Modeling rare gene variation to gain insight into the oldest biomarker in autism: construction of the serotonin transporter Gly56Ala knock-in mouse

Abstract

Alterations in peripheral and central indices of serotonin (5-hydroxytryptamine, 5-HT) production, storage and signaling have long been associated with autism. The 5-HT transporter gene (HTT, SERT, SLC6A4) has received considerable attention as a potential risk locus for autism-spectrum disorders, as well as disorders with overlapping symptoms, including obsessive-compulsive disorder (OCD). Here, we review our efforts to characterize rare, nonsynonymous polymorphisms in SERT derived from multiplex pedigrees carrying diagnoses of autism and OCD and present the initial stages of our effort to model one of these variants, Gly56Ala, in vivo. We generated a targeting vector to produce the Gly56Ala substitution in the Slc6a4 locus by homologous recombination. Following removal of a neomycin resistance selection cassette, animals exhibiting germline transmission of the Ala56 variant were bred to establish a breeding colony on a 129S6 background, suitable for initial evaluation of biochemical, physiological and behavioral alterations relative to SERT Gly56 (wild-type) animals. SERT Ala56 mice were achieved and exhibit a normal pattern of transmission. The initial growth and gross morphology of these animals is comparable to wildtype littermate controls. The SERT Ala56 variant can be propagated in 129S6 mice without apparent disruption of fertility and growth. We discuss both the opportunities and challenges that await the physiological/behavioral analysis of Gly56Ala transgenic mice, with particular reference to modeling autism-associated traits.

Keywords: Autism; Polymorphism; Protein kinase G; Serotonin; Transgenic mouse; Transporter; p38 mitogen activated protein kinase.

Fig. 1

Location and 5-HT transport activity of autism-associated SERT coding variants. A) Autism-associated variants are overlayed 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 black whereas those in membrane domains are shaded white. B) Altered activity of SERT variants is evident in native lymphocytes. Lymphocytes were genotyped and cultured 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 analyzed 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. Data reflect mean values ± SEM of 3 separate experiments. Means were compared to 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 reproduced from authors’ prior work [79]

Fig. 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 analog [¹²⁵I]RTI-55 (5 nM). A) Total binding values as defined with paroxetine (10 µM) as displacer. B) Surface labeling by [¹²⁵I]RTI-55 as defined with 5-HT (100 µM) as displacer. In vehicle-treated cells, hSERT total binding (fmol/10⁵) was 583.7 ± 20.6, and the surface binding was 175.7 ± 11.7. Results for A) and B) reflect mean values ± SEM of three separate experiments normalized to hSERT (100%). Binding levels were analyzed 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. 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 ± SEM. Means were compared with a One-Way ANOVA followed by Dunnett’s test to compare variant surface expression to that achieved with hSERT, with p < 0.05 taken as significant (* Significantly elevated vs hSERT, p < 0.05; ⋕ Significantly reduced vs hSERT, p < 0.05). Figure reproduced from authors’ prior work [79]

Fig. 3

Impact of 8BrcGMP 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 following pretreatments of cells with either 100 µM 8BrcGMP or vehicle for 1 hr. 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. Cells transfected with hSERT or autism-associated hSERT coding variants were examined for 5-HT transport activities 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 values ± SEM of three separate experiments normalized to each mutant’s control measured under vehicle treated conditions (100%). Results in A and B reflect mean values ± SEM of three separate experiments normalized to each mutant’s level under vehicle treated conditions (100%). Data were analyzed by a One-Way ANOVA with post-hoc Bonferonni tests comparing variant to hSERT 8BrcGMP/anisomycin responses with p < 0.05 taken as significant. Figure reproduced from authors’ prior work [79]

Fig. 4

Altered activity of SERT variants is differently exhibited in CHO-Flip-In™ stable cells. A) 5-HT transport activity of autism-associated SERT coding variants in CHO-Flip-In™ stable cells. All variants were assayed for 5-HT transport activity. Data reflect mean values ± SEM of 3 separate experiments. Means for variants were compared to 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-Flip-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. 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 ± SEM. Means were compared with a One-Way ANOVA followed by Dunnett’s test to compare variant surface expression to that achieved with hSERT, with p < .05 taken as significant. F) Gly56Ala and Ile425leu proteins exhibit enhanced catalytic function, with a turnover rate of  ~ 250% or more than that of wildtype SERT. Data reflect mean values of three separate experiments ± SEM. Means were compared with a One-Way ANOVA followed by Dunnett’s test. Figure reproduced from authors’ prior work [79]

Fig. 5

Diagram for Knock-in of Gly56Ala allele in toSlc6a4locus. The targeting vector with the Ala56 knock-in mutation contained a floxed positive selection Neomycin-resistance cassette and a negative selection thymidine kinase cassette. Recombination with 129S6 embryonic stem cell DNA resulted in an Slc6a4 gene containing the Ala56 knock-in mutation, as well as two loxP sites surrounding the Neomycin-resistance cassette in intron 4. Breeding with Protamine (Prm) Cre transgenic mice yielded excision of the floxed Neomycin-resistance cassette with retention of a residual loxP site in intron 4

Fig. 6

Growth curves for wildtype and Ala56/Ala mice. Weight was recorded weekly from one week to eight weeks of life. Male growth is shown. Repeated measures ANOVA revealed a significant effect of week (F = 996, p < 0.001) but no significant effects of genotype (F = 0.44, p = 0.65) or genotype x week interaction (F = 0.47, p = 0.95). Female growth also did not differ between genotypes (data not shown)