Research ReportRs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue
Highlights
► The mRNA expression of 5HT1AR in human hippocampal tissue is affected by rs6295. ► Rs6295 determines the binding affinity of the transcription factor cJun. ► c-Jun has an impact on the promoter activity of the 5HT1AR promoter variants.
Introduction
Impaired serotonin homeostasis is a frequent pathogenetic aspect in many neuropsychiatric disorders including depression, migraine and epilepsy (Trindade-Filho et al., 2008, Bahremand et al., 2011, Richerson and Buchanan, 2011, Theodore et al., 2012). The episodic, sustained onset of symptoms in these disorders suggests transient alterations of serotonin signaling due to factors such as transcriptional changes of relevant receptors (Lambru and Matharu, 2011, Richerson and Buchanan, 2011, Theodore et al., 2012). The serotonin-1A receptor (5HT1AR) is a central component in activity regulation of the serotonin system, and is expressed in many neuronal structures including the hippocampus where it serves as major postsynaptic receptor of neurons (Le Francois et al., 2008). Association studies suggest that genetic variation of the HTR1a gene influences the risk for a broad range of neuropsychiatric disorders (Lemonde et al., 2003, Marziniak et al., 2007, Le Francois et al., 2008). Lack of common missense variations in the HTR1a gene may suggest that regulatory sequence variations affecting gene expression of the HTR1a gene exert the functional effects.
Here, we have investigated the functional role of the single nucleotide polymorphism (SNP) rs6295 located in the promoter of HTR1a for the expression of the respective gene in human brain tissue. Rs6295 shows prominent associations in several neuropsychiatric disorders. In migraineurs, the rs6295 GG-genotype is associated with avoidance of physical activity during migraine attacks (Marziniak et al., 2007). The G-allele of rs6295 is linked to major depression, panic disorders and neuroticism (Lemonde et al., 2003, Le Francois et al., 2008). The G-allele of rs6295 is further related to reduced response to antidepressant or antipsychotic treatment (Le Francois et al., 2008). Rs6295 has been demonstrated as a functional promoter variant affecting binding sites for transcriptional repressors such as NUDR/Deaf1 and Hes5 (Albert et al., 2011). Yeast one-hybrid analyses suggested repression of the HTR1a promoter activity by NUDR and Hes5 preferentially at the rs6295 C-allele (Lemonde et al., 2003). These data implied that the rs6295 G-allele mediates a reduced repression of the HTR1a promoter, which leads to higher HTR1a mRNA levels in brain tissue and thereby increases individual risk of neuropsychiatric disorders.
However, SNPs can have ambiguous roles for transcription factor binding. We have therefore used bioinformatic promoter analysis in order to find transcriptional activator binding affected by the rs6295 variants. Based on respective results, we have analyzed a potential functional role of the transcriptional activator c-Jun on rs6295 in vitro. Given the association of rs6295 with episodic brain disorders and the in vitro evidence for an allele-specific regulation of the HTR1a promoter activity, we have subsequently explored whether the rs6295 alleles influence HTR1a mRNA expression in human brain tissue. Therefore, we used our unique access to fresh frozen hippocampal tissue of temporal lobe epilepsy (TLE) patients that underwent epilepsy surgery for seizure control. A previous association study did not support a risk-conferring effect of the HTR1a promoter SNP rs6295 in TLE (Stefulj et al., 2010). Due to the high number of neuropsychiatric co-morbidities in TLE, we further addressed potential clinico-genetic associations of rs6295 in TLE patients as addressed in a previous study in our present TLE patient series (Theodore et al., 2012).
Section snippets
Bioinformatic promoter analysis reveals a c-Jun binding site at rs6295 with allele specific binding affinities
We have used bioinformatic promoter analysis in order to find transcriptional activator binding affected by the rs6295 variants. We predicted the position and conservation of a putative binding site for c-Jun using position-specific-scoring matrices in the HTR1a promoter region (Fig. 1A). The rs6295 C-variant exhibits a potential c-Jun binding motif with a higher ‘motif similarity score’, i.e., binding affinity compared to an ‘ideal’ binding motif, compared to the c-Jun binding motif in the
Discussion
Our present results suggest that the transcription factor c-Jun has significantly different activating effects on rs6295 SNP promoter HTR1a variants, i.e., c-Jun binds in vivo to the HTR1a promoter and more strongly activates the C-variant (Fig. 1A and B). Furthermore, we confirmed the previously reported repressing effects of NUDR and Hes5 on the promoter (Lemonde et al., 2003, Albert et al., 2011). Our own bioinformatic TF binding site analyses at rs6295 for NUDR (C-allele: 61% and G-allele:
Patient criteria and surgical specimens
We included hippocampal biopsy specimens of 140 pharmacoresistant TLE patients. The ascertainment scheme and clinical characteristics have been described in detail previously (Kral et al., 2002, Pernhorst et al., 2011) (Fig. 3A). Informed written approval was obtained from all patients and procedures were in accordance with the Declaration of Helsinki and approved by the local ethics committee. Depressive symptoms of patients were assessed according to Beck Depression Inventory (BDI; threshold
Acknowledgment
The mammalian expression vector c-Jun was kindly provided by Dirk Bohrmann (Rochester). We would also like to thank Dr. Tetsuya Taga (Tokyo, Japan) for providing the pEF-Bos-FlagHes5 expression vector and Dr. Jodi Huggenvik (Illinois, USA) for providing the h6NUDR expression vector. Our work is supported by Deutsche Forschungsgemeinschaft (KForG “Innate Immunity” TP2, AJB; Emmy Noether program: SS; SFB-645: SS), Bundesministerium für Bildung und Forschung (NGFNplus EMINet; AJB, SS, TS;
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