The Mitochondrial Unfolded Protein Response Is Mediated Cell-Non-autonomously by Retromer-Dependent Wnt Signaling

Abstract

The mitochondrial unfolded protein response (UPR^mt) can be triggered in a cell-non-autonomous fashion across multiple tissues in response to mitochondrial dysfunction. The ability to communicate information about the presence of mitochondrial stress enables a global response that can ultimately better protect an organism from local mitochondrial challenges. We find that animals use retromer-dependent Wnt signaling to propagate mitochondrial stress signals from the nervous system to peripheral tissues. Specifically, the polyQ40-triggered activation of mitochondrial stress or reduction of cco-1 (complex IV subunit) in neurons of C. elegans results in the Wnt-dependent induction of cell-non-autonomous UPR^mt in peripheral cells. Loss-of-function mutations of retromer complex components that are responsible for recycling the Wnt secretion-factor/MIG-14 prevent Wnt secretion and thereby suppress cell-non-autonomous UPR^mt. Neuronal expression of the Wnt ligand/EGL-20 is sufficient to induce cell-non-autonomous UPR^mt in a retromer complex-, Wnt signaling-, and serotonin-dependent manner, clearly implicating Wnt signaling as a strong candidate for the "mitokine" signal.

Keywords: EGL-20; UPR(mt); VPS-35; Wnt signaling; mitochondrial unfolded protein response; mitokine; retromer complex.

Figure 1. The retromer complex is required for cell-non-autonomous UPR^mt induction in animals expressing Q40∷YFP in neurons

(A) Representative photomicrographs demonstrating: a) bright field images of aligned, WT animals; b) Q40∷yfp expression in neurons; c) hsp-6p∷gfp expression in WT animals; d) hsp-6p∷gfp was up-regulated in the intestine in day2 adult animals expressing neuronal Q40∷YFP; e) hsp-6p∷gfp expression was suppressed in vps-35 mutants. The posterior region of the intestine where hsp-6p∷gfp is induced or suppressed is highlighted in (d) and (e). Scale bar: 250μm. (B) Quantification of hsp-6p∷gfp expression of the entire intestine in animals expressing Q40∷YFP in neurons with the presence (d) or absence of the vps-35 mutation (e) as shown in (A). (C) Immunoblots of GFP expression in animals as indicated. (D) Representative photomicrographs of hsp-6p∷gfp animals with the presence or absence of the vps-35 mutation grown on empty vector (EV) or with cco-1 RNAi from hatching. (E) Quantification of hsp-6p∷gfp expression. The genotypes are as in (D). (F) Representative photomicrographs of neuronal cco-1 knockdown; sid-1(qt9); hsp-6p∷gfp animals with the presence or absence of vps-35 mutation. (G) Quantification of hsp-6p∷gfp expression. The genotypes are as in (F). (H) Representative photomicrographs of animals expressing neuronal Q40∷YFP; hsp-6p∷gfp in WT, vps-26, and vps-29 animals. (I) Quantification of hsp-6p∷gfp expression. The genotypes are as in (H). (J) Immunoblots of GFP expression in animals as indicated. YFP can be recognized by the GFP antibody. Anti-tubulin serves as a loading control. *** p < 0.0001, ns denotes p > 0.05 via t-test. Error bars, SEM. n ≥15 worms. See also Figure S1.

Figure 2. Retromer-dependent Wnt secretion is required for cell-non-autonomous UPR^mt induction in animals expressing Q40∷YFP in neurons

(A) Representative photomicrographs of animals expressing neuronal Q40∷YFP; hsp-6p∷gfp in WT, mig-14, dpy-23, or mom-1 RNAi animals. (B) Quantifications of the hsp-6p∷gfp expression as shown in (A). *** p < 0.0001 via t test. Error bars, SEM. n ≥20 worms. (C) Immunoblots of GFP expression in animals as indicated. See also Figure S2.

Figure 3. Wnt ligand/EGL-20 is required for cell-non-autonomous UPR^mt induction in animals with Q40∷YFP expression in neurons

(A) Representative photomicrographs of D2 adult animals expressing neuronal Q40∷YFP; hsp-6p∷gfp in WT or egl-20 animals. (B) Quantification of hsp-6p∷gfp expression. The genotypes are as in (A). (C) Immunoblots of GFP expression. The genotypes are as in (A). (D) Representative photomicrographs of D2 adult animals expressing neuronal Q40∷YFP; DVE-1∷GFP in WT and egl-20 animals. Arrows highlight the DVE-1∷GFP signal localized in the intestinal nuclei. (E) Quantification of the number of intestinal nuclei with DVE-1 puncta. The genotypes are as in (D). (F) Representative photomicrographs of D1 animals expressing hsp-6p∷gfp in WT or egl-20 animals grown on EV or cco-1 RNAi from hatching. (G) Quantification of hsp-6p∷gfp expression. The genotypes are as in (F). (H) Representative photomicrographs of hsp-6p∷gfp expression in neuronal specific cco-1 RNAi animals in WT or egl-20 background. (I) Quantification of hsp-6p∷gfp expression. The genotypes are as in (H). *** p< 0.0001, ns denotes p > 0.05 via t-test. Error bars, SEM. n ≥ 15 worms. See also Figure S3.

Figure 4. Expression of Wnt ligand/EGL-20 is sufficient to induce UPR^mt in both cell-autonomous and cell-non-autonomous manners

(A) Representative photomicrographs of hsp-6p∷gfp reporter animals in WT, rgef-1p∷egl-20 (neuronal), and gly-19p∷egl-20(intestinal) background. (B) Quantification of hsp-6p∷gfp expression. The genotypes are as in (A). (C) Immunoblot of hsp-6p∷gfp expression. The genotypes are as in (A). (D) Representative photomicrographs of DVE-1∷GFP reporter animals in WT, rgef-1p∷egl-20, and gly-19p∷egl-20 background. (E) Quantification of the number of intestinal nuclei with DVE-1 puncta. The genotypes are as in (D). (F) Immunoblot of DVE-1∷GFP expression. The genotypes are as in (D). (G) Representative photomicrographs of hsp-6p∷gfp reporter animals in WT or egl-20p∷egl-20∷mCherry background. (H) Quantification of hsp-6p∷gfp expression. The genotypes are as in (G). (I) Immunoblot of hsp-6p∷gfp expression. The genotypes are as in (G). (J) Representative photomicrographs of rgef-1p∷egl-20; hsp6p∷gfp reporter animals grown on EV, atfs-1, dve-1, lin-65, or jmjd-1.2 RNAi from hatching. (K) Immunoblot of hsp-6p∷gfp expression. The genotypes are as in (J). (L) Representative photomicrographs of hsp-6p∷gfp reporter animals in WT, rgef-1p∷egl-20N(1-720)∷mCherry, or rgef-1p∷egl-20C(721-1182)∷mCherry background. (M) Quantification of hsp-6p∷gfp expression. The genotypes are as in (L). (N) Overexpression of egl-20 in neurons or in the intestine extends C. elegans lifespan. Lifespan analysis of two independent transgenic lines of rgef-1p∷egl-20 or gly-19p∷egl-20 expressing animals compared to WT animals. See also Table S1. *** p< 0.0001 via t-test. Error bars, SEM. n ≥ 20 worms. See also Figure S4.

Figure 5. The canonical Wnt/β-catenin signaling pathway is required for cell-non-autonomous UPR^mt induction in animals expressing Q40∷YFP in neurons

(A) Representative photomicrographs of neuronal Q40∷YFP; hsp-6p∷gfp expressing animals in WT, mig-1, bar-1 RNAi, or pop-1 RNAi background. (B) Quantification of hsp-6p∷gfp expression. The genotypes are as in (A). *** p< 0.0001 via t-test. Error bars, SEM. n ≥ 20 worms. (C) Immunoblots of GFP expression. The genotypes are as in (A). (D) Representative photomicrographs of neuronal Q40∷YFP; hsp-6p∷gfp expressing animals transferred to RNAi plates either from L3/L4 stages or D1 adulthood. See also Figure S5.

Figure 6. The induction of cell-non-autonomous UPR^mt signaling upon neuronal Wnt/EGL-20 expression is dependent on the retromer complex and canonical Wnt signaling

(A) Representative photomicrographs of rgef-1p∷egl-20; hsp-6p∷gfp reporter animals in WT, vps-35, bar-1 RNAi, or pop-1 RNAi background. (B) Quantification of hsp-6p∷gfp expression. The genotypes are as in (A). (C) Immunoblot of GFP expression. The genotypes are as in (A). (D) Representative photomicrographs of gly-19p∷egl-20; hsp-6p∷gfp reporter animals in WT, vps-35, bar-1 RNAi, or pop-1 RNAi background. (E) Quantification of hsp-6p∷gfp expression. The genotypes are as in (D). (F) Immunoblot of GFP expression. The genotypes are as in (D). *** p< 0.0001 via t-test. Error bars, SEM. n ≥ 15 worms. See also Figure S6.

Figure 7. Serotonin is required for the cell-non-autonomous UPR^mt induction upon neuronal Wnt/EGL-20 expression

(A) Representative photomicrographs of rgef-1p∷egl-20; hsp-6p∷gfp animals in WT or tph-1 background. (B) Quantification of hsp-6p∷gfp expression. The genotypes are as in (A). (C) Immunoblot of hsp-6p∷gfp expression. The genotypes are as in (A). (D) Representative photomicrographs of gly-19p∷egl-20; hsp-6p∷gfp transgenic animals in WT or tph-1 background. (E) Quantification of hsp-6p∷gfp expression. The genotypes are as in (D). (F) Immunoblot of hsp-6p∷gfp expression. The genotypes are as in (D). (G) Representative photomicrographs of hsp-6p∷gfp expression in animals treated with vehicle control or 50mM serotonin (5-HT). (H) Quantification of hsp-6p∷gfp expression. The genotypes are as in (G). (I) Representative photomicrographs of hsp-6p∷gfp expression in neuronal Q40; tph-1 expressing animals treated with vehicle control or 50mM 5-HT. (J) Quantification of hsp-6p∷gfp expression. The genotypes are as in (I). (K) Representative photomicrographs of hsp-6p∷gfp reporter animals in WT and tph-1p∷egl-20 background. (L) Quantification of hsp-6p∷gfp expression. The genotypes are as in (K). (M) Model of the mitokine signaling pathway. Q40 specifically binds to mitochondria in neurons, initiating a signaling cascade across tissues that requires retromer-dependent Wnt secretion, canonical Wnt signaling, serotonin, and functional components of the UPR^mt to ensure cell-non-autonomous UPR^mt induction in peripheral tissues. *** p< 0.0001 via t-test. Error bars, SEM. n ≥ 15 worms. See also Figure S7.