Regulation of alpha-synuclein expression in alcohol-preferring and -non preferring rats

Abstract

The alpha-synuclein (Snca) gene is expressed at higher levels in alcohol-naïve, inbred alcohol-preferring (iP) rats than in alcohol-non preferring (iNP) rats. Snca modulates dopamine transmission and the dopamineregic system, which play a role in mediating the rewarding properties of alcohol consumption. Thus, understanding regulation of Snca gene expression could provide insight into the relationship of Snca and alcohol consumption. To study regulation of rat Snca expression, 1,912 bp of the iP and iNP 5'-regions were cloned and sequenced. 5'-rapid amplification of cDNA ends (RACE), primer extension and RT-PCR mapped three transcription start site clusters (clusters TSS1, TSS2 and TSS3), suggesting that the Snca proximal promoter region has a complex architecture. This proximal promoter region has three TATA-less core promoters containing SP1 binding sites, initiator elements and downstream core promoter elements, which are often located in such promoters. Snca-luc constructs transiently transfected into SK-N-SH neuroblastoma cells showed that the region from - 1,912 to - 1,746 contained a strong core promoter, and that the entire approximately 2 kb region had significant promoter activity. Five polymorphisms identified between the iP and iNP in the proximal promoter region did not influence differential expression between the strains. In contrast, a single nucleotide polymorphism (SNP) at + 679 in the 3'-untranslated region (UTR) resulted in a 1.3-fold longer half-life of iP mRNA compared with iNP mRNA, which is consistent with the differential expression observed between the iP and iNP strains. These results suggest that regulation of rat Snca gene expression is complex and may contribute to alcohol preference in the iP rats.

Fig. 1

P2 luciferase mRNA is more stable than NP2 luciferase mRNA. Constructs P2 and NP2, created previously for transient transfection luciferase reporter assays (Liang et al. 2003), were used to transfect SK-N-SH cells. The relative amount of mRNA was quantified using quantitative RT-PCR. Luciferase mRNA decay rate was determined at 30, 60, 90 and 120 min following DRB administration; the mean and SEM are indicated. Inset: the average luciferase mRNA half-life times were 51 ± 4.89 min (mean ± SEM) and 35 ± 4.89 min for the P2 and NP2 constructs, respectively.

Fig. 2

Northern blot of total RNA from rat brain. White lines on the blot are size markers and designate the sizes of the mRNA transcripts as indicated.

Fig. 3

Identification of TSSs and Exon1. First Choice RLM-RACE was performed two times with different primer sets using total brain RNA from the iP and iNP rats. All the PCR products were run on 2% agarose gels. (a) Genomic structure of exons 1, 2 and 3, and intron 1, of the rat Snca gene. Open boxes represent exons, intron 1 boundaries are noted, and arrows represent forward and reverse primers used in the 5′-RACE assay and confirmation studies. The translation start site, ATG, is designated as + 1. (b) First 5′-RACE assay: 400 bp denotes the size of the primary PCR product in iP and iNP rats, using the 5−-RACE outer adapter primer and LC583; 300 bp denotes the nested PCR product using the 5′-RACE inner adapter primer and the gene-specific nested primer LC582. A larger nested PCR product is barely visible at approximately 700 bp. The 300 bp fragment included part of the published exon 1 sequence (exon 2 in this paper) and also 162 bp of a new exon 1, which mapped from − 1305 to − 1144 of the genomic sequence. (c) Verification of exon 1 and intron 1. PCR was performed, using primers LC461 and LC468, with iP and iNP cDNA and genomic DNA as templates; (−) denotes a PCR reaction without template. (d) Second 5′-RACE assay. This assay was identical to the first 5′-RACE assay except that the outer reverse gene-specific primer was LC460 and the inner reverse gene-specific primer was LC519. The nested iP PCR products are noted at 110 bp and 220 bp; iNP data (not shown) were identical to the iP data. (e) Confirmation of TSS2. iP and iNP cDNA were amplified in parallel using primers LC612 and LC468 to confirm TSS2; 524 bp denote the size of the cDNA product. (f) TSS3. Forward primers LC516 and LC627, and reverse primer LC468, were used to amplify iP and iNP genomic DNA and cDNA. The LC627/LC468 cDNA product is 777 bp and includes 735 bp, indicated as exon 1 and 42 bp from exon 2. The genomic product is 1894 bp. The LC516/LC468 genomic PCR products are 1947 bp and there was no amplification, using cDNA for the template.

Fig. 4

Nucleotide sequence of the rat Snca 5′-region. The translation start site is indicated as + 1. Initiation start sites (clusters) identified by 5′-RACE are designated by arrows and the number of clones above the sequence. The initiation start sites identified by primer extension are indicated by bold letters and underlining. For numbering purposes of subsequent figures, the three rat TSSs, rTSS1, rTSS2 and rTSS3, are indicated by a bold letter. Putative cis-acting elements are shaded in gray with the transcription factor above the sequence. Initiator (Inr) and downstream core promoter element (DPE) consensus sequences are boxed. Reverse primers and forward primers are shown as horizontal lines, with arrows below the sequence and above the sequence, respectively. Lower case letters represent intron canonical GT-AG splice-site sequences flanking intron 1.

Fig. 5

Primer extension assays confirm putative TSS clusters. Primers LC640, LC638 and LC539 (Fig. 4) were end-labeled with [γ³²P]-ATP and T4 polynucleotide kinase. Primers were annealed to total rat brain RNA in (a), (c) and (d), and to total RNA from SK-N-SH cells transfected with the P5-luc construct in (b). Extension products were resolved on a 6% sequencing gel and compared with a sequencing ladder generated by the respective end-labeled primers. Location of the primer extension products are indicated.

Fig. 6

Comparison of 5′-region sequences in rat, human and mouse to identify conserved binding sites. The numbering is relative to the + 1 translation start sites. Binding sites identical between the species and binding sites, with mismatches that bind the transcription factor, are bold or not bold below the sequence, respectively. Sequences shaded in gray are identical between at least two of the species. Rat (rTSS), mouse (mTSS) and human (hMSS) transcription start sites are shown as arrows below the sequence.

Fig. 7

α-Synuclein promoter activity in transient transfection assays. (a) A schematic diagram corresponds to the Snca gene from − 1912 to − 19 relative to translation start site (+ 1). Restriction sites are noted. Arrows indicate three putative transcription initiation sites within the three identified TSS clusters. Various deletion promoter fragments designated P3/NP3 to P7/NP7 from the iP and iNP 5′-regions were fused to the luciferase reporter gene. Vertical lines are polymorphic sites between the iP and iNP rats (see Table 1). (b) The activity of each construct was expressed as fold change compared with the activity of the pGL3-basic vector. The bars and fold change show the mean ± SEM of the results from at least five independent transfection experiments performed in triplicate.