Limits...
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

Autophagy inhibition by chloroquine determines an increased degeneration of the larval midgut.(a,b) TEM analysis of midgut cells in control (a) and chloroquine-treated (b) insects showing a significant accumulation of vesicles (arrows) after inhibition of autophagy; (c) detail of autophagic compartments in midgut cells of treated larvae; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) demonstrating the inhibition of autophagy in midgut cells by chloroquine; (f,g) morphology of the larval and pupal epithelium in control (f) and treated (g) pupae; (h) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with chloroquine. A: autophagosomes; Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium. Values represent mean ± s.e.m. (*p < 0.05 using Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016986&req=5

f6: Autophagy inhibition by chloroquine determines an increased degeneration of the larval midgut.(a,b) TEM analysis of midgut cells in control (a) and chloroquine-treated (b) insects showing a significant accumulation of vesicles (arrows) after inhibition of autophagy; (c) detail of autophagic compartments in midgut cells of treated larvae; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) demonstrating the inhibition of autophagy in midgut cells by chloroquine; (f,g) morphology of the larval and pupal epithelium in control (f) and treated (g) pupae; (h) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with chloroquine. A: autophagosomes; Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium. Values represent mean ± s.e.m. (*p < 0.05 using Student’s t-test).

Mentions: TEM analysis showed that many vesicles accumulated in midgut cells after administering chloroquine (Fig. 6a–c). According to their size, morphology, and content, they could be classified as autophagic compartments. Moreover, Atg8–PE levels (Fig. 6d) and acid phosphatase activity both (Fig. 6e) increased in treated larvae. These results demonstrate the efficacy of chloroquine treatment in blocking autophagic flux and the consequent accumulation of autophagosomes and lysosomes in midgut cells. While the morphology of the new pupal epithelium did not change in larvae injected with chloroquine, the organization of the larval epithelium was completely different. In fact, the old epithelium appeared more degenerated than in control animals, and no tissue structure was visible after the treatment (Fig. 6f,g). Furthermore, Western blot analysis showed increased levels of cleaved BmCaspase-1 after administration of the autophagic inhibitor (Fig. 6h).


Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis
Autophagy inhibition by chloroquine determines an increased degeneration of the larval midgut.(a,b) TEM analysis of midgut cells in control (a) and chloroquine-treated (b) insects showing a significant accumulation of vesicles (arrows) after inhibition of autophagy; (c) detail of autophagic compartments in midgut cells of treated larvae; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) demonstrating the inhibition of autophagy in midgut cells by chloroquine; (f,g) morphology of the larval and pupal epithelium in control (f) and treated (g) pupae; (h) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with chloroquine. A: autophagosomes; Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium. Values represent mean ± s.e.m. (*p < 0.05 using Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Autophagy inhibition by chloroquine determines an increased degeneration of the larval midgut.(a,b) TEM analysis of midgut cells in control (a) and chloroquine-treated (b) insects showing a significant accumulation of vesicles (arrows) after inhibition of autophagy; (c) detail of autophagic compartments in midgut cells of treated larvae; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) demonstrating the inhibition of autophagy in midgut cells by chloroquine; (f,g) morphology of the larval and pupal epithelium in control (f) and treated (g) pupae; (h) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with chloroquine. A: autophagosomes; Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium. Values represent mean ± s.e.m. (*p < 0.05 using Student’s t-test).
Mentions: TEM analysis showed that many vesicles accumulated in midgut cells after administering chloroquine (Fig. 6a–c). According to their size, morphology, and content, they could be classified as autophagic compartments. Moreover, Atg8–PE levels (Fig. 6d) and acid phosphatase activity both (Fig. 6e) increased in treated larvae. These results demonstrate the efficacy of chloroquine treatment in blocking autophagic flux and the consequent accumulation of autophagosomes and lysosomes in midgut cells. While the morphology of the new pupal epithelium did not change in larvae injected with chloroquine, the organization of the larval epithelium was completely different. In fact, the old epithelium appeared more degenerated than in control animals, and no tissue structure was visible after the treatment (Fig. 6f,g). Furthermore, Western blot analysis showed increased levels of cleaved BmCaspase-1 after administration of the autophagic inhibitor (Fig. 6h).

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