Effect of polymorphism on expression of the neuropeptide Y gene in inbred alcohol-preferring and -nonpreferring rats

Abstract

Using animal models of alcoholism, previous studies suggest that neuropeptide Y (NPY) may be implicated in alcohol preference and consumption due to its role in the modulation of feeding and anxiety. Quantitative trait loci (QTL) analysis previously identified an interval on rat chromosome 4 that is highly associated with alcohol preference and consumption using an F2 population derived from inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats. NPY mapped to the peak of this QTL region and was prioritized as a candidate gene for alcohol-seeking behavior in the iP and iNP rats. In order to identify a potential mechanism for reduced NPY protein levels documented in the iP rat, genetic and molecular components that influence NPY expression were analyzed between iP and iNP rats. Comparing the iP rat to the iNP rat, quantitative real-time polymerase chain reaction detected significantly decreased levels of NPY mRNA expression in the iP rat in the six brain regions tested: nucleus accumbens, frontal cortex, amygdala, hippocampus, caudate-putamen, and hypothalamus. In addition, the functional significance of three previously identified polymorphisms was assessed using in vitro expression analysis. The polymorphism defined by microsatellite marker D4Mit7 in iP rats reduced luciferase reporter gene expression in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the NPY gene resulting from the D4mit7 marker polymorphism may contribute to reduced levels of NPY in discrete brain regions in the iP rats.

Fig. 1

NPY mRNA expression is decreased in alcohol-naive P rats compared with NP rats. qRT-PCR analysis was utilized to compare the relative levels of NPY mRNA between alcohol-naive iP and iNP rats in the nucleus accumbens, the frontal cortex, the amygdala, the hippocampus, the caudate putamen, and the hypothalamus. All values were determined using the standard curve method and compared with mRNA expression in the iP hypothalamus, which was arbitrarily designated 1. The graph depicts the mean±S.E.M. of the results from five independent iP and four independent iNP experiments performed in triplicate, using separate preparations of cDNA. Significant differences in regional NPY mRNA expression between iP and iNP rats was determined using the Student’s t-test. * P<0.05; ** P<0.0005.

Fig. 2

Polymorphisms identified in the NPY gene. Sequence analysis was previously performed on the NPY gene and identified three polymorphisms in the 2nd intron, 3rd intron and 3′UTR (Bice et al., 1998). (A) The diagram depicts the NPY gene. NPY’s four exons are represented with white boxes, while a black line depicts the intronic regions. The microsatellite marker D4Mit7 is represented with a black box. The three polymorphisms that have been identified in the NPY gene are labeled with arrows and their position relative to the translational start point (Tsp) that was designated +1. (B) Three polymorphisms are defined by their regional location in the NPY gene, their position relative to the Tsp (+1), and the sequence difference detected between iP and iNP rats.

Fig. 3

Functional importance of the NPY polymorphisms identified in the 2nd intron, 3rd intron, and 3′UTR. The Luc constructs were transiently transfected into SK-N-SH cells, and the resulting effect on luciferase expression was subsequently determined. SV40, luc+, and poly(A) denote the SV40 promoter, the luciferase gene, and the SV40 late poly(A) signal, respectively. The polymorphisms, located at +4666, +6871, and +7010, are noted in addition to the relative insertion site of each fragment into the pGL-3 Promoter vector. The activity of each construct was normalized to the internal control plasmid, pRL-CMV, and was expressed as fold change compared with the activity of the pGL-3 Promoter vector, which was designated as 1. The bars and fold change show the mean±S.E.M. of the results from seven independent transfection experiments performed in triplicate, using two different plasmid preparations. The significance of differences in the resulting mean values within and between multiple constructs was analyzed using ANOVA. An asterisk denotes a significant reduction in luc expression.