Autophagy and Self-Preservation: A Step Ahead from Cell Plasticity?

Abstract

Silencing the SPINK-related gene Kazal1 in hydra gland cells induces an excessive autophagy of both gland and digestive cells, leading to animal death. Moreover, during regeneration, autophagosomes are immediately detected in regenerating tips, where Kazal1 expression is lowered. When Kazal1 is completely silenced, hydra no longer survive the amputation stress (Chera S, de Rosa R, Miljkovic-Licina M, Dobretz K, Ghila L, Kaloulis K, Galliot B. Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype. J Cell Sci 2006; 119:846-57). These results highlight the essential digestive and cytoprotective functions played by Kazal1 in hydra. In mammals, autophagy of exocrine pancreatic cells is also induced upon SPINK1/ Spink3 inactivation, whereas Spink3 is activated in injured pancreatic cells. Hence SPINKs, by preventing an excessive autophagy, appear to act as key players of the stress-induced self-preservation program. In hydra, this program is a prerequisite to the early cellular transition, whereby digestive cells of the regenerating tips transform into a head-organizer center. Enhancing the self-preservation program in injured tissues might therefore be the condition for unmasking their potential cell and/or developmental plasticity. HYDRA, A MODEL SYSTEM FOR DUODENAL DIGESTION AND REGENERATIONHydra is a freshwater cnidarian, made up of two cell layers, the ectoderm and the endoderm, separated by an extracellular matrix named mesoglea. This animal exhibits a tube shape, mostly a gastric cavity limited by a single opening at the top, the mouth, circled by a ring of tentacles, forming together the head region. At the other end, the basal disk secretes mucous to attach to the substratum (Fig. 1A). Hence cnidarian polyps display an oral-aboral polarity, with differentiated tissues and structures at the extremities, including a sophisticated neuromuscular system, but no organs as recognized in bilaterians.The digestive function requires the cooperation of gland cells and endodermal epithelial cells (also named digestive cells) of the gastric cavity. The gland cells, packed with secretory granules full of zymogens, display a cellular organization very similar to that of the vertebrate exocrine pancreatic cells and the proteases they release in the gastric cavity participate in the enzymatic digestion. Hence, hydra gland cells are considered as "pancreatic cells." 1 Beside its physiology, hydra provides a unique model to investigate cell and developmental plasticity. 2,3 Upon regular feeding, hydra continuously reproduce asexually through budding; after amputation they regenerate the missing part of their body in few days; after dissociation of their tissues, they can reaggregate and regenerate. The molecular work carried out over the last 15 years, showed a tremendously high level of conservation between hydra and mammalian genes 3-5 supporting the paradigmatic value of this simple animal.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…In the latter case, an excess of autophagy in the regenerating tip immediately after amputation is deleterious. [1354][1355][1356] Most components of the autophagy and MTOR pathways are evolutionarily conserved in Hydra. 1353 For steady-state measurements, autophagy can be monitored by western blot for ATG8/LC3, by immunofluorescence (using antibodies to ATG8/LC3, lysobisphosphatidic acid or RPS6KA/ RSK), or with dyes such as MitoFluor Red 589 and LysoTracker Red.…”

“…It is also possible to monitor MTOR activity with phosphospecific antibodies to RPS6KB and EIF4EBP1 or to examine gene expression by semiquantitative RT-PCR, using primers that are designed for Hydra. Autophagy can be induced by RNAi-mediated knockdown of Kazal1, 1354,1355 or with rapamycin treatment, and can be inhibited with wortmannin or bafilomycin A 1 . 1352,1353…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…Hydra is a freshwater cnidarian animal that provides a unique model system to test autophagy either in the context of nutrient deprivation, as these animals easily survive several weeks of starvation, 817,818 or in the context of regeneration, because in the absence of protease inhibitors, bisection of the animals leads to an uncontrolled wave of autophagy; in the latter case, an excess of autophagy in the regenerating tip immediately after amputation is deleterious. [819][820][821] Most components of the autophagy and MTOR pathways are evolutionarily conserved in Hydra. 818 For steady-state measurements, autophagy can be monitored by western blot for ATG8/LC3, by immunofluorescence (using antibodies to ATG8/LC3, LBPA or RSK), or with dyes such as MitoFluor Red 589 and LysoTracker Red.…”

“…It is also possible to monitor MTOR activity with phosphospecific antibodies to RPS6KB kinase and EIF4EBP1, or to examine gene expression by semiquantitative RT-PCR, using primers that are designed for Hydra. Autophagy can be induced by RNAi-mediated knockdown of Kazal1, 819,820 or with rapamycin treatment, and can be inhibited with wortmannin or bafilomycin A 1 .…”

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

“…Induction of autophagy has been demonstrated during the early neonatal starvation in mice (Kuma et al 2004). Mutant yeast cells that have a reduced autophagic capability rapidly perish in nutritiondeficient conditions (Tsukada and Ohsumi 1993) and a tight control of autophagy is also required for survival of Hydra and C. elegans after starvation-induced stress (Chera et al 2009;Galliot 2006;Sigmond et al 2008). HSP90 is a key component of the chaperone-mediated autophagy (CMA) mechanism, with a possible inhibitory role in the molecular chaperone complex (Dice 2007;Majeski and Dice 2004).…”