GPX4 degradation via chaperone-mediated autophagy contributes to antimony-triggered neuronal ferroptosis.

Exposure to antimony (Sb), recently identified as a nerve pollutant, can result in neuron damage; but, associated-neurotoxicological mechanisms were still not clear. Herein, we assessed the role of ferroptosis in Sb-mediated neurotoxicity and clarified the underlying mechanism. Following Sb exposure, ferroptosis was significantly promoted in vivo and in vitro. Moreover, following use of ferrostatin-1 (fer-1) to inhibit ferroptosis, Sb-induced ferroptosis in PC12 cells was effectively attenuated. Sb accelerated lysosomal transport and subsequent degradation of glutathione peroxidase 4 (GPX4), resulting in ferroptosis. Furthermore, chaperone-mediated autophagy (CMA) was activated following treatment with Sb, while inhibition of CMA by lysosomal associated protein 2 A (LAMP2A) knockdown attenuated Sb-induced GPX4 degradation. Sb treatment also increased expression of the chaperones heat shock cognate protein 70 (HSC70) and heat shock protein 90 (HSP90) and the lysosome receptor LAMP2A, and increased binding of HSP90, HSC70, and LAMP2A with GPX4 was observed, indicating increased formation of the chaperone-GPX4 complex. Finally, GPX4 overexpression significantly protected PC12 cells from activation of Sb-stimulated ferroptosis and subsequent cytotoxicity. Collectively, our results provide a original mechanism by which Sb triggers neurotoxicity, to concluded that Sb stimulates neuronal ferroptosis through CMA-mediated GPX4 degradation.

Perfluorooctane sulfonate interferes with non-genomic estrogen receptor signaling pathway, inhibits ERK1/2 activation and induces apoptosis in mouse spermatocyte-derived cells.

Perfluorooctane sulfonate (PFOS) is a widespread persistent organic pollutant. Both epidemiological survey and our previous in vivo study have revealed the associations between PFOS exposure and spermatogenesis disorder, while the underlying mechanisms are far from clear. In the present study, GC-2 cells, a mouse spermatocyte-derived cell line, was used to investigate the toxic effects of PFOS and its hypothetical mechanism of action. GC-2 cells were treated with PFOS (0, 50, 100 and 150 µM) for 24 h or 48 h. Results demonstrated that PFOS dose-dependently inhibited cell viability, induced G0/G1 cell cycle arrest and triggered apoptosis, which might be partly explained by the decrease in cyclin D1, PCNA and Bcl-2 protein expression; increase in Bax protein expression; and activation of caspase-9, -3. In addition, PFOS did not directly transactivate or repress estrogen receptors (ERs) in gene reporter assays, whereas the protein levels of both ERα and ERß were significantly altered and the downstream ERK1/2 phosphorylation was inhibited by PFOS. Furthermore, pretreatment with specific ERα agonist PPT (1 µM) significantly attenuated the above PFOS-induced effects while specific ERß agonist DPN (1 µM) accelerated them. These results suggest that PFOS may induce growth inhibition and apoptosis via non-genomic estrogen receptor/ERK1/2 signaling pathway in GC-2 cells, which provides a novel insight regarding the potential role of ERs in mediating PFOS-triggered spermatocyte toxicity.

The effects of antimony on Alzheimer's disease-like pathological changes in mice brain.

We have previously identified antimony (Sb) as a newly nerve poison which leads to neuronal apoptosis. However, the relationship between Sb exposure and Alzheimer's disease (AD) process lacks direct evidence. HE staining and Nissl staining showed significant nerve damage after Sb exposure. Therefore, we further evaluated Sb-associated AD risk by detecting accumulation of ß-amyloid protein (Aß) and neurofibrillary tangles (NFTs) in the brains of mice exposed to Sb for 4 and 8 weeks, and even 1 year. The results showed that dose of 20 mg/kg induced Aß accumulation, but not tau hyperphosphorylation after exposure for 4 week. Eight weeks later, both 10 and 20 mg/kg dramatically triggered Aß accumulation and increased tau phosphorylation at ser199. At the same time, 20 mg/kg could also increase tau phosphorylation at ser396 and number of NFTs. One years later, we found all of AD hallmarks detected in present study showed positive results in the brains of mice exposed to Sb at 10 and 20 mg/kg. In summary, our data provided direct evidence of Sb-associated AD risk, drawing more attention to Sb-triggered neurotoxicity.

Positive regulation of the CREB phosphorylation via JNK-dependent pathway prevents antimony-induced neuronal apoptosis in PC12 cell and mice brain.

Antimony (Sb) is a potentially toxic chemical element abundantly found in the environment. We previously reported that Sb promoted neuronal deathvia reactive oxygen species-dependent autophagy. Here, we assessed the role of cyclic adenosine monophosphate response element-binding protein (CREB) in Sb-induced neuronal damage. We found that Sb treatment induced CREB phosphorylation and neuronal apoptosis both in vitro and in vivo. Interestingly, inhibition of CREB's transcriptional activity with 666-15 dramatically enhanced apoptosis in PC12 cells by downregulating B-cell lymphoma 2 (Bcl-2). Additionally, Sb activated ERK, JNK, and p38 signaling ; however, only JNK promoted CREB phosphorylation. In conclusion, our findings suggest that CREB phosphorylation by JNK attenuates Sb-induced neuronal apoptosis via Bcl-2 upregulation. These data suggest that JNK-dependent CREB activation prevents neurons from Sb-induced apoptosis and guides the development of novel strategies to prevent Sb-induced neurotoxicity.

Akt inhibition-dependent downregulation of the Wnt/ß-Catenin Signaling pathway contributes to antimony-induced neurotoxicity.

Antimony (Sb), as a newly identified nerve poison, can lead to neuronal apoptosis. However, its neurotoxicological mechanisms remain largely unclear. Here, we evaluated the role and regulation of Wnt/ß-catenin pathway in Sb-mediated neurotoxicity. Under Sb treatment, ß-catenin was dramatically downregulated in vivo and in vitro. Moreover, overexpression of ß-catenin effectively attenuated Sb-induced survivin gene expression suppression and subsequent apoptosis in PC12 cells. In addition, Sb stimualted glycogen synthase kinase-3ß (GSK-3ß) activation, shown as decreased phosphorylation levels at Ser 9 both in PC12 cells and mice brain. Paramacological inhibition of GSK-3ß using lithium chloride (LiCl) significantly rescued ß-catenin expression. For upstream pathway analysis, we found Sb treatment decreased protein kinase B (Akt) phosphorylation, and Akt activator protected PC12 cells from GSK-3ß activation and subsequent ß-catenin suppression. In summary, our data provided a novel molecular mechanism of Sb-associated neurotoxicity, namely that Sb induces Wnt/ß-catenin pathway suppression through Akt inhibition, thus resulted in neuronal apoptosis.

p62/SQSTM1 accumulation due to degradation inhibition and transcriptional activation plays a critical role in silica nanoparticle-induced airway inflammation via NF-κB activation.

BACKGROUND: Most nanoparticles (NPs) reportedly block autophagic flux, thereby upregulating p62/SQSTM1 through degradation inhibition. p62 also acts as a multifunctional scaffold protein with multiple domains, and is involved in various cellular processes. However, the autophagy substrate-independent role of p62 and its regulation at the transcriptional level upon NPs exposure remain unclear. RESULTS: In this work, we exposed BEAS-2b cells and mice to silica nanoparticles (SiNPs), and found that SiNPs increased p62 protein levels in vivo and vitro. Then, we further explored the role and mechanism of SiNPs-stimulated p62 in vitro, and found that p62 degradation was inhibited due to autophagic flux blockade. Mechanistically, SiNPs blocked autophagic flux through impairment of lysosomal capacity rather than defective autophagosome fusion with lysosomes. Moreover, SiNPs stimulated translocation of NF-E2-related factor 2 (Nrf2) to the nucleus from the cytoplasm, which upregulated p62 transcriptional activation through direct binding of Nrf2 to the p62 promoter. Nrf2 siRNA dramatically reduced both the mRNA and protein levels of p62. These two mechanisms led to p62 protein accumulation, thus increasing interleukin (IL)-1 and IL-6 expression. SiNPs activated nuclear factor kappa B (NF-κB), and this effect could be alleviated by p62 knockdown. CONCLUSION: SiNPs caused accumulation of p62 through both pre- and post-translational mechanisms, resulting in airway inflammation. These findings improve our understanding of SiNP-induced pulmonary damage and the molecular targets available to mitigate it.

PKA- and Ca2+-dependent p38 MAPK/CREB activation protects against manganese-mediated neuronal apoptosis.

Although manganese (Mn) is an essential trace element, its excessive consumption may lead to neuronal death and neurodegenerative disorders. Human cells launch adaptive responses to attenuate Mn-induced neurotoxicity. However, the regulation of the responsive proteins and their function during Mn-stimulated neurotoxicity remain largely unknown. We report the role of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in Mn-induced neuronal apoptosis. Mn increased CREB phosphorylation and cellular apoptosis in both PC12 cells and mouse brain tissue. Furthermore, downregulation of CREB with shRNA plasmid transfection significantly worsened the PC12 cell apoptosis by decreasing mRNA and protein expression of brain-derived neurotrophic factor (BDNF). Moreover, Mn enhanced protein kinase A (PKA) activation and activation of the p38 MAPK and JNK pathways. Inhibition of p38 MAPK rather than JNK effectively reduced the CREB phosphorylation. Subsequent analysis showed that a PKA inhibitor blocked p38 MAPK and CREB phosphorylation. Moreover, the intracellular Ca2+ chelator BAPTA-AM decreased the phosphorylation of p38 MAPK and CREB but failed to reduce PKA activation. In summary, p38 MAPK/CREB activation via PKA activation and increased cellular Ca2+ helped to alleviate Mn-induced neuronal apoptosis via BDNF regulation. These findings improve our understanding of Mn-induced neurotoxicity and the molecular targets to antagonise it.

SIRT1 downregulation mediated Manganese-induced neuronal apoptosis through activation of FOXO3a-Bim/PUMA axis.

Manganese (Mn) is an essential trace element. Excessive exposure to Mn may lead to neuronal death and neurodegenerative disorders. Accumulating evidence has shown that silent mating type information regulation 2 homolog 1 (SIRT1) plays a vital role in brain damage. However, whether aberrant SIRT1 levels contribute to Mn-induced neurotoxicity remains unknown. In this study, we report the important role of SIRT1 downregulation during Mn-induced neuronal apoptosis. Mn was found to downregulate SIRT1 protein levels in the rat pheochromocytoma (PC12) cells and mouse brain tissues. Mn enhanced SIRT1 protein degradation and downregulated its gene expression. Furthermore, Mn induced cell apoptosis in a dose-dependent manner both in vitro and in vivo, and resulted in an increase in forkhead box O (FOXO) 3a expression and acetylation. SIRT1 activation by resveratrol clearly attenuated Mn-triggered apoptosis and FOXO3a activation. Mn markedly increased the expression of Bcl-2 interacting mediator of cell death (Bim) and p53-up-regulated modulator of apoptosis (PUMA), whereas downregulation of FOXO3a significantly inhibited their upregulation and subsequent apoptosis. In summary, we determined that Mn downregulated SIRT1 by multiple mechanisms, thus led to apoptosis via activation of the FOXO3a-Bim/PUMA axis in PC12 cells. These findings on the impact of Mn on SIRT1 may lead to an improved understanding of Mn-induced neurotoxicity and provide a molecular target to antagonise Mn-associated neuronal damage.

Manganese exposure facilitates microglial JAK2-STAT3 signaling and consequent secretion of TNF-a and IL-1ß to promote neuronal death.

Chronic manganese (Mn) exposure can lead to neuroinflammation and neurological deficit, which resemble idiopathic Parkinson's disease (IPD). However, the precise mechanisms underlying Mn exposure-induced neurotoxicity remain incompletely understood. Microglia can become hyperactivated and plays a vital role in neuroinflammation and consequent neurodegeneration in response to pro-inflammatory stimuli. In the present study, we found that HAPI microglial cells exhibited increased secretion of pro-inflammatory TNF-α and IL-1ß following Mn exposure in dose- and time-dependent manners. In addition, we showed that Mn exposure could trigger the activation of JAK2/STAT3 signaling pathway in microglia. Notably, Mn-induced secretion of TNF-α and IL-1ß was significantly attenuated by the treatment of JAK2 inhibitor. Finally, through incubating PC12 neuronal cells with Mn-treated microglial conditioned medium, we demonstrated that Mn-induced secretion of microglial TNF-α and IL-1ß facilitated neuronal apoptosis. Thus, we speculate that Mn exposure might trigger JAK2-STAT3 signal pathway in microglia, leading to resultant neuroinflammation and neuronal loss.

Downregulation of Mfn2 participates in manganese-induced neuronal apoptosis in rat striatum and PC12 cells.

Manganese (Mn) is a widely distributed trace element that is essential for normal brain function and development. However, chronic exposure to excessive Mn has been known to lead to neuronal loss and manganism, a disease with debilitating motor and cognitive deficits, whose clinical syndrome resembling idiopathic Parkinson's disease (IPD). However, the precise molecular mechanism underlying Mn neurotoxicity remains largely unclear. Accumulating evidence indicates that abnormal mitochondrial functionality is an early and causal event in Mn-induced neurodegeneration and apoptosis. Here, we investigated whether Mitofusin 2 (Mfn2), a highly conserved dynamin-related protein (DRP), played a role in the regulation of Mn-induced neuronal apoptosis. We revealed that Mfn2 was significantly dysregulated in rat striatum and PC12 neuronal-like cells following Mn exposure. Western blot analysis revealed that the expression of Mfn2 was remarkably decreased following different concentrations of Mn exposure. Immunohistochemistry analysis confirmed a remarkable downregulation of Mfn2 in rat striatum after Mn exposure. Immunofluorescent staining showed that Mfn2 was expressed predominantly in neurons, and neuronal loss of Mfn2 was associated with the expression of active caspase-3 following Mn exposure. Importantly, overexpression of Mfn2 apparently attenuated Mn-induced neuronal apoptosis. Notably, treatment with caspase-3 inhibitor Ac-DEVD-CH could not rescue Mn-induced downregulation of Mfn2, suggesting that Mn-induced mfn2 occurs prior to neuronal apoptosis. Taken together, these results indicated that down-regulated expression of Mfn2 might contribute to the pathological processes underlying Mn neurotoxicity.

Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice.

Perfluorooctane sulfonate (PFOS, CAS#1763-23-1) causes male reproductive toxicities, but the underlying mechanisms are still unclear. In this study, 0, 0.5 and 10mg/kg/day PFOS were given by oral gavage to adult mice for 5 weeks. In the 10mg/kg group, serum testosterone levels decreased significantly. Sperm counts declined which might be associated with the decreased proliferation and increased apoptosis of germ cells. In relation to increased apoptosis, bax, cleaved caspase-9 and cleaved caspase-3 levels elevated significantly, indicating that PFOS induced germ cell apoptosis by activating the mitochondrial pathway. In addition, the increase in levels of testicular estrogen receptor (ER) ß was observed in both 0.5 and 10mg/kg group, whereas a decrease in ERα expression was only observed in 10mg/kg group. These results suggested that the alterations in testicular ERs expression, together with decreased proliferation and increased apoptosis of germ cells, might be involved in PFOS-induced testicular toxicity.

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. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1068-1079, 2016.

Zinc deficiency impairs the renewal of hippocampal neural stem cells in adult rats: involvement of FoxO3a activation and downstream p27(kip1) expression.

Zinc plays an important role in the development and maintenance of central neural system. Zinc deficiency has been known to alter normal brain function, whose molecular mechanism remains largely elusive. In the present study, we established a zinc deficiency-exposed rat model, and, using western blot and immunohistochemical analyses, found that the expression of FoxO3a and p27(kip1) was remarkably up-regulated in the rat brain hippocampus. Immunofluorescence assay showed that FOXO3a and p27(kip1) were significantly co-localized with nestin, the marker of neural stem cells (NSCs). Furthermore, we identified that the proportion of proliferating NSCs was markedly decreased in zinc-deficient rat hippocampaus. Using C17.2 neural stem cells, it was revealed that exposure to zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethy) ethylenediamine induced the expression of FoxO3a and p27(kip1) , which coincided with reduced NSC proliferation. Furthermore, depletion of FoxO3a inhibited p27(kip1) expression and restored the growth of NSCs. On the basis of these data, we concluded that FoxO3a/p27(kip1) signaling might play a significant role in zinc deficiency-induced growth impairment of NSCs and consequent neurological disorders. We describe here that zinc deficiency induces the proliferative impairment of hippocampal neural stem cells partially through the activation of FOXO3a-p27 axis in rats. Neural progenitor cells exhibited significantly up-regulated expression of FOXO3a and p27 after zinc deficiency in vivo and in vitro. Depletion of FOXO3a ameliorates zinc deficiency-induced expression of p27 and growth impairment of neural stem cells. We provide novel insight into the mechanisms underlying zinc deficiency-induced neurological deficits.

Critical Role of TAK1-Dependent Nuclear Factor-κB Signaling in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Astrocyte Activation and Subsequent Neuronal Death.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been recently shown to elicit inflammatory response in a number of cell-types. However, whether TCDD could provoke inflammation in astrocytes, the most abundant glial cells in central nervous system (CNS), remains virtually unknown. In the present study, we showed that TCDD exposure could induce evident astrocyte activation both in vivo and in vitro. Further, we found that TGF-ß-activated kinase 1 (TAK1), a critical regulator of NF-κB signaling, was rapidly phosphorylated in the process of TCDD-induced reactive astroglia. Exposure to TCDD led to rapid TAK1 and NF-κB p65 phosphorylation, as well as IKBα degradation. Moreover, blockage of TAK1 using siRNA oligos or TAK1 inhibitor 5Z-7-oxozeaenol significantly attenuated TCDD-induced astrocyte activation as well as the release of TNF-α. Finally, we showed that the conditioned medium of TCDD-treated astrocytes promoted the apoptosis of PC12 neuronal cells, which could be blocked with the pre-treatment of TAK1 inhibitor. Taken together, these findings suggested that TCDD could promote the inflammatory activation of astrocytes through modulating TAK1-NF-κB cascade, implicating that reactive astrocytes might contribute to TCDD-induced adverse effects on CNS system.

The downregulation of Wnt/ß-catenin signaling pathway is associated with zinc deficiency-induced proliferative deficit of C17.2 neural stem cells.

Zinc is an essential nutrient that is important for normal brain development. Zinc deficiency has been linked to aberrant neurological development and functioning. However, the molecular mechanisms underlying Zinc deficiency-induced neurological disorders remain largely elusive. In the present study, we showed that the proliferation of C17.2 neural stem cells (NSCs) was evidently impaired after exposed to low levels of Zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethy) ethylenediamine (TPEN). In addition, we found that TPEN-induced proliferative deficit of NSCs was related with significant downregulation of Wnt/ß-catenin signaling. Zinc deficiency impaired the proliferation of neural stem cells in dose- and time-dependent manners. Western blot revealed that the levels of p-Ser9-glycogensynthase kinase-3ß (p-GSK-3ß) and ß-catenin were remarkably downregulated during TPEN-induced C17.2 proliferative impairment. Moreover, immunofluorescent analysis indicated that the level of nuclear ß-catenin was apparently decreased following TPEN exposure. Furthermore, application with GSK-3ß inhibitor lithium chloride (LiCl) reversed TPEN-induced downregulation of ß-catenin and impairment of cell proliferation. Flow cytometry analysis also showed that TPEN-induced impairment of NSC proliferation could be reversed by LiCl. Taken together, these findings suggested that the disturbance of canonical Wnt/ß-catenin signaling pathway partially accounted for Zinc deficiency-induced proliferative impairment of NSCs.

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/ß-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 ß-galactosidase (SA-ß-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)/ß-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/ß-catenin signaling pathway, ameliorated the effect of TCDD on cellular ß-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/ß-catenin and ROS-dependent mechanisms.

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/ß-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.

Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis.

Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-µ (PFT-µ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-µ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H2O2 production. Finally, we observed that pretreatment with PFT-α and PFT-µ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death.

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.