Reversal of Neurological Defects in a Mouse Model of Rett Syndrome

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Science  23 Feb 2007:
Vol. 315, Issue 5815, pp. 1143-1147
DOI: 10.1126/science.1138389


Rett syndrome is an autism spectrum disorder caused by mosaic expression of mutant copies of the X-linked MECP2 gene in neurons. However, neurons do not die, which suggests that this is not a neurodegenerative disorder. An important question for future therapeutic approaches to this and related disorders concerns phenotypic reversibility. Can viable but defective neurons be repaired, or is the damage done during development without normal MeCP2 irrevocable? Using a mouse model, we demonstrate robust phenotypic reversal, as activation of MeCP2 expression leads to striking loss of advanced neurological symptoms in both immature and mature adult animals.

Mutations in the X-linked MECP2 gene are the primary cause of Rett syndrome (RTT), a severe autism spectrum disorder with delayed onset that affects 1 in 10,000 girls (1). MECP2 mutations are also found in patients with other neurological conditions, including learning disability, neonatal encephalopathy, autism, and X-linked mental retardation (2). RTT patients show abnormal neuronal morphology, but not neuronal death (3), which implies that it is a neurodevelopmental rather than a neurodegenerative disorder. MeCP2 is expressed widely, but is most abundant in neurons of the mature nervous system (4). Conditional deletion and neuron-specific expression of Mecp2 in mice showed that the mutant phenotype is specifically due to absence of MeCP2 in neurons (57). The persistent viability of mutant neurons in RTT patients raises the possibility that reexpression of MeCP2 might restore full function and, thereby, reverse RTT. Alternatively, MeCP2 may be essential for neuronal development during a specific time window, after which damage caused by its absence is irreversible. To distinguish these possibilities, we created a mouse in which the endogenous Mecp2 gene is silenced by insertion of a lox-Stop cassette (8), but can be conditionally activated under the control of its own promoter and regulatory elements by cassette deletion (9) (fig. S1). Western blots (Fig. 1A) and in situ immunofluorescence (Fig. 1B) confirmed absence of detectable MeCP2 protein in Mecp2lox-Stop/y (Stop/y) animals. Like Mecp2-null mice (6), Stop/y males developed symptoms at ∼6 weeks and survived for 11 weeks, on average, from birth (Fig. 1C). We concluded that the Mecp2lox-Stop allele behaves as a null mutation.

Fig. 1.

Insertion of a lox-Stop cassette into intron 2 of the mouse Mecp2 gene creates an allele that is effectively null, but can be activated by TM treatment. (A) Western blot analysis of MeCP2 protein (solid arrow) in brains of wild-type (wt), Stop/y, and Stop/y,cre mice before and after TM. Antibodies against MeCP2 were from J. Pevsner (left panel) and Upstate (right panel). Internal controls are nonspecific cross-reacting bands (asterisk) and bands generated by a histone H4–specific antibody (open arrow). (B) Detection of MeCP2 by in situ immunofluorescence in dentate gyrus of wild-type (wt), Stop/y, and TM-treated Stop/y,cre mice. White scale bar, 50 μm. Green cells that did not stain with DAPI (4′,6′-diamidino-2-phenylindole) in the upper Stop panel are nonnucleate erythrocytes showing background fluorescence. The DAPI channel was changed from blue to red using Adobe Photoshop to contrast with the green MeCP2 signal. (C) Comparison of the survival of Stop/y mice with and without the cre-ER transgene. (D) A Southern blot assay for deletion of the lox-Stop cassette in brains of heterozygous Stop/+,cre females (f) aged 10 months (lanes 2 and 3) that had not been exposed to TM. Restriction fragments from the Mecp2 lox-Stop (Stop; see male Mecp2lox-Stop/y, lane 1), Mecp2Δ with Stop deleted (Δ, see lane 4), and the wild-type (wt) alleles are indicated. (E) Southern blot assay for conversion of the Stop allele to the Mecp2Δ allele (Δ) in male mouse brains after five daily TM injections. Lanes 2 and 5 show the wt allele.

To control the activation of Mecp2, we combined a transgene expressing a fusion between Cre recombinase and a modified estrogen receptor (cre-ER) with the Mecp2lox-Stop allele (10). The Cre-ER protein remains in the cytoplasm unless exposed to the estrogen analog tamoxifen (TM), which causes it to translocate to the nucleus. To verify that the Cre-ER molecule did not spuriously enter the nucleus in the absence of TM and cause unscheduled deletion of the lox-Stop cassette, we looked for signs of lox-Stop deletion in Mecp2lox-Stop/+, cre-ER (Stop/+,cre) females by Southern blotting. Even after 10 months in the presence of cytoplasmic Cre-ER, there was no sign of the deleted allele (Fig. 1D). The absence of spontaneous deletion of the lox-Stop cassette was independently confirmed by the finding that Stop/y males showed identical survival profiles in the presence or absence of Cre-ER (Fig. 1C). Therefore, in the absence of TM, the Cre-ER molecule does not cause detectable deletion of the lox-Stop cassette.

We next tested the ability of TM to delete the lox-Stop cassette in Mecp2lox-Stop/y, cre-ER (Stop/y,cre) male mice. Five daily injections 3 to 4 weeks after birth caused 75 to 81% deletion of the cassette in brain (Fig. 1E, lanes 3, 4, and 6) and led to reexpression of the Mecp2 gene as measured by Western blotting (Fig. 1A, lanes 4 and 5) and MeCP2 immunostaining of neurons (Fig. 1B). Activation of Mecp2 in Stop/y,cre males at this stage [(Fig. 2A), bracket TM-1], before symptom onset, revealed toxicity associated with abrupt Mecp2 reactivation, as 9 out of 17 mice developed neurological symptoms and died soon after the daily TM injection series (fig. S2). The remaining eight mice, however, did not develop any detectable symptoms, showed wild-type survival (fig. S2), and were able to breed. Four retained mice have survived for >15 months. Death of about half of animals was not due to intrinsic TM toxicity, because injected controls, including mice that had either the Stop allele or the cre-ER transgene (but not both), were unaffected. The toxic effects resembled those caused by overexpression of an Mecp2 transgene in mice (7, 11), although the reactivated Mecp2 gene retains its native promoter. The data indicate that sudden widespread activation of the Mecp2 gene leads to either rapid death or complete phenotypic rescue.

Fig. 2.

Reversal of the neurological phenotype by activation of the Mecp2 gene in Stop/y,cre males. (A) Time course of the Stop/y phenotype. (B, C, and D) Plots of the phenotypic scores (⚫) and weights (x) of individual wild-type (wt)(B), Stop/y (Stop)(C), and Mecp2lox-Stop/y, cre-ER (Stop-cre)(D) animals after TM injections (vertical arrows). (See also fig. S2.) Stars in (D) indicate when the clips shown in movies S1 and S2 were recorded. (E) Aggregate symptom score profiles following TM injection of Stop/y,cre (⚫, n =3 to 6, except *, which was a single animal) and Stop/y (▲, n = 4 to 5; except ## and #, whichare 2and 1datapoints, respectively)mice. (F) Survival profiles of TM-treated Stop/y,cre mice and control Stop/y mice. (G) Southern blot showing deletion of the lox-Stop cassette (lanes 3 and 5) after a weekly TM injection regime + booster injections.

We found that a more gradual Mecp2 activation induced by weekly TM injections followed by three daily booster treatments eliminated toxicity. Using this scheme, we asked whether Stop/y,cre male mice with advanced symptoms [(Fig. 2A), bracket TM-2] could be rescued by restoration of MeCP2. To monitor the specific features of the RTT-like mouse phenotype, we devised simple observational tests for inertia, gait, hind-limb clasping, tremor, irregular breathing, and poor general condition. Each symptom was scored weekly as absent, present, or severe (scores of 0, 1, and 2, respectively). Wild-type mice always scored zero (Fig. 2B), whereas Stop/y animals typically showed progression of aggregate symptom scores (e.g., from 3 to 10) during the last 4 weeks of life (Fig. 2, C and E). By contrast, five out of six symptomatic Stop/y,cre animals were rescued by TM treatment. These animals initially had symptom scores of 2 or 3 and would be expected to survive for up to 4 weeks from the date of the first injection. Instead, they showed mild symptoms (see fig. S3 for examples of detailed scores) and survived well beyond the maximum-recorded life span of Mecp2lox-Stop/y animals (17 weeks) (Fig. 2, D to F, and fig. S4; see movies S1 and S2). The weekly TM injection regime, plus booster injections, gave the same level of lox-Stop cassette deletion as five daily TM injections (∼80%) (Fig. 2G). The one animal that died had reduced lox-Stop cassette deletion (∼50% compared with ∼80%), which may explain failure to rescue.

RTT results from mosaic expression of mutant and wild-type MECP2 alleles in the brain caused by the random inactivation of one X-linked MECP2 allele during early female development. Heterozygous female mice may be the most appropriate model for human RTT (12), because both Mecp2+/– (6) and Stop/+ females (Fig. 3, A, B, and E) develop RTT-like symptoms, including inertia, irregular breathing, abnormal gait, and hind-limb clasping, at 4 to 12 months of age. As in humans, the phenotype stabilizes, and the animals have an apparently normal life span. The mice often become obese, which is not a feature of the human condition. In an attempt to reverse the neurological phenotype in mature female heterozygotes, we TM-treated Stop/+,cre females with clear neurological symptoms. These mice progressively reverted to a phenotype that scored at or close to wild type (Fig. 3, C to E, and fig. S5 and movie S3; see fig. S3 for examples of detailed scores), including normalized weight (Fig. 3D and fig. S6). Mouse 5, for example, had a phenotypic score close to the usual plateau level and was obese at commencement of the weekly TM injection regime, but these features were both reversed (Fig. 3D). On the other hand, Stop/+ females lacking Cre-ER did not respond to TM. Southern blots showed levels of cassette deletion in Stop/+,cre females that were consistently close to 50% (Fig. 3F). As the great majority of neurons became MeCP2-positive after TM treatment (fig. S7), we suspect that recombination predominantly occurs on the active X-chromosome (see legend to fig. S7). The results demonstrate that late-onset neurological symptoms in mature adult Stop/+, cre heterozygotes are reversible by de novo expression of MeCP2.

Fig. 3.

Reversal of late-onset neurological symptoms by Mecp2 gene induction in mature adult Stop/+,cre females. (A) Time course of symptom onset. TM administration began during the bracketed period (TM3). (B, C, and D) Phenotype (⚫) and weight (x) profiles for a Stop/+ female (B) and two Stop/+,cre females (C and D). All animals shown were subjected to either five daily TM injections or five weekly plus three booster TM injections (vertical arrows). Animals subject to weekly injection regimes were scored blind as part of a mixed genotype cohort. (E) Plot of average symptom scores for females with wt (♦, n = 5 to 6), Stop/+ (▲, n = 6 to 7), and Stop,+,cre (⚫, n = 5 to 11) genotypes. Repeated measures analysis of variance (ANOVA) compared Stop/+ and Stop/+,cre female scores in weeks 11 to 16. (F) Southern blot analysis of the effects of TM treatment on a cohort including six Stop/+,cre (lanes 5 to 10), six wt (lanes 11 to 16) and six Stop/+ (lanes 17 to 22) females. All three genotypes received TM. Restriction fragments derived from Mecp2 lox-Stop (Stop), deleted Mecp2Δ (Δ) and wild type (wt) are marked with arrows. Brain DNA from animals 32 and 5 shown above are in lanes 9 and 6, respectively. Lanes 1 to 4 show blots of wt male, Stop/y male, Stop/+ female, and Mecp2Δ/+ female, respectively.

We also assessed the effect of Mecp2 activation on neuronal signaling. Long-term potentiation (LTP) is reduced in the hippocampus of Mecp2-mutant male mice (13, 14), but heterozygous females have not been tested. We performed electrophysiological analysis of Mecp2+/– heterozygous females (6) before and after onset of overt symptoms using both high-frequency stimulation and theta-burst (TBS) LTP induction protocols. Stimulation-response curves showed that the strength of basal synaptic transmission did not differ between symptomatic or presymptomatic Mecp2+/– female mice and wild-type littermate controls (Fig. 4A). In addition, no significant difference in hippocampal LTP between wild-type and presymptomatic females was detected. After symptom onset, however, LTP was significantly reduced in Mecp2+/– females with both protocols (Fig. 4, B and C). The magnitude of the defect was similar to that reported in Mecp2-null mice (13). To test for reversal of this effect, we measured LTP in six Stop/+,cre females that were TM-treated following the appearance of symptoms. LTP was measured 18 to 26 weeks after commencement of TM treatment. Control Stop/+ and wild-type animals were also TM-treated and analyzed. The hippocampal LTP deficit was evident in symptomatic Stop/+ mice lacking the cre-ER transgene, but in TM-treated Stop/+,cre mice, LTP was indistinguishable from wild type (Fig. 4D), which demonstrates that this pronounced electrophysiological defect is abolished in mature adults by restoration of MeCP2.

Fig. 4.

A deficit in long-term potentiation (LTP) accompanies onset of symptoms in mature adult Mecp2lox-Stop/+ heterozygous females and is reversed by Mecp2 reactivation. (A) Stimulation-response curves in symptomatic (blue) or presymptomatic (red) Mecp2+/– female mice and wt littermate controls (black; all P >0.05). (B) Measurements of LTP using a high-frequency stimulation (HFS) paradigm in presymptomatic (n = 9; P > 0.05), symptomatic (n =9; P < 0.05) Mecp2+/– mice, and wt female littermate control groups (n = 7 and 8; pooled data plotted). (C) Measurements of LTP using theta-burst stimulation in presymptomatic (n = 9; P > 0.05), symptomatic (n = 9; P < 0.05) Mecp2+/– mice, and wt female littermate control groups (n = 7 and 8). (D) HFS-induced LTP measurements in TM-treated symptomatic Stop/+ mice (n = 11; P < 0.05), Stop/+,cre mice (n =10; P >0.05),and wt mice (n =9; P > 0.05). Recombination data are shown in Fig. 3F. Insets in (B) to (D) show representative voltage traces before (1) and after (2) LTP induction. Two-way repeated measures ANOVA was used to assess significance throughout.

Our data show that developmental absence of MeCP2 does not irreversibly damage neurons, which suggests that RTT is not strictly a neurodevelopmental disorder. The delayed onset of behavioral and LTP phenotypes in Mecp2+/– females emphasizes the initial functional integrity of MeCP2-deficient neurons and fits with the proposal that MeCP2 is required to stabilize and maintain the mature neuronal state (4, 6). Consistent with the maintenance hypothesis, the time taken for major symptoms to appear post-natally in females heterozygous for an MECP2 mutation is similar in humans (6 to 18 months) and mice (4 to 12 months), despite fundamental interspecies differences in developmental maturity at this time. The restoration of neuronal function by late expression of MeCP2 suggests that the molecular preconditions for normal MeCP2 activity are preserved in its absence. To explain this, we propose that essential MeCP2 target sites in neuronal genomes are encoded solely by patterns of DNA methylation that are established and maintained normally in cells lacking the protein. According to this hypothesis, newly synthesized MeCP2 molecules home to their correct chromosomal positions as dictated by methyl-CpG patterns and, once in place, resume their canonical role as interpreters of the DNA methylation signal (15, 16).

Our study shows that RTT-like neurological defects due to absence of the mouse Mecp2 gene can be rectified by delayed restoration of that gene. The experiments do not suggest an immediate therapeutic approach to RTT, but they establish the principle of reversibility in a mouse model and, therefore, raise the possibility that neurological defects seen in this and related human disorders are not irrevocable.

Supporting Online Material

Materials and Methods

Figs. S1 to S7


Movies S1 to S3

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