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

      Results type: novel result
  • Shangru Lyu (a1), Atbin Doroodchi (a2), Yi Sheng (a3), Mark P. DeAndrade (a1), Youfeng Yang (a2), Yuning Liu (a1), Michael A. Miller (a2), Rui Xiao (a3) and Yuqing Li (a1)...

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.

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Copyright

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Corresponding author

*Corresponding author. E-mail: yuqing.li@neurology.ufl.edu

References

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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.

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Supplementary materials

Lyu et al. supplementary material
Lyu et al. supplementary material

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

      Results type: novel result
  • Shangru Lyu (a1), Atbin Doroodchi (a2), Yi Sheng (a3), Mark P. DeAndrade (a1), Youfeng Yang (a2), Yuning Liu (a1), Michael A. Miller (a2), Rui Xiao (a3) and Yuqing Li (a1)...

Reviewing Editor: Michael Nevels University of St Andrews, Biomolecular Sciences Building, Fife, United Kingdom of Great Britain and Northern Ireland, KY16 9ST

This article has been accepted because it is deemed to be scientifically sound, has the correct controls, has appropriate methodology and is statistically valid, and met required revisions.

Review 1: Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse

  • Aaro Salminen Helmholtz Zentrum München, Institute of Neurogenomics, Neuherberg, Germany
Date of review: 05 February 2020
 

Conflict of Interest Statement

Reviewer declares none

Comments

Comments to the Author: The manuscript details mouse and worm experiments on BTBD9 and MEIS1, two major candidate genes of RLS. As RLS is a polygenic disease, the double knock-out experiment is of great value in determining the relationship as well as a possible additive effect of these genes to the susceptibility to RLS. However, there are concerns relating to the analysis of the data which should be addressed.

In Figures 2 and 3, individual measurements resulting from repeated measurements in the same animal should not be displayed as individual data points, but should be first averaged within animal.

In statistical tests relating to Figs 2-3, was repeated measures ANOVA used? The test used should be stated in the legend. If so, the appropriate way to analyse the data would be to average first within animal and then use for example one-way ANOVA.

Division into light and dark period is used for wheel running activity, as is often done for this kind of data. Rather, the period right before and after the lights-on should be looked at. This would correspond better to human RLS and allow comparison with previous data in MEIS1 knock-out mice.

In the introduction, the sentence describing the human genetics of RLS is somewhat confusing and should be reformulated.

What does “4 repeats for each genotype” mean? Does this mean the last 4 days that were used in the analysis according to Lyu et al. 2019 or did the mice spend four times seven days in the running wheel?

Scorecard

Presentation

Overall Score 3.2 out of5

Is the article written in clear and proper English? (30%)
Score 4out of5
Is the data presented in the most useful manner? (40%)
Score 2out of5
Does the paper cite relevant and related articles appropriately? (30%)
Score 4out of5

Context

Overall Score 4.2 out of5

Does the title suitably represent the article? (25%)
Score 4out of5
Does the abstract correctly embody the content of the article? (25%)
Score 5out of5
Does the introduction give appropriate context? (25%)
Score 4out of5
Is the objective of the experiment clearly defined? (25%)
Score 4out of5

Analysis

Overall Score 3.8 out of5

Does the discussion adequately interpret the results presented? (40%)
Score 4out of5
Is the conclusion consistent with the results and discussion? (40%)
Score 4out of5
Are the limitations of the experiment as well as the contributions of the experiment clearly outlined? (20%)
Score 3out of5

Review 2: Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse

  • Guy A. Rouleau McGill University, Neurology and Neurosurgery, Montreal, Canada, H3A 0G4
Date of review: 05 February 2020
 

Conflict of Interest Statement

Reviewer declares none.

Comments

Comments to the Author: In this paper Lyu et al. investigated the impact of two RLS candidate genes in the sensory-motor characteristics of C. elegans and mice. They also probed the potential interactions of these two genes based on the sensory-motor effects of their double KOs in the animals. This is an interesting paper that can provide useful information on the roles of MEIS1 and BTBD9 as two significant genes in RLS genetics.

The paper is well written, and the experiments are well conducted. The Western blot analysis results are not included in the supplemental data, and only graphs with quantitative measurement of the protein levels are provided.

Minor revisions:

  1. 1. Lines 39-41 "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." Based on publications by Salminen et al. 2017, Meneely et al. 2018 and Spieler et al. 2014, the sensitivity to heat stimuli was not observed in mice with Meis1 deficiency. The authors could provide references supporting the observation of increased sensitivity to heat stimuli in BOTH KOs.

  2. 2. Could the authors elaborate on why Meis1 was only measured at protein level (WB), but Btbd9 at mRNA level (q-RT-PCR)? Would have been ideal if both genes were measured at both levels. The reason and limitations should be discussed in the paper.

  3. 3. Lines 84-85 "With or without unc-62 RNAi, hpo-9(tm3719) retained fewer eggs than N2." According to Figure 1, “N2” should be “N2(unc-62)”.

  4. 4. To make it easier for the readers, in the results section, it should be more clearly stated that which paragraph refers to which animal.

  5. 5. Line 87 "fo" is a typo?

  6. 6. Lines 90-91. The two sentences should be separated more clearly. The second sentence seems to continue the previous sentence about dark phase activity.

  7. 7. Line 122. "the two RLS risk genes work independently and have functional interactions in both worms and mice." The authors are suggesting that there are functional interactions between the two proteins. A protein-protein interaction assay would be ideal to confirm this interaction. If not feasible, the limitations of this conclusion should be discussed in the manuscript.

  8. 8. Please provide all the original Western blot figures in the supplemental data.

Scorecard

Presentation

Overall Score 4.6 out of5

Is the article written in clear and proper English? (30%)
Score 5out of5
Is the data presented in the most useful manner? (40%)
Score 4out of5
Does the paper cite relevant and related articles appropriately? (30%)
Score 5out of5

Context

Overall Score 4.8 out of5

Does the title suitably represent the article? (25%)
Score 4out of5
Does the abstract correctly embody the content of the article? (25%)
Score 5out of5
Does the introduction give appropriate context? (25%)
Score 5out of5
Is the objective of the experiment clearly defined? (25%)
Score 5out of5

Analysis

Overall Score 4.0 out of5

Does the discussion adequately interpret the results presented? (40%)
Score 4out of5
Is the conclusion consistent with the results and discussion? (40%)
Score 4out of5
Are the limitations of the experiment as well as the contributions of the experiment clearly outlined? (20%)
Score 4out of5

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