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. 2014 Jul;19(7):801-10.
doi: 10.1038/mp.2013.104. Epub 2013 Sep 3.

Evidence From Mouse and Man for a Role of Neuregulin 3 in Nicotine Dependence

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Free PMC article

Evidence From Mouse and Man for a Role of Neuregulin 3 in Nicotine Dependence

J R Turner et al. Mol Psychiatry. .
Free PMC article

Abstract

Addiction to nicotine and the ability to quit smoking are influenced by genetic factors. We used functional genomic approaches (chromatin immunoprecipitation (ChIP) and whole-genome sequencing) to identify cAMP response element-binding protein (CREB) targets following chronic nicotine administration and withdrawal (WD) in rodents. We found that chronic nicotine and WD differentially modulate CREB binding to the gene for neuregulin 3 (NRG3). Quantitative analysis of saline, nicotine and nicotine WD in two biological replicates corroborate this finding, with NRG3 increases in both mRNA and protein following WD from chronic nicotine treatment. To translate these data for human relevance, single-nucleotide polymorphisms (SNPs) across NRG3 were examined for association with prospective smoking cessation among smokers of European ancestry treated with transdermal nicotine in two independent cohorts. Individual SNP and haplotype analysis support the association of NRG3 SNPs and smoking cessation success. NRG3 is a neural-enriched member of the epidermal growth factor family, and a specific ligand for the receptor tyrosine kinase ErbB4, which is also upregulated following nicotine treatment and WD. Mice with significantly reduced levels of NRG3 or pharmacological inhibition of ErbB4 show similar reductions in anxiety following nicotine WD compared with control animals, suggesting a role for NRG3 in nicotine dependence. Although the function of the SNP in NRG3 in humans is not known, these data suggest that Nrg3/ErbB4 signaling may be an important factor in nicotine dependence.

Conflict of interest statement

Conflict of Interest: The authors have no conflict of interest in relation to the work described.

Figures

Figure 1
Figure 1. Chronic treatment with nicotine and/or 24hWD significantly increases phosphorylation of CREB in the hippocampus and increases CREB binding to the promoter for Nrg3
Animals were treated in vivo with either saline, nicotine (18 mg/kg/day), or undergoing 24h withdrawal (WD) from nicotine. Hippocampal tissues from treated animals were used in A) Western blot analysis to evaluate phosphorylation levels of CREB as well as in B) ChIP analysis to evaluate binding to the promoter for Nrg3. A) Both chronic nicotine as well as 24h WD from chronic nicotine elicited significant increases in pCREB relative to saline in wildtype (WT) animals. No change was observed in CREB mutant mice, which exhibit a 90% reduction in total CREB levels (N=4–5). Representative blots are shown below panel A. B) CREB binding to Nrg3 was significantly increased following chronic treatment with nicotine (18 mg/kg/day) (N=6–7). *, p<0.05; **, p<0.01.
Figure 2
Figure 2. Chronic treatment with nicotine and/or 24hWD significantly increases neuregulin 3, but not neuregulin 1, in the hippocampus
Animals were treated in vivo with either saline, nicotine (18 mg/kg/day), or undergoing 24h withdrawal (WD) from nicotine. Hippocampal tissues from treated animals were used in qPCR or Western blot experiments to evaluate mRNA or protein levels. As shown, chronic treatment with nicotine or 24hWD does not alter neuregulin 1 (NRG1) (A) mRNA or (D) protein levels in the hippocampus. In contrast, mRNA (B) and protein (E) levels of ErbB4 are increased following chronic treatment with nicotine and/or during 24hWD. Additionally, mRNA levels of the CREB target neuregulin 3 are increased following chronic nicotine treatment and 24hWD (C). Furthermore, protein levels of NRG3 (F) are increased following 24hWD in wildtype (WT), but not CREB mutant, mice. Representative blots are shown below each panel. *, p<0.05; **, p<0.01. A–C: N=6–11; D–F: 6–10.
Figure 3
Figure 3. Smoking cessation with transdermal nicotine, by study and NRG3 rs1896506 genotype. A)
Eight-week quit rate was significantly lower in A/* carriers of the rs1896506 genotype in both the discovery cohort and the replication cohort. B) Six-month quit rate in the pooled sample of participants from both cohorts who received standard 8-week duration therapy was significantly lower in A/* carriers of the rs1896506 genotype. The number of subjects in each group is indicated above its respective bar.
Figure 4
Figure 4. Chronic treatment with nicotine and withdrawal results in differential biochemical responses in the F1 wildtype and NRG3ska mice
F1 hybrid and NRG3ska mice were treated in vivo with either saline, nicotine (18 mg/kg/day), or undergoing 24h withdrawal (WD) from nicotine. A) Hippocampal tissues from saline treated animals were used in Western blot analysis to evaluate basal levels of NRG3 in the two strains of mice. NRG3ska mice demonstrated a signficant reduction in NRG3 levels compared to F1 hybrids (N=8). Representative blots are shown below. B) Hippocampal tissues from NRG3ska mice treated with saline, nicotine, or 24h WD were used in Western blot analysis to evaluate treatment changes in NRG3 levels. NRG3ska mice did not show an induction of NRG3 following chronic nicotine or 24h WD (N=8). Representative blots are shown below. C) Hippocampal tissues from F1 hybrid mice treated with saline, nicotine, or 24h WD were used for homogenate-binding experiments with a saturating concentration of [3H]epibatidine ([3H]EB, 1.5 nM). F1 hybrid mice chronically treated with either nicotine or 24h WD displayed an increased density of nAChRs compared to saline controls (N=4). D) Hippocampal tissues from NRG3ska mice treated with saline, nicotine, or 24h WD were used for homogenate-binding experiments with a saturating concentration of [3H]epibatidine ([3H]EB, 1.5 nM). NRG3ska mice chronically treated with either nicotine or 24h WD displayed an increased density of nAChRs compared to saline controls (N=4). *, p<0.05; **, p<0.01; ***, p<0.005.
Figure 5
Figure 5. Chronic treatment with nicotine and withdrawal results in differential behavioral responses in the F1 wildtype and NRG3ska mice
NRG3ska mice were treated in vivo with either saline, nicotine (18 mg/kg/day), or undergoing 24h withdrawal (WD) from nicotine. F1 hybrid mice were treated in vivo with either saline, nicotine (18 mg/kg/day), or undergoing 24h withdrawal (WD) from nicotine; additionally, these mice also received daily injections of either vehicle or afatinib at the concentrations indicated for 10 days. A) NRG3ska mice treated with saline, nicotine, or 24h WD were tested in the NIH test. Mice chronically treated with either nicotine or 24h WD displayed a reduced latency to feed on novel test day compared to saline controls (N=16). B) NRG3ska mice treated with saline, nicotine, or 24h WD were tested in the marble-burying test. Mice chronically treated with either nicotine or 24h WD buried fewer marbles compared to saline controls (N=10). C) Locomotor activity was tested in NRG3ska mice treated with saline, nicotine, or 24h WD. No treatment effects were observed (N=4–6). D) F1 hybrid mice treated with saline, nicotine, 24h WD, or 24h WD + Afatinib were tested in the NIH test. Mice chronically treated with either nicotine or 24h WD + Afatinib displayed a reduced latency to feed on novel test day compared to the saline group or the 24hWD group (N=6–7). E) F1 hybrid mice treated with saline, nicotine, 24h WD, or 24h WD + Afatinib were tested in the marble-burying test. Mice chronically treated with either nicotine or 24h WD + 20mg/kg Afatinib buried fewer marbles compared to saline controls (N=5–7). F) Locomotor activity was tested in treated F1 Hybrid mice. No treatment effects were observed (N=4–6). Significant compared to vehicle control - **, p<0.01; ***, p<0.005. Significant compared to 24hWD - #, p<0.05; ###, p<0.005.

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