Advances in the pathogenesis of Rett syndrome using cell models

Rett syndrome(RTT)is a progressive neurodevelopmental disorder that occurs mainly in girls with a range of typical symptoms of autism spectrum disorders.MeCP2 protein loss-of-function in neural lineage cells is the main cause of RTT pathogenicity.As it is still hard to understand the mechanism of RTT on the basis of only clinical patients or animal models,cell models cultured in vitro play indispensable roles.Here we reviewed the research progress in the pathogenesis of RTT at the cellular level,summarized the preclinical-research-related applications,and prospected potential future development.

Functional Recovery with Electro-Acupuncture Stimulation in an Mecp2-Knockout Rat Model of Rett Syndrome

Rett syndrome is a progressive neurodevelopmental disorder that lacks effective treatments.Although deep-brain stimulation can alleviate some symptoms in Rett model mice,this interventional manipula-tion requires deliberate surgical operations.Here,we report that electro-acupuncture stimulation(EAS)can ameliorate symptoms of an Mecp2-knockout rat model of Rett syndrome from the remote acupoints Baihui(GV 20),Yongquan(KI 1),and Shenmen(HT 7).We find that EAS not only prolongs the survival time of Rett rats,but also improves their behavior ability,including locomotion,motor coordination,and social interaction.Neural activation was observed in the substantia nigra of the midbrain,corpus striatum,and cerebral cortex of wild-type and Rett model rats,as reflected by the increased expression of the c-Fos protein.Hence,EAS provides a potential promising therapeutic tool for treating neurodevel-opmental diseases.

The neural circuit basis of Rett syndrome

Rett syndrome is an Autism Spectrum Disorder caused by mutations in the gene encoding methyl-CpG binding protein （MeCP2）. Following a period of normal development, patients lose learned communication and motor skills, and develop a number of symptoms including motor disturbances, cognitive impairments and often seizures. In this review, we discuss the role of MeCP2 in regulating synaptic function and how synaptic dysfunctions lead to neuronal network impairments and alterations in sensory information processing. We propose that Rett syndrome is a disorder of neural circuits as a result of non-linear accumulated dysfunction of synapses at the level of individual cell populations across multiple neurotransmitter systems and brain regions.

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

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

Modeling Rett syndrome using TALEN-edited MECP2 mutant cynomolgus monkeys

Subject Code:H09With the support by the National Natural Science Foundation of China,a collaborative study by the research group led by Prof.Chen Yongchang(陈永昌)and Ji Weizhi from the Yunnan Key Laboratory of Primate Biomedicine Research&Institute of Primate Translational Medicine,Kunming University

Deep learning-based activity recognition and fine motor identification using 2D skeletons of cynomolgus monkeys

Video-based action recognition is becoming a vital tool in clinical research and neuroscientific study for disorder detection and prediction.However,action recognition currently used in non-human primate(NHP)research relies heavily on intense manual labor and lacks standardized assessment.In this work,we established two standard benchmark datasets of NHPs in the laboratory:Monkeyin Lab(Mi L),which includes 13 categories of actions and postures,and MiL2D,which includes sequences of two-dimensional(2D)skeleton features.Furthermore,based on recent methodological advances in deep learning and skeleton visualization,we introduced the Monkey Monitor Kit(Mon Kit)toolbox for automatic action recognition,posture estimation,and identification of fine motor activity in monkeys.Using the datasets and Mon Kit,we evaluated the daily behaviors of wild-type cynomolgus monkeys within their home cages and experimental environments and compared these observations with the behaviors exhibited by cynomolgus monkeys possessing mutations in the MECP2 gene as a disease model of Rett syndrome(RTT).Mon Kit was used to assess motor function,stereotyped behaviors,and depressive phenotypes,with the outcomes compared with human manual detection.Mon Kit established consistent criteria for identifying behavior in NHPs with high accuracy and efficiency,thus providing a novel and comprehensive tool for assessing phenotypic behavior in monkeys.

Dysregulated cortical synaptic plasticity under methyl-CpG binding protein 2 deficiency and its implication in motor impairments

Caused by the mutation of methyl-CpG binding protein 2(MeCP2),Rett syndrome leads to a battery of severe neural dysfunctions including the regression of motor coordination and motor learning.Current understanding has revealed the motor cortex as the critical region mediating voluntary movement.In this review article,we will summarize major findings from human patients and animal models regarding the cortical synaptic plasticity under the regulation of MeCP2.We will also discuss how mutation of MeCP2 leads to the disruption of cortical circuitry homeostasis to cause motor deficits.Lastly,potential values of physical exercise and neuromodulation approaches to recover neural plasticity and motor function will be evaluated.All of this evidence may help to accelerate timely diagnosis and effective interventions for Rett syndrome patients.

Structural investigation of Rett-inducing MeCP2 mutations

X-ray structure of methyl-CpG binding domain(MBD)of MeCP2,an intrinsically disordered protein(IDP)involved in Rett syndrome,offers a rational basis for defining the spatial distribution for most of the sites where mutations responsible of Rett syndrome,RTT,occur.We have ascribed pathogenicity for mutations of amino acids bearing positively charged side chains,all located at the protein-DNA interface,as positive charge removal cause reduction of the MeCP2-DNA adduct lifetime.Pathogenicity of the frequent proline replacements,outside the DNA contact moiety of MBD,can be attributed to the role of this amino acid for maintaining both unfolded states for unbound MeCP2 and,at the same time,to favor some higher conformational order for stabilizing structural determinants required by protein activity.These hypotheses can be extended to transcription repressor domain,TRD,the other MeCP2-DNA interaction site and,in general,to all the IDP that interact with nucleic acids.

Generation of a monkey with MECP2 mutations by TALEN-based gene targeting

Gene editing in model organisms has provided critical insights into brain development and diseases. Here, we report the generation of a cynomolgus monkey （Macaca fascicularis） carrying MECP2 mutations using transcription activator-like effector nucleases （TALENs）-mediated gene targeting. After injecting TALENs mRNA into monkey zygotes achieved by in vitro fertilization and embryo transplantation into surrogate monkeys, we obtained one male newborn monkey with an MECP2 deletion caused by frame- shifting mutation in various tissues. The monkey carrying the MECP2 mutation failed to survive after birth, due to either the toxicity of TALENs or the critical requirement of MECP2 for neural development. The level of MeCP2 protein was essentially depleted in the monkey＇s brain. This study demonstrates the feasibility of introducing genetic mutations in non-human primates by site-specific gene-editing methods.

MeCP2:multifaceted roles in gene regulation and neural development

Methyl-CpG-binding protein 2 （MeCP2） is a classic methylated-DNA-binding protein, dysfunctions of which lead to various neurodevelopmental disorders such as Rett syndrome and autism spectrum disorder. Initially recognized as a transcriptional repressor, MeCP2 has been studied extensively and its functions have been expanded dramatically in the past two decades. Recently, it was found to be involved in gene regulation at the post-transcriptional level. MeCP2 represses nuclear microRNA processing by interacting directly with the Drosha/DGCR8 complex. In addition to its multifaceted functions, MeCP2 is remarkably modulated by post- translational modifications such as phosphorylation, SUMOylation, and acetylation, providing more regulatory dimensions to its functions. The role of MeCP2 in the central nervous system has been studied extensively, from neurons to glia. Future investigations combining molecular, cellular, and physiological methods are necessary for defining the roles of MeCP2 in the brain and developing efficient treatments for MeCP2-related brain disorders.

Comprehensive Evaluation on Effect of IMPX977 on Expression of Methyl-CpG-binding Protein 2 in Rats

Objective To investigate the effect of IMPX977 on methyl-Cp G-binding protein 2（MeCP2） expression in rats. Methods Forty-eight SD rats were randomly divided into four groups： normal control group, olive oil（negative control, 5 mL/kg oil） group, and 10 mg/kg and 30 mg/kg IMPX977 administration groups. All rats were given corresponding dose of drugs each other day and administered orally for two weeks. Tissues including cortex and cerebellum were collected from rats to assay the expression of MeCP2 by quantitative RT-PCR and Western blotting. Results The IMPX977 supplement showed no significant effect on the body weight of rats. In normal rats, MeCP2 was highly expressed in cerebellum, cortex and hippocampus, and less expressed in heart, spleen and lung. In addition to male rats, compared with the control group, the expression of MeCP2 mRNA was significantly increased in cerebellum after 30 mg/kg IMPX977 treatment and contrarily, absolutely decreased in cortex of all treatment groups. Furthermore, in female rats MeCP2 mRNA was reduced in cortex of both olive oil and 30 mg/kg IMPX977 treatment groups compared with control group. Meanwhile, MeCP2 protein level was significantly elevated in cerebellum of treated male rats compared to the control group. In contrast to the control group, the expression of MeCP2 protein in both cerebellum and cortex of female rats in other three groups was increased. Conclusion IMPX977 treatment（10 mg/kg） may elevate the expression of MeCP2, which establishes experimental foundation for the further research on rat models of Rett syndrome.

Regulation and function of stimulus-induced phosphorylation of MeCP2

DNA methylation-dependent epigenetic regulation plays important roles in the development and function of the mammalian nervous system. MeCP2 is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. Mutations in the MECP2 gene cause Rett syndrome, a devastating neurological disease that shares many features with autism. One interesting aspect of MeCP2 function is that it can be phosphorylated in response to diverse stimuli. Insights into the regulation and function of MeCP2 phosphorylation will help improve our understanding of how MeCP2 integrates environmental stimuli in neuronal nuclei to generate adaptive responses and may eventually lead to treatments for patients.

Regulatory functions and pathological relevance of the MECP23′UTR in the central nervous system

Methyl-CpG-binding protein 2(MeCP2),encoded by the gene MECP2,is a transcriptional regulator and chromatinremodeling protein,which is ubiquitously expressed and plays an essential role in the development and maintenance of the central nervous system(CNS).Highly enriched in post-migratory neurons,MeCP2 is needed for neuronal maturation,including dendritic arborization and the development of synapses.Loss-of-function mutations in MECP2 cause Rett syndrome(RTT),a debilitating neurodevelopmental disorder characterized by a phase of normal development,followed by the progressive loss of milestones and cognitive disability.While a great deal has been discovered about the structure,function,and regulation of MeCP2 in the time since its discovery as the genetic cause of RTT,including its involvement in a number of RTT-related syndromes that have come to be known as MeCP2-spectrum disorders,much about this multifunctional protein remains enigmatic.One unequivocal fact that has become apparent is the importance of maintaining MeCP2 protein levels within a narrow range,the limits of which may depend upon the cell type and developmental time point.As such,MeCP2 is amenable to complex,multifactorial regulation.Here,we summarize the role of the MECP23'untranslated region(UTR)in the regulation of MeCP2 protein levels and how mutations in this region contribute to autism and other non-RTT neuropsychiatric disorders.