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. 2021 Jul 26;12(1):53.
doi: 10.1186/s13229-021-00458-2.

MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders

Sarah C Borrie  1 , Ellen Plasschaert  1 , Zsuzsanna Callaerts-Vegh  2 , Akihiko Yoshimura  3 , Rudi D'Hooge  2 , Ype Elgersma  4   5 , Steven A Kushner  4   6 , Eric Legius  1 , Hilde Brems  7
Affiliations

MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders

Sarah C Borrie et al. Mol Autism. .

Abstract

Background: RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model.

Methods: We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901.

Results: Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice.

Limitations: This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn.

Conclusions: These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

Keywords: Autism spectrum disorder; Neurofibromatosis type 1; RASopathy; Social dominance; Spred1; Ultrasonic vocalization.

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Conflict of interest statement

E.L. received consulting fees from SpringWorks Therapeutics. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Social and communicative impairments in Spred1-/- mice. A, B Automated tube test for social dominance. A Mean percentage of matches won per day in the automated tube test across 5 days of tournaments between WT and Spred1-/- mice, 129T2/SEMvJ background. Significant effect of genotype on days 1–4 (Two tailed binomial test compared to chance (50%): day 1 p < 0.0001; day 2 p < 0.0001; day 3 p = 0.011; day 4 p < 0. 0001). Box and whisker plots of median and quartiles of 2 independent experiments, pooled; n = 8 male mice/genotype. B Mean percentage of matches won per day in the tube test across 5 days of tournaments between WT and Spred1-/- female mice on the 129T2/SVEmJ background. Significant effect of genotype on days 1, 3, 4 and 5 (Two tailed binomial test compared to chance (50%): day 1 p = 0.0011; day 3 p = 0.0011; day 4 p = 0.0251; day 5 p = 0. 01). Box and whisker plots of median and quartiles of 2 independent experiments, pooled; n = 8 mice/genotype. C–F USVs from WT and Spred1-/- male mice (B6 background) to a novel B6 female: C Number of USV calls/min made by male mice (unpaired t-test, t = 4.481, df = 27 p = 0.0001). D Mean call duration of male mice in response to a novel female (Mann Whitney test, p = 0.0002). E Mean peak frequency of USV calls (unpaired t-test, t = 1.001, df = 27, p = 0.3258). F Mean peak amplitude of USV calls (unpaired t-test, t = 0.8998, df = 27, p = 0.3762). n = 14–15 mice/genotype, mean ± SEM. G Nest building score of female Spred1-/- and WT mice (B6 background) after 24 h, indicating quality of nest building. 1 = no nest shredding; 5 = high-sided nest with all material shredded (unpaired t-test, t = 3.496, df = 34, p = 0.0013). n = 17–19 mice/genotype, mean ± SEM. For all figures, asterisks indicate: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 2
Fig. 2
Spred1-/- mice exhibit deficits in neonatal ultrasonic vocalization. Neonatal USVs upon maternal separation in WT and Spred1-/- mice were recorded from P4 to P12. A Mean call rate per minute (2-way ANOVA with repeated measures: main effect of age, F(4,156) = 12.28, ****p < 0.0001, no effect of genotype, no interaction). Mean ± SEM. B Mean call duration in milliseconds (2-way ANOVA with repeated measures: effect of age, F(4,156) = 7.347, p < 0.0001; no effect of genotype; no interaction). C Mean peak frequency of USV calls (2-way ANOVA with repeated measures: effect of age F(4,150) = 5.866, p = 0.0002; effect of genotype F(1,150) = 55.13, p < 0.0001; no interaction). D Mean peak amplitude of USV calls (2-way ANOVA with repeated measures: effect of age F(4,205) = 10.77, p < 0.0001, effect of genotype F(1,205) = 9.181, p = 0.0028; no interaction). For all analyses, n = 22–24 mice/genotype. Violin plots show median and quartiles. For C and D, asterisks indicate post-hoc Bonferroni multiple comparisons tests: *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 3
Fig. 3
MEK inhibition abolishes the social dominance phenotype in Spred1-/- mice. A Mean percentage of matches won per day in the tube test across 5 days of tournaments between WT treated with DMSO vehicle and Spred1-/- mice treated with PD325901. Two tailed binomial tests compared to chance (50%): no significant differences day 1–5. n = 8 mice/genotype. B Mean percentage of matches won per day in the tube test across 5 days of tournaments between Spred1-/- treated with DMSO vehicle and Spred1-/- mice treated with PD325901, with significant differences on day 1–3 (Two tailed binomial test compared to chance (50%): day 1 p = 0.0011; day 2 p = 0.0011; day 3 p = 0.025). n = 6 mice/genotype. C Mean percentage of matches won per day in the tube test across 5 days of tournaments between WT treated with DMSO vehicle and WT mice treated with PD325901. Two tailed binomial tests compared to chance (50%): significant differences day 2–5; Two tailed binomial tests compared to chance (50%): day 2 p = 0.0003; day 3 p = 0.0003; day 4 p < 0.0001; day 5 p < 0.0001. n = 8 mice/genotype. All graphs are box and whisker with median and quartiles. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
MEK inhibition rescues impairments in nesting behavior in Spred1-/- mice. A WT and Spred1-/- mice were treated with PD325901 or DMSO vehicle for 3 days, then nest building was assessed using a qualitative nest building score. PD325901 significantly increased nesting score in Spred1-/- mice (2-way ANOVA: effect of PD325901 treatment F(1,26) = 28.81, p < 0.0001; effect of genotype F(1,26) = 6.679, p = 0.0157; no interaction). Post-hoc comparisons show a significant difference between WT + DMSO and Spred1-/- + DMSO groups (Sidak multiple comparison test, **p = 0.0066), whereas no difference was seen between WT + PD325901 and Spred1-/- + PD325901 groups (Sidak multiple comparison test, p = 0.8955). B After a 3-week washout period, nest building was assessed again (2-way ANOVA: no effect of treatment, effect of genotype F(1,26) = 15.99, p = 0.0005; no interaction). Mean ± SEM, n = 7–8 mice/genotype
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