Phenotypic characterization of the new Kctd13+/- mouse model and comparison with the 16p11.2 Del/+ mouse model
We created a new Kctd13 KO mouse model with the deletion of exon 3 and 4, leading to a frameshift at the transition of exon 2 and 5 (Sup Fig. 1A). We have validated the new allele by DNA analysis for the deletion, the decrease expression of KCDT13 in mutant hippocampi (Sup Fig. 1) and by phenotyping at the IMPC (www.mousephenotype.org). Then, we evaluated its learning and memory behaviour at 12 weeks of age in the open field, NOL and NOR, and compared the results obtained with the characterization of 16p11.2 Del/+ model (Fig. 2). First, we did not detect significant differences in the open field test (12 weeks), when we measured the total distance travelled by Kctd13+/− mice compared to wt littermates. This result was different from the phenotype of the Del/+ mice (Fig. 2A). Indeed Del/+ mutant mice were more active with a significant increase in the distance travelled compared to their wt littermates as previously observed . Then, we analysed the distance travelled in 5-minute intervals, to see the habituation to a new environment during the test. We found that the Kctd13+/− mice experience a similar habituation to the control individuals (Sup Fig. 2A). Here too, a significant difference was seen in the 16p11.2 Del/+ carrier mice. Athough being more active, the mutant mice showed normal habituation compared to their wild-type littermates.
Afterwards, we analysed the object location memory (Fig. 2B). For this test, animals were scored for their ability to distinguish between a previously presented object that stays in the same location and another object whose position was changed, after a retention delay of 5 min. The recognition indexes for the novel location object showed that, contrary to their wt littermates, both Kctd13+/− and 16p11.2 Del/+ mice were not able to differentiate between the novel and familiar location.
Finally, we investigated whether Kctd13+/− mice could discriminate a novel object from a previously explored set of two objects after a retention delay of 3 hours in the NOR task (Fig. 2C). Whereas wt animals were able to differentiate objects showing a novel object preference, Kctd13+/− mice were not able to discriminate the novel from the familiar object. The deficit was like the one seen in the 16p11.2 Del/+ mice. Overall, our behavioural analysis showed that the Kctd13 haploinsufficiency, in the pure C57BL/6N genetic background, phenocopied the object location and recognition memory deficits seen in the 16p11.2 deletion model. However, the increased exploration activity found in the 16p11.2 Del/+ mice was not seen in the Kctd13+/− mutant mice.
Fasudil treatment partially reverses the cognitive impairment in the Kctd13+/- and in the 16p11.2 Del/+ mouse models
Thus, after the behavioural characterization of the Kctd13+/− and 16p11.2 Del/+ mouse models, at 14 weeks of age, individuals from both genotypes were randomly assigned to a subgroup that was treated with fasudil or an untreated control group for 4 weeks prior to further behaviour testing (Fig. 1). First, we noticed that the fasudil treatment did not change the locomotor exploration activity of the different genotypes (Fig. 3A; Sup Fig. 3A) with Del/+ individuals travelling more than the control wt with no effect of the treatment (Fig. 3B, Sup Fig. 3B). Similar to earlier stage, the 18weeks old Kctd13 mutant mice were no more active in the open field than their control littermates. Interestingly, one month after the first phenotypic characterization, Kctd13+/− mice performed in the NOL (Fig. 3B) but with a lower recognition index compared to wt. In contrast, no improvement was observed at 18 weeks of age in 16p11.2 Del/+ mice (Fig. 3B). However, fasudil treatment significantly improved the NOR defect found in both Kctd13+/− and 16p11.2 Del/+ mice at 19 weeks of age (Fig. 3C). Here the altered recognition memory, found previously at 12 weeks, was seen again in mutants at 19 weeks,and the fasudil treatment had a rescuing effect both in the Kctd13+/− and the 16p11.2 Del/+ models. It also highlights that, at least one gene, deleted in the 16p11.2 Del/+ model, and different from Kctd13, contributes to the NOL phenotype in this model at this latter age.
Molecular analyses of RHOA / ROCK signalling pathway in the Kctd13+/- and the 16p11.2 Del/+ mouse models
Then we checked whether the RHOA / ROCK signalling pathway was changed in both 16p11.2 deletion and Kctd13 mouse models. Neither the loss of one copy of the complete chromosomic region in 16p11.2 Del/+ mice nor the deficiency of Kctd13 resulted in increased levels of RHOA in the hippocampal region at 18 weeks of age (Fig. 4A-B), nor at a change of MLC level in Kctd13 mutant versus wt control (Sup. Figure 4) Nevertheless, both models showed an over activation of the RHOA / ROCK pathway. Indeed, the level Myosin Light Chain (MLC), a protein targeted by the RHOA/ROCK pathway, was not showed increased levels of phosphorylation in the hippocampus of these mice, supporting the idea that this pathway could be associated to the cognitive phenotype observed (Fig. 4C and D; Sup table 4). Therefore, we verified whether fasudil therapeutic effect was acting through the inhibition of this RHOA/ROCK signalling pathway. Interestingly, the fasudil treatment reduced MLC phosphorylation levels in Kctd13 mutant individuals. As for the Kctd13+/- model, we found that in 16p11.2 deficient mice fasudil restored a normal MLC phosphorylation in treated mutant mice but surprisingly induced increased MLC phosphorylation in wt mice.