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.

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

a) ENU-treated C57BL/6J males were mated to 129.Mecp2^tm1.1Bird/+ females. G₁ Mecp2^tm1.1Bird/Y males were aged and assessed for fore- and hindlimb clasping, tremors, body size, cage activity, and longevity. b) N₂ animals from five lines (352 (Sum1^m1Jus; blue), 856 (Sum2^m1Jus; green), 895 (Sum3^m1Jus; red), 1395 (Sum4^m1Jus; brown) and 1527 (Sum5^m1Jus; purple)) produced N₃ offspring that exhibited increased longevity. A closed circle represents offspring of male N₂ parents, and an open circle represents offspring of female N₂ parents. The longevity of the G₁ founder of each line is indicated by the colored square.

Figure 2

Survival curves for each line with a confirmed modifier map location are shown assessed at the N₃ generation. Survival of Mecp2^tm1.1Bird/Y mice is significantly increased by the presence of each suppressing mutation: a) Line 352 (Sum1^m1Jus); p=.001. b) Line 856 (Sum2^m1Jus); p=.005. c) Line 895 (Sum3^m1Jus); p=.002. d) Line 1395 (Sum4^m1Jus); p=.016.

Figure 3. A stop codon mutation in Sqle confers rescue at Sum3^m1Jus

a) The ENU-induced Sqle R399× mutation occurs in exon 7 in the squalene epoxidase domain. b) Sqle is not expressed in homozygous E8.0 mutant embryos. SqleΔ7 is the short predicted transcript lacking exon 7. c) Western blot on stage-matched E8.0 embryos shows that the expected 64kDa protein and 36kDa degradation product are absent in homozygous mutant embryos. d) Expression of Sqle but not Hmgcr is decreased in Sqle^Sum3/+ brain; e) gene expression is unchanged by Sqle^Sum3/+ in liver. i) Brain concentration of cholesterol precursors lanosterol and desmosterol is also decreased by Sqle^Sum3/+. f) Serum cholesterol concentration is unchanged by Sqle^Sum3/+. Tissue (d–g) analysis performed at P70; N=6 per group. Mecp2^tm1.1Bird/Y Sqle^Sum3/+ animals at backcross generation N₇ to 129S6/SvEvTac show g) significantly improved rotarod performance at P56 (p=.0001), h) improved open field activity at P70. All error bars represent s.e.m.

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

a) A simplified schematic of the enzymes and products in cholesterol biosynthesis via desmosterol is shown. b) Expression of Hmgcr, Sqle and Cyp46a1 in Mecp2^tm1.1Bird/Y and Mecp2^tm1.1Jae/Y are similar in brain. c) Lanosterol (Lan), desmosterol (Des) and total cholesterol (TC) concentrations are displayed per gram of brain tissue at P56 (N=8 per group) and P70 (N=4 per group). d) Cholesterol synthesis is decreased in Mecp2^tm1.1Jae/Y brain at P56 (wild type N=4; null N=5). e) Expression of Hmgcr and Sqle in Mecp2^tm1.1Bird/Y and Mecp2^tm1.1Jae/Y differ in liver. f) Triacylglyceride (TAG) and TC concentrations are displayed per gram of liver tissue at P56 (N=6 per group). g) Cholesterol synthesis is slightly increased in Mecp2^tm1.1Jae/Y liver per gram of tissue at P56 (wild type N=4; null N=5). Serum h) total cholesterol, i) LDL-cholesterol and j) triglyceride levels are significantly higher in Mecp2^tm1.1Bird/Y mice by P56 (N=8–11 per group), but unchanged in Mecp2^tm1.1Jae/Y mice (N=6 per group). For gene expression data (b,e) Bird: N=6 per genotype at P28, and 12 per genotype at P56; Jae: N=4 per genotype at P28, and 6 per genotype at P56. Tissue data (b–g) represent percentage change from wild type levels. *p≤0.05; All error bars represent s.e.m.

Figure 5. Statin treatment improves health in 129.Mecp2^tm1.1Bird/Y males

Total animals assessed were 37 Mecp2^tm1.1Bird/Y fluvastatin-treated, 12 Mecp2^tm1.1Bird/Y lovastatin-treated, 31 Mecp2^tm1.1Bird/Y vehicle-treated, 29 wild type +/Y fluvastatin-treated, 8 +/Y lovastatin-treated, and 29 wild type +/Y vehicle-treated mice for the following tests. a) Fluvastatin treatment of 129.Mecp2^tm1.1Bird/Y confers increased longevity: median 122 days compared to 87 days with 57% survival beyond 120 days (p<.0001). Three animals were sacrificed due to dermatitis (boxes) while active and otherwise healthy. b) Rotarod performance improves in P56 treated null males (fluvastatin p=.015; lovastatin p=.009), c) Open field activity is increased in P70 treated null males as assessed by beam breaks (fluvastatin: p=.011, lovastatin: p=.049). d) Statin treatment lowers plasma cholesterol by P70 (fluvastatin: p=.001, lovastatin: p=.001). e) Statin treatment ameliorates elevated lipid concentration in 129.Mecp2^tm1.1Bird/Y livers at P70 (fluvastatin p=.020; lovastatin: p=.386). The concentration of f) lanosterol slightly increases and g) desmosterol significantly increases in the brains of fluvastatin-treated 129.Mecp2^tm1.1Bird/Y mice at P70 (N=4 per group; p=.042). All error bars represent s.e.m.

Figure 6. Fluvastatin treatment improves health in 129.Mecp2^tm1.1Bird/+ females

a) No fluvastatin-treated 129.Mecp2^tm1.1Bird/+ females died prior to eight months, but three vehicle-treated females died. b) Rotarod performance improves in five-month-old fluvastatin-treated 129.Mecp2^tm1.1Bird/+ females (p=.001). c) Open field activity assessed at four months shows no significant differences in fluvastatin- or vehicle-treated groups. d) Fluvastatin treatment does not significantly change serum cholesterol levels at eight months. e) Fluvastatin treatment ameliorates elevated lipid concentration in 129.Mecp2^tm1.1Bird/+ livers assessed at eight months (p=.045). All error bars represent s.e.m.