A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety

Abstract

Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities.

Trial registration: ClinicalTrials.gov NCT01323556.

Conflict of interest statement

T.K. received fees for educational programs from Janssen-Cilag, Eli Lilly, Servier, Lundbeck, Bristol Myers Squibb, Pfizer, and Astra-Zeneca; travel support/sponsorship for congresses from Servier; speaker’s honoraria from Janssen-Cilag; and research grants from Pfizer and Lundbeck. H.-U.W. has been a member of advisory boards of several pharmaceutical companies. He received travel reimbursements and research grant support from Essex Pharma, Sanofi, Pfizer, Organon, Servier, Novartis, Lundbeck, and Glaxo Smith Kline. V.A. is a member of advisory boards and/or gave presentations for the following companies: Astra-Zeneca, Janssen-Organon, Lilly, Lundbeck, Servier, Pfizer, and Wyeth. He also received research grants from Astra-Zeneca, Lundbeck, and Servier. He chaired the committee for the “Wyeth Research Award Depression and Anxiety”. A.S. received research from Lundbeck and speaker honoraria from AstraZeneca, Boehringer Ingelheim, Eli Lilly & Co, Lundbeck, Pfizer, Wyeth, and UCB. Educational grants were given by the Boehringer Ingelheim Fonds and the Eli Lilly International Foundation. A.R. has received a research grant from Medice and speaker’s honoraria from Medice and neuraxpharm. The other authors declare that they have no conflict of interest.

Figures

Fig. 1. MicroRNA-mediated regulation of SLC6A2 expression.

Data show mean repression of normalized firefly luciferase activity upon cotransfection of a dual luciferase reporter vector containing the 3’UTR of SLC6A2 with the microRNA as indicated and 95% confidence intervals. Data were normalized to same-well renilla activity as well as to the activity of a no 3’UTR-containing vector (n = 5 technical triplicates). The 95% confidence intervals of all microRNAs shown were outside the 95% confidence interval of the negative control microRNA ath-miR-159a (95% CI [98.2, 109.7]) and thus considered to be significantly regulating SLC6A2

Fig. 2. Dual luciferase reporter assay for modulation of MIR579 expression by upstream genetic variation and hsa-miR-579-3p-mediated regulation of anxiety candidate gene expression.

a Data show mean relative firefly luciferase activity under a minimal promoter sequence containing the (A)- and (T)-allele of rs2910931 upstream of MIR579 as indicated. Data were normalized to same-well renilla activity and are shown as mean and standard error (n = 7 technical triplicates, *p < 0.05 using a two-sided t test). b Data show mean repression of normalized firefly luciferase upon cotransfection with hsa-miR-579-3p and ath-miR159a (negative control) as indicated. Dual luciferase reporter assays contained the 3’UTR of the corresponding receptor genes as indicated. Data were normalized to negative-control-3’UTR relative firefly activity for each microRNA as indicated (n = 5 technical triplicates, *p < 0.05, ***p < 0.005)

Fig. 3. Neurofunctional activation patterns of fear-related brain structures in healthy controls during fear conditioning and extinction.

Healthy controls (n = 40) were subjected to an experimental fear conditioning and extinction task. Activation of brain regions was analyzed by means of fMRI as a function of minor (T)-alleles of rs2910931 (TT+AT: n = 25; AA: n = 15). Beta values from significant clusters were extracted and used for bar graph visualization (a.u.). a Activation of midbrain/periaqueductal gray, amygdala, and hippocampus regardless of conditioned stimulus presented during the second half of the extinction phase as a function of rs2910931 genotype group. b Interaction of rs2910931 genotype group with the conditioned stimulus presented during the first half of the extinction phase. *p < 0.05, **p < 0.01, and ***p < 0.001. CS+ conditioned stimulus that was followed by the unconditioned stimulus (US); CS− conditioned stimulus that was never followed by the US

Fig. 4. Increase of heart rate in PD patients during the behavioral avoidance test.

Patients with panic disorder and agoraphobia (n = 276: n(AA) = 95, n(AT) = 129, n(TT) = 52) were subjected to experimental panic exposition using the behavioral avoidance test. Data show the mean difference with standard error of heart rates between the last minute of the anticipation phase and the first minute of the exposure phase during the behavioral avoidance test as a function of rs2910931 genotype. *p < 0.05