Although many smokers attempt to quit each year, few are successful. The real world success rate for smoking abstinence at one year following a cessation attempt is an abysmal 4%. One of the reasons why smokers relapse is the cognitive impairment produced by nicotine withdrawal. In fact, this nicotine withdrawal symptom is predictive of relapse. Another phenomenon of chronic nicotine exposure is the up-regulation of ?2 nAChRs in brain. However, the role of this up-regulation in nicotine dependence is not clear. In animal models, nicotine withdrawal also produces cognitive impairment as well as up-regulation of ?2* nAChRs. Recent data have suggested that withdrawal-induced learning deficits in mice are dependent upon the up-regulation of ?2* nAChRs in the hippocampus. The mechanisms for nAChR up-regulation are not fully elucidated and hypotheses vary considerably and depend upon the in vitro system used for the mechanistic studies. Importantly, in brain, up-regulation is not uniform: it occurs in some brain regions but not others. Therefore, whatever the mechanism of up-regulation, it must be cell-type specific. It is known that modifications of serine residues in the large intracellular loop of the ?nAChR subunit are phosphorylated by PKA and PKC and modulate the expression and function of ?2 nAChR. Since phosphorylation can be cell-type specific, it is plausible that differential phosphorylation f the ?2* nAChR contributes to the cell-type specific up- regulation. Consistent with this possibility, at least one phosphorylation appears to be important for up- regulation of ?2 nAChRs in vitro. Our preliminary data also indicate that the kinase CDK5 alters ?2 nAChR expression and a putative CDK5 phosphorylation site in the ?subunit significantly affects function of ?2 nAChRs expressed in HEK293T cells. This site has not previously been implicated in ?2* expression or function. In this proposal, we will assess the role of the three putative serine phosphorylation sites in the intracellular loop of the ?nAChR-subunit for effects on cellular distribution, up-regulation, function and withdrawal-induced learning deficits. These experiments will completed by re-expressing wild type and phosphorylation site mutant ?subunits in primary cultured neurons derived from ?knockout mice (Specific Aim 1) and in the hippocampus of ?knockout mice (Specific Aim 2).

R21DA036673

2015-02-01

2017-01-31