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The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a newWe produced transgenicLoss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 inAntagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFβ/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.

International audience; Polyglutamine diseases are nine dominantly inherited neurodegenerative pathologies caused by the expansion of a polyglutamine domain in a protein responsible for the disease. This expansion leads to protein aggregation, inclusion formation and toxicity. Despite numerous studies focusing on the subject, whether soluble polyglutamine proteins are responsible for toxicity or not remains debated. To focus on this matter, we evaluated the level of soluble and insoluble truncated pathological Ataxin-3 in vivo in Drosophila, in presence or absence of two suppressors (i.e. Hsp70 and non-pathological Ataxin-3) and along aging. Suppressing truncated Ataxin-3-induced toxicity resulted in a lowered level of aggregated polyglutamine protein. Interestingly, aggregates accumulated as flies aged and reached a maximum level when cell death was detected. Our results were similar with two other pathological polyglutamine proteins, namely truncated Ataxin-7 and full-length Ataxin-3. Our data suggest that accumulation of insoluble aggregates beyond a critical threshold could be responsible for toxicity.

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.

Background After more than 40 years of research, the mechanisms underlying the association of HLA-B27 with spondyloarthritis (SpA) remain poorly understood. We hypothesised that Drosophila might be a relevant model to study HLA-B27 and particularly for deciphering the cellular cascade and the genetic pathways affected by HLA-B27 mutation. To do so, we developed HLA-B2705, HLA-B0702 (control) and human Beta-2-microglobulin (hB2m) transgenic Drosophila. Methods Gateway Technology and UAS-Gal4 system used for developing transgenic HLA-B/human hB2m Drosophila. UAS-hB2m transgene was inserted in the long arm of chromosome 3 and UAS-HLA-B2705 and UAS-HLA-B0702 were alternatively inserted at another position in the short arm of the same chromosome. For each construct, various transgenic lines were obtained and crossed with several tissue specific driver strains, to induce the expression of the sequence coding for HLA-B0702 and hB2m or HLA-B2705 and hB2m placed under the control of UAS sequences. Results HLA-B2705/hB2m transgenes were first expressed in Drosophila thanks to the vestigialGa4 driver line allowing to produce targeted proteins in the Vestigial domain (dorso-ventral frontier of the wing epithelia). We observed positive staining with HC10 antibodies (class I heavy chain) and W6/32 antibody (HLA-A, HLA-B and HLA-C conformation) for both tested HLA-B/hB2m but only HLA-B27/hB2m was labelled with ME1 (anti-HLA B/C) antibodies, suggesting a different conformation of HLA-B27 and HLA-B7 with hB2m in the wing epithelia. Furthermore, by mean of the nubbin or engrailed drivers which drive the expression in a larger part of the wing, we observed specific loss of the posterior cross-vein following HLA-B27/hB2m expression but not HLA-B7/hB2m. Conclusion Drosophila lines were established allowing tissue specific expression of different HLA-B alleles. Our data suggest that the expression of HLA-B/hB2m transgenes in epithelial cells leads to a plasma membrane localization for HLA-B2705/hB2m but not for HLA-B0702/hB2m. Furthermore, we observed that tissue specific expression of HLA-B27/hB2m but not HLA-B7/hB2m induced specific loss of cross-vein suggesting it interferes with developmental pathways involved in differentiation. This is the first time that differences in localization between HLA-B2705, a subtype associated with SpA, and HLA-B0702, which is not associated with the disease are reported. These results show that transgenic Drosophila might be a pertinent model to decipher molecular mechanisms involved in HLA-B27 trafficking and to better understand potential different behaviours of HLA-B subtypes.

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.

Les genes impliques dans l’execution par apop-tose de la mort cellulaire programmee ont ete conserves au cours de l’evolution. Toutefois, le controle de l’engagement dans ce processus varie selon les organismes. Chez Caenorhabditis elegans , l’approche genetique a permis de montrer que l’execution de l’apoptose est essentiellement assuree par le gene ced-3 , qui code pour une pro-tease a cysteine appartenant a la famille des caspases dont l’activite est controlee par les proteines CED-4 et CED-9. Ces genes ont des homologues chez les mammiferes, mais le controle de l’activation des caspases y est plus complexe. Un autre organisme modele, la drosophile, presente une complexite intermediaire entre le nematode et les mammiferes pour la regulation et l’execution de l’apoptose. L’objectif de cet article est de faire le point sur l’apport de la drosophile a notre comprehension de l’apoptose, et de montrer comment cet organisme pourrait permettre d’identifier des voies de signalisation et de regulation conservees chez les mammiferes.

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.

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 Deltavps15 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 Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 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.

Dietary restriction (DR) extends life span of many organisms, from yeast to mammals. The question of whether or not the SIR2 protein functions to mediate life span extension in response to DR remains debated. In this paper, we studied the relationship between SIR2 and DR in the filamentous fungus Podospora anserina. We show that the loss of PaSir2, PaHst2 or PaPnc1 does not alter life span under standard conditions. PaHst2 is the closest paralog of PaSir2 and the ortholog of yeast HST2 and PaPnc1 is the ortholog of the yeast PNC1 which encodes a nicotinamidase that deaminates nicotinamide, a natural inhibitor of SIR2. As observed for other organisms, overexpression of PaSir2 weakly increases life span under standard condition. Under DR conditions, deletion of the PaSir2 or PaHst2 genes induce a significant reduction in life span extension, while the double mutant strongly reduces life span extension. However, a clear response to DR subsists in the double mutant, demonstrating that DR acts through a SIR2/HST2 independent pathway.

International audience; 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.

International audience; Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.