Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome

Abstract

Mutations in MECP2, encoding methyl CpG-binding protein 2, cause Rett syndrome, the most severe autism spectrum disorder. Re-expressing Mecp2 in symptomatic Mecp2-null mice markedly improves function and longevity, providing hope that therapeutic intervention is possible in humans. To identify pathways in disease pathology for therapeutic intervention, we carried out a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis suppressor screen in Mecp2-null mice and isolated five suppressors that ameliorate the symptoms of Mecp2 loss. We show that a stop codon mutation in Sqle, encoding squalene epoxidase, a rate-limiting enzyme in cholesterol biosynthesis, underlies suppression in one line. Subsequently, we also show that lipid metabolism is perturbed in the brains and livers of Mecp2-null male mice. Consistently, statin drugs improve systemic perturbations of lipid metabolism, alleviate motor symptoms and confer increased longevity in Mecp2 mutant mice. Our genetic screen therefore points to cholesterol homeostasis as a potential target for the treatment of patients with Rett syndrome.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A dominant suppressor screen shows inheritance of longevity in five lines.
Figure 2: Survival curves for each line with a confirmed map location for the modifier are shown assessed at the N3 generation.
Figure 3: A stop codon mutation in Sqle confers rescue at Sum3m1Jus.
Figure 4: Cholesterol metabolism is disrupted in Mecp2-null male mice.
Figure 5: Statin treatment improves health in 129.
Figure 6: Fluvastatin treatment improves health in 129.

Similar content being viewed by others

References

  1. Amir, R.E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG–binding protein 2. Nat. Genet. 23, 185–188 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Bienvenu, T. & Chelly, J. Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Natl. Rev. Genet. 7, 415–426 (2006).

    Article  CAS  Google Scholar 

  3. Guy, J., Hendrich, B., Holmes, M., Martin, J.E. & Bird, A. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat. Genet. 27, 322–326 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Shepherd, G.M. & Katz, D.M. Synaptic microcircuit dysfunction in genetic models of neurodevelopmental disorders: focus on Mecp2 and Met. Curr. Opin. Neurobiol. 21, 827–833 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kavalali, E.T., Nelson, E.D. & Monteggia, L.M. Role of MeCP2, DNA methylation, and HDACs in regulating synapse function. J. Neurodev. Disord. 3, 250–256 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Guy, J., Gan, J., Selfridge, J., Cobb, S. & Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 315, 1143–1147 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Collins, A.L. et al. Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Hum. Mol. Genet. 13, 2679–2689 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224–1229 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Stancheva, I., Collins, A.L., Van den Veyver, I.B., Zoghbi, H. & Meehan, R.R. A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol. Cell 12, 425–435 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. St Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nature Rev. Genet. 3, 176–188 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Carpinelli, M.R. et al. Suppressor screen in Mpl−/− mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling. Proc. Natl. Acad. Sci. USA 101, 6553–6558 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Matera, I. et al. A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy. Hum. Mol. Genet. 17, 2118–2131 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Justice, M.J., Siracusa, L.D. & Stewart, A.F. Technical approaches for mouse models of human disease. Dis. Model. Mech. 4, 305–310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Derecki, N.C. et al. Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature 484, 105–109 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Neuhaus, I.M. & Beier, D.R. Efficient localization of mutations by interval haplotype analysis. Mamm. Genome 9, 150–154 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Moran, J.L. et al. Utilization of a whole genome SNP panel for efficient genetic mapping in the mouse. Genome Res. 16, 436–440 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fairfield, H. et al. Mutation discovery in mice by whole exome sequencing. Genome Biol. 12, R86 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jurevics, H.A., Kidwai, F.Z. & Morell, P. Sources of cholesterol during development of the rat fetus and fetal organs. J. Lipid Res. 38, 723–733 (1997).

    CAS  PubMed  Google Scholar 

  19. Zlokovic, B.V. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Gill, S., Stevenson, J., Kristiana, I. & Brown, A.J. Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase. Cell Metab. 13, 260–273 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Cory, E.J., Russey, W.E. & Ortiz de Montellano, P.R. 2,3-oxidosqualene, an intermediate in the biological synthesis of sterols from squalene. J. Am. Chem. Soc. 88, 4750–4751 (1966).

    Article  CAS  PubMed  Google Scholar 

  22. Yamamoto, S. & Bloch, K. Studies on squalene epoxidase of rat liver. J. Biol. Chem. 245, 1670–1674 (1970).

    CAS  PubMed  Google Scholar 

  23. Shibata, N. et al. Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis. Proc. Natl. Acad. Sci. USA 98, 2244–2249 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Astruc, M., Tabacik, C., Descomps, B. & de Paulet, A.C. Squalene epoxidase and oxidosqualene lanosterol-cyclase activities in cholesterogenic and non-cholesterogenic tissues. Biochim. Biophys. Acta 487, 204–211 (1977).

    Article  CAS  PubMed  Google Scholar 

  25. Ingham, P.W., Nakano, Y. & Seger, C. Mechanisms and functions of Hedgehog signalling across the metazoa. Natl. Rev. Genet. 12, 393–406 (2011).

    Article  CAS  Google Scholar 

  26. Posé, D. & Botella, M.A. Analysis of the Arabidopsis dry2/sqe1-5 mutant suggests a role for sterols in signaling. Plant Signal. Behav. 4, 873–874 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Posé, D. et al. Identification of the Arabidopsis dry2/sqe1-5 mutant reveals a central role for sterols in drought tolerance and regulation of reactive oxygen species. Plant J. 59, 63–76 (2009).

    Article  PubMed  Google Scholar 

  28. Nieweg, K., Schaller, H. & Pfrieger, F.W. Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. J. Neurochem. 109, 125–134 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Dietschy, J.M., Turley, S.D. & Spady, D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J. Lipid Res. 34, 1637–1659 (1993).

    CAS  PubMed  Google Scholar 

  30. Dietschy, J.M. Central nervous system: cholesterol turnover, brain development and neurodegeneration. Biol. Chem. 390, 287–293 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Russell, D.W., Halford, R.W., Ramirez, D.M., Shah, R. & Kotti, T. Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain. Annu. Rev. Biochem. 78, 1017–1040 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chen, R.Z., Akbarian, S., Tudor, M. & Jaenisch, R. Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat. Genet. 27, 327–331 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Pfrieger, F.W. & Ungerer, N. Cholesterol metabolism in neurons and astrocytes. Prog. Lipid Res. 50, 357–371 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Xie, C., Lund, E.G., Turley, S.D., Russell, D.W. & Dietschy, J.M. Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration. J. Lipid Res. 44, 1780–1789 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Ko, M. et al. Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons. J. Biol. Chem. 280, 42759–42765 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Jolley, C.D., Dietschy, J.M. & Turley, S.D. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: effect on cholesterol responsiveness. Am. J. Physiol. 276, G1117–G1124 (1999).

    CAS  PubMed  Google Scholar 

  37. Bellosta, S., Paoletti, R. & Corsini, A. Safety of statins: focus on clinical pharmacokinetics and drug interactions. Circulation 109, III50–III57 (2004).

    Article  PubMed  CAS  Google Scholar 

  38. García-Sabina, A., Gulin-Davila, J., Sempere-Serrano, P., Gonzalez-Juanatey, C. & Martinez-Pacheco, R. Specific considerations on the prescription and therapeutic interchange of statins. Farm. Hosp. 36, 97–108 (2012).

    Article  PubMed  Google Scholar 

  39. Osterweil, E.K. et al. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron 77, 243–250 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ardern-Holmes, S.L. & North, K.N. Therapeutics for childhood neurofibromatosis type 1 and type 2. Curr. Treat. Options Neurol. 13, 529–543 (2011).

    Article  PubMed  Google Scholar 

  41. Keber, R. et al. Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14α-demethylase (Cyp51) resembles Antley-Bixler syndrome. J. Biol. Chem. 286, 29086–29097 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Waterham, H.R. Defects of cholesterol biosynthesis. FEBS Lett. 580, 5442–5449 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Björkhem, I. & Hansson, M. Cerebrotendinous xanthomatosis: an inborn error in bile acid synthesis with defined mutations but still a challenge. Biochem. Biophys. Res. Commun. 396, 46–49 (2010).

    Article  PubMed  CAS  Google Scholar 

  44. Lund, E.G. et al. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover. J. Biol. Chem. 278, 22980–22988 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Lioy, D.T. et al. A role for glia in the progression of Rett's syndrome. Nature 475, 497–500 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Vance, J.E. Dysregulation of cholesterol balance in the brain: contribution to neurodegenerative diseases. Dis. Model. Mech. 5, 746–755 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Paolicelli, R.C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–1458 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Cibičkova, L. Statins and their influence on brain cholesterol. J. Clin. Lipidol. 5, 373–379 (2011).

    Article  PubMed  Google Scholar 

  49. Stranahan, A.M., Cutler, R.G., Button, C., Telljohann, R. & Mattson, M.P. Diet-induced elevations in serum cholesterol are associated with alterations in hippocampal lipid metabolism and increased oxidative stress. J. Neurochem. 118, 611–615 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Day, C.P. & James, O.F. Steatohepatitis: a tale of two “hits?”. Gastroenterology 114, 842–845 (1998).

    Article  CAS  PubMed  Google Scholar 

  51. Fyffe, S.L. et al. Deletion of Mecp2 in Sim1-expressing neurons reveals a critical role for MeCP2 in feeding behavior, aggression, and the response to stress. Neuron 59, 947–958 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Percy, A.K. Rett syndrome: exploring the autism link. Arch. Neurol. 68, 985–989 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Lyst, M.J. et al. Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT transcriptional co-repressor. Nat. Neurosci. 16, 898–902 (2013).

    Article  CAS  PubMed  Google Scholar 

  54. Ebert, D.H. et al. Activity-dependent phosphorylation of MECP2 threonine 308 regulates interaction with NcoR. Nature published online; doi:10.1038/nature12348 (16 June 2013).

  55. Knutson, S.K. et al. Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks. EMBO J. 27, 1017–1028 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Sun, Z. et al. Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration. Nat. Med. 18, 934–942 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Feng, D. et al. A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism. Science 331, 1315–1319 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kile, B.T. et al. Functional genetic analysis of mouse chromosome 11. Nature 425, 81–86 (2003).

    Article  CAS  PubMed  Google Scholar 

  59. McDonald, J.G., Smith, D.D., Stiles, A.R. & Russell, D.W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53, 1399–1409 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method. Methods 25, 402–408 (2001).

    CAS  PubMed  Google Scholar 

  61. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    PubMed  PubMed Central  Google Scholar 

  62. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  63. Broman, K.W., Wu, H., Sen, S. & Churchill, G.A. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19, 889–890 (2003).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The Genetics Analysis Facility (T. Patton and C. Marshall) at the Centre for Applied Genomics, Toronto Hospital for Sick Kids, Toronto, Ontario, Canada performed the Illumina Goldengate SNP analysis. We thank J. Crowe, M. Schrock, J. Borkey, M. Hill, A. Willis and J. Shaw (Justice laboratory), C. Lee, A. MacKenzie and L. Felker (Shendure laboratory), I. Adams (Katz laboratory), B. Thompson (McDonald and Russell laboratory) and K.S. Posey and A.M. Lopez (Turley laboratory) for technical assistance. We thank C. Spencer and R. Paylor for advice on assessing mouse behavior, which was carried out in the Baylor College of Medicine (BCM) Mouse Neurobehavior Core, and C. Reynolds for advice on plethysmography, which was carried out in the BCM Mouse Phenotyping Core. We thank H. Zoghbi and J. Neul (BCM) for Mecp2 mutant mice. We also thank H. Zoghbi (BCM) and R. Behringer (University of Texas MD Anderson Cancer Center) for valuable discussions during revision of the manuscript. M. Coenraads of the Rett Syndrome Research Trust (RSRT) provided crucial moral and uninterrupted financial support while she aided intellectually through literature searches and advice.

The work was supported by grants from the RSRT, the Rett Syndrome Research Foundation, the International Rett Syndrome Foundation (ANGEL award 2608 to M.J.J. and ANGEL award 2583 to D.M.K.), Autism Science Foundation predoctoral fellowship #11-1015, US National Institutes of Health (NIH) grants NIH T32 GM08307 to C.M.B., NIH U54 GM69338 to D.W.R., NIH R01 HL09610 to S.D.T. and NIH R01 CA115503 to M.J.J. and the National Institute of Neurologic Diseases and Stroke, including funding from the American Recovery and Reinvestment Act (D.M.K.). Grants to the BCM Diabetes and Endocrinology Research Center (2P30DK079638-05) and the BCM Intellectual and Developmental Disabilities Research Center (5P30HD024064-23) from the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development also supported this work. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health and Human Development or the NIH.

Author information

Authors and Affiliations

Authors

Contributions

M.J.J. conceived of the work, carried out the genetic screen and dissected embryos. J.S. and W.H. carried out the capture sequencing and analysis. C.M.B. confirmed map locations and lesions, performed statin injections and carried out behavior and plethysmography testing and quantitative RT-PCR (qRT-PCR). S.M.K. performed protein blotting and liver histopathology. H.M.B. performed preliminary qRT-PCR. J.G.M., B.L. and S.D.T. analyzed sterols and performed synthesis studies. S.D.T. evaluated liver cholesterol and triglycerides. A.A.P. and D.M.K. provided Jaenisch mice and laboratory facilities. D.M.K. helped analyze plethysmography data. M.J.J., D.W.R., D.M.K., S.D.T., S.M.K. and C.M.B. wrote the manuscript with input from the other coauthors.

Corresponding author

Correspondence to Monica J Justice.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary figures 1-10 and supplementary tables 1-5 (PDF 6794 kb)

Statin treatment improves home cage activity

30 second videos of mice treated with lovastatin and vehicle at P56 showing an increase in home cage activity in statin treated Mecp2tm1.1Bird/Y mice immediately following removal of the cage lid. (MOV 104247 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Buchovecky, C., Turley, S., Brown, H. et al. A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome. Nat Genet 45, 1013–1020 (2013). https://doi.org/10.1038/ng.2714

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.2714

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research