Progerin elicits disease phenotypes of progeria in mice whether or not it is farnesylated - PubMed (original) (raw)

Progerin elicits disease phenotypes of progeria in mice whether or not it is farnesylated

Shao H Yang et al. J Clin Invest. 2008 Oct.

Abstract

Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.

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Figures

Figure 1. Production of mice that express a nonfarnesylated version of progerin.

(A) The mutant allele yielding nonfarnesylated progerin, LmnanHG, was generated by deleting intron 10, intron 11, and the last 150 bp of exon 11 and introducing a point mutation in exon 12 that changes the cysteine of the CaaX motif to a serine. (B) Southern blot identification of the targeting event in 2 different mouse ES cell clones. The genomic DNA was cleaved with _Eco_RI, and the blot was hybridized with a 5′ flanking probe (location of probe shown in A). (C) Western blot identification of progerin in extracts of LmnanHG/+ MEFs with a lamin A/C–specific polyclonal antibody. Extracts of Lmna+/+, LmnaHG/+, and LmnaHG/HG MEFs were included as controls. (D) Sequencing chromatograms of PCR products amplified from the mutant alleles of LmnaHG/+ and LmnanHG/+ mice. The single nucleotide substitution in the LmnanHG allele (a thymine to adenine substitution) changes the cysteine of the CaaX motif to a serine.

Figure 2. Assessing protein farnesylation in LmnanHG/+ MEFs.

(A) Western blot of MEF extracts performed with an Odyssey Infrared Imaging System. The top panel shows the merged images of Western blots with an antibody specific for lamin A/C (red) and an antibody specific for the farnesol analogue AG (green); the middle panel shows the signal for the antibody against AG; the bottom panel shows a loading control (actin). In this experiment, Lmna+/+, LmnaHG/+, and LmnanHG/+ MEFs were incubated with AG (30 μM) in the presence (+) or absence (–) of an FTI (ABT-100, 5 μM). The electrophoretic mobility of progerin, lamin B1, and lamin B2 are virtually identical, and all are farnesylated; hence, the antibody against AG detected a farnesylated protein in all cell lines. When progerin and lamin A/C were immunoprecipitated with an antibody against lamin A/C, the progerin in LmnaHG/+ MEFs — but not LmnanHG/+ MEFs — stained with the antibody against AG. As expected, an FTI blocked the incorporation of AG into the lamin proteins. (B) Western blots of Lmna+/+, LmnaHG/+, and LmnanHG/+ MEFs with an HDJ-2–specific monoclonal antibody. Most of the HDJ-2 in FTI-treated MEFs migrated more slowly, a characteristic of nonfarnesylated HDJ-2.

Figure 3. Phenotypes of LmnanHG/+ mice.

(A) Body weight curves of male LmnanHG/+ (n = 17), Lmna+/+ (n = 15), and LmnaHG/+ mice (n = 16). (B) Body weight curves of female LmnanHG/+ (n = 16), Lmna+/+ (n = 12), and LmnaHG/+ mice (n = 15). Body weights were lower in male and female LmnanHG/+ mice than in Lmna+/+ mice (P < 0.0001). However, body weights were higher in male and female LmnanHG/+ mice than in LmnaHG/+ mice (P < 0.0001). (C) Kaplan-Meier survival plots for LmnanHG/+ mice (n = 17 males, 16 females), LmnaHG/+ mice (n = 16 males, 15 females), and Lmna+/+ mice (n = 15 males, 12 females). Male and female LmnanHG/+ mice survived longer than LmnaHG/+ mice (P < 0.0001). (D) Number of rib fractures in LmnanHG/+ mice (n = 17 males, 16 females) and LmnaHG/+ mice (n = 16 males, 15 females). Rib fractures normally increase with age (14). The number of fractures was lower in LmnanHG/+ mice than in LmnaHG/+ mice (P < 0.0001), even though LmnanHG/+ mice were older than the LmnaHG/+ mice (36.1 ± 0.69 weeks vs. 21.3 ± 0.84 weeks). (E) Body fat in Lmna+/+, LmnaHG/+, and LmnanHG/+ mice at the conclusion of the study. Body fat in LmnanHG/+ mice was lower than in Lmna+/+ mice (P < 0.0001) but higher than in LmnaHG/+ mice (P < 0.0001). Error bars indicate SEM.

Figure 4. Reduced bone abnormalities in LmnanHG/+ mice at 6 months of age, as judged by surface renderings of μCT analyses.

(A–F) μCT scans of the thoracic spine illustrating reduced number of rib fractures in LmnanHG/+ mice. Red arrowheads indicate rib fractures and surrounding callus. Red arrows indicate thinning ribs along with a small amount of callus. (A and D) Lmna+/+ mouse; (B and E) LmnaHG/+ mouse; (C and F) LmnanHG/+ mouse. (D–F) Lateral view of the thoracic spine illustrating reduced kyphosis of the spine in LmnanHG/+ mouse. Bone density (G) and cortical thickness (H) were improved in the ribs of LmnanHG/+ mice compared with LmnaHG/+ mice (n = 4 mice/genotype; P < 0.0001). Error bars indicate SEM.

Figure 5. Analysis of nuclear shape in primary MEFs from Lmna+/+, LmnanHG/+, LmnanHG/nHG, LmnaHG/+, and LmnaHG/HG embryos by immunofluorescence microscopy.

(A) The frequency of misshapen nuclei (folds, black bars; blebs, white bars) was greater in LmnanHG/+ and LmnanHG/nHG MEFs than in Lmna+/+ MEFs, but the frequency of misshapen nuclei was lower in LmnanHG/+ and LmnanHG/nHG MEFs than in LmnaHG/+ and LmnaHG/HG MEFs, respectively (n = 3–8 cell lines/genotype, >1000 cells counted for 3 fibroblast cell lines of each genotype_; P <_ 0.0001, χ2 test). Error bars indicate SEM for results with independently isolated cells of the same genotype. The fraction of abnormal nuclei that had nuclear folds was higher in LmnanHG/+ MEFs than in LmnaHG/+ MEFs and higher in LmnanHG/nHG MEFs than in LmnaHG/HG MEFs (P < 0.0001). (B) Frequency of misshapen nuclei in Lmna+/+, LmnaHG/+, and LmnanHG/+ MEFs in the presence and absence of an FTI (10 μM, ABT-100). The FTI reduced the number of misshapen nuclei in LmnaHG/+ MEFs, and an FTI had no effect on Lmna+/+ MEFs (n = 4 cell lines/genotype; P < 0.0001, χ2 test). In LmnanHG/+ and LmnanHG/nHG MEFs, the FTI treatment had no significant effect on nuclear shape (n = 4 cell lines/genotype; 4,000 cells/genotype counted). Ratios in each bar represent the number of cells with misshapen nuclei divided by the total number of cells evaluated (from all cell lines of each genotype). Error bars indicate SEM. Similar results, with identical levels of statistical significance, were obtained by 3 independent observers — all blinded to genotype. (C) Immunostaining of wild-type MEFs with a lamin A–specific antibody (red). (D) Immunostaining of LmnanHG/nHG MEFs with a lamin A–specific antibody (red). Blebs are indicated by white arrows, and folds are indicated by white arrowheads. (E) The localization of progerin (red) and LAP2β (green) in LmnanHG/nHG MEFs; both proteins were concentrated in nuclear folds. Images were recorded with a ×63 oil immersion objective (C–E).

Figure 6. Western blots documenting relative levels of progerin in LmnaHG/+ and LmnanHG/+ tissues and MEFs.

(A–D) Western blot (with antibodies against lamin A/C and actin) showing progerin, lamin A, lamin C, and actin in the heart (A), liver (B), kidney (C), and skull (D) of LmnaHG/+ and LmnanHG/+ mice (n = 3/genotype; each sample loaded in duplicate). (E) Western blot analysis (with antibodies against lamin A/C and actin) showing progerin, lamin A, lamin C, and actin in LmnaHG/+ and LmnanHG/+ MEFs (n = 4 cell lines/genotype). (F) Quantitative analysis of progerin expression in the tissues (n = 3 mice/genotype) and MEFs (n = 4 cell lines/genotype) of LmnaHG/+ and LmnanHG/+ mice. Each sample was analyzed on 4 separate Western blots; the variation in the progerin/actin ratio averaged 5.6%. Progerin/actin ratios in LmnanHG/+ samples were expressed relative to those in LmnaHG/+ samples (which were set at a value of 1). In both mouse tissues and the MEFs, the progerin/actin ratio was lower in LmnanHG/+ mice than in LmnaHG/+ mice (P < 0.0001).

Figure 7. Western blots assessing the levels of progerin relative to actin in LmnaHG/+ MEFs and in the hearts of LmnaHG/+ mice in the presence and absence of an FTI.

(A) Western blot (with antibodies against lamin A/C and actin) showing progerin, prelamin A, lamin A, lamin C, and actin in LmnaHG/+ MEFs (n = 4; the extracts of FTI-treated cells were loaded in duplicate). Cells were treated with an FTI (10 μM, ABT-100) or vehicle (DMSO) alone for 2 weeks. (B) Quantitative analysis of progerin expression in LmnaHG/+ MEFs (n = 4) relative to actin in the presence and absence of an FTI. The progerin/actin ratios in FTI-treated cell were expressed relative to those in DMSO-treated cells (where the ratio was set at 1.0). Each cell extract was analyzed in 3 independent experiments; the variation in the progerin/actin ratio averaged 10.6%. The progerin/actin ratio was lower in the FTI-treated cells (P < 0.0001). (C) Western blot analysis showing progerin, prelamin A, lamin A, lamin C, and actin in the hearts of LmnaHG/+ mice treated with the FTI or vehicle alone (n = 3 mice/group; each sample loaded in duplicate). Mice were given the FTI (or vehicle alone) for 4 months, beginning at 4 weeks of age. (D) Quantitative analysis of progerin expression in the hearts of FTI-treated LmnaHG/+ mice and LmnaHG/+ mice treated with vehicle alone (n = 3; each sample loaded in duplicate). Progerin/actin ratios in the hearts of FTI-treated mice were expressed relative to those in vehicle-treated mice (where the ratio was set at 1.0). Each sample was analyzed in 3 independent experiments; the variation in the progerin/actin ratios averaged 11.3%. The progerin/actin ratio was lower in the FTI-treated mice (P < 0.0001). Error bars indicate SEM.

Figure 8. Progerin transcript levels in livers of LmnaHG/+ and LmnanHG/+ mice (n = 4/genotype).

(A) Syto-60–stained agarose gel of progerin RT-PCR products (at cycle 20) from RNA of livers from LmnaHG/+ and LmnanHG/+ mice (n = 4/genotype, each sample run in duplicate). Image intensity was quantified with the Odyssey Infrared Imaging System. Trace amounts of wild-type prelamin A (from the wild-type allele) were observed in all samples. The RT-PCR reaction spanned Lmna exons 10–12. Arbp was used as an internal control. (B) Quantitative analysis of progerin mRNA levels, corrected for Arbp expression. Progerin mRNA levels in LmnanHG/+ samples were expressed relative to those in LmnaHG/+ samples (which were set at a value of 1). Error bars indicate SEM.

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Progerin elicits disease phenotypes of progeria in mice whether or not it is farnesylated - PubMed (original) (raw)