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

Journal name:
Nature Genetics
Volume:
45,
Pages:
1013–1020
Year published:
DOI:
doi:10.1038/ng.2714
Received
Accepted
Published online

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.

At a glance

Figures

  1. A dominant suppressor screen shows inheritance of longevity in five lines.
    Figure 1: A dominant suppressor screen shows inheritance of longevity in five lines.

    (a) ENU-treated C57BL/6J males were mated to 129.Mecp2tm1.1Bird/+ females. The G1 Mecp2tm1.1Bird/Y males (boxed) were aged and assessed for forelimb and hindlimb clasping, tremors, body size, cage activity and longevity. +/+, female wild-type mice; +/Y, male wild-type mice. (b) N2 mice from five lines (352 (Sum1m1Jus; blue), 856 (Sum2m1Jus; green), 895 (Sum3m1Jus; red), 1395 (Sum4m1Jus; brown) and 1527 (Sum5m1Jus; purple)) produced N3 offspring that showed increased longevity. Closed circles represent offspring of a male N2 parent, and open circles represent offspring of a female N2 parent. The longevity of the G1 founder of each line is indicated by the colored squares. Mecp2tm1.1Bird/Y mice rarely survive past 120 d (horizontal dashed line).

  2. Survival curves for each line with a confirmed map location for the modifier are shown assessed at the N3 generation.
    Figure 2: Survival curves for each line with a confirmed map location for the modifier are shown assessed at the N3 generation.

    (ad) Survival of Mecp2tm1.1Bird/Y mice is significantly increased by the presence of each suppressing mutation in lines 352 (Sum1m1Jus; P = 0.001) (a), 856 (Sum2m1Jus; P = 0.005) (b), 895 (Sum3m1Jus; P = 0.002) (c) and 1395 (Sum4m1Jus; P = 0.016) (d). (Kaplan-Meier analysis followed by log-rank comparisons.)

  3. A stop codon mutation in Sqle confers rescue at Sum3m1Jus.
    Figure 3: A stop codon mutation in Sqle confers rescue at Sum3m1Jus.

    (a) The ENU-induced Sqle p.Arg399X mutation occurs in exon 7 in the squalene epoxidase domain. (b) Sqle is not expressed in homozygous E8.0 mutant embryos (SqleSum3Jus/SqleSum3Jus). SqleΔ7 is the short predicted transcript lacking exon 7. (c) Protein blot of stage-matched E8.0 embryos showing that the expected 64-kDa protein and the 36-kDa degradation product are absent in homozygous mutant embryos. (d) Expression of Sqle, but not Hmgcr, is decreased in SqleSum3Jus/+ brains. (e) Gene expression is unchanged by SqleSum3Jus/+ in the liver. (f) Brain concentration of the cholesterol precursors lanosterol and desmosterol is decreased by SqleSum3Jus/+. (g) Serum cholesterol concentration is unchanged by SqleSum3Jus/+. All tissue analyses (dg) were performed at P70 (n = 6 mice per group). (h,i) Mecp2tm1.1Bird/Y; SqleSum3Jus/+ mice at backcross generation N7 to 129S6/SvEvTac mice show significantly improved rotarod performance at P56 (P = 0.0001) (h) and improved open-field activity at P70 (i). *P ≤ 0.05, #P ≤ 0.08. One-way analysis of variance (ANOVA) with Bonferroni adjustment was used for the data analyses in b, dg and i. The data in h were analyzed using repeated measures ANOVA. All error bars represent the s.e.m.

  4. Cholesterol metabolism is disrupted in Mecp2-null male mice.
    Figure 4: Cholesterol metabolism is disrupted in Mecp2-null male mice.

    (a) A simplified schematic of the enzymes and products in cholesterol biosynthesis through desmosterol is shown. (b) Expression of Hmgcr, Sqle and Cyp46a1 is similar in the brains of Mecp2tm1.1Bird/Y and Mecp2tm1.1Jae/Y mice. (c) Lanosterol (Lan), desmosterol (Des) and total cholesterol (TC) concentrations are shown per gram of brain tissue at P56 (n = 8 mice per group) and P70 (n = 4 mice per group). (d) Cholesterol synthesis is decreased in Mecp2tm1.1Jae/Y brains at P56 (wild type, n = 4; null, n = 5). (e) Expression of Hmgcr and Sqle differs in the livers of Mecp2tm1.1Bird/Y and Mecp2tm1.1Jae/Y mice. (f) Triacylglyceride (TAG) and total cholesterol concentrations are shown per gram of liver tissue at P56 (n = 6 mice per group). (g) Cholesterol synthesis is slightly increased in Mecp2tm1.1Jae/Y livers per gram of tissue at P56 (wild type, n = 4; null, n = 5). In bg, gray represents the Mecp2tm1.1Bird line, and brown represents the Mecp2tm1.1Jae line. (hj) Total cholesterol (h), LDL cholesterol (i) and triglyceride (j) concentrations in serum are significantly higher in Mecp2tm1.1Bird/Y mice by P56 but are unchanged in Mecp2tm1.1Jae/Y mice (n = 6 mice per group). In hj, black represents 129 +/Y mice, brown represents B6 +/Y mice, gray represents 129.Mecp2tm1.1Bird/Y mice, and light tan represents B6.Mecp2tm1.1Jae/Y mice. For the gene expression data in b and e, n = 6 mice per genotype at P28 and 12 mice per genotype at P56 for Mecp2tm1.1Bird, and n = 4 mice per genotype at P28 and 6 mice per genotype at P56 for Mecp2tm1.1Jae. Tissue data (bg) show the percentage change from wild-type levels. *P ≤ 0.05. Statistical analyses were performed using Student's t test. All error bars represent the s.e.m.

  5. Statin treatment improves health in 129.
    Figure 5: Statin treatment improves health in 129.

    Mecp2tm1.1Bird/Y male mice. (a) Fluvastatin treatment of 129.Mecp2tm1.1Bird/Y mice confers increased longevity (median lifespan of 122 d compared to 87 d, with 57% survival beyond 120 d; P < 0.0001). Three mice were euthanized because of dermatitis (boxes) while active and otherwise healthy. (b) Rotarod performance improves in P56 statin-treated null male mice (fluvastatin, P = 0.015; lovastatin, P = 0.009). (c) Open-field activity is increased in P70 statin-treated null males as assessed by beam breaks (fluvastatin, P = 0.011; lovastatin, P = 0.049). (d) Statin treatment lowers plasma cholesterol concentrations by P70 (fluvastatin, P = 0.001; lovastatin, P = 0.001). (e) Statin treatment ameliorates elevated lipid concentrations in 129.Mecp2tm1.1Bird/Y livers at P70 (fluvastatin, P = 0.020; lovastatin, P = 0.386). The numbers of total mice assessed in ad were 37 Mecp2tm1.1Bird/Y fluvastatin-treated mice, 12 Mecp2tm1.1Bird/Y lovastatin-treated mice, 31 Mecp2tm1.1Bird/Y vehicle-treated mice, 29 wild-type +/Y fluvastatin-treated mice, 8 +/Y lovastatin-treated mice and 29 wild-type +/Y vehicle-treated mice. (f,g) The concentration of lanosterol slightly increases (f) and that of desmosterol significantly increases (g) in the brains of fluvastatin-treated 129.Mecp2tm1.1Bird/Y mice at P70 (n = 4 mice per group; P = 0.042). The data in b were analyzed using repeated measures ANOVA. One-way ANOVA was used for the data analyses in cg. *P ≤ 0.05 determined using Dunnett's post-hoc test to compare statin- with vehicle-treated groups. All error bars represent the s.e.m.

  6. Fluvastatin treatment improves health in 129.
    Figure 6: Fluvastatin treatment improves health in 129.

    Mecp2tm1.1Bird/+ female mice. (a) No fluvastatin-treated 129.Mecp2tm1.1Bird/+ female mice died before 8 months of age, but three vehicle-treated female mice died. (b) Rotarod performance improves in 5-month-old fluvastatin-treated 129.Mecp2tm1.1Bird/+ female mice (P = 0.001). (c) Open-field activity assessed at 4 months of age shows no significant differences in the fluvastatin- and vehicle-treated groups. (d) Fluvastatin treatment does not significantly change serum cholesterol concentrations at 8 months of age. (e) Fluvastatin treatment ameliorates elevated lipid concentrations in 129.Mecp2tm1.1Bird/+ livers assessed at 8 months of age (*P = 0.045). The data in b were analyzed using repeated measures ANOVA. One-way ANOVA was used for the data analyses in ce. *P ≤ 0.05 determined using Dunnett's post-hoc test to compare statin- with vehicle-treated groups. All error bars represent the s.e.m.

Videos

  1. Statin treatment improves home cage activity
    Video 1: 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.

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Author information

Affiliations

  1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

    • Christie M Buchovecky,
    • Hannah M Brown,
    • Stephanie M Kyle &
    • Monica J Justice
  2. Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas, USA.

    • Stephen D Turley &
    • Benny Liu
  3. Department of Molecular Genetics, University of Texas Southwestern Medical School, Dallas, Texas, USA.

    • Jeffrey G McDonald &
    • David W Russell
  4. Department of Psychiatry, University of Texas Southwestern Medical School, Dallas, Texas, USA.

    • Andrew A Pieper
  5. Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas, USA.

    • Andrew A Pieper
  6. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Wenhui Huang &
    • Jay Shendure
  7. Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

    • David M Katz
  8. Present addresses: Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, South Australia, Australia (H.M.B.), Department of Psychiatry, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA (A.A.P.), Department of Neurology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA (A.A.P.) and Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (W.H.).

    • Hannah M Brown,
    • Andrew A Pieper &
    • Wenhui Huang

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.

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The authors declare no competing financial interests.

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Supplementary information

Video

  1. Video 1: Statin treatment improves home cage activity (101.8 MB, Download)
    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.

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  1. Supplementary Text and Figures (6,957.7 KB)

    Supplementary figures 1-10 and supplementary tables 1-5

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