Peipei Gong

An IDH1 mutation inhibits growth of glioma cells via GSH depletion and ROS generation

The isocitrate dehydrogenase 1 (IDH1) gene mutation occurs frequently in glioma. While some studies have demonstrated that IDH1 mutations are associated with prolonged survival, the mechanism remains unclear. In this study, we found that growth was significantly inhibited in glioma cells overexpressing the mutated IDH1 gene. Furthermore, these cells were characterized by decreased intracellular NADPH levels accompanied by glutathione (GSH) depletion and reactive oxygen species (ROS) generation. Moreover, the increased apoptosis and the decreased proliferation were found in the glioma cells overexpressing the mutant IDH1 gene. Accordingly, our study demonstrates that using H2O2-regulated mutant IDH1 glioma cells could obviously increase the inhibition of cell growth; nevertheless, GSH had the opposite result. Our study provides direct evidence that mutation of IDH1 profoundly inhibits the growth of glioma cells, and we speculate that this is the major factor behind its association with prolonged survival in glioma. Finally, our study indicates that depletion of GSH and generation of ROS are the primary cellular events associated with this mutation.

Cyclic AMP Response Element Modulator-1 (CREM-1) Involves in Neuronal Apoptosis after Traumatic Brain Injury

The cyclic AMP response element-binding protein (CREB) family can regulate biological functions of various types of cells by forming homo- or heterodimers to bind the target DNA sequences; it plays an essential role in individual neuronal function and entire neuronal circuits. One attractive activity of the CREB family is regulating the transcription of apoptosis-suppressor gene bcl-2. Cyclic AMP response element modulator-1 (CREM-1) is one member of the family with limited acquaintance. To investigate whether CREM-1 is involved in central nervous system injury and repair, we performed an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of CREM-1 in ipsilateral peritrauma cortex. Immunofluorescent labeling indicated that CREM-1 was localized mainly in the nuclei of neurons; co-localization of CREM-1 and active-caspase-3 in the ipsilateral cortex suggested that CREM-1 might participate in neuronal apoptosis. To further investigate the function of CREM-1, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of CREM-1 with p-CREB on PC12 cells by Western blot, immunofluorescent labeling, and co-immunoprecipitation. The result implied that the association of CREM-1 with p-CREB was enhanced in apoptotic cells. Additionally, knocking CREM-1 down with siRNA demonstrated the probable pro-apoptotic role played by CREM-1 in neuronal apoptosis. Together with our data, we hypothesized that CREM-1 might play an important role in regulating neuronal death after TBI by interacting with CREB.

PAX3 is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells.

Paired box 3 (PAX3) is overexpressed in glioma tissues compared to normal brain tissues, however, the pathogenic role of PAX3 in human glioma cells remains to be elucidated. In this study, we selected the human glioma cell lines U251, U87, SHG-44, and the normal human astrocytes, 1800, which have differential PAX3 expression depending upon the person. SiRNA targeting PAX3 and PAX3 overexpression vectors were transfected into U87 and SHG-44 glioma cell lines, and cell proliferation, invasion, apoptosis, and differentiation were examined by CCK-8 assays, transwell chamber assays, tunnel staining, Annexin V/PI analysis, and Western blotting, respectively. In addition, we used subcutaneous tumor models to study the effect of PAX3 on the growth of glioma cells in vivo. We found that PAX3 was upregulated in the three glioma cell lines. PAX3 knockdown inhibited cell proliferation and invasion, and induced apoptosis in the U87MG glioblastoma cell line, whereas PAX3 upregulation promoted proliferation, inhibited apoptosis, and increased invasion in the SHG-44 glioma cell line. Moreover, we found that targeting PAX3 expression in glioma cell lines together with chemotherapeutic treatment could increase glioma cell susceptibility to the drug. In subcutaneous tumor models in nude mice using glioma cell lines U-87MG and SHG-44, inhibition of PAX3 expression in glioblastoma U-87MG cells suppressed tumorigenicity, and upregulation of PAX3 expression in glioma SHG-44 cells promoted tumor formation in vivo. These results indicate that PAX3 in glioma is essential for gliomagenesis; thus, targeting PAX3 or its downstream targets may lead to novel therapies for this disease.

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.

O-GlcNAc Glycosylation of nNOS Promotes Neuronal Apoptosis Following Glutamate Excitotoxicity

Ischemic stroke is a dominant health problem with extremely high rates of mortality and disability. The main mechanism of neuronal injury after stroke is excitotoxicity, during which the activation of neuronal nitric oxide synthase (nNOS) exerts a vital role. However, directly blocking N-methyl-d-aspartate receptors or nNOS can lead to severe undesirable effects since they have crucial physiological functions in the central nervous system. Here, we report that nNOS undergoes O-linked-β-N-acetylglucosamine (O-GlcNAc) modification via interacting with O-GlcNAc transferase, and the O-GlcNAcylation of nNOS remarkably increases during glutamate-induced excitotoxicity. In addition, eliminating the O-GlcNAcylation of nNOS protects neurons from apoptosis during glutamate stimulation by decreasing the formation of nNOS–postsynaptic density protein 95 complexes. Taken together, our data suggest a novel function of the O-GlcNAcylation of nNOS in neuronal apoptosis during glutamate excitotoxicity, suggesting a novel therapy strategy for ischemic stroke.

Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines

Purpose It is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases, although the mechanisms through which this occurs remain unclear. Among mitogen-activated protein kinase (MAPK) targets, mitogen- and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression. In this study, the roles of MSK1 activation in neuroinflammation were investigated. Methods The bacterial lipopolysaccharide (LPS)-induced brain injury model was performed on Sprague-Dawley rats. The dynamic expression changes and the cellular location of p-MSK1 in the brain cortex were detected by Western blot and immunofluorescence staining. The synthesis of inflammatory cytokines in astrocytes was detected by enzyme-linked immunosorbent assay (ELISA). Results Phosphorylated MSK1 (p-MSK1 Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1 in astrocytes was also observed in inflamed cerebral cortex. At 1 day after LPS stimulation, iNOS, TNFα expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1 (Thr-581) may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS in primary astrocytes stimulated the synthesis of inflammatory cytokines, through MAPKs signaling pathways. In cultured primary astrocytes, both knock-down of total MSK1 by small interfering RNAs (siRNA) or specific mutation of Thr-581 resulted in higher production of certain cytokines, such as TNFα and IL-6. Conclusions Collectively, these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of inflammation.

Increased expression of actin filament-stabilizing protein tropomyosin after rat traumatic brain injury

Tropomyosin (TM), is a coiled-coil dimmer which modulates actin filament properties, has been implicated in the control of actin filament dynamics during cell migration, morphogenesis, and cytokinesis. However, the expressions and possible functions of tropomyosin in central nervous system (CNS) lesion remain unknown. In this study, we found the expression of tropomyosin gradually increased in rat brains subjected to traumatic brain injury (TBI). Double immunofluorescence staining showed tropomyosin was expressed in neurons and reactive astrocytes following TBI but not in quiescent astrocytes in normal brains. Furthermore, we detected that proliferating cell nuclear antigen (PCNA) had the co-localization with GFAP, and tropomyosin. In conclusion, this was the first description of tropomyosin expression in rat traumatic brain. Our date suggested that tropomyosin might be involved in the astrocytes proliferation following TBI.

Erratum to: O-GlcNAc Glycosylation of nNOS Promotes Neuronal Apoptosis Following Glutamate Excitotoxicity

Ischemic stroke is a dominant health problem with extremely high rates of mortality and disability. The main mechanism of neuronal injury after stroke is excitotoxicity, during which the activation of neuronal nitric oxide synthase (nNOS) exerts a vital role. However, directly blocking N-methyl-D-aspartate receptors or nNOS can lead to severe undesirable effects since they have crucial physiological functions in the central nervous system. Here, we report that nNOS undergoes O-linked-b-Nacetylglucosamine (O-GlcNAc) modification via interacting with O-GlcNAc transferase, and the O-GlcNAcylation of nNOS remarkably increases during glutamate-induced excitotoxicity. In addition, eliminating the O-GlcNAcylation of nNOS protects neurons from apoptosis during glutamate stimulation by decreasing the formation of nNOS– postsynaptic density protein 95 complexes. Taken together, our data suggest a novel function of the O-GlcNAcylation of nNOS in neuronal apoptosis during glutamate excitotoxicity, suggesting a novel therapy strategy for ischemic stroke.

Combination Glioma Therapy Mediated by a Dual-Targeted Delivery System Constructed Using OMCN-PEG-Pep22/DOX

Accumulating studies have investigated the efficacy of receptor-mediated delivery of hydrophobic drugs in glioma chemotherapy. Here, a delivery vehicle comprising polyethylene glycol (PEG) and oxidized nanocrystalline mesoporous carbon particles (OMCN) linked to the Pep22 polypeptide targeting the low-density lipoprotein receptor (LDLR) is designed to generate a novel drug-loaded system, designated as OMCN-PEG-Pep22/DOX (OPPD). This system effectively targets glioma cells and the blood-brain barrier and exerts therapeutic efficacy through both near-infrared (NIR) photothermal and chemotherapeutic effects of loaded doxycycline (DOX). Pathological tissue microarrays show an association of LDLR overexpression in human glioma tissue with patient survival.NIR irradiation treatment and magnetic resonance imaging results show that OPPD reaches the effective glioma-killing temperature in a glioma-bearing rat with a skull bone removal model and considerably reduces glioma sizes relative to the drug-loaded system without the Pep22 peptide modification and the control respectively. Thus, OPPD not only effectively targets LDLR-overexpressing glioma but also exerts a dual therapeutic effect by transporting DOX into the glioma and generating thermal effects with near-infrared irradiation to kill tumor cells. These collective findings support the utility of the novel OPPD drug-loaded system as a promising drug delivery vehicle for clinical application in glioma therapy.

Up-regulation of FOS-like antigen 1 contributes to neuronal apoptosis in the cortex of rat following traumatic brain injury

Neuronal apoptosis is an important process of secondary brain injury which is induced by neurochemical signaling cascades after traumatic brain injury (TBI). Present study was designed to investigate whether FOS-like antigen 1 (Fra-1) is involved in the neuronal apoptosis. Western blot analysis and immunohistochemistry in a rat TBI model revealed a significant increase in the expression of Fra-1 in the ipsilateral brain cortex, which was in parallel with increase in the expression of active caspase-3. With immunofluorescence double-labeling, Fra-1 was colocalized with active caspase-3 and with NeuN, a neuronal marker. In an in vitro cell injury model, H2O2 exposure induced cell apoptosis and reduced cell viability and at the same time, a similar increased expression of active caspase-3, p53 and Fra-1 was found in PC12 cells. Down-regulation of Fra-1 through transfection with Fra-1 siRNA remarkably elevated cell viability, reduced the expression of active caspase-3 and p53, and decreased apoptosis of PC12 cells after H2O2 exposure. Taken together, present findings suggest that Fra-1 may be involved in the induction of neuronal apoptosis through up-regulating p53 signaling pathway and that this action may contribute to the secondary neuropathological process after TBI.