CONICET | Buscador de Institutos y Recursos Humanos

Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in cestoda.The best-characterized functions of autophagy include maintenance of cellular quality control and provision of an alternate source ofenergy during starvation, a physiological condition of Echinococcus granulosus larval stages (permanent nutritional dependence on thehosts and low cellular energy generation in the parasite life cycle -Zheng et al., 2013), which produces cystic echinococcosis disease orhydatidosis in humans. In this project, the autophagy has been cellular and molecularly analyzed under basal conditions in the larvalstages (metacestode and protoscolex forms) as well as under the pharmacological induction of rapamycin (positive control) andmetformin, arsenic trioxide, bortezomib and octreotide (study cases). Metacestode sensitivity to rapamycin (Rm) and metformin (Met)as well as TORC1 and AMPK expression in protoscoleces and metacestodes have been demonstrated (Cumino et al., 2010; Loos &Cumino 2015).Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involvedin autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 inthe drug treatments. A single FoxO transcription factor was identified in E. granulosus (Eg-FoxO), the first transcription factor that isnecessary and sufficient to induce autophagy in the larvae of Drosophila melanogaster and Caenorhabditis elegans (Klionsky et al.,2012). Eg-FoxOs share similar DNA binding specificity, with the core binding motif being defined as TTGTTTAC (Furuyama et al.,2000). We thus analyzed Eg-FoxO transcriptional target candidates predicted by the 1 kb upstream survey of each Echinococcus atggene and we detected that the conserved binding motif described for FoxO-activated genes was present in upstream sequences from Egatg8and Eg-atg12 putative promoters. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition throughTOR and by transcription-dependent up-regulation via FoxO-like protein.The presence of Eg-Atg8 punctate images and the high gene expression level of atg genes from control parasites suggest theoccurrence of basal autophagy in the larval stages (Loos et al., 2014). Our experiments also showed an Eg-Atg8 level incrementproportional to drug concentration, and a punctate pattern was detected in the syncytial tegument and in several parenchymal cells ofthe soma in treated parasites. Another important observation was the presence of Eg-Atg8 punctate dot formations in the terminalvesicle system and vesicularised protoscoleces, which suggests that autophagy has a particular role in development towards vesiculardifferentiation. On the other hand, SEM/EDAX images and high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix ofbiomineralized cells as the calcareous corpuscles were observed, which owe their origin and cellular activity to the occurrence ofautophagic events in cestode tissues (Loos et al., 2014) leading to reconsideration of the theory proposed by McCullough andFairweather (1987) on corpuscle autophagic development. The presence of multilamellar structures and focal deposition of excessiveamounts of calcium into calcareous corpuscles could be a reason for autophagy after endoplasmic reticulum stress, being different fromthose cells deprived of nutrients (Ogata et al., 2006). In addittion, ultrastructural studies showed that Rm treated parasites had anisolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. As the endoplasmic reticulum isfrequently observed in close proximity to autophagosomes, it has been proposed as the source of membrane for the autophagosome orelse as the plataform.Actually we are interested in describing the lipophagy and glycophagy as homeostatic processes that could occur in theparasite, which could also be pharmacologically inferred by Met. Since starvation and acute lipid stimulus increase autophagicsequestration of lipid droplets (LDs), we find that providing a physiological fatty acid such as oleic acid (OA), the number and totalarea of LDs detected by bodipy 493/503 (a lipid droplet marker) were significantly higher compared to the control. Given thedifferences in organelle size between autophagosme-lysosome and LDs, it is likely that autophagosome assembly occurs at the surfaceof the lipid droplet. In this line evidence, the proximity and colocalization of LDs with Eg-Atg8 were determined by confocalmicroscopy. In support of this notion, other proteins that upstream regulator of autophagy as Atg5, Atg6 and Atg7 could be analyzed onthe LD surface in order to study the lipophagy in the parasite. Additionally, it has been revealed that starvation-induced activation ofthe transcription factor FoxO modulates LDs by upregulation of lysosomal acid lipase-mediated autophagy via TFEB, other masterregulator of autophagy and lysosoma biogenesis also identified in the parasite. Thus, in the following experiment we analyzed thefunctional activity of FoxO and TFBE in Echinococcus sp. regarding in autophagic degradation of neutral lipids and glycogen. Thedetection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under bothphysiological and stress conditions which will allow to identificate new strategies for drug discovery against neglected parasiticdiseases caused by cestoda.Proposal Projections: Study the activation pathway of autophagy through AMPK and elucidate the activation state of Eg-Atg1, raptor and FoxO inthe absence of TSC1/2 and Rheb. Carry a functional study of the FoxO and TFBE transcription factor from the gene promoters of the autophagic proteins andlysosomal lipases of the parasite. Analyze the events of selective autophagy of lipid droplets and glycogen as homeostatic mechanisms that support theintermediary metabolism in the cestode Study as the lack of expression of specific Atg proteins by RNAi could be disturb the autophagy process and then evaluatehow much this disruption affects the parasite differentiation and cell death.

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