Expression of MeCP2 in postmitotic neurons rescuesRett syndrome in miceSandra Luikenhuis*†, Emanuela Giacometti*, Caroline F. Beard*, and Rudolf Jaenisch*†‡*Whitehead Institute for Biomedical Research,†Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142Contributed by Rudolf Jaenisch, March 8, 2004Mutations in MECP2 are the cause of Rett syndrome (RTT) inhumans, a neurodevelopmental disorder that affects mainly girls.MeCP2 is a protein that binds CpG dinucleotides and is thought toact as a global transcriptional repressor. It is highly expressed inneurons, but not in glia, of the postnatal brain. The timing ofMeCP2 activation correlates with the maturation of the centralnervous system, and recent reports suggest that MeCP2 may beinvolved in the formation of synaptic contacts and may function inactivity-dependent neuronal gene expression. Deletion or targetedmutation of Mecp2 in mice leads to a Rett-like phenotype. Selectivemutation of Mecp2 in postnatal neurons leads to a similar, al-though delayed, phenotype, suggesting that MeCP2 plays a role inpostmitotic neurons. Here we test the hypothesis that the symp-toms of RTT are exclusively caused by a neuronal MeCP2 deficiencyby placing Mecp2 expression under the control of a neuron-specificpromoter. Expression of the Mecp2 transgene in postmitotic neu-rons resulted in symptoms of severe motor dysfunction. Transgeneexpression in Mecp2 mutant mice, however, rescued the RTTphenotype.Rett syndrome (RTT), a neurodevelopmental disorder, is aleading cause of mental retardation in females with anestimated prevalence of 1 in 10,000–15,000 female births. RTTpatients develop normally until 6–18 months of age, when theystart to show symptoms including respiratory irregularities,progressive loss of motor skills, stereotypic hand movements,seizures, and features of autism. Examination of the brain revealsprofound microencephaly due, at least in part, to smaller, moredensely packed neurons. Other abnormalities include a reduc-tion in dendritic arborization (1, 2). In ⬇80% of cases, RTT isassociated with mutations in the X-linked MECP2 gene that issubject to inactivation when located on the inactive X chromo-some (3). Therefore, heterozygous mutant females are mosaicfor MeCP2 deficiency and show a wide range of phenotypes.Males, however, show a more severe phenotype, usually involv-ing encephalopathy, motor abnormalities, and respiratory dys-function. They rarely live beyond 2 years (2).Mecp2 encodes a protein that binds specifically to methylatedCpG dinucleotides and recruits chromatin remodeling com-plexes that contain the transcriptional repressor Sin3A andhistone deacetylases 1 and 2 (4). In mouse, the protein localizesto highly methylated pericentromeric heterochromatin (5). Al-though MeCP2 is found in most tissues and cell types, highestexpression levels are detected in the brain, where it is primarilypresent in neurons but not in glia (5–7). The timing of Mecp2expression correlates with the maturation of the CNS (5, 8), andrecent reports suggest that MeCP2 may be involved in theformation of synaptic contacts (9). Although biochemical evi-dence suggests that MeCP2 acts as a global silencer, transcrip-tional profiling has failed to detect global changes in geneexpression (10). A candidate approach has identified BDNF,agene involved in neuronal survival, development, and plasticity,as a target for MeCP2 (11). These findings are consistent withMeCP2 playing a role in the maintenance and modulation ofneuronal maturity. In particular, MeCP2 may function as a keyregulator of activity-dependent neuronal gene expression.Complete or partial deletion of Mecp2 in mice leads to aneurological phenotype that is similar but less severe than humanRTT (12, 13). Heterozygous females remain healthy into adult-hood. In contrast, Mecp2 mutant males appear normal andhealthy at birth but begin to show a phenotype that resembles thehuman condition at 3–8 weeks of age, and die at 6–10 weeks ofage. Mutant brains show a reduction in brain weight andneuronal cell size but no obvious structural defects or signs ofneurodegeneration. Conditional mutation of Mecp2 in the neuralprogenitor cells at embryonic day 12 results in a phenotypeidentical to that of the null mutation (12). Mutation of Mecp2 inthe postnatal neurons of restricted regions in the brain leads toa similar although delayed neuronal phenotype, suggesting thatMeCP2 plays a role in postmitotic neurons (12). Here we test thehypothesis that the phenotype is exclusively caused by a neuronalMeCP2 deficiency by placing Mecp2 expression under the con-trol of a neuron-specific promoter. Overexpression of the Mecp2transgene in postmitotic neurons proved to be detrimental andled to symptoms of severe motor dysfunction. Transgene expres-sion in Mecp2 mutant mice, however, resulted in a rescue of theRTT phenotype.Materials and MethodsGene Targeting Construct. To introduce the Mecp2 coding se-quence as an in-frame fusion into exon 1 of the tau locus, we firstcloned a 3.8-kb KpnI兾EcoRI fragment from pHV, which con-tains 14-kb of tau genomic sequence (kindly provided by K.Tucker, University of Heidelberg, IZN, Heidelberg, Germany)into pBluescript (Stratagene) generating pTau-KR with a uniqueNcoI cloning site. Next, we eliminated a unique SpeI site in thepolylinker of pTau-KR by cutting the vector with SpeI, treatingit with the DNA polymerase I Klenow fragment (New EnglandBiolabs), and religating it. We created pTau-KR-linker by in-troducing suitable restriction sites to allow the in-frame fusion.We inserted an adapter that destroyed the NcoI cloning sitewhile introducing a new NcoI site that was shifted by 2 bp, plusa SpeI and an EcoRV site. The primers used were TAUadapt-F(5⬘-TTT GGT CAT GAT GCC ATG GAC TAG TCG ATATCT CAT GAG ATT A-3⬘) and TAU-link-R (5⬘-TAA TCTCAT GAG ATA TCG ACT AGT CCA TGG CAT CAT GACCAA A-3⬘). The 1,455-nt-long coding sequence of Mecp2 wasamplified by PCR from IMAGE clone 1395411 (GenBankaccession no. AI181668) and confirmed by sequencing. The PCRprimers introduced a modified Kozak sequence including anNcoI site (ATTCCATGG was changed to CCACCATGG) andrestriction sites that facilitated cloning. Primer RI-MeCP2-F(5⬘-CGGAATTCCGCCACCATGGTAGCTGGGATGTT-AGGG-3⬘) added an EcoRI site 5⬘ of the sequence, and primerXba-MeCP2-R (5⬘-GCTCTAGAGCTCAGCTAACTCTC-TCGGTCACG-3⬘) added an XbaI site to the 3⬘ end. To providethe construct with a simian virus