Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse

Abstract

Restless legs syndrome (RLS) is a neurological disorder characterized by an urge to move and uncomfortable sensations. Genetic studies have identified polymorphisms in up to 19 risk loci, including MEIS1 and BTBD9. Rodents deficient in either homolog show RLS-like phenotypes. However, whether MEIS1 and BTBD9 interact in vivo is unclear. Here, with C. elegans, we observed that the hyperactive egg-laying behavior caused by loss of BTBD9 homolog was counteracted by knockdown of MEIS1 homolog. This was further investigated in mutant mice with Btbd9, Meis1, or both knocked out. The double knockout mice showed an earlier onset of the motor deficit in the wheel running test but did not have increased sensitivity to the heat stimuli as observed in single KOs. Meis1 protein level was not influenced by Btbd9 deficiency, and Btbd9 transcription was not affected by Meis1 haploinsufficiency. Our results demonstrate that MEIS1 and BTBD9 do not regulate each other.

Introduction

RLS is characterized by a strong urge to move and uncomfortable sensations in lower limbs, which can be relieved by movements. Genome-wide association studies have implicated up to 19 risk loci for RLS, across which two of the candidate genes are MEIS1 and BTBD9 (Schormair et al., 2017).

Knockout animals of BTBD9 or MEIS1 homologs exhibit RLS-like phenotypes. For instance, homozygous Btbd9 knockout (KO) mice have motor restlessness, disrupted sleep, and altered sensory perception (DeAndrade et al., 2012). Loss of BTBD9 homolog in Drosophila melanogaster results in increased motor activity, decreased dopamine levels, and disrupted sleep (Freeman et al., 2012). Heterozygous Meis1 KO mice are hyperactive (Meneely et al., 2018). C. elegans with decreased MEIS1 homolog show increased expression of ferritin (Catoire et al., 2011). Therefore, both BTBD9 and MEIS1 may play a role in the development of RLS, yet whether and how the two genes interact is not known.

Objective

Our goal is to define the relationships between BTBD9 and MEIS1 in the pathogenesis of RLS. Egg retention assay in C. elegans was used to determine if there are genetic interactions between hpo-9, a BTBD9 homolog, and unc-62, a MEIS1 homolog. Furthermore, we created mouse models by knocking out BTBD9 homolog, Btbd9, MEIS1 homolog, Meis1, or both. Their motor-sensory responses were compared by wheel-running and tail-flick tests. Homozygous Meis1 KO was not included because of the embryonic lethality (Spieler et al., 2014). The transcription of Btbd9 in Meis1 KO and the level of Meis1 protein in Btbd9 KO were studied.

Methods

C. elegans were maintained using standard methods (Catoire et al., 2011). The wildtype (WT) used was Bristol N2. The hpo-9 KO, hpo-9(tm3719), was obtained from the National BioResource Project (Japan) and backcrossed four times to the N2 background. RNAi against unc-62 (unc-62 RNAi) and the empty vector (EV) were used according to a standard feeding method with HT115 bacterial strain. Egg retention assay was performed according to (Chase & Koelle, 2004) and analyzed by a Students’ t-test (supplementary material).

Heterozygous Btbd9 KO (Lyu et al., 2019) were bred with Meis1 KO (Meneely et al., 2018) to generate double heterozygotes, which were bred with heterozygous Btbd9 KO mice to generate experimental mice. Behavioral tests were conducted as described (Lyu et al., 2019) and analyzed by SAS GENMOD or mixed model ANOVA. Western blot and quantitative RT-PCR were performed and analyzed as described (Yokoi et al., 2015) using striatal tissues (supplementary material).

Results

Worms

Figure 1 shows that unc-62 knockdown led to an increased number of eggs retained in both N2 and hpo-9(tm3719) as (Kamath et al., 2003). The hpo-9 mutation caused fewer eggs retained in the presence or absence of unc-62 RNAi. Additionally, hpo-9(tm3719) treated with unc-62 RNAi retained a similar number of eggs as N2.

Figure 1.

Egg retention assay. Bars represent the mean ± standard error of the mean (SEM) for 12 animals for each strain. ***, p < 0.001.

Mice

During the light phase of the wheel running test, neither single KOs exhibited significant difference compared with the WT (Figure 2). However, the double KO showed a robust increase from the WT and a lesser extent of increase from both single KOs. During the dark phase, activity levels were similar among the four groups. Figure 3 shows that the double KO did not have changes in the tail-flick response although both single KOs had reduced latency. Moreover, Meis1 protein levels and Btbd9 mRNA levels were unaffected by Btbd9 knockout and Meis1 deficiency, respectively (Figure 4).

Figure 2.

Wheel running during the light phase (A), and the dark phase (B). The data was not normally distributed and analyzed by SAS GENMOD with a negative binomial distribution. In the scatter plot, each dot is an average value calculated from 4 days’ data for each mouse. Bars represent the median with 95% confidence intervals (CIs). Hourly activity is presented next to the scatter plot. Each dot is an average value calculated from 4 days’ data for each genotype. The activity of the double KO mice shot up right after the light was turned on and right before the light was turned off. In addition, they also showed high levels of activity around the middle of the rest period. The results indicate that the double KO mice may have difficulty in falling asleep and tend to wake up early. WT, n=7; Btbd9 KO, n=5; Meis1 KO, n=4; double KO, n=6. *, p < 0.05.

Figure 3.

Tail-flick test. The data were normally distributed and analyzed by mixed model ANOVA with repeated measurements. Each dot is an average value calculated from 3 trials for each mouse. Single KO had reduced latency compared with the WT but did not show a significant difference compared with the double KOs. The double KO did not have significant changes compared with the WT. Bars represent the mean ± SEM. WT, n=7; Btbd9 KO, n=5; Meis1 KO, n=4; double KO, n=6. *, p < 0.05.

Figure 4.

Molecular analysis. (A) Western blot to measure the amount of Meis1, normalized to β-actin, in Btbd9 KO (n=6) and WT (n=7) mice. (B) Quantitative real-time RT-PCR to test the level of Btbd9 mRNA, normalized to β-actin, in Meis1 KO (n=4) and WT (n=4) mice. Bars represent the mean ± SEM.

Discussion

Wheel-running data from day and night were analyzed separately because RLS symptoms mostly happen at night, which is the day for rodents. With animals at an average age of 3 months, we did not observe increased activity in either single KO as suggested before (DeAndrade et al., 2012; Meneely et al., 2018), indicating that the double KO had early-onset deficit while the single KOs were still asymptomatic. It has been shown that Btbd9 expression did not change by Meis1 deficiency (Spieler et al., 2014). This was confirmed by molecular analyses, suggesting that Btbd9 and Meis1 do not regulate each other.

Conclusion

In worms, the augmentation effect of unc-62 knockdown is independent of hpo-9 manipulation and it is also true the other way around. Moreover, hpo-9 knockout and unc-62 knockdown counteract with each other. In mice, the wheel running test suggests that there is an additive effect of Meis1 and Btbd9 mutations in the double KO mice. Btbd9 does not influence the Meis1 protein level, and Meis1 cannot alter Btbd9 gene expression. Hence, the two RLS risk genes work independently and have functional interactions in both worms and mice. Protein-protein interaction assays would be ideal to confirm this conclusion in the future.