Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Pauline Beckers,Ines Belo Do Nascimento,Nathalie Desmet,Emmanuel Hermans,Olaya Lara,Ann Massie

doi:10.3389/fncel.2021.815771

Pauline Beckers, Ines Belo Do Nascimento + Show 4 more

Open Access

https://doi.org/10.3389/fncel.2021.815771

Copy DOI

Abstract

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

Highlights

Proposed in the early fifties, the role of glutamate as a neurotransmitter was demonstrated 20 years later
Astrocytes are best known for their capacity to take up excitatory amino acids through diverse excitatory amino acid transporters (EAATs) which recognize both glutamate and aspartate whereas system xc− only achieves the transport of glutamate
Regarding the influence of these inhibitors on the transport of [3H]-L-glutamate, data presented in Figure 1C showed that LTHA considerably reduced the uptake in the Na+-containing buffer whereas only HCA was inhibiting the uptake in the Na+-free buffer (Figure 1D). These data confirm that astrocytes achieve glutamate uptake through both system xc− and EAATs

Summary

Introduction

Proposed in the early fifties, the role of glutamate as a neurotransmitter was demonstrated 20 years later. Glutamate participates in the formation of glutathione (GSH), the most abundant antioxidant in the CNS (Had-Aissouni, 2012) and may serve as an alternative source of energy for diverse cells of the nervous system (McKenna, 2013). Besides these roles in physiological activities in the CNS, glutamate is known for its implication in a large variety of nervous disorders (Lewerenz and Maher, 2015). High concentrations of this excitatory transmitter can cause damage to neuronal cells, a process known as excitotoxicity (Dong et al, 2009)

Methods

Results

Conclusion