Repression of the Antioxidant NRF2 Pathway in Premature Aging

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.

Keywords: HGPS; NRF2; aging; oxidative stress; progerin.

Fig 1. High-throughput RNAi screen identifies CAND1 as a mediator of progerin-induced aging

(A) Schematic and (B) heatmap representation of RNAi screen, indicating identified candidates (Fig S1J, Table S2). (C) Representative IF images for CAND1 RNAi and controls in progerin inducible fibroblasts (See Experimental procedures). Scale bar: 25μm. (D) IF quantification in inducible or (E) WT and HGPS fibroblasts. p<0.05: ^& ONorHGPS/siCTRL vs. OFForWT/siCTRL; ^# ONorHGPS/siCAND1 vs. OFForWT/siCTRL; * ONorHGPS/siCAND1 vs. ONorHGPS/siCTRL. N>300; values represent averages ± SD from at least 3 experiments.

Fig 2. NRF2 transcriptional activity is impaired in HGPS

(A) Western blot (WB) analysis of total progerin inducible fibroblasts (P1 cells, See Experimental Procedures) extracts (2 biological replicates). Dotted line indicates separate WBs. (B) WB of total cell extract of WT and HGPS fibroblast (2 biological replicates for 4 cell lines, see Experimental Procedures). (C) WB of nuclear P1 cell extract (3 biological replicates of P1 cells). α-tubulin serves as cytoplasmic control. (D) NRF2 IF staining (Abcam 62352) in formaldehyde fixed WT and HGPS fibroblasts. N>300; Scale bar: 10μm. (E) ARE-luc assay (See Experimental procedures). *p<0.05. (F) Line graph of Log2 mRNA expression changes between WT and HGPS fibroblasts for indicated transcriptional targets. A left-shift of the NRF2-targets plot indicates an increased frequency of NRF2 regulated target genes that are downregulated in HGPS patients (p<0.0001).

Fig 3. Progerin mislocalizes NRF2

(A) NRF2 IF staining (Sc-722) in methanol fixed progerin indicuble fiborblasts (P1 cells, See Experimental Procedures). Arrows indicate overlapping IF signals. Scale bar: 10μm. (B) IF stain for NRF2 (Sc-722) and lamin B1 (Sc-6217) in methanol fixed WT and HGPS fibroblasts. Arrows indicate overlapping IF signals. Scale bar: 10μm. (C) Line graphs indicating IF signal intensity across the dotted lines in panel A and B. (D) WB analysis of interaction between immunoprecipitated OST-NLS-mCherry-NRF2 with HA-lamin A or HA-progerin, or (E) immunoprecipitated OST-lamin A and OST-progerin with FLAG-NLS-NRF2 in HEK293FT cells (3 biological replicates; See Experimental Procedures). *p<0.05. (F) WB analysis to probe the interaction between WT immunoprecipitated OST-lamin A or OST-progerin with recombinant HIS6x- NRF2. * p<0.05.

Fig 4. NRF2 impairment causes oxidative stress and recapitulates the progeroid phenotype

(A) IF analysis in WT and HGPS fibroblasts. p<0.05: ^& WT/shCTRL vs. HGPS/shCTRL; * WT/shCTRL vs. WT/shNRF2; ^#, HGPS/shCTRL vs. HGPS/shNRF2. (B) WB analysis of HGPS fibroblasts (2 biological replicates). (C) DCFDA-based ROS quantification in WT fibroblasts expressing indicated shRNAs or (D) in WT and HGPS fibroblasts (N>300; *p<0.05). Scale bar: 10 μm. (E) IF analysis in WT and HGPS fibroblasts treated with H₂O₂ (250μM; 4 days). p<0.05: ^&WT/vehicle vs. HGPS/vehicle; *WT/vehicle vs. WT/H₂O₂; ^#HGPS/vehicle vs. HGPS/H₂O₂. (F) WB analysis of HGPS fibroblasts treated with H₂O₂ (250μM; 4 days) (2 biological replicates).*, p<0.05. For all IF-based panels: N>300; values represent averages ± SD from at least 3 experiments.

Fig 5. Reactivation of NRF2 restores aging defects in HGPS patient fibroblasts

(A) IF analysis in WT or HGPS fibroblasts expressing control or caNRF2 for 96 hours. p<0.05: ^& WT/CTRL vs. HGPS/CTRL; *HGPS/CTRL vs. HGPS/caNRF2; ^#WT/CTRL vs. HGPS/caNRF2. (B) WB analysis of HGPS fibroblasts (2 biological replicates). *p<0.05 HGPS vs HGPS/caNRF2 (C) Representative IF stain for panel A. Scale bar: 10μm. (D) DCFDA-based ROS quantification in CTRL or caNRF2 expressing WT and HGPS fibroblasts. (E) IF analysis of 96 hour vehicle or NAC-treated WT and HGPS fibroblasts. p<0.05: ^&WT/vehicle vs. HGPS/vehicle. *HGPS/vehicle vs. HGPS/NAC. ^#WT/vehicle vs. HGPS/NAC. For all IF-based panels: N>300; values represent averages ± SD from at least 3 experiments.

Fig 6. NRF2 activation alleviates HGPS mesenchymal stem cell viability defects

(A) Heatmap of NRF2-target mRNA expression levels. Values represent averages from at least 3 experiments. (B) NRF2 target mRNA expression. *p<0.05, GC/shCTRL vs. GC/shNRF2. (C) H2DCFDA-based ROS quantification. (D) IF analysis of lamin B1 (manual count of low lamin expressing B1 cells), LAP2 and HP1γ (See experimental Procedures). Scale bar: 10 μm. N>300. P<0.05: *GC/shCRTL vs. GC/shNRF2; ^#GC/shCRTL vs. HGPS/shCTRL. (E) Frequency of SA-β-gal positive cells and (F) relative cell survival rate (See Experimental Procedures). p<0.05: *GC/shCRTL vs. GC/shNRF2; ^#GC/shCTRL vs. HGPS/shCTRL. (G) NRF2-target mRNA expression levels. *p<0.05, HGPS/CTRL vs. HGPS/caNRF2. (H) H2DCFDA-based ROS quantification for indicated cell types. (I) IF quantification (See panel D) in vehicle or Oltipraz treated (20 μM; 3 weeks) HGPS-iPSC-MSCs. N>100. *p<0.05. (J) Relative amounts of apoptotic cells and (K) SA-β-galactosidase positive cells in GC- and HGPS-iPSC-MSC expressing caNRF2 or treated with Oltipraz (20 μM; 3 weeks). N>300. *p<0.05. (L) In vivo MSC implantation assay for indicated conditions (Oltipraz: 20 μM; 3 wks) (N=3–5). *p<0.05 for indicated comparisons: HGPS vs. GC; GC/shNRF2 vs. GC/shCTRL; HGPS/caNRF2 vs. HGPS/CTRL; HGPS/Oltipraz vs. HGPS/Vehicle. For all bar-graphs values represent averages ± SD from at least 3 experiments.