Nuclear membrane ruptures underlie the vascular pathology in a mouse model of Hutchinson-Gilford progeria syndrome
Paul H. Kim, Natalie Y. Chen, Patrick J. Heizer, Yiping Tu, Thomas A. Weston, Jared L.-C. Fong, Navjot Kaur Gill, Amy C. Rowat, Stephen G. Young, Loren G. Fong
Paul H. Kim, Natalie Y. Chen, Patrick J. Heizer, Yiping Tu, Thomas A. Weston, Jared L.-C. Fong, Navjot Kaur Gill, Amy C. Rowat, Stephen G. Young, Loren G. Fong
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Research Article Vascular biology

Nuclear membrane ruptures underlie the vascular pathology in a mouse model of Hutchinson-Gilford progeria syndrome

Abstract

The mutant nuclear lamin protein (progerin) produced in Hutchinson-Gilford progeria syndrome (HGPS) results in loss of arterial smooth muscle cells (SMCs), but the mechanism has been unclear. We found that progerin induces repetitive nuclear membrane (NM) ruptures, DNA damage, and cell death in cultured SMCs. Reducing lamin B1 expression and exposing cells to mechanical stress — to mirror conditions in the aorta — triggered more frequent NM ruptures. Increasing lamin B1 protein levels had the opposite effect, reducing NM ruptures and improving cell survival. Remarkably, raising lamin B1 levels increased nuclear compliance in cells and was able to offset the increased nuclear stiffness caused by progerin. In mice, lamin B1 expression in aortic SMCs is normally very low, and in mice with a targeted HGPS mutation (LmnaG609G), levels of lamin B1 decrease further with age while progerin levels increase. Those observations suggest that NM ruptures might occur in aortic SMCs in vivo. Indeed, studies in LmnaG609G mice identified NM ruptures in aortic SMCs, along with ultrastructural abnormalities in the cell nucleus that preceded SMC loss. Our studies identify NM ruptures in SMCs as likely causes of vascular pathology in HGPS.

Authors

Paul H. Kim, Natalie Y. Chen, Patrick J. Heizer, Yiping Tu, Thomas A. Weston, Jared L.-C. Fong, Navjot Kaur Gill, Amy C. Rowat, Stephen G. Young, Loren G. Fong

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Figure 1

Progerin expression causes NM ruptures in cultured SMCs.

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Progerin expression causes NM ruptures in cultured SMCs. (A) Western blo...
(A) Western blot analysis of SMCs expressing prelamin A (PreA) and progerin (Prog), and in the aorta and kidney of WT and LmnaG609G/+ mice. The bar graph shows the expression of lamin A plus progerin, relative to total protein (Supplemental Figure 1C). (B) Bar graph showing that progerin increases the frequency of abnormally shaped nuclei in SMCs, as judged by live-cell microscopy (mean ± SEM, n = 4 experiments; Student’s t test, *P < 0.001). (C) Illustration depicting a NM rupture, defined as the escape of nuclear-targeted GFP (Nuc-GFP; green) into the cytoplasm (tan). (D) Bar graph showing that progerin causes NM ruptures in SMCs (mean ± SEM, n = 3 experiments; Student’s t test, *P < 0.01). NM ruptures were measured in fixed cells 48 hours after adding Dox (blue). (E) Fluorescence microscopy images of live SMCs expressing progerin and Nuc-GFP (green) at 1-hour intervals. A DIC image (grey) is superimposed. The yellow arrow points to a cell followed for 14 hours by time-lapse microscopy (see Supplemental Video 1; images were captured every 10 minutes). The cell had several NM ruptures and eventually died. Scale bar: 20 μm. (F–I) Characteristics of NM ruptures in PreA-SMC and Progerin-SMC derived from time-lapse microscopy studies. Bar graphs show the percentage of cells with a NM rupture; the number of NM ruptures per cell; the percentage of cells with a NM rupture that die; and the percentage of cells with a NM rupture and abnormal-shaped nuclei (mean ± SEM, n = 4 experiments; Student’s t test, *P < 0.02, **P < 0.002, ***P < 0.001). The results from the individual experiments are shown in Supplemental Figure 1E.