Loss Of methyl-CpG-binding Protein 2 Research Articles

In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system

The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2's function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post-transcriptional and post-translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals.

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.

Neural development of methyl-CpG-binding protein 2 null embryonic stem cells: A system for studying Rett syndrome

Mutations in methyl-CpG-binding protein 2 (MeCP2) gene cause the neurodevelopmental disorder Rett syndrome (RTT). Here, we describe a new experimental system that efficiently elucidates the role of MeCP2 in neural development. MeCP2-null and control ES cells were generated by adenoviral conditional targeting and examined for maintenance of the undifferentiated ES cell state, neurogenesis, and gliogenesis during in vitro differentiation. In addition, dopamine release and electrophysiological features of neurons differentiated from these ES cells were examined. Loss of MeCP2 did not affect undifferentiated ES cell colony morphology and growth, or the timing or efficiency of neural stem cell differentiation into Nestin-, TuJ- or TH-positive neurons. In contrast, gliogenesis was drastically accelerated by MeCP2 deficiency. Dopamine production and release in response to a depolarizing stimulus in MeCP2-null ES-derived dopaminergic neurons was intact. However, MeCP2-null differentiated neurons showed significantly smaller voltage-dependent Na + currents and A-type K + currents, suggesting incomplete maturation. Thus, MeCP2 is not essential for maintenance of the undifferentiated ES cell state, neurogenesis, or dopaminergic function; rather, it is principally involved in inhibiting gliogenesis. Altered neuronal maturity may indirectly result from abnormal glial development and may underlie the pathogenesis of RTT. These data contribute to a better understanding of the developmental roles of MeCP2 and the pathogenesis of RTT.

MeCP2 Is Required for Normal Development of GABAergic Circuits in the Thalamus

Methyl-CpG binding protein 2 (MeCP2) is highly expressed in neurons in the vertebrate brain, and mutations of the gene encoding MeCP2 cause the neurodevelopmental disorder Rett syndrome. This study examines the role of MeCP2 in the development and function of thalamic GABAergic circuits. Whole cell recordings were carried out in excitatory neurons of the ventrobasal complex (VB) of the thalamus and in inhibitory neurons of the reticular thalamic nucleus (RTN) in acute brain slices from mice aged P6 through P23. At P14-P16, the number of quantal GABAergic events was decreased in VB neurons but increased in RTN neurons of Mecp2-null mice, without any change in the amplitude or kinetics of quantal events. There was no difference between mutant and wild-type mice in paired-pulse ratios of evoked GABAergic responses in the VB or the RTN. On the other hand, unitary responses evoked by minimal stimulation were decreased in the VB but increased in the RTN of mutants. Similar changes in the frequency of quantal events were observed at P21-P23 in both the VB and RTN. At P6, however, quantal GABAergic transmission was altered only in the VB not the RTN. Immunostaining of vesicular GABA transporter showed opposite changes in the number of GABAergic synaptic terminals in the VB and RTN of Mecp2-null mice at P18-P20. The loss of MeCP2 had no significant effect on intrinsic properties of RTN neurons recorded at P15-P17. Our findings suggest that MeCP2 differentially regulates the development of GABAergic synapses in excitatory and inhibitory neurons in the thalamus.

Postnatal changes in serotonergic innervation to the hippocampus of methyl-CpG-binding protein 2-null mice

Rett syndrome is a progressive neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Previous reports have revealed serotonergic function to be altered in the medullas of patients with Rett syndrome and in an animal model of the disease. However, it has remained unclear whether a genetic loss of MeCP2 disrupts serotonergic innervation to the forebrain. In this study, we measured levels of monoamines by high-performance liquid chromatography with electrochemical detection in selected regions of the forebrains of Mecp2-null mice (Mecp2−/y) and wild-type mice (Mecp2+/y) on postnatal day (P) 14, P28, P42 and P56. The levels of hippocampal serotonin (5-HT) and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were significantly lower in Mecp2-null mice than in age-matched wild-type mice on P28, P42 and P56. Immunohistochemical analysis revealed a loss of 5-HT-immunoreactive fibers in the Mecp2-null hippocampus on P56. By contrast, in the raphe region of Mecp2-null mice, there were significant decreases in 5-HT and noradrenaline levels, but these differences later disappeared and there was no change in the number of 5-HT-immunoreactive neuronal cell bodies. Furthermore, we conducted an experiment comparing HPLC measurements in presymptomatic heterozygous females (Mecp2+/−) and wild-type female littermates (Mecp2+/+) on P56. Significant decreases in hippocampal 5-HT and 5-HIAA contents in Mecp2-heterozygous mice were revealed, and these were not accompanied by changes in 5-HT or noradrenaline contents in the raphe region. Therefore, these results indicated decreases in serotonergic innervation to the hippocampus in Mecp2-null males and Mecp2 heterozygous females. We speculate that disturbances in serotonergic neurotransmission in the hippocampus may be linked to the behavioral abnormalities seen in Rett syndrome, such as increased anxiety-like behaviors and reduced exploratory locomotion. MeCP2 may be required for stable serotonergic homeostasis and serotonergic innervation to the hippocampus during postnatal development.

The yin and yang of MeCP2 phosphorylation

Studies of the childhood neurological disorder Rett syndrome and methyl-CpG-binding protein 2 (MeCP2) taught us that MeCP2 performs a balancing act in modulating neurological functions. Rett syndrome, characterized by cognitive deficits, motor impairments, autistic-like features, seizures, and stereotyped repetitive hand movements is caused by loss of MeCP2 function (1). On the other hand, a syndrome with overlapping features including autism, mental retardation, seizures, motor impairments, and repetitive movements results from gain of MeCP2 function due to doubling or tripling the protein level (2–4). In animal models, neuronal loss of MeCP2 leads to reduced glutamatergic synaptic strength due to reduced synapse numbers and gain of MeCP2 in neurons results in increased synaptic strength due to increased synapse numbers (5). Until recently it was believed that MeCP2 was exclusively expressed in neurons in the central nervous system (6, 7), we now know MeCP2 is also expressed in astrocytes and that MeCP2 deficient astrocytes cannot support neuronal dendritic arborization (8). At the molecular level, several studies have shown that MeCP2 functions as a transcriptional repressor by binding to methylated CpG dinucleotides and recruiting co-repressor proteins to silence gene expression (9, 10). However, in vivo studies showed that loss of MeCP2 leads to reduced expression of thousands of genes suggesting that MeCP2 may be a transcriptional modulator important for decreasing the expression of some genes and enhancing the expression of others (11). In this issue of PNAS, we learn about yet other balancing forces in modulating MeCP2 function: the differential phosphorylation of MeCP2 in response to neuronal activity (12). Such phosphorylation events may be one key mechanism by which MeCP2 modulates gene expression. Fig. 1. MeCP2 phosphorylation: a balancing act in neurons. A. MeCP2 in resting neurons … 1To whom correspondence should be addressed. E-mail: hzoghbi{at}bcm.edu

Non–cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology

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, resulting from a lack of functional MeCP2 in neurons, whether non-cell autonomous factors contribute to the disease is unknown. We found that the loss of MeCP2 occurs not only in neurons but also in glial cells of RTT brains. Using an in vitro co-culture system, we found that mutant astrocytes from a RTT mouse model, and their conditioned medium, failed to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. Our studies suggest that astrocytes in the RTT brain carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, probably as a result of aberrant secretion of soluble factor(s).

Deletion of Mecp2 in Sim1-Expressing Neurons Reveals a Critical Role for MeCP2 in Feeding Behavior, Aggression, and the Response to Stress

Rett Syndrome (RTT) is an autism spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). In order to map the neuroanatomic origins of the complex neuropsychiatric behaviors observed in patients with RTT and to uncover endogenous functions of MeCP2 in the hypothalamus, we removed Mecp2 from Sim1-expressing neurons in the hypothalamus using Cre-loxP technology. Loss of MeCP2 in Sim1-expressing neurons resulted in mice that recapitulated the abnormal physiological stress response that is seen upon MeCP2 dysfunction in the entire brain. Surprisingly, we also uncovered a role for MeCP2 in the regulation of social and feeding behaviors since the Mecp2 conditional knockout (CKO) mice were aggressive, hyperphagic, and obese. This study demonstrates that deleting Mecp2 in a defined brain region is an excellent approach to map the neuronal origins of complex behaviors and provides new insight about the function of MeCP2 in specific neurons.

The MeCP2‐null mouse hippocampus displays altered basal inhibitory rhythms and is prone to hyperexcitability

Rett syndrome is an autism-spectrum disorder caused by loss of function mutations within the gene encoding methyl CpG-binding protein 2 (MeCP2). While subtle decreases in synaptic plasticity have been detected within cortical and hippocampal neurons of Mecp2-null mice, only minimal information exists regarding how the loss of MeCP2 affects network activity in the brain. To address this issue, we compared the intrinsic network activities of Mecp2-null hippocampal slices derived from symptomatic mice to wild-type slices. Extracellular and whole-cell patch recordings revealed that although spontaneous, IPSP-based rhythmic activity is present in Mecp2-null slices; its frequency is significantly reduced from wild-type. This reduction was not associated with alterations in the gross electrophysiological properties of hippocampal neurons, but was associated with a decreased level of spontaneous glutamate receptor-mediated synaptic currents in hippocampal CA3 neurons. Paradoxically, however, repetitive sharp wave-like discharges were readily induced in the Mecp2-null hippocampal slices by a brief train of high-frequency stimulation commonly used to establish long-term potentiation at wild-type slices. Taken together, our data indicate that the Mecp2-null hippocampal CA3 circuit has diminished basal inhibitory rhythmic activity, which in turn renders the circuitry prone to hyperexcitability.

Homeostatic regulation of MeCP2 expression by a CREB-induced microRNA

Both increases and decreases in methyl CpG-binding protein 2 (MeCP2) levels cause neurodevelopmental defects. We found that MeCP2 translation is regulated by microRNA 132 (miR132). Block of miR132-mediated repression increased MeCP2 and brain-derived neurotrophic factor (BDNF) levels in cultured rat neurons and the loss of MeCP2 reduced BDNF and miR132 levels in vivo. This feedback loop may provide a mechanism for homeostatic control of MeCP2 expression.

Dysregulation of Brain-Derived Neurotrophic Factor Expression and Neurosecretory Function inMecp2Null Mice

Disruptions in brain-derived neurotrophic factor (BDNF) expression are proposed to contribute to the molecular pathogenesis of Rett syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein-2 (MeCP2). Although MeCP2 is a transcriptional regulator of BDNF, it is unknown how MeCP2 mutations affect transsynaptic BDNF signaling. Our findings demonstrate an early, abnormal neurosecretory phenotype in MeCP2-deficient neurons characterized by significant increases in the percentage of cellular BDNF content available for release. However, loss of MeCP2 also results in deficits in total cell BDNF content that are developmentally regulated in a cell-type-specific manner. Thus, the net effect of MeCP2 loss on absolute BDNF secretion changes with age and is determined by both the amount of BDNF available for release and progressive declines in total cellular BDNF. We propose, therefore, that loss of MeCP2 function disrupts transsynaptic BDNF signaling by perturbing the normal balance between BDNF protein levels and secretion. However, mutant neurons are capable of secreting wild-type levels of BDNF in response to high-frequency electrical stimulation. In addition, we found elevated exocytic function in Mecp2(-/y) adrenal chromaffin cells, indicating that the Mecp2 null mutation is associated with alterations of neurosecretion that are not restricted to BDNF. These findings are the first examples of abnormal neuropeptide and catecholamine secretion in a mouse model of RTT.

MeCP2-Dependent Transcriptional Repression Regulates Excitatory Neurotransmission

Mutations in the transcriptional repressor, methyl-CpG binding protein 2 (MeCP2), result in a neurodevelopmental disorder called Rett Syndrome (RTT) . Based on the neurological phenotypes observed in Rett patients, we examined the potential role of MeCP2 in synaptic function. We compared elementary properties of synaptic transmission between cultured hippocampal neurons from MeCP2 knockout and wild-type littermate control mice and found a decrease in the frequency of spontaneous excitatory synaptic transmission (mEPSCs) in neurons lacking MeCP2. We also detected a significant increase in the rate of short-term synaptic depression. To explore whether these functional effects can be attributed to MeCP2's role as a transcriptional silencer, we treated cultures with a drug that impairs histone deacetylation and examined spontaneous synaptic transmission. Treatment with this compound induced a similar decrease in mEPSC frequency in wild-type control cultures, but this decrease was occluded in MeCP2-deficient neurons. Interestingly, neither the loss of MeCP2 nor the drug treatment resulted in changes in mIPSC properties. Finally, by means of a lentivirus expressing Cre recombinase, we show that loss of MeCP2 function after neurodevelopment and synaptogenesis was sufficient to mimic the decrease in mEPSC frequency seen in constitutive MeCP2 KO neurons. Taken together, these results suggest a role for MeCP2 in control of excitatory presynaptic function through regulation of gene expression.

Postnatal Loss of Methyl-CpG Binding Protein 2 in the Forebrain is Sufficient to Mediate Behavioral Aspects of Rett Syndrome in Mice

Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues. To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice. These mice display behavioral abnormalities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits. These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT.

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.