Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities.

Rett syndrome (RTT) is a neurodevelopmental disorder with X-linked dominant inheritance caused mainly by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. The effects of various Mecp2 mutations have been extensively assessed in mouse models, but none adequately mimic the symptoms and pathological changes of RTT. In this study, we assessed the effects of Mecp2 gene deletion on female rats (Mecp2+/-) and found severe impairments in social behavior [at 8 weeks (w), 12 w, and 23 w of age], motor function [at 16 w and 26 w], and spatial cognition [at 29 w] as well as lower plasma insulin-like growth factor (but not brain-derived neurotrophic factor) and markedly reduced acetylcholine (30%-50%) in multiple brain regions compared to female Mecp2+/+ rats [at 29 w]. Alternatively, changes in brain monoamine levels were relatively small, in contrast to reports on mouse Mecp2 mutants. Female Mecp2-deficient rats express phenotypes resembling RTT and so may provide a robust model for future research on RTT pathobiology and treatment.

Association of CaMK2A and MeCP2 signaling pathways with cognitive ability in adolescents.

The glutamatergic signaling pathway is involved in molecular learning and human cognitive ability. Specific single variants (SNVs, formerly single-nucleotide polymorphisms) in the genes encoding N-methyl-D-aspartate receptor subunits have been associated with neuropsychiatric disorders by altering glutamate transmission. However, these variants associated with cognition and mental activity have rarely been explored in healthy adolescents. In this study, we screened for SNVs in the glutamatergic signaling pathway to identify genetic variants associated with cognitive ability. We found that SNVs in the subunits of ionotropic glutamate receptors, including GRIA1, GRIN1, GRIN2B, GRIN2C, GRIN3A, GRIN3B, and calcium/calmodulin-dependent protein kinase IIα (CaMK2A) are associated with cognitive function. Plasma CaMK2A level was correlated positively with the cognitive ability of Taiwanese senior high school students. We demonstrated that elevating CaMK2A increased its autophosphorylation at T286 and increased the expression of its downstream targets, including GluA1 and phosphor- GluA1 in vivo. Additionally, methyl-CpG binding protein 2 (MeCP2), a downstream target of CaMK2A, was found to activate the expression of CaMK2A, suggesting that MeCP2 and CaMK2A can form a positive feedback loop. In summary, two members of the glutamatergic signaling pathway, CaMK2A and MeCP2, are implicated in the cognitive ability of adolescents; thus, altering the expression of CaMK2A may affect cognitive ability in youth.

Collapse of complexity of brain and body activity due to excessive inhibition and MeCP2 disruption.

Complex body movements require complex dynamics and coordination among neurons in motor cortex. Conversely, a long-standing theoretical notion supposes that if many neurons in motor cortex become excessively synchronized, they may lack the necessary complexity for healthy motor coding. However, direct experimental support for this idea is rare and underlying mechanisms are unclear. Here we recorded three-dimensional body movements and spiking activity of many single neurons in motor cortex of rats with enhanced synaptic inhibition and a transgenic rat model of Rett syndrome (RTT). For both cases, we found a collapse of complexity in the motor system. Reduced complexity was apparent in lower-dimensional, stereotyped brain-body interactions, neural synchrony, and simpler behavior. Our results demonstrate how imbalanced inhibition can cause excessive synchrony among movement-related neurons and, consequently, a stereotyped motor code. Excessive inhibition and synchrony may underlie abnormal motor function in RTT.

Perineuronal net degradation rescues CA2 plasticity in a mouse model of Rett syndrome.

Perineuronal nets (PNNs), a specialized form of extracellular matrix, are abnormal in the brains of people with Rett syndrome (RTT). We previously reported that PNNs function to restrict synaptic plasticity in hippocampal area CA2, which is unusually resistant to long-term potentiation (LTP) and has been linked to social learning in mice. Here we report that PNNs appear elevated in area CA2 of the hippocampus of an individual with RTT and that PNNs develop precociously and remain elevated in area CA2 of a mouse model of RTT (Mecp2-null). Further, we provide evidence that LTP could be induced at CA2 synapses prior to PNN maturation (postnatal day 8-11) in wild-type mice and that this window of plasticity was prematurely restricted at CA2 synapses in Mecp2-null mice. Degrading PNNs in Mecp2-null hippocampus was sufficient to rescue the premature disruption of CA2 plasticity. We identified several molecular targets that were altered in the developing Mecp2-null hippocampus that may explain aberrant PNNs and CA2 plasticity, and we discovered that CA2 PNNs are negatively regulated by neuronal activity. Collectively, our findings demonstrate that CA2 PNN development is regulated by Mecp2 and identify a window of hippocampal plasticity that is disrupted in a mouse model of RTT.

Reviewing Evidence for the Relationship of EEG Abnormalities and RTT Phenotype Paralleled by Insights from Animal Studies.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that is usually caused by mutations of the MECP2 gene. Patients with RTT suffer from severe deficits in motor, perceptual and cognitive domains. Electroencephalogram (EEG) has provided useful information to clinicians and scientists, from the very first descriptions of RTT, and yet no reliable neurophysiological biomarkers related to the pathophysiology of the disorder or symptom severity have been identified to date. To identify consistently observed and potentially informative EEG characteristics of RTT pathophysiology, and ascertain areas most worthy of further systematic investigation, here we review the literature for EEG abnormalities reported in patients with RTT and in its disease models. While pointing to some promising potential EEG biomarkers of RTT, our review identify areas of need to realize the potential of EEG including (1) quantitative investigation of promising clinical-EEG observations in RTT, e.g., shift of mu rhythm frequency and EEG during sleep; (2) closer alignment of approaches between patients with RTT and its animal models to strengthen the translational significance of the work (e.g., EEG measurements and behavioral states); (3) establishment of large-scale consortium research, to provide adequate Ns to investigate age and genotype effects.

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.

Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons.

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.

MeCP2 mediates transgenerational transmission of chronic pain.

Pain symptoms can be transmitted across generations, but the mechanisms underlying these outcomes remain poorly understood. Here, we identified an essential role for primary somatosensory cortical (S1) glutamate neuronal DNA methyl-CpG binding protein 2 (MeCP2) in the transgenerational transmission of pain. In a female mouse chronic pain model, the offspring displayed significant pain sensitization. In these mice, MeCP2 expression was increased in S1 glutamate (GluS1) neurons, correlating with increased neuronal activity. Downregulation of GluS1 neuronal MeCP2 in maternal mice with pain abolished offspring pain sensitization, whereas overexpression of MeCP2 in naïve maternal mice induced pain sensitization in offspring. Notably, single-cell sequencing and chromatin immunoprecipitation analysis showed that the expression of a wide range of genes was changed in offspring and maternal GluS1 neurons, some of which were regulated by MeCP2. These results collectively demonstrate the putative importance of MeCP2 as a key regulator in pain transgenerational transmission through actions on GluS1 neuronal maladaptation.

Rett syndrome-causing mutations compromise MeCP2-mediated liquid-liquid phase separation of chromatin.

Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2. Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.

Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2.

The neurodevelopmental disorder Rett syndrome is caused by mutations in the gene Mecp2 Misexpression of the protein MECP2 is thought to contribute to neuropathology by causing dysregulation of plasticity. Female heterozygous Mecp2 mutants (Mecp2het ) failed to acquire a learned maternal retrieval behavior when exposed to pups, an effect linked to disruption of parvalbumin-expressing inhibitory interneurons (PV) in the auditory cortex. Nevertheless, how dysregulated PV networks affect the neural activity dynamics that underlie auditory cortical plasticity during early maternal experience is unknown. Here we show that maternal experience in WT adult female mice (WT) triggers suppression of PV auditory responses. We also observe concomitant disinhibition of auditory responses in deep-layer pyramidal neurons that is selective for behaviorally relevant pup vocalizations. These neurons further exhibit sharpened tuning for pup vocalizations following maternal experience. All of these neuronal changes are abolished in Mecp2het , suggesting that they are an essential component of maternal learning. This is further supported by our finding that genetic manipulation of GABAergic networks that restores accurate retrieval behavior in Mecp2het also restores maternal experience-dependent plasticity of PV. Our data are consistent with a growing body of evidence that cortical networks are particularly vulnerable to mutations of Mecp2 in PV neurons. Moreover, our work links, for the first time, impaired in vivo cortical plasticity in awake Mecp2 mutant animals to a natural, ethologically relevant behavior.SIGNIFICANCE STATEMENT Rett syndrome is a genetic disorder that includes language communication problems. Nearly all Rett syndrome is caused by mutations in the gene that produces the protein MECP2, which is important for changes in brain connectivity believed to underlie learning. We previously showed that female Mecp2 mutants fail to learn a simple maternal care behavior performed in response to their pups' distress cries. This impairment appeared to critically involve inhibitory neurons in the auditory cortex called parvalbumin neurons. Here we record from these neurons before and after maternal experience, and we show that they adapt their response to pup calls during maternal learning in nonmutants, but not in mutants. This adaptation is partially restored by a manipulation that improves learning.

Brain protein changes in Mecp2 mouse mutant models: Effects on disease progression of Mecp2 brain specific gene reactivation.

Rett syndrome (RTT) is a leading cause of severe intellectual disability in females, caused by de novo loss-of function mutations in the X-linked methyl-CpG binding protein 2 (MECP2). To better investigate RTT disease progression/pathogenesis animal models of Mecp2 deficiency have been developed. Here, Mecp2 mouse models are employed to investigate the role of protein patterns in RTT. A proteome analysis was carried out in brain tissue from i) Mecp2 deficient mice at the pre-symptomatic and symptomatic stages and, ii) mice in which the disease phenotype was reversed by Mecp2 reactivation. Several proteins were shown to be differentially expressed in the pre-symptomatic (n = 18) and symptomatic (n = 20) mice. Mecp2 brain reactivated mice showed wild-type comparable levels of expression for twelve proteins, mainly related to proteostasis (n = 4) and energy metabolic pathways (n = 4). The remaining ones were found to be involved in redox homeostasis (n = 2), nitric oxide regulation (n = 1), neurodevelopment (n = 1). Ten out of twelve proteins were newly linked to Mecp2 deficiency. Our study sheds light on the relevance of the protein-regulation of main physiological process in the complex mechanisms leading from Mecp2 mutation to the RTT clinical phenotype. SIGNIFICANCE: We performed a proteomic study of a Mecp2stop/y mouse model for Rett syndrome (RTT) at the pre-symptomatic and symptomatic Mecp2 deficient mice stage and for the brain specific reactivated Mecp2 model. Our results reveal major protein expression changes pointing out to defects in proteostasis or energy metabolic pathways other than, to a lesser extent, in redox homeostasis, nitric oxide regulation or neurodevelopment. The Mecp2 mouse rescued model provides the possibility to select target proteins more susceptible to the Mecp2 gene mutation, potential and promising therapeutical targets.

Accelerated Hyper-Maturation of Parvalbumin Circuits in the Absence of MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) mutations are the primary cause of Rett syndrome, a severe neurodevelopmental disorder. Cortical parvalbumin GABAergic interneurons (PV) make exuberant somatic connections onto pyramidal cells in the visual cortex of Mecp2-deficient mice, which contributes to silencing neuronal cortical circuits. This phenotype can be rescued independently of Mecp2 by environmental, pharmacological, and genetic manipulation. It remains unknown how Mecp2 mutation can result in abnormal inhibitory circuit refinement. In the present manuscript, we examined the development of GABAergic circuits in the primary visual cortex of Mecp2-deficient mice. We identified that PV circuits were the only GABAergic interneurons to be upregulated, while other interneurons were downregulated. Acceleration of PV cell maturation was accompanied by increased PV cells engulfment by perineuronal nets (PNNs) and by an increase of PV cellular and PNN structural complexity. Interestingly, selective deletion of Mecp2 from PV cells was sufficient to drive increased structure complexity of PNN. Moreover, the accelerated PV and PNN maturation was recapitulated in organotypic cultures. Our results identify a specific timeline of disruption of GABAergic circuits in the absence of Mecp2, indicating a possible cell-autonomous role of MeCP2 in the formation of PV cellular arbors and PNN structures in the visual cortex.

MeCP2 triggers diabetic cardiomyopathy and cardiac fibroblast proliferation by inhibiting RASSF1A.

Diabetes causes cardiomyopathy and increases the risk of heart failure independent of hypertension and cardiac fibrosis disease. However, the molecular mechanism of cardiomyopathy caused by diabetic (DCM) is currently unknown. Here we explore the role of the Methyl CpG binding protein 2 (MeCP2) in DCM patients and a type 1 DM (T1DM) rat model. In this study, we employed streptozotocin (STZ)-induced rats DCM and DCM patient and found that MeCP2 triggers cardiac fibroblast proliferation in DCM by inhibiting of RASSF1A expression. Moreover, the in vitro study demonstrated that high glucose inhibited RASSF1A expression, accompanied by the increases of MeCP2 expression and DNA hypermethylation in RASSF1A promoter region. MeCP2 inhibition or knockdown reversed the decrease of RASSF1A transcription induced by high glucose in cardiac fibroblasts. MeCP2 triggers cardiac fibroblasts proliferation through the activation of RASSF1A/ERK1/2 signaling pathways. Our results demonstrated that MeCP2 plays a key role in RASSF1A mediated ERK1/2 activation in DCM. Taken together, these indicate that MeCP2 acts as a key regulator of DCM and cardiac fibroblasts proliferation.

The role of MeCP2 in learning and memory.

Gene transcription is a crucial step in the sequence of molecular, synaptic, cellular, and systems mechanisms underlying learning and memory. Here, we review the experimental evidence demonstrating that alterations in the levels and functionality of the methylated DNA-binding transcriptional regulator MeCP2 are implicated in the learning and memory deficits present in mouse models of Rett syndrome and MECP2 duplication syndrome. The significant impact that MeCP2 has on gene transcription through a variety of mechanisms, combined with well-defined models of learning and memory, make MeCP2 an excellent candidate to exemplify the role of gene transcription in learning and memory. Together, these studies have strengthened the concept that precise control of activity-dependent gene transcription is a fundamental mechanism that ensures long-term adaptive behaviors necessary for the survival of individuals interacting with their congeners in an ever-changing environment.

Rett Syndrome in Males: The Different Clinical Course in Two Brothers with the Same Microduplication MECP2 Xq28.

Rett syndrome (RTT) is a neurodevelopmental disorder with a genetic basis that is associated with the mutation of the X-linked methyl-CpG binding protein 2 (MECP2) gene in approximately 90% of patients. RTT is characterized by a brief period of normal development followed by loss of acquired skills and evolution towards impairment of brain and motor functions and multi-organ dysfunction. Originally, RTT was considered lethal in males as it has an X-linked dominant inheritance. However, although this syndrome has a higher incidence in females, rare cases are also documented in males. Here, we describe the case of an 11-year-old male patient with a microduplication MECP2 Xq28. Our patient is currently living, while his older brother with the same mutation died at the age of 9 years. We showed that the role of MECP2 as an epigenetic modulator and the X-chromosome inactivation pattern can explain the lethal clinical form of the older brother with the same microduplication MECP2 Xq28 presented by our patient who is still alive. Given the limited case history of RTT in males, further studies are needed to better characterize this syndrome in males and consequently improve the currently available therapeutic strategies.

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth.

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2-/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system.

The protein doublecortin is mainly expressed in migrating neuroblasts and immature neurons. The X-linked gene MECP2, associated to several neurodevelopmental disorders such as Rett syndrome, encodes the protein methyl-CpG-binding protein 2 (MeCP2), a regulatory protein that has been implicated in neuronal maturation and refinement of olfactory circuits. Here, we explored doublecortin immunoreactivity in the brain of young adult female Mecp2-heterozygous and male Mecp2-null mice and their wild-type littermates. The distribution of doublecortin-immunoreactive somata in neurogenic brain regions was consistent with previous reports in rodents, and no qualitative differences were found between genotypes or sexes. Quantitatively, we found a significant increase in doublecortin cell density in the piriform cortex of Mecp2-null males as compared to WT littermates. A similar increase was seen in a newly identified population of doublecortin cells in the olfactory tubercle. In these olfactory structures, however, the percentage of doublecortin immature neurons that also expressed NeuN was not different between genotypes. By contrast, we found no significant differences between genotypes in doublecortin immunoreactivity in the olfactory bulbs. Nonetheless, in the periglomerular layer of Mecp2-null males, we observed a specific decrease of immature neurons co-expressing doublecortin and NeuN. Overall, no differences were evident between Mecp2-heterozygous and WT females. In addition, no differences could be detected between genotypes in the density of doublecortin-immunoreactive cells in the hippocampus or striatum of either males or females. Our results suggest that MeCP2 is involved in neuronal maturation in a region-dependent manner.

The array of clinical phenotypes of males with mutations in Methyl-CpG binding protein 2.

Mutations in the X-linked gene MECP2 are associated with a severe neurodevelopmental disorder, Rett syndrome (RTT), primarily in girls. It had been suspected that mutations in Methyl-CpG-binding protein 2 (MECP2) led to embryonic lethality in males, however such males have been reported. To enhance understanding of the phenotypic spectrum present in these individuals, we identified 30 males with MECP2 mutations in the RTT Natural History Study databases. A wide phenotypic spectrum was observed, ranging from severe neonatal encephalopathy to cognitive impairment. Two males with a somatic mutation in MECP2 had classic RTT. Of the remaining 28 subjects, 16 had RTT-causing MECP2 mutations, 9 with mutations that are not seen in females with RTT but are likely pathogenic, and 3 with uncertain variants. Two subjects with RTT-causing mutations were previously diagnosed as having atypical RTT; however, careful review of the clinical history determined that an additional 12/28 subjects met criteria for atypical RTT, but with more severe clinical presentation and course, and less distinctive RTT features, than females with RTT, leading to the designation of a new diagnostic entity, male RTT encephalopathy. Increased awareness of the clinical spectrum and widespread comprehensive genomic testing in boys with neurodevelopmental problems will lead to improved identification.

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.

Regulation of neural differentiation, synaptic scaling and animal behavior by MeCP2 phophorylation.

Highly expressed in the mammalian brain and widely distributed across the genome, MeCP2 is a key player in recognizing modified DNA and interpreting the epigenetic information encoded in different DNA methylation/hydroxymethylation patterns. Alterations in sequence or copy number of the X-linked human MECP2 gene cause either Rett syndrome (RTT) or MECP2 duplication syndrome. Alterations in MECP2 levels have also been identified in patients with autism. To fully understand the significant role of MECP2 in regulating the development and function of the nervous system, it is important to study all aspects of MeCP2 function. Stimulus-induced MeCP2 phosphorylation has been shown to influence the proliferation and differentiation of neural progenitor cells, synaptic scaling, excitatory synaptogenesis, and animal behavior. However, all of the previous functional evidence is from studying phospho-dead mutations. In addition, the relationship between phosphorylation events at multiple sites on the MeCP2 protein is not well understood. Here, we report the generation of a phospho-mimic knockin Mecp2 mouse line. At the synaptic and behavioral levels, the phospho-mimic Mecp2 mice show phenotypes opposite to those observed in phospho-dead mutation at the same phosphorylation site. Moreover, we report opposite phenotypes between phospho-mutants of two sites on the MeCP2 protein. Our new data further confirm the functional significance of specific MeCP2 phosphorylation event and support the opposing regulatory role between different MeCP2 phosphorylation events.