Functional identification of activity‐regulated, high‐affinity glutamine transport in hippocampal neurons inhibited by riluzole

Abstract

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 +/− 0.5µM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K+-stimulated MeAIB transport displays an affinity (Km) for MeAIB of 37 +/− 1.2 µM, saturates at ~200 µM, is dependent on extracellular Ca2+, and is blocked by inhibition of voltage gated Ca2+-channels (VGCCs). Spontaneous MeAIB transport is also dependent on extracellullar Ca2+ and VGCCs, but is also blocked by the Na+ channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca2+, but on Na+ ions, and is pH-sensitive. Activity-regulated, riluzole-sensitive spontaneous and K+-stimulated transport is minimal at 7–8 days in vitro (DIV), coordinantly induced during the next two weeks, and is maximally expressed by DIV>20; the known period for maturation of the Glu/Gln cycle and regulated presynaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity stimulated presynaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity.

Mitochondrial dysfunction increases fatty acid β‐oxidation and translates into impaired neuroblast maturation

“…Got2 catalyzes the same transamination in the mitochondria to fuel TCA cycle (oxidative pathway) [8,24,25]. Aspartate, glutamine, and glutamate directly sustain cell proliferation, being a source of nitrogen and carbon for nucleotide biosynthesis, but also cell differentiation, being important precursors for microtubule and synapse formation, as well as neurotransmitter biosynthesis [26,27]. Recent findings also prove that proper cell nutrients catabolism through TCA cycle and ETC are instrumental to sustain aspartate production, and hence cell growth [28][29][30][31][32].…”

Riluzole attenuates glutamatergic tone and cognitive decline in AβPP/PS1 mice

“…N = number of mice; C57BL/6J vehicle (n = 13), AβPP/PS1 vehicle (n = 12); AβPP/PS1 riluzole (n = 9) mice evidence supports enhanced astrocytic glutamine synthetase with riluzole treatment (Deng et al, 2009), which breaks down glutamate to glutamine and may help to stimulate glutamate uptake. Glutamine shuttling from astrocytes to neurons is also increased with riluzole treatment (Chowdhury et al, 2008), however, riluzole also potently inhibits the high-affinity glutamine transport system (Gln/ 2-(methylamino) isobutyrate) responsible for transporting glutamine into neurons (Erickson, 2017), effectively decreasing the glutamine pool in neurons available to produce glutamate. This is further confounded by an increase in glutamate metabolism with more glutamate entering into the tricarboxylic acid cycle (Chowdhury et al, 2008).…”

Hippocampal hyperglutamatergic signaling matters: Early targeting glutamate neurotransmission as a preventive strategy in Alzheimer's disease

“…Riluzole inhibits persistent and fast Na + currents, voltage-gated Ca 2+ and K + currents, repetitive firing, and neurotransmitter release, while it potentiates Ca 2+ -dependent K + current and Glu transporters, promotes neuronal survival and expression of growth factors (Figure 1). A high-affinity neuron-specific glutamine transport system inhibited by riluzole is described in developing and mature neuron-enriched hippocampal cultures (Erickson, 2017). Riluzole is approved by FDA for clinical use in patients with amyotrophic lateral sclerosis and is promising for spinal cord injury (both preclinical evidence, and clinical data are available) as a safe and well-tolerated neuroprotective treatment (Zhou et al, 2019).…”