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

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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.


Rapamycin fails to activate a full autophagic response.(a) Western blot analysis of p-Bm4ebp1 in midgut of larvae injected with rapamycin; (b,c) qRT-PCR analysis of BmATG8 (b) and BmATG1 (c) expression after rapamycin treatment; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) after administration of rapamycin. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
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f4: Rapamycin fails to activate a full autophagic response.(a) Western blot analysis of p-Bm4ebp1 in midgut of larvae injected with rapamycin; (b,c) qRT-PCR analysis of BmATG8 (b) and BmATG1 (c) expression after rapamycin treatment; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) after administration of rapamycin. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).

Mentions: To verify the involvement of Torc1 (Tor complex 1) in 20E-induced autophagy in midgut cells, we evaluated the activity of this complex by monitoring the phosphorylation levels of its target, 4ebp1. Western blot analyses showed that Torc1 activity substantially decreased after the treatment (Fig. 3e). To verify whether 20E activates autophagy by acting exclusively on Torc1, we evaluated the effects of inhibiting this complex on autophagy induction by treating larvae with rapamycin. Following rapamycin administration, the levels of phosphorylated 4ebp1 showed a strong decrease, similar to that observed after 20E injection (Fig. 4a). Indeed, the inhibition of Torc1 during the last larval instar increased the expression of BmATG8 at 6 and 24 h after rapamycin was administered (Fig. 4b). However, rapamycin was not able to induce BmATG1 expression (Fig. 4c). Surprisingly, BmAtg8–PE levels decreased following treatment with rapamycin (Fig. 4d), while acid phosphatase activity did not increase significantly after the injection (Fig. 4e), two results confirming the occurrence of an incomplete autophagic response in rapamycin-treated larvae.


Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis
Rapamycin fails to activate a full autophagic response.(a) Western blot analysis of p-Bm4ebp1 in midgut of larvae injected with rapamycin; (b,c) qRT-PCR analysis of BmATG8 (b) and BmATG1 (c) expression after rapamycin treatment; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) after administration of rapamycin. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Rapamycin fails to activate a full autophagic response.(a) Western blot analysis of p-Bm4ebp1 in midgut of larvae injected with rapamycin; (b,c) qRT-PCR analysis of BmATG8 (b) and BmATG1 (c) expression after rapamycin treatment; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) after administration of rapamycin. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).
Mentions: To verify the involvement of Torc1 (Tor complex 1) in 20E-induced autophagy in midgut cells, we evaluated the activity of this complex by monitoring the phosphorylation levels of its target, 4ebp1. Western blot analyses showed that Torc1 activity substantially decreased after the treatment (Fig. 3e). To verify whether 20E activates autophagy by acting exclusively on Torc1, we evaluated the effects of inhibiting this complex on autophagy induction by treating larvae with rapamycin. Following rapamycin administration, the levels of phosphorylated 4ebp1 showed a strong decrease, similar to that observed after 20E injection (Fig. 4a). Indeed, the inhibition of Torc1 during the last larval instar increased the expression of BmATG8 at 6 and 24 h after rapamycin was administered (Fig. 4b). However, rapamycin was not able to induce BmATG1 expression (Fig. 4c). Surprisingly, BmAtg8–PE levels decreased following treatment with rapamycin (Fig. 4d), while acid phosphatase activity did not increase significantly after the injection (Fig. 4e), two results confirming the occurrence of an incomplete autophagic response in rapamycin-treated larvae.

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.