[Research Advances in the Association Between Alzheimer's Disease and Double-Stranded RNA-Dependent Protein Kinase].

Alzheimer's disease (AD) is a severe threat to human health and one of the three major causes of human death.Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein kinase involved in innate immunity.In the occurrence and development of AD,PKR is upregulated and continuously activated.On the one hand,the activation of PKR triggers an integrated stress response in brain cells.On the other hand,it indirectly upregulates the expression of ß-site amyloid precursor protein cleaving enzyme 1 and facilitates the accumulation of amyloid-ß protein (Aß),which could activate PKR activator to further activate PKR,thus forming a sustained accumulation cycle of Aß.In addition,PKR can promote Tau phosphorylation,thereby reducing microtubule stability in nerve cells.Inflammation in brain tissue,neurotoxicity resulted from Aß accumulation,and disruption of microtubule stability led to the progression of AD and the declines of memory and cognitive function.Therefore,PKR is a key molecule in the development and progression of AD.Effective PKR detection can aid in the diagnosis and prediction of AD progression and provide opportunities for clinical treatment.The inhibitors targeting PKR are expected to control the activity of PKR,thereby controlling the progression of AD.Therefore,PKR could be a target for the development of therapeutic drugs for AD.

Ctenopharyngodon idella IKKß interacts with PKR and IκBα.

Inhibitor of nuclear factor kappa-B kinase ß (IKKß) is a subunit of the IKK complex. It can activate the NF-κB pathway through phosphorylating IκB in response to a wide range of stimuli. In the present study, an IKKß gene from grass carp (Ctenopharyngodon idella; KT282114) was cloned and identified by homologous cloning and rapid-amplification of cDNA ends (RACE) technique. The complete CiIKKß cDNA is 3428 bp in length, with the longest open reading frame (ORF) of 2337 bp encoding a polypeptide of 778 amino acids. The deduced amino acid sequence of CiIKKß has similar domain distribution to those of mammalian. For example, CiIKKß consists of a serine/threonine kinase domain at the N-terminal, a basic region leucin zipper (BRLZ) domain in the middle, a homeobox associated leucin zipper (HALZ) domain and an IKKß NEMO (NF-κB essential modulator) binding domain at the C-terminal. Phylogenetic tree analysis also showed that CiIKKß is highly homologous to zebrafish IKKß (DrIKKß) and clearly distinct from the mammalian and amphibian counterparts. The expression of CiIKKß was ubiquitously found in the liver, intestine, kidney, gill, spleen, heart, and brain tissues of grass carp and significantly up-regulated in CIK cells under the stimulation with Poly I:C and UV-inactivated grass carp hemorrhagic virus. To investigate the activation mechanism of NF-κB pathway in fish and the role of CiIKKß in the pathway, we explored the protein interactions of protein kinase R (PKR) with IKKß and IKKß with IκBα by co-immunoprecipitation and GST-pull down assays. The interaction between each pair was confirmed. The results suggest that CiIKKß may be a primary member in the activation of NF-κB pathway in fish.

Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate in vitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.

Ctenopharyngodon idella PKZ facilitates cell apoptosis through phosphorylating eIF2α.

PKZ, protein kinase containing Z-DNA binding domains, is a novel member of eIF2α kinase in fish. PKZ can be up-regulated in response to Poly I:C or heat stress, and it has a typical eIF2α kinase activity. However, the relationship between fish PKZ and apoptosis remains unclear. In the present study, effects of PKZ on apoptosis were explored. Initially, we found that PKZ and PKR were up-regulated by poly I:C in a time-dependent manner. Q-PCR analysis showed that the change of Caspase-3 mRNA was consistent with that of PKZ under the stimulation of poly I:C or transfection with PKZ siRNA in CIK cells. It suggested that PKZ might mediate the induction of apoptosis. Knockdown and overexpression assays indicated that a significant increase of apoptotic cell number was observed in the wild type PKZ (PKZ-wt) transfected PKZ-deficient cells, while mutant PKZ-K198R lost this ability. MTT also showed that overexpression of PKZ-wt in CIK cells resulted in a striking decrease of cell viability rate to about 32.5%, whereas the viability of cell with PKZ-K198R was about 88.1%. Likewise, when transfected eIF2α-wt or phosphomimetic eIF2α (S51D), CIK cells displayed higher apoptotic rate than the controls. In contrast, overexpression of phosphodeficient eIF2α (S51A) blocked induction of apoptosis. In addition, levels of eIF2α phosphorylation were significantly related to apoptosis in these CIK cells. Furthermore, when mutant eIF2α (S51A) was transiently transfected into the PKZ/PKR knockdown cells that transfected with wildtype PKZ previously, the apoptotic rates and levels of eIF2α phosphorylation were dramatically decreased. Overall, these results suggested that PKZ could facilitate apoptosis via eIF2α phosphorylation.

SiRNA-mediated knockdown of CiGRP78 gene expression leads cell susceptibility to heavy metal cytotoxicity.

Heavy metal ion is one of the critical environmental pollutants accumulated in living organisms and causes toxic or carcinogenic effects once passed threshold levels. As an important member of Hsp70 (heat shock protein 70) family, the 78-kDa glucose-regulated protein (GRP78) can enhance cell survival rates remarkably under thermal stress. Recent studies also demonstrated that the expression of GRP78 enhances the cell survival under heavy metal stress. In this study, three most representative heavy metal ions, Pb(2+), Hg(2+) and Cd(2+), were used to stimulate Ctenopharyngodon idella kidney (CIK) cells. The results showed that cell viability under Pb(2+), Hg(2+) and Cd(2+) stress decreased significantly. The longer and the greater the concentrations of stimulation from heavy metal ions, the higher the rate of cell death was observed. Among them, Hg(2+) is the most hazardous to cells. Under the same stress condition, Hg(2+) resulted in 50% of cell death, Cd(2+) (or Pb(2+)) led to 45% (or 35%) of cell death, respectively. Western immunoblotting indicated that C. idella GRP78 (CiGRP78) protein expression level was enhanced obviously in CIK cells under Pb(2+), Hg(2+) and Cd(2+) stress, meaning CiGRP78 is involved in heavy metal cytotoxicity. To further study the role of CiGRP78 in cytoprotection, we designed the siRNA against CiGRP78 (from nucleotides +788 to +806) and transfected it into CIK cells to silence endogenous CiGRP78. The viability rate of CIK cells transfected with or without siRNA incubated with HgCl2 for 12h showed a significant decrease from 50% to 21%. Our results showed that CiGRP78 protects cells against heavy metal stimuli to some extent.

ATF4 (activating transcription factor 4) from grass carp (Ctenopharyngodon idella) modulates the transcription initiation of GRP78 and GRP94 in CIK cells.

GRP78 and GRP94, belong to GRP (glucose-regulated protein) family of endoplasmatic reticulum (ER) chaperone superfamily, are essential for cell survival under ER stress. ATF4 is a protective protein which regulates the adaptation of cells to ER stress by modulating the transcription of UPR (Unfolded Protein Response) target genes, including GRP78 and GRP94. To understand the molecular mechanism of ATF4 modulates the transcription initiation of CiGRP78 and CiGRP94, we cloned ATF4 ORF cDNA sequences (CiATF4) by homologous cloning techniques. The expression trend of CiATF4 was similar to CiGRP78 and CiGRP94 did under 37 °C thermal stress, namely, the expression of CiATF4 was up-regulated twice at 2 h post-thermal stress and at 18 h post recovery from thermal stress. In this paper, CiATF4 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. On the basis of the cloned CiGRP78 and CiGRP94 cDNA in our laboratory previously, we cloned their promoter sequences by genomic walking approach. In vitro, gel mobility shift assays revealed that CiATF4 could bind to CiGRP78 and CiGRP94 promoter with high affinity. Subsequently, the recombinant plasmid of pGL3-CiGRPs and pcDNA3.1-CiATF4 were constructed and transiently co-transfected into Ctenopharyngodon idella kidney (CIK) cells. The impact of CiATF4 on CiGRP promoter sequences were measured by luciferase assays. These results demonstrated that CiATF4 could activate the transcription of CiGRP78 and CiGRP94. What's more, for better understanding the molecular mechanism of CiATF4 modulate the transcription initiation of CiGRP, three mutant fragments of CiGRP78 promoter recombinant plasmids (called CARE-mut/LUC, CRE1-mut/LUC and CRE2-mut/LUC) were constructed and transiently co-transfected with CiATF4 into CIK cells. The results indicated that CRE or CARE elements were the regulatory element for transcription initiation of CiGRP78. Between them, CRE element would play more important role in it.

Cloning, expression and functional analysis of PKR from grass carp (Ctenopharyngodon idellus).

The interferon-induced, dsRNA-activated protein kinase (PKR) is considered as an important component of innate immune system and as a representative effector protein of interferon system. In the present study, PKR gene (CiPKR, JX511974) from grass carp (Ctenopharyngodon idellus) was isolated and identified using homology-based PCR. CiPKR shares high sequence identity with the counterparts of goldfish (Crucian carp) and zebrafish (Danio rerio). The full-length cDNA of CiPKR was found to be 2436 bp, with an ORF of 2067 bp that encodes a polypeptide of 688 amino acids. The deduced polypeptide CiPKR contains three tandem dsRNA-binding motifs (dsRBMs) at the N-terminus and a conserved Ser/Thr kinase domain at the C-terminus. CiPKR was expressed ubiquitously at a low-level under normal conditions, but it could be up-regulated after intraperitoneal (ip) injection with grass carp haemorrhagic virus (GCHV). CiPKR was dramatically up-regulated at 6 h post-injection and then recovered rapidly to normal levels within 24 h; however, it was obviously up-regulated once again at 48 h or 72 h post-injection. It seemed that CiPKR could respond to GCHV infection in an IFN-independent as well as an IFN-dependent pathway. To further investigate its mechanism of biological actions, we constructed a series of recombinant plasmids including pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C (deletion of dsRBD sequence) and pcDNA3.1/PKR-C-K430R, and then each recombinant plasmid was transfected into CIK cells. In comparison with those of controls, a 79% and a 64% decrease of luciferase activities were detected in the tested cells transfected with CiPKR and CiPKR-C, respectively; however, luciferase activities were increased in those cells transfected with PKR-K430R and PKR-C-K430R, with a 160% and 115% up-regulation, respectively. Similarly, MTT colorimetric assay indicated that cell viabilities of CIK cells transfected with pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C and pcDNA3.1/PKR-C-K430R were 49%, 90%, 54% and 100%, respectively. Our observations suggested that the expression of CiPKR could be up-regulated following viral infection, and then resulted in the inhibition of protein synthesis and the induction of potential apoptosis.

Gig1 and Gig2 homologs (CiGig1 and CiGig2) from grass carp (Ctenopharyngodon idella) display good antiviral activities in an IFN-independent pathway.

The virus-induced genes, Gig1 and Gig2, were identified first as IFN-stimulated genes (ISGs) from CAB cells. Previous studies suggested that Gig protein may have some potential antiviral functions. In this study, we cloned and identified the full-length cDNA sequences of Gig1 and Gig2 homologs (designated as CiGig1 and CiGig2, respectively) from grass carp (Ctenopharyngodon idella). The complete cDNA sequences of Gig1 and Gig2 contain 1231 bp and 690 bp, encoding for a 194 amino acid protein and a 158 amino acid protein, respectively. Their structure characteristics of CiGig1 and CiGig2 are highly similar to the corresponding homologues in crucian carp. The tissue-specific expressions of CiGig1 and CiGig2 in liver, spleen, kidney, intestine, gill and heart were significantly up-regulated following GCHV challenge. The results indicated that CiGig1 and CiGig2 may be involved in the antiviral immune responses in cells. To better understand the antiviral functions of CiGig1 and CiGig2 in vivo, CiGig1 or CiGig2 ORF cDNA were inserted into the plasmid pcDNA3.1, respectively. Subsequently, the recombinant plasmids were transfected into C. idellus kidney (CIK) cells. The over-expressions of CiGig1 and CiGig2 were observed in the CIK cells after treatment with GCHV. Cells with pcDNA3.1-CiGig1 or pcDNA-CiGig2 exhibited a relatively higher survival rate of (70.84% or 69.24%) than non-transfection (22.16%) and mock-vehicle controls (24.38%) following the virus infection. Our data showed that both CiGig1 and CiGig2 could exert antiviral effects effectively in vivo. Cycloheximide blocking protein synthesis demonstrated that both CiGig1 and CiGig2 mRNA expression could be induced by GCHV rather than by recombinant grass carp IFN (rCiIFN) directly, suggesting that CiGig1 and CiGig2 may not be IFN-stimulated genes since they display their antivirus activities in an IFN-independent pathway.