Project Summary Persistent pain states, arising from inflammatory conditions, such as in arthritis, diabetes, HIV, and chemotherapy among others, have an extraordinary negative impact on quality of life. A common feature of these initiating events is the release of damage-associated molecular pattern (DAMP) molecules, which can activate Toll-like receptor-4 (TLR4). Our previous studies suggest that TLR4 is critical in mediating the transition from acute to persistent pain. TLR4 as well as other inflammatory receptors localize to lipid raft microdomains on the plasma membrane. Lipid rafts, enriched with cholesterol and sphingomyelin, facilitate ligand-mediated receptor dimerization and downstream signaling. Removal of cholesterol from the plasma membrane reduces lipid rafts and often results in inhibition of receptor function. We have found that the secreted apoA-I binding protein (AIBP) accelerates cholesterol removal, disrupts lipid rafts, prevents TLR4 dimerization and inhibits microglia inflammatory responses to LPS. Furthermore, because AIBP binds to TLR4 and its affinity increases when TLR4 is activated by an agonist, we propose that AIBP targets cholesterol removal to lipids rafts harboring activated TLR4. In our recent work, we have also found that this mechanism is relevant to regulation of neuropathic pain states. Intrathecal injections of recombinant AIBP prevented LPS-induced tactile allodynia and, remarkably, reversed established cisplatin-induced allodynia. Based on these findings, we propose that targeted, AIBP- mediated disruption of lipid rafts and its effects upon TLR4 signaling can be a potential therapeutic strategy in treating neuropathic pain states. The Specific Aims of this proposal are: (1) to test the hypothesis that AIBP targets lipid rafts harboring activated TLR4; (2) to test the hypothesis that AIBP reduces glial activation and neuroinflammation in mouse models of neuropathic pain; and (3) to identify the origin and function of endogenous AIBP in the spinal cord. To test these hypothesis, we propose experiments, in vivo and in isolated glial cells, to elucidate the AIBP-TLR4 binding and lipid raft mechanism, and to characterize glial activation and neuroinflammation states. We will also make AIBP variants with mutated apoA-I or TLR4 binding interfaces to validate the proposed mechanism. Using our unique AIBP knockout and AIBP flox/flox mice, we will identify the role of endogenous AIBP in lipid raft regulation and neuroinflammation. In summary, our proposed experiments will elucidate the mechanisms by which AIBP reduces neuroinflammation and alleviates neuropathic pain. Our studies may also suggest that raising AIBP levels in the CNS may be a novel therapeutic approach to treat persistent pain states.

Project Narrative Neuropathic pain states affect millions of Americans and have an extraordinary negative impact on quality of life. We have identified the apoA-I binding protein (AIBP) as a regulator of cellular membrane organization and inflammatory receptor function, which reduces persistent pain in a mouse model. This project will elucidate the mechanisms of AIBP action and may lead to the development of new therapy for persistent pain states.