Insights into the regulation of two caspase-activating platforms, the inflammasome and the PIDDosome

Résumé: Les organismes multicellulaires ont adopté diverses stratégies pour répondre aux stress auxquels ils sont exposés. Cette étude a exploré deux de ces stratégies l'inflammation en réponse à une invasion par un pathogène, et l'apoptose ou la survie en réponse aux dommages à l'ADN. L'interleukine-lß (IL-lß) est une importante cytokine inflammatoire. Elle est synthétisée sous forme d'un précurseur inactif et nécessite un clivage par la caspase-1 pour être activée. La caspase-1 elle-même est activée dans un complexe appelé inflammasome. Certains NLRs (Nod-like receptors), IPAF et les NALPs, sont capables de former des inflammasomes fonctionnels. Cette étude s'est intéressée au rôle d'un autre NLR structurellement proche, la protéine NAIP, dans la régulation de la caspase-1 et la maturation de l'IL-1 ß. NAIP est incorporé à l'inflammasome contenant NALP3 et est capable d'inhiber l'activation de la caspase-1 et la maturation de l'IL-lß. Cette fonction inhibitrice dépend des ses domaines BIR et est inhibée par ses LRRs. Le mécanisme exact d'inhibition reste à définir et la régulation de l'activation de NAIP est discutée. La deuxième partie de cette étude concerne la protéine PIDD. Cette protéine est impliquée avec RAIDD dans l'activation de la caspase-2, et est aussi capable, avec l'aide de RIP et de NEMO, d'activer NF-κB en réponse aux dommages à l'ADN. Deux isoformes de PIDD ont déjà été décrites dans la littérature, PIDD (isoforme 1) et LRDD (isoforme 2) et une troisième isoforme est rapportée ici. L'étude de l'expression de ces isoformes a montré qu'elles sont exprimées différemment dans les tissus et dans les lignées cellulaires, et que l'isoforme 3 est induite en réponse à un stress génotoxique. La caractérisation fonctionnelle a établi que les trois isoformes sont capables d'activer NF-κB, donc la survie, mais que seule l'isoforme 1 peut interagir avec RAIDD pour activer la caspase-2 et sensibiliser les cellules à la mort induite par un stress génotoxique. Le domaine intermédiaire de PIDD, situé entre le deuxième ZU5 et le DD est essentiel pour l'interaction entre PIDD et RAIDD et l'activation de la caspase-2 qui en découle. En conclusion, l'épissage différentiel de l'ARNm de PIDD permet la production d'au moins trois protéines possédant des fonctions agonistes ou antagonistes et qui peuvent participer au choix cellulaire entre survie et apoptose en réponse aux dommages à l'ADN. Summary: Multicellular organisms have evolved several strategies to cope with the stresses they encounter. The present study has explored two of these strategies: inflammation in response to a pathogenic invasion, and apoptosis or repair/survival in response to DNA damage. Interleukin-lß (IL-lß) is a key mediator of inflammation. It is synthesized as an inactive precursor and requires cleavage by caspase-1 to be activated. caspase-1 itself is activated in molecular platforms called inflammasomes, which can be formed by members of the NOD-like receptors (NLR) family, like IPAF and NALPs. This study has investigated the role of another NLR, the structurally related protein NAIP, in the regulation of caspase-1 activation and IL-lß maturation. An inhibitory role of NAIP on caspase-1 activation and IL-lß maturation was demonstrated, as well as NAIP incorporation in the NALP3 inflammasome. This inhibitory property relies on NAIP BIR domains and is inhibited by NAIP LRRs. The exact mechanism of NAIP-mediated caspase-1 activation remains to be elucidated and the regulation of NAIP activation is discussed. The second part of this study focused on the caspase-2 activating protein PIDD. This protein is known to mediate caspase-2 activation via RAIDD and to signal NF-κB via RIP and NEMO in response to DNA damage. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and a third isoform is described here. Investigation of the expressional regulation of these isoforms indicated that they are differentially expressed in tissues and cell lines, and that isoform 3 mRNA levels are upregulated in response to genotoxic stress. Functional studies demonstrated that all three isoforms can activate NF-κB in response to DNA damage, but only isoform 1 is able to interact with RAIDD and activate caspase-2, sensitizing cells to genotoxic stress-induced cell death. The intermediate domain located between the second ZUS and the DD is essential for the interaction of PIDD and RAIDD and the subsequent caspase-2 activation. Thus the differential splicing of PIDD mRNA leads to the formation of at least thrée proteins with antagonizing/agonizing functions that could participate in determining cell fate in response to DNA damage.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

c-Jun N-terminal kinase has a key role in Alzheimer disease synaptic dysfunction in vivo.

Altered synaptic function is considered one of the first features of Alzheimer disease (AD). Currently, no treatment is available to prevent the dysfunction of excitatory synapses in AD. Identification of the key modulators of synaptopathy is of particular significance in the treatment of AD. We here characterized the pathways leading to synaptopathy in TgCRND8 mice and showed that c-Jun N-terminal kinase (JNK) is activated at the spine prior to the onset of cognitive impairment. The specific inhibition of JNK, with its specific inhibiting peptide D-JNKI1, prevented synaptic dysfunction in TgCRND8 mice. D-JNKI1 avoided both the loss of postsynaptic proteins and glutamate receptors from the postsynaptic density and the reduction in size of excitatory synapses, reverting their dysfunction. This set of data reveals that JNK is a key signaling pathway in AD synaptic injury and that its specific inhibition offers an innovative therapeutic strategy to prevent spine degeneration in AD.

Low doses of ionizing radiation promote tumor growth and metastasis by enhancing angiogenesis.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies.

APO866 inhibits nicotinamide phosphoribosyltransferase (NMPRTase), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Intracellular NAD is essential for cell survival, and NAD depletion resulting from APO866 treatment elicits tumor cell death. Here, we determine the in vitro and in vivo sensitivities of hematologic cancer cells to APO866 using a panel of cell lines (n = 45) and primary cells (n = 32). Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma), but not normal hematopoietic progenitor cells, were sensitive to low concentrations of APO866 as measured in cytotoxicity and clonogenic assays. Treatment with APO866 decreased intracellular NAD and adenosine triphosphate (ATP) at 24 hours and 48 to72 hours, respectively. The NAD depletion led to cell death. At 96 hours, APO866-mediated cell death occurred in a caspase-independent mode, and was associated with mitochondrial dysfunction and autophagy. Further, in vivo administration of APO866 as a single agent prevented and abrogated tumor growth in animal models of human AML, lymphoblastic lymphoma, and leukemia without significant toxicity to the animals. The results support the potential of APO866 for treating hematologic malignancies.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Age-dependent impairment of spine morphology and synaptic plasticity in hippocampal CA1 neurons of a presenilin 1 transgenic mouse model of Alzheimer's disease.

Presenilin 1 (PS1) mutations are responsible for a majority of early onset familial Alzheimer's disease (FAD) cases, in part by increasing the production of Abeta peptides. However, emerging evidence suggests other possible effects of PS1 on synaptic dysfunction where PS1 might contribute to the pathology independent of Abeta. We chose to study the L286V mutation, an aggressive FAD mutation which has never been analyzed at the electrophysiological and morphological levels. In addition, we analyzed for the first time the long term effects of wild-type human PS1 overexpression. We investigated the consequences of the overexpression of either wild-type human PS1 (hPS1) or the L286V mutated PS1 variant (mutPS1) on synaptic functions by analyzing synaptic plasticity and associated spine density changes from 3 to 15 months of age. We found that mutPS1 induces a transient increase observed only in 4- to 5-month-old mutPS1 animals in NMDA receptor (NMDA-R)-mediated responses and LTP compared with hPS1 mice and nontransgenic littermates. The increase in synaptic functions is concomitant with an increase in spine density. With increasing age, however, we found that the overexpression of human wild-type PS1 progressively decreased NMDA-R-mediated synaptic transmission and LTP, without neurodegeneration. These results identify for the first time a transient increase in synaptic function associated with L286V mutated PS1 variant in an age-dependent manner. In addition, they support the view that the PS1 overexpression promotes synaptic dysfunction in an Abeta-independent manner and underline the crucial role of PS1 during both normal and pathological aging.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Inflammation-induced alteration of astrocyte mitochondrial dynamics requires autophagy for mitochondrial network maintenance.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo.

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy.

AIMS/HYPOTHESIS: Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia. METHODS: Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor. RESULTS: PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death. CONCLUSIONS: In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.