CDK5 protects from caspase‐induced Ataxin‐3 cleavage and neurodegeneration

Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease‐specific proteins. In case of SCA3, mutation of Ataxin‐3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin‐dependent kinase‐5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase‐dependent Ataxin‐3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease‐propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin‐3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin‐3 and other polyglutamine proteins.

     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Over‐expression of HSP70 attenuates caspase‐dependent and caspase‐independent pathways and inhibits neuronal apoptosis

HSP70 is a member of the family of heat‐shock proteins that are known to be up‐regulated in neurons following injury and/or stress. HSP70 over‐expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over‐expression by transfection with HSP70‐expression plasmids in primary cortical neurons and the SH‐SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2‐ceramide, and β‐Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease‐activating factor 1, as well as apoptosis‐inducing factor, key molecules involved in development of caspase‐dependent and caspase‐independent PCD, respectively. Markers of caspase‐dependent PCD, including active caspase‐3, caspase‐9, and cleaved PARP were attenuated in neurons over‐expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase‐dependent and caspase‐independent PCD pathways.     

Bone marrow‐derived mesenchymal stem cells mitigate caspase‐3 and 8‐hydroxy proline induced via β‐adrenergic agonist in pulmonary injured rats

Estimating the ability of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to alleviate pulmonary injury induced via isoproterenol (ISP). ISP was injected in a dose of (100 mg/kg, subcutaneously twice at an interval of 24 h). One month post BM‐MSCs transplantation by intravenous injection, pulmonary oxidative stress was assessed, and Western blot analyses and histopathological investigations were conducted. Compared with the normal control group, BM‐MSCs transplantation significantly decreased the expression of pulmonary anti‐oxidative stress marker. Western blot analysis revealed that ISP significantly reduced the protein expression of the anti‐oxidative stress marker nuclear related factor‐2 (Nrf2). However, the apoptotic marker (caspase‐3) and collagen content marker (8‐hydroxyproline) were markedly elevated. These biochemical markers were confirmed by histopathological investigations. Finally, it was demonstrated that BM‐MSCs transplantation showed a superior effect in improving pulmonary function through alleviating oxidative stress, apoptosis, and collagen content.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

Fluoride and diethylnitrosamine coexposure enhances oxido‐inflammatory responses and caspase‐3 activation in liver and kidney of adult rats

The present study investigated the impact of coexposure to fluoride and diethylnitrosamine (DEN) on hepatorenal function in adult rats. The animals were exposed to fluoride (15 mg/L in drinking water) and DEN (10 mg/kg) singly or coexposed to both compounds for 14 days. Results demonstrated that the fluoride or DEN mediated increase in hepatorenal toxicity was intensified in the coexposure group. Additionally, the decrease in antioxidant enzyme activities as well as the elevation in reactive oxygen and nitrogen species, and lipid peroxidation was markedly aggravated in rats coexposed to DEN and fluoride. Furthermore, the increase in levels of nitric oxide, tumor necrosis factor‐α and interleukin‐1β, myeloperoxidase and caspase‐3 activities as well as histological lesions was more pronounced in the liver and kidney of rats coexposed to DEN and fluoride. Conclusively, coexposure to fluoride and DEN exacerbated hepatorenal damage via enhancement of oxido‐inflammatory responses and caspase‐3 activation in rats.     

Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non‐apoptotic caspase‐8 is required for inflammasome priming

A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co‐deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase‐8, a caspase essential for death‐receptor‐mediated apoptosis, is required for efficient Toll‐like‐receptor‐induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non‐apoptotic role for caspase‐8 in regulating inflammasome activation and pro‐inflammatory cytokine levels.     

Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF‐κB p65 and Bcl‐2 and upregulation of caspase‐3

Propofol is widely used in paediatric anaesthesia and intensive care unit because of its essentially short‐acting anaesthetic effect. Recent data have shown that propofol induced neurotoxicity in developing brain. However, the mechanisms are not extremely clear. To gain a better insight into the toxic effects of propofol on hippocampal neurons, we treated cells at the days in vitro 7 (DIV 7), which were prepared from Sprague–Dawley embryos at the 18th day of gestation, with propofol (0.1–1000 μM) for 3 h. A significant decrease in neuronal proliferation and a remarkable increase in neuroapoptosis were observed in DIV 7 hippocampal neurons as measured by 3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis assay respectively. Moreover, propofol treatment decreased the nuclear factor kappaB (NF‐κB) p65 expression, which was accompanied by a reduction in B‐cell lymphoma 2 (Bcl‐2) mRNA and protein levels, increased caspase‐3 mRNA and activation of caspase‐3 protein. These results indicated that downregulation of NF‐κB p65 and Bcl‐2 were involved in the potential mechanisms of propofol‐induced neurotoxicity. This likely led to the caspase‐3 activation, triggered apoptosis and inhibited the neuronal growth and proliferation that we have observed in our in vitro systems. Copyright © 2014 John Wiley & Sons, Ltd.     

Disabled‐1 is down‐regulated in clinical breast cancer and regulates cell apoptosis through NF‐κB/Bcl‐2/caspase‐9

Disabled‐1 (Dab1) is best known as an adaptor protein regulating neuron migration and lamination during development. However, the exact function of Dab1 in breast cancer is unknown. In this study, we examined the expression of Dab1 in 38 breast cancer paraffin sections, as well as 60 paired frozen breast cancer and their adjacent tissues. Our results showed that Dab1 was reduced in breast cancer, and its compromised expression correlated with triple negative breast cancer phenotype, poor differentiation, as well as lymph node metastasis. Functional analysis in breast cancer cell lines demonstrated that Dab1 promoted cell apoptosis, which, at least partially, depended on its regulation of NF‐κB/Bcl‐2/caspase‐9 pathway. Our study strongly suggests that Dab1 may be a potential tumour suppressor gene in breast cancer.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Entomotoxic action of Sambucus nigra agglutinin i in Acyrthosiphon pisum aphids and Spodoptera exigua caterpillars through caspase‐3‐like‐dependent apoptosis

In this project, the toxicity and mechanism of action of the ricin‐B‐related lectin SNA‐I from elderberry (Sambucus nigra) in the pea aphid (Acyrthosiphon pisum) and the beet armyworm (Spodoptera exigua), two important pest insects in agriculture, were studied. SNA‐I is a chimeric lectin belonging to the class of ribosome‐inactivating proteins and consists of an A‐chain with N‐glycosidase activity and a carbohydrate‐binding B‐chain. Incorporation of 2 mg/ml of SNA‐I in the diet of neonates and adults of A. pisum caused 40–46% mortality within 2 days, while in third instars of S. exigua, the larval biomass was significantly reduced by 12% after feeding for 3 days on a diet containing 5 mg/g of SNA‐I. Interestingly, extracts of the (mid)gut of treated A. pisum and S. exigua demonstrated DNA fragmentation and this was accompanied with an increase in caspase‐3‐like activity. The involvement of cell death or apoptosis in the entomotoxicity of SNA‐I through induction of caspase‐3‐like activity was also confirmed by addition of the permeable caspase‐3 inhibitor III in the diet, leading to a rescue of the treated aphid neonates. Finally, similar to the chimeric lectin SNA‐I, the hololectin SNA‐II, consisting of two carbohydrate‐binding B‐chains caused high mortality to neonate A. pisum aphids with an LC₅₀ of 1.59 mg/ml, suggesting that the entomotoxic action of the lectins under study mainly relies on their carbohydrate‐binding activity. © 2010 Wiley Periodicals, Inc.     

Dimer‐specific immunoprecipitation of active caspase‐2 identifies TRAF proteins as novel activators

Caspase‐2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase‐2 bimolecular fluorescence complementation (BiFC) system with fluorophore‐specific immunoprecipitation to isolate and study the active caspase‐2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor‐associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase‐2 dimer. TRAF2 in particular is necessary for caspase‐2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase‐2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase‐2 activation and consequent cell death by driving its activation through dimer‐stabilizing ubiquitylation.     

Overexpression of fibroblast growth factor‐21 (FGF‐21) protects mesenchymal stem cells against caspase‐dependent apoptosis induced by oxidative stress and inflammation

The clinical application of stem cells offers great promise as a potential avenue for therapeutic use in neurodegenerative diseases. However, cell loss after transplantation remains a major challenge, which currently plagues the field. On the basis of our previous findings that fibroblast growth factor 21 (FGF‐21) protected neurons from glutamate excitotoxicity and that upregulation of FGF‐21 in a rat model of ischemic stroke was associated with neuroprotection, we proposed that overexpression of FGF‐21 protects bone marrow‐derived mesenchymal stem cells (MSCs) from apoptosis. To test this hypothesis, we examined whether the detrimental effects of apoptosis can be mitigated by the transgenic overexpression of FGF‐21 in MSCs. FGF‐21 was transduced into MSCs by lentivirus and its overexpression was confirmed by quantitative polymerase chain reaction. Moreover, FGF‐21 overexpression did not stimulate the expression of other FGF family members, suggesting it does not activate a positive feedback system. The effects of hydrogen peroxide (H₂O₂), tumor necrosis factor‐α (TNF‐α), and staurosporine, known inducers of apoptosis, were evaluated in FGF‐21 overexpressing MSCs and mCherry control MSCs. Caspases 3 and 7 activity was markedly and dose‐dependently increased by all three stimuli in mCherry MSCs. FGF‐21 overexpression robustly suppressed caspase activation induced by H₂O₂ and TNF‐α, but not staurosporine. Moreover, the assessment of apoptotic morphological changes confirmed the protective effects of FGF‐21 overexpression. Taken together, these results provide compelling evidence that FGF‐21 plays a crucial role in protecting MSCs from apoptosis induced by oxidative stress and inflammation and merits further investigation as a strategy for enhancing the therapeutic efficacy of stem cell‐based therapies.