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The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.

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.

Homer, also designated Vesl, is one member of the newly found postsynaptic density scaffold proteins, playing a vital role in maintaining synaptic integrity, regulating intracellular calcium mobilization, and being critical for the regulation of cellular apoptosis. However, its function in the inflamed central nervous system (CNS) is not fully elucidated. Here, we investigated the role of Homer1b/c, a long form of Homer1, in lipopolysaccharide (LPS) induced neuroinflammation in CNS. Western blot analysis indicated that LPS administration significantly increased the expression of Homer1b/c in rat brain. Moreover, double immunofluorescent staining suggested Homer1b/c was mainly distributed in the cytoplasm of neurons and had a close association with cleaved caspase-3 level in neurons in rat brain after LPS injection. In vitro studies indicated that up-regulation of Homer1b/c might be related to the subsequent apoptosis in neurons treated by conditioned media (CM), collected from LPS-stimulated mixed glial cultures (MGC). We also found down-regulation of Homer1b/c partly blocked the increase of cleaved caspase-3 and the proportion of Bax/Bcl-2 in neurons induced by MGC-CM. Taken together, these findings suggested that Homer1b/c might promote neuronal apoptosis via the Bax/Bcl-2 dependent pathway during neuroinflammation in CNS, and inhibiting Homer1b/c expression might provide a novel neuroprotective strategy against the inflammation-related neuronal apoptosis.

Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins involved in the regulation of actin filaments, was recently reported to play a role in the pathology of sciatic nerves injury. However, the distribution and function of CAP1 in the central nervous system (CNS) remain unclear. To investigate whether CAP1 is involved in CNS injury and repair, we used an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of CAP1 in ipsilateral peritrauma cortex compared with the contralateral and sham-operated ones. Double immunofluorescence staining showed that CAP1 was co-expressed with glial fibrillary acidic protein (GFAP). In addition, we detected that Ki-67 had colocalization with GFAP and CAP1 after TBI. In vitro, during the process of lipopolysaccharide (LPS)-induced primary astrocyte proliferation, we observed enhanced expression of CAP1. Specially, CAP1-specific siRNA-transfected primary astrocytes show significantly decreased ability for proliferation. Together, all these data indicated that the change of CAP1 protein expression was associated with astrocyte proliferation after the trauma of the central nervous system (CNS).

Podoplanin (PDPN) is a mucin-type transmembrane sialoglycoprotein expressed in multiple tissues in adult animals, including the brain, lungs, kidney, and lymphoid organs. Studies of this molecule have demonstrated its great importance in tumor metastasis, platelet aggregation, and lymphatic vessel formation. However, information regarding its regulation and possible function in the central nervous system is still limited. In this study, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats and detected increased expression of PDPN in the brain cortex. Immunofluorescence indicated that PDPN was located in the neurons, but not astrocytes. Moreover, there was a concomitant up-regulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, the expression of these three proteins in cortical primary neurons was decreased after knocking down PDPN by siRNA. Collectively, all these results suggested that the up-regulation of PDPN might be involved in neuronal apoptosis in neuroinflammation after LPS injection.

Krüppel-like zinc-finger transcription factor 5 (KLF5), known as BTEB2 or IKLF, has several biological functions that involve cell proliferation, development and apoptosis. Previous studies demonstrated that BTEB2 had anti-apoptotic effect in multiple diseases such as esophageal cancer and non-small cell lung cancers (NSCLCs). However, the distribution and function of BTEB2 in CNS diseases remain unknown. In this study, we show that BTEB2 down-regulates neuronal apoptosis during pathophysiological processes of intracerebral hemorrhage (ICH). A rat ICH model was established by behavioral tests. Western blot and immunohistochemistry revealed a remarkable up-regulation of BTEB2 expression surrounding the hematoma after ICH. Double-labeled immunofluorescence showed BTEB2 was mostly co-localized with neurons, rarely with activated astrocytes and microglia. Furthermore, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with BTEB2. In addition, KLF5 knockdown in vitro specifically resulted in increasing neuronal apoptosis coupled with reduced Bad phosphorylation at both ser112 and ser136 residues. All our findings suggested that BTEB2 down-regulated neuronal apoptosis via promoting Bad phosphorylation after ICH. Electronic supplementary material The online version of this article (doi:10.1007/s12031-014-0305-8) contains supplementary material, which is available to authorized users.

To investigate whether Nischarin participated in neuronal apoptosis induced by neuroinflammation and via the phosphatidylinositol 3-kinase (PI3K) and PKB-dependent pathway. Use of male Sprague–Dawley rats, rat pheochromocytoma (PC12), and murine microglial cells (BV-2). Treatment lipopolysaccharides (LPS) were injected into the brain lateral ventricle of the rat. The BV-2 cells were treated by LPS. The PC12 cells were pretreated by or not pretreated by conditioned media and siRNA. Western blotting was used for analyzing the expression level of Nischarin, pAKT, BAD and Bcl-2. Immunohistochemistry and immunofluorescence were used to perform the morphology and localization of Nischarin. The siRNA could down-regulate the protein level of endogenous Nischarin. The expression level of Nischarin was elevated after LPS injection; meanwhile, Nischarin was located in the neuron. Nischarin was involved in regulating the PI3K/PKB patway. Nischarin might be involved in mediating the process of PI3K/PKB pathway-dependent neuronal apoptosis. After the silencing of Nischarin in cultured PC12 (pheochromocytoma) by siRNA, these results showed that it would induce a reduction of pAKT and Bcl-2 proteins expression; meanwhile, it induces an increase of BAD and active caspase-3.

Intrahepatic cholestasis of pregnancy (ICP) is associated with increased perinatal mortality and morbidity. Circulating cell-free fetal DNA has been a useful parameter for monitoring of pregnancy-associated diseases. The purpose of this study was to determine the concentrations of hypermethylated RAS-association domain family 1, isoform A (RASSF1A) gene sequences in the plasma of pregnant women with intrahepatic cholestasis. This study included 56 women in their third trimester of pregnancy, of whom 26 had ICP (study group) and 30 were healthy (control group). Real time PCR was performed to detect RASSF1A concentrations after methylation-sensitive restriction digestion with HinpII and HhaI to measure cell-free fetal DNA. Beta-actin was detected as an internal control to confirm complete enzyme digestion. The data show a significant increase in the circulating hypermethylated RASSF1A levels regarding the pregnancies complicated with ICP as compared with normal pregnancies. Circulating hypermethylated RASSF1A levels in maternal plasma related to total bile acid. Based on these observations, we suggest that the circulating hypermethylated RASSF1A levels in maternal plasma may be used as a diagnostic marker for ICP.

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.

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.

Previous studies have suggested that bone morphogenetic protein-6 (BMP-6) has a pronounced upregulation in rat brains subjected to traumatic brain injury. Bone morphogenetic protein-10 (BMP-10) is a newly identified cardiac-specific peptide growth factor that belongs to the TGF-β superfamily. To elucidate the dynamic expression changes and cellular localization of BMP-10 during traumatic brain injury (TBI), we performed an acute traumatic brain injury model in adult rats. Western blot analysis, immunohistochemistry and RTPCR revealed that BMP-10 expression in impaired cerebral cortex was more strongly induced not only at protein level but also at mRNA level compared to that in normal group. Double immunofluorescence labeling suggested that BMP-10 was localized mainly in the cytoplasm of neurons, microglias, and astrocytes within 3 mm from the lesion site at day 3 post-injury. And there was a specific upregulation of BMP-10 in astrocytes following brain injury. Besides, co-localization of BMP-10 and proliferating cell nuclear antigen (PCNA) was detected in Glial fibrillary acidic protein (GFAP) (+) cells. We also examined the expression profiles of PCNA and GFAP whose change was correlated with the expression profiles of BMP-10 in the incised injury model used here. Another experiment in which astrocytes were treated with BMP-10 was also performed to confirm the relationship between the upregulation of BMP-10 and proliferation of astrocytes following TBI. Taken together, this is the first description of BMP-10 expression during the central nervous system (CNS) lesion and repair. Thus, the present data suggested that BMP-10 may be implicated in CNS pathophysiology after TBI. But, further studies are needed to understand the cell signal pathway which can direct the exact role of BMP-10 following 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.

Numblike (Numbl) plays an important role in ependymal wall integrity and subventricular zone neuroblast survival. And Numbl is specifically expressed in the brain. 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 Numbl expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Numbl was present in normal brain. It gradually decreased, reached the lowest point at day 3 after TBI, and then increased during the following days. Double immunofluorescence staining showed that Numbl immunoreactivity 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. All these results suggested that Numbl may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.

Cultured neural stem cells (NSCs) provide a powerful means for investigating central nervous system disease, neuron development, differentiation, and regeneration. To obtain sufficient neurospheres, subculturing is essential following establishment of the primary NSC culture. Passaging the primary neurospheres is a key issue that is often ignored. We evaluated the influence of different passaging schedules on primary cultured NSCs. Passaging was performed on day 5, 7 or 9. We observed more neurospheres with diameters of 200-250 μm on day 7 than on day 5 or 9. Prolonging the time of primary culture reduced the cell metabolic activity by the MTT assay and cell proliferation by colony-forming assay and the differentiation to neurons from cells at P2 and later decreased. Additionally, more cells were in G0/G1 phase, and higher expression of p16 ( INK4a ) and lower expression of cyclin D1 was found when the time of primary culture was prolonged to 9 days compared to 7-days cultures. Thus, in this study, we established that the optimal time for subculturing aggregated NSCs was on day 7 based on the primary culture.