Inactivation of the catalytic phosphatase domain of PTPRT/RPTPρ increases social interaction in mice

Abstract

Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a transmembrane protein expressed at high levels in the developing hippocampus, olfactory bulb, cortex, and cerebellum. It has an extracellular domain that interacts with other cell adhesion molecules, and it has two intracellular phosphatase domains, one of which is catalytically active. In a recent genome-wide association study, PTPRT was identified as a potential candidate gene for autism spectrum disorder (ASD) susceptibility. Mutation of a critical aspartate to alanine (D1046A) in the PTPRT catalytic domain inactivates phosphatase function but retains substrate binding. We have generated a knockin mouse line carrying the PTPRT D1046A mutation. The D1046A mutation in homozygous knockin mice did not significantly change locomotor activities or anxiety-related behaviors. In contrast, male homozygous mice had significantly higher social approach scores than wild-type animals. Our results suggest that PTPRT phosphatase function is important in modulating neural pathways involved in mouse social behaviors relevant to the symptoms in human ASD patients.

Keywords: PTPRT; RPTPρ; animal model; social interaction.

Figure 1.

Targeting strategy for generating PTPRT knockin mouse and confirmation of correct homologous recombination. (A) Targeting strategy. Thin lines represent wild-type mouse genomic DNA between exons 20 and 23; thick lines represent sequences included in the targeting construct. Open boxes represent exons; the large box represents exon 22, which contains a line representing the mutation in exon 22. Open arrows represent the positive selection marker (Neo cassette) and the negative selection marker (thymidine kinase gene). Triangles represent LoxP sites. Small arrows represent polymerase chain reaction (PCR) primers. (B) Agarose gel image of PCR screening for mice with correct homologous recombination using primers P2f annealing to the Neo gene and P2r annealing to the cell genomic sequence just outside of the short arm (see panel A). The presence of a 1.35-kb band indicates positive mice. (C) Image of a preparative agarose gel of PCR using primers P1r annealing to the Neo gene and P1f annealing to the mouse genomic sequence just outside of the long arm. The presence of a 5.7-kb band confirms that the two mice have correct recombination at the long arm side. (D, E, F) portions of chromatograms of the sequencing results of the PTPRT mRNAs (cDNAs) from wild-type (+/+), heterozygous (+/m), and homozygous (m/m) knockin mice. The three mutation sites are highlighted.

Figure 2.

Open field Locomotor activity. The total distance traveled in 20 min by male and female wild-type (+/+) and homozygous PTPRT mutant mice (m/m) are shown. No significant difference in the total locomotor activity was observed between +/+ and m/m mice (Fig. 2A and C). Distance traveled over time is shown for males (Fig. 2B) and females (Fig. 2D); a significant effect of time but no effect of genotype was observed. Data are presented as mean ± standard error of the mean (SEM).

Figure 3.

Elevated plus maze. The number of entries into the open arms are shown for male (Fig. 3A) and female (Fig. 3C) wild-type (+/+) and homozygous PTPRT mutant mice (m/m). Time spent on the open arms for males and females is shown in Figure 3B and 3D. Data are presented as mean ± standard error of the mean (SEM).

Figure 4.

Social approach. Time spent in the center chamber, the side chamber containing an unfamiliar mouse (Sstranger 1), or the side chamber containing an empty wire cage for male (Fig. 4A) and female (Fig. 4B) wild-type (+/+) and homozygous PTPRT mutant mice (m/m). In males, both genotypes spent significantly more time in the chamber containing stranger 1 than the chamber containing the wire cage (*P < 0.05). Social approach scores (Fig. 4C) were significantly higher in male m/m mice (n = 8) compared wth +/+ (n = 16) mice (*P < 0.05). Both +/+ and m/m female mice showed preference for the chamber containing stranger 1 over wire cage (*P < 0.05). No differences in social approach scores were observed between female +/+ (n = 13) and m/m (n = 11) mice (Fig. 4D). Data are presented as mean ± standard error of the mean (SEM).

Figure 5.

Social novelty. Time spent with stranger 1 and 2 for male (Fig. 5A) and female (Fig. 5B) wild-type (+/+) and homozygous PTPRT mutant mice (m/m). In males, both +/+ and m/m mutant mice spent significantly more time with stranger 2 than stranger 1 (*P < 0.05). In females, only +/+ and not m/m mutant mice showed a significant preference for the chamber containing stranger 2 over stranger 1 (*P < 0.05). Figure 5C and 5D show the difference in time spent with stranger 2 vs. stranger 1 (social novelty score) by (C) male and (D) female +/+ and m/m mice. Data are presented as mean ± standard error of the mean (SEM).