Wallenda regulates JNK-mediated cell death in Drosophila

The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. Here, we show that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, we find that Wnd triggers JNK activation and cell death via its kinase domain. Moreover, we show that both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of scribble-induced JNK-dependent cell death. Thus, our data suggest that wnd encodes a novel essential cell death regulator in Drosophila.Programmed cell death (PCD) is a fundamental biological process required for normal organ development and tissue homeostasis in multicellular organisms.¹ Disruption of PCD would result in a variety of diseases including neurodegenerative diseases, autoimmune disorders and cancers.² Drosophila melanogaster, with its well-established genetic techniques and compact genome size, has been regarded as an excellent model organism to study PCD and its related signaling pathways.^{3, 4} The c-Jun N-terminal kinase (JNK) signaling has been implicated as one of the most important pathways that regulates various fundamental cell behaviors, such as proliferation, migration and cell death.^{5, 6}Rac1 belongs to the Rho family of small GTPase that regulates many aspects of physiological activities ranging from immune response to wound healing and migration.^{7, 8, 9, 10, 11} For instance, Rac1 has been implicated in JNK-mediated dorsal closure via Slpr (Slipper) in fly,⁷ osteoclast differentiation through TAK1-mediated NF-κB signaling¹² and myocyte hypertrophy via Ask1 (apoptotic signal-regulating kinase 1) in mammals.¹³ However, despite the reported role of Rac1 in cell death,¹⁴ its underlying mechanism and downstream components remain largely elusive.Here by using Drosophila compound eye as a model, we found Rac1 expression induces JNK-dependent cell death and identified Wallenda (Wnd), a MAPKKK (mitogen-activated protein kinase kinase kinase) member as an essential downstream mediator. Furthermore, we found that Wnd is sufficient to induce JNK-mediated cell death through both Hep and MKK4. Finally, we established Wnd as a general modulator of cell death in Drosophila by showing that it is also required for ectopic Egr or Rho1 and loss of Scribble (Scrib)-induced cell death.     

klumpfuss regulates cell death in the Drosophila retina

Programmed cell death (PCD) plays a central role in the sculpting and maturation of developing epithelia. In adult tissue, PCD plays a further role in the prevention of malignancy though removal of damaged cells. Here, we report that mutations in klumpfuss result in an excess of support cells during maturation of the developing Drosophila pupal retina. These ectopic cells are the result of a partial and specific failure of apoptotic death during normal cell fate selection. klumpfuss is required and differentially expressed in the cells that choose the life or death cell fate. We also provide genetic and biochemical evidence that klumpfuss regulates this process through down-regulation of the Epidermal Growth Factor Receptor/dRas1 signaling pathway. Based on its sequence Klumpfuss is an EGR-class nuclear factor, and our results suggest a mechanism by which mutations in EGR-class factors such as Wilms' Tumor Suppressor-1 may result in oncogenic events such as pediatric kidney tumors.     

Tumor suppressor CYLD regulates JNK-induced cell death in Drosophila

CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, we generated Drosophila CYLD (dCYLD) mutant and transgenic flies expressing wild-type and mutant dCYLD proteins. Here we show that dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. We show that dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death.     

Contribution of Atg1-dependent autophagy to TOR-mediated cell growth and survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.     

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.     

Drosophila in the study of neurodegenerative disease

As populations benefit from increasing lifespans, neurodegenerative diseases have emerged as a critical health concern. How can the fruit fly, Drosophila melanogaster, contribute to curing human diseases of the nervous system? A growing number of neurodegenerative diseases, as well as other human diseases, are being modeled in Drosophila and used as a platform to identify and validate cellular pathways that contribute to neurodegeneration and to identify promising therapeutic targets by using a variety of approaches from screens to target validation. The unique properties and tools available in the Drosophila system, coupled with the fact that testing in vivo has proven highly productive, have accelerated the progress of testing therapeutic strategies in mice and, ultimately, humans. This review highlights selected recent applications to illustrate the use of Drosophila in studying neurodegenerative diseases.     

Drosophila models of human neurodegenerative disease

Drosophila has provided a powerful genetic system in which to elucidate fundamental cellular pathways in the context of a developing and functioning nervous system. Recently, Drosophila has been applied toward elucidating mechanisms of human neurodegenerative disease, including Alzheimer's, Parkinson's and Huntington's diseases. Drosophila allows study of the normal function of disease proteins, as well as study of effects of familial mutations upon targeted expression of human mutant forms in the fly. These studies have revealed new insight into the normal functions of such disease proteins, as well as provided models in Drosophila that will allow genetic approaches to be applied toward elucidating ways to prevent or delay toxic effects of such disease proteins. These, and studies to come that follow from the recently completed sequence of the Drosophila genome, underscore the contributions that Drosophila as a model genetic system stands to contribute toward the understanding of human neurodegenerative disease.     

p73 regulates DRAM-independent autophagy that does not contribute to programmed cell death

Evading programmed cell death is a common event in tumour development. The p53 family member, p73, is a potent inducer of death and a determinant of chemotherapeutic response, but different to p53, is rarely mutated in cancer. Understanding cell death pathways downstream of p53 and p73 is therefore pivotal to understand both the development and treatment of malignant disease. Recently, p53 has been shown to modulate autophagy--a membrane trafficking process, which degrades long-lived proteins and organelles. This requires a p53 target gene, DRAM, and both DRAM and autophagy are critical for p53-mediated death. We report here that TA-p73 also regulates DRAM and autophagy, with different TA-p73 isoforms regulating DRAM and autophagy to varying extents. RNAi knockdown of DRAM, however, revealed that p73's modulation of autophagy is DRAM-independent. Also, p73's ability to induce death, again different to p53, is neither dependent on DRAM nor autophagy. In contrast to TA-p73, deltaN-p73 is a negative regulator of p53-induced and p73-induced autophagy, but does not affect autophagy induced by amino-acid starvation. These studies, therefore, represent not only the first report that p73 modulates autophagy but also highlight important differences in the mechanism by which starvation, p53 and p73 regulate autophagy and how this contributes to programmed cell death.     

Role of autophagy in disease resistance and hypersensitive response-associated cell death

Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum stress. At the same time, autophagy can contribute to cellular suicide. The concurrent engagement of autophagy in these processes during infection may sometimes mask its contribution to differing pro-survival and pro-death decisions. The importance of autophagy in innate immunity in mammals is well documented, but how autophagy contributes to plant innate immunity and cell death is not that clear. A few research reports have appeared recently to shed light on the roles of autophagy in plant-pathogen interactions and in disease-associated host cell death. We present a first attempt to reconcile the results of this research.     

The therapeutic importance of understanding mechanisms of neuronal cell death in neurodegenerative disease

Background

The low-density lipoprotein receptor-related protein 1 (LRP1) plays critical roles in lipid metabolism, cell survival, and the clearance of amyloid-β (Aβ) peptide. Functional soluble LRP1 (sLRP1) has been detected in circulating human placenta; however, whether sLRP1 is also present in the central nervous system is unclear.

Results

Here we show that abundant sLRP1 capable of binding its ligands is present in human brain tissue and cerebral spinal fluid (CSF). Interestingly, the levels of sLRP1 in CSF are significantly increased in older individuals, suggesting that either LRP1 shedding is increased or sLRP1 clearance is decreased during aging. To examine potential effects of pathological ligands on LRP1 shedding, we treated MEF cells with Aβ peptide and found that LRP1 shedding was increased. ADAM10 and ADAM17 are key members of the ADAM family that process membrane-associated proteins including amyloid precursor protein and Notch. We found that LRP1 shedding was significantly decreased in MEF cells lacking ADAM10 and/or ADAM17. Furthermore, forced expression of ADAM10 increased LRP1 shedding, which was inhibited by ADAM-specific inhibitor TIMP-3.

Conclusion

Our results demonstrate that LRP1 is shed by ADAM10 and ADAM17 and functional sLRP1 is abundantly present in human brain and CSF. Dysregulated LRP1 shedding during aging could alter its function and may contribute to the pathogenesis of AD.     

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.     

Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.     

PP2A regulates autophagy in two alternative ways in Drosophila

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.     

PP2A regulates autophagy in two alternative ways in Drosophila

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.     

自体吞噬在细胞死亡中的角色

一直以来人们认为自主性细胞死亡就是凋亡。然而 ,最近的研究表明 ,自主性细胞死亡不仅包括凋亡 ,还包括自体吞噬。自体吞噬通过与凋亡不同的降解机制 (膜包被降解 )和分子机制 (特定基因的激活 ) ,使细胞在某些特殊环境如饥饿、发育、分化等条件下自主死亡。在自主性细胞死亡中 ,自体吞噬与凋亡是两个既相互独立又紧密相关的过程。对自体吞噬机制的了解 ,必将为自主性细胞死亡机制的阐明及其相关疾病的治疗提供新的思路。