Calcineurin-Mediated Immunosuppressants in Binge Ethanol Drinking and Stress Responsivity — 93p — Caroline Titze¹, Emily Dorn², Jennifer Agar², Liam Porter², Brock Goeden², Thomas P. Beresford³, P.J. Ronan²

1. University of Sioux Falls, Sioux Falls, SD, 2. Sioux Falls VA/USD Sch. of Med., Sioux Falls, SD; 3. RMRVAMC-SOM U Colorado, Denver, CO

We have found that the calcineurin mediated immunosuppressants cyclosporine A (CsA) and tacrolimus inhibit binge alcohol drinking in mice. Further, we have shown that this effect is mediated directly in brain, as intracerebroventricular administration also significantly decreases drinking. As these immunosuppressants have severe systemic toxic effects along with dangerous inhibition of immune function, our goal is to determine proximal mechanisms by which these immunosuppressants are working in order to develop effective treatments for alcohol use disorder (AUD) with fewer side effects. To this end, we are employing genomic, molecular, transcriptomic, metabolomic, anatomic, and behavioral approaches to explore the relationship between binge alcohol drinking, stress, and calcineurin mediated immunosuppressants. Calcineurin is a somewhat ubiquitous phosphatase, involved in a wide range of signaling pathways – both in neurons and glia. One major question is whether immunosuppressants are acting through neuronal signaling pathways, regulating reward and stress/anxiety pathways, or in glia, mediating neuroinflammatory effects.  To address this, we have developed multiple transgenic models using a floxed calcineurin line (C57BL/6-Ppp3r1tm1Stl/J) crossed with various Cre driver lines to knockout CN in various neuronal or glial populations.  Results will be presented from two neuronal CN knockout lines: a “panneuronal” CN knockout line (CamKIIα-Cre) and a corticotropin releasing factor specific CN knockout line (CRH-Cre).  We are also investigating the effects of CsA on candidate brain signaling pathways in models of both binge drinking and stress, as stress is a primary factor driving drinking behaviors.  The CeA and PVN were microdissected from 300 μm frozen sections and qRT-PCR was performed.  Overall, CsA inhibited the stress-induced expression of a wide range of neuroinflammatory markers in these regions including cytokines such as IL-2, IL-1β, IL-6, TNFα; markers of glial activation: CD45 and Iba-1; chemokine and chemoattractant molecules such as CCR2 and CCL2; as well as other inflammatory signaling molecules such as COX-2.  Some of the largest effects were seen on IL-1β and IL-6 expression in both CeA and PVN. While CsA inhibited the expression of CD45 and Iba-1 in the CeA, in the PVN these effects were striking.  Together, these data suggest that immunosuppressants are acting through glial mediated neuroinflammatory mechanisms to reduce binge-like alcohol consumption in mice.

University of Sioux Falls
Dr. Patrick Ronan