Mechanisms of Claudin-5 Regulation during Neuroinflammation - PROJECT SUMMARY The blood-brain barrier (BBB) is a vital defender of the central nervous system (CNS) that allows for nutrient exchange while blocking the entry of toxic factors or immune cells. However, its disruption is common in CNS disorders and can drive their disease progression. As such, our long-term goals are to identify the mechanisms governing BBB function and evaluate their therapeutic potential to preserve or restore BBB integrity and limit disease progression. The overarching objective of this proposal is to investigate the role of FoxO1, nmMLCK, and HDAC6 in BBB dysfunction and disease progression using animal and cell culture models of multiple sclerosis (MS), a chronic CNS disorder affecting an estimated one million adults in the US. During barrier maturation, the transcription factors FoxO1 and β-catenin are inactivated and removed from a silencer region in the Cldn5 promoter. However, we have shown that inflammation-induced BBB-dysfunction requires their concurrent re-activation and occupancy at the silencer region. This process involves nmMLCK. In a follow-up study, we defined an insulin receptor (IR)-Akt2-FoxO1 axis that can be targeted with an IR agonist to limit FoxO1 activation, claudin-5 loss, and barrier dysfunction in BBB-ECs and mice induced with experimental autoimmune encephalomyelitis (EAE), a model of MS. Here, we provide preliminary data showing the importance of nmMLCK and its regulation by histone deacetylase 6 (HDAC6) in barrier dysfunction. Global knockout mice are resilient to EAE, including paralysis, BBB dysfunction, and claudin-5 loss. In tandem, we explored mechanisms for nmMLCK activation and identified that it is robustly deacetylated in IL-1ꞵ-stimulated BBB-ECs. Using inhibitors, we found that tubastatin A attenuated barrier dysfunction, implying that HDAC6 is responsible. Thus, we hypothesize that FoxO1 and β-catenin are essential for mediating inflammation-induced claudin-5 repression, driving BBB dysfunction. To address this, we have developed a two-part approach that focuses on the relative contributions of FoxO1 deficiency in BBB-ECs (Aim 1) and HDAC6/nmMLCK/β-catenin signaling-dependent downregulation of claudin-5 (Aim 2) to BBB disruption and disease progression. The multi-faceted approach combines in vivo physiological analyses in EAE with complementary co-culture systems using BBB-ECs, astrocytes, pericytes, and myelin-reactive T cells. Multiple innovative models are proposed, including transgenic mice with inducible BBB-EC-specific knockout of FoxO1, nmMLCK, or HDAC6 and pharmacologic approaches to inhibit them; new molecular tools like TetOn overexpression systems for FoxO1 and HDAC6; and state-of-the-art histopathology techniques to detect and characterize perivenular inflammatory lesion formation which occurs around CNS microvessels in white matter lesions. In sum, the proposed research is significant, as data derived from these studies will provide new mechanistic insights into the pathophysiology of BBB dysfunction during inflammation, which has the potential to provide a basis for developing new therapeutic targets for CNS disorders that involve BBB dysfunction.
