Abstract 20574: Selective Activation of the PI3K/Akt Pathway With the Insulin Mimetic Demethylasterriquinone B1 Reverses Neuroinflammatory-Mediated Blood-Brain Barrier Dysfunction in Mice

Blood-brain barrier (BBB) dysfunction is a hallmark of neuroinflammation brought on by pathologies such as stroke and amyloid angiopathy. Inflammatory cytokines that act directly on cerebral endothelium can impair tight junction (TJ) stability. We previously identified a mechanism for IL-1β-mediated dysfunction involving inactivation of the PI3K/Akt pathway, activation of the transcription factor FoxO1, and FoxO1-dependent transcriptional repression of the TJ gene claudin-5 (Cldn5). Here we used an insulin mimetic, Demethylasterriquinone B1 (DMAQ-B1), to test the hypotheses that targeted activation of the PI3K/Akt pathway: 1) enhances cerebral endothelial barrier function, 2) reverses IL-1β-mediated dysfunction and 3) attenuates neuroinflammation-induced BBB dysfunction. In Vitro: Primary cerebral microvascular endothelial cells (CMVECs) were grown in collagen-coated dishes, transwell inserts (0.4 μm pores), or ECIS Arrays (Applied Biophysics). Culture media was conditioned with vehicle control, IL-1β (100 ng/mL), and/or DMAQ-B1 (2.5-10 μM). DMAQ-B1 dose-dependently activated Akt (increased pT308), inactivated FoxO1 (decreased nuclear localization), increased Cldn5 expression (WB, ICC), increased transendothelial electrical resistance (TER), and decreased CMVEC monolayer permeability (Ps). Akt silencing (siRNA), PI3K inhibition (LY294002), or IL-1β treatment downregulated Cldn5 and induced CMVEC barrier dysfunction. DMAQ-B1 (5 μM) reversed IL-1β-mediated Akt inactivation, FoxO1 activation, Cldn5 downregulation and CMVEC barrier dysfunction. In Vivo: We used a murine model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE). Cerebral microvascular enriched fractions from EAE-induced mice had less pT308 Akt, increased FoxO1 nuclear accumulation, and decreased Cldn5 expression compared to control. Decreased Cldn5 expression and increased BBB hyperpermeability (sodium fluorescein extravasation) observed in EAE-induced mice was attenuated by 24 hour DMAQ-B1 (5 mg/kg; oral gavage) treatment. These results indicate that promoting PI3K/Akt pathway activation with DMAQ-B1 during neuroinflammatory pathologies may provide a novel therapeutic approach to attenuate BBB dysfunction.