Mouse Neuroblastoma Neuro2a Cells Research Articles

Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway.

ObjectiveTo investigate the protective effects of dexmedetomidine (DEX) in oxygen-glucose deprivation/reoxygenation (OGD/R) injury, which is involved in a number of ischaemic diseases.MethodsAn in vitro OGD/R injury model was generated using mouse Neuro 2A neuroblastoma (N2A) cells. Different concentrations of DEX were administrated to OGD/R cells. CV-65 was used to inhibit p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling. Cell proliferation, cell cycle, apoptosis, and the levels of proteins related to p38 MAPK/ERK signalling and apoptosis were evaluated using Cell Counting Kit-8, flow cytometry, TdT-UTP nick end labelling and Western blot analysis, respectively.ResultsDEX treatment of OGD/R cells promoted cell survival and attenuated OGD/R-induced cell apoptosis. It also activated the p38 MAPK/ERK signalling pathway, increased the levels of Bcl-2, and decreased the levels of Bax and cleaved caspase-3. Treatment with the p38 MAPK/ERK inhibitor CV-65 inhibited the activation of p38 MAPK/ERK and abrogated the DEX-induced effects on cell survival and apoptosis.ConclusionsDEX protects N2A cells from OGD/R-induced apoptosis via the activation of the p38 MAPK/ERK signalling pathway. DEX might be an effective agent for the treatment of ischaemic diseases.

Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner.

ABSTRACTPorcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes neurological dysfunction. Neural cells are its targets for viral progression. However, the detailed mechanism underlying PHEV entry and trafficking remains unknown. PHEV is the etiological agent of porcine hemagglutinating encephalomyelitis, which is an acute and highly contagious disease that causes numerous deaths in suckling piglets and enormous economic losses in China. Understanding the viral entry pathway will not only advance our knowledge of PHEV infection and pathogenesis but also open new approaches to the development of novel therapeutic strategies. Therefore, we employed systematic approaches to dissect the internalization and intracellular trafficking mechanism of PHEV in Neuro-2a cells. This is the first report to describe the process of PHEV entry into nerve cells via clathrin-mediated endocytosis in a dynamin-, cholesterol-, and pH-dependent manner that requires Rab5 and Rab7.

Piperine-like alkamides from Piper nigrum induce BDNF promoter and promote neurite outgrowth in Neuro-2a cells.

Black pepper (Piper nigrum) contains a variety of alkamides. Among them, piperine has been reported to have antidepressant-like effects in chronically stressed mice, but little is known about the biological activity of other alkamides. In this study, we investigated the effects of alkamides from white pepper (P. nigrum) on neuronal cells. Twelve alkamides were isolated from white pepper MeOH extracts, and their chemical structures were identified by NMR and MS analyses. The compounds were subjected to assays using the luciferase-reporter gene under the control of the BDNF promoter or cAMP response element in mouse neuroblastoma Neuro-2a cells. In both assays, marked reporter-inducing activity was observed for piperine (1), piperettine (2) and piperylin (7), all of which have in common an (E)-5-(buta-1,3-dien-1-yl)benzo[d] [1, 3] dioxole moiety. Piperettine (2) and piperylin (7) tended to increase endogenous BDNF protein levels. Furthermore, piperylin (7) promoted retinoic acid-induced neurite outgrowth. These results suggest that piperylin (7), or analogues thereof, may have a beneficial effect on disorders associated with dysregulation of BDNF expression, such as depression.

From the Cover: Comparative Proteomics Reveals Silver Nanoparticles Alter Fatty Acid Metabolism and Amyloid Beta Clearance for Neuronal Apoptosis in a Triple Cell Coculture Model of the Blood–Brain Barrier

Silver nanoparticles (AgNPs) enter the central nervous system through the blood-brain barrier (BBB). AgNP exposure can increase amyloid beta (Aβ) deposition in neuronal cells to potentially induce Alzheimer's disease (AD) progression. However, the mechanism through which AgNPs alter BBB permeability in endothelial cells and subsequently lead to AD progression remains unclear. This study investigated whether AgNPs disrupt the tight junction proteins of brain endothelial cells, and alter the proteomic metabolism of neuronal cells underlying AD progression in a triple cell coculture model constructed using mouse brain endothelial (bEnd.3) cells, mouse brain astrocytes (ALT), and mouse neuroblastoma neuro-2a (N2a) cells. The results showed that AgNPs accumulated in ALT and N2a cells because of the disruption of tight junction proteins, claudin-5 and ZO-1, in bEnd.3 cells. The proteomic profiling of N2a cells after AgNP exposure identified 298 differentially expressed proteins related to fatty acid metabolism. Particularly, AgNP-induced palmitic acid production was observed in N2a cells, which might promote Aβ generation. Moreover, AgNP exposure increased the protein expression of amyloid precursor protein (APP) and Aβ generation-related secretases, PSEN1, PSEN2, and β-site APP cleaving enzyme for APP cleavage in ALT and N2a cells, stimulated Aβ40 and Aβ42 secretion in the culture medium, and attenuated the gene expression of Aβ clearance-related receptors, P-gp and LRP-1, in bEnd.3 cells. Increased Aβ might further aggregate on the neuronal cell surface to enhance the secretion of inflammatory cytokines, MCP-1 and IL-6, thus inducing apoptosis in N2a cells. This study suggested that AgNP exposure might cause Aβ deposition and inflammation for subsequent neuronal cell apoptosis to potentially induce AD progression.

Selenoprotein S is required for clearance of C99 through endoplasmic reticulum-associated degradation

Amyloid beta precursor protein (APP) is normally cleaved by α-secretase, but can also be cleaved by β-secretase (BACE1) to produce C99 fragments in the endoplasmic reticulum (ER) membrane. C99 is subsequently cleaved to amyloid β (Aβ), the aggregation of which is known to cause Alzheimer's disease. Therefore, C99 removing is for preventing the disease. Selenoprotein S (SelS) is an ER membrane protein participating in endoplasmic reticulum-associated degradation (ERAD), one of the stages in resolving ER stress of misfolded proteins accumulated in the ER. ERAD has been postulated as one of the processes to degrade C99; however, it remains unclear if the degradation depends on SelS. In this study, we investigated the effect of SelS on C99 degradation. We observed that both SelS and C99 were colocalized in the membrane fraction of mouse neuroblastoma Neuro2a (N2a) cells. While the level of SelS was increased by ER stress, the level of C99 was decreased. However, despite the induction of ER stress, there was no change in the amount of C99 in SelS knock-down cells. The interaction of C99 with p97(VCP), an essential component of the ERAD complex, did not occur in SelS knock-down cells. The ubiquitination of C99 was decreased in SelS knock-down cells. We also found that the extracellular amount of Aβ1−42 was relatively higher in SelS knock-down cells than in control cells. These results suggest that SelS is required for C99 degradation through ERAD, resulting in inhibition of Aβ production.

Potential Role for a β-Catenin Coactivator (High-Mobility Group AT–Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/β-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148-3159, 2016). In the present study, a β-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and β-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing β-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and β-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate β-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing β-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate β-catenin-dependent transcription. Since the canonical Wnt/β-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and β-catenin may be important for certain aspects of the latency-reactivation cycle.IMPORTANCE The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant numbers of infected sensory neurons survive infection and maintain normal functions. Consequently, we hypothesize that viral products expressed during latency cooperate with neuronal factors to maintain latency. Our studies revealed that a β-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was readily detected in a subset of trigeminal ganglion neurons in latently infected calves but not in uninfected calves. A viral protein (ORF2) expressed in latently infected neurons interacted with β-catenin and HMGA1 in transfected cells, which resulted in the nuclear localization of β-catenin. This interaction correlated with the ability of ORF2 to stimulate the coactivator functions of HMGA1. These findings are significant because the canonical Wnt/β-catenin signaling pathway promotes neurogenesis and inhibits neurodegeneration.

Bovine herpesvirus 1 can efficiently infect the human (SH-SY5Y) but not the mouse neuroblastoma cell line (Neuro-2A)

Bovine herpesvirus 1 (BoHV-1) is a significant bovine pathogen that establishes a life-long latent infection in sensory neurons. Previous attempts to develop immortalized bovine neuronal cells were unsuccessful. Consequently, our understanding of the BoHV-1 latency-reactivation cycle has relied on studying complex virus-host interactions in calves. In this study, we tested whether BoHV-1 can infect human (SH-SY5Y) or mouse (Neuro-2A) neuroblastoma cells. We provide new evidence that BoHV-1 efficiently infects SH-SY5Y cells and yields virus titers approximately 100 fold less than bovine kidney cells. Conversely, virus titers from productively infected Neuro-2A cells were approximately 10,000 fold less than bovine kidney cells. Using a β-Gal expressing virus (gC-Blue), we demonstrate that infection of Neuro-2A cells (actively dividing or differentiated) does not result in efficient virus spread, unlike bovine kidney or SH-SY5Y cells. Additional studies demonstrated that lytic cycle viral gene expression (bICP4 and gE) was readily detected in SH-SY5Y cells: conversely bICP4 was not readily detected in productively infected Neuro-2A cells. Finally, infection of SH-SY5Y and bovine kidney cells, but not Neuro-2A cells, led to rapid activation of the Akt protein kinase. These studies suggest that the Neuro-2A cell line may be a novel cell culture model to identify factors that regulate BoHV-1 productive infection in neuronal cells.

Thymoquinone inhibits the migration of mouse neuroblastoma (Neuro-2a) cells by down-regulating MMP-2 and MMP-9

Thymoquinone (TQ), an active component derived from the medial plant Nigella sativa, has been used for medical purposes for more than 2 000 years. Recent studies have reported that TQ blocked angiogenesis in animal model and reduced migration, adhesion, and invasion of glioblastoma cells. We have recently shown that TQ could exhibit a potent cytotoxic effect and induce apoptosis in mouse neuroblastoma (Neuro-2a) cells. In the present study, TQ treatment markedly decreased the adhesion and migration of Neuro-2a cells. TQ down-regulated MMP-2 and MMP-9 protein expression and mRNA levels and their activities. Furthermore, TQ significantly down-regulated the protein expression of transcription factor NF-κB (p65) but not significantly altered the expression of N-Myc. Taken together, our data indicated that TQ's inhibitory effect on the migration of Neuro-2a cells was mediated through the suppression of MMP-2 and MMP-9 expression, suggesting that TQ treatment can be a promising therapeutic strategy for human malignant neuroblastoma.

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization.

Inhibition of sphingomyelin synthase 1 affects ceramide accumulation and hydrogen peroxide-induced apoptosis in Neuro-2a cells.

Oxidative stress plays a key role in brain injury after cerebral ischemia-reperfusion, which contributes toward excessive apoptosis of nerve cells. Therefore, it would be beneficial to identify a therapy that could interfere with the progression of apoptosis and protect the brain from ischemia-reperfusion injury. As ceramide, a well-known second messenger of apoptosis, can be metabolized by sphingomyelin synthase 1 (SMS1), recent research has focused on the link between SMS1 and apoptosis in different cells. To investigate whether SMS1 is involved in the process of oxidative stress-induced apoptosis in neurons and to explore the possible underlying mechanism, we treated mouse neuroblastoma Neuro-2A (N2a) cells with hydrogen peroxide (H2O2). Incubation with H2O2 significantly upregulated the expression of SMS1, increased the intracellular levels of ceramide and sphingomyelin synthase activity, and induced apoptosis. Moreover, pretreatment of N2a cells with D609, an sphingomyelin synthase inhibitor, or SMS1-silencing RNA (siRNA) further increased ceramide and potentiated H2O2-induced apoptosis which could be reversed by SB203580 (a p38 inhibitor). Thus, our study has shown that SMS1 regulates ceramide levels in N2a cells and plays a potent protective role in this oxidative stress-induced apoptosis partly through the p38 pathway.

Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors

Phospholipase A2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3–10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC50 of about 50 μM and 250 μM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC50 of about 3 μM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 μM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC50 of 20.6 ± 3.93 μM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 μM, the value being close to IC50 on α7 nAChR. These data are in accord with non-competitive inhibition as follows from α-bungarotoxin binding experiments. The described peptides are the shortest peptides without disulfide bridges isolated from animal venom and capable to inhibit nAChR by non-competitive way.

Antioxidant properties of atypical antipsychotic drugs used in the treatment of schizophrenia

The aim of this study was to compare the antioxidant activities of six atypical antipsychotic drugs: clozapine (CLZ), quetiapine, olanzapine (OLA), risperidone, ziprasidone, aripiprazole (ARI), as well as a typical antipsychotic drug, haloperidol. Several tests of antioxidant activity were used: protection of thiol groups against oxidation by peroxynitrite (PN) and 3-morpholinosydnonimine (SIN-1, generator of PN), oxidation of dihydrorhodamine 123 by PN, SIN-1 and hypochlorite (NaOCl), bleaching of fluorescein fluorescence by PN, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH, generator of peroxyl radicals) and NaOCl, radical-scavenging activity with respect to 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) radical, 2,2-diphenyl-1-picrylhydrazyl free radical and the Ferric Reducing Antioxidant Potential. In most of the tests, OLA showed the highest antioxidant activity, followed by CLZ and in some cases ARI, other compounds being much less active or not active. OLA and CLZ exerted limited toxicity on mouse neuroblastoma Neuro-2A (N2A) cells and protected the cells against the toxic action of SIN-1, AAPH and NaOCl in the physiologically relevant concentration range of these oxidants. Both drugs reduced the PN-induced nitration of intracellular proteins. Given that schizophrenia is associated with oxidative and nitrosative stress, the direct antioxidant activity OLA and CLZ may contribute to the therapeutic action of these compounds.

Bovine herpesviruses induce different cell death forms in neuronal and glial-derived tumor cell cultures.

Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40% of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40% in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80% of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

The voltage-gated sodium ion channel inhibitory activities of a new tetrodotoxin analogue, 4,4a-anhydrotetrodotoxin, and three other analogues evaluated by colorimetric cell-based assay

The voltage-gated sodium ion channel inhibitory activities of four tetrodotoxin analogues were evaluated for their ability to reduce the cytotoxicity of ouabain and veratridine in mouse neuroblastoma Neuro-2a cells. EC50 of the novel analogue, 4,4a-anhydrotetrodotoxin purified from pufferfish, was 750 fold larger than that of tetrodotoxin, supporting the implication of 4-OH in activity. The high activity of 11-oxotetrodotoxin was confirmed. Modification of C-6 of 11-nortetrodotoxin-6,6-diol to form an oxime derivative decreased the activity to 1/22.

In Vitro Anti-Neuroblastoma Activity of Thymoquinone Against Neuro-2a Cells via Cell-cycle Arrest.

We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose- dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 μM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and β) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation.

Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders.

Efficacy of Favipiravir (T-705) in Rabies Postexposure Prophylaxis.

Rabies is a fatal encephalitis caused by rabies virus (RABV), and no antiviral drugs for RABV are currently available. We report for the first time the efficacy of favipiravir (T-705) against RABV in vitro and in vivo. T-705 produced a significant, 3–4 log10 reduction in the multiplication of street and fixed RABV strains in mouse neuroblastoma Neuro-2a cells, with half-maximal inhibitory concentrations of 32.4 µM and 44.3 µM, respectively. T-705 significantly improved morbidity and mortality among RABV-infected mice when orally administered at a dose of 300 mg/kg/day for 7 days, beginning 1 hour after inoculation. T-705 significantly reduced the rate of virus positivity in the brain. Furthermore, the effectiveness of T-705 was comparable to that of equine rabies virus immunoglobulin for postexposure prophylaxis. Collectively, our results suggest that T-705 is active against RABV and may serve as a potential alternative to rabies immunoglobulin in rabies postexposure prophylaxis.

UCH-L1 Inhibition Decreases the Microtubule-Binding Function of Tau Protein.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is critical for protein degradation and free ubiquitin recycling. In Alzheimer's disease brains, UCH-L1 is negatively related to neurofibrillary tangles whose major component is hyperphosphorylated tau protein, but the direct action of UCH-L1 on tau has not been reported. In the current study, mouse neuroblastoma Neuro2a (N2a) cells were treated by the different concentrations of UCH-L1 inhibitor LDN (2.5, 5 and 10 μM) to inhibit the hydrolase activity of UCH-L1. In addition, we also used UCH-L1 siRNA to treat the HEK293/tau441 cells to decrease the expression of UCH-L1. After LDN and UCH-L1 siRNA treatment, we used immunofluorescence, immunoprecipitation, and tau-microtubule binding assay to measure the microtubule-binding ability and post-translational modifications of tau protein. All the results presented that both inhibition of the activity and expression of UCH-L1 induced the decreased microtubule-binding ability and increased phosphorylation of tau protein. Abnormal aggregation and ubiquitination of tau protein was also observed after UCH-L1 inhibition. The above results suggested that aggregation of tau protein might be devoted to the abnormal post-translational modifications of tau protein. Our study first indicates that dysfunction of UCH-L1 most likely affected normal biological function of tau protein through decreasing degradation of ubiquitinated and hyperphosphorylated tau.

Toxic acrolein production due to Ca2+ influx by the NMDA receptor during stroke

Background and purposeN-Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca2+, contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. MethodsA unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. ResultsWhen the size of brain infarction was decreased by N1, N4, N8-tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca2+, the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca2+ increase. ConclusionThe results indicate that toxicity of Ca2+ during brain infarction is correlated with the increase of acrolein.