HGPS (Hutchinson–Gilford progeria syndrome) is a rare genetic disease affecting children causing them to age and die prematurely. The disease is typically due to a point mutation in the coding sequence for the nuclear intermediate-type filament protein lamin A and gives rise to a dominant-negative splice variant named progerin. Accumulation of progerin within nuclei causes disruption to nuclear structure, causes and premature replicative senescence and increases apoptosis. Now it appears that accumulation of progerin may have more widespread effects than previously thought since the demonstration that the presence and distribution of some nucleolar proteins are also adversely affected in progeria cells. One of the major breakthroughs both in the lamin field and for this syndrome is that many of the cellular defects observed in HGPS patient cells and model systems can be restored after treatment with a class of compounds known as FTIs (farnesyltransferase inhibitors). Indeed, it is demonstrated that FTI-277 is able to completely restore nucleolar antigen localization in treated progeria cells. This is encouraging news for the HGPS patients who are currently undergoing clinical trials with FTI treatment.
- farnesyltransferase I inhibitor
- Hutchinson–Gilford progeria syndrome (HGPS)
HGPS (Hutchinson–Gilford progeria syndrome)
HGPS is an extremely rare disorder that affects children, causing them to age prematurely. Clinical features of this disease include alopecia, growth retardation, an aged appearance, loss of subcutaneous fat, progressive atherosclerosis, bone deformities and cardiovascular diseases . HGPS is typically caused by an autosomal dominant de novo mutation in the LMNA gene that encodes the nuclear intermediate filament proteins lamins A and C [2,3], both of which are components of the nuclear lamina (a structure that has roles in DNA replication, transcription, chromatin organization and remodelling, maintenance of nuclear shape and integrity and cell division) . The most common mutation associated with HGPS is a single base-substitution in codon 608 of exon 11 on the LMNA gene, which results in the formation of a cryptic splice site producing a truncated prelamin A protein (progerin) that lacks 50 amino acids within the C-terminus end. Progerin acts in a dominant-negative manner regardless of whether its expression occurs naturally through alternative splicing or it is activated using anti-sense oligonucleotides . This single base-pair mutation not only leads to disruption of the nuclear lamina and nuclear shape but also has perhaps more clinically relevant effects on major processes taking place in the cell nucleus, such as the epigenetic control of chromatin, gene expression, cell division, interphase chromosome positioning, DNA replication and the DNA damage response [4,6,7].
In normal cells, prelamin A, a precursor protein from which mature lamin A is derived, contains a CaaX (where a is an aliphatic residue) motif at the C-terminal end that signals farnesylation of the cysteine residue by the enzyme farnesyltransferase . The presence of a farnesyl group at the C-terminal end along with the CaaX motif promotes the association of prelamin A with the nuclear membrane and is needed for correct localization of the mature protein . The protein then loses the three terminal aaX amino acids followed by methyl esterification of the C-terminal cysteine residue. Finally, the protein undergoes endoproteolytic cleavage by an enzyme ZMPSTE24 (FACE1) metalloproteinase , resulting in a loss of 15 amino acids at the C-terminal end, including the farnesylated cysteine, thus generating mature lamin A protein . The final step of this post-transcriptional modification process whereby the farnesyl group is cleaved is thought to be important for insertion of the mature lamin A into the nuclear lamina . In progerin, the deletion of 50 amino acids within the C-terminus does not affect the CaaX motif and the protein undergoes normal farnesylation. However, the endoproteolytic site normally cleaved by ZMPSTE24 is missing and hence progerin remains permanently farnesylated . Retention of the farnesyl group and accumulation of the farnesylated protein at the nuclear envelope are thought to compromise nuclear integrity, disrupt the nuclear lamina including the underlying heterochromatin and lead to the formation of abnormally shaped nuclei, a prominent characteristic seen in HGPS [14,15]. Thus a concept that blocking farnesylation of progerin might help ameliorate disease pathology seen in HGPS cells was put forward in 2003, shortly after the discovery of the gene involved in causing HGPS. To test this hypothesis, a class of drugs called FTIs (farnesyltransferase inhibitors) were used. FTIs inhibit attachment of a farnesyl group to proteins by irreversibly binding to the CaaX domain .
FTI treatment for HGPS patients
Encouragingly, the first few studies demonstrated that treating HGPS patient cells in culture with FTI prevented an accumulation of progerin at the nuclear envelope and reduced the frequency of abnormally shaped nuclei [12,17–20]. This finding was also demonstrated in HeLa HGPS model cells  and in culture in fibroblasts derived from a mouse model . A study by Glynn and Glover  demonstrated an improved morphology and a reduction in percentage of abnormal nuclei, by 70% in HGPS cells after treatment with low doses of FTIs (10 nM), over a 14 day period. Significant dose-dependent reduction in nuclear blebbing, as well as redistribution of mutant protein, was reported within 48–72 h of FTI treatment on HGPS cells , ZMPSTE24−/− mouse embryonic fibroblasts  and LmnaHG/+ and LmnaHG/HG mouse fibroblasts . HGPS cells treated with FTIs for 72 h also show improved nuclear stiffness to levels almost comparable with normal cells and significant restoration of directional persistence with regard to cell migration and thus improvements in wound healing ability . However, FTI treatment was unable to restore the DNA damage response mechanism, which is affected in HGPS cells .
With promising results from in vitro studies with regard to the ability of FTIs to reverse nuclear abnormalities, various laboratories then focused on animal models of HGPS to test these drugs further. The most common models available for progeria are ZMPSTE24−/− and LmnaHG/+ mouse models. Treatment of ZMPSTE24−/− mice with FTI, beginning at 5 weeks of age, showed the presence of non-farnesylated prelamin A, improved growth and survival rates, better bone integrity and increased grip strength . In LmnaHG/+ mice, also treated with FTIs, there is an increase in survival rates, an increased body weight (adipose) and reduced rib fractures [24,25]. A more recent study that uses a transgenic mouse model carrying the human G608G LMNA mutation and displaying a cardiovascular phenotype, a characteristic reason that causes death in most HGPS patients, demonstrated that FTI treatment reduces VSMC (vascular smooth-muscle cell) loss and proteoglycan accumulation and thus retards the onset, as well as progression, of cardiovascular diseases in these mice .
Nucleoli in HGPS cells before and after FTI treatment
The studies have so far been concentrated on the defects of the nuclear lamina and functions related to the lamina in HGPS cells. Since lamin A is not only present at the nuclear lamina but is also a component of the nuclear matrix [29–31] and is found within the nuclear interior , with sites of replication , transcription factories  and splicing speckles , mutations in the LMNA gene might affect other nuclear structures and bodies. In particular, we wondered whether impaired lamin A function may affect the structure and/or functions of the nucleolus. It has been demonstrated recently that lamin B1 does indeed play a role in nucleolar integrity .
The nucleolus is a membraneless, crucial nuclear compartment involved in ribosomal biogenesis. Ultrastructural analysis of nucleoli has defined three subcompartments: the FCs (fibrillar centres), the DFC (dense fibrillar component) and the granular component , each of which has distinct but related functions . Some nucleolar proteins are constrained within these nucleolar compartments; for example, fibrillarin and Ki67 are located in the DFC [39,40], nucleophosmin-B23 is localized in the granular component [41,42], and RNA polymerase I is localized within the FC [43,44]. Apart from transcription of rDNA (ribosomal DNA) genes and processing of pre-ribosomal particles, the nucleolus is also involved in many diverse fundamental cellular processes .
In order to question whether nucleolar structure was affected in HGPS cells, both normal and HGPS fibroblasts were stained with antibodies reacting with pKi67, nucleolin and fibrillarin. Each of these antigens has a distinct role in nucleolar function but all three occupy regions of the DFC. Ki67 is present only in proliferating cells and is therefore a robust marker of both proliferative state and nucleolar integrity . Nucleolin is involved in a number of processes including rDNA transcription, rRNA processing, nucleocytoplasmic transport and regulation of apoptosis . Fibrillarin is involved in pre-rRNA processing and ribosome assembly [47,48]. Representative images of staining patterns in normal and HGPS cells are shown in Figure 1. Ki67 staining in both control and HGPS cells appeared normal with typical staining patterns revealed in all Ki67-positive (proliferating) nuclei (Figures 1A, 1E, 1I, 1M, 1Q and 1U). In these Ki67-positive cells, staining for both fibrillarin (Figures 1B, 1F and 1J) and nucleolin (Figures 1N, 1R and 1V) appeared normal. However, in Ki67-negative cells (non-proliferating), staining patterns for fibrillarin in control cells were normal (Figure 1D), and in HGPS cells, they appeared abnormal with reduced intensity and lack of any punctate structure (Figure 1H). Similarly, staining for nucleolin in Ki67-negative cells was normal in control cells (Figure 1P) but abnormal in HGPS cells (Figure 1T). Tables 1 and 2 show the percentages of cells displaying typical, atypical and negative staining for fibrillarin and nucleolin in Ki67-positive and Ki67-negative cells for normal fibroblasts (2DD) and three HGPS fibroblast cultures. The table includes data for both early passage and late passage cultures, showing how the relative proportions of staining class change with progression of replicative senescence. We have previously shown that with an increase in cellular age, cellular abnormalities predominate within HGPS cultures . Indeed, atypical staining for both nucleolin and fibrillarin in Ki67-negative cells increases between early and late passage for all three HGPS cultures. Furthermore, the proportion of cells with an absence of nucleolin staining increases with cellular age in HGPS cultures, whereas the absence of nucleolin staining is never observed in control cultures. The total proportion of cells (Ki67-positive plus Ki67-negative) displaying typical nucleolar staining for both fibrillarin and nucleolin remains high in early and late passage control cells. In contrast, there is a dramatic decline in the proportion of typically stained cells in all three HGPS cultures with an increase in cellular age for both fibrillarin and nucleolin. Remarkably, the proportion of HGPS cells displaying typical nucleolar staining for both fibrillarin and nucleolin is partially or fully restored respectively after treatment for 48 h with 2.5 μM of the FTI, FTI-277 (Figures 1L and 1X).
It seems that accumulation of progerin in cells may have even more widespread consequences than already discussed in the literature. The observations reported here that certain aspects of nucleolar structure are also altered in HGPS cells may provide further insight into the cellular pathology of the disease. On a positive note, it appears that FTI treatment can effectively restore these nucleolar abnormalities, a finding that provides more encouragement for the efficacy of FTI treatments currently undergoing clinical trials on HGPS patients.
We thank the Brunel Progeria Research Fund for financial support of the present studies. I.S.M. is partially funded by an Overseas Research Student Award Scheme.
We thank Dr John Aris (University of Florida, Gainesville, FL, U.S.A.) for the monoclonal anti-fibrillarin antibody.
Nuclear Envelope Disease and Chromatin Organization 2009: Independent Meeting held at College of St Hild and St Bede, University of Durham, Durham, U.K., 22–23 April 2009. Organized and Edited by Chris Hutchison (Durham, U.K.).
Abbreviations: DFC, dense fibrillar component; FC, fibrillar centre; FTI, farnesyltransferase inhibitor; HGPS, Hutchinson–Gilford progeria syndrome; rDNA, ribosomal DNA
- © The Authors Journal compilation © 2010 Biochemical Society