ACSL6 is associated with the number of cigarettes smoked and its expression is altered by chronic nicotine exposure

Abstract

Individuals with schizophrenia tend to be heavy smokers and are at high risk for tobacco dependence. However, the nature of the comorbidity is not entirely clear. We previously reported evidence for association of schizophrenia with SNPs and SNP haplotypes in a region of chromosome 5q containing the SPEC2, PDZ-GEF2 and ACSL6 genes. In this current study, analysis of the control subjects of the Molecular Genetics of Schizophrenia (MGS) sample showed similar pattern of association with number of cigarettes smoked per day (numCIG) for the same region. To further test if this locus is associated with tobacco smoking as measured by numCIG and FTND, we conducted replication and meta-analysis in 12 independent samples (n>16,000) for two markers in ACSL6 reported in our previous schizophrenia study. In the meta-analysis of the replication samples, we found that rs667437 and rs477084 were significantly associated with numCIG (p = 0.00038 and 0.00136 respectively) but not with FTND scores. We then used in vitro and in vivo techniques to test if nicotine exposure influences the expression of ACSL6 in brain. Primary cortical culture studies showed that chronic (5-day) exposure to nicotine stimulated ACSL6 mRNA expression. Fourteen days of nicotine administration via osmotic mini pump also increased ACSL6 protein levels in the prefrontal cortex and hippocampus of mice. These increases were suppressed by injection of the nicotinic receptor antagonist mecamylamine, suggesting that elevated expression of ACSL6 requires nicotinic receptor activation. These findings suggest that variations in the ACSL6 gene may contribute to the quantity of cigarettes smoked. The independent associations of this locus with schizophrenia and with numCIG in non-schizophrenic subjects suggest that this locus may be a common liability to both conditions.

Figure 1. Association analyses of the MGS_EA controls for the SPEC2, PDZ-GEF2, FNIP1 and ACSL6 region.

Of the 145 markers tested in this interval, 68 and 12 markers respectively reached nominal significance for numCIG and FTND. The two markers selected for replication were highlighted as red.

Figure 2. Meta-analyses of rs667437 and rs477086 for numCIG and FTND.

A) rs667437, numCIG; B) rs477086, numCIG; C) rs667437, FTND; and D) rs477086, FTND.

Figure 3. ACSL6 mRNA expression in rat cortical primary culture after nicotine treatment.

The expression levels of ACSL6 mRNA were significantly increased by 5 days of nicotine exposure for both doses. *p<0.01 compared to saline controls; n = 3. mRNAs were measured by real-time-PCR, and were presented as normalized ΔCt values. Bars represent ± SD.

Figure 4. Western blot analyses of ACSL6 protein in mouse brain regions after chronic nicotine exposure and mecamylamine challenge.

Nicotine (NIC+SAL) led to a significant increase in ACSL6 protein levels in A) the prefrontal cortex and B) the hippocampus [*p<0.01 compared to saline controls (SAL+SAL)]. These increases were suppressed by the administration of mecamylamine (NIC+MEC; # p<0.01 compared to nicotine treated NIC-SAL mice). There was no effect of nicotine on levels of ACSL6 in C) the NAC and D) VTA. (n sizes = 3–8 per group).