Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function

Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in?vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.     

MeCP2 mutation results in compartment-specific reductions in dendritic branching and spine density in layer 5 motor cortical neurons of YFP-H mice

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 Mecp2(tm1.1Jae) 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.     

X-chromosome inactivation patterns are unbalanced and affect the phenotypic outcome in a mouse model of rett syndrome

Rett syndrome (RTT), a neurodevelopmental disorder affecting mostly females, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Although the majority of girls with classic RTT have a random pattern of X-chromosome inactivation (XCI), nonbalanced patterns have been observed in patients carrying mutant MECP2 and, in some cases, account for variability of phenotypic manifestations. We have generated an RTT mouse model that recapitulates all major aspects of the human disease, but we found that females exhibit a high degree of phenotypic variability beyond what is observed in human patients with similar mutations. To evaluate whether XCI influences the phenotypic outcome of Mecp2 mutation in the mouse, we studied the pattern of XCI at the single-cell level in brains of heterozygous females. We found that XCI patterns were unbalanced, favoring expression of the wild-type allele, in most mutant females. It is notable that none of the animals had nonrandom XCI favoring the mutant allele. To explore why the XCI patterns favored expression of the wild-type allele, we studied primary neuronal cultures from Mecp2-mutant mice and found selective survival of neurons in which the wild-type X chromosome was active. Quantitative analysis indicated that fewer phenotypes are observed when a large percentage of neurons have the mutant X chromosome inactivated. The study of neuronal XCI patterns in a large number of female mice carrying a mutant Mecp2 allele highlights the importance of MeCP2 for neuronal viability. These findings also raise the possibility that there are human females who carry mutant MECP2 alleles but are not recognized because their phenotypes are subdued owing to favorable XCI patterns.     

Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析

MeCP2(Methyl CpG binding protein 2)基因突变可导致Rett综合征(Rett syndrome, RTT)。目前已报道的MeCP2敲除小鼠表型与RTT病人症状存在显著差异。为探索MeCP2在脑发育中的作用及其导致RTT的机制,本研究利用CRISPR/Cas9技术构建了MeCP2基因敲除大鼠模型。通过构建靶向敲除MeCP2基因的载体,体外将Cas9 mRNA和sgRNA显微注射到SD大鼠受精卵中,在MeCP2基因exon2中造成移码突变,从而获得MeCP2基因敲除大鼠。利用测序和Western blotting方法鉴定MeCP2敲除大鼠,并对其表型和行为学特征进行分析,发现MeCP2敲除大鼠体重降低,存在焦虑倾向和认知缺陷。本研究成功构建了MeCP2基因敲除大鼠模型,其表型类似人类RTT患者的症状,为后续MeCP2功能研究提供了更好的动物模型。     

Cell-autonomous alterations in dendritic arbor morphology and connectivity induced by overexpression of MeCP2 in Xenopus central neurons in vivo

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes.     

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.     

MeCP2 deficiency is associated with impaired microtubule stability

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Previous studies performed on Mecp2-deficient brain showed striking changes in neuronal maturation. We recently showed that MeCP2 deficiency affects microtubule (MT) dynamics in RTT astrocytes. Here, we analyze MT stability in primary fibroblast cultures from patients with RTT syndrome and identify a significant decrease in stability compared to controls. Furthermore, we found that MT stability was reduced both in cells expressing the mutant or the wild-type allele in RTT fibroblasts, suggesting that mutated cells could damage wild-type ones through a non-cell-autonomous pathway. These results suggest that MeCP2 has a stabilizing role on MT dynamics and that its deficiency could lead to impaired MT stability that may explain in part the dendritic abnormalities observed in RTT brains.     

Dynamic Changes in Histone H3 Lysine 9 Acetylation Localization Patterns During Neuronal Maturation Require MeCP2

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.      

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

A case report: bone marrow mesenchymal stem cells from a Rett syndrome patient are prone to senescence and show a lower degree of apoptosis

Rett syndrome (RTT) is one of the most common genetic diseases responsible for a progressive disabling neurodevelopmental disorder. Mutations in the MeCP2 gene were identified in the great majority of RTT patients. MeCP2 protein binds to methylated DNA and produces changes in chromatin structure. This is a key event in regulation of gene expression. It has been suggested that MeCP2 might be important for neuronal development. Moreover, the frequent occurrence of osteoporosis and scoliosis in RTT patients suggests impaired bone formation and/or remodeling. Mesenchymal stem cells (MSCs) can differentiate as mesodermal cells such as bone, cartilage cells, and adipocytes. MSCs have been shown to possess great somatic plasticity; in fact, they can differentiate as neurons and astrocytes. We studied RTT patients' MSCs because they are progenitors of osteocytes, and it has been suggested that RTT patients' osteogenesis could be impaired. Moreover, MSCs might represent a useful model for the study of neurogenesis. MSCs from RTT patient showed precocious signs of senescence in a comparison with the MSCs of healthy-patient control groups. This was in agreement with the reduced gene-expression in the control of stem cell self-renewal and upregulation of lineage specific genes, such as those involved in osteogenesis and neural development. Control groups enabled us to observe a lower degree of apoptosis in RTT patient cells. This means that aberrant stem/progenitor cells, instead of being eliminated, can survive and become senescent. Our research provides a new insight into RTT syndrome. Senescence phenomena could be involved in triggering RTT syndrome-associated diseases.