Mice Lacking Brain/Kidney Phosphate-Activated Glutaminase Have Impaired Glutamatergic Synaptic Transmission, Altered Breathing, Disorganized Goal-Directed Behavior and Die Shortly after Birth

Glutamate is the principal excitatory neurotransmitter in the central nervous system (CNS). At birth, the first behavior essential to survival is respiration, and this is mediated by brain stem glutamatergic circuits. Later on in development, and continuing into adulthood, glutamatergic synaptic transmission is involved in every CNS circuit. Despite extensive investigations, the source of neurotransmitter glutamate has not been resolved. Because de novo synthesis from α-ketoglutarate is minimal in neurons (Hertz et al., 1999; Peng et al., 1993), metabolic studies have suggested that most neurotransmitter glutamate is recycled via the glutamine-glutamate neuron-astrocyte shuttle, involving brain/kidney phosphate-activated glutaminase type 1 (GLS1; EC 3.5.1.2). A review of studies ranging from physiology to biochemistry and magnetic resonance spectroscopy concluded that the majority (70%) of neurotransmitter glutamate is recycled by GLS1, with the remainder reflecting de novo synthesis (Hertz, 2004). The predominant means of inactivation of released glutamate is by rapid uptake into glia via transporters (Danbolt et al., 1998). Glutamine synthetase which is expressed exclusively in glia rapidly converts the glutamate to glutamine. Glutamine is then released from astrocytes via System N transporters and taken up by neurons via System A transporters (Chaudhry et al., 2002; Varoqui et al., 2000). This provides a major flux of glutamine into neurons, which convert glutamine into glutamate via the action of GLS1. The critical role of GLS1 has been supported further by pharmacological experiments in which a near complete depletion of neuronal glutamate was observed after inhibition of glutamine synthesis (Ottersen et al., 1992). Furthermore, direct inhibition of GLS1 by the suicide inhibitor 6-diazo-5-oxo-L-norleucine (DON) decreased the release of glutamate from cerebrocortical synaptosomes (Bradford et al., 1989), glutamate immunoreactivity in neurons (Conti and Minelli, 1994; Sulzer et al., 1998), and glutamate-dependent secretion of gonadotropin-releasing hormone from hypothalamic explants (Bourguignon et al., 1995). GLS1 was cloned in 1988 (Banner et al., 1988) and shown to be expressed with high baseline activity in brain (Beitz and Ecklund, 1988; Chatziioannou et al., 2003; Farb et al., 1992), but also in kidney, intestinal endothelium, fetal liver, lymphocytes, adipocytes and tumor cells. GLS1 was (other than glutamate itself) the only known marker for the glutamatergic status of neurons (Kaneko and Mizuno, 1988) till recently, when three families of vesicular glutamate transporters were identified, with strikingly different distributions in subpopulations of glutamatergic neurons (Bai et al., 2001; Bellocchio et al., 2000; Gras et al., 2002). Here we have asked how important GLS1 is for glutamatergic synaptic function by knocking out the GLS1 gene in mice. We inserted a STOP cassette ahead of the initiating ATG codon in the GLS1 gene on mouse chromosome 1. GLS1 null mice do not feed properly, show a respiratory deficit and die shortly after birth. Glutamate release from GLS1 null neurons is impaired, presumably accounting for the lethal phenotype, and revealing the relative importance of the pathway as the source of neurotransmitter glutamate.