Yajing Mi

Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H2O2). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H2O2 treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure–function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290–562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.

The Inverse F-BAR Domain Protein srGAP2 Acts through srGAP3 to Modulate Neuronal Differentiation and Neurite Outgrowth of Mouse Neuroblastoma Cells

The inverse F-BAR (IF-BAR) domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA)-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1R542A and srGAP2R527A exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.

LEF1 regulates glioblastoma cell proliferation, migration, invasion, and cancer stem-like cell self-renewal

Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common primary tumors of the central nervous system. This disease remains one of the incurable human malignancies because the molecular mechanism driving the GBM development and recurrence is still largely unknown. Here, we show that knockdown of lymphocyte enhancer factor-1 (LEF1), a major transcription factor of Wnt pathway, inhibits U251 cell migration, invasion, and proliferation. Furthermore, downregulation of LEF1 expression inhibits the self-renewal capacity of U251 GBM stem-like cells and decreases the expression level of the GBM stem-like cell (GSC) markers such as CD133 and nestin. Our findings reveal that LEF1 maintains the GBM cell proliferation, migration, and GBM stem-like cell self-renewal. Taken together, these results suggest that LEF1 may be a novel therapeutic target for GBM suppression.

Dynamics of ten-eleven translocation hydroxylase family proteins and 5-hydroxymethylcytosine in oligodendrocyte differentiation

The ten‐eleven translocation (TET) family of methylcytosine dioxygenases catalyze oxidation of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and TET proteins in the brain, little is known about their role in oligodendrocytes (OLs). Here, we analyzed TET expression during OL development in vivo and in vitro, and found that three TET family members possess unique subcellular and temporal expression patterns. Furthermore, the level of 5hmC exhibits dynamic changes during OL maturation, which implies that 5hmC modification may play a role in the expression of critical genes necessary for OL maturation. siRNA‐mediated silencing of the TET family proteins in OLs demonstrated that each of the TET proteins is required for OL differentiation. However, based on their unique domain structures, we speculate that the three TET members may function by different mechanisms. In summary, we have established the temporal expression of TET proteins and the dynamic level of 5hmC during OL development and demonstrate that all three TET members are necessary for OL differentiation. GLIA 2014;62:914–926

The mental retardation associated protein, srGAP3 negatively regulates VPA-induced neuronal differentiation of Neuro2A cells.

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.

M9, A Novel Region of Amino-Nogo-A, Attenuates Cerebral Ischemic Injury by Inhibiting NADPH Oxidase-Derived Superoxide Production in Mice

In acute stroke, neurological damage is due to oxidative stress and neuronal apoptotic death. This study investigated whether Nogo‐A 290‐562 residues region (M9), fused to the transduction domain of the HIV trans‐activator (TAT) protein, is neuroprotective against cerebral ischemia and the mechanisms.

A Novel Function of TET2 in CNS: Sustaining Neuronal Survival.

DNA dioxygenases Ten-Eleven Translocation (TET) proteins can catalyze the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), and thereby alter the epigenetic state of DNA. The TET family includes TET1, TET2 and TET3 members in mammals. Recently, accumulative research uncovered that TET1–3 occur abundantly in the central nervous system (CNS), and their biological functions have just begun to be investigated. In the present study, we demonstrated that mRNA and protein of TET2 were highly expressed in the cerebral cortex and hippocampus along the whole brain-development process. Further studies showed that TET2 was expressed in various types of cells, especially in most neurons. Subcellular distribution pattern implicated that TET2 is localized in both nucleus and cytoplasm of neurons. Down-regulation of TET2 in cultured cortical neurons with RNA interference implied that TET2 was required for cell survival. In all, our results indicate that neuronal TET2 is positively involved in the regulation of cell survival.

The PHLDB1 rs498872 (11q23.3) polymorphism and glioma risk: A meta-analysis.

The association between the rs498872 single nucleotide polymorphism (SNP) and glioma risk has been studied, but these studies have yielded conflicting results. In order to explore this association, we performed a meta‐analysis. A comprehensive literature search was performed using PubMed and EMBASE database, with the last search up to August 23, 2013. Six articles including 10 case‐control studies in English with 18 002 controls and 8434 cases were eligible for the meta‐analysis. Subgroup analyses were conducted by source of controls and ethnicity. The combined results showed that rs498872 polymorphism was significantly associated with glioma risks (TT vs CC: OR = 1.337, 95% CI = 1.222–1.462; TC vs CC: OR = 1.173, 95% CI = 1.081–1.272; dominant model: OR = 1.199, 95% CI = 1.101–1.306; recessive model: OR = 1.237, 95% CI = 1.135–1.347; additive model: OR = 1.156, 95% CI = 1.085–1.232). Moreover, there was increased cancer risk in all genetic models after stratification of the SNP data by the source of controls and ethnicity, and no evidence of publication bias was produced. Our meta‐analysis suggested that rs498872 polymorphism was associated with increased risk of glioma. However, additional studies exploring the combined effects of rs498872 polymorphisms in Asian population should be investigated.

A link between the nuclear-localized srGAP3 and the SWI/SNF chromatin remodeler Brg1

The Slit-Robo GTPase activating protein 3 (srGAP3) is an important modulator of actin cytoskeletal dynamics and has an important influence on a variety of neurodevelopmental processes. Mutations in the SRGAP3 gene on chromosome 3p25 have been found in patients with intellectual disability. Genome-wide association studies and behavioral assays of knockout mice had also revealed SRGAP3 as a risk gene for schizophrenia. We have recently shown that srGAP3 protein undergoes regulated shuttling between the cytoplasm and the nucleus during neuronal development. It is shown here that nuclear-localized srGAP3 interacts with the SWI/SNF remodeling factor Brg1. This interaction is mediated by the C-terminal of srGAP3 and the ATPase motif of Brg1. In the primary cultured rat cortical neurons, the levels of nuclear-localized srGAP3 and its interaction with Brg1 have a significant impact on dendrite complexity. Furthermore, the interaction between srGAP3 and Brg1 was also involved in valproic acid (VPA) -induced neuronal differentiation of Neuro2a cells. We then show that GTP-bound Rac1 and GAP-43 may be potential mediators of nuclear srGAP3 and Brg1. Our results not only indicate a novel signaling pathway that contributes to neuronal differentiation and dendrite morphology, but also implicate a novel molecular mechanism underlying srGAP3 regulation of gene expression.

Disrupted in schizophrenia 1 (DISC1) inhibits glioblastoma development by regulating mitochondria dynamics

Glioblastoma(GBM) is one of the most common and aggressive malignant primary tumors of the central nervous system and mitochondria have been proposed to participate in GBM tumorigenesis. Previous studies have identified a potential role of Disrupted in Schizophrenia 1 (DISC1), a multi-compartmentalized protein, in mitochondria. But whether DISC1 could regulate GBM tumorigenesis via mitochondria is still unknown. We determined the expression level of DISC1 by both bioinformatics analysis and tissue analysis, and found that DISC1 was highly expressed in GBM. Knocking down of DISC1 by shRNA in GBM cells significantly inhibited cell proliferation both in vitro and in vivo. In addition, down-regulation of DISC1 decreased cell migration and invasion of GBM and self renewal capacity of glioblastoma stem-like cells. Furthermore, multiple independent rings or spheres could be observed in mitochondria in GBM depleted of DISC1, while normal filamentous morphology was observed in control cells, demonstrating that DISC1 affected the mitochondrial dynamic. Dynamin-related protein 1 (Drp1) was reported to contribute to mitochondrial dynamic regulation and influence glioma cells proliferation and invasion by RHOA/ ROCK1 pathway. Our data showed a significant decrease of Drp1 both in mRNA and protein level in GBM lack of DISC1, indicating that DISC1 maybe affect the mitochondrial dynamic by regulating Drp1. Taken together, our findings reveal that DISC1 affects glioblastoma cell development via mitochondria dynamics partly by down regulation of Drp1.