Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 15;25(20):4389-4404.
doi: 10.1093/hmg/ddw269.

Exclusive Expression of MeCP2 in the Nervous System Distinguishes Between Brain and Peripheral Rett Syndrome-Like Phenotypes

Affiliations
Free PMC article

Exclusive Expression of MeCP2 in the Nervous System Distinguishes Between Brain and Peripheral Rett Syndrome-Like Phenotypes

Paul D Ross et al. Hum Mol Genet. .
Free PMC article

Abstract

Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked MECP2 gene. MeCP2 protein is highly expressed in the nervous system and deficiency in the mouse central nervous system alone recapitulates many features of the disorder. This suggests that RTT is primarily a neurological disorder, although the protein is reportedly widely expressed throughout the body. To determine whether aspects of the RTT phenotype that originate in non-neuronal tissues might have been overlooked, we generated mice in which Mecp2 remains at near normal levels in the nervous system, but is severely depleted elsewhere. Comparison of these mice with wild type and globally MeCP2-deficient mice showed that the majority of RTT-associated behavioural, sensorimotor, gait and autonomic (respiratory and cardiac) phenotypes are absent. Specific peripheral phenotypes were observed, however, most notably hypo-activity, exercise fatigue and bone abnormalities. Our results confirm that the brain should be the primary target for potential RTT therapies, but also strongly suggest that some less extreme but clinically significant aspects of the disorder arise independently of defects in the nervous system.

Figures

Figure 1.
Figure 1.
Generation of a CNS rescue or ‘peripheral knockout’ mouse. (A) Plot showing levels of native MeCP2 protein relative to whole brain levels (and standardised to histone H3 levels) in wild type (WT) mice as revealed by quantitative immunoblot (mean ± S.D., n = 3). (B) Cartoon showing MeCP2 expression profile in three experimental cohorts of mice including WT, knockout (KO) in which Mecp2 is silenced globally using a stop cassette, and a peripheral knockout (PKO) mouse in which Mecp2 is silenced in the peripheral tissues but reactivated in the nervous system using nestin-cre mediated recombination of an Mecp2-stop allele. (C) Representative Southern blots showing recombination of the Stop/y allele (5.1 kb) to delete the Stop cassette (open triangle, 4.3 kb) in PKO mice. (D) Plot showing recombination efficiency in brain and peripheral tissues as revealed by Southern blot analysis (n = 2–4 mice; mean ± S.D.). (E) Representative quantitative western blots showing MeCP2 expression in WT, PKO and null mice. WT whole brain reference samples were used to enable comparison of MeCP2 levels between different gels and different tissues. Histone H3 was used as a loading control. (F) MeCP2 protein levels in PKO mice relative to WT levels across representative tissues (mean ± S.D., n = 3). Abbreviations: W brain, whole brain; F brain, forebrain; M/H brain, Mid/hindbrain; Sk muscle, skeletal muscle; Cer, Cerebellum.
Figure 2.
Figure 2.
Normal survival and absence of RTT-like signs in peripheral KO mice. (A) Survival plot showing normal survival of WT (blue circles, n = 17) and PKO mice (green squares, n = 7) compared to reduced lifespan in KO mice (red triangles, n = 8). The median survival period was significantly reduced in KO mice (***P < 0.001, log-rank test). (B) Plot showing aggregate phenotype severity (mean ± SEM) based on an established observational scoring system. There was a highly significant difference in score between WT and PKO animals from 8 weeks onwards when compared to KO (***P <0.001, Kruskal-Wallis test with Dunn’s post hoc analysis). No significant difference was seen between WT and PKO at the time of behavioural testing (15 weeks) but these groups differed significantly at 1 year (*P < 0.05). (C) Plot showing body weight changes over time (mean ± SEM). No significant differences were seen between genotypes at the time of behavioural testing (15 weeks; P > 0.05, one-way ANOVA) but PKO mice had significantly lower bodyweight than WT mice when compared at 1 year (***P < 0.001, student’s unpaired t-test). In B) and C) group sizes at the start of the experiment are as given in A).
Figure 3.
Figure 3.
PKO mice show mild hypoactivity but absence of behavioural or gait defects. (A–B) Spontaneous motor and exploratory activity assessed using open-field. Results show (A) total distance moved and (B) number of rearing events/session. There was a significant difference from WT in both PKO and KO mice. (C) Nesting behaviour was normal in PKO mice but impaired in KO mice. (D–F) Gait assessed using motorised treadmill. Results show (D) Proportion of mice capable of performing to criterion (E) stride frequency and (F) stance width. All data other than (D) are mean ± SEM. Numbers of animals per genotype are shown within each bar. Groups were compared using one-way ANOVA with Tukey’s post hoc analysis. # indicates not determined due to mice being unable to perform to criterion. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4.
Figure 4.
PKO mice show no difference in balance but marked deficiency in exercise capacity. (A–B) Balance beam task showing time to traverse medium (A) and narrow (B) beams. (C) Rotarod performance. (D) Exercise capacity measured using an elevated treadmill, with a steadily increasing speed. Results show time lasted on treadmill. Plots show mean ± S.E.M. Number of animals per genotype are shown within each bar. Groups were compared using one-way ANOVA and Tukey’s post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5.
Figure 5.
RTT-like respiratory phenotypes are absent in PKO mice. (A) Representative whole-body plethysmograph traces showing regular and erratic breathing patterns/apnoeas (arrows) in WT, stop-cre mice and in stop mice, respectively. (B) breathing frequency at rest, (C) breathing frequency variability and (D) apnoea frequency. In (B–D), data are plotted as mean values ± S.E.M. Numbers of animals per genotype are shown within each bar. Groups were compared using one-way ANOVA and Tukey’s post hoc comparisons. ***P < 0.001.
Figure 6.
Figure 6.
Echocardiography parameters unchanged in PKO mice. (A) Example M-mode images from (i) WT, (ii) PKO and (iii) KO mice. The left ventricular internal diameter is indicated in each image by the white dashed line. (B) Mean ± SEM for M-mode measured parameters. (C) Example pulse-wave Doppler images from each of the experimental and control groups. Arrows indicate peak heights for both E (early diastolic filling) and A (atrial contribution to diastolic filling) waves. (D) Mean ± SEM for pulse-wave Doppler measured parameters. Numbers of animals per genotype are shown within each bar. Groups were compared using one-way ANOVA and Tukey’s post hoc comparisons. *P < 0.05.
Figure 7.
Figure 7.
No significant muscle abnormalities in PKO mice. Representative images of gastrocnemius muscle cross section (A) stained with haematoxylin and eosin (H&E) for measurement of myofiber cross sectional area; (B) stained with picrosirius red for measurement of collagen fibers; and (C) immunolabelled with an antibody against the endothelial cell marker Griffonia simplicifolia lectin I (red) for measurement of capillary density. Black arrow indicates a centrally-located nucleus. Graphs show (D) myofiber cross sectional area in µm2(E) the proportion of fibers with a centrally-located nucleus (F) the proportion of the section composed of collagen fibers and (G) the capillary density per mm2. Data are plotted as mean ± SEM. Groups were compared using one-way ANOVA with Tukey’s post hoc comparisons. *P < 0.05, **P < 0.01.
Figure 8.
Figure 8.
PKO mice show characteristic RTT-like bone phenotypes. Three-point bending test reveals reduced (A) ultimate load and (B) stiffness of tibia in PKO and KO mice compared to WT. (C) Microindentation test in polished femur reveals significantly reduced cortical bone hardness in PKO and KO mice when compared with WT controls. Plots show mean ± S.E.M. Numbers of animals per genotype are shown within each bar. Groups were compared using one-way ANOVA with Tukey’s post hoc comparisons. ***P < 0.001, **P < 0.01, *P < 0.05.

Similar articles

See all similar articles

Cited by 16 articles

See all "Cited by" articles

References

    1. Neul J.L., Kaufmann W.E., Glaze D.G., Christodoulou J., Clarke A.J., Bahi-Buisson N., Leonard H., Bailey M.E., Schanen N.C., Zappella M., et al. (2010) Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol., 68, 944–950. - PMC - PubMed
    1. Hagberg B., Aicardi J., Dias K., Ramos O. (1983) A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett's syndrome: report of 35 cases. Ann Neurol., 14, 471–479. - PubMed
    1. Percy A.K., Lee H.S., Neul J.L., Lane J.B., Skinner S.A., Geerts S.P., Annese F., Graham J., McNair L., Motil K.J., et al. (2010) Profiling scoliosis in Rett syndrome. Pediatr. Res., 67, 435–439. - PMC - PubMed
    1. Guidera K.J., Borrelli J., Jr., Raney E., Thompson-Rangel T., Ogden J.A. (1991) Orthopaedic manifestations of Rett syndrome. J. Pediatr. Orthop., 11, 204–208. - PubMed
    1. Zysman L., Lotan M., Ben-Zeev B. (2006) Osteoporosis in Rett syndrome: A study on normal values. ScientificWorldJournal, 6, 1619–1630. - PMC - PubMed

Publication types

Substances

Feedback