Proteomics of Altered Signaling in Nicotine Addiction

Overview: Over the last funding period we focused on identifying the high affinity nicotinic acetylcholine receptor (b2*-nAChR)-associated proteome, the phosphorylation sites on the a4 and b2 nAChR subunits, and the effects of nicotine on both. In pilot studies, we also started to evaluate sex differences in the proteome of mouse brain areas important for behaviors related to nicotine addiction, including the ventral tegmental area (VTA), nucleus accumbens (NAc) and the basolateral amygdala (BLA), as well as the effects of either locomotor stimulating or rewarding regimens of nicotine administration in vivo. These pilot studies have shown that we can use isobaric labeling with a TMT 10-plex kit (ThermoFisher), then high pH reverse phase peptide fractionation (5 fractions), followed by whole proteome analysis by LC-MS/MS to obtain highly reliable protein identifications that allow sex differences and nicotine-dependent differences in the VTA proteome to be identified with high reliability and sensitivity. Our proposal going forward will be to establish the baseline and nicotine-dependent proteome in male and female mice of 2 different genetic backgrounds with opposite locomotor responses to acute nicotine challenge (C57BL/6, decreased and C3H, increased) to identify baseline sex differences in the VTA, NAc and BLA that are robust across mouse strains and that differ depending on sensitivity to acute nicotine challenge. We will also evaluate the effects of chronic nicotine exposure, a regimen that leads to nicotine dependence, b2*-nAChR upregulation and locomotor activation in mice, or a subchronic regimen that leads to nicotine conditioned place preference, on the sex-dependent proteome in these structures.

Our preliminary data implicate pathways associated with dopamine signaling and protein kinase A (PKA) signaling in the VTA with nicotine-dependent effects in male, but not female, mice. A number of different pathways are emerging as different between male and female VTA. These analyses will be carried forward in the next funding period.