The Regulation of Astrocytic Glutamate Transporters in Health and Neurodegenerative Diseases

Abstract

The astrocytic glutamate transporters excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2) play a key role in nervous system function to maintain extracellular glutamate levels at low levels. In physiology, this is essential for the rapid uptake of synaptically released glutamate, maintaining the temporal fidelity of synaptic transmission. However, EAAT1/2 hypo-expression or hypo-function are implicated in several disorders, including epilepsy and neurodegenerative diseases, as well as being observed naturally with aging. This not only disrupts synaptic information transmission, but in extremis leads to extracellular glutamate accumulation and excitotoxicity. A key facet of EAAT1/2 expression in astrocytes is a requirement for signals from other brain cell types in order to maintain their expression. Recent evidence has shown a prominent role for contact-dependent neuron-to-astrocyte and/or endothelial cell-to-astrocyte Notch signalling for inducing and maintaining the expression of these astrocytic glutamate transporters. The relevance of this non-cell-autonomous dependence to age- and neurodegenerative disease-associated decline in astrocytic EAAT expression is discussed, plus the implications for disease progression and putative therapeutic strategies.

Keywords: Astrocyte; EAAT1; EAAT2; excitotoxicity; glutamate; neurodegenerative disease.

Figure 1

Glutamate-glutamine cycle. Glutamate (Glu⁻) released after excitatory transmission is collected by astrocytic EAAT transporters 1 and 2. The glutamate is then either converted into α-ketoglutarate (α-KG) via glutamate dehydrogenase (GDH) or transaminase reaction and enters the TCA cycle, or else is converted into glutamine (Gln) by glutamine synthetase (GS). Astrocytes excrete Gln back into the extracellular environment via the Na⁺ driven SNAT3 transporter, which is then taken up by an as yet unconfirmed neuronal Gln transporter. Neurons then convert Gln back to Glu⁻ via a phosphate-activated glutaminase (PAG) reaction to replenish their vesicular Glu⁻ stores.

Figure 2

The Notch signalling pathway. Notch is a contact dependent signalling pathway. When a Notch ligand contacts a Notch receptor this initiates a cleavage event through the enzyme γ-secretase, releasing the Notch intracellular domain (NICD). The NICD then translocates into the cell nucleus, where it pulls down various proteins, such as MAML, and associates with the Notch effector, CBF1. This association turns on transcription, with the Hes and Hey family of genes prominent examples of genes transcribed by this cascade. The γ-secretase inhibitor DAPT is able to prevent activation of the Notch signalling pathway as the NICD is unable to be cleaved.

Figure 3

Notch signalling is needed to maintain astrocytic EAAT function. Example traces of EAAT mediated currents in response to 200 μM L-Asp in (i) DIV13 neuronal cocultured (CC) astrocyte, (ii) DIV24 CC astrocyte, (iii) DIV24 monocultured (MC) astrocyte, (iv) DIV24 CC astrocyte +DAPT from DIV0, and (v) DIV24 astrocyte +DAPT from DIV14. Cells were voltage-clamped at −80 mV and all recordings were done in the presence of 100 μM AP-5. (vi) At DIV24 there was a significantly larger EAAT mediated response in control CC astrocytes compared to MC astrocytes (p = 0.009, LME ANOVE, df = 29), as well as CC astrocytes treated with DAPT from either DIV0 or DIV14 (p = 0.028 & 0.027, for DIV0 and DIV14 respectively, LME ANOVA, df = 29). There was no difference in EAAT response in CC astrocytes treated with DAPT from DIV0 or from DIV14 after currents had been established. Recordings were taken from cells across at least three independent culture batches. * p < 0.05, ** p < 0.001.