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Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs
- Source :
- Biochemistry and Biophysics Reports, Vol 9, Iss C, Pp 266-272 (2017), Biochemistry and Biophysics Reports
- Publication Year :
- 2017
- Publisher :
- Elsevier, 2017.
-
Abstract
- The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System xC- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC50 values in agreement with previous studies using established cell lines.<br />Highlights • Conditions to screen for SXC inhibitors were optimized in 96-well format. • Assay enables higher throughput screens for SXC inhibitors with primary microglia. • Screening assay was used to evaluate prototype SXC inhibitors and erastin analogs.
- Subjects :
- 0301 basic medicine
CCF-STTG-1 cells
Biophysics
Cystine
Excitotoxicity
Pharmacology
(S)-4-CPG
medicine.disease_cause
Biochemistry
Proinflammatory cytokine
lcsh:Biochemistry
03 medical and health sciences
chemistry.chemical_compound
Primary microglia
medicine
lcsh:QD415-436
System xc
lcsh:QH301-705.5
Neuroinflammation
chemistry.chemical_classification
Reactive oxygen species
Microglia
Chemistry
Erastin
Glutamate receptor
3. Good health
Sulfasalazine
030104 developmental biology
medicine.anatomical_structure
lcsh:Biology (General)
System X
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 24055808
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- Biochemistry and Biophysics Reports
- Accession number :
- edsair.doi.dedup.....4f6f042f45b0550b9112cda01d673321