Tian-Lin Cheng

Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as compared to wild-type monkeys of similar age. Together, these results indicate the feasibility and reliability of using genetically engineered non-human primates to study brain disorders.

Conditional deletion of Mecp2 in parvalbumin-expressing GABAergic cells results in the absence of critical period plasticity

Mutations in the X-linked gene encoding the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2) impair postnatal development of the brain. Here we use neuronal-type specific gene deletion in mice to show that conditional Mecp2 deletion in GABAergic parvalbumin-expressing (PV) cells (PV-Mecp2(-/y)) does not cause most Rett-syndrome-like behaviours, but completely abolishes experience-dependent critical period plasticity of primary visual cortex (V1) that develops normal visual functions. However, selective loss of Mecp2 in GABAergic somatostatin-expressing cells or glutamatergic pyramidal cells does not affect the critical period plasticity. MeCP2-deficient PV cells exhibit high intrinsic excitability, selectively reduced efficacy of recurrent excitatory synapses in V1 layer 4 circuits, and decreased evoked visual responses in vivo. Enhancing cortical gamma-aminobutyric acid (GABA) inhibition with diazepam infusion can restore critical period plasticity in both young and adult PV-Mecp2(-/y) mice. Thus, MeCP2 expression in inhibitory PV cells during the critical period is essential for local circuit functions underlying experience-dependent cortical plasticity.

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 posttranslational 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.

Role of the PTEN signaling pathway in autism spectrum disorder.

Autism is an etiologically heterogeneous group of neurodevelopmental disorders, diagnosed mostly by the clinical behavioral phenotypes. The concept that the tumor-related gene PTEN plays a critical role in autism spectrum disorder has emerged over the last decade. In this review, we focus on the essential role of the PTEN signaling pathway in neuronal differentiation and the formation of neural circuitry, as well as genetic mouse models with Pten manipulations. Particularly, accumulated data suggest that the effect of PTEN on neural stem-cell development contributes significantly to the pathophysiology of autism spectrum disorders.

Reciprocal regulation of autism-related genes MeCP2 and PTEN via microRNAs

MeCP2 encodes a methyl-CpG-binding protein that plays a critical role in repressing gene expression, mutations of which lead to Rett syndrome and autism. PTEN is a critical tumor suppressor gene that is frequently mutated in human cancers and autism spectrum disorders. Various studies have shown that both MeCP2 and PTEN proteins play important roles in brain development. Here we find that MeCP2 and PTEN reciprocally regulate expression of each other via microRNAs. Knockdown of MeCP2 leads to upregulation of microRNA-137, which in turn represses expression of PTEN, thus PTEN would be down-regulated when MeCP2 is knockdown. Furthermore, we find that deletion of PTEN leads to phosphorylation of Serine 133 of CREB, then increases the expression of microRNA-132. miR-132 inhibits the expression of MeCP2 by targeting on the 3′UTR of MeCP2 mRNA. Our work shows that two critical disorders-related gene MeCP2 and PTEN reciprocally regulate expression of each other by distinct mechanisms, suggesting that rare mutations in various disorders may lead to dysregulation of other critical genes and yield unexpected consequences.

A case report of Chinese brothers with inherited MECP2-containing duplication: autism and intellectual disability, but not seizures or respiratory infections

BackgroundAutistic spectrum disorders (ASDs) are a family of neurodevelopmental disorders with strong genetic components. Recent studies have shown that copy number variations in dosage sensitive genes can contribute significantly to these disorders. One such gene is the transcription factor MECP2, whose loss of function in females results in Rett syndrome, while its duplication in males results in developmental delay and autism.Case presentationHere, we identified a Chinese family with two brothers both inheriting a 2.2u2009Mb MECP2-containing duplication (151,369,305 – 153,589,577) from their mother. In addition, both brothers also had a 213.7u2009kb duplication on Chromosome 2, inherited from their father. The older brother also carried a 48.4u2009kb duplication on Chromosome 2 inherited from the mother, and a 8.2u2009kb deletion at 11q13.5 inherited from the father. Based on the published literature, MECP2 is the most autism-associated gene among the identified CNVs. Consistently, the boys displayed clinical features in common with other patients carrying MECP2 duplications, including intellectual disability, autism, lack of speech, slight hypotonia and unsteadiness of movement. They also had slight dysmorphic features including a depressed nose bridge, large ears and midface hypoplasia. Interestingly, they did not exhibit other clinical features commonly observed in American-European patients with MECP2 duplication, including recurrent respiratory infections and epilepsy.ConclusionsTo our knowledge, this is the first identification and characterization of Chinese Han patients with MECP2-containing duplications. Further cases are required to determine if the above described clinical differences are due to individual variations or related to the genetic background of the patients.

Regulation of mRNA splicing by MeCP2 via epigenetic modifications in the brain

Mutations of X-linked gene Methyl CpG binding protein 2 (MECP2) are the major causes of Rett syndrome (RTT), a severe neurodevelopmental disorder. Duplications of MECP2-containing genomic segments lead to severe autistic symptoms in human. MECP2-coding protein methyl-CpG-binding protein 2 (MeCP2) is involved in transcription regulation, microRNA processing and mRNA splicing. However, molecular mechanisms underlying the involvement of MeCP2 in mRNA splicing in neurons remain largely elusive. In this work we found that the majority of MeCP2-associated proteins are involved in mRNA splicing using mass spectrometry analysis with multiple samples from Mecp2-null rat brain, mouse primary neuron and human cell lines. We further showed that Mecp2 knockdown in cultured cortical neurons led to widespread alternations of mRNA alternative splicing. Analysis of ChIP-seq datasets indicated that MeCP2-regulated exons display specific epigenetic signatures, with DNA modification 5-hydroxymethylcytosine (5hmC) and histone modification H3K4me3 are enriched in down-regulated exons, while the H3K36me3 signature is enriched in exons up-regulated in Mecp2-knockdown neurons comparing to un-affected neurons. Functional analysis reveals that genes containing MeCP2-regulated exons are mainly involved in synaptic functions and mRNA splicing. These results suggested that MeCP2 regulated mRNA splicing through interacting with 5hmC and epigenetic changes in histone markers, and provide functional insights of MeCP2-mediated mRNA splicing in the nervous system.

The protein phosphatase activity of PTEN is essential for regulating neural stem cell differentiation

BackgroundThe tumor suppressor gene Phosphatase and tensin homolog (PTEN) is highly expressed in neural progenitor cells (NPCs) and plays an important role in development of the central nervous system. As a dual-specificity phosphatase, the loss of PTEN phosphatase activity has been linked to various diseases.ResultsHere we report that the protein phosphatase activity of Pten is critical for regulating differentiation of neural progenitor cells. First we found that deletion of Pten promotes neuronal differentiation. To determine whether the protein or lipid phosphatase activity is required for regulating neuronal differentiation, we generated phosphatase domain-specific Pten mutations. Interestingly, only expression of protein phosphatase-deficient mutant Y138L could mimic the effect of knocking down Pten, suggesting the protein phosphatase of Pten is critical for regulating NPC differentiation. Importantly, we showed that the wild-type and lipid phosphatase mutant (G129E) forms of Pten are able to rescue neuronal differentiation in Pten knockout NPCs, but mutants containing protein phosphatase mutant cannot. We further found that Pten-dependent dephosphorylation of CREB is critical for neuronal differentiation.ConclusionOur data indicate that the protein phosphatase activity of PTEN is critical for regulating differentiation of NSCs during cortical development.

Mir505–3p regulates axonal development via inhibiting the autophagy pathway by targeting Atg12

ABSTRACT In addition to the canonical role in protein homeostasis, autophagy has recently been found to be involved in axonal dystrophy and neurodegeneration. Whether autophagy may also be involved in neural development remains largely unclear. Here we report that Mir505–3p is a crucial regulator for axonal elongation and branching in vitro and in vivo, through modulating autophagy in neurons. We identify that the key target gene of Mir505–3p in neurons is Atg12, encoding ATG12 (autophagy-related 12) which is an essential component of the autophagy machinery during the initiation and expansion steps of autophagosome formation. Importantly, axonal development is compromised in brains of mir505 knockout mice, in which autophagy signaling and formation of autophagosomes are consistently enhanced. These results define Mir505–3p-ATG12 as a vital signaling cascade for axonal development via the autophagy pathway, further suggesting the critical role of autophagy in neural development.

Long non-coding RNA tagging and expression manipulation via CRISPR/Cas9-mediated targeted insertion

Long non-coding RNAs (lncRNAs), defined as RNA transcripts longer than 200 nucleotides without the protein-coding ability (Carninci et al., 2005), share many features with protein-coding messenger RNAs (mRNAs) such as polyadenylated 5′ ends and multi-exonic structures (Guttman et al., 2010). Though expression levels are less abundant, lncRNAs outnumber mRNAs with more diverse regulatory functions (Quinn and Chang, 2016). They may serve as decoys, sponges, signals or scaffolds in regulating chromatin conformation, nuclear organization, gene expression, and protein activity in cis or trans manner (Ulitsky and Bartel, 2013; Quinn and Chang, 2016). LncRNAs are involved in various physiological processes and their lossor gain-offunction mutations have been implicated in the pathogenesis of human diseases (Wapinski and Chang, 2011). Although their functions have been investigated extensively, manipulation of lncRNAs is challenging, limiting further in-depth analysis for lncRNAs. Efficient and convenient tagging method could be helpful for effective lncRNAs immunoprecipitation to explore lncRNAs-DNA/RNA/protein interactions (Engreitz et al., 2014; Chu et al., 2015). Another challenge is lncRNAs expression manipulation with high efficiency and specificity: Point mutations or insertions and deletions (Indels) are usually insufficient to block lncRNAs functions completely (Cong et al., 2013; Mali et al., 2013). Deleting the whole lncRNA loci or changing lncRNA expression with either clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) system have been developed as alternative approaches (Zhu et al., 2016; Liu et al., 2017). However, many lncRNA loci overlap with protein-coding genes and even share common promoter regions, restricting the applications of available tools. In addition, many lncRNAs are cisacting factors, so traditional overexpression strategy may not work in such conditions. Recently it is shown that targeted insertion could be achieved with CRISPR/Cas9 system via canonical non-homologous end joining (c-NHEJ) pathway without the need for homologous or microhomologous sequences (Schmid-Burgk et al., 2016; Suzuki et al., 2016), so it is plausible to achieve targeted insertion at different sites with one universal donor vector using CRISPR/Cas9 system. As gene trap system has been well-established to disrupt gene functions with selection markers/tags for subsequent functional analysis (Stanford et al., 2001), we here modified gene trap vectors and used CRISPR/Cas9 to establish a scalable tool entitled CTRL (CRISPR-mediated tagging and regulation of lncRNAs) for lncRNA tagging and expression manipulation in mammalian cells. With this method, we successfully tagged lncRNAs at either 5′ or 3′ end. And lncRNA expression status was either stimulated or inhibited reversibly depending on the targeted insertion site. CTRL system contains a modified gene trap vector, a plasmid expressing S. pyogenes Cas9 (SpCas9) and two sgRNAs driven by two U6 promoters respectively (one genome-targeting sgRNA and another donor plasmid-targeting sgRNA) (defined as Cas9-2sgRNA) for lncRNA tagging and expression manipulation purposes. In principle, modified gene trap vector and Cas9-2sgRNA were transfected simultaneously into 293T cells for donor DNA plasmid linearization and targeted insertion at desired genome locus (Fig. 1A). For targeted insertion near transcriptional termination site, a modified polyA trap vector containing CMVpuromycin selection cassette without polyA signal, a specific sgRNA targeting site and 4× MS2 or 24× MS2 tagging sequences were designed (Fig. S1A and S1B). With targeted insertion near transcriptional termination site, puromycin expression is induced to serve as selection marker for cells containing established targeted insertion. Initially, to determine the applicability of CTRL system for lncRNA tagging and purification, genome-targeting sgRNA inside transcriptional termination site of phosphatase and tensin homolog pseudogene 1 (PTENP1) was designed. Then modified polyA trap vector containing 24× MS2 tags and Cas9-2sgRNA/PTENP1 were transfected into 293T cells. After 48 h, puromycin was added at a final concentration of 2 μg/mL and cells were cultured for another 4 days. Survival cells were further incubated in normal growth medium without puromycin for about 1 week to obtain sufficient cells for subsequent analysis. Established targeted insertion and