Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane

Abstract

Progerin, a truncated unprocessed lamin A protein, causes tissue aging and degeneration. In this study we explored the role of progerin in the pathogenesis of intervertebral disc degeneration (IDD). We also examined the effect of sulforaphane (SFN) on progerin accumulation and mitochondrial dysfunction in IDD. Methods: The role of progerin in IDD was explored using human nucleus pulposus (NP) tissues, rat NP cells, and Lmna G609G knock-in mice. Immunostaining, X-ray imaging, and Western blotting were performed to assess the phenotypes of intervertebral discs. Alterations in senescence and apoptosis were evaluated by SA-β-galactosidase, immunofluorescence, flow cytometry, and TUNEL assays. Mitochondrial function was investigated by JC-1 staining, transmission electron microscopy, and determination of the level of ATP and the activities of mitochondrial enzymes. Results: The progerin level was elevated in degenerated human NP tissues. Lmna G609G/G609G mice displayed IDD, as evidenced by increased matrix metalloproteinase-13 expression and decreased collagen II and aggrecan expression and disc height. Furthermore, progerin overexpression in rat NP cells induced mitochondrial dysfunction (decreased ATP synthesis, mitochondrial membrane potential, and activities of mitochondrial complex enzymes), morphologic abnormalities, and disrupted mitochondrial dynamic (abnormal expression of proteins involved in fission and fusion), resulting in apoptosis and senescence. SFN ameliorated the progerin-induced aging defects and mitochondrial dysfunction in NP cells and IDD in Lmna G609G/G609G mice. Conclusions: Progerin is involved in the pathogenesis of IDD. Also, SFN alleviates progerin‑induced IDD, which is associated with amelioration of aging defects and mitochondrial dysfunction. Thus, SFN shows promise for the treatment of IDD.

Keywords: Intervertebral disc degeneration; mitochondria; progerin; sulforaphane.

Figure 1

Progerin expression in human NP tissues. (A) IF staining of progerin in human NP tissues from the Grade II/III and Grade IV/V groups. Nuclei were stained with DAPI. Arrows indicate progerin-positive cells. (B) Progerin-positive cells in human NP tissues from the Grade II/III and Grade IV/V groups; We selected 10 random fields in 3 sections per individual for quantitative analysis. A total of 235-600 cells were analyzed from each individual; n = 10. **P < 0.01. (C) qRT-PCR analysis of progerin mRNA levels in NP tissues from the Grade II/III and Grade IV/V groups; n = 10; **P < 0.01. (D) Western blotting analysis of progerin in NP tissues from the Grade II/III and Grade IV/V groups; n = 10; **P < 0.01. Data represent mean ± SEM.

Figure 2

LMNA G609G/G609G mice show accelerated IDD. (A) Representative X-ray images and disc height indices (DHI) of the IVD of WT and G609G/G609G mice; n = 3; *P < 0.05, **P < 0.01. DHI% was calculated as: DHI% = (D1+D2+D3) × 100% / (V1+V2+V3+D1+D2+D3), where D indicates disc height and V indicates vertebra length. (B) HE and safranin O staining and histological scores of the IVDs of WT and G609G/G609G mice; n = 3; **P < 0.01. (C) Representative IF images of progerin, collagen II, and aggrecan expression and TUNEL staining of IVDs in WT and G609G/G609G mice. Nuclei were stained with DAPI. (D) Representative Western blots of collagen II, aggrecan, and MMP-13 in the IVDs of WT and G609G/G609G mice; n = 3; *P < 0.05, **P < 0.01. Data represent mean ± SEM. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick‑end labelling; MMP, matrix metalloproteinase.

Figure 3

Progerin induces aging-related defects and mitochondrial dysfunction in NP cells. (A) Representative IF images and quantification of LAP2, γH2AX, H3K27me3, and lamin B1 expression in the vector con and progerin groups; n = 5; **P < 0.01. (B) TUNEL staining of NP cells in the vector con and progerin groups. Nuclei were stained with DAPI. The number of TUNEL-positive cells was significantly greater in the progerin group than in the vector con group; n = 5; **P < 0.01. (C) Representative flow cytometry dot plots of apoptosis after Annexin V-FITC/PI dual staining. The relative number of apoptotic cells was greater in the progerin group compared to the vector con group; n = 5; **P < 0.01. (D) Representative images and quantification of DCFDA-based ROS levels in the vector con and progerin groups; n = 5; **P < 0.01. (E) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the vector con and progerin groups; n = 5; **P < 0.01. (F) ATP production in the vector con and progerin groups; n = 5; *P < 0.05. (G) Relative activities of mitochondrial complex enzymes in NP cells in the vector con and progerin groups; n = 4; *P < 0.05, **P < 0.01. Data represent mean ± SEM. LAP2, lamina-associated polypeptide2; γH2AX, serine-139 phosphorylated H2AX; H3K27me3, heterochromatin-associated tri-methylated lysine 27 on histone 3; SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5‑,6,6‑tetrachloro-1,1‑,3,3‑tetraethylbenzimidazole-carbocyanine iodide.

Figure 4

Progerin disrupts mitochondrial morphology and mitochondrial dynamics. (A) Representative TEM images of mitochondria in NP cells from the vector con and progerin groups; F, Fragmented mitochondria; S, swollen mitochondria. (B) Representative fluorescence images of mitochondria in NP cells; S, swollen mitochondria. (C) Representative Western blots of OPA1, Drp1, Mfn1, and Mfn2 in the vector con and progerin groups (n = 5). GAPDH was used as the control. *P < 0.05, **P < 0.01. (D) Representative Western blots of PGC1α, pAMPK, and AMPK in the vector con and progerin groups; n = 5; *P < 0.05, **P < 0.01. Data represent mean ± SEM. OPA1, optic atrophy1; Drp1, dynamin-related peptide1; Mfn1, mitofusin 1; Mfn2, mitofusin2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGC1α, peroxisome proliferator-activated receptor-γ coactivator1α; AMPK, AMP-activated protein kinase.

Figure 5

SFN ameliorates progerin-induced aging defects and mitochondrial dysfunction. (A) IF of LAP2, γH2AX, H3K27me3, and lamin B1 in the progerin+vehicle and progerin+SFN groups; n = 5; *P <0.05. (B) Representative images and quantification of ROS levels in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (C) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (D) ATP production in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (E) Western blotting analysis of OPA1, Drp1, Mfn1, and Mfn2 in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5; *P < 0.05, **P < 0.01. (F) Western blotting analysis of PGC1α, pAMPK, and AMPK in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5. β-actin was used as the control. *P < 0.05, **P < 0.01. Data represent mean ± SEM. SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5‑,6,6‑tetrachloro-1,1‑,3,3‑tetraethylbenzimidazole-carbocyanine iodide; OPA1, optic atrophy-1; Drp1, dynamin-related peptide1; Mfn1, mitofusin1; Mfn2, mitofusin2; PGC1α, peroxisome proliferator-activated receptor-γ coactivator1α; AMPK, AMP-activated protein kinase.

Figure 6

SFN attenuates progerin-induced IDD in LMNA G609G/G609G mice. (A) Representative X-ray images and disc height indices (DHI) of the IVDs of vehicle-treated and SFN-treated G609G/G609G mice; n = 3; *P < 0.05. (B) HE and safranin O staining and histological scores of the IVDs of vehicle-treated and SFN-treated G609G/G609G mice; n = 3; *P < 0.05. (C) Representative IF images of progerin, collagen II, and aggrecan expression and TUNEL staining of IVDs from vehicle-treated and SFN-treated G609G/G609G mice. Nuclei were stained with DAPI. (D) Western blotting analysis of collagen II, aggrecan, and MMP-13 in IVDs in vehicle-treated and SFN-treated G609G/G609G mice; n = 3; *P < 0.05. Data represent mean ± SEM. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick‑end labelling; MMP, matrix metalloproteinase.