Biomedical subjects

Aviva M Tolkovsky

Publications and source records attributed to Aviva M Tolkovsky.

12 recordsLinked to original sources

PubMed2006

Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells.

Oxidative stress is central to ischemia-reperfusion injury. The role of the endoplasmic reticulum (ER) in this process is uncertain. In ER signaling, PERK-Nrf2 and Ire-CHOP are two pathways that determine cell fate under stress. PERK-Nrf2 up-regulates antioxidant enzyme expression whereas Ire-CHOP promotes apoptosis. We have identified a novel pathway in ER stress-induced apoptosis after ischemia-reperfusion in vitro involving translational suppression of the survival kinase PKB/Akt (Akt), and elucidated an alternative protective role of antioxidants in the regulation of Akt activity. Using human choriocarcinoma JEG-3 cells, we found that sustained activation of ER stress by tunicamycin or thapsigargin exacerbated apoptosis in oxygen-glucose-deprived cells during reoxygenation. This was mediated via a reduction in phosphorylated Akt secondary to down-regulation of protein translation rather than suppression of phosphorylation. Transient overexpression of wild-type Akt, but not kinase-dead Akt, in JEG-3 cells diminished tunicamycin-OGD reoxygenation-induced apoptosis. The antioxidants Trolox and Edaravone reduced apoptosis, but the protective effect of Trolox was abrogated by the PI3K inhibitor, LY294002. We speculate that sustained ER stress may contribute to the placental dysfunction seen in human pregnancy complications.

Apoptosis↗

PubMed2006

The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons.

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.

Animals↗

PubMed2005

Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins.

We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with (32)Pi, whereas cells in which the kinase is active are labeled with (33)Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both (32)P- and (33)P-labeled proteins; the kinase-specific spots are revealed because of (33)P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic (33)P-labeled proteins while allowing the more energetic (32)P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons.

Acetates↗

PubMed2005

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.

Animals↗

PubMed2005

Osteonectin is a Schwann cell-secreted factor that promotes retinal ganglion cell survival and process outgrowth.

We have investigated the factors made by Schwann cells (SCs) that stimulate survival and neurite outgrowth from postnatal rat retinal ganglion cells (RGCs). These effects are preserved under K252a blockade of the Trk family of neurotrophin receptors and are not fully mimicked by the action of a number of known trophic factors. To identify novel factors responsible for this regenerative activity, we have used a radiolabelling assay. Proteins made by SCs were labelled radioactively and then fed to purified RGCs. The proteins taken up by the RGCs were then isolated and further characterized. Using this assay we have identified a major 40 kDa factor taken up by RGCs, which was microsequenced and shown to be the matricellular protein osteonectin (ON). Using an in vitro assay of purified RGCs we show that ON promotes both survival and neurite outgrowth. We conclude that ON has a potential new role in promoting CNS repair.

Animals↗

PubMed2005

Mutually exclusive subsets of BH3-only proteins are activated by the p53 and c-Jun N-terminal kinase/c-Jun signaling pathways during cortical neuron apoptosis induced by arsenite.

The c-Jun N-terminal protein kinase (JNK)/c-Jun and p53 pathways form distinct death-signaling modules in neurons that culminate in Bax-dependent apoptosis. To investigate whether this signaling autonomy is due to recruitment of particular BH3-only proteins, we searched for a toxic signal that would activate both pathways in the same set of neurons. We show that arsenite activates both the JNK/c-Jun and p53 pathways in cortical neurons, which together account for >95% of apoptosis, as determined by using the mixed-lineage kinase (JNK/c-Jun) pathway inhibitor CEP11004 and p53-null mice. Despite the coexistence of both pathways in at least 30% of the population, Bim mRNA and protein expression was increased only by the JNK/c-Jun signaling pathway, whereas Noxa and Puma mRNA and Puma protein expression was entirely JNK/c-Jun independent. About 50% of Puma/Noxa expression was p53 dependent, with the remaining signal being independent of both pathways and possibly facilitated by arsenite-induced reduction in P-Akt. However, functionally, Puma was predominant in mediating Bax-dependent apoptosis, as evidenced by the fact that more than 90% of apoptosis was prevented in Puma-null neurons, although Bim was still upregulated, while Bim- and Noxa-null neurons died similarly to wild-type neurons. Thus, the p53 and JNK/c-Jun pathways can activate mutually exclusive subclasses of BH3-only proteins in the same set of neurons. However, other factors besides expression may determine which BH3-only proteins mediate apoptosis.

Animals↗

PubMed2004

Aggravation of necrotic death of glucose-deprived cells by the MEK1 inhibitors U0126 and PD184161 through depletion of ATP.

The extracellular-regulated kinases (ERK) modulate cell proliferation and survival in response to several different stimuli and are therefore important drug targets. ERKs are activated by the dual phosphorylation kinase MEK1 and MEK1 inhibitors PD98059, U0126 and CI-1040 are now widely used to inhibit ERKs in cell and animal studies. In an analysis of ERK functions in astrocytes we found that PD98059 (100microM) failed to inhibit ERK phosphorylation but U0126 (50microM) inhibited ERK phosphorylation to approximately 80%. Surprisingly, U0126 also caused profound depletion of ATP in glucose-deprived cells, leading to death by necrosis. Since glucose-deprived cells depend mainly on mitochondrial ATP-synthase for ATP production, we tested whether U0126 or PD184161, a derivative of CI-1040, might inhibit ATP synthase activity, using 143B(Rho0) cells (which lack a functional F0 subunit) to further parse this effect. We found that the F1F0ATPase activity extracted from U0126- or PD184161-treated parental 143B cells or astrocytes was indeed inhibited by >or=80% suggesting a covalent change in the enzyme. However, F1F0ATPase activity extracted from similarly treated 143B(Rho0) cells was spared. Because F1F0ATPase activity in isolated mitochondria was not inhibited directly, we propose that U0126 and PD184161 inhibit ATP-synthase via an indirect action on F0. The MEK1 inhibitors also induced necrosis of other glucose-deprived cell types including primary neurons at the same concentrations required for inhibition of ERK phosphorylation. Thus, the MEK1/ERK signalling pathway may modulate ATP synthase function, and its inhibition may cause cells unable to perform glycolysis to die by necrosis.

Adenosine Triphosphate↗

PubMed2004

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways.

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.

Active Transport, Cell Nucleus↗

PubMed2003

Erasure of kinase phosphorylation in astrocytes during oxygen-glucose deprivation is controlled by ATP levels and activation of phosphatases.

We have examined the relationship between adenosine triphosphate (ATP) concentration and loss of maintenance of kinase-signalling cascades in primary cortical astrocytes during oxygen-glucose deprivation (OGD) as this may constitute an irreversible step that commits astrocytes to cell death. We report that the phosphorylation of Akt, ERK, JNK and p38 kinases, whose activities depend on serine, threonine and tyrosine phosphorylation, were all increased during OGD. All these phosphorylations were reduced to below detection limits when ATP levels were less than 10% of normal levels. Using ERK and Akt as representative examples, we show that this erasure is not irreversible as both ERK and Akt phosphorylations can be partially restored by addition of glucose under anoxic conditions. We further investigated whether OGD caused any change in phosphatase activity. The PP1/PP2A phosphatase inhibitors okadaic acid and caliculyn A, but not cyclosporine A, delayed the removal of ERK and Akt phosphorylation under OGD. By comparing the extent of phosphorylation increase under OGD and normoxic conditions, we calculate that phosphatase activity was increased by approximately 3.6-fold during OGD. These data show that ATP levels control an important checkpoint in kinase function, and that ATP levels may need to be considered when studies of kinase function in relation to OGD are conducted.

Adenosine Triphosphate↗

PubMed2002

Apoptosis-specific protein (ASP 45 kDa) is distinct from human Apg5, the homologue of the yeast autophagic gene apg5.

We have examined whether the apoptosis-specific protein p45ASP and human Apg5 are identical proteins. Like p45ASP, myc-hApg5 cross-reacted with a c-Jun antibody and approximately 50% of myc-hApg5 was bound to a Triton X-100-insoluble fraction in HeLa cells. However, soluble myc-hApg5 was degraded during apoptosis induced by staurosporine or TNFalpha/cycloheximide whilst expression of soluble p45ASP was stabilised. Furthermore, myc-hApg5 degradation was blocked by the caspase inhibitor Boc-Asp(OMe)FMK whilst p45ASP expression was eliminated. Moreover, myc-hApg5 ( approximately 32 kDa) never assumed the size of p45ASP (45 kDa). It is therefore likely that p45ASP and human Apg5 are distinct proteins although they do share some common characteristics.

Apoptosis↗

PubMed2002

Inhibition of mitochondrial permeability transition and release of cytochrome c by anti-apoptotic nucleoside analogues.

We have investigated whether nucleoside drugs that induce or protect neurones against apoptosis might directly activate or inhibit mitochondrial permeability transition (mPT) since opening of the mPT pore can promote release of cytochrome c and apoptosis, while its closure can prevent these changes. We found that the pro-apoptotic pyrimidine analogues cytosine beta-D-arabinofuranoside and cytosine beta-D-arabinofuranoside 5'-triphosphate, which activated apoptosis in post-mitotic neurones without incorporation into nuclear DNA, induced rapid calcium-dependent mitochondrial swelling of isolated liver mitochondria in a dose-dependent manner. Induction of up to 50 and 80%, respectively, of maximal swelling induced by high calcium was obtained at 1mM concentrations, which also promoted a 17-fold increase in the release of cytochrome c. Both activities were inhibited by cyclosporine A to unstimulated levels; dCTP had no effect. In contrast, the anti-apoptotic adenine analogues, 3-methyladenine (3-MA) and olomoucine (but not iso-olomoucine), inhibited swelling induced by calcium or phenylarsine oxide in a dose-dependent manner at concentrations that protect neurones from apoptosis. Both compounds also inhibited the release of cytochrome c (by 82%, 20 mM 3-MA and 95%, 0.9 mM olomoucine), similar to the inhibition obtained with cyclosporine A, and 5mM ADP or ATP. Similar inhibitory effects with olomoucine and 3-MA were found in isolated heart mitochondria. These studies identify the mPT as an important target for hitherto untested pro- and anti-apoptotic nucleoside-based drugs and suggest that screening for mPT modulation is an important component in the validation of a drug's mechanism of action.

Adenine↗

PubMedDate not supplied

Mitochondrial disappearance from cells: a clue to the role of autophagy in programmed cell death and disease?

When cells are induced to undergo apoptosis in the presence of general caspase inhibitors and then returned to their normal growth environment, there follows an extended period of life during which the entire cohort of mitochondria (including mitochondrial DNA) disappears from the cells. This phenomenon is widespread; it occurs in NGF-deprived sympathetic neurons, in NGF-maintained neurons treated with cytosine arabinoside, and in diverse cell lines treated with staurosporine, including HeLa, CHO, 3T3 and Rat 1 cells. Mitochondrial removal is highly selective since the structure of all other organelles remains unperturbed. Since Bcl2 overexpression blocks the removal of mitochondria without preventing death-inducing signals, it appears that the mitochondria are responsible for initiating their own demise. Degradation of mitochondria is not in itself a rare event. It occurs in large part by autophagy during normal cell house-keeping, during ecdysis in insects, as well as after induction of apoptosis. However, the complete and selective removal of an entire cohort of mitochondria in otherwise living mammalian cells has not been described previously. These findings raise several questions. What are the mechanisms which remove mitochondria in such a 'clean' fashion? What are the signals that target mitochondria for such selective degradation? How are cells that have lost their mitochondria different from rho0 cells (which retain mitochondria but lack mitochondrial DNA, and cannot carry out oxidative phosphorylation)? Are the cells which have lost mitochondria absolutely committed to die or might they be repaired by mitochondrial therapy? The answers will be especially relevant when considering treatment of diseases affecting long-lived and non-renewable organs such as the nervous system.

Apoptosis↗