Microglial secreted cathepsin B induces neuronal apoptosis

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.     

4-hydroxynonenal contributes to NGF withdrawal-induced neuronal apoptosis

Reactive oxygen species are a necessary triggering event for apoptosis of sympathetic neurons after nerve growth factor (NGF) withdrawal. Reactive oxygen species can lead to the generation of 4-hydroxynonenal (HNE), a highly reactive aldehyde that forms adducts with proteins. This covalent modification can activate or inhibit signal transduction pathways involved in the induction of apoptosis. This process may be clinically relevant because HNE-adduct immunoreactivity increases in several disease states. Here we evaluate the role of HNE-adducts in sympathetic neurons undergoing NGF-deprivation-induced apoptosis, a model of developmental programmed cell death. We show that HNE-adduct immunoreactivity is dramatically increased after NGF-withdrawal in an NADPH oxidase-dependent manner. Moreover, HNE-adducts appear to contribute to NGF-deprivation-induced apoptotic signal transduction because microinjected HNE-adduct antiserum protects sympathetic neurons from NGF withdrawal. In conclusion, this report suggests the direct contribution of endogenously generated HNE in the stimulation of apoptotic signal transduction in neurons.     

The cathepsin B death pathway contributes to TNF plus IFN-gamma-mediated human endothelial injury

Vascular endothelial cells are primary targets of cytokine-induced cell death leading to tissue injury. We previously reported that TNF in combination with LY294002, a PI3K inhibitor, activates caspase-independent cell death initiated by cathepsin B (Cat B) in HUVEC. We report that TNF in the presence of IFN-gamma activates Cat B as well as a caspase death pathway in both HUVEC and human dermal microvascular endothelial cells, but only activates caspase-mediated death in HeLa cells and human embryonic kidney (HEK)293 cells. Like LY294002, IFN-gamma triggers Cat B release from lysosomes in HUVEC. Cat B-triggered death involves mitochondria, indicated by release of cytochrome c, loss of mitochondrial membrane potential and inhibition of death by overexpressed Bcl-2. Cat B effects on mitochondria do not depend upon Bid cleavage. Unexpectedly, overexpression of a dominant negative mutated form of Fas-associated death domain protein (FADD), which blocks caspase activation by TNF, potentiates TNF activation of Cat B and cell death in HUVEC. Similarly, mutant Jurkat cells lacking FADD also show increased susceptibility to TNF-induced Cat B-dependent cell death. These observations suggest that the Cat B death pathway is cell type-specific and may contribute to cytokine-mediated human tissue injury and to the embryonic lethality of FADD gene disruption in mice.     

Role of cathepsin B in dengue virus-mediated apoptosis

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.     

Dysregulation of autophagy by HIV-1 Nef in human astrocytes

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

HIV-1gp120 induces neuronal apoptosis through enhancement of 4-aminopyridine-senstive outward K+ currents

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) usually occurs late in the course of HIV-1 infection and the mechanisms underlying HAD pathogenesis are not well understood. Accumulating evidence indicates that neuronal voltage-gated potassium (Kv) channels play an important role in memory processes and acquired neuronal channelopathies in HAD. To examine whether Kv channels are involved in HIV-1-associated neuronal injury, we studied the effects of HIV-1 glycoprotein 120 (gp120) on outward K+ currents in rat cortical neuronal cultures using whole-cell patch techniques. Exposure of cortical neurons to gp120 produced a dose-dependent enhancement of A-type transient outward K+ currents (IA). The gp120-induced increase of IA was attenuated by T140, a specific antagonist for chemokine receptor CXCR4, suggesting gp120 enhancement of neuronal IA via CXCR4. Pretreatment of neuronal cultures with a protein kinase C (PKC) inhibitor, GF109203X, inhibited the gp120-induced increase of IA. Biological significance of gp120 enhancement of IA was demonstrated by experimental results showing that gp120-induced neuronal apoptosis, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase-3 staining, was attenuated by either an IA blocker 4-aminopyridine or a specific CXCR4 antagonist T140. Taken together, these results suggest that gp120 may induce caspase-3 dependent neuronal apoptosis by enhancing IA via CXCR4-PKC signaling.     

Dysregulation of Mg2+ homeostasis contributes to acquisition of cancer hallmarks

Derangement of magnesium homeostasis underlies the pathophysiology of many diseases, including cancer. Recent advances support the view that aberrant expression of Mg²⁺ channels and other Mg²⁺ homeostatic factors may affect many hallmarks of cancer. The seminal idea of magnesium as a key regulator of cell proliferation has been enriched by novel intriguing findings that link magnesium and Mg²⁺ transporters to distinctive and complementary capabilities that enable tumour growth and metastatic dissemination. In this review, we examine the evidence on the involvement of members from the TRPM, CNNM and SCL41 protein families in cancer progression, and discuss their potential as therapeutic targets.     

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.     

c-Jun contributes to amyloid beta-induced neuronal apoptosis but is not necessary for amyloid beta-induced c-jun induction

The role of gene expression in neuronal apoptosis may be cell- and apoptotic stimulus-specific. Previously, we and others showed that amyloid beta (Abeta)-induced neuronal apoptosis is accompanied by c-jun induction. Moreover, c-Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c-Jun in Abeta toxicity, we compared Abeta-induced apoptosis in neurons from murine fetal littermates that were deficient or wild-type with respect to c-Jun. We report that neurons deficient for c-jun are relatively resistant to Abeta toxicity, suggesting that c-Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Abeta treatment surprisingly led to an apparent activation of the c-jun promoter in both the c-jun-deficient and wild-type neurons, suggesting that c-Jun is not necessary for activation of the c-jun promoter. Indeed, several genes induced by Abeta in wild-type neurons were also induced in c-jun-deficient neurons, including c-fos, fosB, ngfi-B, and ikappaB. In summary, these results indicate that c-Jun contributes to Abeta-induced neuronal death but that c-Jun is not necessary for c-jun induction.     

GM1 ganglioside contributes to retain the neuronal conduction and neuronal excitability in visceral and baroreceptor afferents

GM1 ganglioside has a great impact on the function of nodes of Ranvier on myelinated fiber, suggesting its potential role to maintain the electrical and neuronal excitability of neurons. Here we first demonstrate that visceral afferent conduction velocity of myelinated and unmyelinated fibers are reduced significantly by tetrodotoxin (TTX) or cholera toxin-B subunits (CTX-B), and only the effects mediated by CTX-B are prevented by GM1 pre-treatment. At soma of myelinated A and unmyelinated C-type nodose ganglion neurons (NGNs), the action potential spike frequency reduced by CTX-B is also prevented by GM1. Additionally, the current density of both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na⁺ channels were significantly decreased by CTX-B without changing the voltage-dependent property. These data confirm that endogenous GM1 may play a dominant role in maintaining the electrical and neuronal excitability via modulation of sodium (Na⁺) channel around nodes and soma as well, especially TTX-S Na⁺ channel, which is also confirmed by the reduction of spike amplitude and depolarization. Similar data are also extended to fluorescently identified and electrophysiologically characterized aortic baroreceptor neurons. These findings suggest that GM1 plays an important role in the neural modulation of electric and neuronal excitability in visceral afferent system.     

An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.     

Mitochondria in HIV-1-induced apoptosis

It is now well admitted that HIV infection leading to AIDS is associated with an abnormal susceptibility of T cells to undergo apoptosis. Recent progress in research into programmed cell death has resulted in the identification of the principal pathways involved in this process. Thus the "extrinsic" as well as the "intrinsic" pathways converge to the mitochondria considered as the main sensor of programmed cell death. This review summarizes our knowledge of the influence of mitochondrial control on T cell death during HIV and SIV infections.     

Inactivation of cathepsin B1 by diazomethyl ketones

Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxy-carbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin B₁ of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling.     

Caspase-dependent cleavage of c-Abl contributes to apoptosis

The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.