Junkang Jiang

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.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin induces premature senescence of astrocytes via WNT/β-catenin signaling and ROS production

2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental contaminant that could exert significant neurotoxicity in the human nervous system. Nevertheless, the molecular mechanism underlying TCDD‐mediated neurotoxicity has not been clarified clearly. Herein, we investigated the potential role of TCDD in facilitating premature senescence in astrocytes and the underlying molecular mechanisms. Using the senescence‐associated β‐galactosidase (SA‐β‐Gal) assay, we demonstrated that TCDD exposure triggered significant premature senescence of astrocyte cells, which was accompanied by a marked activation of the Wingless and int (WNT)/β‐catenin signaling pathway. In addition, TCDD altered the expression of senescence marker proteins, such as p16, p21 and GFAP, which together have been reported to be upregulated in aging astrocytes, in both dose‐ and time‐dependent manners. Further, TCDD led to cell‐cycle arrest, F‐actin reorganization and the accumulation of cellular reactive oxygen species (ROS). Moreover, the ROS scavenger N‐acetylcysteine (NAC) markedly attenuated TCDD‐induced ROS production, cellular oxidative damage and astrocyte senescence. Notably, the application of XAV939, an inhibitor of WNT/β‐catenin signaling pathway, ameliorated the effect of TCDD on cellular β‐catenin level, ROS production, cellular oxidative damage and premature senescence in astrocytes. In summary, our findings indicated that TCDD might induce astrocyte senescence via WNT/β‐catenin and ROS‐dependent mechanisms. Copyright

Upregulation of mitochondrial protease HtrA2/Omi contributes to manganese-induced neuronal apoptosis in rat brain striatum.

Manganese (Mn) is an essential trace element that is required for normal brain functioning. However, excessive intake of Mn has been known to lead to neuronal loss and clinical symptoms resembling idiopathic Parkinsons disease (IPD), whose precise molecular mechanism remains largely elusive. In the study, we established a Mn-exposed rat model and identified a mitochondrial protease, the mature form of high temperature requirement A2 (HtrA2/Omi), which was significantly upregulated in rat brain striatum after Mn exposure. Western blot and immunohistochemical analyses revealed that the expression of mature HtrA2 was remarkably increased following Mn exposure. In addition, immunofluorescence assay demonstrated that overexposure to Mn could lead to significant elevation in the number of HtrA2-positive neurons. Accordingly, the expression of X-linked inhibitor of apoptosis protein (XIAP), a well-characterized target of HtrA2-mediated proteolysis, was progressively decreased following Mn exposure, and was correlated with increased level of active caspase-3. Further, we showed that Mn exposure decreased the viability and induced apparent apoptosis of NFG-differentiated PC12 cells. Importantly, the expression of HtrA2 was progressively increased, whereas the level of cellular XIAP was reduced during Mn-induced apoptosis. In addition, blockage of HtrA2 activity with UCF-101 restored Mn-induced reduction in XIAP expression. Finally, we observed that UCF-101 treatment ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings suggested that upregulated HtrA2 played a role in Mn-induced neuronal death in brain striatum.

KHSRP Participates in Manganese-Induced Neurotoxicity in Rat Striatum and PC12 Cells

Manganese (Mn) is an essential micronutrient. However, exposure to high doses of Mn may lead to a neurological disease known as manganism, which is characterized by marked brain neuronal loss. K-homology splicing regulator protein (KHSRP) is a multifunctional RNA-binding protein and has been implicated in the regulation of multiple cellular signaling associated with neuronal apoptosis and survival, such as p38 mitogen-activated protein kinase (MAPK), nuclear factor kappaB (NF-κB), and Wnt/β-catenin pathways. In the present study, the role of KHSRP in Mn-induced neurotoxicity was investigated in vivo using a rat model of chronic Mn exposure and in vitro using differentiated PC12 cell cultures. Western blot and immunohistochemical analyses showed a significant upregulation of KHSRP in rat striatum following Mn exposure. Immunofluorescent labeling indicated that KHSRP was localized mainly in neurons. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end labeling (TUNEL) assay showed that KHSRP was mainly distributed in apoptotic neurons. Increased KHSRP expression was positively correlated with the upregulation of several apoptosis-related proteins, such as p53, bax, and active caspase-3. In addition, significant co-localization of KHSRP and active caspase-3 in neurons after Mn exposure was also observed, suggesting a potential involvement of KHSRP in the regulation of Mn-induced striatal neuronal apoptosis. Importantly, interference with KHSRP apparently decreased the level of p53 and attenuated Mn-induced neuronal apoptosis. Taken together, these results indicate that upregulation of KHSRP may be involved in the pathological process underlying Mn neurotoxicity via the modulation of p53 signaling.

Downregulation of the Wnt/β-catenin signaling pathway is involved in manganese-induced neurotoxicity in rat striatum and PC12 cells

Manganese (Mn) is an essential trace element. However, exposure to excessive Mn may cause neurodegenerative disorders called manganism. Accumulating evidence indicated that dysregulation of Wnt/β‐catenin signaling was tightly associated with the onset of neurodegenerative disorders. However, whether aberrant Wnt/β‐catenin signaling contributes to Mn‐induced neurotoxicity remains unknown. The present study investigates the involvement of Wnt/β‐catenin signaling in Mn‐induced neurotoxicity. Western blot and immunohistochemistry analyses showed a remarkable downregulation of p‐Ser9‐glycogen synthase kinase‐3β (GSK‐3β) and β‐catenin in rat striatum after Mn exposure. TUNEL assay revealed significant neuronal apoptosis following treatment with 25 mg/kg Mn. Immunofluorescent staining showed that β‐catenin was expressed predominantly in neurons, and colocalization of β‐catenin and active caspase‐3 was observed after Mn exposure. Furthermore, Mn exposure resulted in PC12 cells apoptosis, which was accompanied by reduced levels of cellular β‐catenin and p‐GSK‐3β. Accordingly, the mRNA level of the prosurvival factor survivin, a downstream target gene of β‐catenin, was synchronously decreased. More importantly, blockage of GSK‐3β activity with the GSK‐3β inhibitor lithium chloride could attenuate Mn‐induced downregulation of β‐catenin and survivin as well as neuronal apoptosis. Overall, the present study demonstrates that downregulation of Wnt/β‐catenin signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes astrocyte activation and the secretion of tumor necrosis factor-α via PKC/SSeCKS-dependent mechanisms.

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro‐inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src‐Suppressed‐C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)‐mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC‐induced SSeCKS activation in TCDD‐triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD‐induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho‐p65 expression, and tumor necrosis factor‐α secretion in astrocytes. In addition, pre‐treatment with PKC inhibitor also attenuated TCDD‐induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD‐triggered tumor necrosis factor‐α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS‐dependent mechanisms, highlighting that astrocytes might be important target of TCDD‐induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces expression of p27kip1 and FoxO3a in female rat cerebral cortex and PC12 cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27(kip1), in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27(kip1) in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27(kip1) were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27(kip1) expression. Furthermore, knockdown of FoxO3a expression inhibited p27(kip1) transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity.

Autophagy potentially protects against 2,3,7,8‐tetrachlorodibenzo‐p‐Dioxin induced apoptosis in SH‐SY5Y cells

The environmental toxicant TCDD may elicit cytotoxic effects by inducing reactive oxygen species (ROS) generation. Autophagy is one of the first lines of defense against oxidative stress damage. Herein, we investigated whether autophagy played a regulatory role in TCDD‐induced neurotoxicity. Here, we showed that TCDD exposure caused marked autophagy in SH‐SY5Y cells, whose dose range was close to that inducing apoptosis. Electron microscopic and Western blot analyses revealed that TCDD induced autophagy at a starting dose of approximate 100 nM. Interestingly, 100–200 nM TCDD exposure resulted in obviously decreased cell viability and evident apoptotic phenotype. Furthermore, the levels of pro‐apoptotic molecules, Bax and cleaved‐PARP, increased significantly, whereas Bcl2 declined after exposed to 100 nM TCDD. In addition, the apoptosis was verified using flow cytometrical analysis. These data strongly suggested that TCDD induced both autophagy and apoptosis at a similar dose range in SH‐SY5Y cells. Interestingly, pretreatment with ROS scavenger, N‐acetyl‐cysteine (NAC), could effectively block both TCDD‐induced apoptosis and autophagy. More surprisingly, inhibition of autophagy with 3‐methyladenine (3MA), remarkably augmented TCDD‐induced apoptosis. The findings implicated that the onset of autophagy might serve as a protective mechanism to ameliorate ROS‐triggered cytotoxic effects in human SH‐SY5Y neuronal cells under TCDD exposure.

Role of mitogen-activated protein kinase cascades in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptosis in neuronal pheochromocytoma cells.

Mitogen-activated protein kinases (MAPKs) are involved in neuronal death caused by many cytotoxins. Conventional MAPKs consist of three family members: extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38. It has been originally shown that ERK1/2 is important for cell survival, whereas JNK and p38 are deemed stress responsive and thus involved in apoptosis. However, information describing the role of MAPKs in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced neurotoxicity is insufficient. The aim of this study was to identify the role of MAPK cascades in TCDD-induced neurotoxicity using differentiated pheochromocytoma (PC12) cells as a model for neuronal cells. Cell viability assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and flow cytometry analysis showed that TCDD attenuated cell viability with a dose- and time-dependent manner and significantly induced apoptosis in primary cortical neurons and PC12 cells. Western blot analysis indicated that TCDD markedly activated the expression of ERK1/2, JNK and p38 in TCDD-treated PC12 cells. Furthermore, PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor) notably blocked the effect of TCDD on cell apoptosis. Based on the findings above, it is concluded that the activation of MAPK signaling pathways may be associated with TCDD-mediated neuronal apoptosis.

Activation of neuronal nitric oxide synthase (nNOS) signaling pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced neurotoxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been reported to cause alterations in cognitive and motor behavior during both development and adulthood. In this study, the neuronal nitric oxide synthase (nNOS) signaling pathway was investigated in differentiated pheochromocytoma (PC12) cells to better understand the mechanisms of TCDD-induced neurotoxicity. TCDD exposure induced a time- and dose-dependent increase in nNOS expression. High levels of nitric oxide (NO) production by nNOS activation induced mitochondrial cytochrome c (Cyt-c) release and down-regulation of Bcl-2. Additionally, TCDD increased the expression of active caspase-3 and significantly led to apoptosis in PC12 cells. However, these effects above could be effectively inhibited by the addition of 7-nitroindazole (7-NI), a highly selective nNOS inhibitor. Moreover, in the brain cortex of Sprague-Dawley (SD) rats, nNOS was also found to have certain relationship with TCDD-induced neuronal apoptosis. Together, our findings establish a role for nNOS as an enhancer of TCDD-induced apoptosis in PC12 cells.