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Meeting Report
Highlights of the 2007 2007 Progeria Research Foundation Scientific Workshop: Workshop: Progress in Translational Science Science
Leslie B. Gordon,' Christine J. Harling-Berg,2 and Frank G. RothlnanRothman22
IWarren Alpert Medical School of Brown University, 2 Srown University, Providence.Providence, Rhode Island.
N the spring ofof 2007, a 2-year clinical drug trial began at Early in vitro data on a potcntialpotential rolerole of famesyltransferasefamesy!transferase I Children's Hospital Boston (I) with the goal to advance inhibitors (FTls)(FTIs) inin treatingtreating progeria were presented by fourfour the quality of life for children with Hutchinson-GilrordHutchinson-Gilford laboratories, demonstrating thatthat FTIFTl could reversereverse nuclear progeria syndrome (henceforth progeria), a rare (frequency blebbing in cells expressing progeriprogerinn (8-1(8-11). I). Armed with I in 4 million), multisystem, and inevitably l~ltall~llal disease data demonstrating thatthat treatmenttreatment of newly developed that claims young lives due to myocardial infarctions and cellular assays with FTIsFTls improves or reverses some of thethe strokes between ages 7 and 20 years (2). The journey to the errectseffects of progerin accumulation, a clinical trialtrial treatingtreating first clinical trial for progeria has been facilitated by a series children with progeria with an FTlFTI was initiatedinitiated inin May 2007 of collaborative scientific workshops organized by The (I) (www.c1inicaltrials.gov).(www.c1inicaltrials.gov ). Progeria Research Foundation (PRF) and supp0l1eclsupp0l1ed by The presenlpresent article, detailing thethe 2007 PRF Scientifk agencies interested in aging, cardiovascular disease, rare Workshop (November 12-14, Boston), emphasizes thethe disease research, and genetics (http://www.progeriaresearch. complexities of progerin and laminlamin A biology, andand thethe org/2007_prl"-_prl'-workshop_on_progeria.html). These meet need to continue to10 looklook for thethe most effective protocols rorfor ings have provided a concentrated forum 10to facilitate the correcting thethe effects of thethe laminlamin A defect inin progeria. All collectivecolleclive thinking of clinicians and scientists about speakers presented preliminary data thatthat had not been peer progeria, forge collaborations in this lillie-knownlittle-known field, reviewed alat the timelime of presentation. In thethe months sincesince thethe and accelerate the discovery of new ways to push the field workshop, some data have been published (referenced(referenced forward towardtowarcltreatmentstreatments and cure. The firstIlrst PRF Scientific within this article), and we looklook forward toto additional peer Workshop in 2001 helped to identifyidentiry nuclear blebbing as an reviewed publications on the work presented at thisthis meeting imp0l1antimpol1ant phenotypic marker of progeria cells (noted by over the coming year. Anthony Weiss; University of Sydney, Australia), and to The essence of translational research was represented inin recognize a translocation on chromosome 1I in the cells the presentations, which brought forth the most impOltantimpOltant cultured from a progeria patient (W. Ted Brown, New York issues in progeria today-the efrectseffects orof progerin and laminslamins State Institute for Basic Research in Developmental Dis and their binding partners on the functioning of cells, sys abilities), pointing geneticists in the direction of the gene tems, mouse models, aneland humans; the connection between mutation for progeria (3). In 2003, the second workshop progeria.progeria, aging, and cardiovascular disease inin thethe general was held just 3 months after publication of the gene defect population; careful analysis of FTIITI effects on progeria at all in progeria, which is typically a sporadic autosomal domi levels (summarized inin Table 1); and strategies forfor futurefuture nant disease caused by a C --,--). T mutation at nucleotide 1824 disease treatments and cure. In his introductionintroduction at thethe work of the LMNA gene encoding lamin A (4,5). Importanlly,Importantly, the shop, Francis Collins (Director, NHGRI) noted thatthat a 4-year mutation results in a persistence ofor an aberrant form of period between gene discovery and a clinical drug trialtrial isis a famesylated-prelaminramesylaled-prelamin A molecule (4) now called progerin. " .... an IlJlprecedented./£>a1/orunprecedentedfeatfor a rare genetic disease." For The attendees now included expelts in the field of lamin progeria patients and their families, who have struggled toto biology and laminopathies. The 2004 Progeria Workshop, live within a void of infonnation, the clinical trialtrial represents held within the National Human Genome Research Institute " ... a source of light coming up after a long,long, longlong nighl"night" (NHGRI), was held specifically to discuss the role of stem and hope has replaced" ... a sense of fear and helplessness cells and the potential for sternstem cell transplantation in on each passing birthday" (Subbarao, (-~Itherfather of Priya, a 15 progeria, an area of continuing interest forror the field. At the year-old with progeria). general Progeria Workshop in 2005, two mouse models of progeria were unveiled and, although they contained iden GETTING TO KNOW THE eHlU)CHILD WITH PROGERIA ticaltical mutations, they yielded very different phenotypes, For a disease as rare as progeria, scientists often have no mimicking various pOltionsp0l1ions of the human disease (6,7). exposure to the very population they wish to serve. During
777 778 GORDON ET ilL.AI.,.
Table I. Workshop-PrescnledWorkshop-Presenled Studies of Effectiveness of FTIs 011on Progeria Cells*Cclls*
Presel11erPresenter Cell(rissueCell(risslle Type Cellular Defect ITl]T! EffeclEffect
Collins Progeria RAC transgenic HGPS~HHOpS-I-" TissueTI"sue pathology:palhology: progressive loss of Improved carotid alteries:al1enes: more VSMC.VSMC mouse alieriesaliencs VSMC :md:ll1d appearance of PO less PGPC; COl1luiComai Progeria fibroblasts and transfertedtransfected Over-expressingOver-expresslllg progcrinprogerin increased raleraIl' No improvement progerin-expressing nom1,l)noml,l! fibroblastsnbroblast~ of cell death LammcnlingLammerdmg: Progeria fibroblastsfibroblasts IncrcnscdIncrenscd nuclear rigidityngidity and early cell Revers,l)Revers,l! del1hdeath al lalerlalcr passages after mechanicalmcchanlcal streIchslretch LammcrdingLallllllcniing Progeria librobJaslslibroblasls Wound-healing deficitdeficil Reversal LlmmerdingLlrl1menllng Progeria fibroblastsfihroblash ProliferationProhterallon deficit aner nwchanic;]]flwehanical slreldl No improvementImprovcment Paschal Progeria fibroblastsfibroblasts and transferledlransfecled NPC abnormalily-TPRabnormailly-TPR mislocalizatiollmi"locaJlza!loll Reversal progerin-expressingprogerin-ex press ing HcLaHe La cellscc IIs Paschal L647RLf)lnR cells_,celL'>. inIII which cleavagecleavnge by InvertedInverled RAN gradientgradIenl Reversal Zmpste24 isis lackinglackmg 7ZOll..ou Progeria fibroblastsfibroblasts DSBs No improvemeTl1improvement SincnskySinensky TransfectedTrnnsfCcled progcrin-cxpressingprogcrin-expressillg FarnesylmedFarnesyl,l1ed progerinprogcnn was prcsentpresent Geranylgeranylated progerinprogel"lll appeared HeLaHeLn cells Lopez-OtinLope7-0tin Zmpstc24-Zmpslc24 -II tibroblastsfibroblas!.; FamesylatedFamesylaled prelaminprclamin A was presentpreseJl1 FamesylatcdFamcsylaled prelamin A levelsleveb decreased;decreased: geranylgeranylmedgerallylgeranylaled prelamillprclamin A appeared
NOles:Not('s: *Data*Data arcarc listedli'itcd inIII order of appearanceappc FTIFTI "-'- famesyhransferasefamesyllransfcrasc inhibitor;inhibItor; BAC _cc hacterialhaclel"lal artificialartiJiei,,1 chromosome;chromosome: HGPS,,-,HGPS - Hutchinson-GuilfordHUlchmson-Guilford ProgeriaProgeri" Syndrome: VSMC = vascular smoothsmoolh muscle cells;cells: PGPO == progeria;progeria: DSBsJ)SB~ "-'-=- double-stranddouble-strand breaks; NPC " nuclearIluclear pore complexes: TPRTPI~ =-=- translocatedtranslocaled promoter region.reglOll. thethe firstfirst evening of thethe workshop, participants had a unique especially challenging. To thatthat end, a clinical trial using FTI chance toto hear fromli"OITI children with progeria and theirtheir families toto treattreal progeria isis currently being conducted at Children'sChildren '."I during a panel discussion. Dr. ScoffScotf Berns (PRF and Warren Hospital, Boston. Several of thethe trial team members Alpert Medical School of Brown University), thelhe fatherfather of presented baseline clinical studies orof thethe major systems a child with progeria, moderated thethe familyfamily session. affected inin progeria, trialtrial design,design. and measures of efficacy Through intellJrcters,inlellJreters, Sammy (12(12 year old fromfrom Italy)Italy) and thatthat will likelylikely serve as models for futurefuture treatmenttreatment trials in Priya (15(15 year old fromti"om India),India), displayed an uncanny ability Ihethe field. toto describedescribe theirtheir liveslives with eloquence, poise, and wisdom~wisclom Mark Kieran (Dana Farber Cancer Institute), principal theirtheir frailfrail andand agedaged physical appearance belying theirtheir livelylively investigatorinvestigator of thethe clinical trialtrial team,team, reviewed thethe rationale spirits.spirits. Priya sharedshared thatthat sheshe enjoys traveling,traveling, and Sammy forfor thethe clinical trialtrial design,design. drug selection, and parameters likedliked toto skateboard,skateboard, swim,swim, and isis well assimilated intointo forfor assessing drug efficacy. The selection of thethe FTIFTf schoolschool life.life. Sammy's parents, Laura and Amerigo, spokespoke lonaramiblonafamib was made on thethe basis of twotwo lineslines of evidence: aboutabout howhow theirtheir son"son"...... gives themthem thethe strengthstrength toto keep on thethe addition of FTI toto progeria l1broblaslsl1broblasls reverses nuclear andand toto not give up, andand alwaysalways looklook at thethe good sideside ofor thethe blebbing (8-11),(8-11), and itsits lowlow toxicitytoxicity profile inin adults and day." Sammy has given themthem anan ""."... .opportunityopportunity toto enjoy children with cancer (13).(13). However, because 10nafamiblonafamib has lifelife inin aa different way ...... toto enjoyenjoy everyevery dayday of your lifelife not previously been administered toto children with progeria, with your childchild ... ". Keith andand Molly MooreMoore (U.S.A.),(U.S.A.), itit will be impOltantimp0l1ant toto carefully tracktrack thethe toxicitytoxicity profile, parentsparents orof Zachary, who recentlyrecently passed away,away, described thethe pharmacokinetics, and efficacy or lonafarniblonafarnib inin a variety of selflessselfless traitstraits of theirtheir sonson asas well asas other childrenchildren with organ systemssystems toto dete1111inedetem1ine whether therethere isis any benefit toto progeria,progeria, relatingrelating howhow childrenchildren with progeria "" ...... carecare soso usinglIsing thisthis FTI inin progeria clinically. To quantitate thethe effect much for others, and Zach understood that the research much for others, and Zach understood that the research of drug treatmenttreatment inin a measurable way, Dr. Kieran stressedstressed mightmight notnot helphelp himhim directly andand personally, but itit certainlycertainly thethe importanceimportance of firstfirst documenting thethe natural history of wouldwould helphelp others"others'· (12).(12). InIn concluding,concluding, young Sammy thethe disease, includingincluding an understanding of thethe breadth and wantedwanted toto especiallyespecially ""...... thankthank allall thethe doctors andand allall thethe variabilityvariability inin disease phenotype fromfrom patient toto patient. researchersresearchers thatthat areare puttingputting suchsuch anan efforteffort intointo tryingtrying toto help Because thethe number of children with progeria isis often tootoo meme outout andand allall thethe otherother kidskids withwith thethe disease." smallsmall toto seesee population-based changes,changes. forfor somesome clinical measures eacheach childchild must serveserve as hishis or her own control forfor DESIGNINGDESIGNING THETHE FIRSTFIRST CLINICALCLINICAL DRUGDRUG TRIALTRIAL assessingassessing drug efrect,effect, comparingcomparing pretreatment measures toto TheThe ultimateultimate goalsgoals ofof disease-relateddisease-related researchresearch areare toto findfind posttreatmentposUreatment measures. Because disease inin progeria isis treatmentstreatments andand aa cure~il1cure-in thisthis casecase toto improveimprove thethe health progressive, anyany measures improvingimproving with treatmenttreatment would andand increaseincrease thethe lifelife spansspans ofof childrenchildren withwith progeria.progeria. be consideredconsidered drug-related changes.changes. The primary clinical DesigningDesigning aa clinicalclinical trialtrial thatthat hashas thethe greatestgreatest chancechance ofof parameter forfor measuringmeasuring treatmenttreatment effkacyeflkacy isis raterate of weight assessingassessing thethe efficacyefficacy ofof anan interventionintervention onon thethe diseasedisease inin gain,gain, which isis linearlinear forfor eacheach childchild afterafter approximatelyapproximately ageage 2 aa statisticallystatistically signillcantsignificant mannermanner isis aa keykey elementelement inin thisthis yearsyears inin progeria (14),(J 4). andand thereforetherefore provides anan opportunity process.process. ForFor aa diseasedisease suchsuch asas progeria,progeria, inin whichwhich fewerfewer thanthan toto assessassess whetherwhether FTIFrI affectsaffects raterate of weight gain over aa 2 5050 patientspatients areare identifiedidentified worldwideworldwide atat anyoneanyone time,time, thisthis isis yearyear treatmenttreatment period.period. Secondary parametersparameters of efficacyefficacy HIGHLIGHTS'HIGHLIGHFS' OF TIlET/-IE 2007 PI?FPRF SCIENTIFIC WOI?KS/-/OPWORKSHOP 779 include measures of abnormality in other well-known thickening,thickening. c) atherosclerotic plaque (rich lipid core) with features of the disease, such as alopecia, short slature,stature, ruptured cap and superimposed thrombosis, and d) some subcutaneous !~lt.l~lL bone integrity, limb abnormalities.abnormalities, proliferating smooth muscle cells. Instead, Drs. Nabel and auditory and dental abnonnalities, and cardiovascular Gerhard-Herman propose that progeria vascular phenotype disease. While these features arcare important to the disease resembles an arteriosclerosis, which is characterized by process, the ability to statistically assess lrealmenltreatmenl effectellect on hardening of the vessels and vascular stiffness,sti llness, typically thesethese systems is Jess reliable than rate of weight gain. seen in the nOl1nalnonnal aging populations. Vessel cross-sections Although cardiovascular disease is clinically the most at autopsy or one 20-year-old patient revealed no indication importantimportant determinant of health and longevity in progeria, of vasculature inHammationint-lammation (IS). In addition, children with itit isis currently not a feasible primary outcome measure for progeria do not have abnormal serum cholesterol levels, a clinical triaLtrial. but isis followed as a secondary measure. which is a factor in chronic lipid-driven int-lammation (16).(16), Some patients inin thethe study have already haclhad myocardial Clinical studiessludics at both the National InstitutesInstilutes or HealthHeallh infarctionsinfarctions or strokes, and thethe damage incurred cannot (NIH)(NII-J) clinical center (17) and Children's Hospital, Boston, predictably be reversed. In addition, the ages for participants revealed that the intima-media thicknessthickncss of the progeria inin thethe trialtrial range from 3 toto 16 years. Because thethe most carotid artery is normal. Furthennore, in agreement with accurate assessment of cardiovascular improvementimprovement would Stehbens and colleagues (I5),(15). Dr. Nabel presented autopsy be an increaseincrease inin mean age of death,death. thethe clinical trial with cross-sections from a child with genetically confinnedconfirmed a cardiovascular primal)'primary parameter would require a 10- to progeria, and the histology showed that the v,lscularvascular media IS-yearIS-year commitment. Hence, rate of weight gain isis used as no longer contained smooth muscle cells and the elastic a surrogatesurrogate marker of general health inin progeria. structure was destroyed and replaced with extracellular InIn addition toto rate of weight gain, other more focused matrix or fibrosis.Iibrosis. She hypothesized that the primary loss of featuresfeatures suchsuch as bone heallh could change inin a measurable smooth muscle cells initiates vascular remodeling by way within thethe 2-year treatmenttreatment period. Catherine Gordon secondary replacement with matrix in large and small (Children's(Children's Hospital, Boston) presented thethe preliminary data vasculature. on thethe bone phenotype forfor 28 children, measured using twotwo In a study of 28 children with progeria at Brigham and dimensional dual-energy x-ray absorptiometry. Because thethe Women's Hospital (Boston), Dr. Gerhard-Hennan measured body habitushabit us of children with progeria isis drastically global vascular stiffening and, through imaging techniques, different fromfi'om theirtheir age-matched peers, calculation adjust observed vessel tortuosity,lortuosity, profound adventitial thickening,thickening, ments must be made forfor height-age. Remarkably, thethe and a depleted media layer.layer. For children with an occluded clinicalclinical historieshistories indicateindicate thatthat children with progeria do internalinternal carotid artery, therethere was an overall loss of the notnot have a higher riskrisk of fracturingfracturing theirtheir weight-bearing trilaminartrilaminar architecture. Functional studies demonstrated bones,bones, althoughalthough theythey are very playful and active. She high femoral pulse wave velocities, and decreased vaso suspectssuspects thatthat fracturefracture ratesrates inin progeria children are notnol dilatory response toto nitrous oxide stimulation inducedinduced by increasedincreased becausebecause thethe cortex of01" weight-bearing longlong bones isis ankle flexion.Oexion. Overall, thethe vessel stiffness, tortuosity,tortuosity, and of nOl1nalnonnal densitydensity byby x-ray (14),(14), inin contrast toto other lowlow adventitial brightness (the molecular composition not yetyel weight-disorders suchsuch asas anorexia nervosa and cystic identified),identified), as observed inin imagingimaging studies, support a model fibrosis,fibrosis, where fracturelj'acture ratesrates areare increasedincreased and cortical bone of arteriosclerosis similar to10 what isis observed inin aging. isis thinnedthinned onon x-ray.x-ray. Inln supportsupport ofor thisthis hypothesis, pre Remarkably,Remarkably. thethe blood velocities are inin thethe nonnalnOl1nal range liminaryliminary datadata demonstrateddemonstrated thatthat bonebone density z-scores;:-scores move and now isis stillstill laminar,laminar, implyingimplying thatthat therethere isis either fromfrom anan uncorrecteduncorrected osteoporoticosteoporotic rangerange of -2 toto -4,-4, where a tremendoustremendous reserverescrvc capacity inin thelhe vasculature. or therethere we would expectexpect somesome spontaneousspontaneous bonebone breaksbreaks similarsimilar toto are compensatorycompensatory mechanisms helping toto preserve function. osteogenesisosteogenesis imperf'ecta,imperrecta, toto aa height-ageheight-age cOlTectedcOITected osteo Complementary data gathered at thethe NIH studying a cohOl1 penicpenic rangerange of'of -1-1 toto --2.--2. Further studiesstudies of bonebone qualityquality areare of 15 patients revealeclrevealed initiallyinitially n01111aln0l111al vascular compliance inin progress,progress, usingusing peripheralperipheral qualllitativequantitative computedcomputed tomogtomog thatthat decreasesdecreases with increasingincreasing age, and a number of children raphyraphy toto tracktrack thethe three-dimensionalthree-dimensional corticalcortical andand trabeculartrabecular displayeddisplayed lenlen atrial enlargement (17).(17). Blood pressure and characteristicscharacteristics withwith andand without FTI treatmenttreatment. heartheart raterate were inin thethe normal rangerange inin younger children, while thethe older children had increasingincreasing blood pressures and variablevariable heartheart rates.rates. Electrocardiograms were normal inin THETHE CARDIOVASClJLARCAIWIOVASCLJLAR PHENOTYPEPHENOTYPE ININ PROGEIHAPROGEIUA AND most,most. althoughalthough a fewfcw had longlong Q-Q-TT intervalsintervals suggestingsuggesting PROSIJECTSPRO,'WECTS FORFOR VASClJl,ARVASClJLAR TlmATMENTTnl~ATMI~NT WITH FTI fibrosisfibrosis of thethe conductionconduction system.system. InIn summary,summary, thethe progeria CardiovascularCardiovascular eventsevents inin progeriaprogeria includeinclude hypertension,hypertension, vasculaturevasculature isis characterizedcharacterized byby global stiffness,stiffness, tortuosity,tortuosity, myocardialmyocardial infarction,infarction, congestivecongestive heartheart failure,failure, andand stroke.stroke. andand aa lossloss ofor smoothsmooth muscle cells inin thethe media with ElizabethE/izab()th NobelNobel (National(National Heart.Heart. LungLung andand BloodBlood Institute)Institute) subsequentsubsequent extracellularextracellular replacement.replacement. Smooth muscle cell andand MarieMarie Gerhard-HermanGerhard-Herman (Brigham(Brigham andand Women's dropoutdropout isis uniqueunique toto progeria,progeria, while globalglobal stiffnessstiffness and Hospital)Hospital) discusseddiscusscd recentrecent findingsfindings onon thethe vascularvascular phenopheno tortuositytortuosity areare observedobserved inin thethe nonnalnonnal agingaging population. typetype andand functionfunction inin childrenchildren withwith progeria.progeria. ItIt waswas thethe The vascularvascular remodelingremodeling characteristicscharacteristics inin childrenchildren with consensusconsensus ofof bothboth presentingpresenting cliniciansclinicians thatthat thethe diseasedisease progeriaprogeria areare similarsimilar toto vascularvascular eventsevents observedobserved inin aa transtrans progressionprogression ofor progeriaprogeria bloodblood vesselsvessels doesdoes notnot litfit thethe typicaltypical genicgenic progeriaprogeria mouse model thatthat expressesexpresses humanhuman progerin,progerin, atheroscleroticatherosclerotic descriptiondescription thatthat includes:includes: a)a) preservationpreservation orof createdcreated inin Francis Collins' laboratOl)'laboratory (7).(7). Within thethe thethe vascularvascular smoothsmooth musclemuscle cellscells (VSMC),(VSMC), b)b) intimalintimal descendingdescending aortaaorta (cross-section(cross-section histology),histology), smoothsmooth muscle 780 GORDON tTALE1' AL. Normal lam in A processin9proces.o;ing Hutchinson-Gilford progeria ~yndrornesyndrome ( ~I\\ < "/'./ (\IM(:\1\1 IMP<.,IMI'51[}1 lli1 t~ OfOCII, II, 1'I)q(('iI111 ,iem-m)" I/MI''', Iflil Up,I, '>i\fll c!pd,aq'> ,1()l)U,Ii "d ,,' '"'t' _/'''Z/{ <::::~.. -~" .. f ( 01"11, "'--...",.. / Proql>'1IlI'roqerill (C.OPI,riqhl 0p\,r;qht ," )!lOiJ N'HUr<' P"hli~hm Figure I,L Posttran~lation:l!Postlranslatiolla! processing ofor prelamin A to10 lll:llllremalllre laminlamin A (lei!)(Idi) and prcprogcrin toto prog-erinprogerin (rig-hl)(righll (57).(57), C = cyslelne~cystcinc~ S -- serine: I --'" isoleucine:isoleucine: M -"--= mClhionin<~:methionine: FLISCFLlse -"--= ramesyJramesy! lransferase.transferase. IjReproducedReproduced by pellnis~ionpennission or lhethe pubhsher,publishcr.l1 cells begin to "drop-out" by 5 months, and by 20 months DNA replication, the organization of the chromosome there is a paucity to a complete absence of smooth muscle territories, and epigenetic modulations within the nucleus. cells. The remaining endothelial cells are picnotic, similar Lamin A is processed via a series orof posHranslationalposttranslational steps to progeria cells grown in vitro when progerin is over (Figure I):I): prelamin A is farnesylated, carboxymethycarboxymethylated,lated, produced. There is substantial adventitial thickening, and and cleaved twice to produce mature laminlam in A. Elements for the anatomical changes con'elateCOiTclate with functional changes; this pathway include initial famesylation by a farnesyltransfarnesyltrans the blood pressure response to a vasodilator is blunted and lerase,ferase, removal of the three C-tenninalC-terminaI amino acids, and includes a protracted recovery time to achieve baseline methyl esterifkationesterification of the now C-telminal farnesylfarnesyl values. The NIH progeria mouse model mimics the human cysteine. This is followed by integrationintegration of prelamin A into vascular disease,disease. and is therefore a viable model for as the nuclear membrane.membrane, and cleavage of 15 amino acids and sessing treatment effects on progeria. At the 2007 workshop,workshop. the attached famesylramesyl group fromfrom itsits C-tenninal end by Dr. Collins reported on studies fromrrom his laboratory where Zmpste24 to produce mature laminlamin A. Mature laminlamin A isis transgenic progeria mice began daily treatment with ITI at then released from thethe nuclear membrane (19).(19). Typically inin two different phases orof development: at time ofor weaning progeria, a single silent C toto T transitiontransition mutation at (0-9 months) and later between 6-9 months. Whereas the nucleotide 1824 (Gly608Gly)(Gly608Gly) activates a cryptic splice site, carotid a1teriesal1eries ofor untreated mutant mice demonstrated resulting in thethe deletion orof 150 messenger RNA bases inin thethe a progressive loss of VSMC and excessive appearance of 3' portion of exon IIII (4).(4). The aberrant protein,protein. progerin, proteoglycans with age, matched progeria mice treated with lacks thethe cleavage site forfor removalremoval of itsits C-tenninal intermediate to high doses of FTI had significantly more farnesylatedfarnesylated peptide by Zmpsle24Zmpste24 endoprotease, and VSMC, and little evidence ofor proteoglycan accumulation. thereforethererore remains remains bound toto thethe innerinner nuclear membrane. The results with the mice treated between 6 and 9 months 1l1e results with the mice treated between 6 and 9 months Hence, progerillprogerin resemblesresembles an aberrant fOnllfom1 of famesylatedfarnesylated give us the first evidence thaIthat ITTITI may have a beneficial prelamin A. However, whereas prelamin A isis a transienttransient. effect in reversing vasculature change brought about intermediateilllermediate protein, which isis present at very lowlow levelslevels inin by progeria. thethe cell, progerin isis a nontransient protein whose concen trationtration builds with successive cell divisions. EIi'J<'ECTSEIi'FECTS 01<'01" PIWGERIN AND LAMINLAMIN IMHALANCE ON Several workshop presentations addressed progerin's CELL STRliCTlIRE ANDANOFuNCTION FUNCTION influencesinfluences on laminlamin A and itsits binding paltners.pmtners. itsits rolerole inin To continue developing treatment prospects inin progeria, architectural changes inin thethe nuclear membrane, andand subsub and to understand its role in normal aging, itit isis essential toto sequent effects on cell functionfunction thatthat include:include: increaseincrease inin study not only the effectsefrects of f"J'1,I',l, butbUI also the biology ofor apoptosis and disruption of binding with lamin-bindinglamin-binding lamins,lamins. progerin, and related molecules at the cellular level.level. proteins,proteins; increasedincreased nuclear rigidity,rigidity, sluggish wound healing In nOlmal cells, larninslamins Connfonn a concentrated layerlayer along thethe inin vitro, and altered cell proliferation andand chromosomal inner rim of the nucleus, called thethe nuclear lamina,lamina, and are distribution. LucioLudo Comai's (University(University of Southern Cal also found dispersed within thethe nucleoplasm (18).(18). Lamins ifornia)ifornia) studies stressed thethe importanceimportance of detennining thethe play a critical rolerole inin a number of essential cellular cellular effectseHects of progerin andand excessexcess prelamin A on cellcell functions, such as thethe transcriptiontranscription of RNA polymerase n.It growth (20).(20). Dr. Comai used aa lentiviruslentivirus systemsystem toto createcreate HIGHLIGHTS OF THE 2007 PRF SCIEN71FJCSCIENTIFIC WORKSHOP 7RI7XI two cell lines, progerin expressors and prclamin A ovcrover A and lamin B arcare separate yet interdependent; the failure of exexpressors,pressors, from normal human fibroblasts. Over-expression one lamlamin-typein-type to properly form can interfercinterrere with the of either prelamin A or progerin significantly increased the formation of another laminIamin type (24). Progerin build-up with amount orof cell death compared to wild-type cOlltrolscontrols in increasing cell passage leads to progressively increased a passage-independent manner. When progerin-expressing percentages ofor blebbed nuclei (25). Roher!Robert Goldman cells 10.'11losl progerillprogerin expression, apoplasisapoptosis decreased to (Northwestern University) referred to these blebs as wild-Iypewild-type levels. ApoplasisApoplosis also decreased 10to wild-Iypewild-type "sophisticated structures" worthy of close examination to levelslevels when prelarnin A overexpressors were transfected detellllinedetem1ine their inOuenceintluence on cell function,Cunction, and began by with additional Zmpste24 DNA, which decreased prelamin hypothesizing that progerin's affect on other lamin types is A protein levels and increased mature lamin A levels. central to the blebbing defect. In the region of the blebs, As expected, over-expressing Zmpste24 did no1not rescue there is an absence of heterochromatin but euchromatin can progerin-exprcssingprogerin-expressing cells, because progerin lacks the be identified surrounded by lamin A,A. a configuration not Zmpsle24Zmpste24 recagnirecogni lion1ion site. Adding FIIFTI to10 progerin observed in other regions where the nuclear membrane expressing cell cultures did not improve rates of cell death, appears to be formed normally (26). Dr. Goldman and his presumably because the excess nonfarnesylated prelamin A group suggest that the blebs contain transciptionally active createdcrealed with FIIFfI exposure counteracted any improvements DNA and thutthat this is providing clues regarding the role of inin growth thatthat decreasing progerin may have caused. Studies mutant laminlarnin A in progeria. by Stephen Young (UCLA) provided additional evidence for Complementary chromosome positioning studies were thethe toxictoxic effects of farnesylatedfamesylated prelamin A. Dr. Young explored by Joanna Bridger (Brunei(BruneI University, U.K.), who showed thatthat certain clinically used HIV protease inhibitorsinhibitors supports the hypothesis that A-type lamins (and/or emerin) thatthat cause lipodystrophylipodystrophy reminiscent of thatthat seen in progeria are involved in translocating and anchoring chromosomes to also create a Zmpste24 blockade and a build-up of the nuclear membrane or nuclear matrix in nonrandom 1~ll11esylatedfamesylaled prelamin A inin normal fibroblasts (21).(2 I). Thus, distributions according to cell cycle status, and that lamin itit isis crucial toto study not only theIhe effects ofor progerin, but that mutations pcrturbperturb this balance. Normally, upon cell cycle of other proteins thatthat are affected by thethe presence of exit, gene-poor chromosomes move from the nuclear progerin or by treatmentstreatments thatlhat createcreale an imbalanceimbalance of laminlamin periphery to more interior positions (27,28). The distribution A-related proteins. profileprollle of chromosome position within the nucleus during Jan Lammerding (Harvard(Harvard Medical School) studied thethe cell cycling, quiescence, and senescence all differ. effects of accumulated progerin on nuclear rigidity, wound Dr. Bridger had earlier reported that proliferating fibroblasts healing inin vitro, and cell cycling, and asked whether FTls from laminopathylaminopathy patients, including progeria, exhibited reversereverse abnormalities. When strainstrain isis applied toto wild-type chromosome distributions characteristic of nonprolifcratingnonproliferating fibroblastfibroblast nuclei, theylhey remainremain stilTstilT (22). Fibroblasts cultured (senescelll(senescent or quiescent) n0I111alnO/lTIal fibroblasts, where the fromfrom a laminlamin A/C deficient mouse model were twicetwice as largestlargest chromosomes are located at the periphery of the fragilefragile as wild-type cells,cells. and after 24 hours of mechanical nucleus and thethe smallest at thethe interior.interior. At the workshop.workshop, strain,strain, cell death signilkantlysignificantly increased.increased. InIn sharp contrast,conlrast, Dr. Bridger reported new experiments that distinguished the while progeria fibroblastsfibroblasts showedshowed normal rigidityrigidity at early chromosome distribution inin senescent from quiescent cells passagespassages (when(when progerin levelslevels and nuclear blebbingblcbbing are atal based on thethe position of chromosome to,10, which she finds aa minimum), late-passagelate-passage nuclei showshow dramatically inin locatedlocated at thethe periphery inin quiescent cells and at the interior creasedcreased rigidityrigidity over wild-type fibroblasts.fibroblasts. Although thethe inin senescent cells. In proliferating progeria I1broblasts,Ilbroblasls, laminlamin A/C defkientddicient fibroblastsfibroblasts and progeria Ilbroblastsfibroblasts however, chromosome lO10 was locatedlocated at thethe periphery, displayeddisplayed vastlyvastly different nuclear rigidities,rigidities, nuclear strainstrain a position foundround only inin thethe nonproliferating,nonprol iferating, quiescent state elicitedelicited earlyearly cellcell deathdeath inin both cell typestypes over controls. InIn inin nonnaln01111 al cells. Several other proliferating laminopathylaminopathy addition,addition, whereas wild-type cells elllerenter S phase and G2 cultures also displayed thethe quiescent positioning. This phasephase inin responseresponse toto mechanical stretch,stretch, progeria cells did chromosome positioning was not changed when progeria notnot proliferateproliferate inin responseresponse toto stretch.stretch. Serum-starved progeria or laminopathylaminopathy cells became senescent. The consequences hbroblastsflbroblasts hadhad aa slowslow andand incompleteincomplete responseresponse 10to an inin vitro of aberrant chromosome 10 locatlocationion on transcriptiontranscription inin wound healinghealing assayassay asas well. The abilityability of FTls toto affect progeria will be thethe subjectsubject of Dr. Bridger's future studies. thesethese cellularcellular abnormalitiesabnormalitics was mixed.mixcd. Nuclear rigidityrigidity and Bryce Paschal (University(University ofor Virginia) reported on wound-healing deficitsdeficits were reversedreversed inin PrJ-treatedPTJ-treated proge progerin-induced changes inin thethe nuclear pore complexes riaria cultures.cultures. However, FTI treutmentlreatment diddid notnot promotepromote cell (NPCs),(NPCs), channels throughthrough which all transporttransport between thethe proliferationproIi feration afterafter mechanicalmechanical strain.strain. nucleus and thethe cytoplasm takestakes piplaceace (29).(29). He has found WithinWithin thethe nucleus,nucleus. laminlamin A monomers first(jrst dimcrizc,dimcrize, major changes both inin NPC structurestructure and inin thethe transporttransport thcnthen dirnersdimcrs associateassociate inin aa head-tn-tailhcad-to-tail t~lshionhlshion andand finallyIlnally machinery and signals.signals. The structuralstructural change isis inin thethe associateassociate laterallylateralIy (23).(23). The 50-amino50-amino acidacid deletiondeletion inin positionposition of thethe nucleoplasmic basket,basket. a structurestructure locatedlocated at progerinprogerin probablyprobably docsdoes notnot affectaffect itsits abilityability toto dimerize,c1imerize, asas thethe porepore on thethe nucleoplasmic side.side. ItIt consists of twotwo normalnormallaminlamin AA docs,does, becausebecause thethe necessarynecessary componentscomponents forfor proteins:proteins: TPR (translocated(translocated promoter region),region), thethe key thisthis functionfunction areare notnot deleted.deleted. Hence progcrinprogerin cancan formform constituent,constituent, andand Nup 1153,53, a facilitatorfacilitator of itsits assembly. The dimersdimers withwith itsclfitself oror nOllnalnonnal laminlamin A,A, therebythereby inOuencinginfluencing entireentire basketbasket structurestructure isis disassemblecldisassembled and reassembledreassembled at laminlamin A'sA's interactionsinteractions withwith nuclearnuclear pores,pores, chromatin,chromatin, andand everyevery mitosis. When Dr. Paschal lookedlooked forfor thethe localizationlocalization laminlarnin bindingbinding partners.partners. ForFor example,example, thethe fOllllationfOllllation of larninlamin ofor TPR inin progeriaprogeria fibroblastsfibroblasts by immunostaining,immunostaining, he 782 GORDON 1:!-7'ET AI.AL. obtained a striking result: Instead of being in thcthe basket, The percentage of progerin-positive cells increasedincreased with TPR is out in the cytoplasm. The same result is obtained passage number, suggesting a linklink to n0ll11aln0I111al aging. whcnwhen human epithelial cells are transfectedlransfectcd with progerin. A large amount ofor the data presentedpresenled at thisthis workshop Administration of an FTJ to the culture corrected the TPR underscored the value of studying progeria as a cellular and localization back into the nuclear envelope of progeria cells. organismalmodel for some key aspects of the aging process. ProgerinProgcrin also had a major effect on localization or The evidence thatIhat progerin is present in normal aging was a transport regulator. While small molecules can diffuse considerably strengthened at the workshop by Kari117aKarima passively through the NPC channel, macromolecules must Djahali (Columbia University) (35). Dr. Djabali measured enter as a complex with a member of the superfamilies of progerin mRNA and protein (using a monoclonal antibody importin or exportincxportin proteins, depending on the direction of shcshe created thatthat recognizes progerin but notnotlamin lamin A) inin cells transport (29). Once one of these complexes has traversed from skin biopsies taken fromfrom progeria patients and from the pore, it is dissociated with the involvement of a Ras 150 nonnaln011nal participants with ages ranging from newborn to related GTPase called RAN,RAN. In normal cells, there is 97 ycars.years. Dr. Djabali found low levels of progerin mRNA a gradient of RAN concentration across the pore, with more in all tissue samples.samples, but found progerin protein only inin skin RAN in the nuclcusnucleus than in the cytoplasm. samples taken from elderly participants, within dennalde11nal Dr. Paschal found that thcthe RAN protein gradient was libroblasts, and in a few telminaJlytemlinally differentiated keratino disrupted in epithelial cells when lamin A was depleted with cytes. Furthermore, using this same antibody, Dr. Djabali siRNA. He therefore examined thethe effect of several detected a dramatic increaseincrease of progerin-containing cells inin laminopathy mutations on RAN localization. Whereas cells nonnal dermal fibroblast cullurescuHures as passage number fi'omfrom dilated cardiomyopathy and Emery-DreifussEmel),-Dreifuss muscular increased. The discovery of progerin in thethe skin biopsies dystrophy mutations that express a mutant 1'01111f01111 of lamin A provided the first evidence for thethe inin vivo accumulation of had nonnal RAN gradients, the RAN protein gradient is progerin during nonnaln01111al aging. The presence of progerin in dramatically disrupted in both progeria cells and in cells nonnaln01111al individuals implies that individuals without progeria with thcthe mutant laminIamin A L647R,L647R in which cleavage by tolerate a low level of progerin as part of nonnaln01111al aging. With Zmpstc24Zmpste24 is lacking. While in normal cells most of the RAN respect to treating progeria, it is encouraging and suggests is in the nucleus, progeria cultures have an invcrtedinverted that effective therapy may be achieved with a balance gradient-there is more RAN in the cytoplasm than inin thethe between prelamin.prelamin, laminlamin A.A, and progerin rather thanthan nucleus. In thcthe L647R cultures, Ihisthis effccteffect was rescued by a complete elimination of thethe progerin molecule. adding an FTIfTf to the culture. It will be important to assess Francis Collins' latcstlatest research is asking a central whether FTI rescues the RAN progerin gradient in progeria question in the search for influencesinlluences of LMNA or progerin cells. production on longevity in the general population. Prompted by the Djabali evidence of increased amounts of progerin with age, Dr. Collins is looking for variations in DNA PROGERIA AND AGING sequence around the LMNA gene in libroblastsfibroblasts ofof' long-lived Hutchinson-Gilford progeria syndrome isis named for itsits people thatthat may give themthem a selective advantage over thosethose resemblance to aging: "pro" = prematurely and "gcras""geras" = individuals who die at younger ages. InIn an experiment thatthat aged. It isis a segmental progeroid syndrome (30), since examined cells from more thanthan 1000lOon people aged greater some, but not all, of the patients' characteristics resemble than 94 years, he found thaithat four single nucleotide accelerated aging. Over the last several years, a growing polymorphisms (SNPs) differed from the gcneralgeneral population body of evidence for cellular and molecular overlaps (p = 2 X 10")).10""5). Four of four centenarians analyzed also between progeria and normal aging has been uncovcred.uncovered. contained these same SNPs. The probability ofof' this finding x Early investigations compared several properties, for being random is 7 X la-x.IO- . Further study will ask whether example, telomere length (31) and distribution of mRNA increases or decreases in progerin production are linked to species (32). The discoverydiscovel)' that progeria is caused by thesethese SNPs. a mutation in lamin A, which had not previously been Nonnal cellular senescence isis marked by increasingincreasing ratesrates implicated in mechanisms of aging, brought in an entirely of DNA damage and a declincdecline inin thethe ability 10to repair thisthis new question: Are defects in lamin A implicated in normal damage (36). Progeria cells have been shown toto accumulate aging? The IIrslfirst positive evidence was reported by ScamdiScanldi double-stranded DNA (dsDNA) breaks and impaired DNA and Misteli in 2006 (33).(33), who showed thaithaI cell nuclei from repair (37-39). Yu('Yue Zou (East Tennessee SlateState University) nOllnalnonnal individuals have acquired defects similar to progeria presented results on the mechanism of this failurcfailure (40). patients, including changes in histonchistone modifications and Nonnally.Nonnally, in responscresponse to fOlmation of dsDNA breaks, increased DNA damage. Younger cells show considerably histone H2AX is rapidly phosphorylated and then recruits less of thesethese defects. They further demonstrated thatthat thethe age various DNA repair proteins, logethertogether fonningfonning focifoci forfor DNA related effects are the resullresult of the production of low levelslevels repair. In progeria libroblasts,fibroblasts, thethe phosphorylatedphosphOl)'lated protein, of preprogerin mRNA produced by activation of the same called gamma-H2AX, was unable toto recruit thethe DNA repairrepair crypticcl)'ptic splice site that functions at much higher levels in enzymes Rad51 and Rad50. Instead.Instead, the damage recognition progeria, and is reversed by inhibition of transcription at this protein XPA (xerodcnna(xeroden11a pigmentosum group A) was splice site. Cao and colleagues (34), studying the samcsame phe recruited. XPA 110nnallynonnally participates only in excision repair nomenon, showed that, among interphase cells in ll11roblastfibroblast systems and has no known role in dsDNA repair. Similar cultures, only a small fraction of the cells contained progerin. results were obtained from restrictive dermopathy cultures. HIGHLIGHT)IIlGHLlGH1:'i OF TilETHE 2007 PRF SCIENTIFICSCIENl1FIC WORKSHOP 783 Dr. Zou connll11Cdconn1111ed the identity of XPA by immunoprecip dystrophy (EDMD). lipodystrophy (progeria), and lossloss of italion. and XPA siRNA knock-dowilknock-down experiments revcrsedreversed fur and whwhiskers. iskers. Dr. Lopez-OLin'sLopez-Otin's prepreliminaryIi mi nary studies these findings in progeria cultures. Thus, XPA likely plays show that, unlike their wild-type counterparts.counterparts, manylllany hair a key role in defective dsDNA repairrcpair in progeria. Impor follicle-derived stem cells li'OInfrom these mice were quiescent, tantly,tantly. Dr. Zou and colleagues found Ihalthat FII treatment showed profound nuclear blebbing, and thethe accumulation of could not reduce the accumulated DNA breaks in progeria heterochromatin in lwotwo large masses in the centers of many cells,cells. suggesting that FTf may not effectively treat this cell nuclei. In addition, clonogenic assays showed thatthat thesethese aspect of disease (41). stem cells have a profound prolilCrationproliJ'eration defect.defect abnollllalabnOllllal accumulation of pp16,16, and an absence orof beta-catcnin,beta-catenin. suggesting a lossloss orof wnl signaling. ImpOliantly,Imp011antly. when an 1 FUrtlREFUTlJRE TREAT1\'IENTTREATMENT POSSIHII,ITIESPOSSIHII,ITJES THROUGH A BETTER BETTER LMNA allele was crossed intointo Zmpste24- -- mice.mice, levelslevels of UNDERSTANDINGUNDERSTANmNG OF THE BJOLO(;VBIOLOGY OF DISEASE ININ famesylated preJaminprelamin A decreased.decreased, laminlamin A was synthesized,synthesized, PnOGER1A:PIWGERIA: STE!\'ISTEM CELLS, ALTERNATIVE PRENYLATlON,PRENYLATlON, and all measured cellular defects were normalized (39).(39). COMHINATION CHEMOTHERAPYCHEMOTHERAPV,l AND A NOVIU, NOVI.;I, Hence, in this system, stem-cell function was profoundly HIGH-THROUGHPUTHIGH-THROUGHPliT SCREENINGSCimENING SYSTEM SYSTEIVI affected by nuclear defects and the ratio orof farnesylated prelaminprclamin A to mature lamin A. Stem Cells In light of this interest in the possibility that stem-cell For several presenting researchers, stem cells took cenlercenter depletion causes many of progeria's phenotypes.phenotypes, IrinaIrina stage for two reasons. First, there is the question of whether Conboy (University of California at Berkeley) was invitedinvited toto or not progeria is a disease characterized by stem-cell present her work on mechanisms causing thethe decline of depletion.depletion, wherebywhcreby there is an inadequate supply ofor stem tissue regenerative potential inin normal aging skeletalskeletal muscle cells for replenishing prematurely dying mesenchyme in response to injuryinjury and on regeneration using embryonic derived cell populations (VSCM, adipocytes, llbroblasts, stem cells,cells. She demonstrated both thatthat a youthful bio and osteocytes). Second, the lack of laminlam in A expression chemical milieu can restore cell vitality, and thatthat old cells in immature cells presents stem-cell therapy as a viable have regenerative capacity that can be tapped.tapped. strategy forlor treatment,treatment. or even reversal, of disease in Skeletal muscle has its own source of stem cells, called progeria. The therapeutic potential of mesenchymal stem satellite cells. Satellite cells are locatedlocated atat thethe basementbasement cells is being tested in numerous clinical and preclinical membrane of terminallyterminally dilTerentiateddi/Terentiated muscle fibersfibers andand cancan trials (42). be identifiedidentified and purified. Dr. ConConboyboy has carried outout bothboth In his introductory presentation, Huber Warner (Univer in vivo and inin vitro experiments inin which stemstem cellscells Ii'omfrom sity orof Minnesota) advanced his hypothesis that depletion of young animals were exposed toto niche clements fromfrom oldold stem cells is a major factorhlCtor in causing the tissue changes animals and vice versa (51). She has discovered thatthat thethe observed in progeria (43). The depletion may arise from the decline of regenerative capacity with age resultsresults li'omfrom increased turnover of differentiated cells, which are lost at changes in the stem cell niche, not inin thethe stemstem cells an accelerated rate (44). This hypothesis has been further themselves,themselves. A key element isis age-relatedage-related impairmentimpairment ofof thethe analyzed in the literature. Prolla (45) suggested lhatthat thethe up-regulation of thethe Notch ligandligand Delta afterafter muscle injuryinjury regenerative capacity of tissues with high turnover might be (52). Exposure of satellitesatellite cells fromfrom old mice toto youngyoung reduced due to the exhaustion ofor progenitor cells. serum enhanced thethe expression or Notch ligandligand Delta asas Mesenchymal stem cells are believed to be involved inin well as proliferation and regenerativeregenerative capacitycapacity of agedaged expression of symptoms of progeria.progeria, since most mesenchy satellite cells. Forced activation of Notch byby addingadding mal stem cells express lamins AICA/C (46), while some a Notch-speciflc antibody restoredrestored regenerativeregenerative potentialpotential toto committed hematopoietic cell lines do not (47). Gotzmann old muscle. and Foisner (48) point out lhatthat deregulated or impaired Dr. Conboy has extended her studiesstudies toto examine thethe tissue regulation and deregulated cell-type plasticity might eflcctseflects of aged systemicsystemic mileu onon thethe functionrunction ofof humanhuman account for nearly all pathological conditions detected inin embryonic stemstem cellscells (hESCs)(hESCs) (53).(53). Aged mousemouse serumserum laminopathies. IIalaschek-WienerlIalaschek-Wiener and Brooks-Wilson (49) dramatically inhibitedinhibited thethe self-renewalself-renewal andand proliferativeproliferative argue that depletion of mesenchymal stem cells can help potential of hESCs and satellitesatellite cells,cells, asas measured byby account for the speciflcspecific segmental nature of progeria. expression of markers of stem-cellstem-cell functionality,functionality, raterate ofof cellcell The current published literature supports the view thatthat proliferation, and thethe capacity forfor myogenic differentiation.differentiation. lamin A, and hence progerin, is produced by differentiated When hESCs are cultured inin serum-freeserum-free mcdiummedium insteadinstead of cells, and undiiTerentiatedundifTerentiated cells do not transcribe or translate immediateimmediate transfertransJCr toto medium containing agedaged serum,serum, thethe either molecule. However, Carlos Lopez-Orin (Universidad cells are no longerlonger susceptible toto thethe effects of agedaged scrum.serum. de Oviedo, Spain) described experimentsexperiment.s inin which hair This impliesimplies thatthat hESCshESCs havehave producedproduced oneone oror moremore antianti fallfollicle-derived ide-derived stem cells are severely affected by defects inin aging factors.factors. 1 the Jaminlamin processing pathway. His Zmpste24-Zmpste24~1"· progeria Together.Together, thesethese experimentsexperiments suggestsuggest thatthat thethe regenerativeregenerative mouse model (50) produces no mature laminlamin A and suffers outcome of stem-cellstem-cell replacementreplacement will bebe determineddetermined byby from a build-up of famesylated prelamin A. The homozy a balance between negativenegative effectseffects of agedaged tissuestissues onon gous null mouse displays many features of human transplantedtransplanted cells and positivepositive effectseffects of embryonicembryonic cellscells onon laminopathies, including severe growth retardation and thethe endogenous regenerativeregenerative capacity.capacity. This resultresult indicatesindicates premature death, dilated cardiomyopathy (DCM), muscular thatthat a complex interplayinterplay between negativenegative regulationregulation byby thethe 784 GORDON E1' AL.At. aged niche and positiveposItive regulation by hEseshESCs will likely is incorporated into prelamin 10to yield a geranylgeranylateclgeranylgeranylated determine the success of hESe-basedhESC-based cell replacement intermediate. He also finds that Zmpste24 does cleave therapies. Dr. Conboy's observations in striated muscle pro both famesylated and gcranylgeranylated prelamin A, and vide a well-tested pathway for studying the key questions or therefore mature lamin A is produced even in the complete the role of stem cells and aging in progeria, and arcare easily absence ofor farnesylation. However, he llndsfinds that the adaptable to the progeria mouse model. farnesylated prelamin A is a 20-fold bellerbeHer substrate than Tom M;steliMisteli and PaolaPaala 5;cqlfidiSNdfidi (National Cancer In geranylgeranylated prelamin A. Therefore,ThcrcCore, processing of stitute) presented the lirstfirst evidence supporting the hypoth geranylgeranylatedgeranylgeranylaled prelamin A is slow, resulting in esis that stem-cell depletion is a factorractor in organismal abnormal accumulation of nonfarnesylated prelaminprclamin A symptoms orof progeria (53). The first stcpstep of their approach within cells. ProgerinProgcrin also became geranylgeranylated in was to determine what gene expression programs are cells treated with FTI, creating a new prenylated protein. affected by progcrin.progerin. The experimental system consisted Prelamin A accumulation can afrectaffect cell cycling (55). There of an imm0l1alized skin fibroblast cell line that expresses is a paucity orof data available comparing the behavior of inducible green fluorescent protein (GFP) progerin. In this geranylgeranylated versus farnesylated prelamin A or system, signiJkant progerin accumulation was flrst(Jrst observed progerin, and this area of research may be essential to 5 days after induction and reached a plateau after 10 days at designing Futurefuture treatments for progeria since altemative levels similar to those found in cells rromfrom progeria patients. prenylation can alter the behavior and function of cells (56). This111is system allowed analysis of time-dependent changes in In a set of experiments with tremendous breadth, Car/osCarlos transcriptional proiliesproflles in response to progerin expression Lopez-OtinLope::.-OtiJl examined the potential role of lamin intennedi and compared to expression of GFP-tagged wild-type lamin ates in disease, roundfound support for alternative prenylation of A as a control. prelamin A,A. and effectively treated a progeria mouse model Among genes known to influence differentiation, pro using combination chemotherapy that intenuptsinten-upts progerin gerin induction led to up-regulation of several genes in the production at multiple points (57). The effects of lamin Notch signaling pathway, most prominently Hes I,I. HesHe... 5,5. intetmediatesintelmediates were first addressed. Dr. Lopez-OlinLopez-Otin created and Hey I. This up-regulation was also found in progeria a "triple mutant" mouse model lhatthat has the complete cells. Consistent with a direct role of nuclear progerin in absence of both the Zmpste endonuclease and farnesyl their regulation, these genes are all in the downstream transferase postnatally and is therefore the genetic equiva portion of the Notch pathway, and no increase was found in lent of complete famesyltransferase inhibition. I-Ience,Hence, only cleaved Notch expression or in the upstreamupslream cleavage nonfarnesylated prelamin A is created in this mouse after reaction. This result dovetailed with Dr. Conboy's observa bilih.bil1h. Strikingly, the phenotype of these mice mimicked the tions that Notch is importantimporlant for maintenance of stem cells Zmpste24-1---only mice, without phenotype rescue. Dr. (including mesenchymal stem cells) and differentiation Lopez-OlinLopez-Otin hypothesized that the phenotype was caused pathways, and points to the Notch pathway as a key either by the accumulation of nonfarnesylated prelamin A,A. elementelemenl in progeria. or by an alternatively prenylaledprcnylated prelamin A product. To Drs. Misteli and ScarndiScaffidi fUliherfUl1hcr explored the effects of resolve this issue, he tumedfumed to cell culture, and cletell11inedcletemlined 1 progerin on differentiation by inducing stem cells to go down which species of prelamins were present in Zmpste24-1 various differentiation pathways (54). Preliminary results cells with and withoutwithollt FTI treatment. As expected. 1 showed thatthat thethe rate of osteogenesis was increased, adipo famesylated prelamin A was presentpresenl in Zmpste24- - cell genesis was blocked, and chondrogenesis was not affected. extracts. However, when FYI was added to Zmpste24-1- cell They hypothesize thatthat progeria leads to the activation of part cultures, the amount of famesylatedfarnesylated prelamin A decreased, of thethe Notch pathway thatthat causes differentiation,differentiation. and that and geranylgeranylated prelamin A appeared. By extrapo accelerated aging inin progeria patients is largelylargely the result orof lation from FTI treated Zmpste24-1- cultured cells to1.0 triple stem-cell dysfunction. mutant mice, he h~~?thesizedhy~othesized thatIhat theIhe absence orof farnesyl'arnesy lation in Zmpste24 '"--,-, mice does not improve the phenotype in vivo because geranylgeranylated prelamin A is created,created. ALTERNATIVE PI{ENVLATIONPRENVLATION AND THE NEED FORFOI~ and this product may be alat least partly responsible for COMBJNATIONCOMnJNATION CHEMOTHEUAPV ININ PROGERIA disease phenotype in thethe triple mutant mice. 1 Posttranslational processing toto create laminlamin A involvesinvolves Dr. Lopez-Otin went on to treat Zmpste24-1- mice with several fon11sfonns of prelamin A, both farnesylatedfarnesylated and two commercially available drugs, statinsstalins and bisphosphohisphospho unfarnesylated (Figure(Figure 1). FYI treatmenttreatment presumably leadsleads nates. These drugs block two diFferentdifferent steps in the toto unusually increasedincreased cellular levelslevels of nonfarnesylatednonl~lrnesylated mevalonate pathway, upstream from blrnesyltransferasef~lrnesyltranslerase f0I111sfor111s of prelamin A and progerin, and could theoreticallytheoretically and geranylgeranyltransferase (Figure 2). Treatment of F promote alternative prenylation (geranylgeranylation).(geranylgeranylation). The Zmpste24- - mice with statins and hisphosphonaleshisphosphonates in influencesinfluences of nonfarnesylatedllonfarnesylated prelamin A and progerin on creased theirtheir 20-week life span by 80%. There was also cellcell functionfunction andand disease statusstalus have yet toto be intenselyintensely impressiveimpressive rescuerescue of bone phenotypes, and cellular blebbing explored.explored. Michael SinenskySilll'Jlsky (East(East Tennessee State Univer was substantially reducecl.reduced. FU11her research is needed to sity)sity) has begun toto examineexamine alternativealtemative prenylation (ger(ger understand thelhe mechanism responsibleresponsible for extended lifeIi fe anylgeranylation)anylgeranylation) of preJaminprelamin A and progerin under thethe span inin treatedtreated mice, since bisphosphonateshisphosphonates home specifi influenceinfluence of FYI.FTI. He findsfinds thaI,that. inin cells treatedtreated with an FTI,FTL cally toto bone. However, Dr. Lopez-Otin presents us with thethe severalseveral importantimportant cellularcellular eventsevents occur: First, geranylgeranyl Ilrstfirst preliminary evidence thatthat combination chemotherapy HIGHLIGHTS OF THE7lfE 2007 PNFPIiF SCIENTIFIC WORKSHOPWOIiKSHOP 785 using FTI along with drugs Ihalthat affect both progerin J\cpfvICo;\' A POWERFlJL NEW TESTING STI~ATEGYSTRATEGY FOR 11i\'\c;·CoA DRUG DISCOVERY IN PROGERIA ;pel",,,,,,,,, )jf---- Drug discovery is an important direction to pursue in progeria. especially in light of the possibility that drugs thai help these children may also be benetlcialbeneficial to the nonnal!lonnal aging population and a large fraction of men and women afflicted with cardiovascular disease. High-throughput ~ screening (HTS) is an essential first step in drug de 1~()p('nt('n\,ll~op(,nt('nyl··IlP• I'P velopmcnt.velopment. The key to10 HTS is to develop an assay that ~t easily detectsdetccts some visible change in the target cells, due to Cf'lrlllylCerClnyl··PP PP exposure to a chemical compound that can be quickly and reliably read by a sensor. Nuclear blebbing has been used to rH'P f' f' '"'' :L~'~J:;i;;;~r;-i;~;;(~:I~.~] measure improvement in progeria cells in response to """0jl- various drugs such as FTIs.FYIs. However, for testing chemical l-iJn)p~)'!11 ,I 1l',1(' 1'/)\( libraries on a large scale, such morphological assays are V ,'"-'--.... "'-':-"~"''''-~''-'"l unsuitable. Tom Misteli and CordeJle Tanega (National rdrtl("\vl PI' ~ ,FilrnesY!illed protein~: Cancer Institute) have developed what could be a break ",.,...J--l i(e~Q.l.IJllil1 A} i through testing strategy. The Misteli laboratory has pre ~rTr~, viously showed that blocking the cryptic splice site that is / activated by the progeria mutation with a complementary ':.qll Whether this will translate into some, but nolnot complete, Gilford progeria .~yndrollle.syndromc, Proc NaifNal! !lead!lead SciSci USAUSA. 2005:2005;102: 102: clinical improvement with FTlFTI remains to be determined. 12879-12884.12ii79-12ii84. 10. 10. Glynn MW.MW, Glover TW. IncompleteIncomplete processingprocessing ofof mutan[mutant laminlam in A inin Pivotal j~lCtorS for disease phenotype that emerged during lIutchinson-GilfordHutchinson-Gilford progeria leadsleads toto nuclearIluclear abllorllla[itie~,abnormalities, which arcare this workshop included the balance between multiple reversed hyby farncsyhransferasefarnesyltransferase inhibition.inhibition. HI/m MotMo! Gelll'1GI'I/I'I. 2005:2005: prclaminprelamin A [011115,fOllllS, mature lamil1lamin A, and progerin.progerin, the 14:2959-2969. presence of alternative prenylation. and epigenetic changes III. I. Yang 51-1,SH, Bergo MO, TolhToth Jl,JI, etet al.al. Blocking protcmprotein l"al1le~y1transferr 32.:12. Ly DJ-I.DH. Lockhar[Lockhart OJ.DJ. Lerner RARA. Schu1t7Schultz PO. Mitotic lllisregillationmisrcgllialion 47. RoherRober RA. SallterSauter H.J-1, Weber K, OsbolTlOsbom M.M. Cells ofof thethe cellularcellular immuneimmune and human aging. SCle/lcc.Sciellce. 2000;2X7:24R6-2492. and hemopoietic syslemsystem of thelhe mouse lacklack laminslamins A/C: di.~tinctiondistinction 3:1.:1:1. ScaffidiScaffidI P. MisleliMislelr T.T- LaminLaIl11n A-dependent nuclear defect."defects IIIin humanIllllllan versus other somatic celkcells. J.1 CellCel! 5ici.S'd. J1990:95(990:95( p[Pt 4):587-598.4):587-598. aging. Scicl1ce.SClenct'. 2006;112:1059-1063.200(dI2:1059-1063. 48. 48. GO[l.mann(JotI.1l1