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, 103 (7), 2154-9

Hutchinson-Gilford Progeria Mutant Lamin A Primarily Targets Human Vascular Cells as Detected by an anti-Lamin A G608G Antibody

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Hutchinson-Gilford Progeria Mutant Lamin A Primarily Targets Human Vascular Cells as Detected by an anti-Lamin A G608G Antibody

Dayle McClintock et al. Proc Natl Acad Sci U S A.

Abstract

Hutchinson-Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC-->GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
A unique Ab specifically detects the mutant lamin A G608G (progerin) in HGPS cells and demonstrates the accumulation of the mutant protein progerin over time. (A) Immunofluorescence microscopy was performed on fibroblasts from an unaffected individual (40 PPDs) and a subject with HGPS (24, 30, 42, and 52 PPDs). Cells were stained with anti-LMNA G608G (red) and anti-emerin (green); the merged signals are indicated. Note the increase of positively labeled HGPS with increasing PPDs. (B) Western blot analysis of control (38 PPDs) and HGPS (28, 35, and 45 PPDs) extracts probed with anti-LMNA G608G and anti-lamin A/C. The arrow indicates the progerin band. (C) HGPS and control fibroblasts double-labeled with anti-LMNA G608G (red) and either anti-lamin A, anti-emerin, or anti-nucleoporin 414 (green) reveal the similar distribution into cable-like structures of mutant and wild-type proteins. Chromatin was stained with DAPI (blue).
Fig. 2.
Fig. 2.
Progerin accumulation induces NE invaginations as shown by EM on ultrathin sections from control (A and B) and HGPS (CF) cells at 38 PPDs. Low magnification of control (A) and HGPS (C) nuclei. (Scale bar, 5 μm.) (B and DF) High magnifications of the corresponding nuclei at the NE invaginations (NI). NC, nucleoplasm; C, cytoplasm; NPC, nuclear pore complex. Although few, small nuclear invaginations are detected in control fibroblasts; HGPS nuclei exhibit numerous, large nuclear invaginations recognizable by the presence of a high density of clustered nuclear pores. (Scale bar, 0.5 μm.)
Fig. 3.
Fig. 3.
FTI-277 treatment prevents nuclear deformations and delocalizes progerin. (A) Nuclei from control and HGPS cells (35–38 PPDs) were scored for abnormal shape every 24 hours after daily treatment with 20 μM FTI-277 for 3 days and compared against scores for untreated cells. Shown are average percentages of cells with abnormally shaped nuclei for each treatment or time course (error bars at one SD). (B) Immunofluorescence microscopy on FTI-277-treated or untreated cells by using anti-LMNA G608G (red), anti-prelamin A (green), and DAPI (blue) demonstrates the gradual relocation of the mutant protein progerin away from the NE and lamina and into intranuclear foci. (Scale bar, 10 μm.)
Fig. 4.
Fig. 4.
Mutant LMNA G608G accumulation reduces cellular proliferation, induces premature senescence, and impairs cell migration in HGPS fibroblasts. (A) The proliferative index, determined by anti-Ki-67 labeling, was shown to decline faster in HGPS cells than in control cells. (B) Senescence β-gal staining revealed more cells of a 4-week-old HGPS culture (55 PPDs) to be senescent versus those of a 1-week-old control culture (55 PPDs). (C) The average percentages of cells scored as senescent β-gal-positive from HGPS cells after 1, 4, and 6 weeks (55 PPDs) were significantly higher than 1- and 4-week-old control cultures (55 PPDs). (D) HGPS cells (55 PPDs) assayed for senescence-associated β-galactosidase also showed a high level of anti-LMNA G608G signal (red); cells were counterstained with DAPI. (Scale bar, 10 μm.) (E) Immunofluorescent labeling of control and HGPS cells (38 PPDs) subjected to migration stimulus revealed an impaired ability to migrate among cells with a bright anti-LMNA G608G signal (red); emerin is green. Merged images are indicated.
Fig. 5.
Fig. 5.
Mutant lamin A G608G was present mostly in vascular cells on skin sections derived from a subject with HGPS. (A) Three serial skin sections were immunostained with anti-lamin A/C Ab, anti-LMNA G608G Ab, or the corresponding preimmune serum. Low magnification, hematoxylin/eosin (H&E) staining: ep, epidermis; de, dermis; SG, sweat glands; C, capillary; apm, arrector pili muscle. Lamin A/C was detected in the nuclei of most cells from the epidermis and in the dermal compartments. Progerin, as detected by anti-LMNA G608G, was restricted to nuclei within the C, some cells surrounding the SG, and the apm. (Lower Right) Corresponds to staining with the preimmune serum (PIM) of the rabbit immunized with the LMNA G608G peptide. (B) High magnification of blood vessels from HGPS skin sections immunolabeled with anti-LMNA G608G and anti-α-smooth muscle actin (SMA) or anti-CD31 Abs (CD31) and counterstained with DAPI. Triple merged signals are indicated. The mutant lamin A accumulates primarily in the vascular cells, smooth muscle cells, and endothelial cells. (C) Normal skin sections immunolabeled with anti-lamin AG608G, anti-lamin A/C, and SMA Abs or stained with hematoxylin/eosin. Note the presence of A-type lamins in most nuclei from the epidermal and dermal compartments. SBG, sebaceous glands.

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