A novel caspase dependent pathway is involved in apoptosis of human endothelial cells by Shiga toxins

Shiga toxins have been shown to induce apoptosis on primary cultures, but not passaged ones, of human umbilical vein endothelial cells, independent of cytokine pre-treatment. Here, a peculiar pattern of caspase activation was observed; caspase-3 and -2, but not conventional upstream caspases, were activated at the initial phase of 6 hr, whereas a broad range inhibitor of caspases, VAD-fmk, but not mono-specific ones, suppressed DNA fragmentation and cell death. These results suggest additional analogous molecules, which have yet to be delineated, are involved. The requirement of retrograde uptake of toxins was also proved by the intervening effect of brefeldin A.     

Karyoptosis: A novel type of cell death caused by chronic autophagy inhibition

Macroautophagy/autophagy influences onset and progression of several human neurodegenerative diseases, because of its critical role as a regulator of neuronal proteostasis and organelle quality control. In many neurodegenerative diseases, impairment in autophagy is thought to play a fundamental part in the terminal phases of cellular degeneration and death. However, the ultimate mechanism of neuronal cell death remains elusive. In a recent study we have identified a new form of regulated cell death, which arises upon autophagy inhibition.     

Neuronal cell death caused by inhibition of intracellular cholesterol trafficking is caspase dependent and associated with activation of the mitochondrial apoptosis pathway

An elevated level of cholesterol in mitochondrial membranes of Niemann-Pick disease type C1 (NPC1) mouse brains and neural cells has been found to cause mitochondrial dysfunction. In this study, we demonstrate that inhibition of intracellular cholesterol trafficking in primary neurons by class 2 amphiphiles, which mimics the major biochemical and cellular feature of NPC1, led to not only impaired mitochondrial function but also activation of the mitochondrial apoptosis pathway. In activation of this pathway both cytochrome c and Smac/Diablo were released but apoptosis-inducing factor (AIF) was not involved. Treatment of the neurons with taurine, a caspase 9-specific inhibitor, could prevent the amphiphile-induced apoptotic cell death, suggesting that formation of apoptosome, followed by caspase 9 and caspase 3 activation, might play a critical role in the neuronal death pathway. Taken together, the mitochondria-dependent death cascade induced by blocking intracellular cholesterol trafficking was caspase dependent. The findings provide clues for both understanding the molecular basis of neurodegeneration in NPC1 disease and developing therapeutic strategies for treatment of this disorder.     

A caspase-3-dependent pathway is predominantly activated by the excitotoxin pregnenolone sulfate and requires early and late cytochrome c release and cell-specific caspase-2 activation in the retinal cell death

This study investigates the implication of mitochondria- and caspase-dependent pathways in the death of retinal neurones exposed to the neurosteroid pregnenolone sulfate (PS) shown to evoke apoptosis and contribute to amplification and propagation of excitotoxicity. After a brief PS challenge of intact retinas, caspase-3 and caspase-2 activation and cytochrome c release occur early and independent of changes in the oxidative state measured by superoxide dismutase activity. The temporal and spatial relationship of these events suggests that a caspase-3-dependent pathway is activated in response to cytochrome c release and requires caspase-2 activation and a late cytochrome c release in specific cellular subsets of retinal layers. The protection by caspase inhibitors indicates a predominant role of the pathway in PS-induced retinal apoptosis, although a limited use of caspase inhibitors is upheld on a conceivable shift from apoptosis toward necrosis. Conversely, 3alpha-hydroxy-5beta-pregnan-20-one sulfate and 17beta-oestradiol provide complete prevention of PS-induced retinal death.     

Down-regulation of Mcl-1 by inhibition of the PI3-K/Akt pathway is required for cell shrinkage-dependent cell death.

The anti-apoptotic effect of a chloride-bicarbonate exchange blocker has been previously examined in endothelial cells and cardiomyocytes. However, the anti-apoptotic effects of this blocker on epithelial cells and the mechanism of the anti-apoptotic effect remain unknown. We examined the anti-apoptotic effects of a chloride-bicarbonate exchange blocker in a renal epithelial cell line (MDCK cells). Changes in the expression of bcl-2 family proteins, which are known to have anti-apoptotic effects, were also examined. Staurosporine was used to induce apoptotic cell death in the MDCK cells. Staurosporine treatment was sufficient to induce apoptotic cell death, detected by propidium iodide and DNA ladder formation. A chloride-bicarbonate exchange blocker was added 24 h before the staurosporine treatment and during treatment. The chloride-bicarbonate exchange blocker inhibited the staurosporine-induced apoptosis in the MDCK cells in a dose-dependent manner. The expression of bcl-2 family gene products was detected by RT-PCR and Western blotting. No changes in the expression of Bax, Bid and Bik (pro-apoptotic proteins), or Bcl-2 (an anti-apoptotic protein) were detected. However, Mcl-1 expression was reduced by the staurosporine treatment, and this reduction was recovered when the chloride-bicarbonate exchange blocker was added. LY294002, a PI 3-kinase inhibitor, partially inhibited this anti-apoptotic effect. In conclusion, chloride-bicarbonate exchange blockers appear to offer cell-protective effects via Mcl-1 up-regulation.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.     

Inverse and distinct modulation of tau-dependent neurodegeneration by presenilin 1 and amyloid-beta in cultured cortical neurons: evidence that tau phosphorylation is the limiting factor in amyloid-beta-induced cell death

Alzheimer's disease (AD) is characterized by massive neuron loss in distinct brain regions, extracellular accumulations of the amyloid precursor protein-fragment amyloid-beta (A beta) and intracellular tau fibrils containing hyperphosphorylated tau. Experimental evidence suggests a relation between presenilin (PS) mutations, A beta formation, and tau phosphorylation in triggering cell death; however, how A beta and PS affect tau-dependent degeneration is unknown. Using herpes simplex virus 1-mediated gene-transfer of fluorescent-tagged tau constructs in primary cortical neurons, we demonstrate that tau expression exerts a neurotoxic effect that is increased with a construct mimicking disease-like hyperphosphorylation [pseudohyperphosphorylated (PHP) tau]. Live imaging revealed that PHP tau expression is associated with increased perikarya suggesting the development of a 'ballooned' phenotype as a specific feature of tau-mediated cell death. Transgenic expression of PS1 suppressed tau-induced neurodegeneration. In contrast, A beta amplified degeneration in the presence of wt tau but not of PHP tau. The data indicate that PS1 and A beta inversely modulate tau-dependent neurodegeneration at distinct steps. They indicate that the mode by which PHP tau causes neurotoxicity is downstream of A beta and that tau phosphorylation is the limiting factor in A beta-induced cell death. Suppression of tau expression or inhibition of tau phosphorylation at disease-relevant sites may provide an effective therapeutic strategy to prevent neurodegeneration in Alzheimer's disease.     

Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C

PKCtheta plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-kappaB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCtheta to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCtheta, we addressed here the question of whether the translocation and activation of PKCtheta in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCalpha. Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it. These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.     

Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.