Chengwei Duan

Lentiviral Vector-Mediated p27kip1 Expression Facilitates Recovery After Spinal Cord Injury

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction attributable to both mechanical damage and secondary biochemical and physiological responses. Upregulation of cell cycle proteins occurs in both neurons and glia after SCI and may contribute to these changes. Increased cell cycle protein is associated with neuronal and oligodendroglial apoptosis, reactive astrogliosis, glial scar formation, and microglial activation. Here, using lentiviral vectors (LV), we induced the expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in the lesioned spinal cord of adult rat. Treatment with LV-p27kip1 significantly reduced the expression of cell cycle proteins and improved functional recovery. In addition, p27kip1 overexpression also reduced lesion volume, decreased astrocytic reactivity, attenuated microglial activation, reduced cell death, and improved the local microenvironment. We suggest that these effects reflect the ability of p27kip1 to inhibit cell cycle pathways. Thus, the present study provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of SCI.

Toll-Interleukin 1 Receptor domain-containing adaptor protein positively regulates BV2 cell M1 polarization.

Microglial activation, including classical (M1) and alternative (M2) activation, plays important roles in the development of several central nervous system disorders and promotes tissue reconstruction. Toll‐like receptor (TLR)4 is important for microglial polarization. TIR domain‐containing adaptor protein (TIRAP) is an intracellular adaptor protein, which is responsible for the early phase of TLR4 activation. The role of TIRAP in BV2 cell M1 polarization is still unknown. In this study, we showed that TIRAP expression is greatly elevated in lipopolysaccharide (LPS)/interferon (IFN)‐γ‐treated microglia. TIRAP overexpression promoted BV2 microglial M1 polarization by increasing M1‐related marker production (inducible nitric oxide synthase, CD86, interleukin‐6, interleukin‐1β and tumour necrosis factor‐α). In contrast, TIRAP knockdown prevented M1‐related marker production. Mechanistically, TIRAP could interact with TNF Receptor‐Associated Factor 6 (TRAF6) to increase M1‐related marker production in TIRAP overexpressed and LPS/IFN‐γ‐treated BV2 cells. In addition, silencing of TIRAP effectively inhibited the activation of the Transforming Growth Factor‐Beta‐Activated Kinase 1/I‐Kappa‐B Kinase /Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B‐Cells (TAK1/IKK/NF‐κB) signalling pathway and the phosphorylation of Akt and mitogen‐activated protein kinases, which were activated by LPS/IFN‐γ stimulation. Thus, our results suggest that TIRAP positively regulated BV2 microglial M1 polarization through TLR4‐mediated TAK1/IKK/NF‐κB, mitogen‐activated protein kinases and Akt signalling pathways.

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.

Up-Regulation of Interferon Regulatory Factor 3 Involves in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats

Interferon regulatory factor 3 (IRF3) is a member of IRF family which plays a significant role in the innate immune response, apoptosis, and oncogenesis. Mounting evidence has demonstrated that IRF3 was involved in central nervous system disease such as cerebral ischemic injury through promoting neuronal apoptosis. However, it remains unclear about the underlying mechanisms of IRF3 upon neuronal apoptosis following intracerebral hemorrhage (ICH). In the present study, we established an adult rat ICH model by injecting autologous whole blood into the right basal ganglia and evaluated their neurological deficits by behavioral tests. IRF3 protein level was up-regulated adjacent to the hematoma following ICH when compared with the sham brain cortex by western blot and immunohistochemistry. Immunofluorescent staining indicated IRF3 was mainly localized in neurons, a few in astrocytes. In addition, we also detected that IRF3 co-localized with active caspase-3 which is a neuronal apoptosis marker. Furthermore, in vitro study, knocking down IRF3 by using IRF3 interference in primary cortical neurons reduced the expression of active caspase-3 and Bax while increased Bcl-2. In conclusion, we speculated that IRF3 might exert pro-apoptotic function in neurons after ICH.

Expression of Dixdc1 and its Role in Astrocyte Proliferation after Traumatic Brain Injury

DIX domain containing 1 (Dixdc1), a positive regulator of Wnt signaling pathway, is recently reported to play a role in the neurogenesis. However, the distribution and function of Dixdc1 in the central nervous system (CNS) after brain injury are still unclear. We used an acute traumatic brain injury (TBI) model in adult rats to investigate whether Dixdc1 is involved in CNS injury and repair. Western blot analysis and immunohistochemistry showed a time-dependent up-regulation of Dixdc1 expression in ipsilateral cortex after TBI. Double immunofluorescent staining indicated a colocalization of Dixdc1 with astrocytes and neurons. Moreover, we detected a colocalization of Ki-67, a cell proliferation marker with GFAP and Dixdc1 after TBI. In primary cultured astrocytes stimulated with lipopolysaccharide, we found enhanced expression of Dixdc1 in parallel with up-regulation of Ki-67 and cyclin A, another cell proliferation marker. In addition, knockdown of Dixdc1 expression in primary astrocytes with Dixdc1-specific siRNA transfection induced G0/G1 arrest of cell cycle and significantly decreased cell proliferation. In conclusion, all these data suggest that up-regulation of Dixdc1 protein expression is potentially involved in astrocyte proliferation after traumatic brain injury in the rat.

Up-Regulation of Corticocerebral NKD2 in Lipopolysaccharide-Induced Neuroinflammation

Naked2 (NKD2), one member of Naked family, has been shown to negatively regulate Wnt/β-catenin signaling pathway. It has been recognized that NKD2 plays a vital role in cell homeostasis and prevention of tumorigenesis. However, NKD2 expression and its functional role in the brain in neuroinflammatory processes remain unclear. In our study, we investigated NKD2 distribution and role in lipopolysaccharide (LPS)-induced neuroinflammation rat model. The data indicated that NKD2 was up-regulated in LPS-injected brain, and the cellular localization of NKD2 was predominantly in cerebral cortical neurons. Furthermore, we treated primary neurons with conditioned media (CM) collected from LPS-stimulated mixed glial cultures (MGC). We detected that the up-regulation of NKD2 might be associated with the subsequent apoptosis in neurons. We also found knockdown NKD2 partially depressed the increase of cleaved caspase-3 and increased the reduction of β-catenin stimulated by MGC-CM. Taken together, these results suggested that NKD2 might be involved in neuronal apoptosis via the Wnt/β-catenin pathway during neuroinflammation in CNS. Our findings might provide a new therapeutic target for the prevention of neuroinflammation-involved neurological disorders.

Genetic regulatory network analysis reveals that low density lipoprotein receptor-related protein 11 is involved in stress responses in mice

To study whether Lrp11 is involved in stress response and find its expression regulatory network, the model of stress has been built using C57BL/6J (B6) and DBA/2 (D2) mice. Western blotting, qPCR and immunohistochemistry were used to investigate the expression variation of Lrp11 in amygdala tissue after exposure to stress. We found the quantity of Lrp11 was more obvious in stress models than that in normal mice (P<0.05) which suggests Lrp11 might participate in the process of stress response. The expression of Lrp11 is controlled by a cis-acting quantitative trait locus (cis-eQTL). We identified four genes that are regulated by Lrp11 and the expression of 66 genes highly correlated with Lrp11, seven of which have previously been implicated in stress pathways. To evaluate the relationship between Lrp11 and its downstream genes or network members, we transfected HEK 293T cells and SH-SY5Y cells with Lrp11 siRNA leading to down-regulation of Lrp11mRNA and were able to confirm a significant influence of Lrp11 depletion on the expression of Xpnpep1, Maneal, Pgap1 and Uprt. These validated downstream targets and members of Lrp11 gene network provide new insight into the biological role of Lrp11 and may be an important risk factor in the development of stress.

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.

Insulin-like Growth Factor Binding Protein6 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage in Rats

The insulin-like growth factor (IGF) system is linked to CNS pathological states. The functions of IGFs are modulated by a family of binding proteins termed insulin-like growth factor binding proteins (IGFBPs). Here, we demonstrate that IGFBP-6 may be associated with neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant upregulation of IGFBP-6 in neurons adjacent to the hematoma following ICH with the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing IGFBP-6 level was found to be accompanied by the upregulation of Bax, Bcl-2, and active caspase-3. Besides, IGFBP-6 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. Knocking down IGFBP-6 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, IGFBP-6 may play a role in promoting the brain secondary damage following ICH.

Genetic expression analysis of E2F-associated phosphoprotein in stress responses in the mouse.

Dysfunction of the monoaminergic system is critical in stress and anxiety disorders, but the role of each family member in the development of stress-related psychopathologies is not sufficiently understood. Eapp has been reported to be a transcriptional repressor of monoamine oxidase B (Maob) and down-regulates Maob via the Maob core promoter. In the present study, we more specifically examine the role of Eapp in stress responses by testing the hypothesis that Eapp may be involved in the occurrence and development of stress responses. Western blotting, qPCR and immunohistochemistry were used to investigate the expression variation of Eapp in hypothalamus tissue after exposure to stress. The expression of Eapp is controlled by a cis-acting quantitative trait locus (cis-eQTL). Two genes Sphk2 and Nosip, had trans-eQTLs that mapped to the location of Eapp and altered expression of these two genes was shown following siRNA knockdown of Eapp. Additionally, Mmp9, Npy, Npy5r and Maob were shown to have different expression levels in the Eapp knock-down experiments. Our data provide strong evidence that the cis-modulated gene, Eapp, is associated with stress responses, and that validated downstream targets and members of Eapp gene network may also be involved in the development of stress.