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Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

View Article: PubMed Central - PubMed

ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.


Related in: MedlinePlus

Wortmannin impairs autophagy and leads to increased degeneration of the midgut epithelium.(a) Western blot analysis of BmAtg8–PE demonstrating autophagy inhibition in midgut cells treated with wortmannin; (b) acid phosphatase activity; (c,d) morphology of larval and pupal epithelium in control (c) and treated (d) pupae; (e) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with wortmannin. Lm: larval midgut epithelium; Pm: pupal midgut epithelium. Values represent mean ± s.e.m.
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f7: Wortmannin impairs autophagy and leads to increased degeneration of the midgut epithelium.(a) Western blot analysis of BmAtg8–PE demonstrating autophagy inhibition in midgut cells treated with wortmannin; (b) acid phosphatase activity; (c,d) morphology of larval and pupal epithelium in control (c) and treated (d) pupae; (e) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with wortmannin. Lm: larval midgut epithelium; Pm: pupal midgut epithelium. Values represent mean ± s.e.m.

Mentions: Lower Atg8–PE levels were visible in the larvae treated with wortmannin (Fig. 7a) due to the impairment of autophagy activation. Acid phosphatase activity did not show any significant change (Fig. 7b), indicating that, conversely to chloroquine treatment, lysosomes had not accumulated in the midgut cells. The effects of the inhibitor on the larval midgut were comparable to those obtained by chloroquine (Fig. 7c,d), and an increase in cleaved BmCaspase-1 levels could also be observed (Fig. 7e). Of note, although the two inhibitors act at different levels of the autophagic process (autophagosome-lysosome fusion vs. an upstream step of autophagy activation, i.e., class III PI3K activity), a similar morphological effect on the tissue was observed after the treatments. Moreover, increased levels of active BmCaspase-1 were recorded after inhibiting autophagy in both cases. These data demonstrate that inhibition of autophagy determines a stronger degree of degeneration of the midgut epithelium and increased levels of activated caspases in these cells, thus suggesting a pro-survival role of autophagy in this tissue.


Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis
Wortmannin impairs autophagy and leads to increased degeneration of the midgut epithelium.(a) Western blot analysis of BmAtg8–PE demonstrating autophagy inhibition in midgut cells treated with wortmannin; (b) acid phosphatase activity; (c,d) morphology of larval and pupal epithelium in control (c) and treated (d) pupae; (e) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with wortmannin. Lm: larval midgut epithelium; Pm: pupal midgut epithelium. Values represent mean ± s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016986&req=5

f7: Wortmannin impairs autophagy and leads to increased degeneration of the midgut epithelium.(a) Western blot analysis of BmAtg8–PE demonstrating autophagy inhibition in midgut cells treated with wortmannin; (b) acid phosphatase activity; (c,d) morphology of larval and pupal epithelium in control (c) and treated (d) pupae; (e) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with wortmannin. Lm: larval midgut epithelium; Pm: pupal midgut epithelium. Values represent mean ± s.e.m.
Mentions: Lower Atg8–PE levels were visible in the larvae treated with wortmannin (Fig. 7a) due to the impairment of autophagy activation. Acid phosphatase activity did not show any significant change (Fig. 7b), indicating that, conversely to chloroquine treatment, lysosomes had not accumulated in the midgut cells. The effects of the inhibitor on the larval midgut were comparable to those obtained by chloroquine (Fig. 7c,d), and an increase in cleaved BmCaspase-1 levels could also be observed (Fig. 7e). Of note, although the two inhibitors act at different levels of the autophagic process (autophagosome-lysosome fusion vs. an upstream step of autophagy activation, i.e., class III PI3K activity), a similar morphological effect on the tissue was observed after the treatments. Moreover, increased levels of active BmCaspase-1 were recorded after inhibiting autophagy in both cases. These data demonstrate that inhibition of autophagy determines a stronger degree of degeneration of the midgut epithelium and increased levels of activated caspases in these cells, thus suggesting a pro-survival role of autophagy in this tissue.

View Article: PubMed Central - PubMed

ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.


Related in: MedlinePlus