A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody raised against a peptide from human caspase 3 was used to investigate how extracellular signals controlled spatial patterning of cell death. The antibody crossreacted specifically with dying Drosophila cells and labelled the activated effector caspase Drice. It was found that the initiator caspase Dronc and the proapoptotic gene head involution defective were important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affected caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appeared to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway.

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Abstract P419: A Systematic Characterization of Brain Endothelial Cell Death in Intracerebral Hemorrhage

Stroke ◽

10.1161/str.52.suppl_1.p419 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Maulana Ikhsan ◽

Marietta Zille

Keyword(s):

Cell Death ◽

Intracerebral Hemorrhage ◽

Brain Tissue ◽

Vascular Integrity ◽

Regulated Cell Death ◽

Injury Mechanisms ◽

Endothelial Cell Death ◽

Cell Death Mechanisms ◽

Underlying Mechanisms

Introduction: Intracerebral hemorrhage (ICH) is a type of stroke caused by the loss of vascular integrity leading to bleeding within the brain tissue. Hematoma-derived factors cause secondary injury mechanisms such as cell death days to weeks after the event and in regions distant from the primary insult. Increasing evidence suggests that hemoglobin released by the hematoma is one of the major contributors to neuronal injury in ICH. To date, it is unclear whether brain endothelial cells (EC) are similarly vulnerable to hemolysis products and undergo regulated cell death. Hypothesis: We hypothesized that brain EC undergo multiple, different modes of cell death after ICH and that the underlying mechanisms are different compared to neurons. Methods: We systematically investigated cell death mechanisms in brain EC after exposure to the hemolysis product hemin. We used chemical inhibitors of apoptosis, autophagy, ferroptosis, necroptosis, and parthanatos and assessed biochemical markers of these cell death modes. Results: Brain EC viability was concentration-dependently decreased, starting at higher hemin concentrations than neurons. Treatment of EC with ferroptosis inhibitors protective against hemin toxicity in neurons and against ICH in vivo showed that only N-acetylcysteine and deferoxamine protected brain EC, while ferrostatin-1 and U0126 did not abrogate EC death. The autophagy inhibitor bafilomycin A1 also reduced EC death and hemin increased the expression of the autophagy marker LC3. While inhibitors against apoptosis and parthanatos were not effective, the necroptosis inhibitor GSK872 demonstrated a partial protective effect. Conclusions: Our data suggest that ICH induces different mechanisms of death in EC (ferroptosis and autophagy) compared to neurons (ferroptosis and necroptosis) and may thus warrant a combinatorial therapeutic approach. Further investigations in human and ovine ICH brain tissue are ongoing.

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In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis

Development ◽

10.1242/dev.127.8.1641 ◽

2000 ◽

Vol 127 (8) ◽

pp. 1641-1649

Author(s):

B. Diaz ◽

J. Serna ◽

F. De Pablo ◽

E.J. de la Rosa

Keyword(s):

Cell Death ◽

Embryonic Development ◽

Insulin Receptor ◽

Neural Development ◽

Developmental Process ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

In Ovo ◽

Retinal Neurogenesis

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Diaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624–1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.

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Dynamics of in vivo ASC speck formation

The Journal of Cell Biology ◽

10.1083/jcb.201703103 ◽

2017 ◽

Vol 216 (9) ◽

pp. 2891-2909 ◽

Cited By ~ 19

Author(s):

Paola Kuri ◽

Nicole L. Schieber ◽

Thomas Thumberger ◽

Joachim Wittbrodt ◽

Yannick Schwab ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Death ◽

Light And Electron Microscopy ◽

Three Dimensional ◽

Inflammasome Activation ◽

Caspase Activation ◽

Endogenous Gene ◽

Pyrin Domain ◽

Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

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Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000337 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000337 ◽

Cited By ~ 56

Author(s):

Lorenzo Galluzzi ◽

Ilio Vitale ◽

Sarah Warren ◽

Sandy Adjemian ◽

Patrizia Agostinis ◽

...

Keyword(s):

Immune Response ◽

Cell Death ◽

Adaptive Immune Response ◽

Operational Definition ◽

Immunogenic Cell Death ◽

Adaptive Immune ◽

Regulated Cell Death ◽

Definition Of

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.

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Caspase-9 cleavage, do you need it?

Biochemical Journal ◽

10.1042/bj20070617 ◽

2007 ◽

Vol 405 (1) ◽

Cited By ~ 34

Author(s):

Davina Twiddy ◽

Kelvin Cain

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Caspase 9 ◽

Autocatalytic Cleavage ◽

Downstream Effector ◽

Apoptosis Inhibition ◽

Biochemical Journal ◽

Effector Caspase ◽

Effector Caspases

Caspase-9, which is activated by association with the Apaf-1 (apoptotic protease-activating factor-1) apoptosome complex, cleaves and activates the downstream effector caspases-3 and -7, thereby executing the caspase-cascade and cell-death programme. Although caspase-9 does not need to be cleaved to be active, apoptotic cell death is always accompanied by autocatalytic cleavage and by further downstream effector caspase-dependent cleavage of caspase-9. In this issue of the Biochemical Journal, Denault and co-workers evaluate the role of caspase-3-dependent cleavage of caspase-9 and conclude that this mechanism mainly serves to enhance apoptosis by alleviating XIAP (X-linked inhibitor of apoptosis) inhibition of the apical caspase.

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Deletion of the Impg2 gene causes the degeneration of rod and cone cells in mice

Human Molecular Genetics ◽

10.1093/hmg/ddaa062 ◽

2020 ◽

Vol 29 (10) ◽

pp. 1624-1634

Author(s):

Huijuan Xu ◽

Chao Qu ◽

Li Gan ◽

Kuanxiang Sun ◽

Junkai Tan ◽

...

Keyword(s):

Cell Death ◽

Er Stress ◽

Molecular Mechanisms ◽

Histopathological Examination ◽

Apoptotic Cell ◽

Macular Dystrophy ◽

Cone Cell ◽

Rod Cells ◽

Cone Cells

Abstract Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.

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Modulation of the extrinsic cell death signaling pathway by viral Flip induces acute-death mediated liver failure

Cell Death and Disease ◽

10.1038/s41419-019-2115-y ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 1

Author(s):

Miriam Bittel ◽

Andreas E. Kremer ◽

Michael Stürzl ◽

Stefan Wirtz ◽

Iris Stolzer ◽

...

Keyword(s):

Cell Death ◽

Viral Infections ◽

Tissue Injury ◽

Multiple Organ ◽

Severe Liver ◽

Transgenic Expression ◽

Effector Caspase ◽

The Impact

AbstractDuring viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi’s sarcoma-associated herpesvirus interacts and inhibits the central cell death effector, Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins, like vFlip, on liver physiology in vivo, mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level, hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably, embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice, culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis.

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Local induction of patterning and programmed cell death in the developing Drosophila retina

Development ◽

10.1242/dev.125.12.2327 ◽

1998 ◽

Vol 125 (12) ◽

pp. 2327-2335 ◽

Cited By ~ 21

Author(s):

D.T. Miller ◽

R.L. Cagan

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Egf Receptor ◽

Pigment Cells ◽

Cell Fates ◽

Adult Retina ◽

Ras Pathway ◽

Cell Ablation ◽

Cone Cells ◽

Local Cell

Local cell signaling can pattern the nervous system by directing cell fates, including programmed cell death. In the developing Drosophila retina, programmed cell death is used to remove excess cells between ommatidia. Cell ablation revealed the source and position of signals required for regulating the pattern of programmed cell death among these interommatidial cells. Two types of signals regulate this patterning event. Notch-mediated signals between interommatidial precursors result in removal of unneeded cells. Cone cells and primary pigment cells oppose this signal by supplying a ‘life’-promoting activity; evidence is provided that this signal occurs through localized activation of the EGF Receptor/Ras pathway. Together, these signals refine the highly regular pattern observed in the adult retina.

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Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200712091 ◽

2008 ◽

Vol 182 (6) ◽

pp. 1127-1139 ◽

Cited By ~ 108

Author(s):

Ying-Chen Claire Hou ◽

Suganthi Chittaranjan ◽

Sharon González Barbosa ◽

Kimberly McCall ◽

Sharon M. Gorski

Keyword(s):

Cell Death ◽

Drosophila Melanogaster ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Mitogen Activated Protein Kinase ◽

Nuclear Condensation ◽

Effector Caspase ◽

Apoptosis Protein ◽

Egg Chambers

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

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Dynamics of ASC speck formation during skin inflammatory responses in vivo

10.1101/111542 ◽

2017 ◽

Author(s):

Paola Kuri ◽

Nicole L. Schieber ◽

Thomas Thumberger ◽

Joachim Wittbrodt ◽

Yannick Schwab ◽

...

Keyword(s):

Cell Death ◽

Real Time ◽

Inflammatory Responses ◽

Full Length ◽

Inflammasome Activation ◽

Endogenous Gene ◽

Effector Caspase

AbstractActivated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signalling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested this speck-containing pyroptotic debris. A 3D ultrastructural reconstruction based on CLEM of in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas PYD or CARD alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis, but after substantial delay. Therefore, formation of a single compact speck and rapid cell death induction in vivo requires full-length ASC.One Sentence SummaryWith a new endogenous ASC real-time reporter we characterize speck dynamics in vivo as well as the concomitant pyroptosis speck formation causes in keratinocytes.

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