Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies

Abstract

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.

Figure 1. Defects in body weight and longevity in Lmna^−/− and Lmna^L530P/L530P (LmnaΔ9 mice) mice are ameliorated in homozygous Sun1 knockout Lmna^−/−Sun1^−/− and LmnaΔ9Sun1 ^−/− animals

(A) Body weights are averages from mice with the indicated genotypes. The number (n) of animals used is indicated. (B) Kaplan-Meier graph showing increased life span of Lmna^−/−Sun1^−/− compared to Lmna^−/− mice. Median survival of wild type or Sun1^−/− is >210 days in a 7 month follow up; Lmna^−/− mice have median survival of 41 days; Lmna^−/−Sun1^+/− mice have a median of 54 days; Lmna^−/−Sun1^−/− mice have a median of 104 days (P <0.01 comparing Lmna^−/− and Lmna^−/−Sun1^−/−). (C) Body weights of LmnaΔ9 mice that are wild type, heterozygous, or homozygous for Sun1 deficiency. Wild type and Sun1^−/− cohorts are graphed for comparison. Values are averages+/−SEM from animals in each cohort. Number (n) of animals is indicated. (P <0.0001 comparing LmnaΔ9Sun1 ^+/+ and LmnaΔ9Sun1 ^−/−). (D) Kaplan-Meier graph showing increased life span of LmnaΔ9Sun1 ^−/− compared to LmnaΔ9Sun1 ^+/+ mice. LmnaΔ9Sun1 ^+/− mice are also graphed. (P <0.0001 comparing LmnaΔ9Sun1 ^+/+ and LmnaΔ9Sun1 ^−/−). (E) Cell proliferation of the indicated MEFs. Curves are averages+/−SD, representative of >3 independent isolates from embryos of the indicated genotypes. (F) Proliferation curves of MAFs (mouse adult fibroblasts) from WT, Sun1^−/−, LmnaΔ9Sun1 ^+/+ and LmnaΔ9Sun1 ^−/− mice. MAFs were seeded at a density of 1000 cells per well. Growth was measured, and normalized cell indexes (averages+/−SD) are presented.

Figure 2. Correction of the Lmna^−/− skeletal defects in the Lmna^−/−Sun1^−/− double knock out mouse

(A) Micro-CT scans of the indicated mice. Lmna^−/− mice display a lordokyphosis (curvature of the spine) phenotype corrected in Lmna^−/−Sun1^−/− mice. (B) Three-dimensional micro-CT images of the femoral trabeculae from 40-day-old mice (left panels). Thinner trabecular formation was observed in the Lmna^−/− mouse compared to the other genotypes. Right panels quantify bone density+/−SD (upper) and the number of trabeculaes/mm+/−SD (lower). P values are shown. (see also Figure S1)

Figure 3. Extranuclear Sun1 is accumulated in the Golgi of Lmna^−/− MEFs

(A) Cells were immunostained with anti-lamin A (green) and anti-Sun1 (red) antibodies. Extranuclear Golgi localization of Sun1 is seen in Lmna^−/− MEFs (also see Figure S2). (B) Quantification of Sun1 in MEFs. Mean±SD reflects collective results from two separate experiments with n = 29 (WT) and n = 36 (Lmna^−/−) MEFs. Difference between WT and Lmna^−/− is statistically significant (P <0.0001). See also Figure S3. (C) WT, Lmna^−/− and Lmna^−/−Sun1^−/− MEFs were stained with anti-Lamin B1 (red) and DAPI (blue). Lamin B1 nuclear envelope staining is intact in WT and Lmna^−/−Sun1^−/− MEFs, with the staining being irregular with herniations in Lmna^−/− nuclei. Arrows point to disruptions in nuclear envelope. Bars: 10 μm. (D) Quantification of prevalence of cells with nuclear envelope disruptions. Values are averages+/−SD from three independently isolated MEFs of the indicated genotype (each counted for 300 nuclei). Prevalence of nuclear disruptions between Lmna^−/− and Lmna^−/−Sun1^−/− MEFs is significantly different (P < 0.0001). (E) (Upper) Sun1 over expression in the absence of lamin A exacerbates nuclear herniations. WT and Lmna^−/−Sun1^−/− MEFs were transfected with increasing mouse Sun1 (mSun1) expression vector. Nuclei were stained 48 hours later. Values are averages+/−SD from three experiments (each sample was counted for 300 nuclei per experiment). (Lower) Transfected cells were Western blotted for Sun1 expression and actin (as loading control). (F) WT (top) or Lmna^−/−Sun1^−/− (bottom) MEFs were transfected with vector-alone (left) or increasing amounts of mSun1 (right three panels) and analyzed 48 hours later by FACS for propidium iodide (PI; Y-axis) and annexin V (X-axis). Percentage of apoptotic cells (in the lower right quadrant) is indicated.

Figure 4. Over expression of Golgi-targeted Sun1 increased nuclear aberrations and cell death

(A) WT MEFs were transfected with FLAG-tagged mouse Sun1 vector and stained with mouse anti-FLAG (green), rabbit anti-GM130 (red), and goat anti-lamin B1 (grey scale). A representative image of modest nuclear blebs and ruffles seen in some transfected cells is shown. Bars, 10 μm. (B) A Golgi-targeted mouse Sun1 (fused with Tgn38, HA-tagged) expression plasmid was transfected into WT MEFs. Thirty hours later, cells were stained with mouse anti-HA (green), rabbit anti-GM130 (red), and goat anti-lamin B1 (grey scale). Aberrancies were visualized by cytoplasmic lamin B1 staining (see arrow heads) of pmSun1-Tgn38-HA transfected cells. Bars, 10 μm. (C) Quantification of the cytoplasmic release of lamin B1 in MEFs transfected (for 30 hours) with either mSun1 (mSun1-FLAG) or the Golgi-targeted mSun1 (pmSun1-Tgn38-HA). One hundred cells were counted in each case. See also Figure S4.

Figure 5. Brefeldin A and nocodazole, but not latrunculin, treatment reduced nuclear irregularities in Lmna^−/− MEFs

(A) (Left) Staining of Sun1 (red) and GM130 (green) in Lmna^−/− MEFs treated for 24 hours with brefeldin A (BFA, 10 μg/mL); note the reduction of Sun1 and GM130 from the Golgi. (Right) Quantification of BFA treatment on the nuclear morphology of Lmna^−/− MEFs. Untreated and treated cells were stained with mouse Sun1-specific antibody or DAPI in cells passaged 4 (P4), and 8 (P8) times, respectively. The nuclear morphology was evaluated by observers blinded for genotype and by computerized image analyses. Nuclear irregularities are also seen in HGPS cells (see list in Table S1). (B) (Left) Sub-cellular localization of Sun1 in Lmna^−/− MEFs untreated or treated with 5 μM nocodazole for 4 hours. The Golgi complex was stained with mouse antibody against GM130 (green) and rabbit antibody against mouse Sun1 (red). (Middle) Cells untreated and treated with nocodazole and stained for α-tubulin are shown. (Right) Quantification of nocodazole treatment on the nuclear morphology of Lmna−/− MEFs. Difference between untreated and treated cells is P = 0.0058. (C) (Left) Lmna^−/− MEFs were untreated or treated with 40 nM of latrunculin (LAT-B) for 12 hours. Cells were fixed and stained for Sun1 and GM130. (Middle) Cells untreated and treated with latrunculin and visualized with fluorescent phalloidin for actin are shown. (Right) Quantification of LAT-B treatment on the nuclear morphology of Lmna^−/− MEFs. Difference between untreated and treated cells was statistically insignificant (P = 0.8376). All values are mean+/−SD.

Figure 6. Nuclear irregularities in HGPS fibroblasts correlate with SUN1 expression

(A) SUN1 and lamin B1 in normal (AG03512 and AG03258) and HGPS (AG06297 and AG11498) skin fibroblasts are stained with anti-human SUN1 (green) and anti-lamin B1 (red). DAPI is in blue. Yellow arrow heads point to cells expressing high-SUN1, white arrow heads to cells with low-SUN1. (B) Nuclear morphologies and SUN1-staining of control (AG03512) and HGPS (AG11498) skin fibroblasts transfected with control- or SUN1-siRNA for 72 hours. (C) Quantification of SUN1 immunofluorescent intensities in cells treated with Control or SUN1 siRNA. One hundred twenty to two hundred cells from each of the indicated samples were visualized and quantified for staining intensities. The intensities were normalized to the SUN1 intensity in AG03512 cells. Cells with SUN1 intensities less than 2 fold different from average are represented by blue bar; cells that are > 2 fold, but <5 fold are represented by pink bar; cells that stained >5 fold above average are represented by brown bar. *, P < 0.001 when compared to AG03512 cells (t-test). (D) Quantification of the prevalence of cells from (B) with nuclear irregularities. ‡, P < 0.0001, when comparing the same cells treated with control RNAi or SUN1-RNAi (Fisher’s exact test). See also Figure S5. (E) Aberrant nuclear morphology in normal and HGPS fibroblasts transfected with a HA-tagged human SUN1 expression plasmid. Two hundred mock transfected cells per sample and fifty transfected cells per sample were scored. P values, Fisher’s exact test.

Figure 7. Knock down of SUN1 alleviated HGPS-associated loss of NURD complex and cellular senescence

(A) Normal (AG03512) and HGPS (AG11498) skin fibroblasts were stained for heterochromatin markers (RBBP4 or H3K9me3; green) and SUN1 (red). Yellow arrow heads point to high-SUN1 cells; white arrow heads denote low-SUN1 cells. See also Figure S6. (B) Expression levels of RBBP4 or H3K9me3 and SUN1 in two normal and three HGPS skin fibroblasts were quantified by MetaMorph software. Each dot represents fluorescence intensity (in Log₁₀ scale) in a single cell of RBBP4 (left) or H3K9me3 (right) vs. SUN1. Linear curve fitting and correlation coefficient (r) are indicated. In HGPS cells, RBBP4 and H3K9me3 expression correlates negatively with SUN1 expression. C) HGPS fibroblasts (AG03513) treated with control or SUN1 siRNA for 72 hours were stained with antibodies for SUN1 (red) and RBBP4 (green). Increased RBBP4 expression was observed in SUN1 siRNA-treated cells compared to control siRNA-treated cells. Graphic quantification of the staining intensities of RBBP4 vs. SUN1 in individual HGPS fibroblasts treated with control (blue) or SUN1 (brown) siRNA is shown (right); each dot represents a single cell (154 control and 157 SUN1 RNAi treated cells were quantified). (D) Visualization (Left) and quantification (Right) of acidic senescence associated β-galactosidase (SA-β-Gal) in normal (AG03257) and HGPS (AG11498 at passage 8) fibroblasts transfected with Control- or SUN1-RNAi for 96 hours. Standard deviations are from three independent assays counting 1200 to 2000 cells in each experiment. Cell scoring was performed in a blinded fashion by an independent investigator. P value (Chi-square) is indicated. (E) Cell proliferation in normal (AG03257) and HGPS (AG11498) cells transfected with control or SUN1 RNAi. Cells at ~50% confluency were transfected. When cells reached confluency, equal numbers were seeded into dishes and quantified for proliferation using Cell Counting Kit-8 24 hours after cell seeding (day 0) and after another 4, 8, 10, 12 days. Relative absorbance at 460nm was obtained by [(Absorbance_460nm-background Absorbance_460nm) at day N]/[(Absorbance_460nm-background Absorbance_460nm) at day 0]. Standard deviations are from triplicate experiments.