Yilu Gao

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.

Upregulation of Ras homolog enriched in the brain (Rheb) in lipopolysaccharide-induced neuroinflammation.

Ras homolog enriched in the brain (Rheb) is a homolog of Ras GTPase that regulates cell growth, proliferation, and cell cycle via mammalian target of rapamycin (mTOR). Recently, it has been confirmed that Rheb activation not only promotes cellular proliferation and differentiation but also enhances cellular apoptosis in response to diverse toxic stimuli. However, the function of Rheb in the central nervous system (CNS) is still with limited understanding. To elaborate whether Rheb was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. Upregulation of Rheb was observed in the brain cortex by performing western blotting and immunohistochemistry. Double immunofluorescent staining demonstrated that Rheb was mainly in active astrocytes and neurons. PCNA and active caspase-3 were upregulated, and co-labeling with Rheb, which indicated that Rheb might be relevant to astrocytic proliferation and neuronal apoptosis following the inflammatory response by LPS-induced. Furthermore, we also found that the expression profiles of cyclinD1 and CDK4 were parallel with that of Rheb in a time-space dependent manner. Finally, knocking down Rheb by siRNA and treatment with rapamycin or lovastatin showed that not only astrocytic proliferation decreased but also neuronal protection. Based on our data, we suggested that Rheb might play an important role in physiological and pathological functions following neuroinflammation caused by LPS, which might provide a potential target to the treatment of neuroinflammation.

Involvement of CLEC16A in activation of astrocytes after LPS treated.

CLEC16A, C-type lectin domain family 16, member A was recently found to be associated with inflation process in the autoimmune diseases. In this study, we elucidated the dynamic expression changes and localization of CLEC16A in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. CLEC16A expression was strongly induced in active astrocytes in inflamed cerebral cortex. In vitro studies indicated that the up-regulation of CLEC16A may be involved in the subsequent astrocyte activation following LPS challenge. And Knock-down of CLEC16A in cultured primary astrocytes by siRNA showed that CLEC16A was required for the activation of astrocytes induced by LPS. Collectively, these results suggested CLEC16A may be important in host defense in astrocyte-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammtion in CNS.

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.

Knockdown of PFTK1 Inhibits the Migration of Glioma Cells

The prognosis of glioma patients is generally poor, so it is urgent to find out the underlying molecular mechanisms. PFTK1 is a member of cyclin-dependent kinases (Cdks) family and has been reported to contribute to tumor migration and invasion. In this study, we aimed to explore the expression and function in human glioma. Western blot and immunohistochemistry were used to evaluate the expression of PFTK1. PFTK1 expression was higher in glioma tissues compared with normal brain tissues, and its level was associated with the WHO grade in Western blot analysis. The suppression of PFTK1 expression by RNA interference was shown to inhibit the migration of glioma cells. Knockdown of PFTK1 increases E-cadherin expression and decreases vimentin expression. These data show that PFTK1 may participate in the pathogenic process of glioma, suggesting that PFTK1 can become a potential therapeutic strategy for gastric cancer.

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.

Expression of G-protein-coupled receptor kinase 6 (GRK6) after acute spinal cord injury in adult rat

Spinal cord injury frequently results in permanent loss of neurological function. It includes many complex molecular and biochemical mechanisms. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors. Some studies reported GRK2 and GRK5 modulate the NFκB pathway in macrophages. Additionally, GRK2 is referred to as regulating activation of spinal cord microglia and GRK6 expression is significantly elevated in most brain regions in the MPTP-lesioned parkinsonian monkeys. However, the expression and function of GRK6 in nervous system lesion and repair are not well understood. In this study, we performed an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed the expression of GRK6 was upregulated significantly at protein level in spinal cord after SCI. Immunohistochemistry and immunofluorescence revealed wide expression of GRK6 in the normal spinal cord. After injury, GRK6 expression was increased predominantly in microglia, which expressed F4/80 (marker of macrophages and activated microglia) strongly. To understand whether GRK6 played a role in microglia activation, we applied lipopolysaccharide (LPS) to induce microglia activation in vitro. Western blot analysis demonstrated up-regulation in GRK6 protein expression after LPS stimulation was time- and dose-dependent and that up-regulation in F4/80 expression was concomitant with GRK6. These data suggested that GRK6 might be involved in the pathophysiology of SCI.

Involvement of early growth response-2 (Egr-2) in lipopolysaccharide-induced neuroinflammation.

Early growth response-2 (Egr-2) protein is a transcription factor, which belongs to Egr family which involve in modulating the peripheral immune response, by means of the induction of differentiation of lymphocyte precursors, activation of T and B cells. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, tissue repair and fibrosis, immune tolerance; however, its regulation and role in central nervous system (CNS) remain poorly understood. In contrast to Egr-1, which has been extensively investigated, the regulation and function of Egr-2 remains less well characterized. To elaborate whether Egr-2 was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. Egr-2 expression was strongly induced in active glia cells (astrocytes and microglias) in inflamed brain cortex. In vitro studies indicated that the upregulation of Egr-2 may be involved in the subsequent glia cellular activation following LPS exposure; and knock down of Egr-2 in primary mixed glial cultures (MGC) by siRNA showed that Egr-2 promoted the synthesis of TNF-α. Collectively, these results suggested Egr-2 may be important in host defense in CNS immune response, which might provide a potential target to the treatment of neuroinflammation.

Up-regulated expression of Bnip3L after intracerebral hemorrhage in adult rats

Bnip3L, also known as NIX, is a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Bnip3 which can bind to Bcl-2 to elaborate that effect. In tumor cells, Bnip3L played a role in tumor growth inhibition, but some studies argued hypoxia-induced autophagy via Bnip3L was a survival mechanism that promoted tumor progression. In heart muscle, it related to decreased myocardial function. However, its function in intracerebral hemorrhage (ICH) is still not clear. In this frame, we found the Bnip3L expression increased in the perihematomal region in adult rats after performed ICH. Double immunofluorenscence staining manifested that Bnip3L co-located with neurons, not astrocytes or oligodendrocytes. Furthermore, we detected that neuronal apoptosis marker active caspase-3 had colocalizations with Bnip3L. In addition, colocalizations and co-immunoprecipitation between Bnip3L and Bcl-2, consistent with previous study, were also found. All our findings suggested that Bnip3L might be involved in the pathophysiology of ICH.