A partial loss of function allele of methyl-CpG-binding protein 2 predicts a human neurodevelopmental syndrome

Abstract

Rett Syndrome, an X-linked dominant neurodevelopmental disorder characterized by regression of language and hand use, is primarily caused by mutations in methyl-CpG-binding protein 2 (MECP2). Loss of function mutations in MECP2 are also found in other neurodevelopmental disorders such as autism, Angelman-like syndrome and non-specific mental retardation. Furthermore, duplication of the MECP2 genomic region results in mental retardation with speech and social problems. The common features of human neurodevelopmental disorders caused by the loss or increase of MeCP2 function suggest that even modest alterations of MeCP2 protein levels result in neurodevelopmental problems. To determine whether a small reduction in MeCP2 level has phenotypic consequences, we characterized a conditional mouse allele of Mecp2 that expresses 50% of the wild-type level of MeCP2. Upon careful behavioral analysis, mice that harbor this allele display a spectrum of abnormalities such as learning and motor deficits, decreased anxiety, altered social behavior and nest building, decreased pain recognition and disrupted breathing patterns. These results indicate that precise control of MeCP2 is critical for normal behavior and predict that human neurodevelopmental disorders will result from a subtle reduction in MeCP2 expression.

Figure 1.

The expression of Mecp2 is decreased in Mecp2^Flox/y mice at both the mRNA and the protein level. The mRNA level as measured by qRT-PCR (A) is decreased in mutant animals. Notably, the e1 isoform (measured by a probe spanning the exons 1–3 boundary, e1–3), the e2 isoform (measured by a probe spanning exons 2–3 boundary, e2–3) and the common exon 3 (probe e3) all are decreased by ∼50% in Mecp2^Flox/y mice compared with wild-type littermates. The overall protein levels of MeCP2 measured by western blot (B) are decreased in Mecp2^Flox/y animals compared with wild-type littermates. MeCP2 was detected by an antibody specific to the common carboxy-terminus (α-C-term). The bottom blot in (B) is a loading control probed with an antibody to Gapdh. Each lane represents biological replicates of the respective genotypes. The decrease in MeCP2 protein level is also observed by immunofluorescence (C, D). Whereas the expression of MeCP2 is clearly discernable as relatively bright nuclear foci in wild-type animals (C), in Mecp2^Flox/y animals the signal is weaker (D). The small dashed box shows the cortical region represented at higher magnification in the inset in (C) and (D). The higher magnification reveals decreased MeCP2 levels, but a similar cellular distribution of MeCP2. The scale bar in the large figure in (C) represents 1 mm and within the inset represents 10 µm.

Figure 2.

Mecp2^Flox/y mice are heavier and perform poorly on motor tasks. Mecp2^Flox/y mice (F1 129S6.B6, n = 16 for each genotype) are ∼1 g heavier than littermate wild-type (WT) controls, although this finding is only significant at 9 and 16 weeks of life (A). The mice perform poorly on a variety of motor tasks including accelerating rotarod (B), dowel walking (C and D) and wire hanging (C). On the accelerating rotarod, the mutant mice perform poorly on Day 1, which indicates an inherent coordination deficit. The Mecp2^Flox/y mice fall off the wire sooner than controls (C, P = 0.002, Mann–Whitney) but not on the dowel (P = 0.12, Mann–Whitney). Additionally, the Mecp2^Flox/y animals have fewer side touches in both the dowel task (D, P = 0.013 Mann–Whitney) and the wire hang task (not shown, P = 0.002 Mann–Whitney). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Mecp2^Flox/y mice have altered pain sensitivity, prepulse inhibition, social interactions and nest building. Mecp2^Flox/y mice (F1 129S6.B6, n = 16 for each genotype) have increased latency of paw withdrawal on the hotplate (A, P = 0.002); however, they show normal response to tail flick at 40 (A), 50 and 60°C (not shown). The Mecp2^Flox/y animals have a diminished startle response (not shown, P = 0.001) but increased inhibitory gating when presented with a 74 or 82 dB sound prepulse (B). Mecp2^Flox/y mice (F1 129.B6, n = 16 for each genotype) spent a greater percentage of time at the partition than WT littermate controls when both an unfamiliar mouse was placed in the adjacent chamber or when the mice were re-exposed to a familiar mouse (C, *P < 0.05, **P < 0.01). When singly housed with a fresh nesting material, many Mecp2^Flox/y mice (n = 15) did not show any attempt to form a nest and only a small percentage had a fully formed nest after 14 h, in contrast to the large percentage (D, χ² P = 0.02) of WT littermate control animals (n = 16).

Figure 4.

Mecp2^Flox/y mice have altered learning and decreased anxiety. The Mecp2^Flox/y mice (F1 129S6.FVB, n = 16 per genotype) have decreased learning in a fear conditioning task both when exposed to the context (A, P = 0.006) or to the cue stimulus (A, P = 0.004). Additionally, they appear to be less anxious as measured by the distance traveled within the center of an open field chamber compared with the total distance (B) during the first and third 10 min intervals. This decreased anxiety is also apparent by the increased vertical exploratory movements that the mutant animals under took during the first and third 10 min intervals in the open field chamber (C). *P < 0.05, **P < 0.01.

Figure 5.

Respiration is altered in Mecp2^Flox/y mice. Representative plethysmographic recordings showing the irregular rhythm and periods of apnea in F1 129S6.FVB Mecp2^Flox/y mice (B) relative to WT mice (A). Population data showing the incidence of apneas (C, n = 4 for each genotype). Population data showing the coefficients of variability of respiratory frequency (D, n = 4 for each genotype). *P < 0.05.