Mitochondrial Complex I Activity Is Required for Maximal Autophagy

Abstract

Cells adapt to nutrient and energy deprivation by inducing autophagy, which is regulated by the mammalian target of rapamycin (mTOR) and AMP-activated protein kinases (AMPKs). We found that cell metabolism significantly influences the ability to induce autophagy, with mitochondrial complex I function being an important factor in the initiation, amplitude, and duration of the response. We show that phenformin or genetic defects in complex I suppressed autophagy induced by mTOR inhibitors, whereas autophagy was enhanced by strategies that increased mitochondrial metabolism. We report that mTOR inhibitors significantly increased select phospholipids and mitochondrial-associated membranes (MAMs) in a complex I-dependent manner. We attribute the complex I autophagy defect to the inability to increase MAMs, limiting phosphatidylserine decarboxylase (PISD) activity and mitochondrial phosphatidylethanolamine (mtPE), which support autophagy. Our data reveal the dynamic and metabolic regulation of autophagy.

Keywords: AMPK; autophagy; mTOR; metabolism; mitochondria associated membrane; mitophagy; phenformin; phosphatidylethanolamine; phosphatidylserine decarboxylase; phospholipids.

Figure 1.. Phenformin Inhibits Autophagy Induced by mTOR Inhibitors

(A and B) Time course of GST-BHMT autophagy assay (A) and plotted ratio of the BHMT fragment/ GFP-myc (B) in HEK293 cells treated with vehicle (V), 5 nM RAD001 and 20 nM BEZ235 (RB), or 1 mM phenformin (P¹). (C) GST-BHMT assay in HEK293 cells treated for 6 hr with 0–5 mM phenformin alone, or in combination with RB. (D and E) LC3-II flux assay (D) and ratio of LC3-II/ACTIN (E) in HEK293 cells treated as indicated. (F and G) Representative confocal images (F) and quantitation of LC3-B puncta/cell (G) in HEK293T cells. Treatments as in (D) and (E). Scale bar is 10 μm. Data represented as mean ± 1 SD.

Figure 2.. Phenformin Inhibits Mitophagy Induced by mTOR Inhibitors

(A) GST_LSCSGFP-ActA mitophagy assay in cells treated for 24 hr with V, RB, P¹, or RBP¹. (B) Mitochondrial DNA content, as measured by RT-qPCR. Statistics by one-way ANOVA. (C) Representative confocal images from Mito-Keima expressing cells, treated for 4 hr. (D) Complex I activity in mitochondria purified from HEK293 cells. 16 hr treatments. **Adjusted p value = 0.0074. Data represented as mean ± 1 SD.

Figure 3.. Autophagy Is Inhibited by Severe Defects in Mitochondrial Complex I Accessory Subunits

(A) GST-BHMT assay in control and NDUFS6 or NDUFA10 knockout (KO) cells. 6 hr treatments. A numerical value for the BHMT fragment (frag)/GFP ratio, normalized to RB, is shown. (B) GST-BHMT assay in HEK293 cells treated for 6 hr, with or without 5 mM nicotinamide (NM). (C) GST_LSCSGFP-ActA mitophagy assay in HEK293T control and NDUFA1^KO cells treated for 20 hr with V, RB, P¹, RBP¹, or 10 μM CCCP (CC).

Figure 4.. Phenformin Suppresses the Initiation, Amplitude, and Duration of Autophagy

(A) GST-BHMT assay in cells treated for 6 hr with V, RB, P¹, RBP¹, with or without 1 mM adenosine. (B) Bar graph of AMP/ATP ratios in HEK293 cells treated with V, RB, P¹, RBP¹, or starved of glucose. (C and D) Immunofluorescence of WIPI2 in HEK293T cells treated for 2 hr. Representative confocal images (C) and quantification of WIPI2 puncta (D). Scale bar is 10 μm. (E–G) GST-BHMT time course assay in cells treated with V, RB, or RBP^1.5, as shown by western blotting (E) and graphs of BHMT frag/GFP ratios (F) and LC3-II/ACTIN ratios (G).

Figure 5.. Glucose Starvation Increases Autophagy and Mitophagy

(A and B) GST-BHMT assay time course in cells treated with V or RB, in media with 25 or 0 mM glucose, as shown by western blotting (A) and graph of BHMT frag/GFP ratio (B). (C) GST-BHMT assay in cells treated for 24 hr with vehicle or RB in DMEM with 25 or 0 mM glucose, 25 mM galactose (Gal), or 100 mM trehalose (Treh). (D) GST_LSCSGFP-ActA mitophagy assay in cells treated for 24 hr with 25, 5, or 0 mM glucose; 25 mM Gal; or P¹, without or with RB.

Figure 6.. Phospholipid Remodeling in Autophagy

(A) Levels of PE (18:0/18:1) and PS (18:0/18:1) in HEK293 cells treated for 24 hr with V, RB, P¹, or RBP¹. ****p < 0.0001, ***p = 0.0002. ns = not significant. Data represented as mean ± 1 SD. (B) GST-BHMT autophagy assay in cells transfected with non-silencing siRNA (siNS), siPISD-6, or siPISD-7. Cells were treated for 6 hr. (C) GST-BHMT autophagy assay in cells treated for 6 hr with drugs as indicated, with 5 mM L-serine and/or 1 mM hydroxylamine as noted. (D) GST-BHMT autophagy assay in cells treated for 6 hr as indicated, with buffer or mixed composition liposomes.

Figure 7.. Defects in Complex I Prevent the Significant Increase in MAMs that Occurs with mTOR Inhibitors

(A) Proximity ligation assay showing MAMs by confocal microscopy. HEK293T cells were treated for 4 hr. Scale bar is 10 μm. (B) Scatter plot graph showing quantified MAMs data. ****p < 0.0001. ns = not significant. Data represented as mean ± 1 SD. (C) Model of conditions that support or inhibit autophagy. Left: mTOR inhibitors (RB) induce active autophagy in cells with healthy mitochondria by increasing MAMs, sites of ATG14 recruitment and phospholipid transport, supporting mtPE biosynthesis and sustained autophagy. RB autophagy is enhanced by conditions that increase TCA intermediates, ATP, or phospholipids. Right: Autophagy is impaired by phenformin or defects in complex I, which prevent the increase of MAMs induced by mTOR inhibitors, local ATP, and the ATP-dependent transfer of PS for mtPE biosynthesis.