Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.     

C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.     

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.     

Caspase activation inhibits proteasome function during apoptosis

The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.     

Regulation of caspase activity in apoptosis

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of neuron death in neurodegenerative diseases.     

Cooperative forms of granule cell and Purkinje cell activity in the frog cerebellar cortex

Spatial and temporal characteristics of functional relations between granule cells and Purkinje cells were studied by multimicroelectrode recording of activity of two or more neurons and statistical analysis of the results. On the arrival of mono- and polymodal afferent volleys, excited granule cells and Purkinje cells were shown to organize themselves into cooperative groups (elementary ensembles) of neurons measuring 200–300 and 300–400 µ, respectively. Elementary ensembles of these neurons are regarded as components of functional units which process information in the frog cerebellar cortex. Some of the special features connected with the cooperative principle of their organization and activity were investigated.Rostov State University. Institute of Cybernetics, Hanoi. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 171–176, March–April, 1977.     

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.     

Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.     

TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB

Tumor necrosis factor receptor-associated factor 6 (TRAF6) functions as an adaptor, positively regulating the NF-kappaB pathway. Here we report a new function of human TRAF6, the direct stimulation of apoptosis. The mechanism of apoptosis induction results from the capacity of human TRAF6 to interact and activate caspase 8. Both the C-terminal TRAF domain of human TRAF6, which directly interacts with the death effector domain of pro-caspase 8, and the N-terminal RING domain, which is required for activation of caspase 8, are necessary for the induction of apoptosis. The role of endogenous TRAF6 in regulating apoptosis was confirmed by extinguishing TRAF6 expression with specific small-hairpin RNA that resulted in diminished spontaneous apoptosis and resistance to induced apoptosis. In contrast to the human molecule, murine TRAF6 displayed less ability to induce apoptosis and a greater capacity to stimulate NF-kappaB activity. Human and murine TRAF6 are similar except in the region between zinc finger 5 and the TRAF domains. Reciprocal transfer of this connecting region completely exchanged the ability of human and murine TRAF6 to induce apoptosis and activate NF-kappaB. Unique regions of TRAF6 therefore play an important role in determining cell fate.     

Caspase 6 regulates B cell activation and differentiation into plasma cells

Caspase (Casp) family proteases regulate not only lymphocyte apoptosis but also lymphocyte activation and development. In this study, we show that Casp6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry. B cells from Casp6 knockout (Casp6 KO) mice examined ex vivo have more cells in G(1) than wild-type B cells, and mitogen-induced G(1) entry of Casp6 KO B cells is much faster than that of wild-type B cells. Even so, S phase entry and proliferation are not increased in Casp6 KO B cells. Rather than proliferating, activated Casp6 KO B cells preferentially differentiate into syndecan-1(+) plasma cells and produce Abs. In Casp6 KO mice compared with WT mice, serum levels of IgG1, IgG2a, and IgG2b are increased and Ag-specific Ab responses are also enhanced along with increased percentages of syndecan-1(+) plasma cells. Casp6 may regulate both B cell activation and differentiation by modifying requirements for G(0) B cells to enter G(1).     

Effects of CB(1) cannabinoid receptor activation on cerebellar granule cell nitric oxide synthase activity.

Cerebellar granule cells (CGCs) express the CB(1) subtype of cannabinoid receptor. CB(1) receptor agonists Win 55212-2, CP55940 and HU210 inhibit KCl-induced activation of nitric oxide synthase (NOS) in CGCs. Win 55212-2 has no effect on either basal NOS activity or on activation by N-methyl-D-aspartate and its effect is abolished by pre-treatment of the cells with pertussis toxin. The CB(1) receptor antagonist/inverse agonist SR141716A both reverses the effects of Win 55212-2 and produces an increase in NOS activity that is additive with KCl. These results support the hypothesis that activation of the CB(1) receptor in CGCs results in a decreased influx of calcium in response to membrane depolarization, resulting in a decreased activation of neuronal NOS.     

Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L -cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O) and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation. J. Cell. Physiol. 177:324–333, 1998. © 1998 Wiley-Liss, Inc.     

6-hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3-dependent activation of protein kinase Cdelta

6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase Cdelta (PKCdelta) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKCdelta (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKCdelta (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.     

Caspase activation and apoptosis in response to proteasome inhibitors

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. In fact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.     

Expression of NALP1 in cerebellar granule neurons stimulates apoptosis

NAcht Leucine-rich-repeat Protein 1 (NALP1) contains a putative nucleotide binding site, a region of leucine-rich repeats, and death domain folds at both termini providing protein/protein association functions such as caspase recruitment. We report here that NALP1 gene expression was induced in primary cerebellar granule neurons (CGN) upon injury. Up-regulation of NALP1 was also observed in a model of transient focal ischemia induced by middle cerebral artery occlusion. We investigated the biological consequence of over-expression of NALP1 in both HeLa cells and in CGN. Expression of recombinant NALP1 stimulated cell death in both HeLa cells and CGN by an apoptotic mechanism, demonstrated by the induction of apoptotic nuclear morphology and activation of the apoptotic enzyme caspase-3. Also described here are studies on the mechanism of action studies including deletion analyses and investigations of nucleotide binding, which begin to elucidate a regulatory function for NALP1 in neuronal apoptosis.