Caspase Cell Death Research Articles

The Caspase-Activated DNase drives inflammation and contributes to defense against viral infection

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection.

Unravelling potential biomedical applications of the edible flower Tulbaghia violacea

Traditionally, edible flowers have been used as foods and for therapeutic purposes, today they have gained importance due to their bioactive compounds such as flavonols, anthocyanins or other phenolic compounds, which give them potential for biomedical applications. This work evaluated a methanolic extract of Tulbaghia violacea. Eleven individual phenolic compounds were found and quantified by mass spectrometry in the extract. Antioxidant activity tests (TEAC, FRAP and DPPH) and other characterization parameters were assayed (total phenolic content and total flavonoid content). In vitro studies showed antitumoral activity against ovarian tumoral cells mediated by the induction of non-dependent caspase cell death and by the activation of reactive oxygen species. The effect of the extract against features of Alzheimer disease was in vivo assayed in Caenorhabditis elegans. Tulbaghia extract led to a reduction in the 1–42 beta amyloid peptide formation and prevented oxidative stress. These results suggested that Tulbaghia violacea could be a new source of phenolic compounds for nutraceuticals and functional food development.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Hippocampal TNF-death receptors, caspase cell death cascades, and IL-8 in alcohol use disorder

The relationship between increased neuroimmune gene expression and hippocampal degeneration in alcohol use disorder (AUD) and other mental diseases is poorly understood. We report here that tumor necrosis factor receptor superfamily death receptor 3 (TNFRSF25, DR3) and Fas receptors (Fas) that initiate caspase cell death cascades are increased in AUD hippocampus and following a rat adolescent binge drinking model. Death receptors are known inducers of apoptosis and cell death that recruit death domain (DD) proteins FADD and TRADD and caspases to form death-inducing signaling complexes (DISC). In postmortem human AUD hippocampus, mRNA and IHC protein are increased for the entire death receptor cascade. In AUD hippocampus, ligand–death receptor pairs, i.e., TL1A-DR3 and FasL–Fas, were increased, as well as FADD and TRADD, and active caspase-8, -7, -9, and caspase-3. Further, pNFκB p65, a key neuroimmune transcription factor, and IL-8, a chemokine, were significantly increased. Interestingly, across AUD patients, increases in DR3 and Fas correlated with TRADD, and TRADD with active caspase+IR and IL-8+IR, consistent with coordinated activation of neuronal DISC mediated death cascades and neuroimmune gene induction in AUD. These findings support a role for DR3 and Fas neuroimmune signaling in AUD hippocampal neurodegeneration.

Caspases in Cell Death, Inflammation, and Pyroptosis.

Caspases are a family of conserved cysteine proteases that play key roles in programmed cell death and inflammation. In multicellular organisms, caspases are activated via macromolecular signaling complexes that bring inactive procaspases together and promote their proximity-induced autoactivation and proteolytic processing. Activation of caspases ultimately results in programmed execution of cell death, and the nature of this cell death is determined by the specific caspases involved. Pioneering new research has unraveled distinct roles and cross talk of caspases in the regulation of programmed cell death, inflammation, and innate immune responses. In-depth understanding of these mechanisms is essential to foster the development of precise therapeutic targets to treat autoinflammatory disorders, infectious diseases, and cancer. This review focuses on mechanisms governing caspase activation and programmed cell death with special emphasis on the recent progress in caspase cross talk and caspase-driven gasdermin D-induced pyroptosis.

Proapoptotic function of deubiquitinase DUSP31 in Drosophila.

Drosophila have been used to identify new components in apoptosis regulation. The Drosophila protein Dark forms an octameric apoptosome complex that induces the initiator caspase Dronc to trigger the caspase cell death pathway and, therefore, plays an important role in controlling apoptosis. Caspases and Dark are constantly expressed in cells, but their activity is blocked by DIAP1 E3 ligase-mediated ubiquitination and subsequent inactivation or proteasomal degradation. One of the regulatory mechanisms that stabilize proapoptotic factors is the removal of ubiquitin chains by deubiquitinases. In this study performed a modified genetic screen for deubiquitinases (dsRNA lines) to identify those involved in stabilizing proapoptotic components. Loss-of-function alleles of deubiquitinase DUSP31 were identified as suppressors of the Dronc overexpression phenotype. DUSP31 deficiency also suppresses apoptosis induced by the RHG protein, Grim. Genetic analysis revealed for the first time that DUSP31 deficiency sufficiently suppresses the Dark phenotype, indicating its involvement in the control of Dark/Dronc apoptosome function in invertebrate apoptosis.

Impact of caspase-1/11, -3, -7, or IL-1\u03b2/IL-18 deficiency on rabies virus-induced macrophage cell death and onset of disease

Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn–Rotnycki–Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1β and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1β and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1β and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection.

Neuroprotective effect of acute melatonin treatment on hippocampal neurons against irradiation by inhibition of caspase-3.

Neuronal cell apoptosis is associated with various factors that induce neurological damage, including radiation exposure. When administered prior to exposure to radiation, a protective agent may prevent cellular and molecular injury. The present study aimed to investigate whether melatonin exerts a neuroprotective effect by inhibiting the caspase cell death pathway. Male Sprague-Dawley rats were administered melatonin (100 mg/kg body weight) 30 min prior to radiation exposure in red light during the evening. In order to elucidate whether melatonin has a neuroprotective role, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling, Nissl staining, reverse transcription-quantitative polymerase chain reaction, reactive oxygen species analysis and western blotting were performed. At 24 h post-melatonin treatment, caspase-3 mRNA and protein expression levels were significantly decreased. These results demonstrated that melatonin may protect hippocampal neurons via the inhibition of caspase-3 when exposed to irradiation. Therefore, caspase-3 inhibition serves a neuroprotective and antioxidant role in the interventional treatment of melatonin. The results of the present study suggested that melatonin may have a potential therapeutic effect against irradiation; however, further studies are required in order to elucidate the underlying antioxidant mechanisms.

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy.

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.

Anticancer activity of sea cucumber triterpene glycosides.

Triterpene glycosides are characteristic secondary metabolites of sea cucumbers (Holothurioidea, Echinodermata). They have hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. These natural products suppress the proliferation of various human tumor cell lines in vitro and, more importantly, intraperitoneal administration in rodents of solutions of some sea cucumber triterpene glycosides significantly reduces both tumor burden and metastasis. The anticancer molecular mechanisms include the induction of tumor cell apoptosis through the activation of intracellular caspase cell death pathways, arrest of the cell cycle at S or G2/M phases, influence on nuclear factors, NF-κB, and up-down regulation of certain cellular receptors and enzymes participating in cancerogenesis, such as EGFR (epidermal growth factor receptor), Akt (protein kinase B), ERK (extracellular signal-regulated kinases), FAK (focal adhesion kinase), MMP-9 (matrix metalloproteinase-9) and others. Administration of some glycosides leads to a reduction of cancer cell adhesion, suppression of cell migration and tube formation in those cells, suppression of angiogenesis, inhibition of cell proliferation, colony formation and tumor invasion. As a result, marked growth inhibition of tumors occurs in vitro and in vivo. Some holothurian triterpene glycosides have the potential to be used as P-gp mediated MDR reversal agents in combined therapy with standard cytostatics.

Phosphorylation Promotes Neurotoxicity in aCaenorhabditis elegansModel of TDP-43 Proteinopathy

Neurodegenerative disorders characterized by neuronal and glial lesions containing aggregated pathological TDP-43 protein in the cytoplasm, nucleus, or neurites are collectively referred to as TDP-43 proteinopathies. Lesions containing aggregated TDP-43 protein are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). In addition, mutations in human TDP-43 cause ALS. We have developed a Caenorhabditis elegans model of TDP-43 proteinopathies to study the cellular, molecular, and genetic underpinnings of TDP-43-mediated neurotoxicity. Expression of normal human TDP-43 in all C. elegans neurons causes moderate motor defects, whereas ALS-mutant G290A, A315T, or M337V TDP-43 transgenes cause severe motor dysfunction. The model recapitulates some characteristic features of ALS and FTLD-U including age-induced decline in motor function, decreased life span, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of TDP-43 protein that accumulates in detergent-insoluble protein deposits. In C. elegans, TDP-43 neurotoxicity is independent of activity of the cell death caspase CED-3. Furthermore, phosphorylation of TDP-43 at serine residues 409/410 drives mutant TDP-43 toxicity. This model provides a tractable system for additional dissection of the cellular and molecular mechanisms underlying TDP-43 neuropathology.

111 Impact of the Caspase-2 Inhibitor Trp601 on Apoptotic Signaling in the Developing Brain During Hyperoxia

Background and aims: Experimental studies show that oxygen, which is widely used in neonatal medicine for resuscitation and treatment of pulmonary hypertension, triggers a disruption of the maintenance of intracellular redox homeostasis. This disturbance can lead to oxidative stress and furthermore to neuronal apoptosis in the developing brain. The role of caspase-2 in apoptosis is poorly defined. Many in vitro studies of caspase-2 knockdown in cultured cells have implicated this caspase in cell death in response to different signaling pathways. To elucidate mechanisms of the caspase-2 inhibitor TRP601 and its mode of functioning in the developing brain in the context of hyperoxia, we investigated its impact on the levels of APAF-1, AIF, cytochrome c, caspase-9 and -3.Methods: Six-day old rats were exposed together with their mothers to 80% oxygen in the presence or absence of the caspase-2 inhibitor TRP601 (1 mg/kg) and were sacrificed after 12 or 24 hrs of hyperoxia following treatment. Dissected brains were either examined histologically to visualize degenerating cells or were subjected to protein studies.Results: Oxygen exposure triggered cell death at 12 to 24 hrs, which was attenuated by TRP601 treatment. Our protein studies demonstrated an upregulation of APAF-1, AIF, cytochrome c, caspase-9 and -3 in the cytosolic fraction of brain homogenates after hyperoxia, which reached control levels following TRP601 treatment.Conclusion: These findings suggest a protective role for the caspase-2 inhibitor TRP601 in the prevention of neonatal oxygen-induced apoptotic brain damage.Supported by the European Commission (Sixth Framework Program, contract no LSHM- CT-2006-036534).

Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFNγ, IL-8, MCP-1, and PD-L1, and decreased IFNβ, IL-10, and TGF-β. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.

Mechanical ventilation enhances lung inflammation and caspase activity in a model of mouse pneumovirus infection

Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1alpha (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection.

Growth Inhibition and Apoptosis Induction by Tanshinone IIA in Human Colon Adenocarcinoma Cells

Tanshinone IIA is the most abundant diterpene quinone in Danshen, Salviae miltiorrhizae Radix, a widely prescribed traditional herbal medicine that is used to treat cardiovascular and inflammatory diseases. Recently, tanshinone IIA was demonstrated to induce cell death and apoptosis in a variety of tumors. However, the effect of tanshinone IIA on human colon cancer cells is not clearly understood yet. In this study, the antigrowth and apoptosis-eliciting effects of tanshinone IIA, as well as its cellular mechanisms of actions, were investigated in Colo-205 human colon cancer cells. Tanshinone IIA reduced cell growth in a concentration-dependent manner, inducing apoptosis accompanied by an increase in TUNEL staining and by an increased percentage of cells in the sub-G1 fraction. The expression of p53 and p21 and mitochondrial cytochrome c release were increased in tanshinone IIA-treated cells. In addition, the expression of Fas proteins was up-regulated by tanshinone IIA. Tanshinone IIA-induced catalytic activation of caspases was confirmed by cleavage of caspase-8 and caspase-3. These findings suggest that tanshinone IIA induces apoptosis in Colo-205 cells through both mitochondrial-mediated intrinsic and Fas-mediated extrinsic caspase cell-death pathways. Accordingly, the chemotherapeutic potential of tanshinone IIA for colon cancer warrants further study.

Reactive-Site Cleavage Residues Confer Target Specificity to Baculovirus P49, a Dimeric Member of the P35 Family of Caspase Inhibitors

Baculovirus proteins P49 and P35 are potent suppressors of apoptosis in diverse organisms. Although related, P49 and P35 inhibit initiator and effector caspases, respectively, during infection of permissive insect cells. The molecular basis of this novel caspase specificity is unknown. To advance strategies for selective inhibition of the cell death caspases, we investigated biochemical differences between these baculovirus substrate inhibitors. We report here that P49 and P35 use similar mechanisms for stoichiometric inhibition that require caspase cleavage of their reactive site loops (RSL) and chemical contributions of a conserved N-terminal cysteine to stabilize the resulting inhibitory complex. Our data indicated that P49 functions as a homodimer that simultaneously binds two caspases. In contrast, P35 is a monomeric, monovalent inhibitor. P49 and P35 also differ in their RSL caspase recognition sequences. We tested the role of the P(4)-P(1) recognition motif for caspase specificity by monitoring virus-induced proteolytic processing of Sf-caspase-1, the principal effector caspase of the host insect Spodoptera frugiperda. When P49's TVTD recognition motif was replaced with P35's DQMD motif, P49 was impaired for inhibition of the initiator caspase that cleaves and activates pro-Sf-caspase-1 and instead formed a stable inhibitory complex with active Sf-caspase-1. In contrast, the effector caspase specificity of P35 was unaltered when P35's DQMD motif was replaced with TVTD. We concluded that the TVTD recognition motif is required but not sufficient for initiator caspase inhibition by P49. Our findings demonstrate a critical role for the P(4)-P(1) recognition site in caspase specificity by P49 and P35 and indicate that additional determinants are involved in target selection.

Distinct Mechanisms of Apoptosis-Induced Compensatory Proliferation in Proliferating and Differentiating Tissues in the Drosophila Eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here we show that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

Protective role of Cop in Rip2/Caspase-1/Caspase-4-mediated HeLa cell death

CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-α-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.

Age-Dependent Susceptibility of the Retinal Ganglion Cell Layer to Cell Death

The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR. After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels. Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression.