Neurotransmitter Homeostasis

NeuroMet

The glutamate-glutamine cycle

Contrary to common sense, glutamatergic neurons lack the quantitatively most important anaplerotic enzyme in the brain, namely pyruvate carboxylase (PC), making them incapable of de novo synthesis of neurotransmitter glutamate from glucose (Hertz et al. 1999;Patel 1974;Shank et al. 1985;Schousboe et al. 1997). Furthermore, there is ample evidence that most of the released neurotransmitter glutamate is lost to surrounding astrocytes (Danbolt 2001;Gegelashvili and Schousboe 1998;Rothstein et al. 1996;Schousboe 1981). As glutamate is formed from the tricarboxylic acid (TCA) cycle constituent a-ketoglutarate, this faces the glutamatergic neurons with the paradox of how to replenish the glutamate pool without draining their TCA cycle of intermediates. The solution to this paradox lies in the discovery of intercellular compartmentation of glutamine and glutamate pools, related to astrocytes and neurons, respectively, which led to the suggestion of a glutamate-glutamine cycle working between glutamatergic neurons and astrocytes (Berl and Clarke 1983;Ottersen et al. 1992;Benjamin and Quastel 1972;van den Berg and Garfinkel 1971). As depicted in Fig. 1, released neurotransmitter glutamate is taken up into surrounding astrocytes, transformed into glutamine by glutamine synthetase (GS) and released into the extracellular space from which it is taken up into neurons and transformed back to glutamate by phosphate-activated glutaminase (PAG). Notice in Fig. 1 that for each molecule of glutamate recycled, one molecule of ammonia will be produced in the neurons and one molecule of ammonia assimilated in the astrocytes. This ammonia will have to be translocated out of the neurons and back to the astrocytes for detoxification. This is because an elevated ammonia concentration has detrimental effects on a number of cellular functions, possibly including inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Lai and Cooper 1986;Zwingmann and Leibfritz 2003).

Several enzymes necessary for sustaining glutamate homeostasis are heterogeneously distributed among neurons and astrocytes. The glutamine synthesizing enzyme, GS, is selectively localized in astrocytes (Norenberg and Martinez-Hernandez 1979) and the enzyme transforming glutamine to glutamate, PAG, is preferentially expressed in neurons (Kvamme et al. 2001). However, low levels of PAG activity have been observed in cultured astrocytes as well (Kvamme et al. 1982;Schousboe et al. 1979). PAG has been suggested to be located on the outer surface of the inner mitochondrial membrane (Kvamme et al. 2001). This is, however, controversial (Zieminska et al. 2004). Finally, PC, the quantitatively most active anaplerotic enzyme in the brain (Patel 1974) is confined to the astrocytic compartment as shown by immunohistochemistry, cell culture studies and work on isolated non-synaptic and synaptic mitochondria (Faff-Michalak and Albrecht 1991;Shank et al. 1985;Yu et al. 1983). These are the factors that force neurons to rely on astrocytes for providing precursors for anaplerosis of their TCA cycle constituents.

It should be noted that a number of other substrates besides glutamine have been suggested to be transported from astrocytes to neurons. Citrate, malate, succinate and α-ketoglutarate are released to a higher extent from cultured astrocytes than from neurons (Sonnewald et al. 1991;Westergaard et al. 1994b;Westergaard et al. 1994a). α-Ketoglutarate, however not citrate, has been shown to act as precursor for neurotransmitter glutamate, although to a lesser extent than glutamine (Kihara and Kubo 1989;Peng et al. 1991;Shank et al. 1989;Westergaard et al. 1994b). Additionally, uptake of malate and incorporation of ¹⁴C label into glutamate from ¹⁴C-labeled malate is limited when compared to the need for glutamate synthesis (Hertz et al. 1992;Shank and Campbell 1984). Finally, and very essential for this discussion, the utilization of TCA cycle intermediates for glutamate synthesis requires an amino group donor which is not the case when glutamine acts as a glutamate precursor. The tentative conclusion from the above considerations is that none of the TCA cycle intermediates are likely candidates for intercellular trafficking between astrocytes and neurons as neurotransmitter glutamate precursors.

Shuttling of ammonia between neurons and astrocytes

As noted in the previous section, ammonia generated in the neuronal PAG reaction during neurotransmission activity must somehow be transferred back to the astrocytic compartment. Basically, this might take place by two different means; ammonia itself may simply diffuse (as NH₃) or be transported (as ammonium ion; NH₄⁺) across the cell membranes. Alternatively, a shuttle system involving carrier molecules might be operating. Certainly, ammonia can diffuse across lipid membranes and it has been shown that ammonium can be transported by K⁺/Cl^- co-transporters, as reviewed by Marcaggi and Coles (2001). In addition, ammonium might diffuse through aquaporins (Jahn et al. 2004;Nakhoul et al. 2001). Aquaporins 1, 4 and 9 have been identified in brain cells (Badaut et al. 2001;Nielsen et al. 1997) and aquaporin 4 in cultured astrocytes is up-regulated as a result of ammonia treatment (Rama Rao et al. 2003).

Ammonia might be transferred between neurons and astrocytes by passive and active diffusion and may afford a method for regulating astrocyte metabolism in response to neuronal activation, as suggested by Provent et al. (2007). However, for the following reasons an apparently attractive way of transporting ammonia between neuronal and astrocytic compartments is via an amino acid shuttle: i) As mentioned above, an increase in ammonia levels in the neuronal compartment might have detrimental effects on metabolism; ii) diffusion and facilitated transport of free ammonia across the cell membrane necessitates a pH regulatory response from the cell, as discussed by Marcaggi and Coles (2001); iii) intuitively, employing an amino acid shuttle seems to be a more efficient and controlled way of transferring nitrogen units compared to simple/facilitated diffusion. By way of an amino acid shuttle, ammonia generated in neurons is incorporated into a non-neuroactive amino acid and carried across the extracellular space. Two such amino acid shuttles have so far been proposed to be operating between glutamatergic neurons and astrocytes.

The first one to be suggested is based on the branched-chain amino acids as the amino acids translocated from neurons to astrocytes (Bixel et al. 2001;Bixel et al. 1997;Hutson et al. 1998;Lieth et al. 2001;Yudkoff et al. 1996a;Yudkoff et al. 1996b). This shuttle, as outlined in Fig. 2 was developed on the basis of several observations, among others the distribution of isoforms of the branched-chain amino acid aminotransferase (BCAT) with the cytosolic variant being present in neurons and the mitochondrial in astrocytes (Bixel et al. 1997;Bixel et al. 2001). Furthermore, branched-chain amino acids (BCAAs), not ammonia, were found to be amino group donors for glutamate biosynthesis in astrocytes (Gamberino et al. 1997;Hutson et al. 1998). Several interpretations and roles of this shuttle are possible, including the mentioned role for providing the amino group for glutamate biosynthesis (e.g. as argued by Lieth et al. 2001); however, here the shuttle will be regarded and investigated solely as a mean of shuttling ammonia between neurons and astrocytes to account for the imbalance imposed by the glutamate-glutamine cycle. In the glutamate/glutamine-BCAA shuttle, the ammonia released in the neuronal PAG reaction is fixated by glutamate dehydrogenase (GDH) and transaminated by BCATc into an BCAA which is released and taken up into astrocytes where the process is reversed, the released ammonia and branched-chain keto acid (BCKA) being used by the GS reaction and cycled back to the neurons, respectively. In this way the ammonia is cycled between the neuronal and the astrocytic compartments.

Another shuttle is based on alanine (Waagepetersen et al. 2000;Zwingmann et al. 2000) as the amino acid translocated in the opposite direction of glutamine, i.e. from neurons to astrocytes (see Fig. 3). In this shuttle, the molecule translocated in the opposite direction of alanine is proposed to be lactate, rather than the corresponding keto acid of alanine, namely pyruvate. The sequence of events for the glutamate/glutamine-lactate/alanine shuttle is similar to the above, except that the transamination between the glutamate and the keto acid (pyruvate) is catalyzed by alanine aminotransferase (ALAT). The primary observations leading to the description of this shuttle, was that cultured cerebellar neurons both contained and released more alanine than did cultured cerebellar astrocytes during a 4 hr incubation period plus that incubating cultured neurons in the presence of [5-¹⁵N]glutamine resulted in a modest labeling (3.3%) of extracellular alanine (Waagepetersen et al. 2000). This labeled alanine was proposed to originate from a small, but highly labeled intracellular pool of alanine in close contact with a glutamate pool derived from neuronal GDH activity (Fig. 3, left part). Indeed, this is one of the weak spots of both shuttles, as it is difficult to show that the GDH reaction works in the direction of reductive amination during normal physiological conditions. This is probably because of the high Km for binding of ammonia (10-20 mM; (Chee et al. 1979;Colon et al. 1986;Zaganas et al. 2001). However, neuronal PAG activity may produce a sufficient level of ammonia in a micro-environment close to the relevant GDH reaction, providing the necessary driving force for the enzyme to catalyze reductive amination (Waagepetersen et al. 2000). Finally, inherent in both hypotheses is the fact that there should be an activity-dependent coupling between glutamate-glutamine cycle activity and activity in the ammonia-carrying amino acid shuttles.

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