Activated double-stranded RNA-dependent protein kinase and neuronal death in models of Alzheimer's disease

Neuronal death is a pathological hallmark of Alzheimer's disease. We have shown previously that phosphorylated double-stranded RNA-dependent protein kinase is present in degenerating hippocampal neurons and in senile plaques of Alzheimer's disease brains and that genetically down-regulating double-stranded RNA-dependent protein kinase activity protects against in vitro beta-amyloid peptide neurotoxicity. In this report, we showed that two double-stranded RNA-dependent protein kinase blockers attenuate, in human neuroblastoma cells, beta-amyloid peptide toxicity evaluated by caspase 3 assessment. In addition, we have used the newly engineered APP(SL)/presenilin 1 knock-in transgenic mice, which display a severe neuronal loss in hippocampal regions, to analyze the activation of double-stranded RNA-dependent protein kinase. Western blots revealed the increased levels of activated double-stranded RNA-dependent protein kinase and the inhibition of eukaryotic initiation factor 2 alpha activity in the brains of these double transgenic mice. Phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum-resident kinase was also increased in the brains of these mice. The levels of activated double-stranded RNA-dependent protein kinase were also increased in the brains of patients with Alzheimer's disease. At 3, 6 and 12 months, hippocampal neurons display double stranded RNA-dependent protein kinase labelings in both the nucleus and the cytoplasm. Confocal microscopy showed that almost constantly activated double-stranded RNA-dependent protein kinase co-localized with DNA strand breaks in apoptotic nuclei of CA1 hippocampal neurons. Taken together these results demonstrate that double-stranded RNA-dependent protein kinase is associated with neurodegeneration in APP(SL)/presenilin 1 knock-in mice and could represent a new therapeutic target for neuroprotection.     

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2'-5'-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).     

Mapping of the interacting domains of hepatitis C virus core protein and the double-stranded RNA-activated protein kinase PKR

Hepatitis C virus (HCV) core protein has been shown to exhibit several biological properties which suggest an important role in liver pathogenesis and carcinogenesis. During a previous study, we showed that core mutants, isolated from tumour, could directly interact with PKR and maintain it in an activated form. In the present report, we have further investigated this interaction and mapped the core and PKR domains involved. Using glutathion S-transferase fusion protein harbouring the different domains of core or PKR, we determined that the N-terminal 1-58 amino acid (aa) of core protein and the N-terminal 1-180 aa of PKR are responsible for this direct interaction. Using this system we also confirmed that the core-PKR interaction induced PKR autophosphorylation. Furthermore, we found that core protein co-localized and co-immunoprecipitated with PKR in cells expressing a full-length HCV replicon, thus confirming that this interaction occurs when all HCV proteins are expressed. Considering that the activation of PKR has been observed in some cancer cell lines and tissues, it suggests that, depending on the cellular context, PKR may stimulate or inhibit cell proliferation. The precise mapping of core-PKR interaction provides new data to study the molecular mechanism underlying HCV pathogenesis.     

Regulatable expression of the interferon-induced double-stranded RNA dependent protein kinase PKR induces apoptosis and fas receptor expression

PKR is an interferon-induced dsRNA-dependent protein kinase involved in the antiviral response as well as in cell growth and differentiation. Studies using a transdominant negative mutant of PKR also have implicated the kinase in tumor suppression and apoptosis. However, functional studies of PKR have been hampered by the lack of a suitable expression system. In this study, we used a tetracycline-regulated inducible system in NIH3T3 cells to investigate the involvement of PKR in programmed cell death (apoptosis). We show that expression of wild-type PKR causes apoptosis and correlates with increased mRNA levels for the Fas receptor, a member of the tumor necrosis family of proteins. Expression of an inactive form of PKR (K296R) or the vector alone did not induce apoptosis or elevate Fas mRNA levels. Our results clearly demonstrate that expression of an active form of PKR triggers apoptosis, possibly through upregulation of the Fas receptor.     

Inhibitor of double stranded RNA-dependent protein kinase protects against cell damage induced by ER stress

Endoplasmic reticulum (ER)-stress is known to induce neuronal cell death and to play roles in neurodegenerative diseases. Phosphorylation of double stranded RNA-dependent protein kinase (PKR) has been demonstrated in brain tissues in patients with Alzheimer's, Parkinson's, and Huntington's diseases. Here, we examined the effect of a PKR inhibitor (an imidazolo-oxindole derivative that acts as an ATP-binding site-directed inhibitor of PKR) on the neuronal cell death induced by ER-stress in cultured human neuroblastoma cells (SH-SY5Y). Cell damage was induced by tunicamycin (an ER-stress inducer), and cell viability was measured by Hoechst 33342 and YO-PRO-1 double staining and by the resazurin-reduction test (to evaluate metabolic activity). Treatment with tunicamycin at 2 microg/ml for 24 h induced apoptotic cell death accompanied by nuclear condensation and/or fragmentation, and these cells were positive for YO-PRO-1 (early-phase apoptosis and necrosis indicator). Treatment with the PKR inhibitor at 0.1 or 0.3 microM led to a decrease in the number of apoptotic cells induced by tunicamycin. In the resazurin-reduction test, the PKR inhibitor (at 0.1 and 0.3 microM) concentration-dependently inhibited the tunicamycin-induced decrease in metabolic activity. On the other hand, treatment with the PKR inhibitor alone (at 0.3 microM) had no effect on cell morphology or viability (versus in normal control cells). These results indicate that inhibition of PKR activation may be neuroprotective against ER stress-induced cell damage.     

The murine double-stranded RNA-dependent protein kinase PKR and the murine 2',5'-oligoadenylate synthetase-dependent RNase L are required for IFN-beta-mediated resistance against herpes simplex virus type 1 in primary trigeminal ganglion culture

A study was undertaken to evaluate the efficacy of an adenoviral construct expressing the murine interferon-beta (IFN-beta) transgene (Ad:IFN-beta) against herpes simplex virus type 1 (HSV-1) infection in a primary trigeminal ganglion (TG) cell culture. The transduction efficiency ranged from 0.2 to 11.0% depending on the multiplicity of infection (m.o.i.) of the adenoviral vector (0.5-50.0). Moreover, neurons were the main target of the adenoviral transduction. TG cultures transduced with Ad:IFN-beta displayed up to a 19-fold reduction in viral titers compared with cells transduced with an Ad:Null or nontransduced TG culture controls. Transduction with Ad:IFN-beta up-regulated two critical antiviral genes, double-stranded RNA-dependent protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS). The absence of PKR or RNase L (downstream effector molecule of OAS) attenuated Ad:IFN-beta efficacy against HSV-1 replication, implicating a critical role for PKR and OAS/RNase systems in the establishment of IFN-induced resistance against HSV-1 in TG cells.     

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.     

Pseudosubstrate inhibition of protein kinase PKR by swine pox virus C8L gene product

The interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2alpha on Ser-51 to inhibit protein synthesis in virally infected cells. Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2alpha. We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells. In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells. The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2alpha and the C8L and K3L gene products. Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR.     

In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.     

Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells

Background

Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD.

Methods

Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500.

Results

We show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4⁺ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C5a could protect human CD4⁺ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls.

Conclusions

Our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.     

Regulation of p38 mitogen-activated protein kinase during NK cell activation

The mitogen-activated protein kinase (MAPK) p38 modulates a variety of cellular functions, including proliferation, differentiation and cell death. However, we report here a novel function for p38, i.e. the regulation of cytotoxic lymphocyte-mediated cytotoxicity. Stimulation of NK cells by either cross-linking of their FcgammaRIII receptors or by binding to NK-sensitive target cells induces the phosphorylation and activation of p38, and also of its upstream regulators MKK3/MKK6. Pharmacologic analyses suggest that Src-family and Syk-family protein tyrosine kinases couple the NK cell surface receptors to p38 activation. The role of p38 in the cytotoxic function of NK cells was tested by treatment of NK cells with the cell-permeable, p38-specific inhibitor SB203580. Interestingly, exposure to the drug reduced both antibody-dependent cellular cytotoxicity and natural cytotoxicity, but maximal inhibitory concentrations resulted in only partial inhibition. Collectively, these results suggest that the p38 MAPK pathway is stimulated during the development of NK cell-mediated cytotoxicity and that efficient killing is influenced by both p38-dependent and p38-independent pathways. More broadly, this study identifies the regulation of cell-mediated killing as a novel role for p38 in cytotoxic lymphocytes.     

The protein kinase PKR is critical for LPS‐induced iNOS production but dispensable for inflammasome activation in macrophages

Inflammasomes are multi‐protein platforms that drive the activation of caspase‐1 leading to the processing and secretion of biologically active IL‐1β and IL‐18. Different inflammasomes including NOD‐like receptor (NLR) family pyrin domain‐containing 3 (NLRP3), NLR caspase‐recruitment domain‐containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double‐stranded RNA‐activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase‐1 activation, processing of pro‐IL‐1β/IL‐18 and secretion of IL‐1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.     

Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation.

Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A2 activity, induced the secretion of both granule-associated beta-hexosaminidase and newly generated leukotriene C4 from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of beta-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing beta-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine, an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce beta-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its ability to elevate intracellular free calcium concentrations.