Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase

The PrP(C) protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP(C) in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP(C) decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu(2+)/ascorbate/H(2)O(2)), which was demonstrated by approximately 70% less DMPO/OH(). In cultured PrP(C)-knockout astrocytes from mice, the absence of PrP(C) caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3h of H(2)O(2) treatment. This rapid increase in ROS disrupted the cell cycle in the PrP(C)-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP(C) in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.     

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.     

Stress-protective signalling of prion protein is corrupted by scrapie prions

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.     

Reactive oxygen species-mediated beta-cleavage of the prion protein in the cellular response to oxidative stress

The cellular prion protein (PrP(C)) is critical for the development of prion diseases. However, the physiological role of PrP(C) is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrP(C) is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed beta-cleavage. Here we show that ROS-mediated beta-cleavage of cell surface PrP(C) occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPDeltaoct, failed to undergo ROS-mediated beta-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPdeltaoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated beta-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the beta-cleavage of PrP(C) is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.     

CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.     

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H(2)O(2)-activated calcium signaling pathway in mouse hippocampal neurons

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H(2)S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O(2), 5% CO(2) and 93% N(2) at 37°C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H(2)S) for 30min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca(2+)](i)), and pretreatment with NaHS prevented the increase in [Ca(2+)](i). Additionally, polyethylene glycol (PEG)-catalase (a H(2)O(2) scavenger) but not PEG-SOD (an O(2)(-) scavenger) conferred an inhibitory effect similar to H(2)S on the hypoxia-induced increase in [Ca(2+)](i). Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP(3)) receptor blocker xestospongin C. Taken together, these findings suggest that H(2)S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H(2)O(2))-activated Ca(2+) signaling pathway in mouse hippocampal neurons.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced higher order chromatin degradation: A novel mechanism of oxidative genotoxicity

The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

PrP mutants with different numbers of octarepeat sequences are more susceptible to the oxidative stress

One of the physiological functions of cellular prion protein(PrP C )is believed to work as a cellular resistance to oxidative stress,in which the octarepeats region within PrP plays an important role.However,the detailed mechanism is less clear.In this study,the expressing plasmids of wild-type PrP (PrP-PG5)and various PrP mutants containing 0(PrP-PG0),9(PrP-PG9)and 12(PrP-PG12)octarepeats were generated and PrP proteins were expressed both in E.coli and in mammalian cells.Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E.coli after treatment of H2O2.MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP.Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats.Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H2O2.Furthermore,cells expressing wild-type PrP showed stronger resistant activity to the challenge of H2O2 at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation.Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.     

Loganin protects against hydrogen peroxide-induced apoptosis by inhibiting phosphorylation of JNK, p38, and ERK 1/2 MAPKs in SH-SY5Y cells

We investigated the mechanisms underlying the protective effects of loganin against hydrogen peroxide (H(2)O(2))-induced neuronal toxicity in SH-SY5Y cells. The neuroprotective effect of loganin was investigated by treating SH-SY5Y cells with H(2)O(2) and then measuring the reduction in H(2)O(2)-induced apoptosis using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays. Following H(2)O(2) exposure, Hoechst 33258 staining indicated nuclear condensation in a large proportion of SH-SY5Y cells, along with an increase in reactive oxygen species (ROS) production and an intracellular decrease in mitochondria membrane potential (MMP). Loganin was effective in attenuating all the above-stated phenotypes induced by H(2)O(2). Pretreatment with loganin significantly increased cell viability, reduced H(2)O(2)-induced LDH release and ROS production, and effectively increased intracellular MMP. Pretreatment with loganin also significantly decreased the nuclear condensation induced by H(2)O(2). Western blot data revealed that loganin inhibited the H(2)O(2)-induced up-regulation of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3, increased the H(2)O(2)-induced decrease in the Bcl-2/Bax ratio, and attenuated the H(2)O(2)-induced release of cytochrome c from mitochondria to the cytosol. Furthermore, pretreatment with loganin significantly attenuated the H(2)O(2)-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results suggest that the protective effects of loganin against H(2)O(2)-induced apoptosis may be due to a decrease in the Bcl-2/Bax ratio expression due to the inhibition of the phosphorylation of JNK, p38, and ERK 1/2 MAPKs. Loganin's neuroprotective properties indicate that this compound may be a potential therapeutic agent for the treatment of neurodegenerative diseases.     

Exacerbated vulnerability to oxidative stress in astrocytic C6 glioma cells with stable overexpression of the glutamine transporter slc38a1

We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation.     

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.     

Oxidant stress stimulates phosphorylation of eIF4E without an effect on global protein synthesis in smooth muscle cells. Lack of evidence for a role of H202 in angiotensin II-induced hypertrophy

Reactive oxygen species (ROS) are implicated in the pathogenesis of several proliferative diseases, including atherosclerosis and cancer. Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in cell proliferation and differentiation. To gain insight into molecular mechanisms by which ROS influence the pathogenesis of these diseases, I have studied the effect of H(2)O(2), a ROS, on eIF4E phosphorylation. H(2)O(2) induced eIF4E phosphorylation in a dose- and time-dependent manner in growth-arrested smooth muscle cells (SMC). H(2)O(2)-induced eIF4E phosphorylation occurred on serine residues. PD098059, a specific inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase inhibited ERK activities but had no significant effect on eIF4E phosphorylation induced by H(2)O(2). Similarly, SB203580, a specific inhibitor of p38 MAPK, although inhibiting H(2)O(2)-induced p38 MAPK activity, had no effect on H(2)O(2)-induced eIF4E phosphorylation. Calphostin C, a specific inhibitor of protein kinase C, also had no effect on H(2)O(2)-induced eIF4E phosphorylation. In contrast, trifluoperazine, an antagonist of calcium/calmodulin kinases, completely blocked H(2)O(2)-induced eIF4E phosphorylation. In addition, intracellular and extracellular Ca(2+) chelators significantly inhibited H(2)O(2)-induced eIF4E phosphorylation. Despite its ability to induce eIF4E phosphorylation, H(2)O(2) had no significant effect on protein levels and new protein synthesis as compared with control. In contrast, it induced the expression of c-Fos, c-Jun, and HSP70 in a time-dependent manner in SMC. Together, these results suggest that H(2)O(2), a ROS and a cellular oxidant, induces eIF4E phosphorylation in a manner that is dependent on Ca(2+) and Ca(2+)/calmodulin kinases and independent of ERKs, p38 MAPK, and protein kinase C. These results also suggest that enhanced eIF4E phosphorylation by H(2)O(2) appears to be an important event in SMC in response to oxidant stress and that eIF4E phosphorylation may be associated with the translation of a small subset of mRNAs such as c-fos, c-jun, and HSP70 gene mRNAs, whose products may have a critical role in cell survival.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

Role of membrane lipids in regulation of vulnerability to oxidative stress in PC12 cells: implication for aging

Previously, we reported that PC12 cells showed increased vulnerability to oxidative stress (OS) induced by H2O2 (as assessed by decrements in calcium recovery, i.e., the ability of cells to buffer Ca(2+) after a depolarization event) when the membrane levels of cholesterol (CHL) and sphingomyelin (SPH) were modified to approximate those seen in the neuronal membranes of old animals. The present study was designed to examine whether the enrichment of the membranes with SPH-CHL and increased cellular vulnerability to OS are mediated by neutral SPH-specific phospholipase C (N-Sase) and the intracellular antioxidant GSH. The results showed a significant up-regulation of N-Sase activity by both low (5 microM) and high (300 microM) doses of H2O2. However, under high doses of H2O2 the up-regulation of N-Sase is accompanied by a significant increase in reactive oxygen species and by a decrease in intracellular GSH. The enrichment of membranes with SPH-CHL significantly potentiated the effects of high doses of H2O2, by further reducing the intracellular GSH and further up-regulating the N-Sase activity. Furthermore, repleting intracellular GSH with 20 mM N-acetylcysteine treatment was sufficient to attenuate the effect of a low dose of H2O2 on Ca(2+) recovery in SPH-CHL-treated cells. Thus, these results suggested that age-related alterations in the membrane SPH-CHL levels could be important determinants of the susceptibility of neuronal cells to OS.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Neuregulin rescues PC12-ErbB4 cells from cell death induced by H(2)O(2). Regulation of reactive oxygen species levels by phosphatidylinositol 3-kinase

Neuregulins (NRGs), a large family of transmembrane polypeptide growth factors, mediate various cellular responses depending on the cell type and receptor expression. We previously showed that NRG mediates survival of PC12-ErbB4 cells from apoptosis induced by serum deprivation or tumor necrosis factor-alpha treatment. In the present study we show that NRG induces a significant protective effect from H(2)O(2)-induced death. This effect of NRG is mediated by the phosphatidylinositol 3-kinase (PI3K)-signaling pathway since NRG failed to rescue cells from H(2)O(2) insult in the presence of the PI3K inhibitor, LY294002. Furthermore, the downstream effector of PI3K, protein kinase B/AKT, is activated by NRG in the presence of H(2)O(2), and protein kinase B/AKT activation is inhibited by LY294002. In addition, our results demonstrate that reactive oxygen species (ROS) elevation induced by H(2)O(2) is inhibited by NRG. LY294002, which blocks NRG-mediated rescue, increases ROS levels. Moreover, both H(2)O(2)-induced ROS elevation and cell death are reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress.     

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress

Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES). Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES). Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES) and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.