Mechanisms of TNFa Enhancement of Nicotinic Receptor Upregulation

描述（由申请人提供）：尼古丁通过与神经元尼古丁乙酰胆碱受体（nAChR）的相互作用对细胞功能产生影响。其中一些受体，尤其是由？4？2个亚基，对持续的配体暴露作出反应，通过高亲和力尼古丁结合位点密度的显著增加来定义上调过程。上调的生理后果与许多不同的过程有关，从成瘾到导致阿尔茨海默病早期阶段的病理生理过程。在与促炎细胞因子的相互作用方面，nachr的一个令人兴奋和相对较新的作用正在出现。事实上，尼古丁的抗炎特性，通过各种nachr起作用，现在被广泛报道影响许多炎症性病因疾病。然而，对控制炎症：nAChR相互作用的机制知之甚少。本提案的总体目标是确定调节关键促炎细胞因子之间相互作用的细胞内机制和确定的nAChR亚型组合。具体目标1。假设:肿瘤坏死因子?-激活p38MAPK通路和/或IL-1？PKA通路激活增强？4？2的表达，这些都被PI3K/Akt信号通路拮抗。在这个目标中，我们将探讨由TNF？和/或il - 1 ?增强尼古丁介导的nAChR的上调。具体目标2。假设：直接磷酸化？4和其他nAChR亚基的存在改变了促炎细胞因子信号，介导了4 2的增强上调。该目的将确定以下影响：1)修改PKA的磷酸化位点；2)引入（或删除）其他nachr(具体来说？7或？5)；3)确定TNF？或il - 1 ?细胞内信号传导导致？4？2 .培养神经元的上调。具体目标3。假设：中枢神经系统细胞如神经元和小胶质细胞（Mg）之间的相互作用指导了整体炎症：nAChR反应的结果。神经元和小胶质细胞将以不同比例共培养，以解剖旁分泌、自分泌或近分泌相互作用在调节TNF？和il - 1 ?-介导的？4？混合细胞系统中的2-上调。

DESCRIPTION (provided by applicant): Nicotine imparts its effects on cellular function through interaction with neuronal nicotinic acetylcholine receptors (nAChR). Some of these receptors, especially those composed of ?4?2 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 Alzheimer's 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: TNF?-activation of the p38MAPK pathway and/or IL-1? activation of PKA pathways enhance ?4?2 expression and these are antagonized by pathways involving PI3K/Akt signaling. In this Aim we will pursue the definition of intracellular pathways initiated by TNF? and/or IL-1? to enhance nicotine-mediated upregulation of nAChR??4?2. SPECIFIC AIM 2. Hypothesis: Direct phosphorylation of ?4 and the presence of other nAChR subunits modify the pro-inflammatory cytokine signals mediating enhanced upregulation of ?4?2. This Aim will determine the impact of: 1) modifying PKA phosphorylation sites in ?4; 2) introducing (or deleting) other nAChRs (specifically ?7 or ?5); and 3) determining effects of TNF? or IL-1? intracellular signaling leading to enhanced ?4?2 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 TNF? and IL-1?-mediated enhancement of ?4?2-upregulation in a mixed cell system. PUBLIC HEALTH RELEVANCE: We are interested in understanding the interaction between nicotine and inflammation. We will determine the signal transduction pathways cytokines use to promote or inhibit enhanced upregulation of nicotinic receptors. Two major cytokines, tumor necrosis factor alpha (TNF?) and interleukin-1? (IL-1?) enhance nicotine-induced upregulation of the high affinity nicotine receptor termed nAChR?4?2. The upregulation response is correlated with addiction behaviors and multiple pathologies of the brain such as Alzheimer's disease. Understanding how the inflammatory system and response to nicotine interact will impact upon how we approach these problems with therapeutic agents.