Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

BACKGROUND: Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes. METHODS: To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity. RESULTS: One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior. CONCLUSIONS: Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans

Serotonin Transporter-Independent Actions of the Antidepressant Vortioxetine as Revealed Using the SERT Met172 Mouse

Selective serotonin (5-HT, SERT) reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression. However, they have delayed efficacy and can induce side-effects that can encourage discontinuation. Recently, agents have been developed, including vortioxetine (Trintellix), that augment SERT blockade with interactions at other targets. At therapeutic doses, vortioxetine interacts with SERT as well as 5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors. We assessed the SERT-dependency of vortioxetine action using the SERT Met172 mouse model, which disrupts high-affinity interactions of many antidepressants with the transporter. We demonstrate that the SERT Met172 substitution induces an ∼19-fold loss in vortioxetine potency for SERT inhibition in midbrain synaptosomes. Moreover, in these mice, we observed reduced SERT occupancy, a diminished ability to prolong 5-HT clearance, and a reduced capacity to elevate extracellular 5-HT. Despite reduced interactions with SERT, vortioxetine maintained its ability to enhance mobility in tail suspension and forced swim tests, reduce consumption latency in the novelty induced hypophagia test, and promoted proliferation and survival of subgranular zone hippocampal stem cells. Our findings suggest that the antidepressant actions of vortioxetine may be SERT-independent, and encourage consideration of agents that mimic one or more actions of the drug in the development of improved depression treatments

Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1.

The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development

Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

Abstract Background Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes. Methods To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity. Results One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior. Conclusions Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans

Targeting Construct and Genotyping of <i>Il1r1</i>^{<i>loxP/loxP</i>} and <i>Il1r1</i>^loxP/loxP:CMV Cre Mice.

<p>(A/B) Targeting construct illustrating position of <i>loxP</i> sites flanking exons 3 and 4 of the <i>Il1r1</i> gene and presence of positive (Neo^r) and negative (TK) selection cassettes. Excision of exons 3 and 4 results in an elimination of the expression of downstream exons and thereby <i>Il1r1</i> function. Diagram also includes locations of sense (RB4082 and RB4081) and antisense (RB4048) oligonucleotide primers utilized for both genotyping and the confirmation of excision of <i>Il1r1</i> exons 3 and 4 and subsequent ligation. (C) PCR-based genotyping of <i>Il1r1</i>^{<i>loxP/loxP</i>} and <i>Il1r1</i>^{<i>loxP/loxP</i>}:CMV Cre mice. The WT locus yields an amplicon of 247 bp, whereas PCR of the floxed allele yields an amplicon of 420 bp. Detection of excised floxed <i>Il1r1</i> alleles results from the utilization of primer 1 (RB4082, sense) and primer 3 (RB4048, antisense) to produce an amplicon of 379 bp. (D) Litters born to <i>Il1r1</i>^{<i>wt/loxP</i>} pairings produce offspring at the normal, expected Mendelian frequency.</p

Basic Phenotyping of <i>Il1r1</i>^{<i>loxP/loxP</i>} Mice.

<p>Adult <i>Il1r1</i>^{<i>loxP/loxP</i>} mice display normal (A) development as indicated by weight gain until adulthood as compared to wild type littermate counterparts (two-way repeated measures ANOVA, followed by post hoc Tukey’s multiple comparison tests, N = 7-10/group, <i>P ></i> 0.05) (B), body temperature (C), Rotarod performance and (D) hang time in the inverted screen test (unpaired Student’s t test, N = 12/group, <i>P</i> > 0.05). Additionally, <i>Il1r1</i>^{<i>loxP/loxP</i>} mice exhibit comparable rates of locomotor activity to WT littermates when evaluated as (E) average velocity (Student’s unpaired t test, <i>P</i> > 0.05) or (F) distance traveled, two-way ANOVA, N = 6/group, genoptype effect, P > 0.05).</p

<i>Il1r1</i>^{<i>loxP/loxP</i>} Mice Exhibit Normal IL-1R1-Mediated Increases in Serum IL-6 Following IL-1α Injections.

<p>(A) WT and <i>Il1r1</i>^{<i>loxP/loxP</i>} animals exhibit significant and comparable serum IL-6 elevations 2 hrs after IL-1α injections (1 μg, i.p.), as compared to vehicle treated controls (two-way ANOVA, <i>P</i> < 0.0001 for treatment, genotype and their interaction, followed by post hoc Tukey’s multiple comparison tests, *** = <i>P</i> < 0.0001). IL-1α induced IL-6 elevations were found to be IL-1R1 dependent, as revealed by a lack of response in <i>Il1r1</i>^-/- animals. N = 4-6/group for all assays. (B) Excision of floxed <i>Il1r1</i> alleles by global Cre recombinase expression in <i>Il1r1</i>^{<i>loxP/loxP</i>} mice attenuates the ability of IL-1α treatment to increase serum IL-6 expression. No increases in IL-6 expression were found in IL-1α treated <i>Il1r1</i>^{<i>loxP/loxP</i>}:CMV Cre or <i>Il1r1</i>^<i>-/-</i> mice (one way ANOVA, <i>P</i> < 0.0001, followed by post hoc Tukey’s multiple comparison tests, *** = <i>P</i> < 0.0001) N = 5-7/group for all assays.</p

<i>Il1r1</i>^{<i>loxP/loxP</i>} Mice Exhibit Normal <i>Il1r1</i> mRNA Expression in the CNS, but Lower Splenic <i>Il1r1</i> mRNA Levels without a Concomitant Decrease in IL-1R1 Protein Expression.

<p>qRT-PCR was utilized to determine <i>Il1r1</i> mRNA levels in midbrain and spleen samples from male and female <i>Il1r1</i>^{<i>loxP/loxP</i>} and wild type littermates. Midbrain samples from both male (A) and female (B) <i>Il1r1</i>^{<i>loxP/loxP</i>} mice exhibit comparable levels of <i>Il1r1</i> mRNA expression as wild type littermates (Student’s unpaired t test, N = 14/group, <i>P</i> > 0.05). In contrast, both male (C) and female (D) <i>Il1r1</i>^{<i>loxP/loxP</i>} mice exhibit significantly reduced levels of splenic <i>Il1r1</i> mRNA expression as compared to wild type littermates (Student’s unpaired t test, N = 6-8/group, * = <i>P</i> < 0.05). Western blot analysis however, revealed <i>Il1r1</i>^{<i>loxP/loxP</i>} spleen samples to express normal levels of IL-1R1 protein (E) as compared to their wild type littermates (quantified in F, Student’s unpaired t test, N = 6/group, <i>P</i> > 0.05).</p

Excision of Floxed <i>Il1r1</i> Alleles by Cre Recombinase and Elimination of <i>Il1r1</i> mRNA Expression in <i>Il1r1</i>^{<i>loxP/loxP</i>}:CMV Cre Mice.

<p>(A) <i>Il1r1</i> genotyping of mice born to a cross of <i>Il1r1</i>^{<i>loxP/loxP</i>} and CMV-Cre mice revealed offspring to be of two distinct genotypes, <i>Il1r1</i>^wt/loxP:CMV-Cre or <i>Il1r1</i>^wt/loxP. All pups expressing Cre recombinase displayed excision of floxed <i>Il1r1</i> alleles (lanes animals 2, 3, 6 and 7). M = Molecular weight marker, WT = wildtype control sample, HET = heterozygous (<i>Il1r1</i>^wt/loxP) control sample (lacking Cre), FLX = homozygous (<i>Il1r1</i>^{<i>loxP/loxP</i>}) control sample (lacking Cre). Actual band sizes–unexcised floxed allele = 420 base pairs, WT allele = 247 base pairs. (B) Global expression of Cre recombinase driven through the CMV promoter results in the elimination of splenic <i>Il1r1</i> mRNA expression in <i>Il1r1</i>^{<i>loxP/loxP</i>}:CMV Cre mice. (One way ANOVA, <i>P</i> < 0.0001, post hoc Tukey’s multiple comparison tests, *** = <i>P</i> < 0.0001; N = 5-7/group) (C) Global expression of Cre recombinase driven through the CMV promoter results in the elimination of <i>Il1r1</i> mRNA expression in the midbrain of <i>Il1r1</i>^{<i>loxP/loxP</i>}:CMV Cre mice. (One way ANOVA, P < 0.01, post hoc Tukey’s multiple comparison tests, * = <i>P</i> < 0.01; N = 5-7/group).</p