Mandibuloacral dysplasia type A-associated progeria caused ...
Highlights of the 2007 Highlights ofthe 2007 Progeria ... · Highlights ofthe 2007 Progeria...
11
)'''"',,,,1 '4' ('(I'IJiJ(lIlog,\'.' [IIOLOGICAL SCIDVCES Vol. 11.1;\, Nn. R. Tn·'m7 Meeting Report Highlights of the 2007 Progeria Research Foundation Scientific Workshop: Progress in Translational Science Leslie B. Gordon,' Christine J. Harling-Berg,2 and Frank G. Rothman 2 IWarren Alpert Medical School of Brown University, 2 Srown University, Providence, Rhode Island. I N the spring of 2007, a 2-year clinical drug trial began at Children's Hospital Boston (I) with the goal to advance the quality of life for children with Hutchinson-Gilford progeria syndrome (henceforth progeria), a rare (frequency I in 4 million), multisystem, and inevitably disease that claims young lives due to myocardial infarctions and strokes between ages 7 and 20 years (2). The journey to the first clinical trial for progeria has been facilitated by a series of collaborative scientific workshops organized by The Progeria Research Foundation (PRF) and supp0l1ed by agencies interested in aging, cardiovascular disease, rare disease research, and genetics (http://www.progeriaresearch. org/2007_prl"-workshop_on_progeria.html). These meet- ings have provided a concentrated forum to facilitate the collective thinking of clinicians and scientists about progeria, forge collaborations in this little-known field, and accelerate the discovery of new ways to push the field forward toward treatments and cure. The first PRF Scientific Workshop in 2001 helped to identify nuclear blebbing as an imp0l1ant phenotypic marker of progeria cells (noted by Anthony Weiss; University of Sydney, Australia), and to recognize a translocation on chromosome 1 in the cells cultured from a progeria patient (W. Ted Brown, New York State Institute for Basic Research in Developmental Dis- abilities), pointing geneticists in the direction of the gene mutation for progeria (3). In 2003, the second workshop was held just 3 months after publication of the gene defect in progeria, which is typically a sporadic autosomal domi- nant disease caused by a C --, T mutation at nucleotide 1824 of the LMNA gene encoding lamin A (4,5). Importanlly, the mutation results in a persistence of an aberrant form of a famesylated-prelamin A molecule (4) now called progerin. The attendees now included expelts in the field of lamin biology and laminopathies. The 2004 Progeria Workshop, held within the National Human Genome Research Institute (NHGRI), was held specifically to discuss the role of stem cells and the potential for stern cell transplantation in progeria, an area of continuing interest for the field. At the general Progeria Workshop in 2005, two mouse models of progeria were unveiled and, although they contained iden- tical mutations, they yielded very different phenotypes, mimicking various pOltions of the human disease (6,7). Early in vitro data on a potential role of famesy!transferase inhibitors (FTIs) in treating progeria were presented by four laboratories, demonstrating that FTl could reverse nuclear blebbing in cells expressing progerin (8-11). Armed with data demonstrating that treatment of newly developed cellular assays with FTIs improves or reverses some of the effects of progerin accumulation, a clinical trial treating children with progeria with an FTI was initiated in May 2007 (I) (www.c1inicaltrials.gov ). The present article, detailing the 2007 PRF Scientifk Workshop (November 12-14, Boston), emphasizes the complexities of progerin and lamin A biology, and the need to continue 10 look for the most effective protocols for correcting the effects of the lamin A defect in progeria. All speakers presented preliminary data that had not been peer- reviewed at the lime of presentation. In the months since the workshop, some data have been published (referenced within this article), and we look forward to additional peer- reviewed publications on the work presented at this meeting over the coming year. The essence of translational research was represented in the presentations, which brought forth the most impOltant issues in progeria today-the effects of progerin and lamins and their binding partners on the functioning of cells, sys- tems, mouse models, and humans; the connection between progeria, aging, and cardiovascular disease in the general population; careful analysis of ITI effects on progeria at all levels (summarized in Table 1); and strategies for future disease treatments and cure. In his introduction at the work- shop, Francis Collins (Director, NHGRI) noted that a 4-year period between gene discovery and a clinical drug trial is . an unprecedentedfeatfor a rare genetic disease." For progeria patients and their families, who have struggled to live within a void of infonnation, the clinical trial represents ... a source of light coming up after a long, long night" and hope has replaced" ... a sense of fear and helplessness on each passing birthday" (Subbarao, father of Priya, a 15- year-old with progeria). GETTING TO KNOW THE CHILD WITH PROGERIA For a disease as rare as progeria, scientists often have no exposure to the very population they wish to serve. During 777 .!,)jIl'JI,iI "/ [IIOLOG/C,-\L SClD\'CE,\ (oJJ\Tll.!hJ 2(UH,' h\' Jhe' (ji>1'OHld/Ol!II'(J! ,)Or h'l\ pi -\merl/'" Vol 61;\, N" Tn- 7';J,7 Meeting Report Highlights of the 2007 Progeria Research Foundation Scientific Workshop: Progress in Translational Science Leslie B. Gordon,' Christine J. Harling-Berg,2 and Frank G. Rothlnan 2 IWarren Alpert Medical School of Brown University, 2 Srown University, Providence. Rhode Island. I N the spring of 2007, a 2-year clinical drug trial began at Children's Hospital Boston (I) with the goal to advance the quality of life for children with Hutchinson-Gilrord progeria syndrome (henceforth progeria), a rare (frequency I in 4 million), multisystem, and inevitably disease that claims young lives due to myocardial infarctions and strokes between ages 7 and 20 years (2). The journey to the first clinical trial for progeria has been facilitated by a series of collaborative scientific workshops organized by The Progeria Research Foundation (PRF) and supp0l1ecl by agencies interested in aging, cardiovascular disease, rare disease research, and genetics (http://www.progeriaresearch. org/2007 _prl'- workshop_on_progeria.html). These meet- ings have provided a concentrated forum 10 facilitate the colleclive thinking of clinicians and scientists about progeria, forge collaborations in this lillie-known field, and accelerate the discovery of new ways to push the field forward towarcltreatments and cure. The Ilrst PRF Scientific Workshop in 2001 helped to identiry nuclear blebbing as an impol1ant phenotypic marker of progeria cells (noted by Anthony Weiss; University of Sydney, Australia), and to recognize a translocation on chromosome I in the cells cultured from a progeria patient (W. Ted Brown, New York State Institute for Basic Research in Developmental Dis- abilities), pointing geneticists in the direction of the gene mutation for progeria (3). In 2003, the second workshop was held just 3 months after publication of the gene defect in progeria, which is typically a sporadic autosomal domi- nant disease caused by a C --). T mutation at nucleotide 1824 of the LMNA gene encoding lamin A (4,5). Importantly, the mutation results in a persistence or an aberrant form of a ramesylaled-prelamin A molecule (4) now called progerin. The attendees now included expelts in the field of lamin biology and laminopathies. The 2004 Progeria Workshop, held within the National Human Genome Research Institute (NHGRI), was held specifically to discuss the role of stem cells and the potential for stem cell transplantation in progeria, an area of continuing interest ror the field. At the general Progeria Workshop in 2005, two mouse models of progeria were unveiled and, although they contained iden- tical mutations, they yielded very different phenotypes, mimicking various p0l1ions of the human disease (6,7). Early in vitro data on a potcntial role of famesyltransferase inhibitors (FTls) in treating progeria were presented by four laboratories, demonstrating that FTI could reverse nuclear blebbing in cells expressing progeri n (8-1 I). Armed with data demonstrating that treatment of newly developed cellular assays with FTls improves or reverses some of the errects of progerin accumulation, a clinical trial treating children with progeria with an FTl was initiated in May 2007 (I) (www.c1inicaltrials.gov). The presenl article, detailing the 2007 PRF Scientifk Workshop (November 12-14, Boston), emphasizes the complexities of progerin and lamin A biology, and the need to continue to look for the most effective protocols ror correcting the effects of the lamin A defect in progeria. All speakers presented preliminary data that had not been peer- reviewed al the time of presentation. In the months since the workshop, some data have been published (referenced within this article), and we look forward to additional peer- reviewed publications on the work presented at this meeting over the coming year. The essence of translational research was represented in the presentations, which brought forth the most impOltant issues in progeria today-the efrects or progerin and lamins and their binding partners on the functioning of cells, sys- tems, mouse models, anel humans; the connection between progeria. aging, and cardiovascular disease in the general population; careful analysis of FTI effects on progeria at all levels (summarized in Table 1); and strategies for future disease treatments and cure. In his introduction at the work- shop, Francis Collins (Director, NHGRI) noted that a 4-year period between gene discovery and a clinical drug trial is " " ... an IlJlprecedented./£>a1/or a rare genetic disease." For progeria patients and their families, who have struggled to live within a void of infonnation, the clinical trial represents " " ... a source of light coming up after a long, long nighl" and hope has replaced" ... a sense of fear and helplessness on each passing birthday" (Subbarao, of Priya, a 15- year-old with progeria). GETTING TO KNOW THE eHlU) WITH PROGERIA For a disease as rare as progeria, scientists often have no exposure to the very population they wish to serve. During 777
Transcript of Highlights of the 2007 Highlights ofthe 2007 Progeria ... · Highlights ofthe 2007 Progeria...
)'''"',,,,1 '4' ('(I'IJiJ(lIlog,\'.' [IIOLOGICAL SCIDVCES2On~, Vol. 11.1;\, Nn. R. Tn·'m7
Meeting Report
Highlights of the 2007Progeria Research Foundation Scientific Workshop:
Progress in Translational Science
Leslie B. Gordon,' Christine J. Harling-Berg,2 and Frank G. Rothman 2
IWarren Alpert Medical School of Brown University,2Srown University, Providence, Rhode Island.
I N the spring of 2007, a 2-year clinical drug trial began atChildren's Hospital Boston (I) with the goal to advance
the quality of life for children with Hutchinson-Gilfordprogeria syndrome (henceforth progeria), a rare (frequencyI in 4 million), multisystem, and inevitably l~llal diseasethat claims young lives due to myocardial infarctions andstrokes between ages 7 and 20 years (2). The journey to thefirst clinical trial for progeria has been facilitated by a seriesof collaborative scientific workshops organized by TheProgeria Research Foundation (PRF) and supp0l1ed byagencies interested in aging, cardiovascular disease, raredisease research, and genetics (http://www.progeriaresearch.org/2007_prl"-workshop_on_progeria.html). These meetings have provided a concentrated forum to facilitate thecollective thinking of clinicians and scientists aboutprogeria, forge collaborations in this little-known field,and accelerate the discovery of new ways to push the fieldforward toward treatments and cure. The first PRF ScientificWorkshop in 2001 helped to identify nuclear blebbing as animp0l1ant phenotypic marker of progeria cells (noted byAnthony Weiss; University of Sydney, Australia), and torecognize a translocation on chromosome 1 in the cellscultured from a progeria patient (W. Ted Brown, New YorkState Institute for Basic Research in Developmental Disabilities), pointing geneticists in the direction of the genemutation for progeria (3). In 2003, the second workshopwas held just 3 months after publication of the gene defectin progeria, which is typically a sporadic autosomal dominant disease caused by a C --, T mutation at nucleotide 1824of the LMNA gene encoding lamin A (4,5). Importanlly, themutation results in a persistence of an aberrant form ofa famesylated-prelamin A molecule (4) now called progerin.The attendees now included expelts in the field of laminbiology and laminopathies. The 2004 Progeria Workshop,held within the National Human Genome Research Institute(NHGRI), was held specifically to discuss the role of stemcells and the potential for stern cell transplantation inprogeria, an area of continuing interest for the field. At thegeneral Progeria Workshop in 2005, two mouse models ofprogeria were unveiled and, although they contained identical mutations, they yielded very different phenotypes,mimicking various pOltions of the human disease (6,7).
Early in vitro data on a potential role of famesy!transferaseinhibitors (FTIs) in treating progeria were presented by fourlaboratories, demonstrating that FTl could reverse nuclearblebbing in cells expressing progerin (8-11). Armed withdata demonstrating that treatment of newly developedcellular assays with FTIs improves or reverses some of theeffects of progerin accumulation, a clinical trial treatingchildren with progeria with an FTI was initiated in May 2007(I) (www.c1inicaltrials.gov ).
The present article, detailing the 2007 PRF ScientifkWorkshop (November 12-14, Boston), emphasizes thecomplexities of progerin and lamin A biology, and theneed to continue 10 look for the most effective protocols forcorrecting the effects of the lamin A defect in progeria. Allspeakers presented preliminary data that had not been peerreviewed at the lime of presentation. In the months since theworkshop, some data have been published (referencedwithin this article), and we look forward to additional peerreviewed publications on the work presented at this meetingover the coming year.
The essence of translational research was represented inthe presentations, which brought forth the most impOltantissues in progeria today-the effects of progerin and laminsand their binding partners on the functioning of cells, systems, mouse models, and humans; the connection betweenprogeria, aging, and cardiovascular disease in the generalpopulation; careful analysis of ITI effects on progeria at alllevels (summarized in Table 1); and strategies for futuredisease treatments and cure. In his introduction at the workshop, Francis Collins (Director, NHGRI) noted that a 4-yearperiod between gene discovery and a clinical drug trial is
. an unprecedentedfeatfor a rare genetic disease." Forprogeria patients and their families, who have struggled tolive within a void of infonnation, the clinical trial represents... a source of light coming up after a long, long night"
and hope has replaced" ... a sense of fear and helplessnesson each passing birthday" (Subbarao, father of Priya, a 15year-old with progeria).
GETTING TO KNOW THE CHILD WITH PROGERIA
For a disease as rare as progeria, scientists often have noexposure to the very population they wish to serve. During
777
.!,)jIl'JI,iI "/ (;")'J)IJI"'{)~\ [IIOLOG/C,-\L SClD\'CE,\ (oJJ\Tll.!hJ 2(UH,' h\' Jhe' (ji>1'OHld/Ol!II'(J! ,)Or h'l\ pi -\merl/'"
2nn~, Vol 61;\, N" ~, Tn- 7';J,7
Meeting Report
Highlights of the 2007 Progeria Research Foundation Scientific Workshop:
Progress in Translational Science
Leslie B. Gordon,' Christine J. Harling-Berg,2 and Frank G. Rothlnan 2
IWarren Alpert Medical School of Brown University, 2Srown University, Providence. Rhode Island.
I N the spring of 2007, a 2-year clinical drug trial began at Children's Hospital Boston (I) with the goal to advance
the quality of life for children with Hutchinson-Gilrord progeria syndrome (henceforth progeria), a rare (frequency I in 4 million), multisystem, and inevitably l~ltal disease that claims young lives due to myocardial infarctions and strokes between ages 7 and 20 years (2). The journey to the first clinical trial for progeria has been facilitated by a series of collaborative scientific workshops organized by The Progeria Research Foundation (PRF) and supp0l1ecl by agencies interested in aging, cardiovascular disease, rare disease research, and genetics (http://www.progeriaresearch. org/2007_prl'-workshop_on_progeria.html). These meetings have provided a concentrated forum 10 facilitate the colleclive thinking of clinicians and scientists about progeria, forge collaborations in this lillie-known field, and accelerate the discovery of new ways to push the field forward towarcltreatments and cure. The Ilrst PRF Scientific Workshop in 2001 helped to identiry nuclear blebbing as an impol1ant phenotypic marker of progeria cells (noted by Anthony Weiss; University of Sydney, Australia), and to recognize a translocation on chromosome I in the cells cultured from a progeria patient (W. Ted Brown, New York State Institute for Basic Research in Developmental Disabilities), pointing geneticists in the direction of the gene mutation for progeria (3). In 2003, the second workshop was held just 3 months after publication of the gene defect in progeria, which is typically a sporadic autosomal dominant disease caused by a C --). T mutation at nucleotide 1824 of the LMNA gene encoding lamin A (4,5). Importantly, the mutation results in a persistence or an aberrant form of a ramesylaled-prelamin A molecule (4) now called progerin. The attendees now included expelts in the field of lamin biology and laminopathies. The 2004 Progeria Workshop, held within the National Human Genome Research Institute (NHGRI), was held specifically to discuss the role of stem cells and the potential for stem cell transplantation in progeria, an area of continuing interest ror the field. At the general Progeria Workshop in 2005, two mouse models of progeria were unveiled and, although they contained identical mutations, they yielded very different phenotypes, mimicking various p0l1ions of the human disease (6,7).
Early in vitro data on a potcntial role of famesyltransferase inhibitors (FTls) in treating progeria were presented by four laboratories, demonstrating that FTI could reverse nuclear blebbing in cells expressing progeri n (8-1 I). Armed with data demonstrating that treatment of newly developed cellular assays with FTls improves or reverses some of the errects of progerin accumulation, a clinical trial treating children with progeria with an FTl was initiated in May 2007 (I) (www.c1inicaltrials.gov).
The presenl article, detailing the 2007 PRF Scientifk Workshop (November 12-14, Boston), emphasizes the complexities of progerin and lamin A biology, and the need to continue to look for the most effective protocols ror correcting the effects of the lamin A defect in progeria. All speakers presented preliminary data that had not been peerreviewed al the time of presentation. In the months since the workshop, some data have been published (referenced within this article), and we look forward to additional peerreviewed publications on the work presented at this meeting over the coming year.
The essence of translational research was represented in the presentations, which brought forth the most impOltant issues in progeria today-the efrects or progerin and lamins and their binding partners on the functioning of cells, systems, mouse models, anel humans; the connection between progeria. aging, and cardiovascular disease in the general population; careful analysis of FTI effects on progeria at all levels (summarized in Table 1); and strategies for future disease treatments and cure. In his introduction at the workshop, Francis Collins (Director, NHGRI) noted that a 4-year period between gene discovery and a clinical drug trial is "" ... an IlJlprecedented./£>a1/or a rare genetic disease." For progeria patients and their families, who have struggled to live within a void of infonnation, the clinical trial represents
"" ... a source of light coming up after a long, long nighl" and hope has replaced" ... a sense of fear and helplessness on each passing birthday" (Subbarao, (-~Ither of Priya, a 15year-old with progeria).
GETTING TO KNOW THE eHlU) WITH PROGERIA
For a disease as rare as progeria, scientists often have no exposure to the very population they wish to serve. During
777
778 GORDON ET ilL.
Table I. Workshop-Prescnled Studies of Effectiveness of FTIs 011 Progeria Cells*
Presel11er
Collins
COl1lui
Lammcnling
LammcrdingLlmmerding
Paschal
Paschal
7..ou
Sincnsky
Lopez-Otin
Cell(rissue Type
Progeria RAC transgenic HGPS~H
mouse alieries
Progeria fibroblasts and transfertedprogerin-expressing nom1,l) fibroblasts
Progeria fibroblasts
Progeria librobJaslsProgeria fibroblasts
Progeria fibroblasts and transferledprogerin-expressing HcLa cells
L647R cells_, in which cleavage by
Zmpste24 is lacking
Progeria fibroblastsTransfected progcrin-cxpressing
HeLa cellsZmpstc24-I tibroblasts
Cellular Defect
Tissue pathology: progressive loss of
VSMC :md appearance of POOver-expressing progcrin increased rale
of cell death
Incrcnscd nuclear rigidity and early celldel1h al laler passages after
mechanical streIchWound-healing deficit
Proliferation deficit aner nwchanic;]] slreldlNPC abnormalily-TPR mislocalizatioll
Inverted RAN gradient
DSBsFarnesylmed progerin was prcsent
Famesylated prelamin A was present
]T! Effecl
Improved carotid alteries: more VSMC
less PGNo improvement
Revers,l)
Reversal
No improvementReversal
Reversal
No improvemeTl1
Geranylgeranylated progerin appeared
Famesylatcd prelamin A levels
decreased; geranylgeranylmed
prelamill A appeared
NOles: *Data arc listed in order of appearance in article texl.
FTI "-' famesyhransferase inhibitor; BAC hacterial artificial chromosome; HGPS,,-, Hutchinson-Guilford Progeria Syndrome: VSMC vascular smooth muscle
cells; PG progeria; DSBs "-' double-strand breaks; NPC nuclear pore complexes: TPR translocated promoter region.
the first evening of the workshop, participants had a uniquechance to hear from children with progeria and their familiesduring a panel discussion. Dr. Scoff Berns (PRF and WarrenAlpert Medical School of Brown University), the father ofa child with progeria, moderated the family session.Through intellJrcters, Sammy (12 year old from Italy) andPriya (15 year old from India), displayed an uncanny abilityto describe their lives with eloquence, poise, and wisdom~
their frail and aged physical appearance belying their livelyspirits. Priya shared that she enjoys traveling, and Sammyliked to skateboard, swim, and is well assimilated intoschool life. Sammy's parents, Laura and Amerigo, spokeabout how their son"...gives them the strength to keep onand to not give up, and always look at the good side of theday." Sammy has given them an ...opportunity to enjoylife in a different way .... to enjoy every day of your lifewith your child Keith and Molly Moore (U.S.A.),parents or Zachary, who recently passed away, described theselfless traits of their son as well as other children withprogeria, relating how children with progeria ...care somuch for others, and Zach understood that the researchmight not help him directly and personally, but it certainlywould help others" (12). In concluding, young Sammywanted to especially ...thank all the doctors and all theresearchers that are putting such an effort into trying to helpme out and all the other kids with the disease."
DESIGNING THE FIRST CLINICAL DRUG TRIAL
The ultimate goals of disease-related research are to findtreatments and a cure~il1 this case to improve the healthand increase the life spans of children with progeria.Designing a clinical trial that has the greatest chance ofassessing the efficacy of an intervention on the disease ina statistically signillcant manner is a key element in thisprocess. For a disease such as progeria, in which fewer than50 patients are identified worldwide at anyone time, this is
especially challenging. To that end, a clinical trial using FTIto treat progeria is currently being conducted at Children'sHospital, Boston. Several of the trial team memberspresented baseline clinical studies or the major systemsaffected in progeria, trial design, and measures of efficacythat will likely serve as models for future treatment trials inIhe field.
Mark Kieran (Dana Farber Cancer Institute), principalinvestigator of the clinical trial team, reviewed the rationalefor the clinical trial design, drug selection, and parametersfor assessing drug efficacy. The selection of the FTIlonaramib was made on the basis of two lines of evidence:the addition of FTI to progeria l1broblasls reverses nuclearblebbing (8-11), and its low toxicity profile in adults andchildren with cancer (13). However, because 10nafamib hasnot previously been administered to children with progeria,it will be impOltant to carefully track the toxicity profile,pharmacokinetics, and efficacy or lonafarnib in a variety oforgan systems to dete1111ine whether there is any benefit tousing this FTI in progeria clinically. To quantitate the effectof drug treatment in a measurable way, Dr. Kieran stressedthe importance of first documenting the natural history ofthe disease, including an understanding of the breadth andvariability in disease phenotype from patient to patient.Because the number of children with progeria is often toosmall to see population-based changes, for some clinicalmeasures each child must serve as his or her own control forassessing drug efrect, comparing pretreatment measures toposttreatment measures. Because disease in progeria isprogressive, any measures improving with treatment wouldbe considered drug-related changes. The primary clinicalparameter for measuring treatment effkacy is rate of weightgain, which is linear for each child after approximately age 2years in progeria (14), and therefore provides an opportunityto assess whether FTI affects rate of weight gain over a 2year treatment period. Secondary parameters of efficacy
778 GORDON ET AI.,.
Table I. Workshop-Presenled Studies of Effectiveness of FTIs on Progeria Cclls*
Presenter
Collins
Comai
Lammerdmg:
Lallllllcniing Llrl1menllng Paschal
Paschal
ZOll Sinensky
Lope7-0tin
Cell(risslle Type
Progeria RAC transgenic HOpS-I-" mouse aliencs
Progeria fibroblasts and transfected progerin-expressing noml,l! nbroblast~
Progeria fibroblasts
Progeria libroblasls Progeria fihroblash Progeria fibroblasts and lransfecled
progerin-ex press ing He La cc IIs Lf)lnR celL'>. III which cleavnge by
Zmpste24 is lackmg Progeria fibroblasts TrnnsfCcled progcrin-expressillg
HeLn cells Zmpslc24 -I fibroblas!.;
Cellular Defect
TI"sue palhology: progressive loss of VSMC :ll1d appearance of PO
Over-expresslllg progerin increased raIl'
of cell death Increnscd nuclear ngidity and early cell
death al lalcr passages after mcchanlcal slretch
Wound-healing deficil Prohterallon deficit aner flwehanical slreldl NPC abnormailly-TPR mi"locaJlza!loll
Inverled RAN gradIenl
DSBs Farnesyl,l1ed progcnn was present
Famesylaled prclamin A was preseJl1
ITl Effect
Improved carotid al1enes: more VSMC. less PC;
No improvement
Revers,l!
Reversal No Improvcment Reversal
Reversal
No improvement Geranylgeranylated progel"lll appeared
Famcsylaled prelamin A leveb decreased: gerallylgeranylaled prclamin A appeared
Not('s: *Data arc li'itcd III order of appc<lrance 111 article lexl. FTI - famesyllransfcrasc inhibItor; BAC _cc haclel"lal artiJiei,,1 chromosome: HGPS - HUlchmson-Guilford Progeri" Syndrome: VSMC == vascular smoolh muscle
cells: PO == progeria: J)SB~ -=- double-strand breaks; NPC " Iluclear pore complexes: TPI~ =-=- translocaled promoter reglOll.
the first evening of the workshop, participants had a unique chance to hear li"OITI children with progeria and their families during a panel discussion. Dr. Scotf Berns (PRF and Warren Alpert Medical School of Brown University), lhe father of a child with progeria, moderated the family session. Through inlellJreters, Sammy (12 year old from Italy) and Priya (15 year old ti"om India), displayed an uncanny ability to describe their lives with eloquence, poise, and wisclomtheir frail and aged physical appearance belying their lively spirits. Priya shared that she enjoys traveling, and Sammy liked to skateboard, swim, and is well assimilated into school life. Sammy's parents, Laura and Amerigo, spoke about how their son"...gives them the strength to keep on and to not give up, and always look at the good side or the day." Sammy has given them an ""." .opportunity to enjoy life in a different way .... to enjoy every day of your life with your child ... ". Keith and Molly Moore (U.S.A.), parents of Zachary, who recently passed away, described the selfless traits of their son as well as other children with progeria, relating how children with progeria "" ...care so much for others, and Zach understood that the research might not help him directly and personally, but it certainly would help others'· (12). In concluding, young Sammy wanted to especially "" ...thank all the doctors and all the researchers that are putting such an effort into trying to help me out and all the other kids with the disease."
DESIGNING THE FIRST CLINICAL DRUG TRIAL
The ultimate goals of disease-related research are to find treatments and a cure-in this case to improve the health and increase the life spans of children with progeria. Designing a clinical trial that has the greatest chance of assessing the efficacy of an intervention on the disease in a statistically significant manner is a key element in this process. For a disease such as progeria, in which fewer than 50 patients are identified worldwide at anyone time, this is
especially challenging. To that end, a clinical trial using FTI to treal progeria is currently being conducted at Children '."I Hospital, Boston. Several of the trial team members presented baseline clinical studies of the major systems affected in progeria, trial design. and measures of efficacy that will likely serve as models for future treatment trials in the field.
Mark Kieran (Dana Farber Cancer Institute), principal investigator of the clinical trial team, reviewed the rationale for the clinical trial design. drug selection, and parameters for assessing drug efficacy. The selection of the FTf lonafamib was made on the basis of two lines of evidence: the addition of FTI to progeria l1broblasls reverses nuclear blebbing (8-11), and its low toxicity profile in adults and children with cancer (13). However, because lonafamib has not previously been administered to children with progeria, it will be imp0l1ant to carefully track the toxicity profile, pharmacokinetics, and efficacy or lonafarnib in a variety of organ systems to detem1ine whether there is any benefit to lIsing this FTI in progeria clinically. To quantitate the effect of drug treatment in a measurable way, Dr. Kieran stressed the importance of first documenting the natural history of the disease, including an understanding of the breadth and variability in disease phenotype from patient to patient. Because the number of children with progeria is often too small to see population-based changes. for some clinical measures each child must serve as his or her own control for assessing drug effect, comparing pretreatment measures to posUreatment measures. Because disease in progeria is progressive, any measures improving with treatment would be considered drug-related changes. The primary clinical parameter for measuring treatment eflkacy is rate of weight gain, which is linear for each child after approximately age 2 years in progeria (J 4). and therefore provides an opportunity to assess whether FrI affects rate of weight gain over a 2year treatment period. Secondary parameters of efficacy
HIGHLIGHTS' OF TIlE 2007 PI?F SCIENTIFIC WOI?KS/-/OP 779
include measures of abnormality in other well-knownfeatures of the disease, such as alopecia, short slature,subcutaneous !~lt. bone integrity, limb abnormalities.auditory and dental abnonnalities, and cardiovasculardisease. While these features arc important to the diseaseprocess, the ability to statistically assess lrealmenl effect onthese systems is Jess reliable than rate of weight gain.Although cardiovascular disease is clinically the mostimportant determinant of health and longevity in progeria,it is currently not a feasible primary outcome measure fora clinical triaL but is followed as a secondary measure.Some patients in the study have already hacl myocardialinfarctions or strokes, and the damage incurred cannotpredictably be reversed. In addition, the ages for participantsin the trial range from 3 to 16 years. Because the mostaccurate assessment of cardiovascular improvement wouldbe an increase in mean age of death, the clinical trial witha cardiovascular primal)' parameter would require a 10- toIS-year commitment. Hence, rate of weight gain is used asa surrogate marker of general health in progeria.
In addition to rate of weight gain, other more focusedfeatures such as bone heallh could change in a measurableway within the 2-year treatment period. Catherine Gordon(Children's Hospital, Boston) presented the preliminary dataon the bone phenotype for 28 children, measured using twodimensional dual-energy x-ray absorptiometry. Because thebody habitus of children with progeria is drasticallydifferent from their age-matched peers, calculation adjustments must be made for height-age. Remarkably, theclinical histories indicate that children with progeria donot have a higher risk of fracturing their weight-bearingbones, although they are very playful and active. Shesuspects that fracture rates in progeria children are notincreased because the cortex of weight-bearing long bones isof nOl1nal density by x-ray (14), in contrast to other lowweight-disorders such as anorexia nervosa and cysticfibrosis, where fracture rates are increased and cortical boneis thinned on x-ray. In support of this hypothesis, preliminary data demonstrated that bone density z-scores movefrom an uncorrected osteoporotic range of -2 to -4, wherewe would expect some spontaneous bone breaks similar toosteogenesis imperf'ecta, to a height-age cOlTected osteopenic range of' -1 to --2. Further studies of bone quality arein progress, using peripheral qualllitative computed tomography to track the three-dimensional cortical and trabecularcharacteristics with and without FTI treatment
THE CARDIOVASClJLAR PHENOTYPE IN PROGEIHA AND
PROSIJECTS FOR VASClJl,AR TlmATMENT WITH FTICardiovascular events in progeria include hypertension,
myocardial infarction, congestive heart failure, and stroke.Elizabeth Nobel (National Heart. Lung and Blood Institute)and Marie Gerhard-Herman (Brigham and Women'sHospital) discussed recent findings on the vascular phenotype and function in children with progeria. It was theconsensus of both presenting clinicians that the diseaseprogression of progeria blood vessels does not lit the typicalatherosclerotic description that includes: a) preservation orthe vascular smooth muscle cells (VSMC), b) intimal
thickening, c) atherosclerotic plaque (rich lipid core) withruptured cap and superimposed thrombosis, and d) someproliferating smooth muscle cells. Instead, Drs. Nabel andGerhard-Herman propose that progeria vascular phenotyperesembles an arteriosclerosis, which is characterized byhardening of the vessels and vascular stiffness, typicallyseen in the nOl1nal aging populations. Vessel cross-sectionsat autopsy or one 20-year-old patient revealed no indicationof vasculature inHammation (IS). In addition, children withprogeria do not have abnormal serum cholesterol levels,which is a factor in chronic lipid-driven int-lammation (16).Clinical studies at both the National Institutes or Health(NIH) clinical center (17) and Children's Hospital, Boston,revealed that the intima-media thickness of the progeriacarotid artery is normal. Furthennore, in agreement withStehbens and colleagues (I5), Dr. Nabel presented autopsycross-sections from a child with genetically confinnedprogeria, and the histology showed that the v,lscular mediano longer contained smooth muscle cells and the elasticstructure was destroyed and replaced with extracellularmatrix or fibrosis. She hypothesized that the primary loss ofsmooth muscle cells initiates vascular remodeling bysecondary replacement with matrix in large and smallvasculature.
In a study of 28 children with progeria at Brigham andWomen's Hospital (Boston), Dr. Gerhard-Hennan measuredglobal vascular stiffening and, through imaging techniques,observed vessel tortuosity, profound adventitial thickening,and a depleted media layer. For children with an occludedinternal carotid artery, there was an overall loss of thetrilaminar architecture. Functional studies demonstratedhigh femoral pulse wave velocities, and decreased vasodilatory response to nitrous oxide stimulation induced byankle flexion. Overall, the vessel stiffness, tortuosity, andadventitial brightness (the molecular composition not yetidentified), as observed in imaging studies, support a modelof arteriosclerosis similar to what is observed in aging.Remarkably, the blood velocities are in the nonnal rangeand now is still laminar, implying that there is eithera tremendous reserve capacity in the vasculature. or thereare compensatory mechanisms helping to preserve function.Complementary data gathered at the NIH studying a cohOl1of 15 patients revealecl initially n01111al vascular compliancethat decreases with increasing age, and a number of childrendisplayed len atrial enlargement (17). Blood pressure andheart rate were in the normal range in younger children,while the older children had increasing blood pressures andvariable heart rates. Electrocardiograms were normal inmost, although a few had long Q-T intervals suggestingfibrosis of the conduction system. In summary, the progeriavasculature is characterized by global stiffness, tortuosity,and a loss of smooth muscle cells in the media withsubsequent extracellular replacement. Smooth muscle celldropout is unique to progeria, while global stiffness andtortuosity are observed in the nonnal aging population.
The vascular remodeling characteristics in children withprogeria are similar to vascular events observed in a transgenic progeria mouse model that expresses human progerin,created in Francis Collins' laboratOl)' (7). Within thedescending aorta (cross-section histology), smooth muscle
779 HIGHLIGHFS' OF T/-IE 2007 PRF SCIENTIFIC WORKSHOP
include measures of abnormality in other well-known features of the disease, such as alopecia, short stature, subcutaneous l~lL bone integrity, limb abnormalities, auditory and dental abnonnalities, and cardiovascular disease. While these features are important to the disease process, the ability to statistically assess treatmenl ellect on these systems is Jess reliable than rate of weight gain. Although cardiovascular disease is clinically the most important determinant of health and longevity in progeria, it is currently not a feasible primary outcome measure for a clinical trial. but is followed as a secondary measure. Some patients in the study have already had myocardial infarctions or strokes, and the damage incurred cannot predictably be reversed. In addition, the ages for participants in the trial range from 3 to 16 years. Because the most accurate assessment of cardiovascular improvement would be an increase in mean age of death. the clinical trial with a cardiovascular primary parameter would require a 10- to IS-year commitment. Hence, rate of weight gain is used as a surrogate marker of general health in progeria.
In addition to rate of weight gain, other more focused features such as bone heallh could change in a measurable way within the 2-year treatment period. Catherine Gordon (Children's Hospital, Boston) presented the preliminary data on the bone phenotype for 28 children, measured using twodimensional dual-energy x-ray absorptiometry. Because the body habit us of children with progeria is drastically different fi'om their age-matched peers, calculation adjustments must be made for height-age. Remarkably, the clinical histories indicate that children with progeria do not have a higher risk of fracturing their weight-bearing bones, although they are very playful and active. She suspects that fracture rates in progeria children are nol increased because the cortex 01" weight-bearing long bones is of nonnal density by x-ray (14), in contrast to other low weight-disorders such as anorexia nervosa and cystic fibrosis, where lj'acture rates are increased and cortical bone is thinned on x-ray. ln support or this hypothesis, preliminary data demonstrated that bone density ;:-scores move from an uncorrected osteoporotic range of -2 to -4, where we would expect some spontaneous bone breaks similar to osteogenesis imperrecta, to a height-age cOITected osteopenic range of -1 to --2. Further studies of bone quality are in progress, using peripheral quantitative computed tomography to track the three-dimensional cortical and trabecular characteristics with and without FTI treatment.
THE CAIWIOVASCLJLAR PHENOTYPE IN PROGEIUA AND
PRO,'WECTS FOR VASClJLAR Tnl~ATMI~NT WITH FTI Cardiovascular events in progeria include hypertension,
myocardial infarction, congestive heart failure, and stroke. E/izab()th Nobel (National Heart. Lung and Blood Institute) and Marie Gerhard-Herman (Brigham and Women's Hospital) discusscd recent findings on the vascular phenotype and function in children with progeria. It was the consensus of both presenting clinicians that the disease progression or progeria blood vessels does not fit the typical atherosclerotic description that includes: a) preservation of the vascular smooth muscle cells (VSMC), b) intimal
thickening. c) atherosclerotic plaque (rich lipid core) with ruptured cap and superimposed thrombosis, and d) some proliferating smooth muscle cells. Instead, Drs. Nabel and Gerhard-Herman propose that progeria vascular phenotype resembles an arteriosclerosis, which is characterized by hardening of the vessels and vascular sti llness, typically seen in the nonnal aging populations. Vessel cross-sections at autopsy or one 20-year-old patient revealed no indication of vasculature int-lammation (IS). In addition, children with progeria do not have abnormal serum cholesterol levels, which is a factor in chronic lipid-driven int-lammation (16), Clinical sludics at both the National Instilutes or Heallh (NII-J) clinical center (17) and Children's Hospital, Boston, revealed that the intima-media thickncss of the progeria carotid artery is normal. Furthennore, in agreement with Stehbens and colleagues (15). Dr. Nabel presented autopsy cross-sections from a child with genetically confirmed progeria, and the histology showed that the vascular media no longer contained smooth muscle cells and the elastic structure was destroyed and replaced with extracellular matrix or Iibrosis. She hypothesized that the primary loss of smooth muscle cells initiates vascular remodeling by secondary replacement with matrix in large and small vasculature.
In a study of 28 children with progeria at Brigham and Women's Hospital (Boston), Dr. Gerhard-Hennan measured global vascular stiffening and, through imaging techniques, observed vessel lortuosi ty, profound adventitial thicken ing, and a depleted media layer. For children with an occluded internal carotid artery, there was an overall loss of the trilaminar architecture. Functional studies demonstrated high femoral pulse wave velocities, and decreased vasodilatory response to nitrous oxide stimulation induced by ankle Oexion. Overall, the vessel stiffness, tortuosity, and adventitial brightness (the molecular composition not yel identified), as observed in imaging studies, support a model of arteriosclerosis similar 10 what is observed in aging. Remarkably. the blood velocities are in the nOl1nal range and now is still laminar, implying that there is either a tremendous rescrvc capacity in lhe vasculature. or there are compensatory mechanisms helping to preserve function. Complementary data gathered at the NIH studying a cohOl1 of 15 patients revealed initially n0l111al vascular compliance that decreases with increasing age, and a number of children displayed len atrial enlargement (17). Blood pressure and heart rate were in the normal range in younger children, while the older children had increasing blood pressures and variable heart rates. Electrocardiograms were normal in most. although a fcw had long Q-T intervals suggesting fibrosis of the conduction system. In summary, the progeria vasculature is characterized by global stiffness, tortuosity, and a loss or smooth muscle cells in the media with subsequent extracellular replacement. Smooth muscle cell dropout is unique to progeria, while global stiffness and tortuosity are observed in the nonnal aging population.
The vascular remodeling characteristics in children with progeria are similar to vascular events observed in a transgenic progeria mouse model that expresses human progerin, created in Francis Collins' laboratory (7). Within the descending aorta (cross-section histology), smooth muscle
780
Normal lam in A proces.o;ing
GORDON E1' AL.
Hutchinson-Gilford progeria syndrome
< "/'./ (:\1\1
IMI'51[}1
OCII,
1'I)q(('iI111 ,iem-m)" I/MI''', Iflil
I'roqerill
C.OPI,riqhl
Figure L Postlranslatiolla! processing or prelamin A 10 malllre lamin A (Idi) and prcprogcrin to progerin (righll (57), C cystcinc~ S serine: I -'" isoleucine:M methionine: FLlse ramesy! transferase. jReproduced by pennission or the publishcr.l
cells begin to "drop-out" by 5 months, and by 20 monthsthere is a paucity to a complete absence of smooth musclecells. The remaining endothelial cells are picnotic, similarto progeria cells grown in vitro when progerin is overproduced. There is substantial adventitial thickening, andthe anatomical changes COiTclate with functional changes;the blood pressure response to a vasodilator is blunted andincludes a protracted recovery time to achieve baselinevalues. The NIH progeria mouse model mimics the humanvascular disease. and is therefore a viable model for assessing treatment effects on progeria. At the 2007 workshop.Dr. Collins reported on studies rrom his laboratory wheretransgenic progeria mice began daily treatment with ITI attwo different phases of development: at time or weaning(0-9 months) and later between 6-9 months. Whereas thecarotid al1eries or untreated mutant mice demonstrateda progressive loss of VSMC and excessive appearance ofproteoglycans with age, matched progeria mice treated withintermediate to high doses of FTI had significantly moreVSMC, and little evidence or proteoglycan accumulation.1l1e results with the mice treated between 6 and 9 monthsgive us the first evidence that ITI may have a beneficialeffect in reversing vasculature change brought aboutby progeria.
EIi'FECTS 01" PIWGERIN AND LAMIN IMHALANCE ON
CELL STRliCTlIRE ANOFuNCTION
To continue developing treatment prospects in progeria,and to understand its role in normal aging, it is essential tostudy not only the efrects of I',l, bUI also the biology orlamins. progerin, and related molecules at the cellular level.In nOlmal cells, lamins fonn a concentrated layer along theinner rim of the nucleus, called the nuclear lamina, and arealso found dispersed within the nucleoplasm (18). Laminsplay a critical role in a number of essential cellularfunctions, such as the transcription of RNA polymerase It
DNA replication, the organization of the chromosometerritories, and epigenetic modulations within the nucleus.Lamin A is processed via a series of posttranslational steps(Figure I): prelamin A is farnesylated, carboxymethylated,and cleaved twice to produce mature lam in A. Elements forthis pathway include initial famesylation by a farnesyltransferase, removal of the three C-terminaI amino acids, andmethyl esterification of the now C-telminal farnesylcysteine. This is followed by integration of prelamin A intothe nuclear membrane, and cleavage of 15 amino acids andthe attached ramesyl group from its C-tenninal end byZmpste24 to produce mature lamin A. Mature lamin A isthen released from the nuclear membrane (19). Typically inprogeria, a single silent C to T transition mutation atnucleotide 1824 (Gly608Gly) activates a cryptic splice site,resulting in the deletion of 150 messenger RNA bases in the3' portion of exon II (4). The aberrant protein. progerin,lacks the cleavage site for removal of its C-tenninalfarnesylated peptide by Zmpste24 endoprotease, andthererore remains bound to the inner nuclear membrane.Hence, progerin resembles an aberrant fom1 of famesylatedprelamin A. However, whereas prelamin A is a transient.illlermediate protein, which is present at very low levels inthe cell, progerin is a nontransient protein whose concentration builds with successive cell divisions.
Several workshop presentations addressed progerin'sinfluences on lamin A and its binding pmtners. its role inarchitectural changes in the nuclear membrane, and subsequent effects on cell function that include: increase inapoptosis and disruption of binding with lamin-bindingproteins; increased nuclear rigidity, sluggish wound healingin vitro, and altered cell proliferation and chromosomaldistribution. Ludo Comai's (University of Southern California) studies stressed the importance of detennining thecellular eHects of progerin and excess prelamin A on cellgrowth (20). Dr. Comai used a lentivirus system to create
780 GORDON tTAL
Normal lam in A processin9 Hutchinson-Gilford progeria ~yndrorne
( ~I\\ (\IM
IMP<., lli1 t~
Of II,
Up,I, '>i\fll c!pd,aq'> ,1()l)U,Ii "d ,,'
't' '" _/'''Z/{ <::::~..-~" ..f ( 01"11,
"'--...", ../Proql>'1Il
( 0p\,r;qht ," )!lOiJ N'HUr<' P"hli~hm<J (,I'OUp
Nature Reviews i (,en(>lk'>
Figure I, Posttran~lation:l! processing of prelamin A to lll:llllre lamin A (lei!) and prcprogcrin to prog-erin (rig-hl) (57). C == cyslelne~ S -- serine: I - isoleucine: M -"--= mClhionin<~: = ramesyJ lransferase. IReproduced by pellnis~ionFLISC -"-- or lhe pubhsher, 1
cells begin to "drop-out" by 5 months, and by 20 months there is a paucity to a complete absence of smooth muscle cells. The remaining endothelial cells are picnotic, similar to progeria cells grown in vitro when progerin is overproduced. There is substantial adventitial thickening, and the anatomical changes con'elate with functional changes; the blood pressure response to a vasodilator is blunted and includes a protracted recovery time to achieve baseline values. The NIH progeria mouse model mimics the human vascular disease, and is therefore a viable model for assessing treatment effects on progeria. At the 2007 workshop, Dr. Collins reported on studies from his laboratory where transgenic progeria mice began daily treatment with ITI at two different phases or development: at time of weaning (0-9 months) and later between 6-9 months. Whereas the carotid a1teries of untreated mutant mice demonstrated a progressive loss of VSMC and excessive appearance of proteoglycans with age, matched progeria mice treated with intermediate to high doses of FTI had significantly more VSMC, and little evidence of proteoglycan accumulation. The results with the mice treated between 6 and 9 months give us the first evidence thaI ITT may have a beneficial effect in reversing vasculature change brought about by progeria.
EIi'J<'ECTS 01<' PIWGERIN AND LAMIN IMHALANCE ON
CELL STRliCTlIRE AND FUNCTION
To continue developing treatment prospects in progeria, and to understand its role in normal aging, it is essential to study not only the effects of f"J'1, but also the biology of lamins, progerin, and related molecules at the cellular level. In nOlmal cells, larnins Conn a concentrated layer along the inner rim of the nucleus, called the nuclear lamina, and are also found dispersed within the nucleoplasm (18). Lamins play a critical role in a number of essential cellular functions, such as the transcription of RNA polymerase n.
DNA replication, the organization of the chromosome territories, and epigenetic modulations within the nucleus. Lamin A is processed via a series or posHranslational steps (Figure I): prelamin A is farnesylated, carboxymethy lated, and cleaved twice to produce mature lamin A. Elements for this pathway include initial famesylation by a farnesyltranslerase, removal of the three C-tenninal amino acids, and methyl esterifkation of the now C-telminal farnesyl cysteine. This is followed by integration of prelamin A into the nuclear membrane. and cleavage of 15 amino acids and the attached famesyl group from its C-tenninal end by Zmpste24 to produce mature lamin A. Mature lamin A is then released from the nuclear membrane (19). Typically in progeria, a single silent C to T transition mutation at nucleotide 1824 (Gly608Gly) activates a cryptic splice site, resulting in the deletion or 150 messenger RNA bases in the 3' portion of exon II (4). The aberrant protein, progerin, lacks the cleavage site for removal of its C-tenninal farnesylated peptide by Zmpsle24 endoprotease, and therefore remains bound to the inner nuclear membrane. Hence, progerill resembles an aberrant fOnll of farnesylatedprelamin A. However, whereas prelamin A is a transient intermediate protein, which is present at very low levels in the cell, progerin is a nontransient protein whose concentration builds with successive cell divisions.
Several workshop presentations addressed progerin's influences on lamin A and its binding paltners. its role in architectural changes in the nuclear membrane, and subsequent effects on cell function that include: increase in apoptosis and disruption of binding with lamin-binding proteins, increased nuclear rigidity, sluggish wound healing in vitro, and altered cell proliferation and chromosomal distribution. Lucio Comai's (University of Southern California) studies stressed the importance of detennining the cellular effects of progerin and excess prelamin A on cell growth (20). Dr. Comai used a lentivirus system to create
HIGHLIGHTS OF THE 2007 PRF SCIEN71FJC WORKSHOP 7XI
two cell lines, progerin expressors and prclamin A ovcrexpressors, from normal human fibroblasts. Over-expressionof either prelamin A or progerin significantly increased theamount or cell death compared to wild-type cOlltrols ina passage-independent manner. When progerin-expressingcells 10.'11 progerill expression, apoplasis decreased towild-Iype levels. Apoplasis also decreased to wild-Iypelevels when prelarnin A overexpressors were transfectedwith additional Zmpste24 DNA, which decreased prelaminA protein levels and increased mature lamin A levels.As expected, over-expressing Zmpste24 did no1 rescueprogerin-exprcssing cells, because progerin lacks theZmpsle24 recagni lion site. Adding FII to progerinexpressing cell cultures did not improve rates of cell death,presumably because the excess nonfarnesylated prelamin Acreated with FII exposure counteracted any improvementsin growth that decreasing progerin may have caused. Studiesby Stephen Young (UCLA) provided additional evidence forthe toxic effects of farnesylated prelamin A. Dr. Youngshowed that certain clinically used HIV protease inhibitorsthat cause lipodystrophy reminiscent of that seen in progeriaalso create a Zmpste24 blockade and a build-up of1~ll11esylated prelamin A in normal fibroblasts (21). Thus,it is crucial to study not only the effects of progerin, but thatof other proteins that are affected by the presence ofprogerin or by treatments that create an imbalance of laminA-related proteins.
Jan Lammerding (Harvard Medical School) studied theeffects of accumulated progerin on nuclear rigidity, woundhealing in vitro, and cell cycling, and asked whether FTlsreverse abnormalities. When strain is applied to wild-typefibroblast nuclei, they remain stilT (22). Fibroblasts culturedfrom a lamin A/C deficient mouse model were twice asfragile as wild-type cells, and after 24 hours of mechanicalstrain, cell death signilkantly increased. In sharp contrast,while progeria fibroblasts showed normal rigidity at earlypassages (when progerin levels and nuclear blebbing are ata minimum), late-passage nuclei show dramatically increased rigidity over wild-type fibroblasts. Although thelamin A/C defkient fibroblasts and progeria Ilbroblastsdisplayed vastly different nuclear rigidities, nuclear strainelicited early cell death in both cell types over controls. Inaddition, whereas wild-type cells elller S phase and G2phase in response to mechanical stretch, progeria cells didnot proliferate in response to stretch. Serum-starved progeriahbroblasts had a slow and incomplete response 10 an in vitrowound healing assay as well. The ability of FTls to affectthese cellular abnormalities was mixed. Nuclear rigidity andwound-healing deficits were reversed in PrJ-treated progeria cultures. However, FTI treutment did not promote cellproliferation after mechanical strain.
Within the nucleus, lamin A monomers first dimcrizc,thcn dirners associate in a head-tn-tail t~lshion and finallyassociate laterally (23). The 50-amino acid deletion inprogerin probably docs not affect its ability to dimerize, asnormal lamin A docs, because the necessary components forthis function are not deleted. Hence progcrin can formdimers with itsclf or nOllnal lamin A, thereby inOuencinglamin A's interactions with nuclear pores, chromatin, andlamin binding partners. For example, the fOllllation of larnin
A and lamin B are separate yet interdependent; the failure ofone lam in-type to properly form can interferc with theformation of another lamin type (24). Progerin build-up withincreasing cell passage leads to progressively increasedpercentages of blebbed nuclei (25). Robert Goldman(Northwestern University) referred to these blebs as"sophisticated structures" worthy of close examination todetelllline their inOuence on cell function, and began byhypothesizing that progerin's affect on other lamin types iscentral to the blebbing defect. In the region of the blebs,there is an absence of heterochromatin but euchromatin canbe identified surrounded by lamin A, a configuration notobserved in other regions where the nuclear membraneappears to be formed normally (26). Dr. Goldman and hisgroup suggest that the blebs contain transciptionally activeDNA and thut this is providing clues regarding the role ofmutant lamin A in progeria.
Complementary chromosome positioning studies wereexplored by Joanna Bridger (Brunei University, U.K.), whosupports the hypothesis that A-type lamins (and/or emerin)are involved in translocating and anchoring chromosomes tothe nuclear membrane or nuclear matrix in nonrandomdistributions according to cell cycle status, and that laminmutations pcrturb this balance. Normally, upon cell cycleexit, gene-poor chromosomes move from the nuclearperiphery to more interior positions (27,28). The distributionprofile of chromosome position within the nucleus duringcell cycling, quiescence, and senescence all differ.Dr. Bridger had earlier reported that proliferating fibroblastsfrom laminopathy patients, including progeria, exhibitedchromosome distributions characteristic of nonprolifcrating(senescelll or quiescent) n0I111al fibroblasts, where thelargest chromosomes are located at the periphery of thenucleus and the smallest at the interior. At the workshop.Dr. Bridger reported new experiments that distinguished thechromosome distribution in senescent from quiescent cellsbased on the position of chromosome to, which she findslocated at the periphery in quiescent cells and at the interiorin senescent cells. In proliferating progeria I1broblasts,however, chromosome lO was located at the periphery,a position found only in the nonproliferating, quiescent statein nonnal cells. Several other proliferating laminopathycultures also displayed the quiescent positioning. Thischromosome positioning was not changed when progeriaor laminopathy cells became senescent. The consequencesof aberrant chromosome 10 locat ion on transcription inprogeria will be the subject of Dr. Bridger's future studies.
Bryce Paschal (University of Virginia) reported onprogerin-induced changes in the nuclear pore complexes(NPCs), channels through which all transport between thenucleus and the cytoplasm takes pi ace (29). He has foundmajor changes both in NPC structure and in the transportmachinery and signals. The structural change is in theposition of the nucleoplasmic basket, a structure located atthe pore on the nucleoplasmic side. It consists of twoproteins: TPR (translocated promoter region), the keyconstituent, and Nup 153, a facilitator of its assembly. Theentire basket structure is disassemblecl and reassembled atevery mitosis. When Dr. Paschal looked for the localizationof TPR in progeria fibroblasts by immunostaining, he
HIGHLIGHTS OF THE 2007 PRF SCIENTIFIC WORKSHOP 7RI
two cell lines, progerin expressors and prclamin A overexpressors, from normal human fibroblasts. Over-expression of either prelamin A or progerin significantly increased the amount of cell death compared to wild-type controls in a passage-independent manner. When progerin-expressing cells losl progerin expression, apoptosis decreased to wild-type levels. Apoplosis also decreased 10 wild-type levels when prelarnin A overexpressors were transfected with additional Zmpste24 DNA, which decreased prelamin A protein levels and increased mature lamin A levels. As expected, over-expressing Zmpste24 did not rescue progerin-expressing cells, because progerin lacks the Zmpste24 recogni 1ion site. Adding FTI 10 progerinexpressing cell cultures did not improve rates of cell death, presumably because the excess nonfarnesylated prelamin A crealed with FfI exposure counteracted any improvements in growth that decreasing progerin may have caused. Studies by Stephen Young (UCLA) provided additional evidence for the toxic effects of famesylated prelamin A. Dr. Young showed that certain clinically used HIV protease inhibitors that cause lipodystrophy reminiscent of that seen in progeria also create a Zmpste24 blockade and a build-up of famesylaled prelamin A in normal fibroblasts (2 I). Thus, it is crucial to study not only Ihe effects or progerin, but that of other proteins that are affected by the presence of progerin or by treatments lhat creale an imbalance of lamin A-related proteins.
Jan Lammerding (Harvard Medical School) studied the effects of accumulated progerin on nuclear rigidity, wound healing in vitro, and cell cycling, and asked whether FTls reverse abnormalities. When strain is applied to wild-type fibroblast nuclei, lhey remain stilT (22). Fibroblasts cultured from a lamin A/C deficient mouse model were twice as fragile as wild-type cells. and after 24 hours of mechanical strain, cell death significantly increased. In sharp conlrast, while progeria fibroblasts showed normal rigidity at early passages (when progerin levels and nuclear blcbbing are al a minimum), late-passage nuclei show dramatically increased rigidity over wild-type fibroblasts. Although the lamin A/C ddicient fibroblasts and progeria fibroblasts displayed vastly different nuclear rigidities, nuclear strain elicited early cell death in both cell types over controls. In addition, whereas wild-type cells enter S phase and G2 phase in response to mechanical stretch, progeria cells did not proliferate in response to stretch. Serum-starved progeria flbroblasts had a slow and incomplete response to an in vitro wound healing assay as well. The ability of FTls to affect these cellular abnormalitics was mixcd. Nuclear rigidity and wound-healing deficits were reversed in PTJ-treated progeria cultures. However, FTI lreatment did not promote cell proIi ferat ion after mechan ical strain.
Within the nucleus. lamin A monomers (jrst dimcrize, then dimcrs associate in a hcad-to-tail hlshion and Ilnally associate lateral Iy (23). The 50-amino acid deletion in progerin probably does not affect its ability to c1imerize, as normallamin A does, because the necessary components for this function are not deleted. Hence progerin can form dimers with itself or nonnal lamin A, thereby influencing lamin A's interactions with nuclear pores, chromatin, and larnin binding partners. For example, the fOllllation of lamin
A and lamin B arc separate yet interdependent; the failure of one lamin-type to properly form can interrere with the formation of another Iamin type (24). Progerin build-up with increasing cell passage leads to progressively increased percentages or blebbed nuclei (25). Roher! Goldman (Northwestern University) referred to these blebs as "sophisticated structures" worthy of close examination to detem1ine their intluence on cell Cunction, and began by hypothesizing that progerin's affect on other lamin types is central to the blebbing defect. In the region of the blebs, there is an absence of heterochromatin but euchromatin can be identified surrounded by lamin A. a configuration not observed in other regions where the nuclear membrane appears to be formed normally (26). Dr. Goldman and his group suggest that the blebs contain transciptionally active DNA and that this is providing clues regarding the role of mutant larnin A in progeria.
Complementary chromosome positioning studies were explored by Joanna Bridger (BruneI University, U.K.), who supports the hypothesis that A-type lamins (and/or emerin) are involved in translocating and anchoring chromosomes to the nuclear membrane or nuclear matrix in nonrandom distributions according to cell cycle status, and that lamin mutations perturb this balance. Normally, upon cell cycle exit, gene-poor chromosomes move from the nuclear periphery to more interior positions (27,28). The distribution prollle of chromosome position within the nucleus during cell cycling, quiescence, and senescence all differ. Dr. Bridger had earlier reported that proliferating fibroblasts from laminopathy patients, including progeria, exhibited chromosome distributions characteristic of nonproliferating (senescent or quiescent) nO/lTIal fibroblasts, where the largest chromosomes are located at the periphery of the nucleus and the smallest at the interior. At the workshop, Dr. Bridger reported new experiments that distinguished the chromosome distribution in senescent from quiescent cells based on the position of chromosome 10, which she finds located at the periphery in quiescent cells and at the interior in senescent cells. In proliferating progeria Ilbroblasls, however, chromosome 10 was located at the periphery, a position round only in the nonprol iferating, quiescent state in n01111al cells. Several other proliferating laminopathy cultures also displayed the quiescent positioning. This chromosome positioning was not changed when progeria or laminopathy cells became senescent. The consequences of aberrant chromosome 10 locat ion on transcription in progeria will be the subject of Dr. Bridger's future studies.
Bryce Paschal (University or Virginia) reported on progerin-induced changes in the nuclear pore complexes (NPCs), channels through which all transport between the nucleus and the cytoplasm takes place (29). He has found major changes both in NPC structure and in the transport machinery and signals. The structural change is in the position of the nucleoplasmic basket. a structure located at the pore on the nucleoplasmic side. It consists of two proteins: TPR (translocated promoter region), the key constituent, and Nup 153, a facilitator of its assembly. The entire basket structure is disassembled and reassembled at every mitosis. When Dr. Paschal looked for the localization or TPR in progeria fibroblasts by immunostaining, he
782 GORDON ET AL.
obtained a striking result: Instead of being in the basket,TPR is out in the cytoplasm. The same result is obtainedwhen human epithelial cells are lransfectcd with progerin.Administration of an FTJ to the culture corrected the TPRlocalization back into the nuclear envelope of progeria cells.
Progcrin also had a major effect on localization ora transport regulator. While small molecules can diffusepassively through the NPC channel, macromolecules mustenter as a complex with a member of the superfamilies ofimportin or cxportin proteins, depending on the direction oftransport (29). Once one of these complexes has traversedthe pore, it is dissociated with the involvement of a Rasrelated GTPase called RAN. In normal cells, there isa gradient of RAN concentration across the pore, with moreRAN in the nucleus than in the cytoplasm.
Dr. Paschal found that the RAN protein gradient wasdisrupted in epithelial cells when lamin A was depleted withsiRNA. He therefore examined the effect of severallaminopathy mutations on RAN localization. Whereas cellsfrom dilated cardiomyopathy and Emel),-Dreifuss musculardystrophy mutations that express a mutant f01111 of lamin Ahad nonnal RAN gradients, the RAN protein gradient isdramatically disrupted in both progeria cells and in cellswith the mutant Iamin A L647R in which cleavage byZmpste24 is lacking. While in normal cells most of the RANis in the nucleus, progeria cultures have an invertedgradient-there is more RAN in the cytoplasm than in thenucleus. In the L647R cultures, this effect was rescued byadding an fTf to the culture. It will be important to assesswhether FTI rescues the RAN progerin gradient in progeriacells.
PROGERIA AND AGING
Hutchinson-Gilford progeria syndrome is named for itsresemblance to aging: "pro" prematurely and "geras"aged. It is a segmental progeroid syndrome (30), sincesome, but not all, of the patients' characteristics resembleaccelerated aging. Over the last several years, a growingbody of evidence for cellular and molecular overlapsbetween progeria and normal aging has been uncovered.Early investigations compared several properties, forexample, telomere length (31) and distribution of mRNAspecies (32). The discovel)' that progeria is caused bya mutation in lamin A, which had not previously beenimplicated in mechanisms of aging, brought in an entirelynew question: Are defects in lamin A implicated in normalaging? The first positive evidence was reported by Scanldiand Misteli in 2006 (33), who showed thaI cell nuclei fromnonnal individuals have acquired defects similar to progeriapatients, including changes in histone modifications andincreased DNA damage. Younger cells show considerablyless of these defects. They further demonstrated that the agerelated effects are the result of the production of low levelsof preprogerin mRNA produced by activation of the samecl)'ptic splice site that functions at much higher levels inprogeria, and is reversed by inhibition of transcription at thissplice site. Cao and colleagues (34), studying the same phenomenon, showed that, among interphase cells in fibroblastcultures, only a small fraction of the cells contained progerin.
The percentage of progerin-positive cells increased withpassage number, suggesting a link to n0I111al aging.
A large amount or the data presenled at this workshopunderscored the value of studying progeria as a cellular andorganismalmodel for some key aspects of the aging process.The evidence Ihat progerin is present in normal aging wasconsiderably strengthened at the workshop by KarimaDjahali (Columbia University) (35). Dr. Djabali measuredprogerin mRNA and protein (using a monoclonal antibodyshe created that recognizes progerin but notlamin A) in cellsfrom skin biopsies taken from progeria patients and from150 n011nal participants with ages ranging from newborn to97 years. Dr. Djabali found low levels of progerin mRNAin all tissue samples, but found progerin protein only in skinsamples taken from elderly participants, within de11nallibroblasts, and in a few temlinally differentiated keratinocytes. Furthermore, using this same antibody, Dr. Djabalidetected a dramatic increase of progerin-containing cells innonnal dermal fibroblast cuHures as passage numberincreased. The discovery of progerin in the skin biopsiesprovided the first evidence for the in vivo accumulation ofprogerin during n01111al aging. The presence of progerin inn01111al individuals implies that individuals without progeriatolerate a low level of progerin as part of n01111al aging. Withrespect to treating progeria, it is encouraging and suggeststhat effective therapy may be achieved with a balancebetween prelamin, lamin A, and progerin rather thana complete elimination of the progerin molecule.
Francis Collins' latest research is asking a centralquestion in the search for inlluences of LMNA or progerinproduction on longevity in the general population. Promptedby the Djabali evidence of increased amounts of progerinwith age, Dr. Collins is looking for variations in DNAsequence around the LMNA gene in fibroblasts of' long-livedpeople that may give them a selective advantage over thoseindividuals who die at younger ages. In an experiment thatexamined cells from more than lOon people aged greaterthan 94 years, he found that four single nucleotidepolymorph isms (SNPs) differed from the general population(p 2 X 10""5). Four of four centenarians analyzed alsocontained these same SNPs. The probability of' this findingbeing random is 7 X IO-x. Further study will ask whetherincreases or decreases in progerin production are linked tothese SNPs.
Nonnal cellular senescence is marked by increasing ratesof DNA damage and a decline in the ability to repair thisdamage (36). Progeria cells have been shown to accumulatedouble-stranded DNA (dsDNA) breaks and impaired DNArepair (37-39). Yue Zou (East Tennessee State University)presented results on the mechanism of this failure (40).Nonnally, in response to fOlmation of dsDNA breaks,histone H2AX is rapidly phosphorylated and then recruitsvarious DNA repair proteins, together fonning foci for DNArepair. In progeria fibroblasts, the phosphOl)'lated protein,called gamma-H2AX, was unable to recruit the DNA repairenzymes Rad51 and Rad50. Instead, the damage recognitionprotein XPA (xeroden11a pigmentosum group A) wasrecruited. XPA nonnally participates only in excision repairsystems and has no known role in dsDNA repair. Similarresults were obtained from restrictive dermopathy cultures.
782 GORDON 1:!-7' AI.
obtained a striking result: Instead of being in thc basket, TPR is out in the cytoplasm. The same result is obtained whcn human epithelial cells are transfected with progerin. Administration of an FTJ to the culture corrected the TPR localization back into the nuclear envelope of progeria cells.
Progerin also had a major effect on localization or a transport regulator. While small molecules can diffuse passively through the NPC channel, macromolecules must enter as a complex with a member of the superfamilies of importin or exportin proteins, depending on the direction of transport (29). Once one of these complexes has traversed the pore, it is dissociated with the involvement of a Rasrelated GTPase called RAN, In normal cells, there is a gradient of RAN concentration across the pore, with more RAN in the nuclcus than in the cytoplasm.
Dr. Paschal found that thc RAN protein gradient was disrupted in epithelial cells when lamin A was depleted with siRNA. He therefore examined the effect of several laminopathy mutations on RAN localization. Whereas cells fi'om dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy mutations that express a mutant 1'01111 of lamin A had nonnal RAN gradients, the RAN protein gradient is dramatically disrupted in both progeria cells and in cells with thc mutant lamin A L647R, in which cleavage by Zmpstc24 is lacking. While in normal cells most of the RAN is in the nucleus, progeria cultures have an invcrted gradient-there is more RAN in the cytoplasm than in the nucleus. In thc L647R cultures, Ihis effcct was rescued by adding an FTI to the culture. It will be important to assess whether FTI rescues the RAN progerin gradient in progeria cells.
PROGERIA AND AGING
Hutchinson-Gilford progeria syndrome is named for its resemblance to aging: "pro" == prematurely and "gcras" = aged. It is a segmental progeroid syndrome (30), since some, but not all, of the patients' characteristics resemble accelerated aging. Over the last several years, a growing body of evidence for cellular and molecular overlaps between progeria and normal aging has been uncovcred. Early investigations compared several properties, for example, telomere length (31) and distribution of mRNA species (32). The discovery that progeria is caused by a mutation in lamin A, which had not previously been implicated in mechanisms of aging, brought in an entirely new question: Are defects in lamin A implicated in normal aging? The IIrsl positive evidence was reported by Scamdi and Misteli in 2006 (33). who showed thai cell nuclei from nOllnal individuals have acquired defects similar to progeria patients, including changes in histonc modifications and increased DNA damage. Younger cells show considerably less of these defects. They further demonstrated that the agerelated effects are the resull of the production of low levels of preprogerin mRNA produced by activation of the same cryptic splice site that functions at much higher levels in progeria, and is reversed by inhibition of transcription at this splice site. Cao and colleagues (34), studying the samc phenomenon, showed that, among interphase cells in ll11roblast cultures, only a small fraction of the cells contained progerin.
The percentage of progerin-positive cells increased with passage number, suggesting a link to n0ll11al aging.
A large amount of the data presented at this workshop underscored the value of studying progeria as a cellular and organismalmodel for some key aspects of the aging process. The evidence that progerin is present in normal aging was considerably strengthened at the workshop by Kari117a Djahali (Columbia University) (35). Dr. Djabali measured progerin mRNA and protein (using a monoclonal antibody shc created that recognizes progerin but not lamin A) in cells from skin biopsies taken from progeria patients and from 150 nonnal participants with ages ranging from newborn to 97 ycars. Dr. Djabali found low levels of progerin mRNA in all tissue samples. but found progerin protein only in skin samples taken from elderly participants, within dennal libroblasts, and in a few telminaJly differentiated keratinocytes. Furthermore, using this same antibody, Dr. Djabali detected a dramatic increase of progerin-containing cells in nonnal dermal fibroblast cullures as passage number increased. The discovery of progerin in the skin biopsies provided the first evidence for the in vivo accumulation of progerin during nonnal aging. The presence of progerin in nonnal individuals implies that individuals without progeria tolerate a low level of progerin as part of nonnal aging. With respect to treating progeria, it is encouraging and suggests that effective therapy may be achieved with a balance between prelamin. lamin A. and progerin rather than a complete elimination of the progerin molecule.
Francis Collins' latcst research is asking a central question in the search for influences of LMNA or progerin 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 sequence around the LMNA gene in libroblasts of long-lived people that may give them a selective advantage over those individuals who die at younger ages. In an experiment that examined cells from more than 1000 people aged greater than 94 years, he found thai four single nucleotide polymorph isms (SNPs) differed from the gcneral population (p == 2 X 10")). Four of four centenarians analyzed also contained these same SNPs. The probability of this finding being random is 7 X la-x. Further study will ask whether increases or decreases in progerin production are linked to these SNPs.
Nonnal cellular senescence is marked by increasing rates of DNA damage and a declinc in the ability 10 repair this damage (36). Progeria cells have been shown to accumulate double-stranded DNA (dsDNA) breaks and impaired DNA repair (37-39). Yu(' Zou (East Tennessee Slate University) presented results on the mechanism of this failurc (40). Nonnally. in responsc to fOlmation of dsDNA breaks, histone H2AX is rapidly phosphorylated and then recruits various DNA repair proteins, logether fonning foci for DNA repair. In progeria libroblasts, the phosphorylated protein, called gamma-H2AX, was unable to recruit the DNA repair enzymes Rad51 and Rad50. Instead. the damage recognition protein XPA (xerodcnna pigmentosum group A) was recruited. XPA 110nnally participates only in excision repair systems and has no known role in dsDNA repair. Similar results were obtained from restrictive dermopathy cultures.
IIlGHLlGH1:'i OF THE 2007 PRF SCIENl1FIC WORKSHOP 783
Dr. Zou connll11Cd the identity of XPA by immunoprecipitalion. and XPA siRNA knock-dowil experiments reversedthese findings in progeria cultures. Thus, XPA likely playsa key role in defective dsDNA repair in progeria. Importantly, Dr. Zou and colleagues found Ihal FII treatmentcould not reduce the accumulated DNA breaks in progeriacells, suggesting that FTf may not effectively treat thisaspect of disease (41).
FUrtlRE TREAT1\'IENT POSSIHII,ITIES THROUGH A BETTER
UNDERSTANDING OF THE BIOLOGY OF DISEASE IN
PnOGER1A: STE!\'I CELLS, ALTERNATIVE PRENYLATlON,
COMHINATION CHEMOTHERAPY AND A NOVI.;I,
HIGH-THROUGHPUT SCREENING SYSTEIVI
Stem CellsFor several presenting researchers, stem cells took center
stage for two reasons. First, there is the question of whetheror not progeria is a disease characterized by stem-celldepletion. whereby there is an inadequate supply or stemcells for replenishing prematurely dying mesenchymederived cell populations (VSCM, adipocytes, llbroblasts,and osteocytes). Second, the lack of lamin A expressionin immature cells presents stem-cell therapy as a viablestrategy for treatment, or even reversal, of disease inprogeria. The therapeutic potential of mesenchymal stemcells is being tested in numerous clinical and preclinicaltrials (42).
In his introductory presentation, Huber Warner (University or Minnesota) advanced his hypothesis that depletion ofstem cells is a major hlCtor in causing the tissue changesobserved in progeria (43). The depletion may arise from theincreased turnover of differentiated cells, which are lost atan accelerated rate (44). This hypothesis has been furtheranalyzed in the literature. Prolla (45) suggested that theregenerative capacity of tissues with high turnover might bereduced due to the exhaustion or progenitor cells.Mesenchymal stem cells are believed to be involved inexpression of symptoms of progeria, since most mesenchymal stem cells express lamins A/C (46), while somecommitted hematopoietic cell lines do not (47). Gotzmannand Foisner (48) point out that deregulated or impairedtissue regulation and deregulated cell-type plasticity mightaccount for nearly all pathological conditions detected inlaminopathies. lIalaschek-Wiener and Brooks-Wilson (49)argue that depletion of mesenchymal stem cells can helpaccount for the specific segmental nature of progeria.
The current published literature supports the view thatlamin A, and hence progerin, is produced by differentiatedcells, and undiiTerentiated cells do not transcribe or translateeither molecule. However, Carlos Lopez-Orin (Universidadde Oviedo, Spain) described experiment.s in which hairfollicle-derived stem cells are severely affected by defects inthe lamin processing pathway. His Zmpste24~1" progeriamouse model (50) produces no mature lamin A and suffersfrom a build-up of famesylated prelamin A. The homozygous null mouse displays many features of humanlaminopathies, including severe growth retardation andpremature death, dilated cardiomyopathy (DCM), muscular
dystrophy (EDMD). lipodystrophy (progeria), and loss offur and whiskers. Dr. Lopez-Otin's preliminary studiesshow that, unlike their wild-type counterparts, lllany hairfollicle-derived stem cells from these mice were quiescent,showed profound nuclear blebbing, and the accumulation ofheterochromatin in two large masses in the centers of manycell nuclei. In addition, clonogenic assays showed that thesestem cells have a profound proliJ'eration defect abnOllllalaccumulation of p16, and an absence of beta-catenin.suggesting a loss of wnl signaling. Imp011antly. when anLMNA allele was crossed into Zmpste24-1 mice, levels offamesylated prelamin A decreased, lamin A was synthesized,and all measured cellular defects were normalized (39).Hence, in this system, stem-cell function was profoundlyaffected by nuclear defects and the ratio of farnesylatedprclamin A to mature lamin A.
In light of this interest in the possibility that stem-celldepletion causes many of progeria's phenotypes, IrinaConboy (University of California at Berkeley) was invited topresent her work on mechanisms causing the decline oftissue regenerative potential in normal aging skeletal musclein response to injury and on regeneration using embryonicstem cells. She demonstrated both that a youthful biochemical milieu can restore cell vitality, and that old cellshave regenerative capacity that can be tapped.
Skeletal muscle has its own source of stem cells, calledsatellite cells. Satellite cells are located at the basementmembrane of terminally di/Terentiated muscle fibers and canbe identified and purified. Dr. Conboy has carried out bothin vivo and in vitro experiments in which stem cells fromyoung animals were exposed to niche clements from oldanimals and vice versa (51). She has discovered that thedecline of regenerative capacity with age results fromchanges in the stem cell niche, not in the stem cellsthemselves. A key element is age-related impairment of theup-regulation of the Notch ligand Delta after muscle injury(52). Exposure of satellite cells from old mice to youngserum enhanced the expression or Notch ligand Delta aswell as proliferation and regenerative capacity of agedsatellite cells. Forced activation of Notch by addinga Notch-speciflc antibody restored regenerative potential toold muscle.
Dr. Conboy has extended her studies to examine theeflects of aged systemic mileu on the runction of humanembryonic stem cells (hESCs) (53). Aged mouse serumdramatically inhibited the self-renewal and proliferativepotential of hESCs and satellite cells, as measured byexpression of markers of stem-cell functionality, rate of cellproliferation, and the capacity for myogenic differentiation.When hESCs are cultured in serum-free medium instead ofimmediate transJCr to medium containing aged serum, thecells are no longer susceptible to the effects of aged serum.This implies that hESCs have produced one or more antiaging factors.
Together, these experiments suggest that the regenerativeoutcome of stem-cell replacement will be determined bya balance between negative effects of aged tissues ontransplanted cells and positive effects of embryonic cells onthe endogenous regenerative capacity. This result indicatesthat a complex interplay between negative regulation by the
783 HIGHLIGHT) OF TilE 2007 PRF SCIENTIFIC WORKSHOP
Dr. Zou conn1111ed the identity of XPA by immunoprecipitalion. and XPA siRNA knock-down experiments revcrsed these findings in progeria cultures. Thus, XPA likely plays a key role in defective dsDNA rcpair in progeria. Importantly. Dr. Zou and colleagues found that FII treatment could not reduce the accumulated DNA breaks in progeria cells. suggesting that FTf may not effectively treat this aspect of disease (41).
FUTlJRE TREATMENT POSSIHII,ITJES THROUGH A BETTER
UNDERSTANmNG OF THE BJOLO(;V OF DISEASE IN
PIWGERIA: STEM CELLS, ALTERNATIVE PRENYLATlON,
COMHINATION CHEMOTHERAPV,l AND A NOVIU,
HIGH-THROUGHPliT SCimENING SYSTEM
Stem Cells For several presenting researchers, stem cells took cenler
stage for two reasons. First, there is the question of whether or not progeria is a disease characterized by stem-cell depletion, whcreby there is an inadequate supply of stem cells for replenishing prematurely dying mesenchymederived cell populations (VSCM, adipocytes, llbroblasts, and osteocytes). Second, the lack of lam in A expression in immature cells presents stem-cell therapy as a viable strategy lor treatment. or even reversal, of disease in progeria. The therapeutic potential of mesenchymal stem cells is being tested in numerous clinical and preclinical trials (42).
In his introductory presentation, Huber Warner (University of Minnesota) advanced his hypothesis that depletion of stem cells is a major factor in causing the tissue changes observed in progeria (43). The depletion may arise from the increased turnover of differentiated cells, which are lost at an accelerated rate (44). This hypothesis has been further analyzed in the literature. Prolla (45) suggested lhat the regenerative capacity of tissues with high turnover might be reduced due to the exhaustion of progenitor cells. Mesenchymal stem cells are believed to be involved in expression of symptoms of progeria. since most mesenchymal stem cells express lamins AIC (46), while some committed hematopoietic cell lines do not (47). Gotzmann and Foisner (48) point out lhat deregulated or impaired tissue regulation and deregulated cell-type plasticity might account for nearly all pathological conditions detected in laminopathies. IIalaschek-Wiener and Brooks-Wilson (49) argue that depletion of mesenchymal stem cells can help account for the speciflc segmental nature of progeria.
The current published literature supports the view that lamin A, and hence progerin, is produced by differentiated cells, and undifTerentiated cells do not transcribe or translate either molecule. However, Carlos Lopez-Orin (Universidad de Oviedo, Spain) described experiments in which hair fall ide-derived stem cells are severely affected by defects in the Jamin processing pathway. His Zmpste24-1 · progeria mouse model (50) produces no mature lamin A and suffers from a build-up of famesylated prelamin A. The homozygous null mouse displays many features of human laminopathies, including severe growth retardation and premature death, dilated cardiomyopathy (DCM), muscular
dystrophy (EDMD). lipodystrophy (progeria), and loss of fur and wh iskers. Dr. Lopez-OLin's pre Ii mi nary studies show that, unlike their wild-type counterparts. many hair follicle-derived stem cells li'OIn these mice were quiescent, showed profound nuclear blebbing, and the accumulation of heterochromatin in lwo large masses in the centers of many cell nuclei. In addition, clonogenic assays showed that these stem cells have a profound prolilCration defect. abnollllal accumulation of p 16, and an absence or beta-catcnin, suggesting a loss or wnl signaling. ImpOliantly, when an LMNA allele was crossed into Zmpste24-1-- mice. levels of famesylated preJamin A decreased. lamin A was synthesized, and all measured cellular defects were normalized (39). Hence, in this system, stem-cell function was profoundly affected by nuclear defects and the ratio or farnesylated prelamin A to mature lamin A.
In light of this interest in the possibility that stem-cell depletion causes many of progeria's phenotypes. Irina Conboy (University of California at Berkeley) was invited to present her work on mechanisms causing the decline of tissue regenerative potential in normal aging skeletal muscle in response to injury and on regeneration using embryonic stem cells, She demonstrated both that a youthful biochemical milieu can restore cell vitality, and that old cells have regenerative capacity that can be tapped.
Skeletal muscle has its own source of stem cells, called satellite cells. Satellite cells are located at the basement membrane of terminally dilTerentiated muscle fibers and can be identified and purified. Dr. Con boy has carried out both in vivo and in vitro experiments in which stem cells Ii'om young animals were exposed to niche clements from old animals and vice versa (51). She has discovered that the decline of regenerative capacity with age results li'om changes in the stem cell niche, not in the stem cells themselves, A key element is age-related impairment of the up-regulation of the Notch ligand Delta after muscle injury (52). Exposure of satellite cells from old mice to young serum enhanced the expression or Notch ligand Delta as well as proliferation and regenerative capacity of aged satellite cells. Forced activation of Notch by adding a Notch-speciflc antibody restored regenerative potential to old muscle.
Dr. Conboy has extended her studies to examine the eflccts of aged systemic mileu on the function of human embryonic stem cells (hESCs) (53). Aged mouse serum dramatically inhibited the self-renewal and proliferative potential of hESCs and satellite cells, as measured by expression of markers of stem-cell functionality, rate of cell proliferation, and the capacity for myogenic differentiation. When hESCs are cultured in serum-free mcdium instead of immediate transfer to medium containing aged serum, the cells are no longer susceptible to the effects of aged scrum. This implies that hESCs have produced one or more antiaging factors.
Together. these experiments suggest that the regenerative outcome of stem-cell replacement will be determined by a balance between negative effects of aged tissues on transplanted cells and positive effects of embryonic cells on the endogenous regenerative capacity. This result indicates that a complex interplay between negative regulation by the
784 GORDON E1' AL.
aged niche and positive regulation by hEses will likelydetermine the success of hESe-based cell replacementtherapies. Dr. Conboy's observations in striated muscle provide a well-tested pathway for studying the key questions orthe role of stem cells and aging in progeria, and arc easilyadaptable to the progeria mouse model.
Tom M;steli and Paola 5;cqlfidi (National Cancer Institute) presented the lirst evidence supporting the hypothesis that stem-cell depletion is a factor in organismalsymptoms or progeria (53). The first stcp of their approachwas to determine what gene expression programs areaffected by progcrin. The experimental system consistedof an imm0l1alized skin fibroblast cell line that expressesinducible green fluorescent protein (GFP) progerin. In thissystem, signiJkant progerin accumulation was flrst observed5 days after induction and reached a plateau after 10 days atlevels similar to those found in cells rrom progeria patients.This system allowed analysis of time-dependent changes intranscriptional proilies in response to progerin expressionand compared to expression of GFP-tagged wild-type laminA as a control.
Among genes known to influence differentiation, progerin induction led to up-regulation of several genes in theNotch signaling pathway, most prominently Hes I, Hes 5,and Hey I. This up-regulation was also found in progeriacells. Consistent with a direct role of nuclear progerin intheir regulation, these genes are all in the downstreamportion of the Notch pathway, and no increase was found incleaved Notch expression or in the upstream cleavagereaction. This result dovetailed with Dr. Conboy's observations that Notch is important for maintenance of stem cells(including mesenchymal stem cells) and differentiationpathways, and points to the Notch pathway as a keyelement in progeria.
Drs. Misteli and Scarndi fUliher explored the effects ofprogerin on differentiation by inducing stem cells to go downvarious differentiation pathways (54). Preliminary resultsshowed that the rate of osteogenesis was increased, adipogenesis was blocked, and chondrogenesis was not affected.They hypothesize that progeria leads to the activation of partof the Notch pathway that causes differentiation, and thataccelerated aging in progeria patients is largely the result orstem-cell dysfunction.
ALTERNATIVE PI{ENVLATION AND THE NEED FOR
COMBJNATION CHEMOTHEUAPV IN PROGERIA
Posttranslational processing to create lamin A involvesseveral fon11s of prelamin A, both farnesylated andunfarnesylated (Figure 1). FYI treatment presumably leadsto unusually increased cellular levels of nonfarnesylatedf0I111s of prelamin A and progerin, and could theoreticallypromote alternative prenylation (geranylgeranylation). Theinfluences of nonfarnesylated prelamin A and progerin oncell function and disease status have yet to be intenselyexplored. Michael Sinensky (East Tennessee State University) has begun to examine alternative prenylation (geranylgeranylation) of preJamin A and progerin under theinfluence of FYI. He finds thaI, in cells treated with an FTI,several important cellular events occur: First, geranylgeranyl
is incorporated into prelamin 10 yield a geranylgeranylatedintermediate. He also finds that Zmpste24 does cleaveboth famesylated and gcranylgeranylated prelamin A, andtherefore mature lamin A is produced even in the completeabsence of farnesylation. However, he finds that thefarnesylated prelamin A is a 20-fold beHer substrate thangeranylgeranylated prelamin A. Therefore, processing ofgeranylgeranylated prelamin A is slow, resulting inabnormal accumulation of nonfarnesylated prelamin Awithin cells. Progerin also became geranylgeranylated incells treated with FTI, creating a new prenylated protein.Prelamin A accumulation can afrect cell cycling (55). Thereis a paucity or data available comparing the behavior ofgeranylgeranylated versus farnesylated prelamin A orprogerin, and this area of research may be essential todesigning Future treatments for progeria since altemativeprenylation can alter the behavior and function of cells (56).
In a set of experiments with tremendous breadth, Car/osLopez-Otin examined the potential role of lamin intennediates in disease, round support for alternative prenylation ofprelamin A, and effectively treated a progeria mouse modelusing combination chemotherapy that intenupts progerinproduction at multiple points (57). The effects of laminintetmediates were first addressed. Dr. Lopez-Olin createda "triple mutant" mouse model lhat has the completeabsence of both the Zmpste endonuclease and farnesyltransferase postnatally and is therefore the genetic equivalent of complete famesyltransferase inhibition. I-Ience, onlynonfarnesylated prelamin A is created in this mouse afterbilih. Strikingly, the phenotype of these mice mimicked theZmpste24-1---only mice, without phenotype rescue. Dr.Lopez-Olin hypothesized that the phenotype was causedeither by the accumulation of nonfarnesylated prelamin A,or by an alternatively prenylaled prelamin A product. Toresolve this issue, he tumed to cell culture, and cletell11inedwhich species of prelamins were present in Zmpste24-1
cells with and without FTI treatment. As expected.famesylated prelamin A was present in Zmpste24-1 cellextracts. However, when FYI was added to Zmpste24-1- cellcultures, the amount of famesylated prelamin A decreased,and geranylgeranylated prelamin A appeared. By extrapolation from FTI treated Zmpste24-1 cultured cells to triplemutant mice, he h~~?thesized that the absence or farnesylation in Zmpste24 mice does not improve the phenotypein vivo because geranylgeranylated prelamin A is created,and this product may be al least partly responsible fordisease phenotype in the triple mutant mice.
Dr. Lopez-Otin went on to treat Zmpste24-1 mice withtwo commercially available drugs, statins and bisphosphonates. These drugs block two diFferent steps in themevalonate pathway, upstream from blrnesyltransferaseand geranylgeranyltransferase (Figure 2). Treatment ofZmpste24-F mice with statins and hisphosphonales increased their 20-week life span by 80%. There was alsoimpressive rescue of bone phenotypes, and cellular blebbingwas substantially reducecl. FU11her research is needed tounderstand the mechanism responsible for extended lifespan in treated mice, since bisphosphonates home specifically to bone. However, Dr. Lopez-Otin presents us with theIlrst preliminary evidence that combination chemotherapy
784 GORDON E1' At.
aged niche and posItive regulation by hESCs will likely determine the success of hESC-based cell replacement therapies. Dr. Conboy's observations in striated muscle provide a well-tested pathway for studying the key questions or the role of stem cells and aging in progeria, and are easily adaptable to the progeria mouse model.
Tom Misteli and Paala SNdfidi (National Cancer Institute) presented the first evidence supporting the hypothesis that stem-cell depletion is a ractor in organismal symptoms of progeria (53). The first step of their approach was to determine what gene expression programs are affected by progerin. The experimental system consisted of an imm0l1alized skin fibroblast cell line that expresses inducible green fluorescent protein (GFP) progerin. In this system, signiJkant progerin accumulation was (Jrst observed 5 days after induction and reached a plateau after 10 days at levels similar to those found in cells from progeria patients. 111is system allowed analysis of time-dependent changes in transcriptional proflles in response to progerin expression and compared to expression of GFP-tagged wild-type lamin A as a control.
Among genes known to influence differentiation, progerin induction led to up-regulation of several genes in the Notch signaling pathway, most prominently Hes I. He... 5. and Hey I. This up-regulation was also found in progeria cells. Consistent with a direct role of nuclear progerin in their regulation, these genes are all in the downstream portion of the Notch pathway, and no increase was found in cleaved Notch expression or in the upslream cleavage reaction. This result dovetailed with Dr. Conboy's observations that Notch is imporlant for maintenance of stem cells (including mesenchymal stem cells) and differentiation pathways, and points to the Notch pathway as a key elemenl in progeria.
Drs. Misteli and Scaffidi fUl1hcr explored the effects of progerin on differentiation by inducing stem cells to go down various differentiation pathways (54). Preliminary results showed that the rate of osteogenesis was increased, adipogenesis was blocked, and chondrogenesis was not affected. They hypothesize that progeria leads to the activation of part of the Notch pathway that causes differentiation. and that accelerated aging in progeria patients is largely the result of stem-cell dysfunction.
ALTERNATIVE PRENVLATION AND THE NEED FOI~
COMnJNATION CHEMOTHEUAPV IN PROGERIA
Posttranslational processing to create lamin A involves several fonns of prelamin A, both farnesylated and unfarnesylated (Figure 1). FYI treatment presumably leads to unusually increased cellular levels of nonl~lrnesylated
for111s of prelamin A and progerin, and could theoretically promote alternative prenylation (geranylgeranylation). The influences of llonfarnesylated prelamin A and progerin on cell function and disease stalus have yet to be intensely explored. Michael Silll'Jlsky (East Tennessee State University) has begun to examine altemative prenylation (geranylgeranylation) of prelamin A and progerin under the influence of FTI. He finds that. in cells treated with an FTL several important cellular events occur: First, geranylgeranyl
is incorporated into prelamin to yield a geranylgeranylatecl intermediate. He also finds that Zmpste24 does cleave both famesylated and gcranylgeranylated prelamin A, and therefore mature lamin A is produced even in the complete absence or farnesylation. However, he llnds that the farnesylated prelamin A is a 20-fold beller substrate than geranylgeranylated prelamin A. ThcrcCore, processing of geranylgeranylaled prelamin A is slow, resulting in abnormal accumulation of nonfarnesylated prclamin A within cells. Progcrin also became geranylgeranylated in cells treated with FTI, creating a new prenylated protein. Prelamin A accumulation can affect cell cycling (55). There is a paucity of data available comparing the behavior of geranylgeranylated versus farnesylated prelamin A or progerin, and this area of research may be essential to designing future treatments for progeria since altemative prenylation can alter the behavior and function of cells (56).
In a set of experiments with tremendous breadth, Carlos Lope::.-OtiJl examined the potential role of lamin intennediates in disease, found support for alternative prenylation of prelamin A. and effectively treated a progeria mouse model using combination chemotherapy that inten-upts progerin production at multiple points (57). The effects of lamin intelmediates were first addressed. Dr. Lopez-Otin created a "triple mutant" mouse model that has the complete absence of both the Zmpste endonuclease and farnesyltransferase postnatally and is therefore the genetic equivalent of complete famesyltransferase inhibition. Hence, only nonfarnesylated prelamin A is created in this mouse after bil1h. Strikingly, the phenotype of these mice mimicked the Zmpste24-1---only mice, without phenotype rescue. Dr. Lopez-Otin hypothesized that the phenotype was caused either by the accumulation of nonfarnesylated prelamin A. or by an alternatively prcnylated prelamin A product. To resolve this issue, he fumed to cell culture, and cletemlined which species of prelamins were present in Zmpste24-1
cells with and withollt FTI treatment. As expected. famesylated prelamin A was presenl in Zmpste24-1-- cell extracts. However, when FYI was added to Zmpste24-1- cell cultures, the amount of farnesylated prelamin A decreased, and geranylgeranylated prelamin A appeared. By extrapolation from FTI treated Zmpste24-1-- cultured cells 1.0 triple mutant mice, he hy~othesized Ihat Ihe absence of l'arnesylation in Zmpste24 '"- -,-, mice does not improve the phenotype in vivo because geranylgeranylated prelamin A is created. and this product may be at least partly responsible for disease phenotype in the triple mutant mice.
Dr. Lopez-Otin went on to treat Zmpste24-1-- mice with two commercially available drugs, stalins and hisphosphonates. These drugs block two different steps in the mevalonate pathway, upstream from f~lrnesyltranslerase
and geranylgeranyltransferase (Figure 2). Treatment of Zmpste24-F
- mice with statins and hisphosphonates increased their 20-week life span by 80%. There was also impressive rescue of bone phenotypes, and cellular blebbing was substantially reduced. FU11her research is needed to understand lhe mechanism responsible for extended Ii fe span in treated mice, since hisphosphonates home specifically to bone. However, Dr. Lopez-Otin presents us with the first preliminary evidence that combination chemotherapy
HIGHLIGHTS OF 7lfE 2007 PIiF SCIENTIFIC WOIiKSHOP 785
CerClnyl··PP
~l~op(,nt('nyl··IlP
t
C,opyriqht lOOfl NimJl'C f'lIbli~hiI19CroupNature Reviews I (;",rwries
C('rijllylqprilnyl··I'I'/
Squill!.'l\('
!
H'P
Figure 2. ClllC prenylation rathway-opp0l1tll1ities for therapy (59). Potential
for therapy (i.e.. stat ins. fJisphosphol1mes, and ITls Ifarnesyltransfer,lse
inhibitors!) af three points along the pathway. FPP famesyl pyroph()sphatc~
HMG·CoA hydroxymelhylglutaryl co-cll7.ymc A; PI' pyrophosphme.IRcprodueed by permission of thc puhlisher.l
J\cpfvICo;\' <t(f'1,)MefvICo/\
~j ··Hydnlxy· -3· m('t.I\~'lqhlt;J(VI- (:oJ\
11i\'\c;·CoA )j;pel",,,,,,,,, f----
vicinity of the LMNII gene, a step toward identifying LMNAgenotypes involved in longevity. Links between progerin,progeria, and the cardiovascular disease in aging are still intheir early stages, as most experiments have been conductedon flbroblast cultures and should be extended to vascularcell types in future studies. However, pathological andclinical studies in humans and mice point to a nonlipiddriven vascular stitTening shared by both progeria patientsand elderly individuals.
As the current drug trial for progeria is testing an FTI,there was much focus on the biological mechanisms thatinvolve farnesylation during posttransJational steps that leadto lamin A. In a progeria model mouse, the first evidencewas presented that FYI may reverse the damaging effects ofprogeria in vivo, even when administered after vasculardamage has occurred. The importance of this result becomesobvious when one recalls that progeria is not diagnosed untilsignificant damage to organ systems has taken place. In thenumerous basic science studies on cellular effects in progeria, investigators onen checked whether FTI treatmentaffected the cellular damage under study. As shown inTable I, some but not all were reversed by FTI treatment
A POWERFlJL NEW TESTING STRATEGY FOR
DRUG DISCOVERY IN PROGERIA
Drug discovery is an important direction to pursue inprogeria. especially in light of the possibility that drugs thaihelp these children may also be beneficial to the !lonnalaging population and a large fraction of men and womenafflicted with cardiovascular disease. High-throughputscreening (HTS) is an essential first step in drug development. The key 10 HTS is to develop an assay thateasily detccts some visible change in the target cells, due toexposure to a chemical compound that can be quickly andreliably read by a sensor. Nuclear blebbing has been used tomeasure improvement in progeria cells in response tovarious drugs such as FYIs. However, for testing chemicallibraries on a large scale, such morphological assays areunsuitable. Tom Misteli and CordeJle Tanega (NationalCancer Institute) have developed what could be a breakthrough testing strategy. The Misteli laboratory has previously showed that blocking the cryptic splice site that isactivated by the progeria mutation with a complementaryoligonucleotide restores the phenotype of progeria hbroblasts to normalcy (58). Because satishlctory methods forsystem-wide introduction of oligonucleotides to humans donot yet exist, Misteli has embarked on a search for smallmolecules that specifically inhibit the altemate spicingcreated in exon 11. The work is being carried out at a fullyautomated high-throughput system for drug screening at theNIH Chemical Genome Center.
Dr. Misteli devised a system based on dual fluorescencereporters that indicates whether the cl)'ptic splice site inLMNA is used or not. After development, the assay wascharacterized using the available oligonucleotide andoptimized for HTS. In pilot experiments, a small library of1279 compounds was tested at seven concentrations,thereby producing a titration curve on the first screen. Alarge screen of 150,000 compounds followed, and led tothe identification of 100 compounds based on theirresponse profiles as well as structural and phannacologicalconsiderations. Experiments are now under way to test thesecompounds in progeria fibroblasts, looking at cell phenotypes, splicing activity, and specilkity.
DISCUSSION
The presentations, posters, and discussion at the 2007Progeria Workshop showed extraordinary progress at alllevels-from basic science, mouse and human studies thatbeller define the biological effects of progerin on diseasepathogenesis and on aging and cardiovascular disease in thegeneral population., to the first clinical drug trial forprogeria. The discovery that progerin is found in normalhuman fibroblasts in an age-dependent manner establishesa new link between progeria and aging. This is the bestexample yet of how studies on progeria may be valuable tounderstanding human aging. In addition, preliminary studieson samples from people over the age of 94 years founddistinguishing single-nucleotide polymorph isms in the
using FTI along with drugs that affect both progerinproduction and alternative prenylation could be efficacious.
785 HIGHLIGHTS OF THE 2007 PNF SCIENTIFIC WORKSHOP
using FTI along with drugs Ihal affect both progerin production and alternative prenylation could be efficaciolls.
A POWERFlJL NEW TESTING STI~ATEGY FOR
DRUG DISCOVERY IN PROGERIA
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 benetlcial to the nonnal 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 developmcnt. The key to HTS is to develop an assay that easily detects some visible change in the target cells, due to exposure to a chemical compound that can be quickly and reliably read by a sensor. Nuclear blebbing has been used to measure improvement in progeria cells in response to various drugs such as FTIs. However, for testing chemical libraries on a large scale, such morphological assays are unsuitable. Tom Misteli and CordeJle Tanega (National Cancer Institute) have developed what could be a breakthrough testing strategy. The Misteli laboratory has previously showed that blocking the cryptic splice site that is activated by the progeria mutation with a complementary oligonucleotide restores the phenotype of progeria fibroblasts to normalcy (58). Because satisl~lctory methods for system-wide introduction of oligonucleotides to humans do not yet exist. Misteli has embarked on a search for small molecules that specifically inhibit the altemate spicing created in exon 1 I. The work is being carried out at a fully automated high-throughput system for drug screening at the NIH Chemica] Genome Center.
Dr. Misteli devised a system based on dual fluorescence reporten; that indicates whether the cryptic splice site in LMNA is used or not. After development, the assay was characterized using the available oligonucleotide and optimized for HTS. In pilot experiments, a small library of 1279 compounds was tcsted al seven concentrations, thereby producing a titration curve on the first screen. A large screen of ~~ 150.000 compounds followed, and led to the identification Qf ~~ 100 compounds based on their response proflles as well as structural and phannacological considerations. Experiments are now under way to test these compounds in progeria fibroblasts, looking at cell phenotypes, splicing activity, and specilkity.
DISCUSSION
The presentations, posters, and discussion at the 2007 Progeria Workshop showed extraordinary progress at all levels-from basic science, mouse and human studies that better define the biological effects of progerin on disease pathogenesis and on aging and cardiovascular disease in the general population, to the first clinical drug trial for progeria. The discovery thal progerin is found in normal human fibroblasts in an age-dependent manner establishes a new link between progeria and aging. This is the best example yet of how studies on progeria may be valuable to understanding human aging. In addition, preliminary studies on samples from people over the age of 94 years found distinguishing single-nucleotide polymorphisl11s in the
1~()p('nt('n\,l I'P•~
Cf'lrlllyl PP
r f' f' '"''"""0jl- :L~'~J:;i;;;~r;-i;~;;(~:I~.~] l-iJn)p~)'!11 ,I1l',1(' 1'/)\(
V ,'"-'--.... "'-':-"~"''''-~''-'"l rdrtl("\vl PI' ~ ,FilrnesY!illed protein~:",.,...J--l i(e~Q.l.IJllil1 A} i
~rTr~,
/ ':.qll<lIr·IlP CprilllylqPI anyl 1'1'
I--'~-:----·~-·-:·-··-~-'-"-'·-··.:'-'·'·~~. . ... . '--.' .>
, Cef<lnyl~JerilnY[i:lted protein~j :ClJolesterol: ! (l{h()~·!~ily~,\=(J~,47.)__ ' I.•.... __ _. __ _ ·._ ••••v
C,opyrlqht JO()() NdWH' f'lIbll\hIl19 Croup Nature Reviews I C;"'nNics
Figure 2. '1l1e prenylalion pathway-opportllnitic~for therapy (59). POlential
ror lherapy (i.e.. ~Ialjns. hi.lpho'pholl<l1CS, and r-Tls Ifarnesyltransfer;lse inhibitor, I) at three p()inl~ along lhe pa1hway. FPP =-= famesyl pyrophosphate~
HMG-CoA =-= hydroxymclhylglutaryl CO-Ctl7.YIllC A~ PI' --- pyropho~ph;ltc.
IReproduced by permission of the puhlisher.]
vicinity of the LMNIl. gene, a step toward identifying LMNA genotypes involved in longevity. Links between progerin, progcria, and the cardiovascular disease in aging are still in their early stages. as most experimcnts have been conducted 011 Ilbroblast cultures and should be cxtended to vascular cell types in future studies. However, pathological and clinical studies in humans and mice point to a nonlipiddriven vascular stiffening shared by both progeria paticnts and elderly individuals.
As the current drug trial for progeria is testing an FTL there was much focus on the biological mechanisms that involve farnesylation during posttranslational steps that lead to lamin A. In a progeria model mouse, the first evidence was presented that FTT may reverse the damaging effects of progeria in vivo, even when administered after vascular damage has occurred. The importance of this result becomes obvious when one recalls that progeria is not diagnosed until significant damage to organ systems has taken place. In the numerous basic science studies on cellular effects in progeria, investigators onen checked whether FTJ treatment affected the cellular damage under study. As shown in Table I, some but not all were reversed by FTI treatment.
7R6 GORDON ET AL.
Whether this will translate into some, but not complete,clinical improvement with FTI remains to be determined.
Pivotal j~lCtorS for disease phenotype that emerged duringthis workshop included the balance between multipleprclamin A [011115, mature lamin A, and progerin, thepresence of alternative prenylation. and epigenetic changesmodifying chromatin structure. It is highly likely that, aswith most diseases, improving quality and longevity of lifefor children with progena will reqUire intense andmulti pronged research. The emerging data hold promisethat progeria may be amenable to single or combinationchemotherapy, stem-cell therapy. and genetic therapies. Inaddition. this fleld or study has already brought an entirelynew gene (LMNA) and its protein products (lamin A andprogerin) into the aging and cardiovascular l1eJds. Futureresearch should concentrate 011 understanding the influencesof balancing prelamin A, lamin A, and progerin not only inprogeria, but on health and longevity in all of us.
ACKNOWLEJ)(;r-,lENTS
We thank thc parcnts and childrcn with progcria for their kcyparticipation in the family panel. We thank all those researchers whocontributed to the workshops, and apologize that we could not highlight theexcel1c111 work of every speaker and postcr prcscnter due In spaceconstraints. We thank Audrey Gordon. Esq.. and Susan Rosenblatt fororganizing workshop logistics. We thank the workshop advisory panel.Drs. Rohert Goldman, George Martin, Susan Michaelis, Tom Misteli, andI-Iuber Warner, and also the colleagues who chaired the sessions: ScotlBerns, Yosef Gruenbaum (Hebrew U., Jerusalem. Israel), and SusanMichaelis (Johns Hop~ins School of Medicine).
The workshop was generously funded by the Oftlce of Rare Diseasesand the National I-Iearl, Lllllg and Blood Institue (I R13HL091695-0 I j,the National Cancer Institute, thc Ellison Medical Foundation, and TheProgeria Research Foundation.
COllRESI'ONDENCE
Address correspondence to Leslie B. Gordon. MD. PhD, Warren AlpeltMedical School of Brown University. Providence, RI 02912. E-mail:[email protected]
REFERENCES
I. Kieran MW, Gordon L. Kleinman M. New approaches to progeria.Pedialrics 2007; 120:R34-R41.
2. Hennekam RC. Htltchinson-Gilrord progeria syndrome: review or thcphenotype. AI1/ J Med Gellel A, 2006; 140:2603-2624,
3. Uitto J. Searching for clue~ to premature aging. Trl'nds EndocrillO!Metah,2002;13:140-141.
4. Eriksson M, Brown WT, Gordon LB, et al. Recurrent de novo pointmtltatiom in lam in A cause Hutchinson-Gill"ord progeria syndrome.Nal/lre. 2003:423:293-29R.
5, De Sandre-Giovannoli A, Bernard R, Cau P, et al. Lmnin A tnlllcationin Hutchinson-Gilrord proge-ria. Sciellce. 2003:300:2055.
6. Yang Sl-l, Mela M. Qiao X, et a!. A l~lrnesyltransferase inhibitorimproves disease phenotypes in mice with a Ilutchinson-Gilfordprogeria syndrome mutation. J crill [lIl'e,\'I. 2006; 116:2115-2121.
7. Varga R, Eriksson M, Erdos MR. et al. Progressive vasnliar smoothmuscle cell defects in a mouse model of Hutchinson-Gilford progeriasyndrome. Proc Naif Acad Sci USA. 2006; I03:3250--3255.
R. Mallampalli MP, Huyer G, Bendalc P, Gc1b Mil, Michaelis 5,Inhibiting farnesylation reverses the nuclear morphology defect ina HeLa cell model ror Hutchinson-Gilford progeria syndrome. PrO(Naif Acad Sci USA. 2005;102:14416--14421.
9. Capell BC, Erdos MR, Madigan JP, et al. Inhibiting famesylation ofprogerin prevents the characteristic nUl..'lcar blebbing of I-Iutchinson-
Gilford progeria syndromc, Proc Nal! !lead Sci USA. 2005;102:12ii79-12ii84.
10. Glynn MW, Glover TW. Incomplete processing of mutant lam in A inHutchinson-Gilford progeria leads to Iluclear abnormalities, which arereversed by farnesyltransferase inhibition. HI/m Mo! GI'I/I'I. 2005:14:2959-2969.
II. Yang SH, Bergo MO, Toth JI, et al. Blocking protein r<ll11esyltransferase improves nuclear blebbing in mouse fibrohlasts with a targetedllutchinson-Gilford progeria syndrome mutation. Pme Nat! Aead ScilJ S A. 2005:102:10291-10296.
12. Moore K. Old al Ag(' 3, I st Ed. Stillwater, Oklahoma: Boss Publishing,Inc; 2007.
13. Kieran MW, Packer RJ, Onar A. et 'II. Phase- I and pharmacokineticstudy or the oral rarnesyitransferase inhibitor lonafamih administeredtwice daily to pediatric patients with advanced central nervoussystem tumors using a 1110dilicd continuous reassessment method: aPediatric Brain Tumor Consortium Study. J C/in OIlCO! 2007;25:3137-3143.
14. Gordon LB, McCarten KM, Giobbie-llurder A. et al. Diseaseprogression in Ilutchinson-Gilford progeria syndrome: impact ongrowth and development. Pediatrics, 2007: 12():824·833.
15. Stehbens WE, Wakefield 51. Gilbel1-Barness E. Olson RE, Ackerman1. Histological and ultrastructural features or atherosclerosis in progeria.Cardio\'asc Pat/wi. 1999:R:29-39.
16. Gordon LB, Hallen IA. Pal1i ME, Lichtenstein AJ-l. Reduccdadipolleclin and IIDL cholesterol without elevated C-reactive protein:clues to Ihe biology of premature atherosc1crosi:-; in Ilutchinson-GilfordProgeria Syndrome. J Pedialr. 2005; 146:336-341.
17, Merideth MA, Gordon LB, CIllllsS S. ct al. Phenotype and course ofHutchinson-Gilford progeria syndrome. N Eng! .r Med. 200R;35X:592-604.
18. Gruenbaum Y. Goldman RD, Meyuhas R, et al. The nuclear lamina andits functions in the nucleus. 111/ R('\' Cylol, 2003;226: 1--62.
19, Sinensky M, Fantle K. Trujillo M. McLain T, Kupfer A, Dalton M, Theprocessing pathway of prelamin A . .r ('ell Sd. 1994:I07( Pt 1):61-67.
20, Candelario .1, Sudhakar S, Navarro 5, Reddy S, Comai L. Perturbationof wild-type lam in A metabolism results in a progeroid phenotype.Al-(illg Celf. ln Press.
21. Coffinier C, Hudon SE, Farber EA. e[ a!. H1V protease inhibitors blockthe zinc metalloproleinase ZMPSTE24 and lead to an accumulation ofprelamin A in cell.~. Proc Nail Acad Sci U .'I A. 2007; 104: 13432-13437.
22. Verstraeten VL. .Ii JY, Cummings KS, Lee RT, Lammerding J.Increased mechanosensitivity and nuclear stiffness in HlltchinsonGilford progeria ce1L~: effects of famesyltransrcrase inhibitors. AgingCd!. 2008;7:383-393,
23. Shumaker OK. Kuczmarski ER, Goldman RD. The Ilucleoskeleton:lamins and actin are major players in essential nuclear functions, CI/I"/"Opin Cd! BioI. 2003;15:35R",J66.
24. Moil' RD, Yoon M, Khuon S. Goldman RD. Nuclear lamins A and HI:dirfcrent pathways of assembly during Iltlclear envelope rormation inliving cells. J Celf BioI. 2000;151: 1155-116l-J.
25. Goldman RD, Shumaker OK, Erdos MR, el al. Accumulation ofmlltant lamin A causes progressive changes in nuclear architecture inHutchinson-Gilford progeria syndrome, Proc Nat! Acad 5;ci USA.2004: 101 :R963-R96R.
26. Shumaker OK, Dechat T, Kohlmaier A, et a!. Mtlt<lnt nuclear lamin Aleads to progressive al1erations of epigenetic control in prematureaging. Proc Nail !lead Sci USA. 2006: 103:8703-ii70ii.
27. Meabum KJ, Cabuy E. Bonne G, el al. Primary laminopathy fibroblastsdisplay altered genomc organizalion and apoptosis. Al-(illg Cell. 2()()7:6;139-153.
2ii, BridgerJM, Boyle S. Kill JR, Bickmore WA. RC-l1lodelling nfnudeararchitecture in quiescent and sencscent human fibroblasts. Cllr!" Bio!.2000; 10: 149-152.
29. TelTY LJ, Shows EB, Wente SR. Crossing the nuclear envc!ope:hierarchical regulation or nucleocytoplasmic transport. Se/e/1Cl'. 20D7;3IR:1412-1416.
30. Martin GM. Genetic syndromes in man with potential relevance to thepathobiology of aging, Birth Defecls jOrig Artic Serf. 197R: 14:5-39.
31, Allsopp RC, Vaziri H. Palterson C. et al. Telomere length predictsreplicative capacity of human fibroblasts. Proc Nail Acod Sci USA.1992;X9:l()114--IOI18.
7R6 GORDON kT AL.
Whether this will translate into some, but nol complete, clinical improvement with FTl remains to be determined.
Pivotal j~lCtorS for disease phenotype that emerged during this workshop included the balance between multiple prelamin A fOllllS, mature lamil1 A, and progerin. the presence of alternative prenylation. and epigenetic changes modifying chromatin structure. It is highly likely that, as with most diseases, improving quality and longevity or life for children with progeria will require intense and multiprongcd research. The emerging data hold promise that progeria may be amenable to single or combination chemotherapy, stem-cell therapy, and genetic therapies. In addition. this Ileld or study has already brought an entirely new gene (LMNA) and its protein products (Iamin A and progerin) into the aging and cardiovascular Ilclds. Future research should concentrate on understanding the influences of balancing prelamin A, lamin A. and progerin not only in progeria, but on health alld longevity in all of LIS.
ACKNOWLED(;rvlENTs
We thank [he parent.~ and children with progeria for [heir key par[icipatioll in the family panel. We thank all [hose researcher~ who contributed to the workshops. and apologize that we could not highlight the excellent work of every speaker and poster presenter due In .~pace
conslrainls. We Ihank Audrey Gordon. Esq., and Susan Rosenblatt for organil.ing workshop logistic~. We thank the workshop advisory panel. Drs. Roher[ Goldman. George Mar[lIl. Susan Michaeli~. Tom Misteli. and I-Iuber Warncr, and also the c(lileaguc.~ who chaired [he scssions: Seotl Berns. Yosef Grucnbaulll (Hebrew LJ .• Jerusalem. Israel). and Susan Michac1i~ (John~ Hop~in~ School of Medicinc).
The workshop was generously funded by the Oftlce of Rare Diseases and the National I-Ieart. Lllllg and Blood Illstitue (I R 13HL091695-0 I), thc National Cancer Ins[itute. the Ellison Medical Foundation. and The Progeria Research Foundalion.
COil RES I'ONDENCE
Address correspondcnce [0 Lc~he B. Gordon. MD. PhD, Warren Alpel1 Medical School of Brown University. Providence. RI 02912. E-mail: Ic.~ [email protected]
REFH{ENCI,S
1. Kieran MW, Gordon L. Kleinman M. New approaches to progeria. Pedialric,\' 2007; 120:R34-R41.
2. Ilennekam RC. Hutchinson-Gilford progena syndrome: revicw 01" [he phenotype. Am .T Mcd Gellel A. 2006: 140:2603-2624.
3. Ui[to J. Searchmg for c1ue~ to premature aging. Trl'IJd.1 Fndocrillol Melab, 2002: 13: 140-141.
4. Eriksson M. Brown WT. Gordon LB. et al. Recurrent de 110VO point mutation.~ in lal1lin A cause Hlltchin.~on-Gill"ord progeria syndrome. Nall/re. 2003:423:293-298.
5. De Sandrc-Giovannoh A, Bernard R, Cau P, el a1. Lamin A tnmcation in Hutchinson-Gill"ord proge-ria. Sciellce. 2003:300:2055.
6. Yang 51-1, Me[a M. Qwo X. et al. A L1f!lesyltransferase inhibitor improves disease phenotypes in mice with a Ilu[chinson-Gilford progeria syndrome mutation . .T crill fllI·c.I·I. 2006; 116:2115-2121.
7. Varga R. Eriksson M, Erdo~ MR. el al. Progressive vascular sl1100th muscle ccll dcfect1> in a mouse model of Hutchinson-Gilford progcria syndrome. Proc Naif Acad Sci lJ SA. 2006; 103:3250--3255.
It Mallampalli MP, Huyer G. Bendale P. Gelb Mil, Michaelis S. Inhibiting farnesylation reverses lhc nudcar morphology defect in a HeLa cell model I"or Hu[chinson-Gilford progeria syndrome. Pml
NaIl Acad Sci lJ S A. 2005;102:14416--14421. 9. Capell Be. Erdo~ MR. Madigan JP. cl al. Inhibiting famesylation of
progenn prevents the characteri.~tie nu<..'1car blebbing of I-Iutchinson-
Gilford progeria .~yndrollle. Proc Naif !lead Sci USA 2005: 102: 12879-12884.
10. Glynn MW. Glover TW. Incomplete processing of mutan[ lamin A in lIutchinson-Gilford progeria leads to nuclear abllorllla[itie~, which arc reversed hy farncsyhransferase inhibition. HI/m Mot Gelll'1 2005: 14:2959-2969.
I I. Yang 51-1, Bergo MO, Tolh Jl, et al. Blocking protcm l"al1le~y1transferase improves nuclear b1cbbing in mouse fibroblasts with a targeted Il1Itchinson-Gilford progeria ~yndrome mutatioll. Proc Nat! Aead SCI lJ SA 2005: J02: 10291-10296.
12. Moore K. Old 01 Ag(' 3. 1st Ed. Stillwater. Oklahoma: Boss Publishing, Inc; 2007.
! 3. Kieran MW, Packer RJ, Ona r A. et al. Phase I and pha rmacok inet ic study of lhe oral I"arnesyltransferase inhibitor lonafamib administered twice daily to pediatric patients with advanced central nervoll.\ system tumor.~ using a modiJied continuous reassessment method: a Pediatric Brain Tumor ConsortiuIll Study_ .I C/ill 011("01 2007~25:
3137-314:1. 14. Gordon LB. McCar[en KM, Giobbic-Hurder A. e[ al. Disease
progress ion 1n JIu[ch inson -Gi Iford progeria syn drome: im pact on growth and development. P('dialric.\". 2007: 12():824-R33.
i5. S[ehbens WE, Wakefield 51. GilbeJ1-Barness E. Olson RE. Ackerman J. Histological and uHrastruclural !Catures or a[herosclerosis in progena. CardilH'a.IT PalllOl. 1999:H:29-39_
16_ Gordon LB. Hallen IA. Patti ME, LIChtenstein AJ-I. Reduced adipollcctin and IIDL cholesterol wilhout elevated ('-reactive protein: clues to the biology of prematurc atherosc1erosi~ in Ilu[chinson-Gilford Progeria Syndrome. .I Peditl/r. 2005; 146:336-?>41.
17. Merideth MA. Gordol1 LB, Clauss S. el al. Phellotype and course of Hutchinson-Gilford progeria syndrome. N Engf .I Med. 200R;35R: 592-604.
IR. Gruenbaum Y, Goldman RD, Meyuhas R, el al. The nuclear lamina and its functions in the nucleus. 1m RCI' Cylol, 2003;226: 1--62.
19. Sinensky M, Fantle K. Trujillo M. McLain T. Kupfer A. Dalton M. The proccssing pathway of prelamin A. .I CI,II SI'i. 1994: I07( p[ 1):61-67.
20. Candelario .1, SlIdhakar S, Navarro S, Reddy S. Comai L. Perturb<1lion of wild-type lamin A metabolism results in a progcroid phenolype. A,r,:illg Celf. In Press.
21. Coffinier C. Iludon SE. Farber EA. el al. H1V protease inhibitors block the zinc metalloproteinasc ZMPSTE24 and lead to an accumulation of prelamin A in cel!.,. Proc Nail A("ad S("I USA. 2007; 104: 13432- 13437.
22. Verstraeten VL. .Ii JY. Cummings KS. Lee R1', Lammerding .I. Increased mechanosensi[i vi [y and nuclear sti ffness in H1I teh insonGilford progeria celh: en"ccl.~ of famesyltransfenlse inhibitors. Agillg Ccll. 2008~7:383-393.
23. Shumaker DK. Kuczmarski ER, Goldman RD. The nuclcoskeleton: lamins and actin are major players in essential nuclear functions. Cllrr Opin Cell BioI. 2003: J 5:358·J66.
24. Moir RD, YOOIl M. Khuon S, Goldman RO. Nu clear lamin.~ A and B J :
dirJ"crent pathways of assembly during nuclear envelope formation in living cells. .I Celf BioI. 2000: 151: J ! 55-1 J 6H.
25. Goldman RD, Shumaker OK, Erdm MR, el al. Accumulation of Illutant lamin A causes progressivc changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Pmc Naif Acod 5;('; lJ S A. 2004: 101 :R963-R968.
26. Shumaker OK. Dcchat T. Kohlillaler A. et al. Mu1<mt nuclear lamin A leads [0 progrcs.~ive al1erations of epigenetic control in premature aging. Pr()( Nail Ai'ad Sci lJ S A. 2006: 103:R703-870H.
27. MeabunJ KJ. Cabuy E. Bonne G. et al. Primary lamillopathy fibroblasts display altered genome organization and apoptosis. Agil1!-1 Cell. 2007: 6:139-153.
28. Bridger JM, Boyle S. KilllR. Bickmore WA. RC-Illodelling of nuclear architecture in quiescent and senescent human fibroblasts~ Cllrr BioI. 2000; 10: 149-152.
29. Teny LJ. Shows EB. Wente SR. Crossing the nuclear envelope: hierarchical regulation 01" nucleocytoplasmic transport. Sci£'/)("('. 2007: 31 R: J 412-1416.
30. Martin GM_ Genetic syndromes in man with potential relevance to the pathobiology of aging. Birlh D('fecls /Orig Arlie Serf. ! 97&: 14:5-39.
J I. Allsopp Re. Vaziri H. Patterson C. et a!. Telomere length prcdicts replicative capacity of human fibroblast1>. Pmc NaIl Aeod SCI USA. 1992:89:10114----10118.
IIIGIfUGIIH OF TilE 2007 PRF SCIENTIFIC WORKSIIOP 787
32. Ly DJ-I. Lockhart DJ. Lerner RA Schultz PO. Mitotic misrcgllialionand human aging. Sciellce. 2000;2X7:24R6-2492.
3:1. Scaffidi P. Misleli T- Lamin A-dependent nuclear defects in Illllllanaging. Scicl1ce. 2006;112:1059-1063.
34. Can K. Capell BC Erdos MR, DjabaJi K. Collins FS. A lal11in A proteinisofol1n ovcrcxprcssed in Hutchinson-Gilford progeria syndromeinlcrlCres with mitosis in progeria and nOll11al cells. Prof' Nail AcadSci USA. 2007: I04:4949---4954.
35. McClintock D. Ratner D. Lokugc M, el al. The lllutant form of lalllin Athai causes Hutchinson-Gilford progeria is a biomarker of cellular aginginlllllmm skin. PLoS ONE. 2007:2(l2):eI269.
36. Campisi J. ([,Adda eli Fagagna F. Cellular senescence: when bad thingshappen to good cells. Nat RCI' Mot Cdt Bioi. 2007;R:729-740.
37. Manju K. Muralikrishn1l B, Palllaik VK. Expression of disease-causinglamin A rnuwnls impairs the formation of DNA repair foci.'/ Cell Sci.2006: 119(Pt 13):2704-2714.
3R. Liu B, Wang J, Chan KM, et al. Genomic instahility in laminopathybased premature aging. Nal Med. 2005: II :780--7R5.
39. Varela L Cadinanos J, Pendas AM. et al. Accelerated ageing in micedeficient in Zmpste24 protease is linked to p53 signalling activation.Nalure. 2005;437:564-568.
40. Litl Y, Wang Y, Rusinol AE, et al. Involvement of xerodem1apigmcntosum group A (XPA) in progcria arising from del"cc!ivematuration of prelamin A. FASFB J. 2008;22:603---6 II.
41. Liu Y. Rusinol A, Sincnsky M. Wang Y, ZOLI Y, DNA damageresponses in progeroid syndromes arise from defective maturation ofprelamin A. ,/ Celf Sci. 2006; 119(Pt 22):4644·-4649.
42. Picinich SC Mishra PJ, Mishra PJ, Glod J, Baneljee D. The therapeuticpotential of mesenchymal stem cells. Cel!- & tissue-based therapy.Expert Gpill Bioi Ther. 2007;7:965-97:1.
43, Warner HR. 2()()6 Kent award lecture: is cell death and replacementa factor in aging'!.! Geronfot A Riot S('i MI'd Sn". 2007:62:1228-1232.
44. Bridger JM, Kill JR. Aging of Hutchinson-Gilford progeria syndromefibroblasts is characterised by hyperproliferation and increased apoptosis. EXfJ GCl"{mlot. 2004:39:717-724.
45. Prolla TA. Multiple roads to the aging phenotype: insights hom themolecular dissection of progerias through DNA microamlY analysis.Mcch Ageing Dc\'. 2005;126:461--465.
46. Hutchison CJ, Worman HJ. A-type lamins: guardians of the soma'? NatCdt Bioi. 2004;Ci:I062·, 1067.
47. Rober RA. Sauter J-1, Weber K, Osbom M. Cells of the cellular immuneand hemopoietic system of lhe mouse lack lamins A/C: distinctionversus other somatic cells . .1 Cel! S'd. 1990:95( Pt 4):587-598.
48. (JotI.1l1<lnn 1. Foisner R. A-type lall1in complexcs and regenerativepotential: a slep towards understanding laminopalhic diseases'? Histoc!WIlI Cel! Rio!. 2006; 125:33-41.
49. Halaschek-Wiener J, Brooks-Wilson A. Progeria of stem cells: stemcell exhaustion in Ilutchinson-Gilford progeria syndrome . .r Gerol/lot ABioi Sci Mcd Sci. 2007:62:3-8.
50. Espada J, Varela 1, Flores I, et al. Nuclear envelope defects cause stemcell dy.~function in premature-aging mice . .1 Cetl Bioi. 2008; 181 :27--35.
51. Conboy 1M, Conboy MJ. Wagers AJ, (,inna ER. Weissman It. RandoTA. Rejuvcnation of aged progenitor cells by exposure to a youngsystemic environment. Nalure. 2005:433:760----764.
52. Conboy 1M, Conboy MJ, Smythe GM. Rando TA. Notch-mediatedrestoration of regenerative potenliallO aged muscle. Sciencc. 2003;302:1575-1577.
53. Carlson ME, Conboy 1M. Loss of stem cell regenerative capacity withinaged niches. Agil/g CeI!. 2007;6:371-.182.
54. Scaffidi P, Misteli T. Lamin A-dependent misregulation of adultstem cells associated with accelerated ageing. Nal Cdl Biot. 20()8: I 0:452-459.
55. Ukekawa R, Miki K, Fujii M. Hirano 1-1, Ayusawa D. Accumulation ofmultiple fOlllls of lamin A with down-regulation of FACE-I suppressesgrowlh in senescent human cells. GCl/c'\" Cells. 2007:12:397--406.
56. Rusinol AE. Sinensky MS. Farnesy!ated lamins, progeroid syndromesand famesyl transferase inhibitors. .r Cdl Sci. 2006; 119(Pt 16):32653272.
57. Varela l, Pereira S, Ugalde AP, et al. Combined treatment with stat insand aminohisphosphonates extends longevity in a mouse model ofhuman premature aging. Nal Met!. 2008;14:767-772.
5X. Scaffldi 1', Mi.~teli T. Rcver.~'<ll of the cellular phenotype in thepremature aging disease Hutchinson-Gilford progeria syndrome. NmMed, 2005:! 1:440-445.
51). Capell Be Collins FS. Human laminopathies: nuclei gone geneticallyawry. Nal RI'l' GCII('I, 2006:7:940---952.
Rcccil'cd May 28. 2008Ac("cpled May 30. 20(),'jDecision Editor: HI/hcr R. \VaI'llCl". PhD
787 }-IIGlfLIGHTS' OF THE' 2007 PRF SCIENTIFIC WORKSHOP
:12. Ly DH. Lockhar[ OJ. Lerner RA. Schu1t7 PO. Mitotic lllisregillation and human aging. SCle/lcc. 2000;2X7:24R6-2492.
:1:1. ScaffidI P. Mislelr T. LaIl11n A-dependent nuclear defect." III human aging. SClenct'. 200(dI2:1059-1063.
:14. Cao K. Capell Be Erdos MR, DjabaJi K. Colltns FS. A lamin A protein Isofonn overexpressed in HutchInson-Gilford progeria syndromc interfere" with mitosi.<, in progeria and nomlal cells. Prot' Nail Ai ad Si IUS A. 2007: 104:4949---4954.
35. McClintock D. Ratner D. Lo"-uge M. et al. Thc lllutant form of lalllin A that causes HUlchin ..on-Gilford progeria is a hiomarker of" cellular aging in Illllllan s"-in. PLoS' ONE. 2007:2( 12):e 1269.
36. Campisi.l. d'Adda di Fagagna F. Cellular senescence: when bad [hing... happen to good celL'.. NOI R('I' Mol Celf BIOI. 2007:R:729-740.
37. Manju K. Ml11alikrishn<l B. Pamaik VK. Expression of disease-causing 1a111 111 A rnlltants impairs the formation of DNA rep"ir foci ../ Cell Sci. 2006: I! 9(Pl 13):2704-2714.
3H. Liu B. Wang J. Chan KM. e[ al. Genomic instahility in laminopathybased premature aging. Nat Med. 2005: I I :780- 785.
39. Varela I. Cadinano~ J. Pendas AM. et al. Accelerated ageing in mice deficient II) Zlllp...[c24 protease is linked to p53 ... ignalling activation. Na/I/re. 2005A37:564-568.
40. Llli Y, Wang Y. Rusino[ AE. e[ a1. Involvemen[ of xerodem1a pigmentosulll group A (XPA) in progeria arising from del"eclive maturation of prelalllll) A. FASF,B .I. 2008:22:603---611.
4J. Liu Y. Rusinol A, Sinemky M. Wang Y, ZOLI Y. DNA damage responses in progeroid syndromes arise from defective maturation of prclamin A . .I Cell Sci. 2006; I 19(P[ 22):4-644·4649.
42. Picinich SC, Mishra PJ, Mishra PJ. Glod J, Banerjee D. The thempeutie potential of mesenchymal ~tel11 cells. Cell· & [issue-based therapy. Exper/ GfJill BIOI Ther. 2007:7:965-973.
43. Warner HR. 2006 Kent award lecture: IS cell death and replacement a t~1ctor in a!ling'! .I Gerontol A BioI St'i Mi·d Sn·. 2007:62: 1228-1232.
44. Bridger JM. Kill JR. Aging of Hutchinson-Gilford progeria syndrome fi broblast.~ is characterised by hyperproliferation and incrcased apoptosis. E\p GCI"OII/ol. 2004;39:717-724.
45. Prolla TA. Multiple roads to the aging phenotype: msights from the molecular dissection of progerias through DNA microanay analySIS. Mech Ageing Dc\". 2005;126:461--465.
46. Hu[chison CJ, Worman HJ. A-type lamins: guardians of the soma'? Nat Cell BIOI. 2004:6:1()62 1067.
47. Roher RA. Sallter H. Weber K, OsbolTl M. Cells of the cellular immune and hemopoietic syslem of the mouse lack lamins A/C: di.~tinction
versus other somatic celk J Cell 5ici. J990:95( p[ 4):587-598. 48. GO[l.mann 1. Foisner R. A-type lamin comp1cxc" and regenerative
potential: .t ... tep towards understanding lalllinopalhic diseases? Hislo
('hem Cell BioI. 2()06: 125:33--41. 49. Halaschek-Wiener J, Brooks-Wilson A. Progerta of stem cells: stem
cell exhaustion in Ilutchinson-Gi Iford progeria syndrome. J (;1'1"011/01 A
BIOI Sf"i Mcd Sci. 2007:62:3-850. Espada J. Varela I, Flore.<, I. et 'II. Nuclear envelope defects c<lu ...e ,,[em
cell dysfunctIon in premature-aging mice. J Cell Bioi. 2008; I XI:27--35. 51. Conboy 1M. Conboy MJ. Wagers AJ. (,inna ER. Weissman It. Rando
TA. Rejuvcnallon of aged progell1[or cdls by cxposure (0 a young systemic environmenl. Nall/I"e. 2005:433:760--764.
52. Conboy 1M. Conboy MJ. Smylhe GM. Rando TA. Notch-mediated restoration of regenerative potential to aged IllUSCle. ScicJl('('. 2003J02: 1575-1577.
53. Carlson ME, Conboy 1M. Loss of stem cell regenerative capacity within aged niches. Aging Cell 2007:6::171-.182.
54, ScaffidI P, Mistell T. Lamin A-dependent misregulation of adult stem cell., associated wi1h accelerated ageing. NOI C{'II Biol. 200S: J0: 452-459.
55. Ukekawa R, Miki K. FUJii M. Hirano 1-1, Ayusawa D. Accumulation of" multiple f0l111S of [amin A with down·regulation of FACE-J sllppresse... growth in senescent human cells. Gen('s Crlls. 2007: J 2:397--406.
56. Rusinol AE. Sinensky MS. Farnesylated lam ins, proger01d syndromes and famesyl transferase inhibitors. J Ci'll SCI. 2006; 119(p[ 16):32653272.
57. Varela I. Pereira S. Ugalde AP. et 'II. Combined treatmenl with sta[ins and aminohisphosphonates extends Jongevity in a l11ou"e model of human premature aging. Nm Med. 20()g; 14:767-772.
58. Scaffidi P. Misteli T. Reversal of the cellular phenotype III the premature aging dise'l.~e Hu[chinsoll-G ilford progeria syndrome. Nat Med, 2005: 11 :440-445.
59. Capell Be. Collins FS. Human laminopathic.<,: nuclei gone genetically awry. Nat ReI' Gelll'/, 2006;7:940-952.
R('('('II'N! May 28. 2008 Accepted Mal' .W. 20()8 Decision Edilor' J-l1l1J('1" R. WilmCl". PhD
