Sébastien Gaumer

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

Bcl-2 and Hsp27 act at different levels to suppress programmed cell death

Apoptosis and necrosis, two morphologically distinct forms of cell death, can be induced by common stimuli depending on the doses and the cell type. This study compares the protective effect of oncoprotein Bcl-2 and of the small stress protein Hsp27 on these two types of cell death. We use rat embryo fibroblasts conditionally immortalized by the tsA58 mutant of SV40 large T antigen as parental cells to develop cell lines carrying inducible bcl-2 or hsp27 genes. Two apoptotic stimuli were used: shift to the restrictive temperature that induced p53-mediated apoptosis and treatment with low doses of hydrogen peroxide. Necrosis was induced by high doses of hydrogen peroxide. Although Bcl-2 and Hsp27 protect these cells from necrotic death, only Bcl-2 appears capable of preventing apoptotic death. Bcl-2 protection is not mediated by a negative effect on the induction of the p53 responsive genes bax or waf1 but it slows down at least two stages of apoptosis: decrease of mitochondrial membrane potential and subsequent morphological changes. In contrast, although Hsp27 has been recently shown to inhibit apoptosis induced by various stimuli, its overexpression has no effect on apoptosis in this cell system. It should be also noticed that the apoptotic stimuli (temperature shift or hydrogen peroxide treatment) induce Hsp27, but not Bcl-2 accumulation suggesting that, in parental cells, Hsp27 might already provide some protection. However, taken together these results suggest that Hsp27, as well as Bcl-2, acts at several levels to inhibit cell death, but that their protective functions only partially overlap.

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly. Cell Death and Differentiation (2000) 7, 804–814

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates.

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Δvps15 larvae. Fluorescence microscopy showed that Δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates.. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.

Control of Sigma Virus Multiplication by the ref(2)P Gene of Drosophila melanogaster : An in Vivo Study of the PB1 Domain of Ref(2)P

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.

P62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies

Background: Oligomers of pathogenic proteins are implicated in the pathomechanisms of neurodegenerative diseases. Results: Depletion of p62 delays the degradation of polyglutamine protein oligomers via autophagy and exacerbates neurodegeneration in polyglutamine disease model flies. Conclusion: p62 plays a protective role via autophagic degradation of polyglutamine protein oligomers. Significance: p62 should be a therapeutic target for the polyglutamine diseases. Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. In this study, to elucidate the role of p62 in such pathogenic conditions in vivo, we used Drosophila models of neurodegenerative diseases. We found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. We also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. We therefore conclude that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases.

Mitochondria, Bcl-2 family proteins and apoptosomes: of worms, flies and men.

Initiator caspases are activated within specialized complexes, one of which is the apoptosome. The apoptosome is always constituted by at least an initiator caspase and a caspase activator. Apoptosome activation enables maturation of the associated caspase and constitutes a key step for cell fate. This activating complex is found throughout metazoans but its composition and regulation seem slightly different from one species to another. This review focuses on the composition and activation of the apoptosome in different species and details the role of mitochondrial factors and Bcl-2 family members in this activation.

reaper and bax initiate two different apoptotic pathways affecting mitochondria and antagonized by bcl-2 in Drosophila.

bcl-2 was the first regulator of apoptosis shown to be involved in oncogenesis. Subsequent studies in mammals, in the nematode and in Drosophila revealed wide evolutionary conservation of the regulation of apoptosis. Although dbok/debcl, a member of the bcl-2 gene family described in Drosophila, shows pro-apoptotic activities, no anti-apoptotic bcl-2 family gene has been studied in Drosophila. We have previously reported that the human anti-apoptotic gene bcl-2 is functional in Drosophila, suggesting that the fruit fly shares regulatory mechanisms with vertebrates and the nematode, involving anti-apoptotic members of the bcl-2 family. We now report that bcl-2 suppresses rpr-induced apoptosis in Drosophila. Additionally, we have compared features of bax- and rpr-induced apoptosis. Flow cytometry analysis of wing disc cells demonstrate that both killers trigger mitochondrial defects. Interestingly, bcl-2 suppresses both bax- and rpr-induced mitochondrial defects while the caspase-inhibitor p35 is specific to the rpr pathway. Finally, we show that the inhibition of apoptosis by bcl-2 is associated with the down-regulation of rpr expression.

The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model

The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. Here, using the Drosophila wing imaginal disc as a model system, we validated that Presenilin overexpression induces chronic ER stress in vivo. We observed, in this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis requiring downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation.

zVAD-fmk upregulates caspase-9 cleavage and activity in etoposide-induced cell death of mouse embryonic fibroblasts.

Caspases are key effectors of programmed cell death. Down- and up-regulation of their activity are involved in different pathologies. In most cells, zVAD-fmk prevents apoptosis. However, unexpected effects of zVAD-fmk have been characterized in different laboratories, cell models and cell death processes. We have previously shown that zVAD-fmk accelerates p53-dependent apoptosis in rat embryonic fibroblasts. In this study, we pursued our investigations on zVAD-fmk effects and focused our study at the mitochondrial level in mouse embryonic fibroblasts (MEFs). In both primary and immortalized (by AgT or 3T9 protocol) MEFs, zVAD-fmk increased etoposide-induced loss of ΔΨm. This increase correlated with an increase of the number of apoptotic cells in primary and 3T9 MEFs, but did not in AgT MEFs. In both types of immortalized MEFs, zVAD-fmk regulated neither p53 levels nor transcriptional activities, suggesting that zVAD-fmk acts downstream of p53. In MEFs, zVAD-fmk increased p53-dependent loss of ΔΨm, cytochrome c release and caspase-9 activity. Indeed, zVAD-fmk inhibited effector caspases (caspases-3, -6, -7) as expected but increased caspase-9 cleavage and activity in etoposide-treated MEFs. Q-VD-OPh, another caspase inhibitor, also increased both loss of ΔΨm and caspase-9 cleavage in etoposide-treated MEFs. Invalidation of bax and bak suppressed p53-dependent cell death and zVAD-fmk regulation of this process. Invalidation of caspase-9 did not inhibit mitochondrial membrane depolarization but suppressed zVAD-fmk amplification of this process. Altogether, our data suggest that caspase-9 activity is up-regulated by zVAD-fmk and is involved in an amplification loop of etoposide-induced cell death at the mitochondrial level in MEFs.