Cockayne syndrome group B protein prevents the accumulation of damaged mitochondria by promoting mitochondrial autophagy

Abstract

Cockayne syndrome (CS) is a devastating autosomal recessive disease characterized by neurodegeneration, cachexia, and accelerated aging. 80% of the cases are caused by mutations in the CS complementation group B (CSB) gene known to be involved in DNA repair and transcription. Recent evidence indicates that CSB is present in mitochondria, where it associates with mitochondrial DNA (mtDNA). We report an increase in metabolism in the CSB(m/m) mouse model and CSB-deficient cells. Mitochondrial content is increased in CSB-deficient cells, whereas autophagy is down-regulated, presumably as a result of defects in the recruitment of P62 and mitochondrial ubiquitination. CSB-deficient cells show increased free radical production and an accumulation of damaged mitochondria. Accordingly, treatment with the autophagic stimulators lithium chloride or rapamycin reverses the bioenergetic phenotype of CSB-deficient cells. Our data imply that CSB acts as an mtDNA damage sensor, inducing mitochondrial autophagy in response to stress, and that pharmacological modulators of autophagy are potential treatment options for this accelerated aging phenotype.

Figure 1.

The old CSB^m/m mice show signs of neurodegeneration and universal loss of fat. (A) Quantification of adipose tissue using T₁-weighted MRI in young (2 mo) and old (20 mo) WT and CSB^m/m mice (n = 3; data are represented as mean ± SEM). (B) Representative axial T₁-weighted MRI scans of the CSB^m/m and WT mice. Yellow denotes fat. (C) Respiration rates of mice during the MRI scans. (D) HE staining of abdominal skin in young and old WT and CSB^m/m mice. The bar denotes the subcutaneous fat layer (n = 3). (E) HE staining of the liver. (F) Periodic acid-Schiff staining of the liver of the 20-mo-old mice with and without diastase to show glycogen content. (G) HE staining of the inner ear. The star denotes the spiral ganglion (n = 3). (H) Brain weight of WT and CSB^m/m mice (n = 3; data are represented as mean ± SEM).

Figure 2.

CSB deficiency increases whole body metabolism. (A) 1 wk food intake CSB^m/m compared with age-matched WT mice (n = 3; data are represented as mean ± SEM). (B and C) HE staining of the small (B) and large (C) intestines (n = 3). (D) Mice were placed in metabolic cages for 72 h. O₂ consumption and CO₂ production were measured. D1= dark phase 1, L1 = light phase 1, etc. (n = 3–8; data are represented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (E) Gene ontology terms of a microarray comparing MDFs from WT and CSB^m/m mice (n = 3–4; blue is down-regulated and red is up-regulated).

Figure 3.

CSB deficiency increases cellular metabolism. (A and B) Oxygen consumption rates (OCRd; A) and extracellular acidification rates (ECARd; B) were increased in CSB-deficient cells. CS1AN: CSB patient cell line expressing an empty vector or a vector containing WT CSB. HSMM: HSMMs expressing scrambled shRNA or shRNA targeted to CSB. MDF: MDFs isolated from young and old CSB^m/m mice. Primary CSB patient fibroblasts (GM739, GM1629, and GM2838) and healthy age-/gender-matched controls (GM969, GM38, and GM 8402) are shown. Blue are cells expressing WT CSB. Red are CSB-deficient cells (n = 3–7; data are represented as mean ± SEM). (C) FCCP-uncoupled respiration was measured by sequential addition of oligomycin (green background), FCCP (gray background), and antimycin A (yellow background; n = 3–7; data are represented as mean ± SEM). (D) Mitochondrial membrane potential in CS1AN cells measured by 20 nM TMRM staining and flow cytometry and its responsiveness to treatment with cyclosporin A (CsA). Inset: quantification of the response to cyclosporin A (n = 3; data are represented as mean ± SEM). (E) Growth curve of CS1AN cells expressing either an empty vector or WT CSB. (n = 3; data are represented as mean ± SEM). (F) Glucose concentration was measured in the media of CS1AN cells expressing WT CSB or an empty vector (n = 3; data are represented as mean ± SEM). (G) ATP consumption of CS1AN cells expressing WT CSB or an empty vector after inhibition of ATP production with 100 mM 2-deoxyglucose and 1 µM oligomycin. Inset: half-life of ATP (n = 3; data are represented as mean ± SEM).

Figure 4.

CSB-deficient cells show increased mitochondrial content and decreased mitochondrial biogenesis. (A) Citrate synthase activity in CS1AN cells expressing WT CSB or an empty vector or in MDF from young or old CSB^m/m or WT mice or in HSMM expressing scrambled or CSB-specific shRNA (n = 3–5; data are represented as mean ± SEM). (B) Representative confocal image of CS1AN cells expressing WT CSB or an empty vector transfected with the mitochondrial RFP-tagged protein using organelle lights. (C) Quantification of the mitochondrial RFP signal normalized to cell number (n = 3; data are represented as mean ± SEM). (D) Representative flow cytometry experiment of CS1AN cells expressing WT CSB or empty vector treated with 50 nM MitoTracker green. Inset: quantification of the flow cytometry data (n = 3; data are represented as mean ± SEM). (E–H) Quantitative PCR analysis of genes involved in mitochondrial biogenesis in CS1AN cells expressing WT CSB or empty vector (E), MDFs from young (F) and old (G) mice, and HSMM subjected to lentiviral CSB knockdown (H). The inset in H shows efficiency of lentiviral shRNA knockdown of CSB (n = 3; data are represented as mean ± SEM; *, P < 0.05; **, P < 0.01).

Figure 5.

Mitochondrial autophagy is decreased in CSB-deficient cells. (A and B) Western blots of CS1AN cells expressing WT CSB or an empty vector (A) or HSMM subjected to lentiviral knockdown of CSB treated with vehicle, 5 µM rotenone (autophagy inducer), or 500 ng/ml ethidium bromide for 24 h (B; n = 3, data are represented as mean ± SEM). (C) Representative image of CS1AN cells expressing WT CSB or an empty vector transfected with eGFP-tagged LC3 and quantification of the foci per cell (n = 3; data are represented as mean ± SEM). (D) Representative image of CS1AN cells expressing WT CSB or an empty vector treated with vehicle or rotenone and stained for LC3B and COX4 (mitochondria; bars, 50 µm) and quantification of the Pearson’s coefficient of these cells (n = 4; data are represented as mean ± SEM). (E) Representative image of CS1AN cells expressing WT CSB or an empty vector treated with vehicle or rotenone and stained for P62 and COX4 (bars, 50 µm) and the quantification of the Pearson’s coefficient of these cells (n = 4; data are represented as mean ± SEM). (F) Representative image of CS1AN cells expressing WT CSB or an empty vector treated with vehicle or bafilomycin A and stained for ubiquitin and COX4 (bars, 50 µm) and the quantification of the Pearson’s coefficient of these cells (n = 4; data are represented as mean ± SEM).

Figure 6.

CSB-deficient cells show accumulation of damaged mitochondria. (A) Representative electron microscopy images of CS1AN cells lines after treatment with vehicle or 5 µM rotenone. Red dots denote swollen/damaged mitochondria (n = 12). (B) Quantification of damaged mitochondria in vehicle and rotenone-treated CS1AN cells expressing WT CSB or an empty vector (n = 12; data are represented as mean ± SEM). (C) Mitochondrial diameter in CS1AN cells expressing WT CSB or an empty vector after vehicle or rotenone treatment. Each point represents one mitochondria and cross bar represents mean ± SD. (D) Arrows marking hyperdense membranous material in CS1AN cells expressing an empty vector. (E) Arrows marking mitochondrial onion formation in CS1AN cells expressing an empty vector. (F) ROS formation measured by dihydroethidium in CS1AN cells expressing WT CSB or an empty vector (n = 10; data are represented as mean ± SD).

Figure 7.

Rapamycin rescues the bioenergetic profile of CSB-deficient cells. (A) Mitochondrial content of CS1AN cells expressing WT CSB (left) or an empty vector (right) was measured using flow cytometry and 50 nM MitoTracker green staining after treatment with the autophagy inducer 1 µM rapamycin (rapa), serum starvation (starve), or 10 mM of the autophagy inducer lithium chloride (LiCl; n = 3 representative experiments). (B) The membrane potential of CS1AN cells expressing WT CSB (left) or an empty vector (right) was measured using flow cytometry and 20 nM TMRM staining after treatment with 1 µM rapamycin (rapa) or 10 mM of the autophagy inhibitor 3-MA for 24 h (n = 6 representative experiments). (C) The bioenergetics profile of CS1AN cells expressing WT CSB or an empty vector after treatment with 10 mM lithium chloride, 1 µM rapamycin, or 3-MA for 24 h (n = 3; data are represented as mean ± SEM).