Age-dependent expression of MeCP2 in a heterozygous mosaic mouse model

Functional deficiency of the X-linked methyl-CPG binding protein 2 (MeCP2) leads to the neurodevelopmental disorder Rett syndrome (RTT). Due to random X-chromosome inactivation (XCI), most RTT patients are females who are heterozygous for the MECP2 mutation and therefore mosaic in MeCP2 deficiency. Some MECP2 heterozygote females are found to have unbalanced XCI, which may affect the severity of neurological symptoms seen in these patients; however, whether MeCP2 deficiency affects XCI in the postnatal and adult brain is unclear. Here we developed a novel MeCP2 mosaic mouse model in which the X chromosome containing the wild-type Mecp2 expresses a green fluorescent protein (GFP) transgene, while the X chromosome harboring the mutant Mecp2 does not. Due to random XCI, the neurons in the female MeCP2 mosaic mice express either wild-type MeCP2 (GFP+) or mutant MeCP2 (GFP-), and the two can be distinguished by GFP fluorescence. Using this mouse model, we evaluated XCI in female heterozygote mice from 3 to 9 months after birth. We found that MeCP2 deficiency does not affect XCI at 3 months of age, but does alter the proportion of wild-type MeCP2-expressing neurons at later ages, suggesting that MeCP2 impacts XCI patterns in an age-dependent manner. Given the important function of MeCP2 in neuronal development, our data could shed light on how MeCP2 deficiency affects postnatal brain functions and the dynamic changes in the neurological symptoms of RTT.     

Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder that affects girls due primarily to mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). The majority of RTT patients carry missense and nonsense mutations leading to a hypomorphic MECP2, while null mutations leading to the complete absence of a functional protein are rare. MECP2 is an X-linked gene subject to random X-chromosome inactivation resulting in mosaic expression of mutant MECP2. The lack of human brain tissue motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we isolated human induced pluripotent stem (hiPS) cells from RTT patient fibroblasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, we obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPS cell-derived neurons from the same RTT patient. Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons.     

Reduced proportion of Purkinje cells expressing paternally derived mutant Mecp2308 allele in female mouse cerebellum is not due to a skewed primary pattern of X-chromosome inactivation

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. The pattern of X-chromosome inactivation (XCI) is thought to play a role in phenotypic severity. In the present study, patterns of XCI were assessed by lacZ staining of embryos and adult brains of mice heterozygous for a X-linked Hmgcr-nls-lacZ transgene on a mutant mouse model of RTT. We found that there was no difference between the lacZ staining patterns in the brain of wild-type and heterozygous mutant embryos at embryonic day 9.5 (E9.5) suggesting that Mecp2 has no effect on the primary pattern of XCI. At 20 weeks of age, there was no significant difference between XCI patterns in the Purkinje cells in the cerebellum of heterozygous mutant and wild-type mice when the mutant allele was inherited from the mother. However, when the mutant allele was paternally inherited, a significant difference was detected. Thus, parental origin of the mutation may have a bearing on phenotype through XCI patterns. An estimation of the Purkinje cell precursor number based on XCI mosaicism revealed that, when the mutation was paternally inherited, the precursor number was less than that in the wild-type mice. Therefore, it is likely that the number of precursor cells allocated to the Purkinje cell lineage is affected by a paternally inherited mutation in Mecp2. We also observed that the pattern of XCI in cultured fibroblasts was significantly correlated with patterns in the Purkinje cells in mutant animals but not in wild-type mice.     

Novel de novo nonsense mutation of MECP2 in a patient with Rett syndrome

Because of the recent identification of several mutations of methyl-CpG-binding protein 2 (MECP2) in patients with Rett syndrome (RTT), a patient with suspected RTT from an autism clinic was screened for mutations. She was found to have a novel heterozygous nonsense mutation, 129C>T (Q19X), which leads to the most severely truncated MECP2 protein reported to date. Sequencing of parental DNA revealed the mutation was de novo. The patient was not affected with microcephaly or hyperventilation, but had other features of Rett syndrome including severe mental retardation and symptoms of autistic disorder. Moderately skewed X-chromosome inactivation (XCI) may have contributed to her relatively mild phenotype.     

Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome

Rett syndrome is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 gene (MECP2). Several neural systems are affected in Rett, resulting in an autonomic dysfunction, a movement disorder with characteristic loss of locomotor abilities and profound cognitive impairments. A deregulation of monoamines has been detected in the brain and cerebrospinal fluid of both Rett patients and a Rett syndrome murine model, the Mecp2 knock-out mouse. Our goal was to characterize the onset and progression of motor dysfunction in Mecp2^tm1.1Bird knock-out mice and the possible neurochemical alterations in different brain regions potentially playing a role in Rett-like pathophysiology, at two different time-points, at weaning (3 weeks old) and in young adults when overt symptoms are observed (8 weeks old). Our results revealed significant age- and region-dependent impairments in these modulatory neurotransmitter systems that correspond well with the motor phenotype observed in these mice. At 3 weeks of age, male Mecp2 knock-out mice exhibited ataxia and delayed motor initiation. At this stage, noradrenergic and serotonergic transmission was mainly altered in the prefrontal and motor cortices, whereas during disease progression the neurochemical changes were also observed in hippocampus and cerebellum. Our data suggest that the deregulation of norepinephrine and serotonin systems in brain regions that participate in motor control are involved in the pathophysiology of Rett syndrome motor phenotypes. Moreover, we highlight the contribution of cortical regions along with the brainstem to be in the origin of the pathology and the role of hippocampus and cerebellum in the progression of the disease rather than in its establishment.     

Analysis of neuronal subpopulations in mice over-expressing suppressor of cytokine signaling-2

Developing an understanding of factors that regulate development of the nervous system is important if we hope to be able to repair the nervous system after injury or disease. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of cytokine signaling that blocks the inhibitory effects of growth hormone on neuronal differentiation and promotes neurogenesis. Here we examine the effect of SOCS2 over-expression on brain development by assessing density and soma size of different neuronal populations in the somatosensory cortex and striatum of SOCS2 transgenic mice compared with wildtype C57BL/6 mice. There were no significant differences in brain weight, cortical thickness or striatal area between mice of either genotype. Analysis of NeuN positive neuronal cell density showed a modest but significant 9% increase across layers 2-6 of SOCS2 transgenic cortex, while cortical interneuron subpopulations were variably affected. In the cortex, parvalbumin and somatostatin expressing neuron densities were unaffected, while calretinin and calbindin positive neuronal densities increased by 48% and 45% respectively. There was no apparent difference in glial fibrillary acidic protein positive astrocyte numbers in layers 1 or 6b of cortex. Furthermore, soma sizes of calretinin and calbindin positive cortical neurons were significantly smaller than wildtype, although there was no difference in size of Cresyl Violet-stained layer 5 projection neurons nor of parvalbumin or somatostatin positive cortical neurons. Additionally, synaptic density and dendritic branching were found to be increased in SOCS2 transgenic cortex. These effects on calretinin and calbindin positive cortical neurons and cortical neuronal circuitry were not observed in the striatum of SOCS2-Tg brains. However, striatal cholinergic interneurons were significantly smaller in SOCS2-Tg brains. At embryonic day 14.5, proliferation and apoptosis in the developing telencephalon were similar in each genotype. Therefore, over-expression of SOCS2 variably affects different cortical regions and neuronal populations, with the predominant effect appearing to be on interneurons and neuronal connectivity in the cortex.     

Rett综合征和DNA甲基化

DNA甲基化是一种在生物界普遍存在的生命现象,在染色体失活和基因印记以及基因沉默方面起着重要的作用。甲基化CpG结合蛋白2(methyl-CpG-binding protein2,MeCP2)作为一种转录抑制因子能够与甲基化的DNA结合,在基因的转录过程中发挥主要作用,其编码基因MECP2是神经发育性遗传病-Rett综合征的主要致病基因,现主要介绍DNA甲基化和Rett综合征之间的关系。     

Germline mosaicism for a MECP2 mutation in a man with two Rett daughters

Rett syndrome is a severe neurodevelopmental disorder that is caused by mutations in the X-linked gene, methyl-CpG binding protein 2 (MECP2). The majority of cases are sporadic, but rarely germline mosaicism can lead to familial cases. Here, we report the first case where germline mosaicism for a MECP2 mutation has been shown in a man. He has two affected daughters who are half sisters, and both have the c.808delC mutation. We show that this mutation is present at a low level in DNA extracted from the patient's semen. This case has implications for genetic counseling, and pre-natal testing should be offered for the partners of men who have a daughter with Rett syndrome.     

Remodelling of the respiratory network in a mouse model of Rett syndrome depends on brain-derived neurotrophic factor regulated slow calcium buffering

Rett syndrome caused by MeCP2 mutations is a devastating neurodevelopmental disorder accompanied by severe breathing irregularities. Using transduction of organotypic slices from model MeCP2–/y mice with neuron-specific calcium sensor protein D3cpv, we examined the slow calcium buffering in neurons in pre-Bötzinger complex (preBötC), a component of the complex respiratory network. Examination of wild-type (WT) and MeCP2 null mice showed clear differences in the spatial organisations of neurons in preBötC and also in the disturbances in calcium homeostasis in mutant mice during early postnatal development. Deregulated calcium buffering in MeCP2–/y neurons was indicated by increased amplitude and kinetics of depolarisation-induced calcium transients. Both effects were related to an insufficient calcium uptake into the endoplasmic reticulum that was restored after pretreatment with brain-derived neurotrophic factor (BNDF). Conversely, the inhibition of BDNF signalling in WT neurons produced disturbances similar to those observed in MeCP2–/y mice. Brief hypoxia and calcium release from internal stores induced global calcium increases, after which the processes of many MeCP2–/y neurons were retracted, an effect that was also corrected by pretreatment with BDNF. The data obtained point to a tight connection between calcium homeostasis and long-term changes in neuronal connectivity. We therefore propose that calcium-dependent retraction of neurites in preBötC neurons can cause remodelling of the neuronal network during development and set up the conditions for appearance of breathing irregularities in Rett model mice.     

A segment of the Mecp2 promoter is sufficient to drive expression in neurons

Rett syndrome (RTT) is caused by mutations in the gene encoding methyl CpG-binding protein 2 (MeCP2). Although MeCP2 shows widespread expression in both neuronal and non-neuronal tissues, the symptoms of RTT are largely neurological. Herein, we have identified the regulatory region of the mouse Mecp2 gene that is sufficient for its restricted expression in neurons. A segment of the Mecp2 gene (-677/+56) exhibited strong promoter activity in neuronal cell lines and cortical neurons, but was inactive in non-neuronal cells and glia. The region necessary for neuronal-specific promoter activity was located within a 19 bp region (-63/-45). Several nuclear factors were found to bind to this region and some of these factors were enriched in nuclear extracts prepared from the brain. To examine the activity of the Mecp2 promoter in vivo, we generated transgenic mice expressing the LacZ reporter driven by the -677/+56 region of the Mecp2 gene. The transgene was expressed in the mesencephalon as early as embryonic day 10 and in the hindbrain and spinal cord by E12. Interestingly, a marked induction of transgene expression was observed postnatally throughout the brain, similar to that of endogenous MeCP2. However, expression of the transgene was absent in non-neuronal tissues that are known to express Mecp2. Taken together, these data indicate that the -677/+56 region of the Mecp2 promoter partially recapitulates the native expression pattern of the Mecp2 gene, which possesses restricted expression in neurons of the central nervous system.