A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity

Abstract

Macroautophagy (autophagy) is a critical cellular stress response; however, the signal transduction pathways controlling autophagy induction in response to stress are poorly understood. Here we reveal a new mechanism of autophagy control whose deregulation disrupts mitochondrial integrity and energy homeostasis in vivo. Stress conditions including hypoxia and exercise induce reactive oxygen species (ROS) through upregulation of a protein complex involving REDD1, an mTORC1 inhibitor and the pro-oxidant protein TXNIP. Decreased ROS in cells and tissues lacking either REDD1 or TXNIP increases catalytic activity of the redox-sensitive ATG4B cysteine endopeptidase, leading to enhanced LC3B delipidation and failed autophagy. Conversely, REDD1/TXNIP complex expression is sufficient to induce ROS, suppress ATG4B activity and activate autophagy. In Redd1(-/-) mice, deregulated ATG4B activity and disabled autophagic flux cause accumulation of defective mitochondria, leading to impaired oxidative phosphorylation, muscle ATP depletion and poor exercise capacity. Thus, ROS regulation through REDD1/TXNIP is physiological rheostat controlling stress-induced autophagy.

Figure 1. Loss of REDD1 results in defective autophagy in vitro and in vivo.

(a). Western blot analysis showing reduced lipidated LC3B (LC3B-II, bottom band) in immortalized (top) and primary (bottom) Redd1^−/− cells under basal conditions. Autophagic flux was assessed using CQ at the indicated concentrations for 4 h. β-Tubulin (Tub.) serves as a loading control. At right, densitometry showing the mean values of three independent experiments and statistical comparison with respective wild-type lanes. (b) Representative confocal immunofluorescence images showing decreased LC3B membrane-associated puncta in Redd1^−/− compared with wild-type cells cultured under basal conditions in the presence of CQ (30 μM, 4 h). N≥60 cells per genotype were analysed as described in Methods. At right, quantitation of three independent experiments. (c) Impaired hypoxia-induced autophagy in Redd1^−/− cells. Cells were cultured under normoxia (N) or hypoxia (H, 1% O₂) and treated in the absence or presence of CQ (30 μM, 4 h). (d) Attenuated hypoxia-induced degradation of GFP-LC3 in Redd1^−/− cells. Retrovirally transduced cells were cultured under normoxia or hypoxia (1% O₂, 4 h), and analysed using flow cytometry. (e) Failed p62 degradation under starvation (Krebs medium) in Redd1^−/− cells. (f) Disabled autophagy in tissues from Redd1^−/− mice, evidenced by reduced processing of LC3B post 24-h starvation. Ribosomal protein S6, loading control. (g) Electron microscopy showing decreased number and size of autophagic bodies (arrows) in the brain of Redd1^−/− mice following starvation for 24 h. N indicates nuclei. (h) Reduced sensitivity of Redd1^−/− cells to autophagy inhibition, determined with crystal violet staining at 72 h. For d,h, shown are the means of two independent experiments performed as triplicate cultures. All error bars indicate s.d. ***P<0.001, **P<0.01, n.s. not significant, by paired t-test (d,h) or unpaired t-test (b).

Figure 2. REDD1 mediates autophagy independent of mTORC1.

(a) Impaired starvation-induced autophagy in Redd1^−/− cells despite suppressed mTORC1, assessed by the analysis of phosphorylated p70 S6 Kinase (pS6K, T389). Cells were cultured with Krebs media and treated in the absence or presence of CQ (30 μM, 1 h). At right, densitometry showing the mean values of three independent experiments and statistical comparison with respective wild-type lanes. (b) Rapamycin (100 nM, 4 h) fails to fully restore the defective autophagic flux in Redd1^−/− cells, as assessed by LC3B processing in the absence or presence of CQ (30 μM, 4 h; compare lanes 4 and 8). Activity of mTORC1 was assessed via phosphorylated ribosomal protein S6 (pS6, S235/S236). Right, densitometry quantitation of three independent experiments as in a. (c) Failure of hypoxia (1% O₂, 12 h) to induce autophagy in Redd1^−/− cells (left) but not Tsc2^−/− cells (right), assessed by degradation of GFP–LC3 measured using flow cytometry. (d) Both wild-type REDD1 and the mTORC1-inactive mutant REDD1-RPAA are sufficient to induce autophagy, assessed by GFP-LC3 levels using flow cytometry. Tetracycline (Tet)-inducible (TREX) cells were treated in the presence and absence of rapamycin (100 nM, 4 h) as a positive control. For c,d, shown are the means of two independent experiments, each performed as triplicate cultures; error bars, s.d. (*) show P-values compared with untreated control; (+) compares REDD1 with REDD1-RPAA. For all experiments: ***P<0.001, **P<0.01, *P<0.05; ⁺P<0.05, by paired t-test.

Figure 3. Loss of REDD1 results in accumulation of dysfunctional mitochondria.

Redd1^−/− cells exhibit (a) increased mitochondrial (mt) number, evidenced by increased mitochondrial to nuclear (M/N) DNA ratio (COXII/β-globin) as measured by QRT–PCR. MEF, mouse embryonic fibroblast. PMEF, primary MEF. Shown are the mean from two independent experiments, each performed as triplicate cultures. (b) Increased mitochondrial size as determined using flow cytometry of cell-free, purified mitochondria. (c) Increased mitochondrial number (left) and mass (right) in Redd1^−/− primary splenocytes from starved mice (24 h), assessed, respectively, by M/N DNA ratio (N=4 mice per genotype, analysed in duplicate) and by staining with Mitotracker Green (100 nM) and then flow cytometry analysis (N=3 mice per genotype, analysed in triplicate). (d) Reduced mitochondrial membrane potential (MMP) in Redd1^−/− MEFs (left) and splenocytes (right) as assessed by staining with Tetramethylrhodamine, Ethyl Ester (TMRE, 100 nM) and then flow cytometry (N=2 mice per genotype, analysed in triplicate). (e) Dysmorphic mitochondria (M) in Redd1^−/− cells as assessed using electron microscopy. Arrows indicate abnormal hypodense regions. (f) Decreased basal O₂ consumption rate (OCR), mitochondrial ATP synthesis (ATP) and maximal respiratory capacity (MRC) in Redd1^−/− cells, measured via Seahorse XF24. (g) Impaired oxidative phosphorylation in Redd1^−/− MEFs, shown by a drop in ATP per cell on shift from glucose to galactose-containing media (48 h). All error bars show s.d. ***P<0.001, **P<0.01, by paired t-test (a,d,e,g,h).

Figure 4. REDD1-dependent ROS controls autophagy and ATG4B activity.

(a,b) Reduced H₂O₂ in Redd1^−/− MEFs and splenocytes from Redd1^−/− mice as compared with wild-type controls, assessed by staining with CM-H2DCFDA (3 μM) and then flow cytometry. Shown are the means from three independent experiments/mice measured in triplicate. (c) Tsc2^−/− MEFs exhibit elevated cytosolic H₂O₂. (d) Decreased H₂O₂ induction in Redd1^−/− versus wild-type cells on starvation (0.1% FBS). (e) Suppression of ATG4B activity by H₂O₂ (3 mM, 6 h) in Redd1^−/− cells, assessed by incubation of cellular extracts with the LC3-PLA₂ substrate at the indicated concentrations. Each data point for velocity is derived from 60 sequential measurements, with the mean and 95% confidence intervals (shown as error bars) calculated by regression analysis (see Methods). (f) H₂O₂ (3 mM, 6 h) restores LC3B processing in Redd1^−/− cells to wild-type levels in the presence of CQ (30 μM, 4 h). Note similar levels of ATG4B in Redd1^−/− cells and wild-type cells in the absence or presence of H₂0₂. β-Tubulin (Tub.), loading control. (g) Elevated ATG4B activity in Redd1^−/− versus wild-type cells under basal conditions, assessed as described for e. (h) Persistent increase in ATG4B activity in Redd1^−/− versus wild-type cells under starvation (0.1% FBS, 24 h). (i) Enhanced cleavage of unprocessed LC3, as evidenced by a decreased GFP/DsRed ratio (the mean fluorescence of expressing cells) in Redd1^−/− compared with wild-type cells, transfected with double-tagged DsRed-LC3-GFP and analysed using flow cytometry. H₂O₂ (1 mM, 6 h) inhibits ATG4B to increase the GFP/DsRed ratio in Redd1^−/− cells. (j) The mean and s.d. of four independent experiments as in i, measured in triplicate. ***P<0.001, **P<0.01, *P<0.05, by paired t-test (a–e) or repeated measures ANOVA (g,h).

Figure 5. REDD1 and TXNIP interact to control ROS, ATG4B and autophagy.

(a) Physical association of REDD1 with both transfected GFP-TXNIP (left) and endogenous TXNIP (right) following transfection of HA-REDD1 and IP with α-TXNIP in 293T cells. (b) The endogenous REDD1/TXNIP complex is induced by hypoxia (1% O₂, 16 h) and energy stress (2-deoxyglucose (2-DG), 20 mM, 16 h) in 293T cells. Lysates were subjected to IP with α-REDD1 antibody. (c) Increased thioredoxin (TRX) antioxidant activity in Redd1^−/− and Txnip^−/− cells, measured by reduction of insulin disulfides. Shown is the mean of three independent experiments performed in triplicate. (d) Reduced H₂O₂ in Txnip^−/− MEFs stained with CM-H2DCFDA (3 μM) followed by flow cytometry. (e) TXNIP fails to induce H₂O₂ in Redd1^−/− cells unless co-transfected with REDD1. (f) Co-transfection of REDD1 and TXNIP potently inhibits TRX activity in Redd1^−/− cells. (g) Co-transfection of REDD1 and TXNIP is sufficient to suppress ATG4B activity in Redd1^−/− cells, assessed as in Fig. 4. (h) Impaired autophagy under basal and hypoxic (1% O₂, 4 h) conditions in Txnip^−/− MEFs, shown in the absence or presence of CQ (30 μM, 4 h). (i) Reduced MMP in Txnip^−/− cells, assessed by staining with TMRE (100 nM) followed by flow cytometry. (j) Expression of REDD1 or TXNIP is sufficient to induce autophagy in transfected 293T cells. ***P<0.001, **P<0.01, by paired t-test (c–f, i) or repeated measures ANOVA (g).

Figure 6. Impaired ROS/ATG4B/autophagy compromises exercise capacity in Redd1^−/− mice.

(a) Exercise (treadmill run, 2 h) induces autophagy in the muscle of wild-type mice, as assessed by LC3B processing. RPS6 (S6) protein, loading control. Duplicate panels correspond to individual mice. (b) REDD1 and TXNIP mRNA induction by exercise (treadmill run, 2 h) in the skeletal muscle of wild-type mice, measured using QRT–PCR relative to β-actin. Numbers refer to individual mice. (c) Exercise suppresses ATG4B activity in the heart of wild-type mice, assessed as in Fig. 4. Shown are the mean values from three mice per condition. (d) Hyperactivity of ATG4B in the heart and skeletal muscle of treadmill-exercised Redd1^−/− mice, assessed and quantitated as in c. (N=3 mice per genotype.) (e) Increased TRX activity in the heart of treadmill-exercised Redd1^−/− mice, measured as in Fig. 5. (N=3 mice per genotype.) (f) Impaired autophagy induction by treadmill exercise in the muscle of Redd1^−/−. Duplicate panels correspond to individual mice. (g) Decreased exercise capacity in Redd1^−/− mice (voluntary running wheel, 24 h). Each point represents an individual run; eight mice per genotype were subjected to at least three runs each; error bars, s.e.m. (h) Increased mitochondrial (mt) number in the muscle of treadmill-exercised Redd1^−/− compared with wild-type mice. (N=3 mice per genotype.) (i) Reduced MMP in tissues as in h, assessed in cell-free mitochondrial fractions. (j) ATP concentration measured in lysates of tissues described in h. Except as noted, all error bars indicate s.d. ***P<0.001, **P<0.01, by paired t-test (b,e,h,j), or repeated measures ANOVA (c,d), or Mann–Whitney U-test (g).

Figure 7. REDD1 controls stress-induced autophagy and energy homeostasis.

REDD1 and TXNIP are upregulated by physiologic stress, forming a protein complex required for induction of ROS that inhibit ATG4B-mediated LC3 delipidation and thereby promote autophagosome maturation. In the absence of REDD1, decreased ROS results in hyperactivity of ATG4B and disabled autophagy, leading to accumulation of aged, dysfunctional mitochondria and defective oxidative metabolism that compromises ATP generation and exercise capacity.