Downregulation of respiratory complex I mediates major signalling changes triggered by TOR activation

Abstract

Mitochondrial dysfunctions belong amongst the most common metabolic diseases but the signalling networks that lead to the manifestation of a disease phenotype are often not well understood. We identified the subunits of respiratory complex I, III and IV as mediators of major signalling changes during Drosophila wing disc development. Their downregulation in larval wing disc leads to robust stimulation of TOR activity, which in turn orchestrates a complex downstream signalling network. Specifically, after downregulation of the complex I subunit ND-49 (mammalian NDUFS2), TOR activates JNK to induce cell death and ROS production essential for the stimulation of compensatory apoptosis-induced proliferation within the tissue. Additionally, TOR upregulates Notch and JAK/STAT signalling and it directs glycolytic switch of the target tissue. Our results highlight the central role of TOR signalling in mediating the complex response to mitochondrial respiratory dysfunction and they provide a rationale why the disease symptoms associated with respiratory dysfunctions are often alleviated by mTOR inhibitors.

Figure 1

Increase in TOR activity orchestrates signalling changes after complex I downregulation. (A) Signalling changes caused by downregulation of respiratory complex I (RNAi against ND-49 subunit) are rescued by downregulation of Tor pathway (Tor-RNAi). RNAi was driven only in the posterior part of the wing disc (hh-Gal4, Gal80^ts or en-Gal4 drivers), corresponding to the right halves of the immunostaining pictures. White dotted line defines the boundary between the anterior A and posterior P domains. The anteriror (left) half of the pictures serves as control. (B) The similarities between signalling changes caused by downregulation of respiratory complex I (ND-49-RNAi) and after overactivation of TOR in the posterior compartment (right half) of the wing disc. (C) The schematic overview of the immunostaining experiments. RNAi or other UAS experimental construct is driven in the posterior domain of the wing disc, the anterior domain serves as control. Blue frame depicts the wing pouch region shown in the figure panels. The boundary between the anterior and posterior domains was always determined by immunostaining of the Ci protein. (D) mRNA expression in the wing discs measured by qPCR. The RNA was extracted from the whole wing discs but only the posterior compartments were treated with RNAi, therefore the actual changes in gene expression in the posterior compartment are bigger than values plotted in the graph [***p < 0.001; **p < 0.01, *p < 0.05; ^†p < 0.1]. (E) Western blot with antibody against p-S6K, another TOR target, detected in the en-Gal4, ND-49-RNAi and control (en-Gal4, white-RNAi) wing discs. Total protein load was assessed by fluorescent detection of protein lanes within the TGX Stain-Free gel (Biorad, Figs. S1D and S6). **p < 0.01. (F) Graphical summary of Fig. 1.

Figure 2

The effect of complex I downregulation on apoptosis, proliferation and tissue growth is mediated by Tor. (A) Induction of apoptosis (Dcp1) and proliferation (p-H3) after ND-49-RNAi in the posterior domain (right half) of the wing disc is rescued by TOR-RNAi. (B) Overexpression of Tor in the posterior domain leads to the same phenotype as ND-49-RNAi. (C) Quantifications of apoptosis (Dcp1), (D) the relative mitotic index (p-H3) and (E) the size of posterior wing disc compartment after ND-49-RNAi, Tor-RNAi and Tor overexpression. We detect interaction with the effect of ND-49 knockdown using ANOVA [***p < 0.001; *p < 0.05]. LacZ represents control. (F) Graphical summary of Fig. 2.

Figure 3

Downregulation of complex I mediates JNK driven compensatory apoptosis-induced proliferation, dependent on effector caspases. (A) Proliferation following ND-49-RNAi in the posterior compartment of the wing disc (right half) can be rescued by blocking the JNK pathway (through dominant negative Bsk^DN), the initiator caspase (Dronc-RNAi) or the effector caspases (by overexpression of p35); we detect interaction with the effect of ND-49 knockdown in cell proliferation using ANOVA [***p < 0.001; **p < 0.01; *p < 0.05]; a Duncan test was performed and groups that are statistically different were assigned using letters (p < 0.05; a, b, c). A genotype is assigned to two groups when it is not significantly different from any of them. (B) Blocking the initiator caspase by Dronc-RNAi in the posterior compartment in the context of ND-49-RNAi does not rescue the activity of the JNK pathway reporter puc-lacZ. (C) Blocking the JNK pathway by overexpression of Bsk^DN in the posterior compartment (right half) of the wing disc does not reduce the increased activity of TOR pathway (p-4EBP1) in the context of ND-49-RNAi. (D) Graphical summary of Fig. 3.

Figure 4

Strong formation of ROS after complex I downregulation is connected with apoptosis and is mediated by JNK. (A) Downregulation of ND-49 in the posterior compartment of the wing disc causes ROS production (DCFH) that is rescued by incubation with the ROS scavenger N-acetylcysteine (NAC). Posterior domain is located on the right halves of the pictures (exact position of the A/P boundary could not be determined in this experiment). (B) Downregulation of ND-49 in the posterior compartment of the wing disc causes ROS production (DCFH) as well as apoptosis (Dcp1) that are rescued by simultaneous block of the initiator caspase (Dronc-RNAi), by blocking the effector caspase (overexpression of p35) or the JNK pathway (dominant negative Bsk). (C,D) Quantification of data presented in panel (B). (E) The ROS sensitive reporter GstD1-GFP (grey) shows low level of activity within the whole posterior domain and a burst of signal in the same region of the disc where apoposis is occuring (dCP1, green). Yellow box indicates magnified area of the disc to see cellular resolution of the signal. (F) Graphical summary of Fig. 4.

Figure 5

Downregulation of complex I leads to stimulation of glucose metabolism downstream of TOR. (A) The uptake of fluorescently labelled 2-deoxyglucose (2-NBDG) is increased after ND-49 knockdown in the posterior part of the wing disc. Lower panel shows magnification of the region marked by yellow frame. **p < 0.01 (B) Ldh expression is increased after ND-49 downregulation in the posterior part of the wing disc and this increase is rescued by Tor-RNAi. (C) Ldh expression is induced following Tor overexpression but it is not rescued by Bsk-RNAi or Notch-RNAi. (D) Hif-1 responsive element reporter (Hif1-RE-lacZ) is not active after ND-49-RNAi in the posterior part of the wing disc. (E) RNAi against sima, the Drosophila homologue of mammalian HIF-1, can not rescue Ldh increase after ND-49-RNAi in the posterior domain. (F) Transcription of Hif-1 transcriptional targets (fga, seq) is not changed after ND-49-RNAi in the posterior domain. (G) Downregulation of ND-49 in the posterior compartment stimulates transcription of Hexokinase A (HexA) and lactate dehydrogenase (Ldh), as well as the SdhA subunit of respiratory complex II. Transcription of the TCA cycle genes (kdn, Idh) is not changed. The RNA was extracted from the whole wing disc but only the posterior compartment was affected by RNAi, therefore the actual changes in gene expression in the posterior compartment are bigger than the values plotted in the graph [***p < 0.001; **p < 0.01]. (H) Graphical summary of Fig. 5.

Figure 6

Notch pathway is activated by TOR after downregulation of complex I. (A) The induction of TOR pathway (p-4EBP1) after complex I downregulation in the posterior domain (right half) of the wing disc can not be rescued by Notch-RNAi. (B) Activation of JNK (Bsk) in the posterior domain has a negligible effect on the expression of the Notch target gene E(spl)mβ (compare to Fig. 1A). (C) Activation of Notch pathway by overexpression of Nicd in the posterior compartment does not stimulate JNK activity (puc-lacZ) nor Ldh expression (D). (E) TOR overactivation in the posterior compartment of the wing disc leads to loss of veins in adult wings, a typical Notch gain-of-function phenotype (compare to the RNAi against Notch repressor CtBP). Red dotted line represents the posterior compartment of the adult wing, red arrows point to the missing veins. (F) Overview of the signalling network triggered by ND-49 downregulation in the wing disc.