Qiyun Wu

Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure.

Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NO and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis.

Increased expression of transcription initiation factor IIB after rat traumatic brain injury

The protein TFIIB is a general transcription initiation factor that plays a pivotal role in the preinitiation complex (PIC) and selects the transcription initiation site. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of TFIIB during traumatic brain injury (TBI). Western blot analysis revealed that TFIIB was present in normal rat brain cortex. It gradually increased, reached a peak at the 5th day after TBI, and then decreased. Importantly, more TFIIB was colocalized with astrocytes and microglia, which are largely proliferated. In addition, Western blot detection showed that the 5th day post injury was also the proliferation peak indicated by the elevated expression of PCNA. Importantly, injury-induced expression of TFIIB was colabelled by proliferating cell nuclear antigen (proliferating cells marker). These data suggested that TFIIB may be implicated in the proliferation of astrocytes and microglia and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of TFIIB after brain injury.

Upregulation of p21-activated Kinase 6 in rat brain cortex after traumatic brain injury

Abstractp21-activated Kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK kinases are well-known regulators of a wide variety of cellular functions, including regulation of cytoskeleton rearrangement, cell survival, apoptosis and the mitogen-activated protein kinase signaling pathway. To elucidate the expressions and possible functions of PAK6 in central nervous system (CNS) lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. Western blot analysis revealed that PAK6 level significantly increased at day 3 after damage, and then declined during the following days. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and a few of glial cells in the normal group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes had largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) whose change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells at day 3 after injury. In addition, injury-induced expression of PAK6 was co-labeled by active caspase-3 during neuronal apoptosis after injury. Collectively, we hypothesized PAK6 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.

Involvement of dysregulated Wip1 in manganese-induced p53 signaling and neuronal apoptosis.

Overexposure to manganese (Mn) has been known to induce neuronal death and neurodegenerative symptoms. However, the precise mechanisms underlying Mn neurotoxicity remain incompletely understood. In the present study, we established a Mn-exposed rat model and found that downregulation of wild type p53-induced phosphatase 1 (Wip1) might contribute to p53 activation and resultant neuronal apoptosis following Mn exposure. Western blot and immunohistochemical analyses revealed that the expression of Wip1 was markedly decreased following Mn exposure. In addition, immunofluorescence assay demonstrated that Mn exposure led to significant reduction in the number of Wip1-positive neurons. Accordingly, the expression of Mdm2 was progressively decreased, which was accompanied with markedly increased expression of p53, as well as the ratio of Bax/Bcl-xl. Furthermore, we showed that Mn exposure decreased the viability and induced apparent apoptosis in NFG-differentiated neuron-like PC12 cells. Importantly, the expression of Wip1 decreased progressively, whereas the level of cellular p53 and the ratio of Bax/Bcl-xl were elevated, which resembled the expression of the proteins in animal model studies. Depletion of p53 significantly ameliorated Mn-mediated cytotoxic effect in PC12 cells. In addition, ectopic expression of Wip1 attenuated Mn-induced p53 signaling as well as apoptosis in PC12 cells. Finally, we observed that depletion of Wip1 augmented Mn-induced apoptosis in PC12 cells. Collectively, these findings suggest that downregulated Wip1 expression plays an important role in Mn-induced neuronal death in the brain striatum via the modulation of p53 signaling.

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.

Traumatic Brain Injury Induces an Up-Regulation of Hs1-Associated Protein X-1 (Hax-1) in Rat Brain Cortex

HS1-associated protein X-1 (Hax-1) is an intracellular protein with anti-apoptotic properties that, in addition to suppressing cell death by inhibiting the activation of initiator caspase-9 and death caspase-3, is involved in an increasing number of signaling cascades. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of Hax-1 expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Hax-1 was present in normal brain. It gradually increased, reached a peak at day 3 after TBI, and then declined during the following days. Double immunofluorescence staining showed that Hax-1 immunoreactivity (IR) was found in neurons, but not astrocytes and microglia. Moreover, the 3rd day post injury was the apoptotic peak implied by the alteration of caspase-3, Bcl-2 and TUNEL. All these results suggested that Hax-1 may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.

Arsenic trioxide mediates HAPI microglia inflammatory response and the secretion of inflammatory cytokine IL-6 via Akt/NF-κB signaling pathway

Arsenic is a widely distributed toxic metalloid in around the world. Inorganic arsenic species are deemed to affect astrocytes functions and to cause neuron apoptosis. Microglia are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA and reverse transcriptase PCR (RT-PCR), we showed that Arsenic trioxide up-regulated the expression and secretion of IL-6 in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. These pro-inflammatory responses were inhibited by the Akt blocker, LY294002. Further, Arsenic trioxide exposure could induce phospho rylationand degradation of IкBα, and the translocation of NF-κB p65 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the NF-кB signaling pathway can be activated after Arsenic trioxide treatment. Besides, Akt blocker LY294002 also obviously attenuated NF-кB activation and transnuclear induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of pro-inflammatory cytokine and NF-кB activation induced by Arsenic trioxide can be mediated by elevation of p-Akt in HAPI microglia cells.

Upregulation of SYF2 is associated with neuronal apoptosis caused by reactive astrogliosis to neuroinflammation

SYF2, known as CCNDBP1‐interactor or p29, is likely involved in pre‐mRNA splicing and cell cycle progression. The present study was designed to elucidate dynamic changes in SYF2 expression and distribution in the cerebral cortex in a lipopolysaccharide (LPS)‐induced neuroinflammation rat model. It was found that SYF2 expression was induced strongly in active astrocytes after LPS injection. In vitro studies showed that the upregulation of SYF2 might be involved in the activation of C6 cells after LPS challenge and the neuronal apoptosis after conditioned media challenge. In addition, with silencing of SYF2 in C6 and PC12 cells by siRNA, the results indicated that SYF2 was required for astrocyte activation and neuronal apoptosis induced by LPS. Our findings on the cellular signaling pathway may provide a new therapeutic strategy against neuroinflammation in the CNS.

Dynamic change of Numbl expression after sciatic nerve crush and its role in Schwann cell differentiation.

Numbl, as a conserved homolog of Drosophila Numb, has been implicated in early development of the nervous system, but its expression and roles in nervous system lesion and repair remained unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Numbl expression in the sciatic nerve. Temporally, Numbl expression was sharply decreased after sciatic nerve crush and reached a valley at day 7. Spatially, Numbl was widely expressed in the normal sciatic nerve, including axons and Schwann cells, whereas, after injury, Numbl expression was decreased predominantly in Schwann cells. In vitro, we induced Schwann cell differentiation with cAMP and found that Numbl expression was decreased in the differentiated process. Depletion of Numbl could promote Schwann cell differentiation. In addition, we demonstrated that in vitro myelination was suppressed by overexpression of Numbl in Schwann cells. Collectively, we hypothesized peripheral nerve injury induced a downregulation of Numbl in the sciatic nerve, which was associated with Schwann cell differentiation.

Involvement of CtBP2 in LPS-induced microglial activation

CtBP2 (C-terminal binding protein 2), which is widely expressed during developmental processes and differentiation, acts as a transcriptional repressor following recruitment to target promoters through repressors or other co-repressor proteins. In this study, we elucidated the dynamic expression changes and localization of CtBP2 in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. CtBP2 expression was strongly induced in active glia cells (microglia and astrocytes) in inflamed spinal cord. In vitro studies indicated that the up-regulation of CtBP2 may be involved in the subsequent microglia activation following LPS exposure. And the knock-down of CtBP2 in microglia cell line HAPI by siRNA showed that CtBP2 increased the activation of microglia induced by LPS. Collectively, these results suggested CtBP2 may be important in host defense in microglia-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammation in central nervous system.