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Rett syndrome (RTT) is a devastating neurodevelopmental disorder affecting 1 in 10,000 girls. Approximately 90% of cases are caused by spontaneous mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Girls with RTT suffer from severe motor, respiratory, cognitive and social abnomalities attributed to early deficits in synaptic connectivity which manifest in the adult as a myriad of physiological and anatomical abnormalities including, but not limited to, dimished dendritic complexity. Supplementation with acetyl-L-carnitine (ALC), an acetyl group donor, ameliorates motor and cognitive deficits in other disease models through a variety of mechanisms including altering patterns of histone acetylation resulting in changes in gene expression, and stimulating biosynthetic pathways such as acetylcholine. We hypothesized ALC treatment during critical periods in cortical development would promote normal synaptic maturation, and continuing treatment would improve behavioral deficits in the Mecp21lox mouse model of RTT. In this study, wildtype and Mecp21lox mutant mice received daily injections of ALC from birth until death (postnatal day 47). General health, motor, respiratory, and cognitive functions were assessed at several time points during symptom progression. ALC improved weight gain, grip strength, activity levels, prevented metabolic abnormalities and modestly improved cognitive function in Mecp2 null mice early in the course of treatment, but did not significantly improve motor or cognitive functions assessed later in life. ALC treatment from birth was associated with an almost complete rescue of hippocampal dendritic morphology abnormalities with no discernable side effects in the mutant mice. Therefore, ALC appears to be a promising therapeutic approach to treating early RTT symptoms and may be useful in combination with other therapies.

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder that affects girls due primarily to mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). The majority of RTT patients carry missense and nonsense mutations leading to a hypomorphic MECP2, while null mutations leading to the complete absence of a functional protein are rare. MECP2 is an X-linked gene subject to random X-chromosome inactivation resulting in mosaic expression of mutant MECP2. The lack of human brain tissue motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we isolated human induced pluripotent stem (hiPS) cells from RTT patient fibroblasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, we obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPS cell-derived neurons from the same RTT patient. Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons.

The neurodevelopmental disorder Rett Syndrome (RTT) is caused by sporadic mutations in the transcriptional factor methyl-CpG binding protein 2 (MeCP2). Although it is thought that the primary cause of RTT is cell autonomous due to lack of functional MeCP2 in neurons, whether non-cell autonomous factors contribute to the disease, is unknown. Here, we show that loss of MeCP2 occurs not only in neurons but also in glial cells of RTT brain. Using an in vitro co-culture system, we find that mutant astrocytes from a RTT mouse model, and their conditioned medium, fail to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. Our studies suggest that in RTT brain, astrocytes carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, likely due to aberrant secretion of soluble factor(s).

Functional deficiency of the X-linked methyl-CPG binding protein 2 (MeCP2) leads to the neurodevelopmental disorder Rett syndrome (RTT). Due to random X-chromosome inactivation (XCI), most RTT patients are females who are heterozygous for the MECP2 mutation and therefore mosaic in MeCP2 deficiency. Some MECP2 heterozygote females are found to have unbalanced XCI, which may affect the severity of neurological symptoms seen in these patients; however, whether MeCP2 deficiency affects XCI in the postnatal and adult brain is unclear. Here we developed a novel MeCP2 mosaic mouse model in which the X chromosome containing the wild-type Mecp2 expresses a green fluorescent protein (GFP) transgene, while the X chromosome harboring the mutant Mecp2 does not. Due to random XCI, the neurons in the female MeCP2 mosaic mice express either wild-type MeCP2 (GFP+) or mutant MeCP2 (GFP−), and the two can be distinguished by GFP fluorescence. Using this mouse model, we evaluated XCI in female heterozygote mice from 3 to 9 months after birth. We found that MeCP2 deficiency does not affect XCI at 3 months of age, but does alter the proportion of wild-type MeCP2-expressing neurons at later ages, suggesting that MeCP2 impacts XCI patterns in an age-dependent manner. Given the important function of MeCP2 in neuronal development, our data could shed light on how MeCP2 deficiency affects postnatal brain functions and the dynamic changes in the neurological symptoms of RTT.

Postnatal deficits in Brain-Derived Neurotrophic Factor (BDNF) are thought to contribute to pathogenesis of Rett syndrome (RTT), a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2 null mice, a model of RTT, BDNF deficits are most pronounced in structures important for autonomic and respiratory control, functions that are severely affected in RTT patients. However, relatively little is known about how these deficits affect neuronal function or how they may be linked to specific RTT endophenotypes. To approach these issues we analyzed synaptic function in the brainstem nucleus tractus solitarius (nTS), the principal site for integration of primary visceral afferent inputs to central autonomic pathways and a region in which we found markedly reduced levels of BDNF in Mecp2 mutants. Our results demonstrate that the amplitude of spontaneous miniature and evoked EPSCs in nTS neurons is significantly increased in Mecp2 null mice and, accordingly, that mutant cells are more likely than wildtype to fire action potentials in response to primary afferent stimulation. These changes occur without any increase in intrinsic neuronal excitability and are unaffected by blockade of inhibitory GABA currents. However, this synaptopathy is associated with decreased BDNF availability in the primary afferent pathway and can be rescued by application of exogenous BDNF. On the basis of these findings we hypothesize that altered sensory gating in nTS contributes to cardiorespiratory instability in RTT and that nTS is a site at which restoration of normal BDNF signaling could help reestablish normal homeostatic controls.

Rett syndrome (RTT, MIM#312750) is a neurodevelopmental disorder that is classified as an autism spectrum disorder. Clinically, RTT is characterized by psychomotor regression with loss of volitional hand use and spoken language, the development of repetitive hand stereotypies, and gait impairment. The majority of people with RTT have mutations in Methyl-CpG-binding Protein 2 (MECP2), a transcriptional regulator. Interestingly, alterations in the function of the protein product produced by MECP2, MeCP2, have been identified in a number of other clinical conditions. The many clinical features found in RTT and the various clinical problems that result from alteration in MeCP2 function have led to the belief that understanding RTT will provide insight into a number of other neurological disorders. Excitingly, RTT is reversible in a mouse model, providing inspiration and hope that such a goal may be achieved for RTT and potentially for many neurodevelopmental disorders.

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing (∼twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48hrs of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96hrs was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spines density than controls after 96hrs of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96hrs of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. MECP2 protein is primarily expressed in neurons, and mutations in the gene lead to the clinical features of RTT in human patients and neurological deficits in murine models. Visual function is relatively preserved in RTT patients, but the cause for this is unknown. We analyzed the eyes of two RTT patients who died of the disease, and found no gross or microscopic changes. MECP2 expression was examined using immunohistochemistry, and nuclear protein expression was largely limited to ganglion cells and the portion of the inner nuclear layer populated by amacrine cells. No significant differences in MECP2 protein level or distribution were identified in the two eyes from the RTT patients as compared to eleven controls. The findings were compared to MECP2 expression in the brain of these two subjects and in MeCP2 deficient mice. Our findings suggest that the normally limited expression of MECP2 in visual pathway neurons may underlie intact vision in RTT.

Background

Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) and one of the leading causes of mental retardation in females. RTT is characterized by psychomotor retardation, purposeless hand movements, autistic-like behavior and abnormal gait. We studied the effects of environmental enrichment (EE) on the phenotypic manifestations of a RTT mouse model that lacks MeCP2 (Mecp2−/y).

Principal Findings

We found that EE delayed and attenuated some neurological alterations presented by Mecp2−/y mice and prevented the development of motor discoordination and anxiety-related abnormalities. To define the molecular correlate of this beneficial effect of EE, we analyzed the expression of several synaptic marker genes whose expression is increased by EE in several mouse models.

Conclusions/Significance

We found that EE induced downregulation of several synaptic markers, suggesting that the partial prevention of RTT-associated phenotypes is achieved through a non-conventional transcriptional program.

Rett syndrome (RTT) is a neurodevelopmental disorder that affects girls due primarily to heterozygous mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2). Random X-chromosome inactivation (XCI) results in cellular mosaicism in which some cells express wild-type (WT) MECP2 while other cells express mutant MECP2. The generation of patient-specific human induced pluripotent stem cells (hiPSCs) facilitates the production of RTT-hiPSC-derived neurons in vitro to investigate disease mechanisms and identify novel drug treatments. The generation of RTT-hiPSCs has been reported by many laboratories, however, the XCI status of RTT-hiPSCs has been inconsistent. Some report RTT-hiPSCs retain the inactive X-chromosome (post-XCI) of the founder somatic cell allowing isogenic RTT-hiPSCs that express only the WT or mutant MECP2 allele to be isolated from the same patient. Post-XCI RTT-hiPSCs-derived neurons retain this allele-specific expression pattern of WT or mutant MECP2. Conversely, others report RTT-hiPSCs in which the inactive X-chromosome of the founder somatic cell reactivates (pre-XCI) upon reprogramming into RTT-hiPSCs. Pre-XCI RTT-hiPSC-derived neurons exhibit random XCI resulting in cellular mosaicism with respect to WT and mutant MECP2 expression. Here we review and attempt to interpret the inconsistencies in XCI status of RTT-hiPSCs generated to date by comparison to other pluripotent systems in vitro and in vivo and the methods used to analyze XCI. Finally, we discuss the relative strengths and weaknesses of post- and pre-XCI hiPSCs in the context of RTT, and other X-linked and autosomal disorders for translational medicine.

Rett syndrome (RTT) is a neurodevelopmental disorder predominantly occurring in females with an incidence of 1:10,000 births and caused by sporadic mutations in the MECP2 gene, which encodes methyl-CpG-binding protein-2, an epigenetic transcription factor that binds methylated DNA. The clinical hallmarks include a period of apparently normal early development followed by a plateau and then subsequent frank regression. Impaired visual and aural contact often leads to an initial diagnosis of autism. The characterization of experimental models based on the loss-of-function of the mouse Mecp2 gene revealed that subtle changes in the morphology and function of brain cells and synapses have profound consequences on network activities that underlie critical brain functions. Furthermore, these experimental models have been used for successful reversals of RTT-like symptoms by genetic, pharmacological and environmental manipulations, raising hope for novel therapeutic strategies to improve the quality of life of RTT individuals.

Synopsis

Mutations in MECP2 (methyl CpG binding protein 2) are linked to the severe postnatal neurodevelopmental disorder Rett Syndrome (RTT). MeCP2 was originally characterized as a transcriptional repressor that preferentially bound methylated DNA, however, recent data indicates MeCP2 is a multifunctional protein. MeCP2 binding is now associated with certain expressed genes and involved in nuclear organization as well, indicating its gene regulatory function is context dependent. In addition, MeCP2 is proposed to regulate mRNA splicing and a mouse model for RTT shows aberrant mRNA splicing. To further understand MeCP2 and potential roles in RTT pathogenesis, we have employed a biochemical approach to identify the MeCP2 protein complexes present in the mammalian brain. We show that MeCP2 exists in at least four biochemically distinct pools in the brain and characterize one novel brain-derived MeCP2 complex that contains the splicing factor Prpf3. MeCP2 directly interacts with Prpf3 in vitro and in vivo and many MECP2 RTT truncations disrupt the MeCP2-Prpf3 complex. In addition, MeCP2 and Prpf3 associate in vivo with mRNAs from genes known to be expressed when their promoters are associated with MeCP2. This data supports a role for MeCP2 in mRNA biogenesis and suggests an additional mechanism for RTT pathophysiology.

Objective

Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Despite distinct clinical features, the accumulation of clinical and molecular information in recent years has generated considerable confusion regarding the diagnosis of RTT. The purpose of this work was revise and clarify 2002 consensus criteria for the diagnosis of RTT in anticipation of treatment trials.

Method

RettSearch members, representing the majority of the international clinical RTT specialists, participated in an iterative process to come to a consensus on a revised and simplified clinical diagnostic criteria for RTT.

Results

The clinical criteria required for the diagnosis of classic and atypical RTT were clarified and simplified. Guidelines for the diagnosis and molecular evaluation of specific variant forms of RTT were developed.

Interpretation

These revised criteria provide clarity regarding the key features required for the diagnosis of RTT and reinforce the concept that RTT is a clinical diagnosis based on distinct clinical criteria, independent of molecular findings. We recommend that these criteria and guidelines be utilized in any proposed clinical research.

Mutations within the gene encoding methyl CpG binding protein 2 (MECP2) cause the autism-spectrum neurodevelopmental disorder Rett Syndrome (RTT). MECP2 recruits histone deacetylase to methylated DNA and acts as a long-range regulator of methylated genes. Despite ubiquitous MECP2 expression, the phenotype of RTT and the Mecp2-deficient mouse is largely restricted to the postnatal brain. Since Mecp2-deficient mice have a defect in neuronal maturation, we sought to understand how MECP2/Mecp2 mutations globally affect histone modifications during postnatal brain development by an immunofluorescence approach. Using an antibody specific to acetylated histone H3 lysine 9 (H3K9ac), a bright punctate nuclear staining pattern was observed as MECP2 expression increased in early postnatal neuronal nuclei. As neurons matured in juvenile and adult brain samples, the intensity of H3K9ac staining was reduced. Mecp2-deficient mouse and RTT cerebral neurons lacked this developmental reduction in H3K9ac staining compared to age-matched controls, resulting in a significant increase in neuronal nuclei with bright H3K9ac punctate staining. In contrast, trimethylated histone H3 lysine 9 (H3K9me3) localized to heterochromatin independent of MeCP2, but showed significantly reduced levels in Mecp2 deficient mouse and RTT brain. Autism brain with reduced MECP2 expression displayed similar histone H3 alterations as RTT brain. These observations suggest that MeCP2 regulates global histone modifications during a critical postnatal stage of neuronal maturation. These results have implications for understanding the molecular pathogenesis of RTT and autism in which MECP2 mutation or deficiency corresponds with arrested neurodevelopment.

Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). Mutations in loci other than MECP2 have also been found in individuals that have been labeled as atypical RTT. Among them, a mutation in the gene forkhead box G1 (FOXG1) has been involved in the molecular aetiology of the congenital variant of RTT. The FOXG1 gene encodes a winged-helix transcriptional repressor essential for the development of the ventral telencephalon in embryonic forebrain. Later, FOXG1 continues to be expressed in neurogenetic zones of the postnatal brain. Although RTT affects quasi-exclusively girls, FOXG1 mutations have also been identified in male patients. As far as we know, about 12 point mutations and 13 cases with FOXG1 molecular abnormalities (including translocation, duplication and large deletion on the chromosome 14q12) have been described in the literature. Affected individuals with FOXG1 mutations have shown dysmorphic features and Rett-like clinical course, including normal perinatal period, postnatal microcephaly, seizures and severe mental retardation. Interestingly, the existing animal models of FOXG1 deficiency showed similar phenotype, suggesting that animal models may be a fascinating model to understand this human disease. Here, we describe the impacts of FOXG1 mutations and their associated phenotypes in human and mouse models.

Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5–6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.

Summary

Mutations in MECP2 underlie the neurodevelopmental disorder Rett (RTT) syndrome. One hallmark of RTT is relatively normal development followed by a later onset of symptoms. Growing evidence suggests an etiology of disrupted synaptic function, yet it is unclear how these abnormalities explain the clinical presentation of RTT. Here we investigate synapse maturation in Mecp2-deficient mice at a circuit with distinct developmental phases– the retinogeniculate synapse. We find that synapse development in mutants is comparable to that of WT littermates between postnatal days 9–21, indicating that initial phases of synapse formation, elimination and strengthening are not significantly affected by MeCP2 absence. However, during the subsequent experience-dependent phase of synapse remodeling, the circuit becomes abnormal in mutants as retinal innervation of relay neurons increases and retinal inputs fail to strengthen further. Moreover, synaptic plasticity in response to visual deprivation is disrupted in mutants. These results suggest a crucial role for Mecp2 in experience-dependent refinement of synaptic circuits.

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked MECP2 gene, which encodes a methyl-CpG binding transcriptional repressor. Using the Mecp2-null mouse (an animal model for RTT) and differential display, we found that mice with neurological symptoms overexpress the nuclear gene for ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1). Chromatin immunoprecipitation demonstrated that MeCP2 interacts with the Uqcrc1 promoter. Uqcrc1 encodes a subunit of mitochondrial respiratory complex III, and isolated mitochondria from the Mecp2-null brain showed elevated respiration rates associated with respiratory complex III and an overall reduction in coupling. A causal link between Uqcrc1 gene overexpression and enhanced complex III activity was established in neuroblastoma cells. Our findings raise the possibility that mitochondrial dysfunction contributes to pathology of the Mecp2-null mouse and may contribute to the long-known resemblance between Rett syndrome and certain mitochondrial disorders.

Development of the nervous system proceeds through a set of complex checkpoints which arise from a combination of sequential gene expression and early neural activity sculpted by the environment. Genetic and environmental insults lead to neurodevelopmental disorders which encompass a large group of diseases that result from anatomical and physiological abnormalities during maturation and development of brain circuits. Rett syndrome (RTT) is a neurological disorder of genetic origin, caused by mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2). It features a range of neuropsychiatric abnormalities including motor dysfunctions and mild to severe cognitive impairment. Here, we discuss key questions and recent studies describing animal models, cell-type specific functions of methyl-CpG binding protein 2 (MeCP2), defects in neural circuit plasticity, and attempts to evaluate possible therapeutic strategies for RTT. We also discuss how genes, proteins, and overlapping signaling pathways affect the molecular etiology of apparently unrelated neuropsychiatric disorders, an understanding of which can offer novel therapeutic strategies for a range of autism spectrum disorders (ASDs).

A group of post-natal neurodevelopmental disorders collectively referred to as MeCP2 disorders are caused by aberrations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Loss of MeCP2 function causes Rett syndrome (RTT), whereas increased copy number of the gene causes MECP2 duplication or triplication syndromes. MeCP2 acts as a transcriptional repressor, however the gene expression changes observed in the hypothalamus of MeCP2 disorder mouse models suggest that MeCP2 can also upregulate gene expression, given that the majority of genes are downregulated upon loss of MeCP2 and upregulated in its presence. To determine if this dual role of MeCP2 extends beyond the hypothalamus, we studied gene expression patterns in the cerebellum of Mecp2-null and MECP2-Tg mice, modeling RTT and MECP2 duplication syndrome, respectively. We found that abnormal MeCP2 dosage causes alterations in the expression of hundreds of genes in the cerebellum. The majority of genes were upregulated in MECP2-Tg mice and downregulated in Mecp2-null mice, consistent with a role for MeCP2 as a modulator that can both increase and decrease gene expression. Interestingly, many of the genes altered in the cerebellum, particularly those increased by the presence of MeCP2 and decreased in its absence, were similarly altered in the hypothalamus. Our data suggest that either gain or loss of MeCP2 results in gene expression changes in multiple brain regions and that some of these changes are global. Further delineation of the expression pattern of MeCP2 target genes throughout the brain might identify subsets of genes that are more amenable to manipulation, and can thus be used to modulate some of the disease phenotypes.

Rett Syndrome (RTT) is a neurodevelopmental disorder predominantly caused by mutations in the X-linked gene MECP2. A primary feature of the syndrome is the impaired maturation and maintenance of excitatory synapses in the central nervous system (CNS). Different RTT mouse models have shown that particular Mecp2 mutations have highly variable effects on neuronal architecture. Distinguishing MeCP2 mutant cellular phenotypes therefore demands analysis of specific mutations in well-defined neuronal subpopulations. We examined a transgenically labeled subset of cortical neurons in YFP-H mice crossed with the Mecp2tm1.1Jae mutant line. YFP+ Layer 5 pyramidal neurons in the motor cortex of wildtype and hemizygous mutant male mice were examined for differences in dendrite morphology and spine density. Total basal dendritic length was decreased by 18.6% due to both shorter dendrites and reduced branching proximal to the soma. Tangential dendrite lengths in the apical tuft were reduced by up to 26.6%. Spine density was reduced by 47.4% in the apical tuft and 54.5% in secondary apical dendrites, but remained unaffected in primary apical and proximal basal dendrites. We also found that MeCP2 mutation reduced the number of YFP+ cells in YFP-H mice by up to 72% in various cortical regions without affecting the intensity of YFP expression in individual cells. Our results support the view that the effects of MeCP2 mutation are highly context-dependent and cannot be generalized across mutation types and cell populations.

Background:

Rett syndrome (RTT) is a neurodevelopmental disorder primarily seen in females, most with a mutation in MECP2. Epilepsy has been reported in 50%–80%. Previous reports were based on small sample sizes or parent-completed questionnaires, or failed to consider the impact of specific MECP2 mutations.

Methods:

The Rare Disease Consortium Research Network for RTT is an NIH-funded project to characterize the clinical spectrum and natural history of RTT in advance of clinical trials. Evaluations include clinical status (classic vs atypical RTT), MECP2 mutations, clinical severity, and presence, frequency, and treatment of seizures.

Results:

Enrollment as of June 2008 is 602; 528 (88%) meet clinical criteria for classic RTT. Of these, 493 (93%) have MECP2 mutations. Age range was 8 months to 64 years. A total of 360 (60%) were reported to have seizures, including 315 (60%) classic and 45 (61%) atypical RTT. Physician assessment of the 602 indicated that 48% had seizures. There was no significant difference in seizure occurrence by race/ethnicity. A significant age impact for seizures was seen and seizures were infrequent before age 2 years. MECP2 mutations most frequently associated with epilepsy were T158M (74%) and R106W (78%), and less frequently R255X and R306C (both 49%). Individuals with seizures had greater overall clinical severity, and greater impairment of ambulation, hand use, and communication.

Discussion:

Seizures are common in Rett syndrome, have an age-related onset and occurrence, vary by mutation, and are associated with greater clinical severity. This information represents a key consideration for designing clinical trials.

GLOSSARY

= antiepileptic drug;

= Clinical Severity Scale;

= Motor-Behavioral Assessment;

= Rett syndrome;

= vagus nerve stimulator.

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.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2, encoding methyl-CpG binding protein 2. MeCP2 is a transcriptional repressor elevated in mature neurons and is predicted to be required for neuronal maturation by regulating multiple target genes. Identifying primary gene targets in either Mecp2-deficient mice or human RTT brain has proven to be difficult, perhaps because of the transient requirement for MeCP2 during neuronal maturation. In order to experimentally control the timing of MeCP2 expression and deficiency during neuronal maturation, human SH-SY5Y cells undergoing mature neuronal differentiation were transfected with methylated MeCP2 oligonucleotide decoy to disrupt the binding of MeCP2 to endogenous targets. Genome-wide expression microarray analysis identified all four known members of the inhibitors of differentiation or inhibitors of DNA binding (ID1, ID2, ID3 and ID4) subfamily of helix-loop-helix (HLH) genes as novel neuronal targets of MeCP2. Chromatin immunoprecipitation analysis confirmed binding of MeCP2 near or within the promoters of ID1, ID2 and ID3, and quantitative RT-PCR confirmed increased expression of all four Id genes in Mecp2-deficient mouse brain. All four ID proteins were significantly increased in Mecp2-deficient mouse and human RTT brain using immunofluorescence and laser scanning cytometric analyses. Because of their involvement in cell differentiation and neural development, ID genes are ideal primary targets for MeCP2 regulation of neuronal maturation that may explain the molecular pathogenesis of RTT.

Alterations in dendritic spines have been documented in numerous neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, an X chromosome-linked disorder associated with mutations in MECP2, is the leading cause of intellectual disabilities in women. Neurons in Mecp2-deficient mice show lower dendritic spine density in several brain regions. To better understand the role of MeCP2 on excitatory spine synapses, we analyzed dendritic spines of CA1 pyramidal neurons in the hippocampus of Mecp2tm1.1Jae male mutant mice by either confocal microscopy or electron microscopy (EM). At postnatal-day 7 (P7), well before the onset of RTT-like symptoms, CA1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates. On the other hand, at P15 or later showing characteristic RTT-like symptoms, dendritic spine density did not differ between mutant and wildtype neurons. Consistently, stereological analyses at the EM level revealed similar densities of asymmetric spine synapses in CA1 stratum radiatum of symptomatic mutant and wildtype littermates. These results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic Mecp2-deficient mice. However, they underscore the potential role of MeCP2 in the maintenance of excitatory spine synapses.