methyl-CpG-binding Protein 2 Levels Research Articles

Chronic corticosterone exposure in rats induces sex-specific alterations in hypothalamic reelin fragments, MeCP2, and DNMT3a protein levels

Women are disproportionately affected by stress-related disorders like depression. In our prior research, we discovered that females exhibit lower basal hypothalamic reelin levels, and these levels are differentially influenced by chronic stress induced through repeated corticosterone (CORT) injections. Although epigenetic mechanisms involving DNA methylation and the formation of repressor complexes by DNA methyl-transferases (DNMTs) and Methyl-CpG binding protein 2 (MeCP2) have been recognized as regulators of reelin expression in vitro, there is limited understanding of the impact of stress on the epigenetic regulation of reelin in vivo and whether sex differences exist in these mechanisms. To address these questions, we conducted various biochemical analyses on hypothalamic brain samples obtained from male and female rats previously treated with either 21 days of CORT (40 mg/kg) or vehicle (0.9 % saline) subcutaneous injections. Upon chronic CORT treatment, a reduction in reelin fragment NR2 was noted in males, while the full-length molecule remained unaffected. This decrease paralleled with an elevation in MeCP2 and a reduction in DNMT3a protein levels only in males. Importantly, sex differences in baseline and CORT-induced reelin protein levels were not associated with changes in the methylation status of the Reln promoter. These findings suggest that CORT-induced reelin decreases in the hypothalamus may be a combination of alterations in downstream processes beyond gene transcription. This research brings novel insights into the sexually distinct consequences of chronic stress, an essential aspect to understand, particularly concerning its role in the development of depression.

The Epigenetic Reader Methyl-CpG-Binding Protein 2 (MeCP2) Is an Emerging Oncogene in Cancer Biology

Epigenetic mechanisms are gene regulatory processes that control gene expression and cellular identity. Epigenetic factors include the “writers”, “readers”, and “erasers” of epigenetic modifications such as DNA methylation. Accordingly, the nuclear protein Methyl-CpG-Binding Protein 2 (MeCP2) is a reader of DNA methylation with key roles in cellular identity and function. Research studies have linked altered DNA methylation, deregulation of MeCP2 levels, or MECP2 gene mutations to different types of human disease. Due to the high expression level of MeCP2 in the brain, many studies have focused on its role in neurological and neurodevelopmental disorders. However, it is becoming increasingly apparent that MeCP2 also participates in the tumorigenesis of different types of human cancer, with potential oncogenic properties. It is well documented that aberrant epigenetic regulation such as altered DNA methylation may lead to cancer and the process of tumorigenesis. However, direct involvement of MeCP2 with that of human cancer was not fully investigated until lately. In recent years, a multitude of research studies from independent groups have explored the molecular mechanisms involving MeCP2 in a vast array of human cancers that focus on the oncogenic characteristics of MeCP2. Here, we provide an overview of the proposed role of MeCP2 as an emerging oncogene in different types of human cancer.

Clinical significance of methyl-CpG binding protein 2 in postherpetic neuralgia: an observational study

The present study was aimed to investigate the clinical significance of methyl-CpG binding protein 2 (MECP2) in patients with postherpetic neuralgia (PHN). This prospective case control study enrolled 319 cases of PHN patients from April 2017~December 2019. The patients’ sleep quality and quality of life were evaluated using the Pittsburgh sleep quality score and the SF-36 scale, respectively. The serum levels of MECP2, CRP, IL-6 and TNF-α were tested using enzyme linked immunosorbent assay (ELISA). The pain condition of the patients was evaluated using the visual analogue scale (VAS). The levels of MECP2 were significantly increased in PHN patients compared with the patients without PHN. Serum MECP2 levels were the highest in patients with severe pain, and were the lowest in patients with mild pain. Similarly, the frequency of severe pain in patients with low expression of MECP2 was significantly lower than the patients with higher MECP2 expression. Besides, serum levels of inflammatory factors CRP, IL-6 and TNF-α were markedly increased in PHN patients, which were also increased with the increase of the severity of pain. CRP, IL-6 and TNF-α were positively correlated with serum levels of MECP2 in PHN patients. Before the study, patients with lower MECP2 levels showed a significantly higher SF-36 score and lower Pittsburgh and VAS scores than patients with higher levels of MECP2. However, after one month, no significant difference was found between the patients. ROC curve showed MECP2 had the potential as a diagnostic biomarker for PHN. In conclusion, higher serum MECP2 levels are associated with a more severe pain condition and increased release of inflammatory factors.

Exploration of group II metabotropic glutamate receptor modulation in mouse models of Rett syndrome and MECP2 Duplication syndrome

Rett syndrome (RTT) and MECP2 Duplication syndrome (MDS) have opposing molecular origins in relation to expression and function of the transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2). Several clinical and preclinical phenotypes, however, are shared between these disorders. Modulation of MeCP2 levels has recently emerged as a potential treatment option for both of these diseases. However, toxicity concerns remain with these approaches. Here, we focus on pharmacologically modulating the group II metabotropic glutamate receptors (mGlu), mGlu2 and mGlu3, which are two downstream targets of MeCP2 that are bidirectionally affected in expression in RTT patients and mice (Mecp2Null/+) versus an MDS mouse model (MECP2Tg1/o). Mecp2Null/+ and MECP2Tg1/o animals also exhibit contrasting phenotypes in trace fear acquisition, a form of temporal associative learning and memory, with trace fear deficiency observed in Mecp2Null/+ mice and abnormally enhanced trace fear acquisition in MECP2Tg1/o animals. In Mecp2Null/+ mice, treatment with the mGlu2/3 agonist LY379268 reverses the deficit in trace fear acquisition, and mGlu2/3 antagonism with LY341495 normalizes the abnormal trace fear learning and memory phenotype in MECP2Tg1/o mice. Altogether, these data highlight the role of group II mGlu receptors in RTT and MDS and demonstrate that both mGlu2 and mGlu3 may be potential therapeutic targets for these disorders.

Methyl-CpG binding protein 2 is associated with the prognosis and mortality of elderly patients with hip fractures

To investigate the expression level and clinical significance of Methyl-CpG binding Protein2 (MECP2) in elderly patients with hip fractures. This prospective observational study included367elderly patients with hip fractures between April2016 and December2018. All the patients were treated with internal fixation or joint replacement. In addition, 50healthy elderly individuals were enrolled as healthy controls. The serum levels of MECP2 and inflammatory factors Interleukin (IL)-1β, IL-6, IL-8, and Tumor Necrosis Factor (TNF)-α was determined by enzyme-linked immunosorbent assay. Data on patients' basic characteristics and postoperative complications were collected. The Harris score was used to assess hip function at1-month, 3-months, and 6-months after surgery. Patient quality of life was measured using the Barthel Index (BI) score3-months after surgery. The1-year mortality was analyzed using the Kaplan-Meier curve, and logical regression was used to analyze the risk factors for mortality. No significant differences were observed in the basic clinical characteristics of all patients. The serum MECP2 levels were remarkably high in patients with hip fractures and negatively correlated with serum IL-1β, IL-6, and TNF-α levels. Patients with higher MECP2 predicted higher dynamic Harris scores, lower postoperative complications, lower1-year mortality, and higher BI scores. Logical regression showed that age was the only independent risk factor for postoperative1-year mortality in elderly patients with hip fractures. Lower MECP2 predicted poor prognosis and higher1-year mortality in elderly patients with hip fractures.

Profiling beneficial and potential adverse effects of MeCP2 overexpression in a hypomorphic Rett syndrome mouse model.

De novo loss-of-function mutations in methyl-CpG-binding protein 2 (MeCP2) lead to the neurodevelopmental disorder Rett syndrome (RTT). Despite promising results from strategies aimed at increasing MeCP2 levels, additional studies exploring how hypomorphic MeCP2 mutations impact the therapeutic window are needed. Here, we investigated the consequences of genetically introducing a wild-type MECP2 transgene in the Mecp2 R133C mouse model of RTT. The MECP2 transgene reversed the majority of RTT-like phenotypes exhibited by male and female Mecp2 R133C mice. However, three core symptom domains were adversely affected in female Mecp2R133C/+ animals; these phenotypes resemble those observed in disease contexts of excess MeCP2. Parallel control experiments in Mecp2Null/+ mice linked these adverse effects to the hypomorphic R133C mutation. Collectively, these data provide evidence regarding the safety and efficacy of genetically overexpressing functional MeCP2 in Mecp2 R133C mice and suggest that personalized approaches may warrant consideration for the clinical assessment of MeCP2-targeted therapies.

Expression and Distribution Pattern of DNMT1 and MeCP2 and their Relationship with GnRH and Kisspeptin in the Hypothalamus during Puberty Onset in Ewes

Background: The puberty onset is directly related to the sexual maturity and reproductive performance of ewes and the economic benefits of sheep raising industry. Previous studies have shown that DNA methylation in hypothalamus plays an important role in puberty onset of female mammals, but the mechanism is still unclear. The present study aims to investigate the expression and distribution of DNA methyltransferase 1 (DNMT1) and Methyl-CpG-binding protein 2 (MeCP2) and their relationship with Gonadotropin-releasing hormone (GnRH) and kisspeptin in the hypothalamus during the onset of puberty in ewes.Methods: We choose three female small-tailed Han sheep at peri-puberty, pre-puberty and puberty respectively. There after, hypothalamus tissue samples from each ewe were harvested for Real-Time quantitative PCR and double-labeling immunofluorescence.Result: The mRNA expression of GnRH and KiSS-1 were increased significantly, while the mRNA expression levels of DNMT1 and MeCP2 decreased during puberty onset in ewes. DNMT1 and MeCP2 were uniformly distributed in preoptic area (POA) and arcuate nucleus (ARC) in the hypothalamus and both exhibits co-expression patterns with GnRH and kisspeptin. The rate of co-expressing positive cells increased in ARC, while it decreased in POA during puberty onset in ewes. The results indicate that DNMT1 and MeCP2 might be involved in puberty onset through co-expression with GnRH and kisspeptin in the hypothalamus of ewes. Our findings not only provide a reference for the comprehensive understanding of the mechanism for mammalian puberty onset, but also lay a foundation for improving reproductive capacity of ewes.

Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

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.

MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons.

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.SIGNIFICANCE STATEMENT Neuronal function is critically dependent on methyl-CpG binding protein 2 (MeCP2), a nuclear protein abundantly expressed in neurons. The importance of MeCP2 is underscored by the severe childhood neurologic disorders, Rett syndrome (RTT) and MECP2 multiplication disorders, which are caused by depletion and overabundance of MeCP2, respectively. To clarify the molecular function of MeCP2 and to understand the pathogenesis of MECP2-related disorders, we performed detailed structural analyses of neuronal nuclei by using mouse models and high-resolution microscopy. We show that the level of MeCP2 critically regulates 3D structure of heterochromatic foci, and this is mediated in part by the AT-hook 2 domain of MeCP2. Our results demonstrate that one primary function of MeCP2 is to regulate chromatin structure.

Reversal of Social Recognition Deficit in Adult Mice with MECP2 Duplication via Normalization of MeCP2 in the Medial Prefrontal Cortex

Methyl-CpG binding protein 2 (MeCP2) is a basic nuclear protein involved in the regulation of gene expression and microRNA processing. Duplication of MECP2-containing genomic segments causes MECP2 duplication syndrome, a severe neurodevelopmental disorder characterized by intellectual disability, motor dysfunction, heightened anxiety, epilepsy, autistic phenotypes, and early death. Reversal of the abnormal phenotypes in adult mice with MECP2 duplication (MECP2-TG) by normalizing the MeCP2 levels across the whole brain has been demonstrated. However, whether different brain areas or neural circuits contribute to different aspects of the behavioral deficits is still unknown. Here, we found that MECP2-TG mice showed a significant social recognition deficit, and were prone to display aversive-like behaviors, including heightened anxiety-like behaviors and a fear generalization phenotype. In addition, reduced locomotor activity was observed in MECP2-TG mice. However, appetitive behaviors and learning and memory were comparable in MECP2-TG and wild-type mice. Functional magnetic resonance imaging illustrated that the differences between MECP2-TG and wild-type mice were mainly concentrated in brain areas regulating emotion and social behaviors. We used the CRISPR-Cas9 method to restore normal MeCP2 levels in the medial prefrontal cortex (mPFC) and bed nuclei of the stria terminalis (BST) of adult MECP2-TG mice, and found that normalization of MeCP2 levels in the mPFC but not in the BST reversed the social recognition deficit. These data indicate that the mPFC is responsible for the social recognition deficit in the transgenic mice, and provide new insight into potential therapies for MECP2 duplication syndrome.

DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats.

Alcohol abuse causes tissue and organ damage, and may participate neuropsychiatric diseases. Studies have shown that DNA methylation plays an important role in gene expression and behavioral changes induced by alcohol, however the causative neurobiological mechanisms have not been clarified. In this study, 32 healthy adult male SD rats were randomly divided into a drinking water control group (n=16) and a chronic alcohol exposure group (n=16). The alcohol preference and locomotor activity of rats were evaluated by two-bottle choice test (TBCT) and open-field test (OFT). DNA methylation in the medial prefrontal cortex (mPFC) tissue was detected by the reduced representative bisulfite sequencing (RRBS) technology. The methylation differential genes closely related to alcohol abuse were screened. qRT-PCR was used to verify the mRNA expression patterns of differential genes. qRT-PCR and Western blot were used to detect the expression of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MeCP2). Furthermore, the effect of short-term alcohol exposure (7 days) on DNMTs and MeCP2 in the mPFC of rats was tested (n=8/group). The results indicated that the methylation level of promoter region in the mPFC of rats exposed to chronic alcohol was significantly increased. In addition, the increased methylation levels in the promoter of Ntf3 and Ppm1G were accompanied by down-regulated mRNA levels in the chronic alcohol exposure group. The decreased methylation levels in the promoter of Hap1 and DUSP1 were accompanied by up-regulated mRNA levels. Furthermore, chronic alcohol exposure increased the mRNA and protein levels of DNMT3B and MeCP2. However, short term alcohol exposure did not affect their expression. This present study provides evidence that DNA methylation is associated with the development of alcohol abuse, which may be regulated by DNMT3B and MeCP2. The target genes Ntf3, Ppm1G, Hap1, and DUSP1 related to alcohol abuse were discovered as well, providing new insights into the neurobiological mechanism of alcohol abuse and the potential pharmacological targets for the treatment of alcohol abuse.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

DNA Methylation Contributes to the Differential Expression Levels of Mecp2 in Male Mice Neurons and Astrocytes.

Methyl CpG binding protein-2 (MeCP2) isoforms (E1 and E2) are important epigenetic regulators in brain cells. Accordingly, MeCP2 loss- or gain-of-function mutation causes neurodevelopmental disorders, including Rett syndrome (RTT), MECP2 duplication syndrome (MDS), and autism spectrum disorders (ASD). Within different types of brain cells, highest MeCP2 levels are detected in neurons and the lowest in astrocytes. However, our current knowledge of Mecp2/MeCP2 regulatory mechanisms remains largely elusive. It appears that there is a sex-dependent effect in X-linked MeCP2-associated disorders, as RTT primarily affects females, whereas MDS is found almost exclusively in males. This suggests that Mecp2 expression levels in brain cells might be sex-dependent. Here, we investigated the sex- and cell type-specific expression of Mecp2 isoforms in male and female primary neurons and astrocytes isolated from the murine forebrain. Previously, we reported that DNA methylation of six Mecp2 regulatory elements correlated with Mecp2 levels in the brain. We now show that in male brain cells, DNA methylation is significantly correlated with the transcript expression of these two isoforms. We show that both Mecp2 isoforms are highly expressed in male neurons compared to male astrocytes, with Mecp2e1 expressed at higher levels than Mecp2e2. Our data indicate that higher DNA methylation at the Mecp2 regulatory element(s) is associated with lower levels of Mecp2 isoforms in male astrocytes compared to male neurons.

Tsix–Mecp2 female mouse model for Rett syndrome reveals that low-level MECP2 expression extends life and improves neuromotor function

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by a mutation in the X-linked methyl-CpG-binding protein 2 (MECP2). There is currently no disease-specific treatment, but MECP2 restoration through reactivation of the inactive X (Xi) has been of considerable interest. Progress toward an Xi-reactivation therapy has been hampered by a lack of suitable female mouse models. Because of cellular mosaicism due to random X-chromosome inactivation (XCI), Mecp2+/- heterozygous females develop only mild RTT. Here, we create an improved female mouse model by introducing a mutation in Tsix, the antisense regulator of XCI allelic choice. Tsix-Mecp2 mice show reduced MECP2 mosaicism and closely phenocopy the severely affected Mecp2-null males. Tsix-Mecp2 females demonstrate shortened lifespan, motor weakness, tremors, and gait disturbance. Intriguingly, they also exhibit repetitive behaviors, as is often seen in human RTT, including excessive grooming and biting that result in self-injury. With a Tsix allelic series, we vary MECP2 levels in brain and demonstrate a direct, but nonlinear correlation between MECP2 levels and phenotypic improvement. As little as 5-10% MECP2 restoration improves neuromotor function and extends lifespan five- to eightfold. Our study thus guides future pharmacological strategies and suggests that partial MECP2 restoration could have disproportionate therapeutic benefit.

Role of DNA Methylation in Hypobaric Hypoxia-Induced Neurodegeneration and Spatial Memory Impairment

Hypobaric hypoxia (HH) is a major stress factor that is associated with physiological, biochemical, molecular and genomic alterations. Brain is the organ that reacts sensitively to oxygen deprivation, which leads to oxidative stress and cognitive function impairment. Our previous studies have reported that downregulation of brain derived neurotrophic factor (BDNF) leads to neurodegeneration and memory impairment. The aim of the present study was to investigate the effect of HH exposure on DNA methylation and its regulation in BDNF expression, neurodegeneration and spatial memory impairment. For this purpose, Sprague Dawley rats were exposed to HH at a simulated altitude of 25,000 feet for 14 days. Real-time polymerase chain reaction was used for transcriptional expression of DNA Methyltransferases (DNMTs) including DNMT1, DNMT3a and ­DNMT3b, and immunoblotting was used for the translational expression of DNMT1, DNMT3a, DNMT3b, Methyl CpG binding protein 2 (MeCP2), pMeCP2 and BDNF in rat hippocampus. Additionally, neuronal morphology alteration and neurodegeneration in CA1 region of hippocampus were investigated though Cresyl violet (CV) staining and Fluoro-Jade C staining respectively. Results obtained suggested that HH exposure increased the expression of DNMT1 DNMT3b at the mRNA as well as protein level, whereas no significant change was observed in the level of DNMT3a. Furthermore, the level of pMeCP2 and BDNF were significantly decreased; however, the expression level of MeCP2 was significantly increased. The CV and Fluoro-Jade C-positive cells were significantly enhanced in the CA1 region of hippocampus in the HH exposed group as compared to unexposed rats. Thus, the present study concluded that HH decreases neuronal activation by the upregulation of DNA methylation and MeCP2 and decreased the expression of pMeCP2, which result in the downregulation of BDNF. The decreased BDNF expression is associated with neuronal loss and spatial memory impairment. This study highlights that DNMT inhibition could be an important therapeutic target for neurodegenerative diseases.

Effect of MeCP2 on TGF- β1-induced Extracellular Matrix Production in Nasal Polyp-derived Fibroblasts.

Purpose Methyl-CpG-binding protein 2 (MeCP2), known as a transcriptional regulator, has been suggested to play an important role in myofibroblast differentiation in the lung. The purpose of this study was to investigate the role of MeCP2 in transforming growth factor (TGF)- β1-induced myofibroblast differentiation and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs). Methods To identify the expression of MeCP2 in nasal polyp tissues, immunohistochemistry staining and Western blot were performed. TGF- β1-induced NPDFs were treated with 5-azacytidine, a DNA methylation inhibitor, and the expression levels of α-SMA and fibronectin were determined by semiquantitative reverse transcription polymerase chain reaction, immunofluorescent staining, and Western blotting. The total soluble collagen was analyzed by the Sircol collagen assay. MeCP2 silenced by MeCP2-specific small interference ( si) RNA was verified by Western blot. Results The expression levels of MeCP2 increased in nasal polyp tissues compared to normal inferior turbinate tissues. 5-Azacytidine significantly inhibited the expression of α-SMA and fibronectin mRNA in a dose-dependent manner. In addition, 5-azacytidine suppressed collagen production and the expression of MeCP2 in the same manner. The expression levels of a-SMA and collagen production were significantly blocked by MeCP2 silencing in TGF- β1-induced NPDFs. Conclusions Our data suggest that MeCP2 plays an essential role in TGF- β1-induced myofibroblast differentiation and ECM production in NPDFs.

Site-directed RNA repair of endogenous Mecp2 RNA in neurons

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

The L1 adhesion molecule normalizes neuritogenesis in Rett syndrome-derived neural precursor cells

Therapeutic intervention is an important need in ameliorating the severe consequences of Rett Syndrome (RTT), a neurological disorder caused by mutations in the X-linked gene methyl-CpG-binding protein-2 (MeCP2). Following previously observed morphological defects in induced pluripotent stem cell (iPSC)-derived neurons obtained from female RTT patients, we hypothesized that transfection with the L1 cell adhesion molecule (L1) could contribute to normalizing a pathological male cell system bearing a nonsense mutation of MeCP2. We found a decreased expression of L1 in RTT iPSCs-derived neural precursor cells (RTT NPCs) and decreased neuritogenesis. Expression of wild-type MeCP2 in RTTNPCs revealed a positive correlation between the levels of MeCP2 and L1, and normalization of cell survival. Expression of L1 in RTTNPCs enhanced neuritogenesis and soma size. Knock-down of MeCP2 in wild type NPCs reduced neuritogenesis. L1 expression is regulated by the MeCP2 promoter. These results suggest that a deficiency in L1 may partially account for RTT phenotypes.

DNA methylation mechanism of intracellular zinc deficiency-induced injury in primary hippocampal neurons in the rat brain

Objective: To explore Zn2+ deficiency-induced neuronal injury in relation to DNA methylation, providing valuable data and basic information for clarifying the mechanism of Zn2+ deficiency-induced neuronal injury.Methods: Cultured hippocampal neurons were exposed to the cell membrane-permeant Zn2+ chelator N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (2 μM), and to TPEN (2 μM) plus ZnSO4 (5 μM) for 24 hours. We analyzed intracellular Zn2+ levels, neuronal viability, and protein/mRNA levels for DNA (cytosine-5) methyltransferase 1 (DNMT1), DNA (cytosine-5-) methyltransferase 3 alpha (DNMT3a), methyl CpG binding protein 2 (MeCP2), Brain-derived neurotrophic factor (BDNF), and growth arrest and DNA-damage-inducible, beta (GADD45b) in the treated neurons.Results: We found that exposure of hippocampal neurons to TPEN (2 μM) for 24 hours significantly reduced intracellular Zn2+ concentration and neuronal viability. Furthermore, DNMT3a, DNMT1, BDNF, and GADD45b protein levels in TPEN-treated neurons were significantly downregulated, whereas MeCP2 levels were, as expected, upregulated. In addition, DNMT3a and DNMT1 mRNA levels in TPEN-treated neurons were downregulated, while MeCP2, GADD45b, and BDNF mRNA were largely upregulated. Addition of ZnSO4 (5 μM) almost completely reversed the TPEN-induced alterations.Conclusion: Our data suggest that free Zn2+ deficiency-induced hippocampal neuronal injury correlates with free Zn2+ deficiency-induced changes in methylation-related protein gene expression including DNMT3a/DNMT1/MeCP2 and GADD45b, as well as BDNF gene expression.

Expression of Phospho-MeCP2s in the Developing Rat Brain and Function of Postnatal MeCP2 in Cerebellar Neural Cell Development.

Abnormal expression and dysfunction of methyl-CpG binding protein 2 (MeCP2) cause Rett syndrome (RTT). The diverse phosphorylation modifications modulate MeCP2 function in neural cells. Using western blot and immunohistochemistry, we examined the expression patterns of MeCP2 and three phospho-MeCP2s (pMeCP2s) in the developing rat brain. The expression of MeCP2 and phospho-S80 (pS80) MeCP2 increased while pS421 MeCP2 and pS292 MeCP2 decreased with brain maturation. In contrast to the nuclear localization of MeCP2 and pS80 MeCP2, pS421 MeCP2 and pS292 MeCP2 were mainly expressed in the cytoplasmic compartment. Apart from their distribution in neurons, they were also detected at a low level in astrocytes. Postnatally-initiated MeCP2 deficiency affected cerebellar neural cell development, as determined by the abnormal expression of GFAP, DCX, Tuj1, MAP-2, and calbindin-D28k. Together, these results demonstrate that MeCP2 and diverse pMeCP2s have distinct features of spatio-temporal expression in the rat brain, and that the precise levels of MeCP2 in the postnatal period are vital to cerebellar neural cell development.