Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer's disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.     

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection

Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulation.Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the brain, increases neuronal tolerance to lethal cerebral ischemia. The molecular mechanisms responsible for inducing and maintaining ischemic tolerance in the brain are complex and are not yet fully understood. Among the three isoforms of the Na⁺/Ca²⁺ exchanger, NCX1 and NCX3 represent two new possible molecular effectors involved in the neuroprotective mechanisms of IPC.^{1, 2, 3} Indeed, the increased expression of these two plasma membrane proteins, which have a fundamental role in regulating and maintaining cellular calcium and sodium homeostasis in the brain^{4, 5} during IPC, has been associated with a decrease in the infarct volume following a more severe ischemic insult.¹ However, the molecular mechanisms by which NCX1 and NCX3 upregulation lead to IPC-induced brain tolerance still remain unexplored.In vitro experiments performed in cortical neurons exposed to oxygen and glucose deprivation (OGD) and subsequent reoxygenation have demonstrated that changes in NCX isoform expression during OGD are accompanied by increases in both NCX1 activity and endoplasimc reticulum (ER) Ca²⁺ refilling.⁶ Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation,^{7, 8} we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance.More recently, we have reported that among the three NCX isoforms, only NCX3 is expressed on the outer mitochondrial membrane, where it works mainly by extruding calcium from the matrix.⁹ In this regard, an even more compelling result is that NCX3 gene ablation induces not only the disappearance of the protein from the OMM but also the accumulation of mitochondrial calcium in cortical neurons. Interestingly, NCX3 expression decreases in cortical neurons during OGD, a finding that correlates with an increase in [Ca²⁺]_m.⁹Furthermore, preserving mitochondrial function is relevant for preconditioning-induced neuroprotection. In fact, preconditioning positively affects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia,¹⁰ prevents mitochondrial swelling, protects mitochondrial energy metabolism during cerebral ischemia by avoiding ATP consumption¹¹ and increases Mn-SOD expression and activity through the NO/Ras/ERK1-2 pathway.⁸Although mitochondria are considered to be important mediators of endogenous neuroprotection, the mechanisms by which they might integrate cytoprotective signaling of preconditioning still remain to be fully elucidated. Thus, we investigated the role played by NCX1 and NCX3 in regulating ER and mitochondrial calcium homeostasis as a novel mechanism responsible for IPC-induced neuroprotection.For this aim, cortical neurons were exposed to 30 min of OGD followed by 3-h OGD plus reoxygenation. The expression and activity of NCX1 and NCX3 were observed by means of western blot analysis, confocal microscopy and single cell microfluorimetry. The results showed that IPC-induced neuroprotection occurs through the modulation of calcium homeostasis in ER and mitochondria through NO/Akt-mediated NCX1 and NCX3 upregulation.     

SIRT3 protects from hypoxia and staurosporine-mediated cell death by maintaining mitochondrial membrane potential and intracellular pH

Mitochondrial sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. Here, we show that in mammalian cells subjected to hypoxia and staurosporine treatment SIRT3 prevents loss of mitochondrial membrane potential (ΔΨ_mt), intracellular acidification and reactive oxygen species accumulation. Our results indicate that: (i) SIRT3 inhibits mitochondrial permeability transition and loss of membrane potential by preventing HKII binding to the mitochondria, (ii) SIRT3 increases catalytic activity of the mitochondrial carbonic anhydrase VB, thereby preventing intracellular acidification, Bax activation and apoptotic cell death. In conclusion we propose that, in mammalian cells, SIRT3 has a central role in connecting changes in ΔΨ_mt, intracellular pH and mitochondrial-regulated apoptotic pathways.     

DNA fragmentation factor 45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we measured the expression of DFF45 during mouse development and compared DNA fragmentation and viability of DFF45-deficient cells with wild-type control cells after activation of apoptosis. We found that DFF45 is ubiquitously expressed throughout mouse development. Moreover, DFF45-deficient thymocytes are resistant to DNA fragmentation with in vivo dexamethasone treatment. Furthermore, primary thymocytes from DFF45 mutant mice are also more resistant to apoptosis than wild-type control cells on exposure to several apoptotic stimuli. Dying DFF45-deficient thymocytes exhibit different morphology than wild-type control cells in that they show reduced degree of chromatin condensation, absent nuclear fragmentation, intranuclear cytoplasmic invagination, and striking nuclear chromatin conglutination after release from disintegrating cells. These results indicate that DFF45 is essential during normal apoptosis.     

Uptake of dehydroepiandrosterone-3-sulfate by isolated trophoblasts from human term placenta, JEG-3, BeWo, Jar, BHK cells, and BHK cells transfected with human sterylsulfatase-cDNA

The human placenta lacks the enzyme 17-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens. Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood. To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of [³H]-/[³⁵S]-DHEA-S and [³H]-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied. Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured.

During the first 5 min of incubation with [³H]-DHEA-S or [³⁵S]-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase. Radioactivity of BeWo cells increased slightly. For all cell types, there was no significant difference for uptake of either substrate. During incubation with [³H]-taurocholate, radioactivity of cytotrophoblasts did not increase. Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension. Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension. In BHK and Jar cells sterylsulfatase activity was not detectable.

Cytotrophoblasts take up DHEA-S without prior hydrolysis. BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S. In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited. Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides.     

Glutaminolysis and glycolysis regulation by troglitazone in breast cancer cells: Relationship to mitochondrial membrane potential

We studied the roles of glycolysis and glutaminolysis following an acute reduction in mitochondrial membrane potential (Ψ_m) induced by the thiazolidinedione troglitazone (TRO) and compared the responses with CCCP‐induced depolarization in breast cancer derived MCF‐7 and MDA‐MB‐231 cells as well as in the MCF‐10A normal breast cell line. TRO and CCCP both acutely reduced Ψ_m but after 24 h TRO‐treated cells had restored Ψ_m associated with both increased glycolysis and glutaminolysis. In contrast, CCCP‐treated cells exhibited only a partial restoration of Ψ_m associated with increased glycolysis but decreased glutaminolysis. TRO‐induced glutaminolysis was coupled to increased ammonium (GDH flux) and decreased alanine production (ALT flux) in all three cell lines. Both cancer cell lines exhibited a higher spontaneous GDH/ALT flux than the normal breast cell line associated with a reduced keto‐acid pool. TRO's effect on GDH/ALT fluxes and mitochondrial keto‐acid pool homeostasis was additive with glucose withdrawal suggesting limited intramitochondrial pyruvate availability. The TRO‐induced acceleration in GDH flux supplies substrate for Complex I contributing to the restoration of Ψ_m as well as Krebs cycle intermediates for biosynthesis. Inhibiting mitochondrial proton ATPase with oligomycin or nullifying the proton gradient with CCCP prevented both the TRO‐induced recovery of Ψ_m and accelerated GDH flux but restored ALT flux consonant with important roles for proton pumping in regulating GDH flux and Ψ_m recovery. Blocking enhanced GDH flux reduced DNA synthesis consistent with glutaminolysis via GDH playing an important biosynthetic role in tumorigenesis. J. Cell. Physiol. 226: 511–519, 2011. © 2010 Wiley‐Liss, Inc.     

HSP70 protects H9C2 cells from hypoxia and reoxygenation injury through STIM1/IP3R

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Calcium overload is an important factor in myocardial ischemia–reperfusion injury and can lead to apoptosis of myocardial cells. Therefore, it is of great clinical importance to find ways to regulate calcium overload and reduce apoptosis of myocardial cells, and thus alleviate myocardial ischemia–reperfusion injury. There is evidence that heat shock protein 70 (HSP70) has a protective effect on the myocardium, but the exact mechanism of this effect is not completely understood. Stromal interaction molecule 1 and inositol 1,4,5-triphosphate receptor (STIM/1IP3R) play an important role in myocardial ischemia–reperfusion injury. Therefore, this study aimed to investigate whether HSP70 plays an anti-apoptotic role in H9C2 cardiomyocytes by regulating the calcium overload pathway through STIM1/IP3R. Rat H9C2 cells were subjected to transient oxygen and glucose deprivation (incubated in glucose-free medium and hypoxia for 6 h) followed by re-exposure to glucose and reoxygenation (incubated in high glucose medium and reoxygenation for 4 h) to simulate myocardial ischemia reperfusion-induced cell injury. H9C2 cell viability was significantly decreased, and lactate dehydrogenase (LDH) release and apoptosis were significantly increased after oxygen and glucose deprivation. Transfection of HSP70 into H9C2 cells could reduce the corresponding effect, increase cell viability and anti-apoptotic signal pathway, and reduce the apoptotic rate and pro-apoptotic signal pathway. After hypoxia and reoxygenation, the expression of STIM1/IP3R and intracellular calcium concentration of HSP70-overexpressed H9C2 cells were significantly lower than those of hypoxia cells. Similarly, direct silencing of STIM1 by siRNA significantly increased cell viability and expression of anti-apoptotic protein Bcl-2 and decreased apoptosis rate and expression of pro-apoptotic protein BAX. These data are consistent with HSP70 overexpression. These results suggest that HSP70 abrogates intracellular calcium overload by inhibiting upregulation of STIM1/IP3R expression, thus reducing apoptosis in H9C2 cells and playing a protective role in cardiomyocytes.     

Human and hamster procathepsin D, although equally tagged with mannose-6-phosphate, are differentially targeted to lysosomes in transfected BHK cells

BHK cells transfected with human cathepsin D (CD) cDNA normally segregate the autologous hamster cathepsin D while secreting a large proportion of the human proenzyme. In the present work, we have utilized these transfectants to examine to what extent the mannose-6-phosphate-dependent pathway for lysosomal enzyme segregation contributes to the differential sorting of human and hamster CD. We report that, in recipient control BHK cells, the rate of mannose-6-phosphate-dependent endocytosis of human procathepsin D secreted by transfected BHK cells is lower than that of hamster procathepsin D and much lower than that of human arylsulphatase A. The missorted human enzyme bears phosphorylated oligosaccharides and most of its phosphate residues are “uncovered”, like the autologous enzyme. Thus, despite both the Golgi-associated modifications of oligosaccharides, i.e. the phosphorylation of mannose and the uncovering of mannose-6-phosphate residues, which proceed on human and hamster procathepsin D with comparable efficiency, only the latter is accurately packaged into lysosomes. Ammonium chloride partially affects the lysosomal targeting of cathepsin D in control BHK cells, whereas in transfected cells, this drug strongly inhibits the maturation of human procathepsin D and slightly enhances its secretion. These data indicate that: (1) over-expression of a lysosomal protein does not saturate the Golgi-associated reactions leading to the synthesis of mannose-6-phosphate; (2) a portion of cathepsin D is targeted independently of mannose-6-phosphate receptors in the transfected BHK cells; and (3) whichever mechanism for lysosomal delivery of autologous procathepsin D is involved, this is not saturated by the high rate of expression of human cathepsin D.     

Reactions to children's faces: Males are more affected by resemblance than females are, and so are their brains

The detection of genetic relatedness (i.e., kinship) affects the social, parental, and sexual behavior of many species. In humans, self-referent phenotype matching based on facial resemblance may indicate kinship, and it has been demonstrated that facial resemblance increases perceptions of trustworthiness and attractiveness [Proc. R. Soc. Lond., B Biol. Sci. 269 (2002) 1307–1312; Proc. R. Soc. Lond., B Biol. Sci. (in press)]. However, investigations of sex differences in reaction to facial resemblance have produced mixed results [Evol. Hum. Behav. 25 (2004) 142–154; Evol. Hum. Behav. 23 (2002) 159–166; Evol. Hum. Behav. 24 (2003) 81–87]. Here, we replicate the effects of Platek et al. [Evol. Hum. Behav. 23 (2002) 159–166] using high-resolution color morphing. We also extend these findings using functional magnetic resonance imaging (fMRI) to demonstrate a possible neural mechanism that may account for the observed sex difference. These data support the hypothesis that human males may use and favor facial resemblance as a paternity cue.     

Elevation of resting mitochondrial membrane potential of neural cells by cyclosporin A, BAPTA-AM, and bcl-2

This study testedthe hypothesis that the activity of the mitochondrial membranepermeability transition pore (PTP) affects the resting mitochondrialmembrane potential () of normal, healthy cells and that theanti-apoptotic gene product Bcl-2 inhibits the basal activity of thePTP. was measured by both fluorometric and nonfluorometricmethods with SY5Y human neuroblastoma cells and with GT1-7hypothalamic cells and PC12 pheochromocytoma cells in the absence andpresence of Bcl-2 gene overexpression. The resting of Bcl-2nonexpressing PC12 and wild-type SY5Y cells was increased significantlyby the presence of the PTP inhibitor cyclosporin A (CsA) or byintracellular Ca²⁺ chelation through exposure to theacetoxymethyl ester of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA-AM). The of Bcl-2-overexpressing PC12 cells was largerthan that of Bcl-2-negative cells and not significantly increased byCsA or by Ca²⁺ chelation. CsA did not present a significanteffect on the monitored in unstressed GT1-7 cells but didinhibit the decrease in elicited by the addition oft-butyl hydroperoxide, an oxidative inducer of themitochondrial permeability transition. These results support thehypothesis that an endogenous PTP activity can contribute to loweringthe basal of some cells and that Bcl-2 can regulate theendogenous activity of the mitochondrial PTP.

     

Reconstitution of CKMT1 expression fails to rescue cells from mitochondrial membrane potential dissipation: Implications for controlling RNAi experiments

RNA interference (RNAi) is an essential method in molecular biology to reduce the expression of target genes and thereby determine their function. Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects. We show here that the knock-down of the mitochondrial creatine kinase-1 (CKMT1) by RNA interference causes the dissipation of the mitochondrial membrane potential ΔΨ_m. This was accomplished with 11 different RNAi constructs designed to target 7 distinct exons as well as exon/intron junctions making unspecific off-target effects unlikely. However, all our attempts failed to rescue human cells from ΔΨ_m dissipation by the expression of CKMT1 alleles not targeted by RNAi. This included the transient and stable expression of the murine CKMT1 homologue, the expression of human codon usage-modified alleles, the transfection of a novel splice-isoform of CKMT1, and even the introduction of a human genomic clone for CKMT1 with codon usage changes. Our results indicate that while off-target effects of RNA interference can easily be addressed, the rescue of the knock-down phenotype is not necessarily achievable.     

Plasma membrane potential oscillations in insulin secreting Ins-1 832/13 cells do not require glycolysis and are not initiated by fluctuations in mitochondrial bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

Regulation of mitochondrial morphology by membrane potential,and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.     

Cardioprotection by isosteviol derivate JC105: A unique drug property to activate ERK1/2 only when cells are exposed to hypoxia‐reoxygenation

In the present study, we have investigated potential cardioprotective properties of Isosteviol analogue we recently synthesized and named JC105. Treatment of heart embryonic H9c2 cells with JC105 (10 μM) significantly increased survival of cells exposed to hypoxia‐reoxygenation. JC105 (10 μM) activated ERK1/2, DRP1 and increased levels of cardioprotective SUR2A in hypoxia‐reoxygenation, but did not have any effects on ERK1/2, DRP1 and/or SUR2A in normoxia. U0126 (10 μM) inhibited JC105‐mediated phosphorylation of ERK1/2 and DRP1 without affecting AKT or AMPK, which were also not regulated by JC105. Seahorse bioenergetic analysis demonstrated that JC105 (10 μM) did not affect mitochondria at rest, but it counteracted all mitochondrial effects of hypoxia‐reoxygenation. Cytoprotection afforded by JC105 was inhibited by U0126 (10 μM). Taken all together, these demonstrate that (a) JC105 protects H9c2 cells against hypoxia‐reoxygenation and that (b) this effect is mediated via ERK1/2. The unique property of JC105 is that selectively activates ERK1/2 in cells exposed to stress, but not in cells under non‐stress conditions.     

Characterization of a BHK(TK-) cell clone resistant to postattachment entry by herpes simplex virus types 1 and 2.

BHK(TK-) cells selected for resistance to polyethylene glycol-mediated fusion give rise to clones that are resistant to herpes simplex virus (HSV) infection. We have characterized one such clone, designated 95-19, and found that it is resistant to entry of HSV type 1 (HSV-1), HSV-2, and the related alphaherpesvirus pseudorabies virus (PRV). Single-step growth experiments show no detectable replication of multiple strains of HSV-1 and HSV-2 on 95-19 cells. Three lines of evidence suggest that these cells are resistant to postattachment entry. (i) Measurements of binding of radiolabeled virus show that heparin-sensitive binding of HSV-1 and HSV-2 to 95-19 cells is identical to binding to BHK(TK-) cells, suggesting that the block to replication occurs after attachment to heparan sulfate proteoglycan. (ii) 95-19 cells exposed to HSV-1 or HSV-2 at high multiplicity show no detectable immediate-early (IE) mRNA expression. (iii) Exposure of attached virus and cells to polyethylene glycol results in partial recovery of both IE gene expression and virus yield in single-step growth. The degrees of recovery of single-step yield and IE gene expression are similar, suggesting that the only block to single-step replication is at the point of virus entry and that these cells are deficient in some cellular factor required for efficient postattachment entry of free virus. 95-19 cells are also highly resistant to entry by cell-to-cell spread, suggesting that the same cellular factor participates in both types of entry.     

RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells

17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E(2) rapidly increased both the phosphorylation of ERK2 and Ser(112) in BAD. Ser(112) is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E(2)-induced phosphorylation of Ser(112). Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.     

Human DU145 prostate cancer cells overexpressing mitogen-activated protein kinase phosphatase-1 are resistant to Fas ligand-induced mitochondrial perturbations and cellular apoptosis

Recent studies have suggested that MAP kinase phosphatase 1 (MKP-1) is overexpressed in prostate cancer. To evaluate the role of MKP-1 in regulating cell death and tumor growth in prostate cancer, MKP-1 was conditionally overexpressed in the human prostate cancer cell line DU145. Overexpression of MKP-1 in DU145 cells blocked activation of stress-activated protein kinase (SAPK/JNK). MKP-1 overexpression in DU-145 cells was also found to inhibit Fas ligand (FasL)-induced apoptosis, as well as block the activation of caspases by Fas engagement. In addition, MKP-1 blocked the activation of apoptosis by transfected MEKK-1 and ASK-1, presumably through its inhibition of the SAPK/JNK family of enzymes. MKP-1 blocked the ability of FasL to induce loss of mitochondrial transmembrane potential (_m), suggesting that MKP-1 acts upstream of mitochondrial pro-apoptotic events induced by FasL and that the SAPK/JNK pathway may form the signaling link between Fas receptor and mitochondrial dysfunction. Thus, MKP-1 overexpression in prostate cancer may play a role in promoting prostate carcinogenesis by inhibiting FasL-induced cell death.