Caspase-3 mediates in part hippocampal apoptosis in sepsis

The brain is one of the first organs affected during sepsis development resulting in apoptosis for a short-term and cognitive impairment for a long-term. Despite its importance, the mechanisms of brain dysfunction during sepsis are not fully elucidated. Thus, we here, in an animal model of sepsis, evaluated apoptosis in the dentate gyrus cell layer of the hippocampus to document the involvement of caspase-3 in the pathogenesis of neuronal apoptosis. Wistar rats sham-operated or submitted to the cecal ligation and perforation (CLP) procedure were killed at 12, 24, 48 h, and 10 days after surgery for the determination of caspase-3 and apoptosis rate. In a separate cohort of animals, a caspase-3-specific inhibitor was administered and animals were killed at 12 h after sepsis. An increase in the number of apoptotic cells 12, 24, and 48 h by histopathological evaluations and an increase of caspase-3 apoptotic cells 12 and 24 h after sepsis induction were observed. The caspase-3 inhibitor decreases the number of apoptotic cells by histopathological evaluations but not by immunohistochemistry evaluations. Caspase-3 is involved in part in apoptosis in the dentate gyrus cell layer of the hippocampus in septic rats submitted by CLP.

Epidermal caspase-3 cleavage associated with interferon-gamma-expressing lymphocytes in acute atopic dermatitis lesions

Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is believed to play an important role in the pathogenesis of atopic dermatitis (AD), in particular for the formation of spongiosis. To estimate epidermal caspase activation in normal and AD skin under in vivo conditions, we analysed caspase-3 cleavage by immunohistology. In normal skin as well as non-lesional AD skin, we detected caspase-3 cleavage in single cells of the basal layer. In contrast, in acute lesional AD skin, we not only obtained evidence for increased expression of cleaved caspase-3 in keratinocytes of the basal layer but also observed caspase-3 cleavage in one or more layers of the spinous cell layer, in particular in spongiotic areas. Short-term topical treatment of the skin lesions with tacrolimus or pimecrolimus abolished the expression of cleaved caspase-3 in the spinous layer. Moreover, epidermal caspase-3 cleavage correlated with the numbers of dermal interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ lymphocytes in skin lesions of AD patients, supporting the view that IFN-gamma is important for the activation of proapoptotic pathways in keratinocytes. This is also confirmed by the observation of increased Fas expression on keratinocytes in acute AD lesions that was markedly reduced following topical calcineurin inhibitor treatment. These data suggest that caspase-3 cleavage in the spinous layer of the epidermis is a pathologic event contributing to spongiosis formation in AD, whereas cleavage of caspase-3 in basal cells might represent a physiologic mechanism within the process of epidermal renewal.

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

AIMS: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single-stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. METHODS: We studied detection of active caspase 3 and single-stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. RESULTS: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single-stranded DNA have differential sensitivities in HCC and CRC. CONCLUSION: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single-stranded DNA may be tumour cell type dependent.

Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.

Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.

TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by doxorubicin and TNF-alpha co-treatments

The present dataset contains the source data for Figure 2B of Tentner et al. (2012). The data shows the percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by various doses of doxorubicin (0, 2 and 10 µmole/L) in the presence (100 ng/mL) or absence (0 ng/mL) of TNF-alpha co-treatment. For the six treatment conditions investigated, cell counts were made by flow cytometry at times 6, 12, 24, and 48 h following treatment; CULTURE DETAILS: U2OS cells were obtained from ATCC were maintained at 21% oxygen and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2mM L-glutamine, and used within 15-20 passages. The first thymidine block was released by washing the plates three times with PBS, and incubating them in fresh thymidine-free media for 12 h. A second thymidine block was then performed by re-addition of thymidine to 2.5 mM followed by incubation for an additional 18 h. Media was aspirated, plates were washed 3 with PBS, and replaced with fresh media in the presence or absence of 10 mM aphidicolin; ANALYSIS DETAILS: See supplementary journal publication; RESULT: The authors of the supplementary journal publication conclude that TNF enhances dose-dependent cell death following doxorubicin-induced DNA damage with minimal affect on dose-dependent cell-cycle arrest.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1−/−, and caspase-3−/− hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3−/− hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1−/− apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3−/− hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3−/− thymocytes exhibited similar “abnormal” morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

Epistatic and independent functions of Caspase-3 and Bcl-XL in developmental programmed cell death

The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-XL directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-XL in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-ras-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G1 apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the CASP-3 gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome c into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome c from the mitochondria resulting in caspase 3 activation.

Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp “safety-catch” regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this “safety catch” results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.

Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death

The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.

Apoptosis in experimental cerebral malaria: spatial profile of cleaved caspase-3 and ultrastructural alterations in different disease stages

Cerebral malaria (CM) is associated with high mortality and morbidity as a certain percentage of survivors suffers from persistent neurological sequelae. The mechanisms leading to death and functional impairments are yet not fully understood. This study investigated biochemical and morphological markers of apoptosis in the brains of mice infected with Plasmodium berghei ANKA. Cleaved caspase-3 was detected in the brains of animals with clinical signs of CM and immunoreactivity directly correlated with the clinical severity of the disease. Caudal parts of the brain showed more intense immunoreactivity for cleaved caspase-3. Double-labelling experiments revealed processing of caspase-3 primarily in neurons and oligodendrocytes. These cells also exhibited apoptotic-like morphological profiles in ultrastructural analysis. Further, cleavage of caspase-3 was found in endothelial cells. In contrast to neurons and oligodendrocytes, apoptosis of endothelial cells already occurred in early stages of the disease. Our results are the first to demonstrate processing of caspase-3 in different central nervous system cells of animals with CM. Apoptosis of endothelial cells may represent a critical issue for the development of the disease in the mouse model. Neurological signs and symptoms might be attributable, at least in part, to apoptotic degeneration of neurons and glia in advanced stages of murine CM.