Inhibition of system Xc-In microglia using phramacological inhibitors and RNA interference

  • Marion Dourte

Student thesis: Master typesMaster in Biomedecine, professional focus in preclinical research

Abstract

Background: System Xc- is composed of the xCT protein and the 4F2hc subunit. xCT protein, encoded by the Slc7a11 gene, is the specific subunit and has been described to be located on the plasma membrane of the CNS cells. Its main function is to import cystine intracellularly while exporting non-vesicular glutamate extracellularly, acting thus as an antiporter. Beside homeostatic functions, System Xc- seems to be involved in several pathophysiological processes such as inflammation, glutamate excitotoxicity and oxidative stress implicated in the development of neurological disorders. Cellular distribution of xCT protein is matter of debate and has been so far reported in microglia, neurons, astrocytes and meningeal fibroblasts. Aim: The aim of this master thesis was to characterize System Xc- expression and function in BV2 microglial-like cells in basal and stimulated conditions. The use of pharmacological inhibitors and RNA interference was also investigated. Methods: System Xc- was detected throughout the normal mouse spinal cord, brainstem and specifically in microglial cells using in situ hybridization, immunoblotting and RTqPCR. BV2 microglial-like cells were stimulated by LPS, H2O2 and L-glutamate. Pharmacological inhibitors (SAS and S-4-CPG) and targeted siRNA were applied on LPS-activated BV2 to assess their efficiency in modulating System Xc- function. Among the read-outs, we quantified their effect on glutamate and cystine transport, glutathione levels and cytokine expression profile. Results: xCT expression was significantly increased following LPS stimulation and to a less great extent, after H202 exposure. siRNA interference allowed to decrease xCT expression while the effect of pharmacological inhibitors on system expression was variable. Extracellular glutamate, intracellular cystine and glutathione concentrations were increased following LPS stimulation. SAS tends to restore unstimulated condition while S-4-CPG was effective to decrease glutamate and GSH concentration but ineffective in restoring homeostatic cystine levels. Finally, System Xc- inhibition produced a significant impact on the pro- inflammatory cytokines profile. On the contrary, the effect on the anti-inflammatory cytokines was minimal. Conclusion: Modulation of System Xc- expression should be done preferentially using RNA interference whereas the pharmacological inhibitors tested appear to demonstrate a significant effect on the xCT activity as reported in the literature. The use of these inhibitors and/or siRNA to modulate the cytokine expression profile within microglial cells will require the testing of a larger cytokines panel.
Date of Award18 Jan 2021
Original languageEnglish
Awarding Institution
  • University of Namur
SupervisorCharles Nicaise (Supervisor)

Keywords

  • System Xc-
  • microglia
  • cystine/glutamate antiporter
  • sulfasalazine
  • (S)-4- Carboxyphenyglycine

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