Mechanisms of TNFa Enhancement of Nicotinic Receptor Upregulation

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ABSTRACT Nicotine imparts its effects on cellular function through interaction with neuronal nicotinic acetylcholine receptors (nAChR). Some of these receptors, especially those composed of a4b2 subunits, respond to sustained ligand exposure through the process of upregulation as defined by a substantial increase in the density of high affinity nicotine binding sites. The physiological consequences of upregulation have been linked to many diverse processes ranging from addiction to pathophysiological processes contributing to early phases of Alzheimers disease. An exciting and relatively new role for nAChRs is emerging with regard to the interaction with pro-inflammatory cytokines. In fact, the anti-inflammatory properties of nicotine, acting through various nAChRs, is now widely reported to influence many diseases of inflammatory etiology. However, relatively little is known about the mechanisms controlling the inflammatory:nAChR interaction. The overall goal of this proposal is to define intracellular mechanisms that regulate the interactions between key pro- inflammatory cytokines and defined combinations of nAChR subtypes. SPECIFIC AIM 1. Hypothesis: TNFa-activation of the p38MAPK pathway and/or IL-1b activation of PKA pathways enhance a4b2 expression and these are antagonized by pathways involving PI3K/Akt signaling. In this Aim we will pursue the definition of intracellular pathways initiated by TNFa and/or IL-1b to enhance nicotine-mediated upregulation of nAChRa4b2. SPECIFIC AIM 2. Hypothesis: Direct phosphorylation of a4 and the presence of other nAChR subunits modify the pro-inflammatory cytokine signals mediating enhanced upregulation of a4b2. This Aim will determine the impact of: 1) modifying PKA phosphorylation sites in a4; 2) introducing (or deleting) other nAChRs (specifically a7 or a5); and 3) determining effects of TNFa or IL-1b intracellular signaling leading to enhanced a4b2 upregulation in cultured neurons. SPECIFIC AIM 3. Hypothesis: Interactions between cells of the CNS such as neurons and microglia (Mg) direct the outcome of the overall inflammatory:nAChR response. Neurons and microglia will be co-cultured in different ratios to dissect the relative contribution of paracrine, autocrine or juxtacrine interactions in modulating the TNFa and IL-1b-mediated enhancement of a4b2-upregulation in a mixed cell system.