Completing the glutamate - glutamine cycle: The role of Neurotransmitter Transporter 4 in presynaptic glutamate supply

Abstract

Recycling of glutamate following release from synapses is important for neuronal health and continued synaptic function. The glutamate glutamine cycle is the process by which astrocytes sequester released glutamate and return it to presynaptic terminals in the form of glutamine. Despite extensive research into the mechanisms of this cycle, some aspects are not fully understood. Debate surrounds the necessity of the cycle for replenishing synaptic glutamate, and how glutamine is transported into presynaptic terminals is unknown. Here, we investigate whether Neurotransmitter Transporter 4 (NTT4/SLC6A17) provides presynaptic terminals with glutamine. Accordingly, we generated Slc6a17 / (NTT4 KO) mice to assess this possibility. By tracing labelled glucose and acetate, we show that glutamate glutamine cycling is interrupted in NTT4 KO brain compared to control. To understand the mechanism behind this interruption, electrophysiological recordings were carried out in acutely isolated brain slices from NTT4 KO mice. Postsynaptic responses to presynaptic stimulation were measured in the hippocampus, a region shown to strongly express NTT4. The results show for the first time, that knockout of NTT4 causes a reduction in the amplitude of evoked postsynaptic currents in hippocampal CA3 cells and a reduction in field potentials in CA1 over time, when synaptic activity is increased. Pharmacological inhibition of NTT4 with leucine causes an identical reduction in these amplitudes, providing additional evidence for the role of NTT4 during periods of elevated synaptic activity. To ascertain whether these reductions were due to depletion of presynaptic neurotransmitter, quantal size was measured at CA3 pyramidal cells, where it was shown that the amplitude of spontaneous single vesicle associated postsynaptic currents were reduced in NTT4 KO cells after the application of high frequency stimulation. This confirmed that presynaptic terminals rely on NTT4 for replenishing presynaptic glutamate when activity at the synapse increases. Finally, the functional role of this deletion in learning and behaviour was assessed, with NTT4 KO mice displaying worse memory retention, more anxiety, social deficits, and impaired ability to perform tasks of daily living, compared to controls. This research helps to clarify the role of the glutamate glutamine cycle in recycling neurotransmitter at the synapse, and identifies the missing link in this cycle, the presynaptic glutamine transporter NTT4. As interrupted glutamatergic signalling is a hallmark in a number of pathologies including epilepsy, intellectual disability, Alzheimer's disease and many more, the discoveries outlined here provide a new avenue for the development of novel treatments. Show more