Fanconi Anemia Proteins Function in Mitophagy and Immunity

Abstract

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.

Figure 1. Fancc is Required for Autophagic Targeting of Genetically Distinct Viruses, but Not for Starvation-Induced Autophagy

(A) Western blot of LC3-I/LC3-II in primary murine embryonic fibroblasts (MEFs) during growth in normal (starvation −) or EBSS (starvation +) medium for 4 hr +/− 100 nM bafilomycin A1 (Baf A1). (B) Western blot of p62 in MEFs in culture conditions as in (A). (C) Quantification of GFP-LC3 puncta (autophagosomes) in Fancc^+/+/GFP-LC3 and Fancc^−/−/GFP-LC3 MEFs during growth in normal or starvation medium for 4 hr +/− 100 nM Baf A1. Bars are mean ± SEM for triplicate samples (>100 cells analyzed per sample by an observer blinded to experimental condition). NS, not significant; *, P<0.05; t-test. (D) EM of autophagic structures in MEFs during growth in normal or starvation medium (EBSS, 4 hr). White arrows indicate autolysosomes. Scale bars, 1 μm. (E and F) Representative light micrographs of images (E) used for quantification (F) of colocalization of SIN/mCherry.capsid and GFP-LC3 in MEFs infected with SIN mCherry.capsid/GFP-LC3 (multiplicity of infection [MOI] = 2.5 plaque-forming units [PFUs], 18 hr). In (E), white arrows denote representative colocalized mCherry.capsid and GFP-LC3 signal. Scale bars, 10 μm. In (F), bars are mean ± SEM for triplicate samples (> 50 cells analyzed per sample by an observer blinded to experimental condition). *, P<0.05; t-test. (G) Western blot of FANCC in HeLa WT and HeLa FANCC^KO cells. (H) EM of immunoprecipitated autophagic structures using an anti-LC3 antibody from HeLa FANCC^KO cells transduced with a lentivirus expressing FANCC-Flag and infected with SIN strain SVIA (MOI = 5, 6 hr). White arrows indicate SIN nucleocapsids inside autolysosomes. Asterisks indicate dynabeads used for IP. Scale bars, 200 nm. (I) Western blot of indicated proteins in anti-LC3 immunoprecipitates (representative electron microscopes shown in (H)) from HeLa FANCC^KO cells transduced with lentivirus vector (HeLa FANCC^KO/vector) or lentivirus expressing FANCC-Flag (HeLa FANCC^KO/FANCC) and infected with SIN strain SVIA (MOI = 5, 6 hr). (J) Western blot of indicated proteins in control and SIN-infected HeLa FANCC^KO/FANCC cells subjected to anti-LC3 IP as in (H-I) and then 30 min digestion on ice with 2.5 ng/ml proteinase K +/− 0.5% triton X-100. (K) Co-IP of SIN capsid protein with FANCC-Flag in HeLa FANCC^KO/vector (Vector) or HeLa FANCC^KO/FANCC (FANCC) cells infected with SIN strain dsTE12Q (MOI = 5, 6 hr). (L) Representative EMs of MEFs infected with HSV-1 ICP34.5Δ68-87 (MOI = 5, 6 hr). White arrows denote representative HSV-1 nucleocapsids being degraded inside autolysosomes. Black arrows denote representative HSV-1 nucleocapsid (bottom left) or assembled HSV-1 virion (bottom right) inside the cytoplasm. Scale bars, 200 nm for large panels; 50 nm for insets. (M) Quantification of HSV-1 nucleocapsids and virions within autophagosomes or autolysosomes (inside autophagic structures) or free within the cytoplasm (cytoplasmic) in experiment shown in (L). Twenty-five HSV-1-infected cell profiles were counted per genotype by an observer blinded to experimental conditions. **, P<0.01; Mann-Whitney U test. In A-C, E and F, and I-K, similar results were observed in three independent experiments. See also Figure S1.

Figure 2. Fancc is Required for Host Defense against SIN and HSV-1 CNS Viral infections

(A) Survival of 7 day-old mice infected intracerebrally (i.c.) with 1000 PFUs SIN strain dsTE12Q. Results shown represent combined survival for independent infections of more than 5 litters (similar results obtained for each infection). P-value (log-rank test) and number of mice per genotype indicated in graph. (B and C) Quantitation of TUNEL-positive area per brain section (B) and representative light micrographs (C) after infection of 7 day-old mice of indicated genotype infected as in (A). Bars represent median value for each group. *, P<0.05; Mann-Whitney U test. Scale bars, 500 μm. (D) Survival of 6-8 week-old mice infected i.c. with 5x10⁴ PFUs HSV-1 ICP34.5Δ68-87. Results shown represent combined survival data combined from three independent infections (similar results obtained for each infection). P-value (log-rank test) and number of mice per genotype indicated in graph. (E and F) Quantitation of TUNEL-positive area per brain section (E) and representative light micrographs (F) at indicated time points of 2 month-old mice infected as in (D). Bars represent median value for each group. **, P<0.01; Mann-Whitney U test. Scale bars, 500 μm. See also Figure S2.

Figure 3. FANCC is Required for the Clearance of Damaged Mitochondria in vitro and in vivo

(A and B) Mitophagy analysis in FANCC^KO/FANCC (WT) or FANCC^KO/vector (FANCC^KO) cells stably expressing HA-Parkin assessed by clearance of TOMM20 (A) or ATP5B (B). Cells were treated with OA (Oligomycin, 2.5 μM; Anitmycin A, 250 nM) for 8 hr prior to imaging and automated image analysis. Left, representative images of immunoflourescence staining. Right, quantitative image analysis. Shown are box plots of at least 150 cells analyzed per condition. Similar results were observed in three independent experiments. ***, P<0.001, Mann-Whitney U-test. Scale bars, 20 μm. (C) Mitophagy analysis by western blot detection of HSP60, COXIV or TOMM20 in WT or FANCC^KO cells stably expressing HA-Parkin and treated +/− OA for 8 hr. (D) EM analysis of brain (cerebellum) or heart tissue (left ventricle) from 1 year-old mice. Shown are images from one representative mouse per genotype. Similar results were observed in 6 mice per genotype. White arrows indicate damaged mitochondria. Scale bars, 1 μm. See also Figure S3.

Figure 4. FANCC Interacts with Parkin and Increases its Localization to Mitochondria in a Parkin-Dependent Fashion

(A) Mitochondrial colocalization of FANCC-Flag in HeLa FANCC^KO/FANCC/Parkin cells treated with DMSO or OA for 8 hr, and subjected to immunofluorescence staining to detect TOMM20 and FANCC-Flag. Similar results were observed in three independent experiments. Scale bars, 20 μm. (B) Representative light micrographs of immunofluorescence staining of HeLa FANCC^KO cells transiently transfected with FANCC-Flag and mCherry-Parkin for 24 hr and then treated with DMSO or OA for 4 hr. Scale bars, 10 μm. (C) Co-IP of Parkin with FANCC-Flag in HeLa FANCC^KO cells transiently transfected with mCherry-Parkin and vector or FANCC-Flag for 24 hr. (D) Co-IP of Parkin with endogenous FANCA in HeLa WT/Parkin cells +/− OA for 4 hr. (E) EMs of immunoprecipitated mitochondria using an anti-TOMM20 antibody from HeLa WT/Parkin cells +/− OA for 4 hr. Black arrowhead, ruptured mitochondrial membrane. (F) Western blot of indicated proteins in TOMM20 immunoprecipitates from HeLa WT/Parkin or HeLa FANCC^KO/Parkin cells +/− OA for 4 hr. Input, crude mitochondrial fractions. MMC, whole cell lysate from HeLa FANCC^KO/FANCC/Parkin cells treated with MMC for 24 hr is included as a loading control to show FANCD2 mono-ubiquitination. For mitochondrial samples, protein loading was normalized by densitometry for HSP60 in crude mitochondrial fractions (input). Left and right gel panels for each protein are cropped from the same gel image. See also Figure S4.

Figure 5. FANCC Deficiency in Patient Fibroblasts and Murine BMDMs Results in Abnormal mtROS Production

(A) FANCC-deficient patient fibroblasts are defective in FA core complex activity assessed by western blot of MMC-induced FANCD2 mono-ubiquitination. FANCC-deficient patient fibroblasts were transduced with a FANCC-expressing retrovirus (WT FANCC) or a vector control retrovirus (Vector) and then treated with 1 μM MMC for 24 hr. (B) Mitochondrial morphology in WT FANCC or vector transduced immortalized patient fibroblasts. Cells were treated with 10 μM CCCP for 24 hr prior to immunofluorescence staining with anti-TOMM20. White arrow, representative cell with diffuse accumulation of fragmented mitochondria. Scale bars, 20 μm. (C) MtROS production assessed by flow cytometric analysis of MitoSOX fluorescence levels in WT FANCC or vector transduced immortalized patient fibroblasts treated with DMSO or 10 μM CCCP for 24 hr. (D) MtROS production assessed by flow cytometric analysis of MitoSOX fluorescence levels in primary BMDMs from Fancc^+/+ and Fancc^−/− mice. BMDMs were treated with LPS (100 ng/mL, 4 hr) +/− ATP (5 mM) during the final 30 min prior to flow cytometric analysis. (E) Inflammasome activation measured by IL-1β levels in supernatants of BMDMs of treated with LPS (100 ng/mL, 4 hr) +/− ATP (5 mM) and MitoTEMPO (500 μM) for the final 30 min of LPS treatment. Bars are mean ± SEM for triplicate samples. *P<0.05; t-test. (F) Inflammasome activation measured by ASC speck formation in BMDMs treated with LPS (100 ng/mL, 4 hr), LPS and ATP (5 mM for final 15 min of LPS), or LPS, ATP and MitoTEMPO (500 μM for the final hr of LPS) prior to immunostaining and automate image analysis. At least 500 cells were analyzed per condition. **, P<0.01, ****, P<0.0001, chi-square test. (G-H) Representative images of ATP5B immunoflourescence staining (G) and quantitation of cytoplasmic ATP5B puncta (H) in LPS-treated (100 ng/mL, 4 hr) BMDMs. Shown in (H) are box plots of at least 450 cells analyzed per condition. ****, P<0.0001, Mann-Whitney U-test. Scale bars, 10 μm. (I) Representative light micrographs of immunofluorescence staining of ASC and ATP5B in Fancc^+/+ and Fancc^−/− BMDMs treated with LPS + ATP or LPS + ATP + MT as described in (F). Scale bars, 5 μm. For (A-I), similar results were observed in three independent experiments. MT, mitoTEMPO. See also Figure S5.

Figure 6. The Mitophagy Function of FANCC is Genetically Dissociated from its Role in Nuclear DNA Damage Repair

(A) FA core complex activity assessed by western blot of MMC (1 μM, 24 hr)-induced FANCD2 mono-ubiquitination in parental HeLa cells (WT) or HeLa FANCC^KO cells transduced with indicated FANCC vector. A longer exposure of FANCC reveals FANCC-Flag protein expression in lanes 4-8 (data not shown). (B) Cytokine-induced cell death in HeLa FANCC^KO cells transduced with indicated FANCC vector. Cells were treated with TNF-α and IFN-γ (10 ng/mL each, 48 hr) and cell death was measured by flow cytometric analysis of 7-AAD (a vital dye) staining. ****, P<0.0001, chi-square test. (C) MMC-induced cell death in HeLa FANCC^KO cells transduced with indicated FANCC vector. Cells were treated with MMC (1 μM) for 48 hr and cell death was measured by flow cytometric analysis of 7-AAD (a vital dye) staining. ****, P<0.0001, chi-square test. (D) Representative images of TOMM20 (upper panels) or ATP5B (lower panels) immunoflourescence staining of parental HeLa/Parkin cells (WT) or HeLa FANCC^KO/Parkin cells transduced with indicated FANCC vector treated with OA for 8 hr prior to imaging and automated image analysis (D). Scale bars, 10 μm. (E) Quantitation of cytoplasmic TOMM20 puncta or ATP puncta in cells treated as shown in representative images in (D). Shown are box plots of at least 150 cells analyzed per condition. ****, P<0.0001, Mann-Whitney U-test. (G) Mitophagy analysis in HeLa FANCC^KO/Parkin cells transduced with indicated FANCC vector assessed by western blot of HSP60 or TOMM20 proteins in cells +/− OA for 8 hr. For (A-F), similar results were observed in three independent experiments.

Figure 7. Multiple FA Genes are Required for Mitophagy

(A) Representative light micrographs of images used for quantification of TOMM20 (left panels) in (B) and ATP5B (right panels) clearance in (C) in HeLa/Parkin cells transfected with indicated siRNAs and 48 hr later treated with OA for 8 hr. Scale bars, 20 μm. (B-C) Quantitation of cytoplasmic TOMM20 (B) or ATP5B (C) puncta in cells transfected with indicated siRNAs and treated as in (A). Shown are box plots of at least 150 cells analyzed per condition. Similar results were observed in three independent experiments. **, P<0.01, ****, P<0.0001; Mann-Whitney U-test. (D) Mitophagy analysis in HeLa FANCC^KO/Parkin cells transfected with indicated siRNA assessed by western blot of indicated proteins +/− OA for 8 hr. See also Figure S7.