Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells

Abstract

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans.

Figure 1

Structure and sequence of the rhesus 5-HTTLPR. (a) The serotonin transporter (SERT) gene in rhesus monkeys has been reported to contain a 21-bp insertion-deletion polymorphism in the promoter region termed the rh5-HTTLPR. A 43-bp polymorphism, the h5-HTTLPR, occurs in the human SERT gene. In humans, the presence of the 5-HTTLPR short ‘S' allele has been associated with decreased gene transcription and thus, reductions in SERT protein levels and uptake function. We investigated whether the rh5-HTTLPR ‘S' allele is associated with decreased serotonin system parameters in PBCs, which natively express SERT. (b) Sequencing of the rh5-HTTLPR in the 15 animals studied revealed an insertion/deletion region consisting of 23 bp, as well as two additional sequence discrepancies (shown in red) compared with the originally published sequence.

Figure 2

Confocal images of rhesus PBCs. Cells were incubated with (a) 1 μ IDT307 (30 min) and (b) propidium iodide (5 min). Propidium iodide (excitation 536 nm, emission 617 nm) stains dead cells, whereas IDT307 (excitation 485 nm, emission 520 nm), a substrate for SERTs, fluoresces after being taken up into live cells. (c) A differential interference contrast image shows all cells. (d) There is no overlap between cells predominantly labeled with IDT307 (green) vs propidium iodide-labeled cells (red) in the overlay of all three images demonstrating functional SERTs in living cells. Scale bars are 25 μm.

Figure 3

Serotonin uptake rates and surface SERT binding in rhesus PBCs. (a) Maximal uptake rates were calculated using nonlinear curve fitting for data from individual animals. Mean maximal uptake rates with respect to genotype are shown in the inset. One-way analysis of variance indicated that maximal uptake rates vary with respect to rh5-HTTLPR genotype (F(2,12)=5.9; P<0.05). A priori comparisons of uptake rates using one-tailed Student's t-tests showed significant decreases associated with the ‘S' allele (t=3.4, df=7, P<0.01 L/L vs S/L and t=3.0, df=10, P<0.01 L/L vs S/S). (b) Binding of (¹²⁵I)RTI-55 to intact PBCs was used to determine the levels of SERT located at the plasma membrane. Maximal binding was calculated for data from individual animals by nonlinear curve fitting using one-site saturation isotherms. Mean maximal binding as a function of genotype is shown in the inset. There was a trend toward decreased SERT binding associated with the ‘S' allele (F(2,12)=2.5; P<0.12). Data are means±s.e.m.s with N=6 for L/L, N=3 for S/L, and N=6 for S/S. ^**P<0.01 vs the L/L genotype.

Figure 4

Serotonin system characteristics associated with the rh5-HTTLPR short allele in PBCs. Experimental data from S/L and S/S genotypes were merged and means±s.e.m.s are shown for (a) maximal serotonin uptake, (b) maximal surface SERT binding, (c) SERT mRNA levels, (d) 5-HT concentrations, (e) 5-HIAA concentrations and (f) 5-HIAA to 5-HT ratios. Data for animals having one or two ‘S' alleles (S/L and S/S) were compared with data from animals with the L/L genotype using one-tailed t tests (t=3.6, df=13, P<0.01 for maximal serotonin uptake, t=2.3, df=13, P<0.05 for maximal SERT binding) or two-tailed t tests (t=3.7, df=11, P<0.01 for 5-HIAA concentrations, and t=3.0, df=11, P<0.05 for 5-HIAA/5-HT ratios). Individual values for each animal are shown as scatter plots overlaid on the respective bar graphs. Data are means±s.e.m.s with N=5–6 for L/L and N=8–9 for ‘S' genotypes. ^**P<0.01 and ^*P<0.05 vs the L/L genotype.

Figure 5

Correlations between surface SERT binding vs serotonin uptake or SERT mRNA levels. Correlations are shown with respect to individual animals between (a) SERT function and surface SERT binding and (b) SERT mRNA levels and surface SERT binding. Mean values with respect to genotype are shown as larger symbols with s.e.m.s for each variable indicated. Only SERT function and surface SERT binding are correlated, such that 60% of the variance is shared with a low probability of chance correlation (P<0.001).