Review

HGPS and related premature aging disorders: From genomic identificationto the first therapeutic approaches

Sandrine Pereira a, Patrice Bourgeois b, Claire Navarro a, Vera Esteves-Vieira b, Pierre Cau a,c,Annachiara De Sandre-Giovannoli a,b, Nicolas Levy a,b,*a INSERM U910, Faculte de Medecine la Timone, 27 Bd Jean Moulin, 13385 Marseille, Franceb Laboratory of Molecular Genetics, Department of Medical Genetics, Children’s Hospital la Timone, 264 Rue St Pierre, 13385 Marseille, Francec Laboratory of Cell Biology, Department of Medical Genetics, Children’s Hospital la Timone, 264 Rue St Pierre, 13385 Marseille, France

Mechanisms of Ageing and Development 129 (2008) 449–459

A R T I C L E I N F O

Article history:

Available online 12 April 2008

Keywords:

Progeria

Restrictive Dermopathy

Laminopathies

Premature ageing

Lamins

A B S T R A C T

Progeroid syndromes are heritable human disorders displaying features that recall premature ageing. In

these syndromes, premature aging is defined as ‘‘segmental’’ since only some of its features are

accelerated. A number of cellular biological pathways have been linked to aging, including regulation of

the insulin/growth hormone axis, pathways involving ROS metabolism, caloric restriction, and DNA

repair. The number of identified genes associated with progeroid syndromes has increased in recent

years, possibly shedding light as well on mechanisms underlying ageing in general. Among these,

premature aging syndromes related to alterations of the LMNA gene have recently been identified. This

review focuses on Hutchinson–Gilford Progeria syndrome and Restrictive Dermopathy, two well-

characterized Lamin-associated premature aging syndromes, pointing out the current knowledge

concerning their pathophysiology and the development of possible therapeutic approaches.

� 2008 Elsevier Ireland Ltd. All rights reserved.

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1. Progeroid syndromes and Lamin A-related progeroidsyndromes

Progeroid syndromes are heritable human disorders displayingfeatures that recall premature ageing. In these syndromes,premature aging is defined as ‘‘segmental’’ since only some ofits features are accelerated. A number of cellular biologicalpathways have been linked to aging, including regulation of theinsulin/growth hormone axis, pathways involving ROS metabo-lism, caloric restriction, and DNA repair. Different animal models,ranging from yeast, to nematodes, to mice, have been instrumentalin obtaining evidence for these connections (Hasty et al., 2003).

Several heritable premature aging syndromes have for a longtime been linked to defects in genome maintenance, due to alteredDNA repair mechanisms. These mainly include the followingautosomal recessive syndromes: (i) Werner syndrome, due tomutations in RecQL2 DNA helicase; (ii) Cockayne syndrome (CS)type A and B, due to mutations in the genes encoding the group 8 or6 excision-repair cross-complementing proteins (ERCC8 and

* Corresponding author at: Laboratory of Molecular Genetics, Department of

Medical Genetics, Children’s Hospital la Timone, 264 Rue St Pierre, 13385 Marseille,

France. Tel.: +33 491 38 77 87; fax: +33 491 38 46 76.

E-mail address: nicolas.levy@univmed.fr (N. Levy).

0047-6374/$ – see front matter � 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mad.2008.04.003

ERCC6), respectively; (iii) Rothmund–Thomson syndrome (RTS),due to RecQL4 mutations; (iv) trichothiodystrophy (TTD), due tomutations in the genes ERCC2/XPD and ERCC3/XPB, encoding thetwo helicase subunits of the transcription/repair factor TFIIH, aswell as in TFB5, encoding the tenth subunit of TFIIH (Giglia-Mariet al., 2004); (v) ataxia-telangiectasia, due to mutations in theataxia-telangiectasia mutated gene (ATM); (vi) xeroderma pig-mentosum (XP), a genetically heterogeneous autosomal recessivedisorder in which can be distinguished at least seven comple-mentation groups, due to mutations of different DNA excision-repair proteins (Hasty et al., 2003; Kipling et al., 2004). All theseprogeroid diseases, involving heritable defects in DNA repair,suggest a central role of genome integrity maintenance in theaging process.

The number of identified genes associated with progeroidsyndromes has increased in recent years, possibly shedding light aswell on mechanisms underlying ageing in general.

Among these, premature aging syndromes related to alterationsof the LMNA gene have recently been identified. LMNA encodesLamins A/C, ubiquitous nuclear proteins belonging to the inter-mediate filament superfamily. These premature aging disordershave thus been classified as ‘‘Laminopathies’’, the large group ofdiseases associated to Lamin A/C defects. This group of hetero-geneous disorders includes three main subgroups: (1) neuromus-cular disorders (Emery–Dreifuss muscular dystrophy, limb-girdle

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459450

muscular dystrophy type 1B, dilated cardiomyopathy type 1A,Charcot-Marie-Tooth peripheral polyneuropathy type 2B1 as wellas a phenotype mimicking autosomal dominant proximal spinalmuscular atrophy) (Rudnik-Schoneborn et al., 2007), (2) adiposetissue diseases (Dunnigan familial partial lipodystrophy, LDHCP),and (3) systemic diseases presenting with more or less pronouncedpremature aging features (mandibuloacral dysplasia type A and B(MAD-A/B), Hutchinson–Gilford Progeria Syndrome (HGPS),Restrictive Dermopathy (RD), atypical Werner syndrome) (forreview, see Broers et al., 2006; Navarro et al., 2006; Mattout et al.,2006; Rankin and Ellard, 2006; Worman and Bonne, 2007; Foisneret al., 2007; Vlcek and Foisner, 2007a).

In recent years, the secondary involvement of Lamin A/C in thepathogenesis of several other neuromuscular diseases, includingprimary torsion dystonia (McNaught et al., 2004), Facioscapulo-humeral muscular dystrophy (Masny et al., 2004) and fragile X-associated tremor/ataxia syndrome (Greco et al., 2006; Iwahashiet al., 2006) has been observed, further broadening the contribu-tion of these proteins to different diseases.

Fig. 1. Clinical and radiological findings in two patients affected with HGPS and one pa

contractures (A), aged-looking and transparent skin with prominent superficial vessels (

ankylosis, skin erosion at flexure sites (C), generalized osteopenia and clavicular hypop

The involvement of LMNA in Hutchinson–Gilford ProgeriaSyndrome, a very rare and uniformly fatal segmental progeroidsyndrome (Hennekam, 2006), was identified in 2003 (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). Mutations ofZMPSTE24, encoding an enzyme specifically involved in Lamin Apost-translational processing, have subsequently been linked toRestrictive Dermopathy, a neonatally lethal genodermatosis(Navarro et al., 2004, 2005), and mandibuloacral displasia(MAD) (Agarwal et al., 2003; Shackleton et al., 2005).

Since these discoveries, a lot of research has been done atclinical and basic research levels, identifying other prematureaging syndromes linked to primary or secondary Lamin A/Calteration and beginning to delineate the underlying molecularpathophysiological mechanisms.

Patients affected with rather severe Werner syndromes, butwithout a history of tumor development, carrying wild-typeRecQL2 sequences, were found to carry dominant LMNA mutationsmost often affecting both Lamin A and C isoforms (Chen et al.,2003; for review, see Kudlow et al., 2007), inscribing ‘‘atypical’’

tient affected with RD. (A and B) Typical generalized lipodystrophy, alopecia, joint

B), micrognathia (A, B) in two patients affected with HGPS. (C and D) Multiple joint

lasia (D) in two patients affected with RD.

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459 451

Werner syndrome (i.e. with wild-type RecQL2 sequences) in thenosologic spectrum of Laminopathies.

We will focus our review on HGPS and RD, two well-characterized Lamin-associated premature aging syndromes, focus-ing on the current knowledge concerning their pathophysiology andthe development of possible therapeutic approaches. Indeed,although RD is less known than HGPS and is not generally associatedwith premature aging, in our opinion it deserves its place amongLamin-related premature aging syndromes, in light of its molecularbases. As we will describe, typical RD is due to the massiveaccumulation of a toxic Lamin A precursor, similarly, but at higherlevels, to what is observed in Progeria. In this respect, RD can beconsidered as an ‘‘extreme’’ form of premature aging.

2. HGPS and RD: two premature aging related disorders

Since its first clinical description by Hutchinson (1886),followed by the clinical report by Gilford (1897), Progeria was afascinating disorder for clinicians and scientists, due to theapparent clinical recapitulation of normal aging, although thesimilarities are only partial and evolve in the context of adevelopmental disorder. The discovery of the major mutationcausing HGPS in 2003 (De Sandre-Giovannoli et al., 2003; Erikssonet al., 2003), then of ZMPSTE24 mutations causing RestrictiveDermopathy in 2004 and 2005 (Navarro et al., 2004, 2005) gaveclues to the search into the pathophysiology of HGPS and otherpremature aging disorders related to Lamins A/C.

2.1. Clinical features

HGPS is characterized by the premature development ofsegmental features recalling aging. These become generallyapparent after the first year of life, the children appearing healthyat birth although somewhat small for gestational age. The meanage at diagnosis is 2.9 years, generally following a fall-off from thegrowth curves (55%), loss of scalp hair (40%), skin scleroderma-likechanges (28%), lipodystrophy (20%), and the appearance ofcharacteristic facial dysmorphic traits, including a characteristicvisible vein across the nasal bridge (Hennekam, 2006). The meanfinal height of HGPS children is about 110 cm, the mean finalweight 14.5 kg, children linearly gaining a mean of 0.440 kg/yearfrom the second year of age (Gordon et al., 2007).

Typical facial dysmorphism includes micrognathia, prominentscalp veins, alopecia and a beaked nose (Fig. 1A and B). A high pitchedvoice is common. At the bone level, patients present with clavicularhypoplasia, acro-osteolyses of distal phalanges and generalizedosteopenia. Skin is thin, atrophic and sclerodermatous, withhyperpigmented lesions, sometimes hyperkeratosis or hyperplasticscars can be observed (Feingold and Kidd, 1971; Jimbow et al., 1988).Sweat glands and sebaceous glands are reduced in number andsubcutaneous adipose tissue is atrophic (Ackerman and Gilbert-Barness, 2002). Tooth eruption is delayed and dental crowding isoften observed. The cardiovascular system is severely affected, withsmall diameter of the intima and media and extensive loss ofvascular smooth muscle cells with fibrous replacement (reviewedby Capell et al., 2007). These data suggest that the pathophysiologyunderlying HGPS vascular lesions is somewhat different from that ofcommon age-related atherosclerotic lesions, typical plaque forma-tion being rare or focal (Ishii, 1976; DeBusk, 1972).

Strokes are frequent, at a median age of 9 years, resulting inseizures, hemiplegia, and dysarthria or headaches, vertigo, and limbweakness. Myocardial infarction is the most frequent cause of death,at a mean age of 13.5 years. The left ventricule is almost invariablyenlarged and valves are often calcified. Increasing pain of bone ormuscle origin is a major feature. Cognitive functions are preserved.

The estimated incidence is one in four to eight millions births, with abalanced sex-ratio. For further clinical details, see Hennekam (2006).

Restrictive Dermopathy is a perinatal lethal genodermatosischaracterized by a severe intrauterine growth delay, generalizedarthrogryposis, tight, stiff, translucent skin which is frequentlyeroded on flexure sites (Fig. 1C). Clavicules are hypoplasic and bonedensity is reduced, with fontanel enlargement (Fig. 1D). Facialdysmorphy includes a small pinched nose, the mouth open in afixed ‘‘O’’ position, retromicrognathia, and variable loss of scalphair, eyebrows and eyelashes (Witt et al., 1986; Verloes et al.,1992). Histologically, the skin shows hyperkeratosis, a thin dermis,abnormally dense collagen bundles, with almost completelyabsent elastic fibers (Pierard-Franchimont et al., 1992). Patients’death results in most cases from respiratory failure.

2.2. Genetic bases underlying HGPS and RD

In 2003, the genetic basis of Progeria involving the LMNA genewas identified by two different groups with different methodol-ogies. One was based on clinical overlaps between patientsaffected with mandibuloacral dysplasia (MAD), one of thedisorders caused by mutations in LMNA (Novelli et al., 2002),and HGPS. This approach led to mutational analysis of LMNA in twoaffected children (De Sandre-Giovannoli et al., 2003). The othergroup performed a whole-genome scan using microsatellitemarkers in 12 patients, looking for regions of homozygosity,following a supposed autosomal recessive inheritance, andunexpectedly retrieving two cases with uniparental isodisomyand one case with a 6-megabase paternal interstitial deletion in the1q21 region, directing toward candidate gene sequencing in theinterval (Eriksson et al., 2003).

The major mutation identified by both studies was located inLMNA exon 11. It is a recurrent, heterozygous substitutionpredicted to be synonymous at the protein level (c.1824C > T;p.Gly608Gly), causing, in reality, an aberrant splicing event. In thegreat majority of cases it is a de novo mutation, only one case ofgonadic mosaicism having been reported (Wuyts et al., 2005) andcases of familial recurrence being rare.

While LMNA exons 1–10 encode both Lamins A and C, exons 11and 12 specifically encode the carboxy-terminal tail of Lamin A:the HGPS mutation thus selectively affects this isoform, whichphysiologically undergoes a different production pathway com-pared to Lamin C. Indeed, while Lamin C is directly translated anddirected to the nuclear envelope, Lamin A is produced through fourpost-translational processing steps occurring on the C-terminal tailof a precursor, Prelamin A. Briefly (see Section 3 for more details),these steps include: (i) farnesylation; (ii) cleavage by themetalloprotease ZMPSTE24 (FACE1); (iii) methylation; (iv) secondcleavage by ZMPSTE24.

The HGPS mutation activates a cryptic splice site which gainsthe upper hand on the normal consensus site in most pre-mRNAsissued from the mutated allele (Reddel and Weiss, 2004), causingthe in frame-deletion of the last 150 bp of exon 11. A truncatedPrelamin A, named Progerin, is translated from the incorrectlyspliced mRNA, retaining the CaaX farnesylation motif but lackingthe second endoproteolytic cleavage site recognized by ZMPSTE24.Numerous nuclear alterations were observed in patients’ cellnuclei, a cellular abnormality observed in several Laminopathies,which worsened with culture passages and consequent progres-sive Progerin accumulation (De Sandre-Giovannoli et al., 2003;Eriksson et al., 2003; Goldman et al., 2004; Vigouroux et al., 2001;Muchir et al., 2004).

Mutations in ZMPSTE24 were subsequently identified inpatients affected with B type mandibuloacral dysplasia (MAD-B)(Agarwal et al., 2003). The involved mutations caused a reduced

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metalloprotease activity, leading to the hypothesis of a Prelamin A-linked pathophysiology of the disease (Agarwal et al., 2003). Otherreports confirmed this hypothesis (Shackleton et al., 2005).

Between 2004 and 2005, a new, severe phenotype, RestrictiveDermopathy, was associated to mutations in both LMNA andZMPSTE24 genes. Navarro et al. explored two patients in whom thediagnosis of RD had been evoked at birth but that subsequentlylived longer than typical RD cases, who carried dominant splicingmutations in the LMNA gene, resulting in complete or partial loss ofexon 11. In both patients, truncated Prelamin A forms wereevidenced by western blot, in addition to normal Lamin Aexpressed from the wild-type allele (Navarro et al., 2004).Functional consequences of the LMNA-linked RD mutations aresimilar to HGPS, leading to an accumulation of truncatedfarnesylated Prelamin A forms, which cannot be fully processed.All the other RD patients, presenting with the most typical,perinatal lethal clinical features, carried homozygous or compoundheterozygous mutations in the ZMPSTE24 gene (Navarro et al.,2004, 2005). The most frequent mutation, c.1085_1086insT, is aframeshifting mononucleotide insertion occurring in a T stretchwhich had been previously shown to represent a specific target ofmicrosatellite instability in colorectal tumors (Mori et al., 2001).All the mutations involved in typical RD, including the mostfrequent one, c.1085_1086insT, are ‘‘null’’ type, causing a completeabsence of the ZMPSTE24 enzyme (Navarro et al., 2005). Indeed, allthe patients carrying null ZMPSTE24 mutations that could be testedby western blot analyses on fibroblast cell lines, presented onlyfarnesylated Prelamin A, mature Lamin A being completely absent.Numerous abnormalities in nuclear size and shape were observed,as well as mislocalization of Lamin-associated proteins (Navarroet al., 2004, 2005). Navarro et al. (2005) postulated a correlationamong progeroid ‘‘Lamin-linked’’ syndromes, between the diseaseseverity and the overall quantity of intranuclear farnesylatedPrelamin A, whether truncated or full-length. They also concludedthat a common pathogenetic pathway, involving nuclear accu-mulation of farnesylated Prelamin A, underlied both HGPS and RD,in the context of a clinical spectrum of diseases of variable severity.

This hypothesis has been confirmed by different reports,presenting more or less severe and more or less phenotypicallysimilar Progeria-like syndromes, involving either the typical HGPSmutation (Navarro et al., 2004; Sevenants et al., 2005), differentextents of truncation of Prelamin A forms (Fukuchi et al., 2004;Shalev et al., 2007), or a strong activation of the aberrant splice siteobserved in typical HGPS induced by different Lamin A-specificmutations (Moulson et al., 2007).

Another group described a patient affected with HGPS carryinghomozygous null mutations in the ZMPSTE24 gene in addition to aheterozygous mutation in the LMNA gene. The LMNA mutationresulted in a C-terminal elongation of Lamin A impairing its post-translational modifications (Denecke et al., 2006). Whereas nullZMPSTE24 mutations should have resulted in RD, the LMNA

mutation seemed to be a salvage condition. The reduction in theabsolute quantity of accumulated farnesylated Prelamin Aalleviated the clinical picture to a HGPS-like phenotype.

On the other hand, several progeroid syndromes whosepathogenesis seems to involve both Lamin A and C isoforms,often showing a clinical overlap with other non-progeroidLaminopathies, have been described, including autosomal reces-sive or dominant variants of HGPS (Plasilova et al., 2004; Kirschneret al., 2005), autosomal recessive forms of mandibuloacraldysplasia (Garg et al., 2005; Van Esch et al., 2006; Verstraetenet al., 2006; Lombardi et al., 2007) as well as dominant forms ofatypical Werner syndrome (Chen et al., 2003; Csoka et al., 2004).These observations and, possibly, genotype–phenotype correla-tions, suggest that at least partially different pathophysiological

mechanisms might operate in Lamin A ‘‘only’’-related versusLamin A/C-related progeroid syndromes.

3. Disease mechanisms

In this section, we will focus on pathophysiological mechanismsbelieved to be linked to farnesylated Prelamin A accumulation,either truncated or wild type, involved in Lamin A-relatedprogeroid syndromes, namely including HGPS and RD.

Lamins are intermediate filaments proteins synthesized in thecytoplasm and then imported into the nucleoplasm (Hozak et al.,1995). They are classified as A-type Lamins, including the majorisoforms Lamins A and C, and B-type Lamins including the majorisoforms Lamins B1 and B2. In vertebrates, Lamins A and C areexpressed in all differentiated cells, and are translated fromalternatively spliced transcripts of the LMNA gene (for review, seeBurke and Stewart, 2006), while Lamins B1 and B2 are encoded byLMNB1 and LMNB2, respectively. A- and B-type Lamins formhomodimers (Moir et al., 2000) and assemble into a filamentousmeshwork called the nuclear lamina, which is an interface betweenthe inner nuclear membrane and chromatin (for review, seeBridger et al., 2007). Moreover, Lamins A and C are also locatedthroughout the nucleoplasm (Bridger et al., 1993). At bothlocalizations, Lamins A/C seem to play fundamental roles not onlyin the maintenance of nuclear integrity, but also in DNA replicationand global regulation of gene expression patterns (for review, seeHutchison, 2002; Vlcek and Foisner, 2007a; Verstraeten et al.,2007). Indeed, Lamins A/C interact not only directly withchromosomes but with an increasingly high number of structuraland signaling proteins, including components of the nucleus andproteins linking the nucleoplasm to the cytoplasmic cytoskeleton(for review, see Zastrow et al., 2004; Wagner and Krohne, 2007;Shaklai et al., 2007; Vlcek and Foisner, 2007a).

Lamins A (not C) and B are synthetized as precursors, containinga C-terminal CaaX motif (C = cysteine, a = aliphatic, X = anyaminoacid), and are respectively, subjected to four or threepost-translational processing steps to obtain the mature proteins(reviewed in Rusinol and Sinensky, 2006). Post-translationalmodifications of Prelamin A and Prelamins B first includefarnesylation of the CaaX box cysteine by a farnesyl-transferase.This cytosolic enzyme is composed by two subunits, A and B,encoded by FNTA (OMIM 134635) and FNTB (OMIM 134636),respectively. The A subunit, when associated to a different Bsubunit (OMIM 602031), composes the geranyl-geranyl transfer-ase type I, another cytosolic enzyme involved in the geranyl-geranylation of some CaaX box ending proteins (Lane and Beese,2006). The second step is a proteolytic cleavage of the aaX terminaltripeptide (aminoacids 662–664). The three last residues (aaX) arecleaved off by a zinc metalloprotease: ZMPSTE24/FACE1 (ZincMetalloprotease homologous to STE24 in S. cerevisiae or FArnesy-lated protein Converting Enzyme 1) acting on Prelamin A or FACE2on Prelamins B. FACE2, also known as RCE1 (Ras convertingenzyme) is also involved in the post-translational processing of themonomeric G protein Ras (Wright and Philips, 2006) and manyother isoprenylated proteins. The third modification is themethylation of the prenylated cysteine of Prelamins A and B byiso carboxy-methyl transferase (ICMT). Whereas no further post-translational modification occurs for Lamins B, which remainphysiologically farnesylated and methylated, a final step, resultingin the cleavage of the remaining 15 C-terminal Prelamin A residues(aminoacids 647–661) has been shown to occur, yielding matureLamin A forms. This second cleavage is performed again byZMPSTE24/FACE1, whose only known substrate in mammals isPrelamin A (Rusinol and Sinensky, 2006). FACE2 and ICMT aretransmembrane proteins of the endoplasmic reticulum (more

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459 453

likely located at the cytosolic face of the nuclear envelope), havingtheir active site in the cytosol. The situation is, very likely, identicalfor FACE1, although a direct evidence is still lacking in mammals,being already proven in yeast for STE24. It is not solved yetwhether Lamins are completely processed in the cytoplasm andimported into the nucleus as mature proteins, or whether parts ofthe processing occurs also in the nucleus.

The most common splice site mutation responsible of HGPS(c.1824C > T; p.Gly608Gly) results in the production of a truncatedPrelamin A (progerin or Prelamin AD50), deleted over the secondcleavage site, which thus carries a farnesyl and a methyl moieties onthe last cysteine of the CaaX motif. Besides HGPS, RD is caused eitherby LMNA mutations with similar consequences to those observed inHGPS, or, more often, by null ZMPSTE24 mutations, completelyimpairing the endoproteolytic processing of farnesylated PrelaminA. Consequently, in both cases, farnesylated Prelamin A forms,truncated in HGPS or full-length in RD, accumulate in nuclei.

Significant changes in nuclear shape are observed in a greatpercentage of HGPS or RD cells, typically increasing with thenumber of culture passages, with lobulation of the nuclearenvelope, herniations, nuclear lamina thickening, loss of peripheralheterochromatin, and clustering of nuclear pores (Goldman et al.,2004; Navarro et al., 2004, 2005). These changes are presumed tobe related to the toxic accumulation of farnesylated Prelamin Aforms (Goldman et al., 2004). Some authors (Navarro et al., 2004;Fong et al., 2004) hypothesized that alterations in nuclearstructure, as well as the severity spectrum in this category ofprogeroid syndromes could be correlated to the amount ofaccumulated farnesylated Prelamin A. In such a case, a concentra-tion-dependent dominant-negative effect of Prelamin A wouldlead to disruption of Lamin-related functions ranging from themaintenance of nuclear shape to regulation of gene expression andDNA replication. This hypothesis, in structural terms, has beenlately supported by the findings of Delbarre et al., showing that theintranuclear accumulation of Prelamin A induces an alteredhomopolymer segregation pattern of Lamins A and B (Delbarreet al., 2006), establishing the basis for the ‘‘dominant-negative’’effect observed experimentally (Scaffidi and Misteli, 2005) andclinically (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003).

Many cellular and biological experimental data have beencollected in recent years concerning the different ‘‘downstream’’effects of Progerin (Prelamin A) accumulation in nuclei. We will tryto evoke below the main ones that have been reported. How allthese pathophysiological changes result in specific- and multi-organ dysfunction in HGPS children, namely inducing the majorcardiovascular alterations responsible of death in most cases,remains to be established. Nonetheless, as we will further discuss,since farnesylated Prelamin A is believed to be a main pathophy-siological molecular actor, it has been possible to develop differenttherapeutic approaches targeting it.

3.1. Impairment of DNA damage repair

Lamins A/C are also present into the nucleoplasm, as part of thenuclear matrix, where they interact with major components of thenucleoplasm such as chromatin, histones, transcription factors orsplicing regulators (Zastrow et al., 2004). Consequently, diversefunctions are attributed to Lamins A/C, in addition to their well-established involvement in cellular pathways such as apoptosisand cell division (Shumaker et al., 2003; Vlcek and Foisner, 2007b;Quintyne et al., 2005; Tsai et al., 2006).

It is well known that a link between DNA damage andmammalian ageing exists (Sedelnikova et al., 2004; Karanjawalaand Lieber, 2004; Lans and Hoeijmakers, 2006). Recent studieshave shown that double-strand breaks (DSBs) typically accumulate

in HGPS and RD cells and that the resultant genome instabilitymight contribute to premature aging (Liu et al., 2005; Manju et al.,2006). DNA repair pathway defects were observed in HGPS and in aRD mouse model (Zmpste24�/�). Prelamin A accumulation wasalso associated with impairing of DNA repair factors recruitment atdamage sites (Liu et al., 2005). A second study identified the over-expression of many essential p53 targets in the Zmpste24�/�mouse model, which caused at least part of their Progeria-likephenotype (Bergo et al., 2002; Pendas et al., 2002; Varela et al.,2005). Indeed, double knock-out Zmpste24�/�, p53�/� miceshowed a partially rescued phenotype (Varela et al., 2005). It isknown indeed that p53 activation is triggered by DNA damage(Burma et al., 1999; d’Adda di Fagagna et al., 2003), and that, tosome extent, p53 activation can have deleterious effects on bonedevelopment, as observed in Progeria (Zambetti et al., 2006).Further proofs of the links existing between altered bonedevelopment, DNA repair, accelerated aging, and reduced cancerare the phenotypes of several DNA repair mouse models, as XPDmutant mice (de Boer et al., 2002), Ku80 defective mice(Difilippantonio et al., 2000) and p53 truncation mutants (Tyneret al., 2002). Furthermore, Manju et al. demonstrated that severalLamin mutants causing Progeria and muscle-specific disordersinduce defects in ATR signaling pathways such as reducedphosphorylation of g-H2AX and inadequate recruitment of53BP1 to repair sites in response to DNA damage in cultured cells(Manju et al., 2006). More recently, it has been shown that whereasDSBs repair proteins Rad51 and Rad50 were absent at Lamino-pathy-related DNA damage sites in patients’ cells, xerodermapigmentosum group A (XPA) protein, a unique nucleotide excision-repair protein, colocalizes with DSB sites (Liu et al., 2007), maybepointing to ‘‘unifying’’ pathophysiologic clues between differentdisorders characterized by features of premature ageing.

3.2. Cellular senescence

DNA damage accumulation is believed to be one of the majorcauses of cellular senescence and organismal aging (Kirkwood,2005; Lombard et al., 2005) and the similar ‘‘unrepairability’’ ofDSBs has been reported in senescent human cells (Sedelnikovaet al., 2004; Adams, 2007).

Bridger et al. have shown that, while they accumulatefarnesylated Prelamin A, HGPS fibroblasts entered into senescence,with a characteristic step of hyperproliferation followed bywithdrawal from the cell cycle via the apoptotic pathway and/orsenescence (Bridger and Kill, 2004).

Caron and collaborators demonstrated that several LMNA

mutations observed in lipodystrophic patients, or in HIV patientstreated with some protease inhibitors, resulted in an accumulationof Prelamin A and an increased oxidative stress that triggerspremature cellular senescence (Caron et al., 2007). HIV proteaseinhibitors also blocked the ZMPSTE24 protease, leading tofarnesylated Prelamin A accumulation (Coffinier et al., 2007).

Hence, impairment of DNA repair processes leading to celldeath or cell senescence might be a general and fundamentalmechanism that contributes to the pathogenesis of Progeria andother Laminopathies. At the same time, apoptosis and cellsenescence play a pivotal role as anti-cancer barriers and bothmay contribute to degenerative aspects of aging by impairing cellproliferative capacity while blocking the development of cancer.

3.3. Impairment of mitosis and cell cycle progression

Recently, a study demonstrated the abnormal association ofProgerin with membranes during mitosis. This defect delays theonset and the progression of cytokinesis; furthermore, it impaired

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459454

the targeting of nuclear envelope components to daughter cellnuclei in late telophase/early G1 (Dechat et al., 2007). Furthermore,the authors provided evidence that the Rb-mediated progressioninto S-phase is impaired in HGPS cells, by inhibition of itshyperphosphorylation by cyclin D1/cdk4. These delays veryprobably have a deleterious impact on cell cycle progression inHGPS cells. By different approaches, Cao et al. have also shown thatProgerin mislocalized in soluble cytoplasmic aggregates duringmitosis and caused abnormal chromosome segregation and somebinucleations (Cao et al., 2007). It is likely that a defect in mitosis isone of the pathogenic mechanisms involved in Progeria duringpost-natal developmental stages, once Progerin molecules accu-mulate in nuclei, since A-type Lamins are believed to begin to beexpressed in post-natal stages and, correlatively, early develop-ment is not affected in children with Progeria.

3.4. Alterations of epigenetic control

One of the uncontested roles of the nuclear lamina is themaintenance of the mechanical, structural and morphologicalproperties of nuclei (for review, see Hutchison, 2002). It has beenproposed that it provides a molecular docking site for peripheralchromatin (Gruenbaum et al., 2005). More recently, the lamina hasbeen proposed to serve as both a structural framework and aplatform for genome organization (Bridger et al., 2007). AbnormalLamins A/C and wild-type or truncated Prelamin A accumulationproduces nuclear envelope alterations and chromatin disorganiza-tion, both in HGPS or other Laminopathy patients’ cells and animalmodels (De Sandre-Giovannoli et al., 2003; Vigouroux et al., 2001;Novelli et al., 2002; Muchir et al., 2003; Scaffidi and Misteli, 2005;Meaburn et al., 2007; Pendas et al., 2002; Bergo et al., 2002;Mounkes et al., 2003). HGPS fibroblasts accumulate progerinwhile they age in culture and coincidentally display changes innuclear shape and architecture, and, notably, a loss of peripheralheterochromatin (Goldman et al., 2004).

To determine the mechanisms responsible for the loss ofheterochromatin, epigenetic marks regulating either facultative orconstitutive heterochromatin were examined by Shumaker et al.(2006). For example, from a female HGPS patient, histone H3trimethylated on lysine 27 (H3K27me3), a mark for facultativeheterochromatin, was lost on the inactive X chromosome. Themethyltransferase responsible for this mark was also down-regulated. These alterations were detectable before the changes innuclear shape that are considered as pathological hallmarks ofHGPS cells. Their results also showed a down-regulation of thepericentric constitutive heterochromatin mark, histone H3 tri-methylated on lysine 9, and an altered association of this markwith heterochromatin protein 1a (Hp1a) and the CREST antigen.This loss of constitutive heterochromatin is accompanied by an up-regulation of pericentric satellite III repeat transcripts. In contrastto these decreases in histone H3 methylation states, there is anincrease in the trimethylation of histone H4K20, an epigeneticmark for constitutive heterochromatin. Lattanzi et al. confirmedthese findings and further showed that chromatin organization inHGPS cells was differentially affected depending on the accumula-tion of different intermediates of Prelamin A processing in cells’nuclei. Non-farnesylated Prelamin A caused the redistribution ofLAP2a and of HP1a and trimethyl-K9-histone 3, and triggeredheterochromatin localization in the nuclear interior. In contrast,the farnesylated and carboxy-methylated Lamin A precursoraccumulated at the nuclear periphery and caused loss ofheterochromatin markers and Lap2a in enlarged nuclei (Lattanziet al., 2007). These data globally suggest that Progerin accumula-tion leads to perturbations of epigenetic control of chromatinstructures, and subsequently, to altered gene expression patterns.

3.5. Failure of stem cell growth and maintenance

Another hypothesis is that Progeria stem cells are unable togrow and self-renew, leading to stem cell exhaustion and impairedcell renewal in tissues that physiologically necessitate thismechanism. Indeed, on one hand, there are data showing thataccumulation of DNA damage with age can limit the function ofstem cells (Rossi et al., 2007; reviewed in Ruzankina and Brown,2007). On the other hand, several findings (Dorner et al., 2006;Pekovic et al., 2007; Johnson et al., 2004; Van Berlo et al., 2005;Nitta et al., 2006; Galderisi et al., 2006) suggest that Lamin A/C–LAP2a/pRb complexes control the balance between proliferationand differentiation of adult stem cells in tissue homeostasisand regeneration. These processes are very likely substantiallyperturbed by Prelamin A accumulation.

3.6. Altered regulation of gene expression

Several reports have pointed to a deregulation of geneexpression profiles in HGPS. In 2000, before the identification ofLMNA as HGPS’ causative gene, Ly and colleagues reported alteredexpression of genes involved in mitosis in cells of patients affectedwith HGPS. This is to some extent similar to what is observed incells of middle- or old-aged persons, leading to the suggestion thatan underlying mechanism of the aging process involves increasingerrors in the mitotic machinery of dividing cells in thepostreproductive stage of life (Ly et al., 2000). Scaffidi and Mistelidemonstrated as well that several genes (CCL8, MMP3, MMP14,HAS3, STAT3, TIMP3, etc.) were misregulated in HGPS compared tocontrols, and that their expression levels could be brought tonormal with antisense oligonucleotides allowing to lower Progerinexpression levels (Scaffidi and Misteli, 2005).

On one hand, Lamins are known to bind many nucleartranscription factors, as pRb (Shumaker et al., 2003; Zastrowet al., 2004); on the other hand, A-type Lamins have beenobserved to colocalize with RNA splicing factors in speckleswithin the nucleus (Kumaran et al., 2002; Jagatheesan et al.,1999), and a dominant-negative Lamin mutant lacking the NH2-terminal domain inhibits RNA polymerase II activity in bothmammalian cells and transcriptionally active embryonic nucleifrom Xenopus laevis (Spann et al., 2002). Furthermore, recentstudies indicate that the periphery of the nucleus provides aplatform for sequestering transcription factors away fromchromatin. Several transcriptional regulators, operating indifferent signal-transduction pathways, have been found tointeract physically with the lamina and components of the innernuclear membrane (for review, see Heessen and Fornerod,2007). It has been long known as well that gene-richchromosomes are centrally located, whereas gene-poor onesare bound to the nuclear periphery (for review, see Cremer et al.,2006). Dahl and colleagues showed that structural andmechanical properties of the lamina are altered in HGPS cells,wild-type Lamins A and C becoming trapped at the nuclearperiphery due to the presence of Prelamin A. These alterationsare likely to have repercussions on chromosome positioning andgene silencing patterns. In addition, the lamina network in HGPScells seems to have unique mechanical properties that mightcontribute to disease phenotypes by affecting mechanosensitivegene expression (Dahl et al., 2006).

4. Possible links between normal aging and Lamin A-relatedpremature ageing syndromes

Several recent studies show that Lamin A is involved inphysiological ageing (Scaffidi and Misteli, 2006; McClintock et al.,

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459 455

2007). In particular, it has been shown that progerin is produced inhealthy cells, in absence of LMNA mutation, because of a rare andsporadic use of the cryptic splice site in exon 11. This progerinlocalizes at the nuclear lamina, under the nuclear envelope. Agedbut healthy cell nuclei can exhibit blebs containing geneticmaterial that are characteristic of Laminopathies. Such abnorm-alities lead to functional perturbations that are probably involvedin the ageing process. Therefore, fibroblasts overexpressing mutantLamin A exhibited accelerated telomere shortening and reducedreplicative lifespans, in addition to abnormal nuclear morphology(Huang et al., 2007). Telomeres of skin fibroblasts derived from theHGPS patients were also shown to be shorter than those of age-matched controls (Bridger and Kill, 2004).

In human normal skin, progerin is also synthesized in somekeratinocytes and in a sub-population of dermal fibroblasts,accumulating with age. Progerin could thus be considered as asuitable marker of ageing skin (McClintock et al., 2007).

It thus seems that at least some mechanisms could be similarlyinvolved in both the pathological premature ageing of Progeriapatients, and in non-mutated, healthy people during physiologicalageing, at very low levels.

Fig. 2. Inhibitors of isoprenoids and cholesterol biosynthetic pathway. (A) Chemical str

isoprenoid pathway can be arrested at different steps (red): statins inhibit HMG-CoA redu

of the synthesis of these farnesyl-PP and geranylgeranyl-PP is the farnesyl-PP synthase, se

and geranylgeranyl-transferase (GGT, type 1) are the targets of their respective inhibit

5. Therapeutic approaches in Progeria and related disorders

It seems now more and more clear that most cell and tissueabnormalities observed in Progeria are first of all driven by theaberrant accumulation of a toxic farnesylated Lamin A precursor,Progerin. On this basis, the therapeutic approaches have followedtwo major paths: (i) the reduction of the quantities of Progerinexpressed in cells and tissues; (ii) the modification of thequalitative, chemical properties of this aberrantly farnesylatedand methylated protein.

5.1. Therapeutic approaches on cultured cells

Following the first therapeutic strategy, Scaffidi and Mistelihave targeted the splicing defect observed in Progeria, in order tolower Progerin production. To this end, they used antisensemorpholino oligonucleotides specifically directed against theaberrant exon 11–exon 12 junction contained in mutated pre-mRNAs (Scaffidi and Misteli, 2005).

These studies showed that, upon splicing correction andefficient lowering of Progerin expression levels, several cellular

uctures of farnesyl-PP and geranylgeranyl-PP used in protein prenylation. (B) The

ctase, a polytopic protein inserted into the ER membrane. The rate-limiting enzyme

lectively inhibited by amino-bisphosphonates (NBPs). The farnesyl-transferase (FT)

ors (FTI and GGTI).

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459456

disease phenotypes were reversible in HGPS fibroblasts (morphol-ogy as well as aberrant gene expression patterns), even in absenceof mitosis. Another approach was to decrease mutated LMNA

mRNAs expression levels by siRNA (Huang et al., 2005). Thisalternative strategy confirmed that the reduction of Progerin levelssuffices to revert the abnormal nuclear morphology. However, atthe moment, these techniques seem to be difficult to apply towhole organisms. On the same line of evidence, Fong et al. showedthat Zmpste24�/� mice with one inactivated Lmna copy(Zmpste24�/� Lmna �/�), producing half Prelamin A amounts,had a totally rescued phenotype, introducing the notion of a‘‘threshold’’ level for a toxic effect of farnesylated Prelamin A.

Based on the hypothesis that maintenance of the farnesylmoiety is one of the major mediators of Progerin toxicity, throughaberrant anchorage of Progerin in nuclear membranes (Goldmanet al., 2004; Scaffidi and Misteli, 2005) and dominant-negativeinteractions with wild-type Lamins (Delbarre et al., 2006; Dahlet al., 2006) and other Lamin-interacting partners, a secondapproach aims at inhibiting progerin prenylation. Eukaryotic cellscontain three distinct prenyltransferases that attach either afarnesyl group (15 carbons) or a geranyl-geranyl group (20carbons) (Fig. 2A) in thioether linkage to C-terminal cysteineresidues included in a CaaX prenylation motif, in a variety ofproteins. The biosynthesis of the two isoprenylated groups is partof the steroid biosynthesis pathway (see KEGG, http://www.ge-nome.jp/kegg/pathway/map/map00100.html). The pathway startswith acetyl-CoA and aceto-acetyl-CoA, produced from glycolysisand isoleucin catabolism, and with 3-hydroxy-methyl-glutaryl-CoA produced from leucin catabolism (Fig. 2B). This pathway,beside other important molecules (cholesterol, steroid hormones,Coenzyme Q10, etc.) gives rise to the formation of two metabolites,farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate (Reidet al., 2004). The post-translational modification, known asprenylation, consisting in the attachment of prenyl groups toproteins, is important for proper sub-cellular protein traffickingand fate (Reid et al., 2004). In the human proteome, more than 300different proteins are prenylated (McTaggart, 2006). Prenylationconfers lipophilic characteristics to hydrophilic proteins, allowingthem to be anchored to phospholipid membranes, providing amechanism for membrane targeting of proteins lacking trans-membrane domains. In Fig. 2B the already known inhibitors usedas drugs in medical practice are mentioned in red. Amongprenyltransferases, farnesyltransferases (FTases), attach a farnesylgroup to cysteine (C) residues of CaaX boxes.

The treatment of HGPS and RD cells with farnesyltransferaseinhibitors (FTIs) partially mislocalizes Prelamin A away from thenuclear envelope, and reduces the frequency of nuclear shapeabnormalities (Toth et al., 2005; Glynn and Glover, 2005, Capellet al., 2005; Mallampalli et al., 2005). This effect was also shown inmouse embryonic fibroblasts from Zmpste24�/� and Lmna HG/+,two different mouse models of premature aging syndromes (Tothet al., 2005; Yang et al., 2005). Another approach using mevinolinand the histone deacetylase inhibitor trichostatin A led to rescue ofheterochromatin organization in HGPS cells (Columbaro et al.,2005).

5.2. Therapeutic approaches on mouse models

The in vivo use of FTIs in Zmpste24�/� mice partiallyameliorates the progeroid phenotype (Fong et al., 2006). Theauthors have shown that Zmpste24 deficient mice, a mouse modelof HGPS/RD, treated with an FTI (ABT-100), exhibited improvedbody weight, grip strength, bone integrity, and a better percentageof survival at 20 weeks of age. In the Lmna HG/+ mouse model,expressing FTIs increase also the body weight, weight of major

fat pads and decrease the number of rib fractures (Yang et al.,2005, 2006).

However, although these studies present an interestingpotential for therapeutic approaches, the unique use of FTIs as atreatment for Progeria and related disorders is a matter of debate:first of all, concerning the amount of farnesylated Prelamin A andthe status of other farnesylated proteins such as Lamins B in nucleitreated with FTIs (Rusinol and Sinensky, 2006). A second questionconcerns a possible alternative prenylation of the truncated/wild-type Lamin precursors, bypassing the treatment with FTIs as in theRas pathway, i.e. through geranyl-geranylation (Basso et al., 2006;Kilic et al., 1997; Rusinol and Sinensky, 2006). Furthermore, it hasbeen shown that farnesyl and geranyl-geranyl transferase inhibi-tors lead to cell cycle arrest, by targeting the proteasome (Efuet andKeyomarsi, 2006).

Liu and collaborators recently presented evidence that in HGPSand RD fibroblasts, DNA damage checkpoints are persistentlyactivated because genomic integrity is compromised (Liu et al.,2006). They showed as well that treatment of patients’ cells withan FTI did not result in a reduction of DNA double-strand breaksand damage checkpoint signaling, although the treatmentsignificantly reversed the aberrant nuclear shape. These datasuggests that DNA damage accumulation and aberrant nuclearmorphology are independent phenotypes due to Prelamin Aaccumulation in these premature aging syndromes. Since DNAdamage accumulation is supposed to be an important contributorto the clinical phenotypes, the treatment of HGPS with FTIs alonewas questioned (Liu et al., 2006).

Ideally, the treatment strategies of progeroid syndromes needto combine restoration of nuclear morphology with an improve-ment of DNA damage response and of other defects describedabove.

On the other hand, several other molecules known to beinvolved in cholesterol biosynthesis/prenylation pathways arecurrently used for some therapeutic applications (Fig. 2B).

Statins are widely used as cholesterol-lowering agents, inindividuals with increased risk of cardiovascular disease andatherosclerosis. They inhibit the HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway of cholesterolsynthesis. They are known to induce an inhibition of Lamin Amaturation (Sinensky et al., 1990; Lutz et al., 1992; Baker, 2005).Amino-biphosphonates (N-BPs) are used to prevent osteoporosisafter the menopause, as well as the risk of pathological fracturesin bone malignancies. They act as inhibitors of farnesyl-pyrophosphate synthase, thus reducing the synthesis of bothgeranyl-geranyl and farnesyl groups (Amin et al., 1992; Kellerand Fliesler, 1999) (Fig. 2B). These pharmacological moleculescould be tested as an alternative therapy, in alternation or incombination, in HGPS and related progeroid syndromes.

6. Conclusion

From a pathophysiological point of view, the known Progeroidsyndromes are caused either by mutations in genes encoding DNArepair proteins, such as in WS, Bloom syndrome (BS), Rothmund–Thomson syndrome, Cockayne syndrome, xeroderma pigmento-sum or trichothiodystrophy (Hasty et al., 2003; Wood et al., 2005),or by mutations in genes encoding Lamins A/C or partners involvedin their biological pathway, such as HGPS or RD (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003; Navarro et al., 2004,2005).

All progeroid syndromes, whichever their genetic cause, seemto share as a main pathogenetic mechanism defective/delayedDNA repair mechanics due to primary (DNA repair genes-relatedsyndromes) or secondary (LMNA/ZMPSTE24-related syndromes)

S. Pereira et al. / Mechanisms of Ageing and Development 129 (2008) 449–459 457

alterations. However, these progeroid syndromes also displayfundamental differences. In DNA repair-related syndromes, apredisposition to cancer or CNS disorders is evidenced, whereas inLMNA-related diseases an association between genomic instabilityand CNS involvement or cancer has only been reported in very fewcases (King et al., 1978; Shalev et al., 2007) and, at the opposite, p53pathway seems to be activated and associated with a senescentphenotype (Varela et al., 2005). Remarkable progress has beenmade in the recent few years in understanding the genetic,biochemical and cellular bases of Laminopathies, putting in lightcommon aspects which underlie at least part of Lamin-associatedpremature aging disorders.

The more extensively studied and paradigmatic of thesediseases is Hutchinson–Gilford Progeria syndrome, a rare anduniformly fatal segmental progeroid syndrome, leading to death atan average age of 13 years. A whole amount of evidence indicatesthat the accumulation of farnesylated Prelamin A forms mediatestoxic, dominant-negative, downstream effects in this disease aswell as in related syndromes, such as RD.

Notably, these preclinical achievements have recently driventhe development of potential therapies which have deserved toenter into clinical trials (FTIs in USA; statins and biphosphonatesunder study in France (Varela et al., in press)). Preclinical studiesconducted in HGPS cells as well as in progeroid mice models haveindeed suggested that these treatments could be beneficial forHGPS patients. These therapies differently target the prenylationpathway in order to prevent the formation of farnesylatedPrelamin A forms. There is a clear hope that these molecules willameliorate the clinical course of HGPS patients, also probablydepending on the patients’ age at the beginning of the treatment.Nonetheless, since these therapies circumvent but do noteliminate the splicing defect which is at the basis of Progeria,other approaches should still be considered which would act at thispathophysiological level.

If the aforementioned therapeutic approaches are shown toimprove the HGPS phenotype in clinical trials, they might beapplied to many other related progeroid syndromes showingPrelamin A accumulation as a key pathogenic event (Fukuchi et al.,2004; Shalev et al., 2007; Moulson et al., 2007; Lombardi et al.,2007; Capanni et al., 2005).

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