Abstract TP272: Calcium Release-activated Calcium (CRAC) Channel Inhibition Protects Against Brain Injury and Neuronal Death Induced by Activated Microglia
Inflammatory responses following ischemia can worsen neurological outcome, and represent a potential target for therapeutic intervention. Store-operated Ca2+ entry (SOCE) mediated by CRAC channels contribute to calcium signaling in immune cells. CRAC channels consist of the endoplasmic reticulum resident Ca2+-sensing protein stromal interaction molecule 1 (STIM1) and the calcium channel protein ORAI1 located in the plasma membrane. Prolonged Ca2+ entry through CRAC channels activates, via calcineurin, nuclear factor of activated T cells (NFAT), involved in T cell proliferation and cytokine expression. Microglia mediate inflammation in the injured brain, but little is known about the role of CRAC channels in this process. We studied novel CRAC channel inhibitors to explore their therapeutic potential in microglia-mediated injury. A neuron cell line (Neuro-2A, N-2A) was cultured alone or with microglial BV2 cells then exposed to 2h oxygen glucose deprivation (OGD). Some cultures were treated with a novel CRAC channel inhibitor. Toll-like receptor (TLR) -3, -4 agonists or IFNγ were also used to activate microglia. Western blots revealed the presence of CRAC channel proteins STIM1 and ORAI1 in microglia. CRAC channel inhibition decreased NO release and inflammatory proteins iNOS and COX-2 expression in activated microglia, but did not affect STIM1 or ORAI1 expression. CRAC channel inhibitors also reduced agonist induced intracellular calcium accumulation in BV2 cells. Agonists also activated JNK1/2 kinase, NFAT, NF-κB, CREB & STAT1 in microglia, but only JNK1/2 kinase & NFAT were attenuated by inhibitor. OGD decreased N2A neuronal cell viability, further exacerbated by BV2 cells, but neuronal cells were protected by CRAC channel inhibition (n=5, *p<0.05). We then treated male C57/BL6 mice exposed to experimental brain trauma (TBI) and found that CRAC channel inhibition led to decreased lesion size, brain hemorrhage and improved neurological deficits (n=6-7/grp, *p<0.05). We suggest a novel anti-inflammatory approach for treating acute brain injury. Our observations also shed light on new calcium signaling pathways, not previously described in brain injury models.
Author Disclosures: R. Kacimi: None. J. Kim: None. K. Stauderman: Employment; Significant; Co-founder and Senior Vice President, Research, CalciMedica. M. Dunn: Employment; Significant; President; Senior Vice President, Corporate Development, CalciMedica. S. Hebbar: Employment; Significant; SVP Clinical Development. M.A. Yenari: Other Research Support; Significant; research support from CalciMedica.
- © 2017 by American Heart Association, Inc.