38], which generates an estimated ~1.1 mM glutamate concentration in the synaptic cleft at the time of release [39]. Owing to the powerful action of mainly astroglial EAATs, glutamate is quickly lowered, so that the resting glutamate concentration in the extracellular space of the CNS is maintained at ~25 nM [40]. Presumably because of the presence of GS, the cytosolic glutamate concentration in astrocytes is lower than the neuronal concentration at an estimated 0.1–5 mM [37]. This is sufficient to allow for the operation of VGLUTs to concentrate glutamate inside astrocytic vesicles, for which the intravesicular concentration is estimated at ~20 mM [41]. Consequently, vesicular glutamate release from astrocytes creates localized extracellular glutamate accumulations of 1–100 μM [42].

In the CNS, glutamate is synthesized de novo within astrocytes as a by-product of the TCA (tricarboxylic acid) cycle [43]. Here, as glucose breaks down to pyruvate in the cytosol, pyruvate enters the mitochondria and the TCA cycle via pyruvate carboxylase, a ligase that catalyses the carboxylation of pyruvate to form oxaloacetate (Figure 1). In turn, glutamate is produced from 2-oxoglutarate (α-ketoglutarate), a downstream TCA intermediate, by transamination of aspartate via mitochondrial aspartate aminotransferase. The synthesized glutamate once in the cytosol can then be converted into glutamine by GS, or transported into vesicles via VGLUTs, especially isoform 3 (VGLUT3) [44] (Figure 1). The role of GS and cytosolic glutamate concentrations for exocytotic glutamate release from astrocytes has been demonstrated experimentally [44]. To increase the cytosolic glutamate concentration in astrocytes GS activity was blocked with L-methionine sulfoximine, which led to an augmented exocytotic glutamate release in response to mechanical stimulation [44], whereas, importantly, Ca2+ dynamics were unaffected by this GS blocker. Of note, the mechanical stimulation almost exclusively (97%, based on the pan-VGLUT blocker Rose Bengal) recruits the exocytotic/vesicular release of glutamate [45], but not glutamate release through, e.g., Ca2+-activated anion channels [46]. Therefore the impairment of GS activity implies that the increase in cytosolic glutamate concentration provided more glutamate for VGLUTs to transport across the vesicular membrane into the vesicular lumen, thus increasing the amount of glutamate in vesicles available for release. Furthermore, the modulation of cytosolic glutamate concentration and its release could also originate from changes that might occur at the level of pyruvate carboxylase. Hence astrocytes from a Huntington's disease mouse model showed an augmented glutamate release as de novo glutamate synthesis was increased due to an upsurge in the expression of pyruvate carboxylase, whereas the conversion of glutamate into glutamine was unchanged, as no changes in the expression level of GS were observed [47]. Hypothetically, additional modulation of cytosolic glutamate concentration could sprout from the metabolic pathway between pyruvate carboxylase and GS. This could involve various mitochondrial enzymes, such as the previously mentioned aspartate aminotransferase. Similarly, SLCs redistributing reactants/products across the inner mitochondrial membrane could also be involved, such as perhaps mitochondrial glutamate carrier 1 (SLC family 25 member 22) and mitochondrial aspartate glutamate carrier (SLC family 25 member 13, also called citrin).

Regulation of glutamate in exocytotic glutamate release from astrocytes

Figure 1
Regulation of glutamate in exocytotic glutamate release from astrocytes

Glucose (Glc) is broken down to pyruvate (Pyr) in the cytosol. In the mitochondrion (mito), pyruvate enters the TCA via pyruvate carboxylase (PC) and leads to production of oxaloacetate (OAA) and a downstream intermediate, 2-oxoglutarate (α-KG). In turn, glutamate (Glut) is synthesized in astrocytes de novo from 2-oxoglutarate by transamination of aspartate via mitochondrial aspartate aminotransferase (AAT). The synthesized glutamate, once in the cytosol, can then be converted into glutamine (Gln) by glutamine synthetase (GS), or transported into vesicles via vesicular glutamate transporters, especially isoform 3 (VGLUT3). Drawing is not to scale.

Figure 1
Regulation of glutamate in exocytotic glutamate release from astrocytes

Glucose (Glc) is broken down to pyruvate (Pyr) in the cytosol. In the mitochondrion (mito), pyruvate enters the TCA via pyruvate carboxylase (PC) and leads to production of oxaloacetate (OAA) and a downstream intermediate, 2-oxoglutarate (α-KG). In turn, glutamate (Glut) is synthesized in astrocytes de novo from 2-oxoglutarate by transamination of aspartate via mitochondrial aspartate aminotransferase (AAT). The synthesized glutamate, once in the cytosol, can then be converted into glutamine (Gln) by glutamine synthetase (GS), or transported into vesicles via vesicular glutamate transporters, especially isoform 3 (VGLUT3). Drawing is not to scale.

Astroglial glutamine–glutamate and glutamine–GABA shuttle

Maintenance of glutamatergic transmission requires constant replenishment of glutamate, which in turn relies on astroglial glutamate uptake, glutamate conversion into glutamine and transport of the latter into neuronal presynaptic terminals. This co-ordinated system of fluxes of glutamate and glutamine is generally known as the glutamate–glutamine shuttle (Figure 2). This is an energy-dependent mechanism which requires hydrolysis of one molecule of ATP for a single conversion of glutamate into glutamine. Transport of glutamine from astrocytes and into neurons is mediated by amino acid transporters, which have a specific distribution between neurons and astrocytes [48]. Astrocytes are in possession of the system N transporters (represented by Na+/H+-dependent Na+-coupled neutral amino acid transporters SN1/SNAT3/SLC38A3 and SN2/SNAT5/SLC38A5 [49,50], which mediate glutamine efflux). Importantly, these transporters can be relatively easy switched to the reverse mode after, for example, an increase in cytosolic Na+ concentration. Neurons express another system for glutamine transport, mediated by the system A glutamine transporters [51] (the Na+-coupled neutral amino acid transporters ATA1/SNAT1/SLC38A1 and ATA2/SNAT2/SLC38A2) which act as influx transporters mediating glutamine accumulation into the neuronal compartment [52]. There are some exceptions, however; for example GABAergic neurons express N-transporter SNAT7 [53].

Astrocytes maintain glutamatergic and GABAergic transmissions through glutamate–glutamine and GABA–glutamine shuttles

Figure 2
Astrocytes maintain glutamatergic and GABAergic transmissions through glutamate–glutamine and GABA–glutamine shuttles

After entering the astrocyte, glutamate (Glu) is converted into glutamine (Gln), which is then transported back to the presynaptic terminals, where it is converted into glutamate in excitatory synapses and GABA in inhibitory synapses; these neurotransmitters are subsequently accumulated into synaptic vesicles.

Figure 2
Astrocytes maintain glutamatergic and GABAergic transmissions through glutamate–glutamine and GABA–glutamine shuttles

After entering the astrocyte, glutamate (Glu) is converted into glutamine (Gln), which is then transported back to the presynaptic terminals, where it is converted into glutamate in excitatory synapses and GABA in inhibitory synapses; these neurotransmitters are subsequently accumulated into synaptic vesicles.

In presynaptic terminals, glutamine is hydrolysed to glutamate; incidentally, this conversion, which is catalysed by phosphate-activated glutaminase, does not require energy. The newly synthesized glutamate is subsequently concentrated in synaptic vesicles bearing VGLUTs and thus releasable pool of glutamate is maintained. The supply of glutamate by astrocytes is co-ordinated with neuronal activity: an increase in external concentration of glutamate increases the release of glutamine [54], this being mediated through glutamate transporters [55] possibly through an increase in intracellular Na+ concentration.

Astroglia and GABAergic transmission

Astroglial glutamine is also critical for inhibitory transmission in the CNS mediated by GABA. Presynaptic terminals synthetize GABA from glutamate that arrives through the glutamate–glutamine shuttle; inhibition of the latter rapidly inhibits GABAergic transmission [56]; this mechanism is often also referred to as the GABA–glutamine shuttle or cycle (Figure 2). The cytosolic concentration of GABA in astroglial cells can be quite high, approaching ~2.5 mM [57]. Of note, astrocytes also express GABA transporters GAT-1 and GAT-3, which can be readily reversed upon moderate (~7 mM) increases in cytosolic Na+, thus making astrocytes the source of GABA [58].

Ammonia detoxification

In addition to its importance in neurotransmission, GS also plays a significant role in the assimilation of ammonia by the brain. Ammonia, a metabolite mainly produced within the gastrointestinal system (through protein degradation and amino acid deamination), is primarily regulated by the urea cycle, found exclusively in the liver. Ammonia-rich venous blood from the gastrointestinal tract first passes through the liver, maintaining the circulating concentration of ammonia between 35 and 50 μM. Ammonia, composed of gas (NH3) and ion (NH4+) components, can easily cross all plasma membranes through diffusion, channels and transporters [59]. In the setting of liver disease, blood ammonia levels can increase as high as 1 mM, leading to toxic concentrations in the brain and the onset of hepatic encephalopathy, a neuropsychiatric disorder involving cognitive alterations and motor impairments. Hyperammonaemia also arises in infants with inborn errors of urea cycle enzymes, causing seizures and coma. Surviving children have a high incidence of mental retardation and cerebral palsy. Toxic levels of ammonia trigger changes in both pH and membrane potential and have a profound effect on metabolism [59]. The brain is particularly susceptible to increased concentrations of ammonia and relies heavily on GS in astrocytes to prevent toxicity and neurological dysfunction. It has been demonstrated that an increase in brain ammonia elicits an increase in GS activity [60]; however, Cooper and Plum [61] showed that, under normal physiological conditions, GS activity in the brain operates at near maximal capacity, preventing any further induction. In either case, the observation of elevated concentrations of ammonia in liver disease reveals that the capacity of GS to detoxify ammonia in the brain is limited and therefore cannot compensate to maintain ammonia at physiological levels. Moreover, GS activity in brain has been shown to be inhibited in liver disease [62], since an increase in cerebral nitric oxide stimulates peroxynitrite-mediated nitration of tyrosine residues of GS, resulting in inactivation [63]. In turn, a decrease in capacity to clear ammonia in the brain detrimentally leads to sustained elevation in brain ammonia and thus persisting neurological dysfunction [64].

GS mutations in brain

First described by Häberle et al. [65], homozygous mutations in the GS gene (GLUL), as observed in two newborns, lead to a reduction in GS activity and, consequently, severe brain malformations, seizures, multiorgan failure and early death. Surviving infants develop chronic encephalopathy during the neonatal period. Furthermore, mice with prenatal excisions of the GLUL gene die during early embryonic development [66]. Consequently, in addition to causing alterations in glutamate/glutamine homoeostasis, deficiency in GS consequently impairs the detoxification of ammonia; concentrations of ammonia have been demonstrated to rise to between 100 and 200 μM [67]. These levels of ammonia are comparable with those found in liver disease associated with hepatic encephalopathy [68].

CNS disorders and alterations in glutamine synthetase

As GS is an essential part of a complex astrocyte–neuron signalling process, changes in expression and activity of GS will lead to neurological dysfunction. A number of different brain pathologies are associated with alterations in GS expression/activity (Figure 3). Patients diagnosed with medial temporal lobe epilepsy exhibit markedly reduced GS expression and activity in the hippocampus despite astroglial proliferation/reactive astrocytes [69,70].

Alterations in GS and neuropathologies

However, an up-regulation of GS has been demonstrated in the hippocampal dentate gyrus during seizure acquisition in the amygdala kindling model of epilepsy [71] with, however, no change in GS activity found in frontal cortex of epilepsy patients [72]. Overall, it appears that alterations in GS activity/expression are region-selective, depending on the kind of epilepsy. Similarly, a decrease in GS expression was found in the striatum of rats with Parkinsonian tremor [73]. Post-mortem brain tissue collected from Alzheimer's disease patients have revealed a reduction in GS activity [74,75]. Furthermore, it has been shown that the amount of GS protein in Alzheimer's disease patients’ brains is inversely correlated with the number of β-amyloid plaques [76] and that a decrease in GS is associated with the presence of β-amyloid deposits, as demonstrated in a mouse model of Alzheimer's disease [77]. Moreover, in the brains of patients with schizophrenia, an increase and decrease in GLUL mRNA has been found in specific regions as well [78]. Furthermore, there are several lines of evidence that support retinal GS being implicated in diabetic retinopathy. Down-regulation of GS has been demonstrated to occur during the early stages of diabetes [7981]. The susceptibility of certain brain structures (i.e. the CA1 area of the hippocampus) to hypoxic damage is associated with the disappearance of GS; this observation, however, has not been demonstrated in brain regions less susceptible to hypoxia [82]. Brain GLUL mRNA expression has also been found to be decreased in patients with depression [8385]. In addition, increased expression of GS through ischaemic post-conditioning has been shown to be neuroprotective in ischaemic rats [86]. Overall, changes in GS expression/activity are associated with neurodegenerative diseases and mental disorders (Figure 3).

GS expression in other brain cells?

In addition to GS being particularly localized in astrocytes, GS expression has been measured in other cells of the brain. During pathophysiological conditions, such as in patients with Alzheimer's disease, GS was found to be expressed in neurons, a result that was not replicated in post-mortem tissue from control brains. Furthermore, GS has also been demonstrated to be expressed in microglia of SIV (simian immunodeficiency virus)-infected macaques [87]. However, the role of GS, as well as the cause or effect of GS expression in other cells of the brain, remains unclear [88].

Conclusion

Proper astrocyte function is imperative for glutamatergic/GABA physiology. Alterations in GS, an enzyme found exclusively in astrocytes, have been shown to be associated with a number of neurological disorders. In addition to GS playing a vital role in glutamate homoeostasis, it also bears the brunt of ammonia detoxification in the brain. The precise neurotoxic effects following alterations in GS, whether due to glutamate or ammonia neurotoxicity, deserve to be investigated further.

5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders: A joint Biochemical Society/European Society for Neurochemistry Focused Meeting held at the University of Bath, U.K., 23–26 June 2013. Organized and Edited by Marcus Rattray (University of Bradford, U.K.) and Rob Williams (University of Bath, U.K.).

Abbreviations

     
  • CNS

    central nervous system

  •  
  • EAAT

    excitatory amino acid transporter

  •  
  • GABA

    γ-aminobutyric acid

  •  
  • GS

    glutamine synthetase

  •  
  • SLC

    solute carrier

  •  
  • TCA

    tricarboxylic acid

  •  
  • VGLUT

    vesicular glutamate transporter

Funding

The research was supported by the Canadian Institutes of Health Research (to C.F.R.) the Alzheimer's Research Trust (U.K.) [programme grant number ART/PG2004A/1 (to A.V.)] and by the National Science Foundation [grant number CBET 0943343 (to V.P.)].

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