Wenhao Zhou

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.

Endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF activated Notch and Pten signaling

To investigate whether and how endothelial cells affect neurogenesis, we established a system to co‐culture endothelial cells and brain slices of neonatal rat and observed how subventricular zone cells differentiate in the presence of endothelial cells. In the presence of endothelial cells, neural stem cells increased in number, as did differentiated neurons and glia. The augmentation of neurogenesis was reversed by diminishing vascular endothelial growth factor (VEGF) expression in endothelial cells with RNA interference (RNAi). Microarray analysis indicated that expression levels of 112 genes were significantly altered by co‐culture and that expression of 81 of the 112 genes recovered to normal levels following RNAi of VEGF in endothelial cells. Pathway mapping showed an enrichment of genes in the Notch and Pten pathways. These data indicate that endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF, possibly by activating the Notch and Pten pathways. Developmental Dynamics 239:2345–2353, 2010.

Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress

Epigenetic regulations including DNA methylation and demethylation play critical roles in neural development. However, whether DNA methylation and demethylation may play a role in neuronal cell death remains largely unclear. Here we report that the blockade of DNA methyltransferase inhibits apoptosis of cerebellar granule cells and cortical neurons in response to oxidative stress. We found that knockdown of ten-eleven translocation methylcytosine dioxygenase (Tet1), a critical enzyme for DNA demethylation, significantly increase apoptosis of cerebellar granule cells induced by hydrogen peroxide. Moreover, cerebellar granule cells from tet1null mice appeared to be more sensitive to oxidative stress, suggesting the critical role of Tet1 in neuronal cell death. We further showed that the expression of Klotho, an antiaging protein, in cerebellar granule cells is tightly regulated by DNA methylation. Finally, we found that knockdown of Klotho diminished the rescue effects of DNA methyltransferase inhibitors and Tet1 on neuronal cell death induced by oxidative stress. Our work revealed the role of Tet1-mediated DNA demethylation on neuronal protection against oxidative stress and provided the molecular mechanisms underlying the epigenetic regulation of neuronal cell death, suggesting the role of Klotho in regulating neuronal cell death in response to oxidative stress.

The Epigenetic Switches for Neural Development and Psychiatric Disorders

The most remarkable feature of the nervous system is that the development and functions of the brain are largely reshaped by postnatal experiences, in joint with genetic landscapes. The nature vs. nurture argument reminds us that both genetic and epigenetic information is indispensable for the normal function of the brain. The epigenetic regulatory mechanisms in the central nervous system have been revealed over last a decade. Moreover, the mutations of epigenetic modulator genes have been shown to be implicated in neuropsychiatric disorders, such as autism spectrum disorders. The epigenetic study has initiated in the neuroscience field for a relative short period of time. In this review, we will summarize recent discoveries about epigenetic regulation on neural development, synaptic plasticity, learning and memory, as well as neuropsychiatric disorders. Although the comprehensive view of how epigenetic regulation contributes to the function of the brain is still not completed, the notion that brain, the most complicated organ of organisms, is profoundly shaped by epigenetic switches is widely accepted.

Deep-brain magnetic stimulation promotes adult hippocampal neurogenesis and alleviates stress-related behaviors in mouse models for neuropsychiatric disorders

BackgroundRepetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood.ResultsHere we studied the effects of deep-brain magnetic stimulation to brain on the molecular and cellular level. We examined the adult hippocampal neurogenesis and hippocampal synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation treatment. We found that DMS promotes adult hippocampal neurogenesis significantly and facilitates the development of adult new-born neurons. Remarkably, DMS exerts anti-depression effects in the learned helplessness mouse model and rescues hippocampal long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates the stress response in a mouse model for Rett syndrome and prolongs the life span of these animals dramatically.ConclusionsDeep-brain magnetic stimulation greatly facilitates adult hippocampal neurogenesis and maturation, also alleviates depression and stress-related responses in animal models.

Psychiatric Illness and Intellectual Disability in the Prader–Willi Syndrome with Different Molecular Defects - A Meta Analysis

Background and Objectives Several studies have suggested a difference in clinical features of intellectual ability and psychiatric illness in the Prader–Willi syndrome (PWS) with the 15q11-q13 paternal deletion and maternal uniparental disomy (mUPD). Our objective was to appraise evidence on this association through a meta-analysis. Methods The electronic records PubMed and EMBASE from 1956 to 2012 were extracted for meta-analysis. Meta-analyses were performed by using fixed effect model. Mean difference, odds ratio, and 95% confidence interval were calculated. Results We retrieved a total of 744 PWS cases from 13 studies. These include 423 cases with paternal 15q11-q13 deletions and 318 cases of mUPD. Compare to the PWS cases with mUPD, PWS patients with the paternal 15q11-q13 deletion associated with significantly lower full scale IQ (FSIQ) [mean difference (MD), -2.69; 95%CI, -4.86 to -0.52; p=0.02] and verbal IQ (VIQ) (MD, -7.5; 95%CI, -9.75 to -5.26; p<0.00001) but higher performance IQ (PIQ) (MD, 4.02; 95%CI, 1.13 to 6.91; p=0.006). In contrast, PWS patients with mUPD are associated with significantly higher risk of psychiatric illness [odds rate (OR), 0.14; 95%CI, 0.08 to 0.23; p<0.00001] and higher risk of bipolar disorder (OR, 0.04; 95%CI, 0.01 to 0.23; p=0.0002). Conclusions Significant different clinical features of cognitive development and psychiatric illness are associated with PWS with different molecular defects. These findings provide support for evidence based practice to evaluate and manage the PWS syndrome with different molecular defects.

Neuroprotective Effects of Oligodendrocyte Progenitor Cell Transplantation in Premature Rat Brain following Hypoxic-Ischemic Injury

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.

Effects of hypothermia on oligodendrocyte precursor cell proliferation, differentiation and maturation following hypoxia ischemia in vivo and in vitro

Hypoxic-ischemia (HI) not only causes gray matter injury but also white matter injury, leading to severe neurological deficits and mortality, and only limited therapies exist. The white matter of animal models and human patients with HI-induced brain injury contains increased oligodendrocyte precursor cells (OPCs). However, little OPC can survive and mature to repair the injured white matter. Here, we test the effects of mild hypothermia on OPC proliferation, differentiation and maturation. Animals suffered to left carotid artery ligation followed by 8% oxygen for 2h in 7-day-old rats. They were divided into a hypothermic group (rectal temperature 32-33 °C for 48 h) and a normothermic group (36-37 °C for 48 h), then animals were sacrificed at 3, 7, 14 and 42 days after HI surgery. Our results showed that hypothermia successfully enhanced early OL progenitors (NG2(+)) and its proliferation in the corpus callosum (CC) after HI. Late OL progenitor (O4(+)) accumulation decreased accompanied with increased OL maturation which is detected by myelin basic protein (MBP) and proteolipid protein. (PLP) immunostaining and immunoblotting in hypothermia compared to normothermia. Additionally, using an in vitro hypoxic-ischemia model-oxygen glucose deprivation (OGD), we demonstrated that hypothermia decreased preOL accumulation and promoted OPC differentiation and maturation. Further data indicated that OPC death was significantly suppressed by hypothermia in vitro. The myelinated axons and animal behavior both markedly increased in hypothermic- compared to normothermic-animals after HI. In summary, these data suggest that hypothermia has the effects to protect OPC and to promote OL maturation and myelin repair in hypoxic-ischemic events in the neonatal rat brain. This study proposed new aspects that may contribute to elucidate the mechanism of hypothermic neuroprotection for white matter injury in neonatal rat brain injury.

Ischemia induced neural stem cell proliferation and differentiation in neonatal rat involved vascular endothelial growth factor and transforming growth factor-beta pathways

Brain ischemia is a leading cause of mortality and morbidity in premature infants. Knowing the fate of neural stem cells in the subventricular zone (SVZ) after ischemia and the mechanisms that determine this fate would be useful in manipulating neural stem cell proliferation and differentiation and possibly in reversing ischemic damage. We sought to identify the genes involved in the proliferation and differentiation of neural stem cells after exposure to ischemia in a 3-day-old rat model that approximates ischemia in premature infants. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection. Using immunfluorescence assays, we observed the proliferation and differentiation of neural stem cells. Genes were identified with GeneChip and real-time quantitative polymerase chain reaction analysis. Ischemic rats had more BrdU-positive cells in the SVZ at all four time points and more neural stem cells differentiation into neurons, astrocytes, and oligodendrocytes. GeneChip analysis showed a 3- to 10-fold increase in the mRNA expression of vascular endothelial growth factor, transforming growth factor-beta, and their receptors in the SVZ. PCR assays and Western blot analyses confirmed these results, indicating that vascular endothelial growth factor and transforming growth factor-beta might be two of the factors that involve post-ischemic neural stem cell proliferation and differentiation.

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.