Tat‐antioxidant 1 protects against stress‐induced hippocampal HT‐22 cells death and attenuate ischaemic insult in animal model

Oxidative stress‐induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat‐Atox1 and examined the roles of Tat‐Atox1 in oxidative stress‐induced hippocampal HT‐22 cell death and an ischaemic injury animal model. Tat‐Atox1 effectively transduced into HT‐22 cells and it protected cells against the effects of hydrogen peroxide (H₂O₂)‐induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat‐Atox1 regulated cellular survival signalling such as p53, Bad/Bcl‐2, Akt and mitogen‐activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat‐Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat‐Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat‐Atox1 protects against oxidative stress‐induced HT‐22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat‐Atox1 has potential as a therapeutic agent for the treatment of oxidative stress‐induced ischaemic damage.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Protective effects of astaxanthin on 6‐hydroxydopamine‐induced apoptosis in human neuroblastoma SH‐SY5Y cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although understanding of the pathogenesis of PD remains incomplete, increasing evidence from human and animal studies has suggested that oxidative stress is an important mediator in its pathogenesis. Astaxanthin (Asx), a potent antioxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress‐related diseases. This study examined the protective effects of Asx on 6‐hydroxydopamine (6‐OHDA)‐induced apoptosis in the human neuroblastoma cell line SH‐SY5Y. Pre‐treatment of SH‐SY5Y cells with Asx suppressed 6‐OHDA‐induced apoptosis in a dose‐dependent manner. In addition, Asx strikingly inhibited 6‐OHDA‐induced mitochondrial dysfunctions, including lowered membrane potential and the cleavage of caspase 9, caspase 3, and poly(ADP‐ribose) polymerase. In western blot analysis, 6‐OHDA activated p38 MAPK, c‐jun NH₂‐terminal kinase 1/2, and extracellular signal‐regulated kinase 1/2, while Asx blocked the phosphorylation of p38 MAPK but not c‐jun NH₂‐terminal kinase 1/2 and extracellular signal‐regulated kinase 1/2. Pharmacological approaches showed that the activation of p38 MAPK has a critical role in 6‐OHDA‐induced mitochondrial dysfunctions and apoptosis. Furthermore, Asx markedly abolished 6‐OHDA‐induced reactive oxygen species generation, which resulted in the blockade of p38 MAPK activation and apoptosis induced by 6‐OHDA treatment. Taken together, the present results indicated that the protective effects of Asx on apoptosis in SH‐SY5Y cells may be, at least in part, attributable to the its potent antioxidative ability.     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states

Background

Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results

Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H₂O₂ or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1^S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion

Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.     

Mas receptor mediates cardioprotection of angiotensin‐(1‐7) against Angiotensin II‐induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress

Angiotensin II (Ang II) plays an important role in the onset and development of cardiac remodelling associated with changes of autophagy. Angiotensin1‐7 [Ang‐(1‐7)] is a newly established bioactive peptide of renin–angiotensin system, which has been shown to counteract the deleterious effects of Ang II. However, the precise impact of Ang‐(1‐7) on Ang II‐induced cardiomyocyte autophagy remained essentially elusive. The aim of the present study was to examine if Ang‐(1‐7) inhibits Ang II‐induced autophagy and the underlying mechanism involved. Cultured neonatal rat cardiomyocytes were exposed to Ang II for 48 hrs while mice were infused with Ang II for 4 weeks to induce models of cardiac hypertrophy in vitro and in vivo. LC3b‐II and p62, markers of autophagy, expression were significantly elevated in cardiomyocytes, suggesting the presence of autophagy accompanying cardiac hypertrophy in response to Ang II treatment. Besides, Ang II induced oxidative stress, manifesting as an increase in malondialdehyde production and a decrease in superoxide dismutase activity. Ang‐(1‐7) significantly retarded hypertrophy, autophagy and oxidative stress in the heart. Furthermore, a role of Mas receptor in Ang‐(1‐7)‐mediated action was assessed using A779 peptide, a selective Mas receptor antagonist. The beneficial responses of Ang‐(1‐7) on cardiac remodelling, autophagy and oxidative stress were mitigated by A779. Taken together, these result indicated that Mas receptor mediates cardioprotection of angiotensin‐(1‐7) against Ang II‐induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Seizure‐induced impairment in neuronal ketogenesis: Role of zinc‐α2‐glycoprotein in mitochondria

Ketone bodies (KBs) were known to suppress seizure. Untraditionally, neurons were recently reported to utilize fatty acids and produce KBs, but the effect of seizure on neuronal ketogenesis has not been researched. Zinc‐α2‐glycoprotein (ZAG) was reported to suppress seizure via unclear mechanism. Interestingly, ZAG was involved in fatty acid β‐oxidation and thus may exert anti‐epileptic effect by promoting ketogenesis. However, this promotive effect of ZAG on neuronal ketogenesis has not been clarified. In this study, we performed immunoprecipitation and mass spectrometry to identify potential interaction partners with ZAG. The mechanisms of how ZAG translocated into mitochondria were determined by quantitative coimmunoprecipitation after treatment with apoptozole, a heat shock cognate protein 70 (HSC70) inhibitor. ZAG level was modulated by lentivirus in neurons or adeno‐associated virus in rat brains. Seizure models were induced by magnesium (Mg²⁺)‐free artificial cerebrospinal fluid in neurons or intraperitoneal injection of pentylenetetrazole kindling in rats. Ketogenesis was determined by cyclic thio‐NADH method in supernatant of neurons or brain homogenate. The effect of peroxisome proliferator–activated receptor γ (PPARγ) on ZAG expression was examined by Western blot, quantitative real‐time polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation qRT‐PCR. We found that seizure induced ketogenesis deficiency via a ZAG‐dependent mechanism. ZAG entered mitochondria through a HSC70‐dependent mechanism, promoted ketogenesis by binding to four β‐subunits of long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (HADHB) and alleviated ketogenesis impairment in a neuronal seizure model and pentylenetetrazole‐kindled epileptic rats. Additionally, PPARγ activation up‐regulated ZAG expression by binding to promoter region of AZGP1 gene and promoted ketogenesis through a ZAG‐dependent mechanism.     

Regulation of basal and oxidative stress‐triggered jasmonic acid‐related gene expression by glutathione

Glutathione is a determinant of cellular redox state with roles in defence and detoxification. Emerging concepts suggest that this compound also has functions in cellular signalling. Here, we report evidence that glutathione plays potentially important roles in setting signalling strength through the jasmonic acid (JA) pathway. Firstly, we show that basal expression of JA‐related genes is correlated with leaf glutathione content when the latter is manipulated either genetically or pharmacologically. Secondly, analyses of an oxidative stress signalling mutant, cat2, reveal that up‐regulation of the JA pathway triggered by intracellular oxidation requires accompanying glutathione accumulation. Genetically blocking this accumulation in a cat2 cad2 line largely annuls H₂O₂‐induced expression of JA‐linked genes, and this effect can be rescued by exogenously supplying glutathione. While most attention on glutathione functions in biotic stress responses has been focused on the thiol‐regulated protein NPR1, a comparison of JA‐linked gene expression in cat2 cad2 and cat2 npr1 double mutants provides evidence that glutathione acts through other components to regulate the response of this pathway to oxidative stress. Our study provides new information implicating glutathione as a factor determining basal JA gene expression and suggests novel glutathione‐dependent control points that regulate JA signalling in response to intracellular oxidation.     

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

4‐O‐methylhonokiol protects against diabetic cardiomyopathy in type 2 diabetic mice by activation of AMPK‐mediated cardiac lipid metabolism improvement

Diabetic cardiomyopathy (DCM) is characterized by increased left ventricular mass and wall thickness, decreased systolic function, reduced ejection fraction (EF) and ultimately heart failure. The 4‐O‐methylhonokiol (MH) has been isolated mainly from the bark of the root and stem of Magnolia species. In this study, we aimed to elucidate whether MH can effectively prevent DCM in type 2 diabetic (T2D) mice and, if so, whether the protective response of MH is associated with its activation of AMPK‐mediated inhibition of lipid accumulation and inflammation. A total number of 40 mice were divided into four groups: Ctrl, Ctrl + MH, T2D, T2D + MH. Five mice from each group were sacrificed after 3‐month MH treatment. The remaining animals in each group were kept for additional 3 months without further MH treatment. In T2D mice, the typical DCM symptoms were induced as expected, reflected by decreased ejection fraction and lipotoxic effects inducing lipid accumulation, oxidative stress, inflammatory reactions, and final fibrosis. However, these typical DCM changes were significantly prevented by the MH treatment immediately or 3 months after the 3‐month MH treatment, suggesting MH‐induced cardiac protection from T2D had a memory effect. Mechanistically, MH cardiac protection from DCM may be associated with its lipid metabolism improvement by the activation of AMPK/CPT1‐mediated fatty acid oxidation. In addition, the MH treatment of DCM mice significantly improved their insulin resistance levels by activation of GSK‐3β. These results indicate that the treatment of T2D with MH effectively prevents DCM probably via AMPK‐dependent improvement of the lipid metabolism.     

Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.