MeCP2-deficient Neurons Research Articles

Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs.

Overshooting Subcellular Redox-Responses in Rett-Mouse Hippocampus during Neurotransmitter Stimulation.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with disturbed neuronal responsiveness and impaired neuronal network function. Furthermore, mitochondrial alterations and a weakened cellular redox-homeostasis are considered part of the complex pathogenesis. So far, overshooting redox-responses of MeCP2-deficient neurons were observed during oxidant-mediated stress, hypoxia and mitochondrial inhibition. To further clarify the relevance of the fragile redox-balance for the neuronal (dys)function in RTT, we addressed more physiological stimuli and quantified the subcellular redox responses to neurotransmitter-stimulation. The roGFP redox sensor was expressed in either the cytosol or the mitochondrial matrix of cultured mouse hippocampal neurons, and the responses to transient stimulation by glutamate, serotonin, dopamine and norepinephrine were characterized. Each neurotransmitter evoked more intense oxidizing responses in the cytosol of MeCP2-deficient than in wildtype neurons. In the mitochondrial matrix the neurotransmitter-evoked oxidizing changes were more moderate and more uniform among genotypes. This identifies the cytosol as an important reactive oxygen species (ROS) source and as less stably redox buffered. Fura-2 imaging and extracellular Ca2+ withdrawal confirmed cytosolic Ca2+ transients as a contributing factor of neurotransmitter-induced redox responses and their potentiation in the cytosol of MeCP2-deficient neurons. Chemical uncoupling demonstrated the involvement of mitochondria. Nevertheless, cytosolic NADPH- and xanthine oxidases interact to play the leading role in the neurotransmitter-mediated oxidizing responses. As exaggerated redox-responses were already evident in neonatal MeCP2-deficient neurons, they may contribute remarkably to the altered neuronal network performance and the disturbed neuronal signaling, which are among the hallmarks of RTT.

Graded and pan-neural disease phenotypes of Rett Syndrome linked with dosage of functional MeCP2

Rett syndrome (RTT) is a progressive neurodevelopmental disorder, mainly caused by mutations in MeCP2 and currently with no cure. We report here that neurons from R106W MeCP2 RTT human iPSCs as well as human embryonic stem cells after MeCP2 knockdown exhibit consistent and long-lasting impairment in maturation as indicated by impaired action potentials and passive membrane properties as well as reduced soma size and spine density. Moreover, RTT-inherent defects in neuronal maturation could be pan-neuronal and occurred in neurons with both dorsal and ventral forebrain features. Knockdown of MeCP2 led to more severe neuronal deficits as compared to RTT iPSC-derived neurons, which appeared to retain partial function. Strikingly, consistent deficits in nuclear size, dendritic complexity and circuitry-dependent spontaneous postsynaptic currents could only be observed in MeCP2 knockdown neurons but not RTT iPSC-derived neurons. Both neuron-intrinsic and circuitry-dependent deficits of MeCP2-deficient neurons could be fully or partially rescued by re-expression of wild type or T158M MeCP2, strengthening the dosage dependency of MeCP2 on disease phenotypes and also the partial function of the mutant. Our findings thus reveal stable neuronal maturation deficits and unexpectedly, graded sensitivities of neuron-inherent and neural transmission phenotypes towards the extent of MeCP2 deficiency, which is informative for future therapeutic development.

MECP2 Mutation Interrupts Nucleolin-mTOR-P70S6K Signaling in Rett Syndrome Patients.

Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)–P70S6K signaling. Currently, it is unclear how nucleolin–rRNA–mTOR–P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin–rRNA–mTOR–P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR–P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA–nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2–P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin–rRNA–mTOR–P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.

Loss of MeCP2 in immature neurons leads to impaired network integration.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations or deletions in Methyl-CpG-binding Protein 2 (MeCP2), a brain-enriched transcriptional regulator. MeCP2 is highly expressed during neuronal maturation and its deficiency results in impaired dendritic morphogenesis and reduced dendritic spine numbers in developing neurons. However, whether MeCP2 deficiency impacts the integration of new neurons has not been directly assessed. In this study, we developed a modified rabies virus-mediated monosynaptic retrograde tracing method to interrogate presynaptic integration of MeCP2-deficient new neurons born in the adult hippocampus, a region with lifelong neurogenesis and plasticity. We found that selective deletion of MeCP2 in adult-born new neurons impaired their long-range connectivity to the cortex, whereas their connectivity within the local hippocampal circuits or with subcortical regions was not significantly affected. We further showed that knockdown of MeCP2 in primary hippocampal neurons also resulted in reduced network integration. Interestingly, (1-3) insulin-like growth factor-1 (IGF-1), a small peptide under clinical trial testing for RTT, rescued neuronal integration deficits of MeCP2-deficient neurons in vitro but not in vivo. In addition, (1-3) IGF-1 treatment corrected aberrant excitability and network synchrony of MeCP2-deficient hippocampal neurons. Our results indicate that MeCP2 is essential for immature neurons to establish appropriate network connectivity.

Mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of a child with Rett syndrome

Rett syndrome is an X-linked neurodevelopmental disorder associated with psychomotor impairments, autonomic dysfunctions and autism. Patients with Rett syndrome have loss-of-function mutations in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Abnormal biogenic amine signaling and mitochondrial function have been found in patients with Rett syndrome; however, few studies have analyzed the association between these factors. This study investigated the functional relationships between mitochondria and the neuronal differentiation of the MeCP2-deficient stem cells from the exfoliated deciduous teeth of a child with Rett syndrome. An enrolled subject in this study was a 5-year-old girl carrying a large deletion that included the methyl-CpG-binding domain, transcriptional repression domain, and nuclear localization signal of MECP2. Using the single-cell isolation technique, we found that the two populations of MeCP2-expressing and MeCP2-deficient stem cells kept their MECP2 expression profiles throughout the stages of cell proliferation and neuronal differentiation in vitro. Neurite outgrowth and branching were attenuated in MeCP2-deficient dopaminergic neurons. MeCP2-deficient cells showed reduced mitochondrial membrane potential, ATP production, restricted mitochondrial distribution in neurites, and lower expression of a central mitochondrial fission factor, dynamin-related protein 1 than MeCP2-expressing cells. These data indicated that MeCP2-deficiency dysregulates the expression of mitochondrial factors required for the maturation of dopaminergic neurons. This study also provides insight into the pathogenic mechanism underlying dysfunction of the intracerebral dopaminergic signaling pathway in Rett syndrome.

EEA1 restores homeostatic synaptic plasticity in hippocampal neurons from Rett syndrome mice.

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.

DNA methylation in the gene body influences MeCP2-mediated gene repression

Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein gene (MECP2). MeCP2 is a methyl-cytosine binding protein that is proposed to function as a transcriptional repressor. However, multiple gene expression studies comparing wild-type and MeCP2-deficient neurons have failed to identify gene expression changes consistent with loss of a classical transcriptional repressor. Recent work suggests that one function of MeCP2 in neurons is to temper the expression of the longest genes in the genome by binding to methylated CA dinucleotides (mCA) within transcribed regions of these genes. Here we explore the mechanism of mCA and MeCP2 in fine tuning the expression of long genes. We find that mCA is not only highly enriched within the body of genes normally repressed by MeCP2, but also enriched within extended megabase-scale regions surrounding MeCP2-repressed genes. Whereas enrichment of mCA exists in a broad region around these genes, mCA together with mCG within gene bodies appears to be the primary driver of gene repression by MeCP2. Disruption of methylation at CA sites within the brain results in depletion of MeCP2 across genes that normally contain a high density of gene-body mCA. We further find that the degree of gene repression by MeCP2 is proportional to the total number of methylated cytosine MeCP2 binding sites across the body of a gene. These findings suggest a model in which MeCP2 tunes gene expression in neurons by binding within the transcribed regions of genes to impede the elongation of RNA polymerase.

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise.

Aberrant DNA methylation is a hallmark of various human disorders, indicating that the spatial and temporal regulation of methylation readers and modifiers is imperative for development and differentiation. In particular, the cross-regulation between 5-methylcytosine binders (MBD) and modifiers (Tet) has not been investigated. Here, we show that binding of Mecp2 and Mbd2 to DNA protects 5-methylcytosine from Tet1-mediated oxidation. The mechanism is not based on competition for 5-methylcytosine binding but on Mecp2 and Mbd2 directly restricting Tet1 access to DNA. We demonstrate that the efficiency of this process depends on the number of bound MBDs per DNA molecule. Accordingly, we find 5-hydroxymethylcytosine enriched at heterochromatin of Mecp2-deficient neurons of a mouse model for Rett syndrome and Tet1-induced reexpression of silenced major satellite repeats. These data unveil fundamental regulatory mechanisms of Tet enzymes and their potential pathophysiological role in Rett syndrome. Importantly, it suggests that Mecp2 and Mbd2 have an essential physiological role as guardians of the epigenome.

Loss of MeCP2 disrupts cell autonomous and autocrine BDNF signaling in mouse glutamatergic neurons.

Mutations in the MECP2 gene cause the neurodevelopmental disorder Rett syndrome (RTT). Previous studies have shown that altered MeCP2 levels result in aberrant neurite outgrowth and glutamatergic synapse formation. However, causal molecular mechanisms are not well understood since MeCP2 is known to regulate transcription of a wide range of target genes. Here, we describe a key role for a constitutive BDNF feed forward signaling pathway in regulating synaptic response, general growth and differentiation of glutamatergic neurons. Chronic block of TrkB receptors mimics the MeCP2 deficiency in wildtype glutamatergic neurons, while re-expression of BDNF quantitatively rescues MeCP2 deficiency. We show that BDNF acts cell autonomous and autocrine, as wildtype neurons are not capable of rescuing growth deficits in neighboring MeCP2 deficient neurons in vitro and in vivo. These findings are relevant for understanding RTT pathophysiology, wherein wildtype and mutant neurons are intermixed throughout the nervous system.

Sequence features accurately predict genome-wide MeCP2 binding in vivo.

Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2's affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.

KCC2 rescues functional deficits in human neurons derived from patients with Rett syndrome

Rett syndrome is a severe form of autism spectrum disorder, mainly caused by mutations of a single gene methyl CpG binding protein 2 (MeCP2) on the X chromosome. Patients with Rett syndrome exhibit a period of normal development followed by regression of brain function and the emergence of autistic behaviors. However, the mechanism behind the delayed onset of symptoms is largely unknown. Here we demonstrate that neuron-specific K(+)-Cl(-) cotransporter2 (KCC2) is a critical downstream gene target of MeCP2. We found that human neurons differentiated from induced pluripotent stem cells from patients with Rett syndrome showed a significant deficit in KCC2 expression and consequently a delayed GABA functional switch from excitation to inhibition. Interestingly, overexpression of KCC2 in MeCP2-deficient neurons rescued GABA functional deficits, suggesting an important role of KCC2 in Rett syndrome. We further identified that RE1-silencing transcriptional factor, REST, a neuronal gene repressor, mediates the MeCP2 regulation of KCC2. Because KCC2 is a slow onset molecule with expression level reaching maximum later in development, the functional deficit of KCC2 may offer an explanation for the delayed onset of Rett symptoms. Our studies suggest that restoring KCC2 function in Rett neurons may lead to a potential treatment for Rett syndrome.

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse.

Methyl-CpG-binding protein 2 (Mecp2) is an X-linked gene encoding a methylated DNA-binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome (RTT), a neurodevelopmental disorder. Patients with RTT frequently show abnormal sleep patterns and sleep-associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2-deficient mice. We successfully generated both male and female Mecp2-deficient mice on the wild-type C57BL/6 background and PER2(Luciferase) (PER2(Luc)) knock-in background using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Generated Mecp2-deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Furthermore, real-time bioluminescence imaging showed that the amplitude of PER2(Luc)-driven circadian oscillation was significantly attenuated in Mecp2-deficient SCN neurons. On the other hand, in vitro circadian rhythm development assay using Mecp2-deficient mouse embryonic stem cells (ESCs) did not show amplitude changes of PER2(Luc) bioluminescence rhythms. Together, these results show that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of patients with RTT.

Differential Expression and Regulation of Brain-Derived Neurotrophic Factor (BDNF) mRNA Isoforms in Brain Cells from Mecp2(308/y) Mouse Model.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused by mutations in methyl-CpG-binding protein 2 (MECP2), which encodes a transcriptional modulator of many genes including BDNF. BDNF comprises nine distinct promoter regions, each triggering the expression of a specific transcript. The role of this diversity of transcripts remains unknown. MeCP2 being highly expressed in neurons, RTT was initially considered as a neuronal disease. However, recent studies have shown that MeCP2 was also expressed in astrocytes. Though several studies explored Bdnf IV expression in Mecp2-deficient mice, the differential expression of Bdnf isoforms in Mecp2-deficient neurons and astrocytes was never studied. By using TaqMan technology and a mouse model expressing a truncated Mecp2 (Mecp2(308/y)), we firstly showed in neurons that Bdnf transcripts containing exon I, IIb, IIc, IV, and VI are prominently expressed, whereas in astrocytes, Bdnf transcript containing exon VI is preferentially expressed, suggesting a specific regulation of Bdnf expression at the cellular level. Secondly, we confirmed the repressive role of Mecp2 only on the expression of Bdnf VI in neurons. Our data suggested that the truncated Mecp2 protein maintains its function on Bdnf expression regulation in neurons and in astrocytes. Interestingly, we observed that Bdnf transcripts (I and IXA), regulated by neural activity induced by bicuculline in Mecp2(308/y) neurons, were not affected by histone deacetylase inhibition. In contrast, Bdnf transcripts (IIb, IIc, and VI), regulated by histone deacetylation, were not affected by bicuculline treatment in wild-type and Mecp2(308/y) neurons. All these results reflect the complexity of regulation of Bdnf gene.

Reduced seizure threshold and altered network oscillatory properties in a mouse model of Rett syndrome

Rett syndrome (RTT) is a disorder with a pronounced neurological phenotype and is caused mainly by mutations in the X-linked gene MECP2. A common feature of RTT is an abnormal electroencephalography and a propensity for seizures. In the current study we aimed to assess brain network excitability and seizure propensity in a mouse model of RTT. Mice in which Mecp2 expression was silenced (Mecp2stop/y) showed a higher seizure score (mean=6±0.8 compared to 4±0.2 in wild-type [WT]) and more rapid seizure onset (median onset=10min in Mecp2stop/y and 32min in WT) when challenged with the convulsant drug kainic acid (25mg/kg). Hippocampal slices from Mecp2stop/y brain displayed no spontaneous field potential activities under control conditions but showed higher power gamma frequency field potential oscillations compared to WT in response to kainic acid (400nM) in vitro. Brain slices challenged with the GABAA-receptor antagonist bicuculline (0.1–10μM) and the potassium channel blocker 4-aminopyridine (1–50μM) also revealed differences between genotypes with hippocampal circuits from Mecp2stop/y mouse slices showing enhanced epileptiform burst duration and frequency. In contrast to these network level findings, single cell analysis of pyramidal cells by whole-cell patch clamp recording revealed no detectable differences in synaptic or biophysical properties between methyl-CpG-binding protein 2 (MeCP2)-containing and MeCP2-deficient neurons. These data support the proposal that loss of MeCP2 alters network level excitability in the brain to promote epileptogenesis.

MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2 −/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.

Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2308 mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na+ channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

MeCP2 is required for global heterochromatic and nucleolar changes during activity-dependent neuronal maturation

Mutations in MECP2, encoding methyl CpG binding protein 2, cause the neurodevelopmental disorder Rett syndrome. MeCP2 is an abundant nuclear protein that binds to chromatin and modulates transcription in response to neuronal activity. Prior studies of MeCP2 function have focused on specific gene targets of MeCP2, but a more global role for MeCP2 in neuronal nuclear maturation has remained unexplored. MeCP2 levels increase during postnatal brain development, coinciding with dynamic changes in neuronal chromatin architecture, particularly detectable as changes in size, number, and location of nucleoli and perinucleolar heterochromatic chromocenters. To determine a potential role for MeCP2 in neuronal chromatin maturational changes, we measured nucleoli and chromocenters in developing wild-type and Mecp2-deficient mouse cortical sections, as well as mouse primary cortical neurons and a human neuronal cell line following induced maturation. Mecp2-deficient mouse neurons exhibited significant differences in nucleolar and chromocenter number and size, as more abundant, smaller nucleoli in brain and primary neurons compared to wild-type, consistent with delayed neuronal nuclear maturation in the absence of MeCP2. Primary neurons increased chromocenter size following depolarization in wild-type, but not Mecp2-deficient cultures. Wild-type MECP2e1 over-expression in human SH-SY5Y cells was sufficient to induce significantly larger nucleoli, but not a T158M mutation of the methyl-binding domain. These results suggest that, in addition to the established role of MeCP2 in transcriptional regulation of specific target genes, the global chromatin-binding function of MeCP2 is essential for activity-dependent global chromatin dynamics during postnatal neuronal maturation.

Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes

SummaryMutations in the X-linked MECP2, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome (RTT) and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia, and encephalopathy with early lethality. RTT is characterized by apparently normal early development followed by regression, motor abnormalities, seizures, and features of autism, especially stereotyped behaviors. The mechanisms mediating these striking features are poorly understood. Here we show that mice lacking Mecp2 from γ-amino-butyric-acid-(GABA)-ergic neurons recapitulate numerous RTT and autistic features, including repetitive behaviors. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of RTT. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size consistent with presynaptic reduction in glutamic acid decarboxylase-1 and -2 levels and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal GABAergic neuronal function and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes.