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Comparative Study
. 2005 Jul 19;102(29):10291-6.
doi: 10.1073/pnas.0504641102. Epub 2005 Jul 12.

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation

Shao H Yang  1 , Martin O Bergo, Julia I Toth, Xin Qiao, Yan Hu, Salemiz Sandoval, Margarita Meta, Pravin Bendale, Michael H Gelb, Stephen G Young, Loren G Fong
Affiliations
Comparative Study

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation

Shao H Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.

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Figures

Fig. 1.
Fig. 1.
Production of a mutant Lmna allele, LmnaHG, that yields progerin. (A) Gene-targeting strategy, which involves deleting intron 10, intron 11, and the last 150 nt of exon 11 of Lmna. (B) Southern blot analysis detecting the LmnaHG allele in mouse ES cells, with EcoRI-cleaved genomic DNA and the indicated 5′ flanking probe. (C) PCR identification of the LmnaHG allele. Results with wild-type MEFs (WT), heterozygous MEFs (LmnaHG/+), homozygous ES cells (LmnaHG/HG), and heterozygous MEFs (LmnaHG/+) are shown. (D) Western blotting identification of progerin with a lamin A/C-specific monoclonal antibody. Wild-type cells, LmnaHG/+ MEFs, and LmnaHG/HG cells are shown. On SDS/PAGE gels, the electrophoretic migration of progerin in mouse LmnaHG/+ MEFs and human HGPS fibroblasts was identical (data not shown).
Fig. 2.
Fig. 2.
Immunofluorescence microscopy of wild-type and LmnaHG/+ fibroblasts. (A–E) Immunostaining showing nuclear blebs in LmnaHG/+ MEFs. Blebs are indicated by white arrows. (F) Lmna+/+ MEFs. In this experiment, cells were stained for lamin B1 (red).
Fig. 3.
Fig. 3.
Western blot analysis of wild-type, LmnaHG/+, and Zmpste24-/- MEFs. Cells were grown in the presence and absence of an FTI (10 μM PB-43), and Western blot analyses of cell extracts were performed with antibodies specific for prelamin A (specific for the extreme C terminus), lamin A/C (binds to both lamin A and lamin C), lamin B1, Hdj-2, and actin.
Fig. 4.
Fig. 4.
Bar graph showing increased frequency of nuclear blebbing in LmnaHG/+ MEFs and a reduction in nuclear blebbing in LmnaHG/+ MEFs treated with an FTI (10 μM PB-43). A and B represent two independent experiments. Each black circle shows the frequency of nuclear blebbing with an independently isolated fibroblast cell line. Bars indicate the mean frequency of blebbing. The number of cells with nuclear blebs and the total number of cells examined are recorded within each bar. In both experiments, the FTI did not change the frequency of blebbing in Lmna+/+ MEFs, as determined by the χ2 test. LmnaHG/+ MEFs exhibited more blebbing than Lmna+/+ cells (P < 0.0001, χ2 test). An FTI reduced the frequency of blebbing in LmnaHG/+ MEFs (P < 0.0001, χ2 test). The frequency of blebbing in FTI-treated LmnaHG/+ MEFs was not different from the frequency of blebbing in the treated or untreated Lmna+/+ MEFs. Very similar results, with identical levels of statistical significance, were obtained when the microscopic slides were scored by a second blinded observer.
Fig. 5.
Fig. 5.
Immunofluoresence images showing the distribution of progerin in untreated and FTI-treated LmnaHG/HG cells. DNA was visualized with DAPI (blue), and progerin was visualized with an antibody against lamin A (red). (A and B) Untreated LmnaHG/HG cells, showing progerin along the nuclear envelope. Misshapen nuclei were common (arrow). (C and D) FTI-treated LmnaHG/HG cells, revealing intensely staining progerin aggregates (arrow-heads) in the nucleoplasm.
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