Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function

Abstract

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13 C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13 C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.

Divergent roles of astrocytic versus neuronal EAAT2 deficiency on cognition and overlap with aging and Alzheimer’s molecular signatures

“…Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and oxidized nicotinamide-adenine dinucleotide (NAD), the primary end products of the kynurenine pathway, are also essential for mitochondrial function (83). EAAT2mediated glutamate uptake in axon terminals in neurons has recently been shown to provide glutamate for mitochondrial energy metabolism (84). The sirtuin signaling pathway, a major regulator of mitochondrial function, is significantly positively correlated with cognition in syn-Cre + EAAT2 −/− mice.…”

Entering a new era of quantifying glutamate clearance in health and disease

“…Glutamate uptake into astrocytes can also be metabolized for use in the tricarboxylic acid cycle. Synaptically released glutamate can also be taken up directly by nerve terminals through EAAT2 (depicted by b), where it could potentially be used for subsequent release or for local energy metabolism (Danbolt et al., 2016; McNair et al., 2019). Synaptically released glutamate may also be taken up directly into postsynaptic dendrites through EAAC3 (depicted by c), although evidence suggests that EAAT3 may primarily buffer glutamate, thereby limiting extrasynaptic receptor activation and delaying glutamate uptake by astrocytic EAAT1 and EAAT2 (Scimemi et al., 2009).…”

“…Presynaptically localized EAAT2 has been detected in the hippocampus (Chen et al., 2004; Furness et al., 2008), cortex (Melone et al., 2009), and striatum (Petr et al., 2013), and provides a putative means through which the CNS could bypass the canonical glutamate–glutamine cycle (Hertz, 2013) and directly recover glutamate into axon terminals following neural activity (Zhou et al, 2019). In addition, presynaptic EAAT2 can act as a source of glutamate for energy metabolism and biosynthesis in excitatory presynaptic terminals (McNair et al., 2019). EAAT2, particularly astrocytic EAAT2, is believed to be responsible for the large majority of glutamate clearance in the brain (Danbolt, 2001; Otis & Kavanaugh, 2000; Vandenberg & Ryan, 2013), although additional transporters can indeed make substantial contributions to overall uptake rates (Pinky et al., 2018; Romanos et al., 2019).…”

Regulation of Glutamate, GABA and Dopamine Transporter Uptake, Surface Mobility and Expression

“…In neurons, EAAT2 co-localizes with the α 1 and α 3 isoforms of the Na + /K + -ATPase, but this co-localization is looser than that with the α 2 isoform in astrocytes, suggesting a less efficient interaction between EAAT2 and the Na + /K + -ATPase in neurons ( Melone et al, 2019 ). Neuronal EAAT2 appears to be required to provide glutamate to synaptic mitochondria, and is therefore linked to energy metabolism ( Petr et al, 2015 ; Fischer et al, 2018 ; McNair et al, 2019 , 2020 ; Sharma et al, 2019 ). By contrast, astrocytic EAAT2 is crucial to ensure survival, resistance to epilepsy, and prevent cognitive decline.…”

“…Loss of astrocytic EAAT2 leads to early deficits, whereas loss of neuronal EAAT2 leads to late-onset deficits in long-term memory and spatial reference learning ( Sharma et al, 2019 ). These findings are important because they identify neuronal and astrocytic EAAT2 as contributing to different aspects of cognitive function and, potentially, as different therapeutic targets in cognitive decline ( Petr et al, 2015 ; Fischer et al, 2018 ; McNair et al, 2019 , 2020 ; Sharma et al, 2019 ).…”