The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model

Abstract

The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. Here, using the Drosophila wing imaginal disc as a model system, we validated that Presenilin overexpression induces chronic ER stress in vivo. We observed, in this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis requiring downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation.

Figure 1

The PERK/ATF4 pathway is responsible for Psn overexpression-induced loss of DIAP1 and apoptosis. (a) Third-instar imaginal wing discs with the Psn-expressing domain detected thanks to GFP (green) and TUNEL staining (red) to detect apoptosis. Genotypes are vg-GAL4/+ UAS-EGFP/+ (top), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-EGFP/+ (middle) and UAS-p35/+ vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-EGFP/+ (bottom). (b) Third-instar imaginal wing discs carrying an xbp1::EGFP reporter transgene (green) to detect ER stress and immunostained with an anti-activated Caspase 3 staining (red) to detect apoptosis. Genotypes are vg-GAL4/+ xbp1::EGFP/+ (top row) and vg-GAL4, UAS-Psn, UAS-Psn/+ xbp1::EGFP/+ (middle row). Bottom row images are enlargement of boxed regions. Colocalization between anti-activated Caspase 3 staining and Ire1 activity reporter is highlighted in white thanks to the ImageJ Colocalization plugin in the merge enlargement. Note that developmental apoptosis is only observed in the notum (top). (c) TUNEL staining of Psn-expressing third-instar imaginal wing discs in the absence or presence of dronc mutations. Genotypes are vg-GAL4, UAS-Psn, UAS-Psn/+ (left) and vg-GAL4, UAS-Psn, UAS-Psn/+ dronc^I24/ dronc^I29(right). (d, e) Third-instar imaginal wing discs with the Psn-expressing domain detected thanks to GFP (green), TUNEL stained (red) to detect apoptosis and immunostained with an anti-DIAP1 antibody (blue). (d) Genotypes are vg-GAL4/+ UAS-EGFP/+ (top), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-EGFP/+ (middle) and vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-EGFP/UAS-atf4-RNAi (bottom). (e) Genotypes are vg-GAL4, UAS-Psn, UAS-Psn /UAS-nGFP; UAS-EGFP/+ (top), vg-GAL4, UAS-Psn, UAS-Psn/UAS-nGFP; UAS-p35/+ (bottom) (f) Anti-β-Galactosidase staining to detect diap1^J5C8 reporter expression in vg-GAL4/+ diap1^J5C8/+ (left column) vg-GAL4, UAS-Psn, UAS-Psn/+ diap1^J5C8/+ (center); and vg-GAL4, UAS-Psn, UAS-Psn/+ diap1^J5C8/UAS-atf4-RNAi (right column) third-instar wing imaginal discs. The black lines correspond to the projection of the corresponding enlarged transverse XZ sections shown under disk images. Enlarge areas are oriented with their basal side to the left. The asterisk indicates the diap1 expression reduction (center)

Figure 2

Dilp8-induced developmental delay favors tissue homeostasis after ER stress. (a) Effects of Psn overexpression on pupariation and adult eclosion timing. Hatched bar (vg-GAL4/+) and open bar (vg-GAL4, UAS-Psn, UAS-Psn/+) represent mean time in hours AED of pupariation (left) or adult eclosion (right). Error bars represent the S.E. of the mean. Asterisks indicate a significant difference with the control (Left: n=4, P<10⁻⁵, ANOVA), (Right: n=6, P<10⁻⁷, ANOVA). (b) Effects of 20E food supplementation of 92 h AED old vg-GAL4, UAS-Psn, UAS-Psn/+ larvae on adult eclosion. Error bars represent the S.E.M. of three independent experiments (asterisk: P<5%, ANOVA). (c) Distribution of notched-wing phenotypes after 20E supplementation (gray bars) or not (black bars) according to their strength in a representative experiment (n=3, P<5%, ANOVA). (d, e) Third-instar wing imaginal discs carrying Dilp8::EGFP (green) and immunostained with an antiactivated caspase 3 antibody (red). Genotypes are vg-GAL4/+ dilp8^MI00727/+ (d) and vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/+ (e). The enlargement of the boxed area in e, shows signal colocalization highlighted in white thanks to the ImageJ Colocalization plugin. (f, g) dilp8 RNA levels measured by quantitative reverse PCR. Data represent mean ± S.E.M. of three independent experiments. RNA was extracted from control (vg-GAL4/+, hatched bar, (f) or ER stressed (vg-GAL4, UAS-Psn, UAS-Psn/+, open bar, f and g) or ER-stressed dronc mutant (vg-GAL4, UAS-Psn, UAS-Psn/+ dronc^I24/ dronc^I29, black bar, g) wing imaginal discs. The asterisk indicates a significant difference with the control (P<10⁻⁴, Student's t-test). (h). Effects of dilp8 homozygous mutation (dilp8^MI00727/dilp8^MI00727, gray bars) compared with dilp8 heterozygosity (dilp8^MI00727/rhea^MI00296, white bars) on adult eclosion of vg-GAL4, UAS-Psn, UAS-Psn/+ (plain bars) and vg-GAL4/+ (hatched bars) on mean time of adult eclosion. The rhea^MI00296 strain is commonly used as the control for dilp8^MI00727 genetic background. Asterisks indicate significant difference from the corresponding control that does not express Psn (P<10⁻⁴, ANOVA). Error bars represent the S.E.M. of six independent experiments. (i) Distribution of notched-wing phenotypes of vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/dilp8^MI00727flies (gray bars) compared with vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/rhea^MI00296 flies (open bars) (n=3, P<10⁻⁶, ANOVA)

Figure 3

Psn overexpression induces a JNK pathway-independent apoptosis. (a, a') Anti-MMP1 staining in vg-GAL4/+ (a) and vg-GAL4, UAS-Psn, UAS-Psn/+ (a') third-instar wing imaginal discs. (b–c') Anti-β-Galactosidase staining to detect the expression of msn- (b) or puc-lacZ (c, d) reporters in vg-GAL4/+ msn⁰⁶⁹⁴⁶/+ (b), vg-GAL4, UAS-Psn, UAS-Psn/+ msn⁰⁶⁹⁴⁶/+ (b'), vg-GAL4/+ puc^E69/+ (c) and vg-GAL4, UAS-Psn, UAS-Psn/+ puc^E69/+ (c') third-instar wing imaginal discs. Note MMP1 staining (a) and msn-lacZ expression (b) in the notum (white arrowheads) are constitutive and independent from Psn overexpression. Note that puc-lacZ expression at the tip of the notum reflects the JNK pathway activation required for thorax closure (c, white arrow). (d, d') Colocalization (white) between anti-β-Galactosidase staining (green) to detect puc expression and either TUNEL labeling (d, red) to detect apoptosis or anti-PH3 (d', red) to detect mitotic cells in vg-GAL4, UAS-Psn, UAS-Psn/+ puc^E69/+ third-instar larvae wing imaginal discs. (e–e''') TUNEL staining in vg-GAL4, UAS-Psn, UAS-Psn/+ (e), UAS-bsk^DN/+ vg-GAL4, UAS-Psn, UAS-Psn/+ (e'), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-hep-RNAi/+ (e') and in vg-GAL4, UAS-Psn, UAS-Psn/UAS-cdk5-RNAi (e''') third-instar wing imaginal discs

Figure 4

The JNK pathway regulates a Dilp8-dependent developmental delay. (a) Effects of puc overexpression (UAS-puc) (gray) and puc mutant heterozygosity (puc^E69/+) (black) compared with control (white) in a vg-GAL4, UAS-Psn, UAS-Psn/+ (plain bars) or vg-GAL4/+ (hatched bars) background on the mean time of adult eclosion. Error bars represent the S.E.M. (n=4). Asterisks indicate significant difference (P<10⁻⁴, ANOVA). (b) Distribution of notched-wing phenotypes of vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-puc/+ (gray bars) and vg-GAL4, UAS-Psn, UAS-Psn/+ puc^E69/+ (black bars) flies compared with vg-GAL4, UAS-Psn, UAS-Psn/+ flies (open bars). (c, d) Intensity of GFP reflecting dilp8 expression in vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/+ (c left, d white bars), vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/UAS-puc (c center, d gray bar), and vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/puc^E69(c right, d black bar) third-instar wing imaginal discs. Error bars represent the S.E.M. of at least eight independent experiments. Asterisks indicate significant difference (P<10⁻³, ANOVA)

Figure 5

ATF4 activates the JNK pathway. (a, left) Anti-activated caspase 3 (green) and anti-MMP1 (red) staining in vg-GAL4, UAS-Psn, UAS-Psn/+ (top) and vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-diap1/+ (bottom) third-instar wing imaginal discs. (a, right) Intensity of GFP in vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/+ (top) and vg-GAL4, UAS-Psn, UAS-Psn/+ dilp8^MI00727/UAS-diap1 (bottom) third-instar wing imaginal discs. (b) Quantification of relative dilp8::EGFP expression. Error bars represent the S.E.M. of at least nine independent experiments. The asterisk indicates significant difference between the indicated genotype and the control (P<10⁻⁵, ANOVA). (c) Anti-MMP1 staining (left) and intensity of Dilp8::EGFP detection (right) in vg-GAL4, UAS-Psn, UAS-Psn/+ (top) and vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-atf4-RNAi (bottom) third-instar wing imaginal discs. (d) Quantification of relative dilp8 expression. Error bars represent the S.E.M. of at least four independent experiments. The asterisk indicates significant difference between the indicated genotype and the control (P<10⁻⁴, ANOVA). (e) Effects of ATF4 depletion (gray bars) compared with control (white bars) on vg-GAL4, UAS-Psn, UAS-Psn/+ (solid bars) and vg-GAL4/+ (streaked bars) on the mean time of adult eclosion. Error bars represent the S.E.M. (n=3). The asterisk corresponds to a statistical difference (P<10⁻³, ANOVA)

Figure 6

The Rac1/Slpr JNK pathway triggers an ER stress-induced developmental delay negatively controlled by Msn. (a–c) Distribution of notched-wing phenotypes of vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-slpr-RNAi/+ (a, red), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-msn-RNAi/+ (b, blue) and vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-Rac1-RNAi/+ (c, yellow) flies compared with vg-GAL4, UAS-Psn, UAS-Psn/+ flies (a–c, white). (d–i) Intensity of GFP emitted by the dilp8::EGFP reporter in vg-GAL4, UAS-Psn, UAS-Psn/+ (d–i), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-slpr-RNAi/+ (d, g), vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-msn-RNAi/+ (e, h) and vg-GAL4, UAS-Psn, UAS-Psn/+ UAS-Rac1-RNAi/+ (f, i) third-instar wing imaginal discs. Error bars represent the S.E.M. of at least six independent experiments. Asterisks indicate significant difference between the indicated genotype and its control (P<10⁻⁴). (j–l) Effects of Slpr (j, red), Msn (k, blue) and Rac1 (l, yellow) depletion compared with the control (white) in a vg-GAL4, UAS-Psn, UAS-Psn/+ (plain bars) or vg-GAL4/+ (hatched bars) background on mean time of adult eclosion. Error bars represent the S.E.M. of seven independent experiments. Asterisks correspond to significant difference between controls without Psn (hatched bars) and the indicated genotype (P<10⁻⁴, ANOVA)

Figure 7

Model of tissue homeostasis maintenance after an ER stress. Chronic ER stress activates the PERK pathway, which results in ATF4 expression. ATF4 has two antagonistic functions. On one hand, ATF4 induces a caspase-dependent apoptosis by repressing diap1 expression. On the other hand, it favors tissue homeostasis through the induction of dilp8 expression by Rac1/Slpr/JNK pathway activation. The role of Msn in the control of the observed developmental delay remains to be elucidated. It could either negatively regulate the JNK pathway or have a JNK pathway-independent mechanism of action