Enhanced activity of human serotonin transporter variants associated with autism

Abstract

Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.

Figure 1

Location and 5-HT transport activity of autism-associated SERT-coding variants. (a) Autism-associated variants are overlaid on a 12-TM model of a single SERT subunit, with NH₂ and COOH termini oriented inside the cell. Variants in extramembrane domains are shaded blue, whereas those in membrane domains are shaded white. (b) Altered activity of SERT variants is evident in native lymphocytes. Lymphocytes were genotyped and cultured as described in §2 and assessed for 5-HT uptake. Data presented derive from n=3 individual assays on lymphocyte lines of determined genotype. Findings were replicated in a separate set of pre-genotyped samples with equivalent results. Transport activities were analysed by a one-way ANOVA with post hoc Dunnett's tests, with ^*p<0.05 taken as significant. (c) 5-HT transport activity of autism-associated SERT-coding variants in transfected HeLa cells. All variants were transfected in parallel with reference hSERT cDNA into HeLa cells and assayed for 5-HT transport activity as described in §2. Data reflect mean±s.e.m. of three separate experiments. Means were compared with hSERT cDNA using a one-way ANOVA followed by Dunnett's test of individual means against hSERT values, with ^*p<0.05 taken as significant.

Figure 2

Analysis of protein expression of autism-associated SERT-coding variants. Impact of SERT-coding variants on total and cell surface [¹²⁵I]RTI-55 binding. HeLa cells transiently transfected with hSERT or one of the SERT-coding variants were subjected to intact cell-binding assays at 4°C with the cocaine analogue [¹²⁵I]RTI-55 (5 nM) as described in §2. (a) Total binding values as defined with paroxetine (10 μM) as displacer. (b) Surface labelling by [¹²⁵I]RTI-55 as defined with 5-HT (100 μM) as displacer. In vehicle-treated cells, hSERT total binding (fmol×10⁻⁵) was 583.7±20.6, and the surface binding was 175.7±11.7. Results for (a,b) reflect mean±s.e.m. of three separate experiments normalized to hSERT (100%). Binding levels were analysed via a one-way ANOVA followed by post hoc Dunnett's tests comparing mutant means to hSERT, with ^*p<0.05 taken as significant. (c) Immunoblots of total cell extracts prepared from HeLa cells transfected with hSERT or one of the variants described in the study. (d) Cell surface expression alterations in hSERT Gly56Ala, Ile425Leu, Phe465Leu and Leu550Val. Variants were transfected in parallel with hSERT into HeLa cells and cell surface transporters identified by immunoblotting of biotinylated samples, captured as described in §2. Quantitative estimations of relative (e) total and (f) surface density of hSERT, Gly56Ala, Ile425Leu, Phe465Leu and Leu550Val based on densitometry of biotinylation immunoblots. Data reflect mean values of three separate experiments ±s.e.m. Means were compared with a one-way ANOVA followed by Dunnett's test to compare variant surface expression with that achieved with hSERT, with p<0.05 taken as significant (^*significantly elevated versus hSERT, p<0.05; NT, non-transfected.)

Figure 3

Impact of 8Br-cGMP and p38 MAPK on SERT activity of autism-associated hSERT-coding variants. (a) Activity modulation. HeLa cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described in §2 following pretreatments of cells with either 100 μM 8Br-cGMP or vehicle for 1 hour. Parallel wells were treated with the PKG inhibitor H8 (10 μM) to validate specificity. (b) Altered p38 MAPK-dependent regulation of hSERT in transfected HeLa cells. The cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described in §2 following pretreatments of cells with either 1 μM anisomycin or vehicle for 10 min. Parallel wells were treated with the p38 MAPK inhibitor SB203580 (1 μM) to validate specificity. Results reflect mean±s.e.m. of three separate experiments normalized to each mutant's control measured under vehicle-treated conditions (100%). Results in (a,b) reflect mean ±s.e.m. of three separate experiments normalized to each mutant's level under vehicle-treated conditions (100%). Data were analysed by a one-way ANOVA with post hoc Bonferroni tests comparing variant with hSERT 8Br-cGMP/anisomycin responses, with ^*p<0.05 taken as significant.

Figure 4

Impact of PKC and PP2A on SERT activity of autism-associated hSERT-coding variants. (a) Activity modulation. HeLa cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described in §2 following pretreatments of cells with either vehicle or 0.1 or 1 μM β-PMA for 30 min. Parallel wells were treated with the PKC inhibitors staurosporine (stauro; 10 μM) or bisindolylmaleimide (BIM, 10 μM). Altered PP2A-dependent regulation of hSERT in transfected HeLa cells. (c) The cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described in §2 following pretreatments of cells with increasing concentrations of (b) calyculin A or (c) fostriecin for 10 min. Results reflect mean±s.e.m. of three separate experiments normalized to each mutant's control measured under vehicle-treated conditions (100%). Results in (a–c) reflect mean±s.e.m. of three separate experiments normalized to each mutant's level under vehicle-treated conditions (100%). Data were analysed by a one-way ANOVA with post hoc Bonferroni tests comparing the variant with hSERT responses, with ^*p<0.05 taken as significant.

Figure 5

Altered activity of SERT variants is differently exhibited in CHO-Flp-In stable cells. (a) 5-HT transport activity of autism-associated SERT-coding variants in CHO-Flp-In stable cells. All variants were assayed for 5-HT transport activity as described in §2. Data reflect mean±s.e.m. of three separate experiments. Means for variants were compared with hSERT using a one-way ANOVA followed by Dunnett's test of individual means against hSERT values, with ^*p<0.05 taken as significant. (b) Immunoblots of total cell extracts prepared from CHO-Flp-In stable cells expressing hSERT or one of the variants described in the study. (c) Cell surface expression alterations in hSERT, Gly56Ala and Ile425Leu. Cell surface transporters were identified by immunoblotting of biotinylated samples, captured as described in §2. Quantitative estimations of relative (d) total and (e) surface density of hSERT, Gly56Ala and Ile425Leu based on densitometry of biotinylation immunoblots. Data reflect mean values of three separate experiments±s.e.m. Means were compared with a one-way ANOVA followed by Dunnett's test to compare variant surface expression with that achieved with hSERT, with ^*p<0.05 taken as significant. (f) Gly56Ala and Ile425leu proteins exhibit enhanced catalytic function, with a turnover rate of approximately 250% or more than that of wild-type SERT. Data reflect mean values of three separate experiments±s.e.m. Means were compared with a one-way ANOVA followed by Dunnett's test.