Skip to main content
NCBI home page
Nucleus logoLink to Nucleus
. 2010 Sep-Oct;1(5):432–439. doi: 10.4161/nucl.1.5.12972

Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome

Yuexia Wang 1, Cecilia Östlund 1, Howard J Worman 1,✉
PMCID: PMC3037539  PMID: 21326826

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and “blinded” electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

Key words: lamin, progeria, nuclear envelope, farnesylation, keratinocytes

Introduction

Hutchinson-Gilford progeria syndrome (HGPS; OMIM no. 176670) is a rare, sporadic, dominant genetic disorder characterized by phenotypic features of accelerated aging.1,2 It is caused by de novo mutations in LMNA.3,4 LMNA encodes A-type nuclear lamins, intermediate filament proteins associated with the inner nuclear membrane.5–9 In addition to causing HGPS, mutations in LMNA cause a wide range of human diseases sometimes called “laminopathies” that affect different organ systems depending upon the mutation.10,11

The predominant A-type lamin isoforms of somatic cells, lamin A and lamin C, arise by alternative splicing of RNA at a site encoded by exon 10 of LMNA.9 As a result, lamin A and lamin C share the first 566 amino acids and differ in their carboxyl-termini. Lamin A is synthesized as a precursor, prelamin A, which contains a CaaX (cysteine-aliphatic-aliphatic-any amino acid) motif at its carboxyl-terminus.5,8 CaaX initiates a series of sequential enzymatic modifications that lead to farnesylation and carboxymethylation of the carboxyl-terminal cysteine of prelamin A.10,12,13 Farnesylated prelamin A is then recognized by the endoprotease ZMPSTE24 and cleaved 15 amino acids from the farnesylated carboxyl-terminal cysteine to yield lamin A.11,13,14 As a result of this processing, mature lamin A that incorporates into the nuclear lamina is not farnesylated.

Mutations that cause HGPS create an abnormal splice donor site within exon 11 of LMNA, leading to an in-frame deletion of 50 amino acids from prelamin A including the ZMPSTE24 endoprotease site.3,4 This leads to expression of an abnormal truncated prelamin A termed progerin, which remains farnesylated at its carboxyl-terminal cysteine.3,4,10,13,14 Homozygous deletion of the gene encoding ZMPSTE24 leads to accumulation of uncleaved, farnesylated prelamin A and causes restrictive dermopathy, a perinatal lethal progeroid syndrome.15,16 Similarly, mice with deletion of Zmpste24 and mice with a targeted HGPS mutation in Lmna develop progeriod phenotypes.17–19 In pioneering studies, Fong, Young and colleagues19,20 showed that treatment with a protein farnesyltransferase inhibitor (FTI) improved the progeroid phenotypes in Zmpste24 null mice and mice with a targeted HGPS mutation in Lmna. Subsequently, Varela et al.21 showed that combined treatment with a statin and an aminobisphosphonate, which also inhibit protein prenylation, improved the progeroid phenotype in Zmpste24 null mice.

At the cellular level, a hallmark of HGPS, restrictive dermopathy and most other diseases caused by mutations in LMNA is the presence of misshapen nuclei.10,11,13,14 The initial studies reporting the genetic defect in HGPS noted the abnormal nuclear morphology in cultured cells from affected individuals.3,4 Since then, abnormal nuclear morphology in HGPS and restrictive dermopathy has received considerable attention; several studies have examined this phenomenon in cultured fibroblasts from human subjects and mouse models of the diseases as well as transfected cells expressing progerin.16,18,21–38 The reported abnormalities in nuclear morphology include lobulation or blebbing of the nuclear envelope, increased nuclear surface area, lower nuclear circularity, thickening of the nuclear lamina, decreased peripheral heterochromatin and clustering of nuclear pores complexes. Consistent with the effects on progeroid phenotypes in mice, pharmacological inhibitors of protein farnesylation significantly reverse these nuclear morphological abnormalities in cultured cells expressing progerin or lacking ZMPSTE24.10,11,13,14,21,25–30,35–37

That inhibition of protein farnesylation improves progerin-induced abnormal nuclear morphology and the phenotypes of experimental mice with targeted mutations has lead to the hypothesis that treatment with these drugs will benefit children with HGPS. As a result, clinical trials of FTIs, statins and aminobisphosphonates have been initiated in the United States and Europe.39,40 A “missing link” in the preclinical research; however, is lack of evidence that progerin-induced abnormal nuclear morphology can be reversed in tissues in animals systemically administered these drugs. We have therefore treated transgenic mice that express progerin in epidermis with a FTI or a combination of a statin plus an aminobisphosphonate to determine if they can reverse nuclear morphological abnormalities in intact tissue.

Results

Systemic administration of FTI or statin plus aminobisphosphonate partially inhibits protein prenylation and appears to improve abnormal nuclear morphology on immunohistofluorescence micrographs from mouse skin expressing progerin.

We have generated transgenic mice that express progerin with an amino-terminal FLAG epitope tag in epidermis under control of a keratin14 promoter.37 As a control, we also generated transgenic mice expressing normal human wild type lamin A with a FLAG epitope tag. While the skin and hair of these mice appear normal, nuclear morphology of keratinocytes expressing progerin is grossly abnormal compared to nuclear morphology of keratinocytes in mice expressing wild-type human lamin A. We used these mice to assess the effects of a systemically administered FTI (FTI-276) or a statin (pravastatin) plus an aminobisphosphonate (zoledronate) on progerin-induced abnormalities in nuclear morphology.

Intraperitoneal injection of pravastatin plus zoledronate or FTI-276 partially blocked farnesylation of HDJ-2, inducing approximately 15–20% non-farnesylated HDJ-2 accumulation in skin keratinocytes compared to mice administered PBS (Fig. 1A). To assess keratinocyte nuclear morphology, we labeled mouse skin sections with anti-FLAG antibody and examined the sections by confocal immunohistofluorescence microscopy. Double labeling with anti-keratin 14 antibody confirmed that the nuclei labeled by anti-FLAG antibody were keratinocytes (data not shown). Keratinocyte nuclei of mice expressing FLAG-tagged progerin administered PBS appeared irregular in contour (Fig. 1Ba). Keratinocyte nuclei from these same mice treated with FTI-276 (Fig. 1Bb) or pravastatin plus zoledronate (Fig. 1Bc) appeared more oval with a smoother contour. Keratinocyte nuclei from mice expressing FLAG-tagged wild type human lamin A appeared primarily as smooth ovals whether the mice received PBS, FTI-276 or pravastatin plus zoledronate (Fig. 1Bd–f). These results suggested that abnormal keratinocyte nuclear morphology induced by progerin in transgenic mice was improved by partial pharmacological inhibition of protein prenylation.

Figure 1.

Figure 1

Open in a new tab

Systemic administration of pravastatin plus zoledronate or FTI-276 inhibits protein prenylation in mouse skin and appears to improve abnormal nuclear morphology in mice expressing progerin. (A) Immunoblots showing farnesylated (f-HDJ-2) and non-farnesylated (nf-HDJ-2) HDJ-2 and β-actin (Actin) loading control in protein extracts from keratinocytes of transgenic mice expressing progerin (K14-progerin, showing proteins from two mice for each drug treatment and one mouse for PBS control injection) or wild type human lamin A (K14-lamin A, showing proteins from two mice for each drug treatment) in epidermis. Plus (+) or minus (−−) above each lanes indicates if sample from mouse that received intraperitoneal pravastatin plus zoledronate (IP P + Z) or intraperitoneal FTI-276 (IP FTI). Only one protein sample is shown for mice receiving no drugs (K14-progerin-/-), as no change in HDJ-2 migration would be expected; two protein samples from two mice injected with each drug treatment are shown to demonstrate small variations of the treatment in different mice. (B) Confocal immunofluorescence micrographs of dorsal skin sections from transgenic mice expressing FLAG-progerin (K14-progerin, a–c) or FLAG-wild type human lamin A (K14-lamin A, d–f). Skin samples were from mice injected with PBS (a and d), FTI-276 (b and e) or pravastatin plus zoledronate (c and f). Sections were labeled with anti-FLAG primary antibody and fluorescein isothiocyanate-conjugated secondary antibody. Bar: 10 µm.

Systemic administration of FTI or statin plus aminobisphosphonate improves abnormal nuclear morphology on electronic micrographs from mouse skin keratinocytes expressing progerin.

Our results obtained at the light microscopic level suggesting that systemic administration of drugs that inhibited protein farnesylation improved nuclear morphology in an intact tissue promoted us to carry out a higher resolution analysis of nuclear morphology using electron microscopy. As we have shown previously,37 transgenic mice expressing progerin in epidermis that were administered PBS had marked abnormal keratinocyte nuclear morphology compared to those expressing wild-type human lamin A or non-transgenic littermates of the progerin-expressing mice (Fig. 2). Keratinocyte nuclei in mice expressing progerin administered PBS appeared less round and had increased surface area with multiple lobulations or irregular extensions, deep invaginations of the nuclear envelope and apparent nuclear fragmentation, realizing that a deep invagination could appear to be fragmentation if the invagination of the nuclear envelope occurred perpendicularly to the skin section (Fig. 2A). In contrast, keratinocyte nuclei in mice expressing progerin but treated with FTI-276 (Fig. 2B) or pravastatin plus zoledronate (Fig. 2C) were much more elliptical with decreased surface area; far few nuclei had lobulations, extensions, invaginations or apparent fragmentation. Keratinocyte nuclei from progerin-expressing mice treated with FTI-276 or pravastatin plus zoledronate appeared more similar to those in mice expressing wild-type human lamin A (Fig. 2D–F) and non-transgenic, wild type littermate controls (Fig. 2G–I). Treatment with FTI-276 or pravastatin plus zoledronate had no effect on nuclear shape in mice expressing human wild type lamin A or non-transgenic mice (Fig. 2D–I).

Figure 2.

Figure 2

Open in a new tab

Representative electron micrographs of keratinocyte nuclei in skin sections from transgenic mice expressing progerin, wild type human lamin A and non-transgenic wild type littermate controls administered PBS (PBS), FTI-276 (FTI) or pravastatin plus zoledronate (P + Z). Nuclei of skin keratinocytes in mice expressing progerin (K14-progerin) administered PBS (A) had increased surface area with multiple lobulations or irregular extensions, deep invaginations (black arrow) of the nuclear envelope or apparent nuclear fragmentation(white arrowhead), realizing that a deep invagination could appear to be fragmentation if the invagination of the nuclear envelope occurred perpendicularly to the skin section. Keratinocyte nuclei in mice expressing progerin (K14-progerin) treated with FTI (B) or P + Z (C) were much more elliptical and few nuclei had lobulations, extensions, invaginations or apparent fragmentation. Nuclei of mice expressing wild-type human lamin A (K14-lamin A) administered PBS (D), FTI (E) or P + Z (F) as well as of non-transgenic, wild-type littermate controls (WT littermate) administered PBS (G), FTI (H) or P + Z (I) were also mostly elliptical and few nuclei had lobulations, extensions, invaginations or apparent fragmentation. Bar: 5 µm.

To quantify the effects of treatment with a FTI or a statin plus an aminobisphosphonate on keratinocyte nuclear morphology, we established a scoring system classifying nuclei into three groups: (1) normal or with slight invaginations, (2) mild or medium abnormal with a few (1–2) deep invaginations and (3) abnormal with many (3 or more) deep invaginations and/or apparent nuclear fragmentation. Using this scoring system, an experienced cell biologist unaware of genotype or drug treatment analyzed at least 150 nuclei from 4 mice of each genotype (transgenic expressing progerin, transgenic expressing wild-type human lamin A or non-transgenic wild type) and receiving each treatment (PBS, pravastatin plus zoledronate or FTI-276). In mice expressing progerin that received PBS, the percentage of nuclei of normal shape was 37% but significantly increased to 60% when these mice were treated with FTI-276 and 62% with pravastatin plus zoledronate (Fig. 3). In mice expressing wild-type human lamin A or non-transgenic wild-type controls administered PBS, approximately 80% of keratinocyte nuclei were of normal shape and administration of FTI-276 or pravastatin plus zoledronate had no significant effect (Fig. 3). Hence, partial inhibition of protein prenylation in keratinoctyes of progerin-expressing mice treated with a FTI or statin plus aminobisphosphonate resulted in significant improvement in nuclear morphology but there were still less morphologically normal nuclei than in non-transgenic littermates or transgenic mice expressing wild type human lamin A.

Figure 3.

Figure 3

Open in a new tab

Quantification of effects of treatment with PBS, FTI-276 (FTI) or a statin plus an aminobisphosphonate (P + Z) on keratinocyte nuclear morphology. Nuclear morphology was scored as three groups: (1) normal or with slight invaginations (white bars), (2) mild or medium abnormal with a few [1–2] deep invaginations (gray bars) and (3) abnormal with many [3 or more] deep invaginations and/or apparent nuclear fragmentation (black bars). Top part shows results from transgenic mice expressing progerin in keratinocytes (K14-progerin), middle part results from transgenic mice expressing wild-type human lamin A (K14-lamin A) and bottom part from non-transgenic wild type littermates of the progerin expressing mice (WT littermates). Number of nuclei scored from n = 4 mice for each genotype and treatment group is indicated above the bars. In top part, p-values for statistical comparisons between groups indicated are shown.

Systemic administration of FTI or statin plus aminobisphosphonate increases nuclear circularity in mouse skin keratinocytes expressing progerin.

Goldman et al.22 originally reported that cells expressing progerin have abnormal nuclear circularity/roundness or contour ratio (4π × area/perimeter2). Similarly, we have previously reported significantly decreased nuclear circularity in keratinocytes of the transgenic mice expressing progerin in epidermis.37 We used nuclear circularity as a parameter to further quantitatively assess the effects of a FTI or statin plus aminobisphosphonate on nuclear morphology of keratinocytes expressing progerin in intact epidermis. Figure 4 shows the tracing nuclear periphery for measurement of the circularity. Circularity/contour ratio was measured for 125 to 282 keratinocyte nuclei from 4 mice of each genotype (transgenic expressing progerin, transgenic expressing wild-type human lamin A or non-transgenic wild type) and receiving each treatment (PBS, pravastatin plus zoledronate or FTI-276). In mice expressing progerin that received PBS, the mean nuclear circularity was 0.29 but significantly increased to 0.51 when these mice were treated with FTI-276 and 0.48 with pravastatin plus zoledronate (Fig. 5). In mice expressing wild-type human lamin A or non-transgenic wild type controls administered PBS, FTI-276 or pravastatin plus zoledronate, mean nuclear circularity ranged from 0.56 to 0.65, with drug treatment having no significant effect (Fig. 5). Hence, treatment with a FTI or a statin plus an aminobisphosphonate resulted in significant increases of the nuclear circularity in transgenic mice expressing progerin in epidermis. However, circularity of nuclei expressing progerin in mice treated with the prenylation inhibitors was not as high as nuclear circularity in mice expressing wild-type human lamin A or non-transgenic controls.

Figure 4.

Figure 4

Open in a new tab

Highlighting of nuclear periphery in electron micrographs showing that FTI-276 (FTI) and pravastatin plus zoledronate (P + Z) increases nuclear circularity of keratinocyte nuclei in skin sections from transgenic mice expressing progerin. Nuclei of skin keratinocytes in mice expressing progerin administered PBS (A, D and G) had decreased nuclear circularity compared to mice expressing progerin administered FTI (B, E and H) or P + Z (C, F and I). Contour ratios of nuclei shown are given in the bottom right of each part. Bar: 2 µm.

Figure 5.

Figure 5

Open in a new tab

Quantification of effects of treatment with PBS, FTI-276 (FTI) or a statin plus an aminobisphosphonate (P + Z) on keratinocyte nuclear circularity. Nuclear circularity was measured using Image J and expressed as averaged contour ratio (4π × area/perimeter2). Top part shows results from transgenic mice expressing progerin in keratinocytes (K14-progerin), middle part results from transgenic mice expressing wild type human lamin A (K14-lamin A) and bottom part from non-transgenic wild type littermates of the progerin expressing mice (WT littermates). Number of nuclei scored from n = 4 mice for each genotype and treatment group is indicated within the bars. In top part, p values for statistical comparisons between groups indicated are shown.

Discussion

Abnormal nuclear morphology is considered to be a hallmark of progerin-expressing cells in HGPS. In recent years, many studies have shown that treatment of cultured cells expressing progerin, as well as those with ZMPSTE24 deficiency that accumulate unprocessed prelamin A, with pharmacological inhibitors of protein farnesylation significantly reverse these nuclear morphological abnormalities.10,11,13,14,21,25–30,35–37 However, reversal of abnormal nuclear morphology in progerin-expressing cells has never been demonstrated in intact tissue from whole animals. Our current results show that systemic treatment of animals with pharmacological inhibitors of protein prenylation can indeed reverse progerin-induced abnormalities in nuclear structure in intact tissue. To the best of our knowledge, the only somewhat similar demonstration has been that of reversal of abnormal nuclear morphology in hepatocytes in intact liver of Zmpste24 null mice after treatment with a statin and an aminobisphosphonate.21 Until now, an in vivo reversal of abnormal nuclear morphology has not been demonstrated in any animal expressing progerin or any animal model of HGPS or restrictive dermopathy treated systemically with an FTI.

We used the same doses of pravastatin and zoledronate that Varela et al.21 used in their study to treat Zmpste24 null mice. We used a dose of FTI-276 that was 2.0–2.5% of that used as a lung cancer chemopreventive agent in model mice.41,42 Our goal was to avoid any potential toxicity by using a lower dose.43 Based on inhibition of HDJ-2 farnesylation, we achieved 15–20% inhibition of protein prenylation. We could not detect an accumulation of prelamin A in the epidermis of mice treated with these drugs using antibodies against lamin A and C (data not shown) because of the lower percentage and reduced stability, similar to what has been described in mice by Young et al.14 In mouse tissues, prelamin A has only been detected in tail using a prelamin A-specific antibody20 and in liver using anti-prelamin A or anti-lamin A/C antibodies.19 The level of inhibition of HDJ-2 farnesylation we obtained in epidermal keratinocytes was slightly lower than 20–50% inhibition of HDJ-2 farnesylation in tail extracts, a tissue similar to skin, reported by Fong et al.20 when using the FTI ABC-100 to obtain phenotypic improvement in Zmpste24 null mice. In liver, Yang et al.19 obtained approximately 70–80% inhibition of HDJ-2 farnesylation using ABC-100 to reverse the progeroid phenotype of mice with a targeted HGPS mutation in Lmna. Capell et al.44 used the FTI tipifarnib to ameliorate vascular smooth muscle abnormalities in mice carrying a bacterial artificial chromosome with a human HGPS LMNA mutation and observed 16–53% inhibition of HDJ-2 farnesylation at a dose of 150 mg/kg/day and 45–85% inhibition of HDJ-2 farnesylation at a dose of 450 mg/kg/day in liver. With the dose of FTI-276 we used in the present study, we did not observe any apparent adverse effects in mice. Very high levels of inhibition of protein farnesyltransferase in keratinocytes would likely have detrimental effects in epidermis, as mice with keratinocyte-specific deletion of the β-subunit of protein farnesyltransferase develop severe alopecia with morphologically abnormal hair follicles containing many apoptotic cells.45 Moreover, high levels of inhibition of protein farnesylation may be detrimental in other ways; for example, recent studies have shown that failure to convert prelamin A to lamin A can cause cardiomyopathy in mice.46

There are currently ongoing clinical trials of a FTI and a statin plus aminobisphosphonate in children with HGPS.39,40 Appropriate clinical endpoints for these open labeled trials have been a subject of debate.47 Given the small numbers of subjects, open label design, variable age of entry and uncertain significance of the clinical parameters being assessed, these trials may not provide sufficient conclusive data about the efficacy of protein prenylation inhibitors in HGPS. Our results suggest that skin biopsy and examination of nuclear morphology by electron microscopy before and after initiation of treatment could be used as a measure of efficacy at least at the cellular level. Results from even a few children willing to consent to a minimally invasive procedure could be enlightening.

Finally, our results raise the question as to whether observed abnormal nuclear envelope morphology is actually responsible for the disease phenotypes in HGPS and other laminopathies. The transgenic mice used in this study have essentially normal skin integrity and function,37 so the reversal of abnormal nuclear morphology in keratinocytes obtained by treatment with protein prenylation inhibitors did not correlate with improvement in a higher level tissue or organ abnormality. It is possible that factors other than altered nuclear architecture, such as genetic background or protein expression levels, play a role in generating the skin abnormalities, including alopecia, in mice expressing progerin. This is suggested by the finding that the mice used in the current study, mice with a targeted HGPS mutation in Lmna19 and transgenic mice carrying a human bacterial artificial chromosome containing a mutant LMNA leading to progerin expression48 do not have alopecia, a profound epidermal abnormality, whereas one strain of transgenic expressing progerin in keratinocytes does have abnormal epidermal phenotypes.49,50 Except for the mice used in the present study, keratinocyte nuclear morphology has not been reported in the other genetically modified mice. That abnormal nuclear architecture is not the only factor that determines tissue disease in HGPS and other laminopathies is also suggested by the finding that hepatocyte nuclei have abnormal morphology in Zmpste24 null21 and Lmna null mice51 but no liver abnormalities have been reported. In addition, treatment with a FTI, while significantly reversing abnormal shape of nuclei in cells from human subjects with HGPS and restrictive dermopathy, does not result in reduction of DNA double-strand breaks and damage checkpoint signaling.35 Mice with a targeted Lmna mutation that leads to the direct synthesis of lamin A, bypassing prelamin A processing, also have misshapen fibroblast nuclei but no apparent pathology.52 Despite this unresolved issue regarding the relationship of abnormal nuclear morphology to pathology, our present results further demonstrate that abnormal nuclear morphology results from progerin expression and that the effects of systemically administered protein prenylation inhibitors on this cellular phenotype of HGPS can be assessed in situ in intact tissues.

Materials and Methods

Transgenic mice and genotyping.

Transgenic mice expressing human wild type lamin A or progerin with FLAG epitope tags at their amino-termini in epidermal keratinocytes have been described previously.37 Genotypes of mice used in all experiments were confirmed by PCR using specific primers.37 Transgene expression was also confirmed by real-time RT-PCR with specific primers using total RNA from epidermal keratinocytes and by immunoblotting.37

Systemic drug treatment of mice.

Four mice from each genotype were injected intraperitoneally with a combination of pravastatin (100 mg/kg/d) and zoledronate (100 µg/kg/d) dissolved in phosphate buffered saline (PBS), FTI-276 (1 mg/kg/d) dissolved in PBS or placebo (an equal volume of PBS) daily for 30 days. Animals were euthanized 30 days after starting treatment at an age of 8 weeks. After sacrificing mice, dorsal skin samples were harvested. Portions of skin samples were fixed for electron microscopy or embedded in O.C.T. medium for immunohistofluorescence analysis.

Protein isolation, electrophoresis and immunoblotting.

Keratinocytes were isolated from mouse skin as described previously.37 Proteins were extracted from keratinocytes in urea isolation buffer, separated in SDS-polyacrylamide gels, transferred on nitrocellulose membranes and analyzed by immunoblotting as described by Fong et al.20 Primary antibodies used for immunoblotting were mouse anti-β-actin (Santa Cruz) at 1:5,000 dilution and mouse anti-HDJ-2 (Neo Markers) at 1:500 dilution. Secondary antibodies were ECL-horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare) used at 1:5,000 dilution. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce) and X-ray film (Kodak). To estimate amount of non-farnesylated compared to farnesylated HD2-J, blots were analyzed using Scion Image and the percentage of inhibited HDJ-2 farnesylation was expressed as a ratio of non-farnesylated HDJ-2 to total HDJ-2 (non-farnesylated plus farnesylated HDJ-2).

Immunofluorescence microscopy.

Dorsal skin samples from mice were embedded in Tissue-Tek O.C.T. medium for immunohistofluorescence microscopy. Embedded blocks were sectioned at 6 µm thickness, collected on silane-coated slides and fixed in acetone at −20°C for 10 min after air-drying. Slides were incubated with primary antibodies at room temperature for 1.5 h. Primary antibodies were mouse anti-FLAG at 1:200 dilution and rabbit anti-keratin 14 AF64 (Covance Research Products) at 1:1,000 dilution. The slides were then washed 3 times with PBS and incubated for 30 min in a dark box with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG) and Rhodamine Red™-X-conjugated goat anti-rabbit IgG (Invitrogen) diluted at 1:200. After washing 3 more times in PBS, coverslips were mounted with gel/mount™ (Biomeda) and sealed with clear nail polish. Immunofluorescence microscopy was performed on a LSM 510 META confocal laser scanning system attached to an Axiovert 200 inverted microscope (Carl Zeiss). Images were processed using Photoshop software (Adobe) on a Macintosh G4 computer (Apple Computer).

Electron microscopy and “scoring” of nuclear morphology.

Mouse dorsal skin samples were fixed in 2.5% glutaraldehyde in 0.1 M Sorenson's buffer (pH 7.2) for electron microscopy as previously reported.37 Each skin sample from 36 different mice was processed for examination. Samples were examined using a JEOL JEM-1200 EXII transmission electron microscope and micrographs taken using an ORCA-HR digital camera (Hamamatsu) as previously described.53 All intact nuclei at outer root sheaths of hair follicles within each electron microscopic grid were photographed at 5,000X, 10,000X and 20,000X magnifications. Nuclei in micrographs of each sample were “scored” as belonging to 3 groups depending on the nuclear morphology: (1) normal or with slight nuclear envelope invaginations (length less than ¼ nuclear diameter), (2) mild or medium abnormal with a few (1–2) deep nuclear envelope invaginations (length equal or greater than ¼ nuclear diameter) and (3) abnormal with many (3 or more) deep nuclear envelope invaginations and/or apparent nuclear fragmentation. A blinded independent observer with experience in cell biology (C.Ö.) who was unaware of the genotypes or drug treatments scored the morphology of nuclei. Small nuclei in the micrographs that appeared to be sectioned close to the nuclear surface were not included in the scoring. For estimating nuclear circularity, a blinded observer selected nuclear micrographs at 10,000X magnification in which the keratinocytes appeared to be sectioned close to the center of a nucleus; these micrographs were compared to micrographs of the same field at 5,000X to make sure all were examined and that none were examined twice. Nuclear circularity was expressed as an average contour ratio of one nucleus from 3 repeat measurements performed on each micrograph using ImageJ 1.37v (National Institutes of Health). Means, standard deviations and significances in nuclear morphology and circularity among the different genotypes and treatment groups were calculated using 1-tailed Student's t-tests with unequal variance using Excel (Microsoft). For demonstration purposes (Fig. 4), we traced the nuclear periphery in electron micrographs using the “scribble” command on PowerPoint (Microsoft).

Acknowledgements

We thank Loren G. Fong (University of California at Los Angeles) sharing information on isolation and detection of prenylated proteins and Kristy Brown (Columbia University) for assistance with electron microscopy. The work was supported by NIH grants AG025240 and NS059352.

Abbreviations

HGPS

Hutchinson-Gilford progeria syndrome

FTI

protein farnesyltransferase inhibitor

PBS

phosphate-buffered saline

IgG

immunoglobulin G

Footnotes

References

  • 1.DeBusk FL. The Hutchinson-Gilford progeria syndrome. J Pediatr. 1972;80:697–724. doi: 10.1016/s0022-3476(72)80229-4. [DOI] [PubMed] [Google Scholar]
  • 2.Merideth MA, Gordon LB, Clauss S, Sachdev V, Smith AC, Perry MB, et al. Phenotype and course of Hutchinson-Gilford progeria syndrome. N Engl J Med. 2008;358:592–604. doi: 10.1056/NEJMoa0706898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Eriksson M, Brown WT, Gordon LB, Glynn MW, Singer J, Scott L, et al. Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature. 2003;423:293–298. doi: 10.1038/nature01629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.De Sandre-Giovannoli A, Bernard R, Cau P, Navarro C, Amiel J, Boccaccio I, et al. Lamin A truncation in Hutchinson-Gilford progeria. Science. 2003;300:2055. doi: 10.1126/science.1084125. [DOI] [PubMed] [Google Scholar]
  • 5.Mckeon FD, Kirschner MW, Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986;319:463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  • 6.Aebi U, Cohn J, Buhle L, Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986;323:560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
  • 7.Goldman AE, Maul G, Steinert PM, Yang HY, Goldman RD. Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins. Proc Natl Acad Sci USA. 1986;83:3839–3843. doi: 10.1073/pnas.83.11.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fisher DZ, Chaudhary N, Blobel G. cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci USA. 1986;83:6450–6454. doi: 10.1073/pnas.83.17.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lin F, Worman HJ. Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C. J Biol Chem. 1993;268:16321–16326. [PubMed] [Google Scholar]
  • 10.Worman HJ, Bonne G. “Laminopathies”: a wide spectrum of human diseases. Exp Cell Res. 2007;313:2121–2133. doi: 10.1016/j.yexcr.2007.03.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Worman HJ, Fong LG, Muchir A, Young SG. Laminopathies and the long strange trip from basic cell biology to therapy. J Clin Invest. 2009;119:1825–1836. doi: 10.1172/JCI37679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sinensky M, Fantle K, Trujillo M, McLain T, Kupfer A, Dalton MJ. The processing pathway of prelamin A. J Cell Sci. 1994;107:61–67. doi: 10.1242/jcs.107.1.61. [DOI] [PubMed] [Google Scholar]
  • 13.Rusiñol AE, Sinensky M. Farnesylated lamins, progeroid syndromes and farnesyl transferase inhibitors. J Cell Sci. 2006;119:3265–3272. doi: 10.1242/jcs.03156. [DOI] [PubMed] [Google Scholar]
  • 14.Young SG, Meta M, Yang SH, Fong LG. Prelamin A farnesylation and progeroid syndromes. J Biol Chem. 2006;281:39741–39745. doi: 10.1074/jbc.R600033200. [DOI] [PubMed] [Google Scholar]
  • 15.Moulson CL, Go G, Gardner JM, van der Wal AC, Smitt JH, van Hagen JM, et al. Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy. J Invest Dermatol. 2005;125:913–919. doi: 10.1111/j.0022-202X.2005.23846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Navarro CL, Cadiñanos J, De Sandre-Giovannoli A, Bernard R, Courrier S, Boccaccio I, et al. Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of lamin A precursors. Hum Mol Genet. 2005;14:1503–1513. doi: 10.1093/hmg/ddi159. [DOI] [PubMed] [Google Scholar]
  • 17.Bergo MO, Gavino B, Ross J, Schmidt WK, Hong C, Kendall LV, et al. Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness and a prelamin A processing defect. Proc Natl Acad Sci USA. 2002;99:13049–13054. doi: 10.1073/pnas.192460799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pendás AM, Zhou Z, Cadiñanos J, Freije JM, Wang J, Hultenby K, et al. Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice. Nat Genet. 2002;31:94–99. doi: 10.1038/ng871. [DOI] [PubMed] [Google Scholar]
  • 19.Yang SH, Meta M, Qiao X, Frost D, Bauch J, Coffinier C, et al. A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation. J Clin Invest. 2006;116:2115–2121. doi: 10.1172/JCI28968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fong LG, Frost D, Meta M, Qiao X, Yang SH, Coffinier C, et al. A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria. Science. 2006;311:1621–1623. doi: 10.1126/science.1124875. [DOI] [PubMed] [Google Scholar]
  • 21.Varela I, Pereira S, Ugalde AP, Navarro CL, Suárez MF, Cau P, et al. Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging. Nat Med. 2008;14:767–772. doi: 10.1038/nm1786. [DOI] [PubMed] [Google Scholar]
  • 22.Goldman RD, Shumaker DK, Erdos MR, Eriksson M, Goldman AE, Gordon LB, et al. Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchison-Gilford progeria syndrome. Proc Natl Acad Sci USA. 2004;101:8963–8968. doi: 10.1073/pnas.0402943101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Fong LG, Ng JK, Meta M, Coté N, Yang SH, Stewart CL, et al. Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice. Proc Natl Acad Sci USA. 2004;101:18111–18116. doi: 10.1073/pnas.0408558102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Bridger JM, Kill IR. Aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis. Exp Gerontol. 2004;39:717–724. doi: 10.1016/j.exger.2004.02.002. [DOI] [PubMed] [Google Scholar]
  • 25.Yang SH, Bergo MO, Toth JI, Qiao X, Hu Y, Sandoval S, et al. Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation. Proc Natl Acad Sci USA. 2005;102:10291–10296. doi: 10.1073/pnas.0504641102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Toth JI, Yang SH, Qiao X, Beigneux AP, Gelb MH, Moulson CL, et al. Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes. Proc Natl Acad Sci USA. 2005;102:12873–12878. doi: 10.1073/pnas.0505767102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Capell BC, Erdos MR, Madigan JP, Fiordalisi JJ, Varga R, Conneely KN, et al. Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci USA. 2005;102:12879–12884. doi: 10.1073/pnas.0506001102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Mallampalli MP, Huyer G, Bendale P, Gelb MH, Michaelis S. Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci USA. 2005;102:14416–14421. doi: 10.1073/pnas.0503712102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.McClintock D, Gordon LB, Djabali K. Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody. Proc Natl Acad Sci USA. 2006;103:2154–2159. doi: 10.1073/pnas.0511133103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Glynn MW, Glover TW. Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Hum Mol Genet. 2005;14:2959–2969. doi: 10.1093/hmg/ddi326. [DOI] [PubMed] [Google Scholar]
  • 31.Scaffidi P, Misteli T. Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat Med. 2005;11:440–445. doi: 10.1038/nm1204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Paradisi M, McClintock D, Boguslavsky RL, Pedicelli C, Worman HJ, Djabali K. Dermal fibroblasts in Hutchinson-Gilford progeria syndrome with the lamin A G608G mutation have dysmorphic nuclei and are hypersensitive to heat stress. BMC Cell Biol. 2005;6:27. doi: 10.1186/1471-2121-6-27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Huang S, Chen L, Libina N, Janes J, Martin GM, Campisi J, et al. Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference. Hum Genet. 2005;118:444–450. doi: 10.1007/s00439-005-0051-7. [DOI] [PubMed] [Google Scholar]
  • 34.Fong LG, Ng JK, Lammerding J, Vickers TA, Meta M, Coté N, et al. Prelamin A and lamin A appear to be dispensable in the nuclear lamina. J Clin Invest. 2006;116:743–752. doi: 10.1172/JCI27125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Liu Y, Rusiñol A, Sinensky M, Wang Y, Zou Y. DNA damage responses in progeroid syndromes arise from defective maturation of prelamin A. J Cell Sci. 2006;119:4644–4649. doi: 10.1242/jcs.03263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Verstraeten VL, Ji JY, Cummings KS, Lee RT, Lammerding J. Increased mechanosensitivity and nuclear stiffness in Hutchinson-Gilford progeria cells: effects of farnesyltransferase inhibitors. Aging Cell. 2008;7:383–393. doi: 10.1111/j.1474-9726.2008.00382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wang Y, Panteleyev AA, Owens DM, Djabali K, Stewart CL, Worman HJ. Epidermal expression of the truncated prelamin A causing Hutchinson-Gilford progeria syndrome: effects on keratinocytes, hair and skin. Hum Mol Genet. 2008;17:2357–2369. doi: 10.1093/hmg/ddn136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Fong LG, Vickers TA, Farber EA, Choi C, Yun UJ, Hu Y, et al. Activating the synthesis of progerin, the mutant prelamin A in Hutchinson-Gilford progeria syndrome, with antisense oligonucleotides. Hum Mol Genet. 2009;18:2462–2471. doi: 10.1093/hmg/ddp184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Gordon LB, Harling-Berg CJ, Rothman FG. Highlights of the 2007 Progeria Research Foundation scientific workshop: progress in translational science. J Gerontol A Biol Sci Med Sci. 2008;63:777–787. doi: 10.1093/gerona/63.8.777. [DOI] [PubMed] [Google Scholar]
  • 40.Pereira S, Bourgeois P, Navarro C, Esteves-Vieira V, Cau P, De Sandre-Giovannoli A, et al. HGPS and related premature aging disorders: from genomic identification to the first therapeutic approaches. Mech Ageing Dev. 2008;129:449–459. doi: 10.1016/j.mad.2008.04.003. [DOI] [PubMed] [Google Scholar]
  • 41.Sun J, Qian Y, Hamilton AD, Sebti SM. Ras CAAX peptidomimetric FTI 276 selectively blocks tumor growth in nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion. Cancer Res. 1995;55:4243–4247. [PubMed] [Google Scholar]
  • 42.Zhang Z, Wang Y, Lantry LE, Kastens E, Liu G, Hamilton AD, et al. Farnesyltransferase inhibitors are potent lung cancer chemopreventive agents in A/J mice with a dominant-negative p53 and/or heterozygous deletion of Ink4a/Arf. Oncogene. 2003;22:6257–6265. doi: 10.1038/sj.onc.1206630. [DOI] [PubMed] [Google Scholar]
  • 43.Konstantinopoulos PA, Papavassiliou AG. Multilevel modulation of the mevalonate and protein-prenylation circuitries as a novel strategy for anticancer therapy. Trends Pharmacol Sci. 2007;28:6–13. doi: 10.1016/j.tips.2006.11.005. [DOI] [PubMed] [Google Scholar]
  • 44.Capell BC, Olive M, Erdos MR, Cao K, Faddah A, Tavarez UL, et al. A farnesyltransferase inhibitor prevents both the onset and late progression of cardiovascular disease in a progeria mouse model. Proc Natl Acad Sci USA. 2008;105:15902–15907. doi: 10.1073/pnas.0807840105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Lee R, Chang SY, Trinh H, Tu Y, White AC, Davies BS, et al. Genetic studies on the functional relevance of the protein prenyltransferases in skin keratinocytes. Hum Mol Genet. 2010;19:1603–1617. doi: 10.1093/hmg/ddq036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Davies BS, Barnes RH, 2nd, Tu Y, Ren S, Andres DA, Spielmann P, et al. An accumulation of non-farnesylated prelamin A causes cardiomyopathy but not progeria. Hum Mol Genet. 2010;19:2682–2694. doi: 10.1093/hmg/ddq158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kieran MW, Gordon L, Kleinman M. New approaches to progeria. Pediatrics. 2007;120:834–841. doi: 10.1542/peds.2007-1356. [DOI] [PubMed] [Google Scholar]
  • 48.Varga R, Eriksson M, Erdos MR, Olive M, Harten I, Kolodgie F, et al. Progressive vascular smooth muscle cell defects in a mouse model of Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci USA. 2006;103:3250–3255. doi: 10.1073/pnas.0600012103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Sagelius H, Rosengardten Y, Hanif M, Erdos MR, Rozell B, Collins FS, et al. Targeted transgenic expression of the mutation causing Hutchinson-Gilford progeria syndrome leads to proliferative and degenerative epidermal disease. J Cell Sci. 2008;121:969–978. doi: 10.1242/jcs.022913. [DOI] [PubMed] [Google Scholar]
  • 50.Sagelius H, Rosengardten Y, Schmidt E, Sonnabend C, Rozell B, Eriksson M. Reversible phenotype in a mouse model of Hutchinson-Gilford progeria syndrome. J Med Genet. 2008;45:794–801. doi: 10.1136/jmg.2008.060772. [DOI] [PubMed] [Google Scholar]
  • 51.Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, et al. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol. 1999;147:913–920. doi: 10.1083/jcb.147.5.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Coffinier C, Jung HJ, Li Z, Nobumori C, Yun UJ, Farber EA, et al. Direct synthesis of lamin A, bypassing prelamin A processing, causes misshapen Nuclei in fibroblasts but No detectable pathology in mice. J Biol Chem. 2010;285:20818–20826. doi: 10.1074/jbc.M110.128835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Wang Y, Herron AJ, Worman HJ. Pathology and nuclear abnormalities in hearts of transgenic mice expressing M371K lamin A encoded by an LMNA mutation causing Emery-Dreifuss muscular dystrophy. Hum Mol Genet. 2006;15:2479–2489. doi: 10.1093/hmg/ddl170. [DOI] [PubMed] [Google Scholar]

Articles from Nucleus are provided here courtesy of Taylor & Francis

RESOURCES