Recent developments in Rett syndrome research

Rett Syndrome and MeCP2: Linking Epigenetics and Neuronal Function

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

The lens epithelium derived growth factor p75 (LEDGF/p75) is an emerging oncoprotein identified as a key player in the cellular response to oxidative stress. LEDGF/p75 is induced by oxidative stress and promotes resistance to stress-induced cell death presumably by transcriptionally activating specific stress and survival genes. LEDGF/p75 has a splice variant, p52, which induces cell death when overexpressed. We have shown elevated expression of LEDGF/p75 in advanced stage prostatic adenocarcinomas, most likely as a result of increased oxidative stress in the prostate tumor microenvironment. To understand the mechanisms by which LEDGF/p75 transcriptionally activate stress genes and promotes protection against oxidative stress-induced cell death, it was necessary to identify endogenous cellular interacting partners of this protein. Screening of a transcription factor protein array identified the Methyl CpG binding Protein 2 (MeCP2) as a possible interacting partner of LEDGF/p75. MeCP2 is mutated in patients with Rett syndrome, both activate and repress transcription and has been implicated in prostate tumor growth. This study was designed to validate MeCP2 as an interacting partner of LEDFG/p75 and to explore its influence on LEDGF/p75's function. To examine the interaction of MeCP2 and LEDGF, pull down assays were performed with purified GST-MeCP2 incubated with His-LEDGF/p75 or His-p52, and with GST-MeCP2 or GST-p75 incubated with U2OS cell lysates. The results suggested binding of MeCP2 to both LEDGF/p75 and p52. Co-immunoprecipitation assays using U2OS cell lysates revealed binding of proteins, detected by immunoblotting using specific anti-LEDGF/p75 or MeCP2 antibodies, suggesting interaction of both proteins in the cellular microenvironment. We also examined the intracellular localization of both proteins in U2OS cells by immunofluorescence microscopy. Both MeCP2 and LEDGF/p75 co-localized in the chromatin, showing a similar dense fine speckled staining pattern. To examine if MeCP2 influences LEDGF/p75's transcriptional activity, luciferase reporter assays were performed. Even though LEDGF/p75, p52 and MeCP2 transactivated the Hsp27 promoter (pr) individually, co-expression of LEDGF/p75 with MeCP2 increased Hsp27pr activity, while co-expression of LEDGF/p52 with MeCP2 decreased activity. In addition, LEDGF/p75 overexpression protected PC3 cells from tert-butyl hydrogen peroxide (TBHP) treatment; but siRNA knockdown of MeCP2 in these cells sensitized cells to TBHP treatment compared to control siRNA. In conclusion, our data suggest that LEDGF/p75 and MeCP2 are interacting partners and MeCP2 influences LEDGF/p75's transcription and survival function. Future studies will explore modulating the interaction between LEDGF/p75 and MeCP2 to sensitize prostate cancer cells to therapy-induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2945.

Homozygous c.1160C>T (P38L) in the MECP2 gene in a female Rett syndrome patient

Rett syndrome (RTT) was first described in 1966. Its biological and genetic foundations were not clear until recently when Amiret al reported that mutations in the MECP2 gene were detected...

Rett syndrome, a prototypical neurological disorder caused by loss of function of the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2) gene, is associated with a severely disordered breathing pattern and reduced ventilatory CO2 sensitivity. In a mouse model of Rett syndrome (MeCP2 knockout), re-introduction of the MeCP2 gene selectively in astrocytes rescues normal respiratory phenotype. In the present study we determined whether the metabolic and/or signalling functions of astrocytes are affected by testing the hypotheses that in conditions of MeCP2 deficiency, medullary astrocytes are unable to produce/release appropriate amounts of lactate or detect changes in /[H+], or both. No differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex in brain slices of MeCP2-knockout and wild-type mice were found. In brainstem slices of wild-type mice, respiratory acidosis triggered robust elevations in [Ca2+]i in astrocytes residing near the ventral surface of the medulla oblongata. The magnitude of CO2-induced [Ca2+]i responses in medullary astrocytes was markedly reduced in conditions of MeCP2 deficiency, whereas [Ca2+]i responses to ATP were unaffected. These data suggest that (i) metabolic function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in /[H+]. Taken together with the evidence of severely blunted ventilatory sensitivity to CO2 in mice with conditional MeCP2 deletion in astroglia, these data support the hypothesis of an important role played by astrocytes in central respiratory CO2/pH chemosensitivity.Key pointsRett syndrome is a prototypical neurological disorder characterised by abnormal breathing pattern and reduced ventilatory CO2 sensitivity. Medullary astrocytes are a crucial component of central CO2/pH chemosensitivity.This study tested the hypotheses that methyl-CpG-binding protein 2 (MeCP2) deficient medullary astrocytes are (i) unable to produce/release appropriate amounts of lactate, and/or (ii) unable to sense changes in /[H+].We found no differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex between MeCP2-knockout and wild-type mice.Respiratory acidosis triggered robust [Ca2+]i responses in wild-type astrocytes residing near the ventral surface of the medulla oblongata. CO2-induced [Ca2+]i responses in astrocytes were dramatically reduced in conditions of MeCP2 deficiency.These data suggest that (i) ‘metabolic’ function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in /[H+].

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome. MECP2 codes for methyl CpG binding protein 2 (MECP2), a transcriptional regulator that activates genetic programs for experience-dependent plasticity. Many neural and behavioral symptoms of Rett syndrome may result from dysregulated timing and thresholds for plasticity. As a model of adult plasticity, we examine changes to auditory cortex inhibitory circuits in female mice when they are first exposed to pups; this plasticity facilitates behavioral responses to pups emitting distress calls. Brainwide deletion of Mecp2 alters expression of markers associated with GABAergic parvalbumin interneurons (PVins) and impairs the emergence of pup retrieval. We hypothesized that loss of Mecp2 in PVins disproportionately contributes to the phenotype. Here, we find that deletion of Mecp2 from PVins delayed the onset of maternal retrieval behavior and recapitulated the major molecular and neurophysiological features of brainwide deletion of Mecp2 We observed that when PVin-selective mutants were exposed to pups, auditory cortical expression of PVin markers increased relative to that in wild-type littermates. PVin-specific mutants also failed to show the inhibitory auditory cortex plasticity seen in wild-type mice on exposure to pups and their vocalizations. Finally, using an intersectional viral genetic strategy, we demonstrate that postdevelopmental loss of Mecp2 in PVins of the auditory cortex is sufficient to delay onset of maternal retrieval. Our results support a model in which PVins play a central role in adult cortical plasticity and may be particularly impaired by loss of Mecp2 SIGNIFICANCE STATEMENT Rett syndrome is a neurodevelopmental disorder that includes deficits in both communication and the ability to update brain connections and activity during learning (plasticity). This condition is caused by mutations in the gene MECP2 We use a maternal behavioral test in mice requiring both vocal perception and neural plasticity to probe the role of Mecp2 in social and sensory learning. Mecp2 is normally active in all brain cells, but here we remove it from a specific population (parvalbumin neurons). We find that this is sufficient to delay learned behavioral responses to pups and recreates many deficits seen in whole-brain Mecp2 deletion. Our findings suggest that parvalbumin neurons specifically are central to the consequences of loss of Mecp2 activity and yield clues as to possible mechanisms by which Rett syndrome impairs brain function.

Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are “pervasive developmental disorders” and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.

Brain protein changes in Mecp2 mouse mutant models: Effects on disease progression of Mecp2 brain specific gene reactivation

Methyl CpG-binding protein 2 (MeCP2) is an epigenetic reader of DNA methylation with high abundance in the brain. While genetic mutations occur across different protein domains of MeCP2, the T158M mutation is amongst the most frequent MeCP2 mutations. MeCP2 is encoded by the MECP2/Mecp2 gene located on the X chromosome. In humans, MECP2 mutations cause Rett Syndrome, a debilitating neurodevelopmental disorder in females, with very rare cases presenting in males. Despite the generation of different transgenic mouse lines with MeCP2 mutations, the sex-dependent phenotypic and molecular impact of common MeCP2 mutations in mouse models of disease remains largely unexplored. Here, we focus on the MeCP2 T158M mutation using Mecp2tm4.1Bird/J transgenic mice (referred to as Mecp2T158M), and report that Mecp2T158M mutant mice display sex-specific molecular, behavioural, and phenotypic characteristics when compared to wild-type controls. Our data indicates sex- and brain-region-dependent impacts on the expression of MeCP2, synaptic proteins, cytoskeletal markers, and autophagy factors. Our findings demonstrate that the phenotypic and molecular characteristics of this mouse model may relate to the clinical manifestation in human patients with Rett Syndrome.

Objective To investigate the mutations in methyl-CpG-binding protein 2 gene(MECP2 gene) from typical sporadic Rett syndrome patients, explore the correlations between their genotype and phenotype, assist in genetic counseling. Methods Genomic DNA was extracted from peripheral blood leukocytes from 2 patients and their parents using standard protocols.Polymerase chain reaction and direct sequencing were performed using specific primers from 4 exons in MECP2 gene. Results No mutations were found in exon 1, 2, 3.Two different heterozygous missense mutations in exon 4 within MECP2 gene were identified from 2 patients.Their nuclear acid changes were: c.C473T and c. C397T, leading to amino acid change accordingly: p.T158M and p. R133C.There were no same mutations from their parents.Phenotype of patient with c. C397T was milder than patient with c. C473T. Conclusions Most of typical Rett syndrome patients had mutations in MECP2 gene.Gene test should be performed.Their biological parents should be detected accordingly if the patient had positive found to support genetic counseling. Key words: Rett syndrome; Methyl-CpG-binding protein 2; Gene mutation; Phenotype; Genetic counseling

Rett syndrome: Expanding the phenotype

Rett syndrome is a neurodevelopmental disorder and an important cause of mental retardation and autistic behavior in girls and in a small group of boys. In 1999, mutation of the methyl-CpG binding protein 2 (MECP2) gene encoding a transcriptional repressor on the X chromosome was found to cause Rett syndrome. Since this discovery, significant research has focused on the elucidation of its specific role in the central nervous system. Recent studies revealed that MECP2 is expressed in more differentiated neurons rather than in less differentiated neuroblasts and that MECP2 is involved in the maturation and maintenance of neurons, including dendritic arborization and axonal projections, rather than in early cell fate decisions in the mammalian brain. In this review, we summarize recent findings regarding regional, temporal, and cell type-specific MECP2 expression in the central nervous system; neurobiologic abnormalities in MECP2 -mutant mice; and MECP2 target genes in the central nervous system.

Rett syndrome (RTT) is a neurodevelopmental disorder caused predominantly by loss-of-function mutations in the gene Methyl-CpG-binding protein 2 (MECP2), which encodes the MeCP2 protein. RTT is a MECP2-related disorder, along with MECP2 duplication syndrome (MDS), caused by gain-of-function duplications of MECP2. Nearly two decades of research have advanced our knowledge of MeCP2 function in health and disease. The following review will discuss MeCP2 protein function and its dysregulation in the MECP2-related disorders RTT and MDS. This will include a discussion of the genetic underpinnings of these disorders, specifically how sporadic X-chromosome mutations arise and manifest in specific populations. We will then review current diagnostic guidelines and clinical manifestations of RTT and MDS. Next, we will delve into MeCP2 biology, describing the dual landscapes of methylated DNA and its reader MeCP2 across the neuronal genome as well as the function of MeCP2 as a transcriptional modulator. Following this, we will outline common MECP2 mutations and genotype-phenotype correlations in both diseases, with particular focus on mutations associated with relatively mild disease in RTT. We will also summarize decades of disease modeling and resulting molecular, synaptic, and behavioral phenotypes associated with RTT and MDS. Finally, we list several therapeutics in the development pipeline for RTT and MDS and available evidence of their safety and efficacy.

Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in MECP2, the gene encoding the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Mecp2 deletion in mice results in an imbalance of excitation and inhibition in hippocampal neurons, which affects 'Hebbian' synaptic plasticity. We show that Mecp2-deficient neurons also lack homeostatic synaptic plasticity, likely due to reduced levels of EEA1, a protein involved in AMPA receptor endocytosis. Expression of EEA1 restored homeostatic synaptic plasticity in Mecp2-deficient neurons, providing novel targets of intervention in Rett syndrome. Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in MECP2, the gene encoding the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Deletion of Mecp2 in mice results in an imbalance of synaptic excitation and inhibition in hippocampal pyramidal neurons, which affects 'Hebbian' long-term synaptic plasticity. Since the excitatory-inhibitory balance is maintained by homeostatic mechanisms, we examined the role of MeCP2 in homeostatic synaptic plasticity (HSP) at excitatory synapses. Negative feedback HSP, also known as synaptic scaling, maintains the global synaptic strength of individual neurons in response to sustained alterations in neuronal activity. Hippocampal neurons from Mecp2 knockout (KO) mice do not show the characteristic homeostatic scaling up of the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and of synaptic levels of the GluA1 subunit of AMPA-type glutamate receptors after 48h silencing with the Na+ channel blocker tetrodotoxin. This deficit in HSP is bidirectional because Mecp2 KO neurons also failed to scale down mEPSC amplitudes and GluA1 synaptic levels after 48h blockade of type A GABA receptor (GABAA R)-mediated inhibition with bicuculline. Consistent with the role of synaptic trafficking of AMPA-type of glutamate receptors in HSP, Mecp2 KO neurons have lower levels of early endosome antigen 1 (EEA1), a protein involved in AMPA-type glutamate receptor endocytosis. In addition, expression of EEA1 in Mecp2 KO neurons reduced mEPSC amplitudes to wild-type levels, and restored synaptic scaling down of mEPSC amplitudes after 48h blockade of GABAA R-mediated inhibition with bicuculline. The identification of a molecular deficit in HSP in Mecp2 KO neurons provides potentially novel targets of intervention for improving hippocampal function in Rett syndrome individuals.