Skip to main content Accessibility help
×
Home

Information:

  • Access
  • Open access

Figures:

Actions:

      • Send article to Kindle

        To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

        Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        Probing the relationship between BTBD9 and MEIS1 in C. elegans and mouse
        Available formats
        ×

        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

        Probing the relationship between BTBD9 and MEIS1 in C. elegans and mouse
        Available formats
        ×

        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

        Probing the relationship between BTBD9 and MEIS1 in C. elegans and mouse
        Available formats
        ×
Export citation

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 a wheel running test but did not have increased sensitivity to heat stimuli as observed in single knock outs. 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., Reference Schormair, Zhao, Bell, Tilch, Salminen, Putz, Dauvilliers, Stefani, Högl, Poewe, Kemlink, Sonka, Bachmann, Paulus, Trenkwalder, Oertel, Hornyak, Teder-Laving, Metspalu and Winkelmann2017).

Knockout (KO) animals of BTBD9 or MEIS1 homologs exhibit RLS-like phenotypes. For instance, homozygous Btbd9 KO mice have motor restlessness, disrupted sleep, and altered sensory perception (DeAndrade et al., Reference DeAndrade, Johnson, Unger, Zhang, L., van Groen, Gamble and Li2012). Loss of BTBD9 homolog in Drosophila melanogaster results in increased motor activity, decreased dopamine levels, and disrupted sleep (Freeman et al., Reference Freeman, Pranski, Miller, Radmard, Bernhard, Jinnah, Betarbet, Rye and Sanyal2012). Heterozygous Meis1 KO mice are hyperactive (Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018). C. elegans with decreased MEIS1 homolog show increased expression of ferritin (Catoire et al., Reference Catoire, Dion, Xiong, Amari, Gaudet, Girard, Noreau, Gaspar, Turecki, Montplaisir, Parker and Rouleau2011). 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 was 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 embryonic lethality (Spieler et al., Reference Spieler, Kaffe, Knauf, Bessa, Tena, Giesert, Schormair, Tilch, Lee, Horsch, Czamara, Karbalai, Toerne, Waldenberger, Gieger, Lichtner, Claussnitzer, Naumann, Müller-Myhsok and Winkelmann2014). The transcription of Btbd9 in Meis1 KO and the level of Meis1 protein in Btbd9 KO animals were studied.

Methods

C. elegans were maintained using standard methods (Catoire et al., Reference Catoire, Dion, Xiong, Amari, Gaudet, Girard, Noreau, Gaspar, Turecki, Montplaisir, Parker and Rouleau2011). 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 published protocols (Chase & Koelle, Reference Chase and Koelle2004) and analyzed by a Students’ t-test (supplementary material).

Heterozygous Btbd9 KO (Lyu et al., Reference Lyu, Xing, DeAndrade, Liu, Perez, Yokoi, Febo, Walters and Li2019) were bred with Meis1 KO animals (Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018) 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., Reference Lyu, Xing, DeAndrade, Liu, Perez, Yokoi, Febo, Walters and Li2019) and analyzed by SAS GENMOD or mixed model ANOVA. Western blot and quantitative RT-PCR were performed and analyzed as described (Yokoi et al., Reference Yokoi, Dang, Liu, Gandre, Kwon, Yuen and Li2015) 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 described (Kamath et al., Reference Kamath, Fraser, Dong, Poulin, Durbin, Gotta, Kanapin, Le Bot, Moreno, Sohrmann, Welchman, Zipperlen and Ahringer2003). 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 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., Reference DeAndrade, Johnson, Unger, Zhang, L., van Groen, Gamble and Li2012; Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018), indicating that the double KO had early-onset deficit while the single KOs were still asymptomatic. It has been shown that Btbd9 expression does not change by Meis1 deficiency (Spieler et al., Reference Spieler, Kaffe, Knauf, Bessa, Tena, Giesert, Schormair, Tilch, Lee, Horsch, Czamara, Karbalai, Toerne, Waldenberger, Gieger, Lichtner, Claussnitzer, Naumann, Müller-Myhsok and Winkelmann2014). 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 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.

Acknowledgments

We would like to thank Drs. Neil Copeland and Hesham Sadek for supplying Meis1 loxP mice, Dr. Shohei Mitani for the hpo-9(tm3719) strain, and Fumiaki Yokoi, Lin Zhang, Chad C. Cheetham, Sung Min Han, Jack Vibbert, Pauline Cottee, Jessica Winek, and Hieu Hoang for their technical assistance and stimulating discussions.

Author Contributions

SL, MPD, MAM, RX and YLi conceived and designed the study. SL, AD, YS, MPD, YY and YLiu conducted data gathering. SL and YLiu performed statistical analyses. SL and YLi wrote the article.

Funding Information

This work was supported by a grant from Restless Legs Syndrome Foundation; startup funds from the Departments of Neurology at UAB and UF; and the National Institutes of Health (grant numbers NS37409, NS47466, NS47692, NS54246, NS57098, NS65273, NS72872, NS74423, and NS82244). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publishing Ethics

The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.

Conflicts of Interest

All authors declare none.

Data Availability Statements

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Materials

To view supplementary material for this article, please visit http://dx.doi.org/10.1017/exp.2020.12.

References

Catoire, H., Dion, P. A., Xiong, L., Amari, M., Gaudet, R., Girard, S. L., Noreau, A., Gaspar, C., Turecki, G., Montplaisir, J. Y., Parker, J. A., & Rouleau, G. A. (2011). Restless legs syndrome-associated MEIS1 risk variant influences iron homeostasis. Annals of Neurology, 70, 170175.
Chase, D. L., & Koelle, M. R. (2004). Genetic analysis of RGS protein function in Caenorhabditis elegans. Methods in Enzymology, 389, 305320.
DeAndrade, M. P., Johnson, R. L., Jr., Unger, E. L., Zhang, , L., van Groen, T., Gamble, K. L., & Li, Y. (2012). Motor restlessness, sleep disturbances, thermal sensory alterations and elevated serum iron levels in Btbd9 mutant mice. Human Molecular Genetics , 21, 39843992.
Freeman, A., Pranski, E., Miller, R. D., Radmard, S., Bernhard, D., Jinnah, H. A., Betarbet, R., Rye, D. B., & Sanyal, S. (2012). Sleep fragmentation and motor restlessness in a Drosophila model of restless legs syndrome. Current Biology, 22, 11421148.
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P., & Ahringer, J. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421, 231237.
Lyu, S., Xing, H., DeAndrade, M. P., Liu, Y., Perez, P. D., Yokoi, F., Febo, M., Walters, A. S., & Li, Y. (2019). The role of BTBD9 in striatum and restless legs syndrome. eNeuro, 6, 0277-19.2019.
Meneely, S., Dinkins, M. L., Kassai, M., Lyu, S., Liu, Y., Lin, C. T., Brewer, K., Li, Y., & Clemens, S. (2018). Differential dopamine D1 and D3 receptor modulation and expression in the spinal cord of two mouse models of restless legs syndrome. Frontiers in Behavioral Neuroscience, 12, 199.
Schormair, B., Zhao, C., Bell, S., Tilch, E., Salminen, A. V., Putz, B., Dauvilliers, Y., Stefani, A., Högl, B., Poewe, W., Kemlink, D., Sonka, K., Bachmann, C. G., Paulus, W., Trenkwalder, C., Oertel, W. H., Hornyak, M., Teder-Laving, M., Metspalu, A., & Winkelmann, J. (2017). Identification of novel risk loci for restless legs syndrome in genome-wide association studies in individuals of European ancestry: A meta-analysis. The Lancet Neurology, 16, 898907.
Spieler, D., Kaffe, M., Knauf, F., Bessa, J., Tena, J. J., Giesert, F., Schormair, B., Tilch, E., Lee, H., Horsch, M., Czamara, D., Karbalai, N., von, Toerne, C., Waldenberger, M., Gieger, C., Lichtner, P., Claussnitzer, M., Naumann, R., Müller-Myhsok, B., Winkelmann, J. (2014). Restless legs syndrome-associated intronic common variant in MEIS1 alters enhancer function in the developing telencephalon. Genome Research, 24, 592603.
Yokoi, F., Dang, M. T., Liu, J., Gandre, J. R., Kwon, K., Yuen, R., & Li, Y. (2015). Decreased dopamine receptor 1 activity and impaired motor-skill transfer in Dyt1 DeltaGAG heterozygous knock-in mice. Behavioural Brain Research, 279, 202210.