male and female lmna , lmna , and lmna mice were bred as described ( 12 , 15 ) .
genotyping of mice was performed by pcr with genomic dna from tail biopsies ( 12 , 15 ) .
mice were fed a chow diet and housed in a virus - free barrier facility with a 12 h light - dark cycle .
an fti , abt-100 ( 18 ) , was administered to groups of male and female lmna , lmna , and lmna mice ( n = 12 mice / group ) .
abt-100 was mixed in drinking water containing 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol at a concentration of 0.4 mg / ml , so as to deliver an approximate dose of 52 mg / kg / day .
vehicle - treated mice were given drinking water with 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol .
the fti was initiated at 4 weeks of age and was continued for up to 38 weeks of age ( at that time point , any mouse that had not yet succumbed to the disease was euthanized ) .
body weights were assessed weekly , and body weight curves were compared with repeated - measures anova and the log rank test .
the number of surviving mice was recorded weekly and expressed as a percentage of the total number of mice .
body fat depots ( reproductive , inguinal , and mesenteric ) were measured when each mouse died or was euthanized .
the number of spontaneous rib fractures in lmna and lmna mice was documented when each mouse died .
after opening the thoracic cavity and removing the heart and lungs , the interior of the thorax was photographed and rib fractures were counted ( 12 , 15 ) .
numbers of rib fractures in fti- and vehicle - treated lmna and lmna mice were compared with a two - tailed student 's t - test .
mean bone density and bone cortical thickness in the ribs were measured with computer - assisted tomographic scans .
procedures for preparing liver extracts and western blotting techniques have been described previously ( 12 , 14 , 15 ) . to detect lamins a and c and progerin
, we used a 1:400 dilution of a goat anti - lamin a / c igg ( santa cruz biotechnology , santa cruz , ca ) and a 1:5000 dilution for an irdye800-conjugated donkey anti - goat igg ( li - cor ; lincoln , ne ) .
to detect prelamin a , we used a 1:2000 dilution of a rabbit anti - prelamin a antiserum ( 6 , 8 , 13 ) and a 1:5000 dilution of an hrp  labeled anti - rabbit igg ( ge healthcare , piscataway , nj ) . to detect hdj-2
, we used a 1:500 dilution of a mouse monoclonal antibody against hdj-2 ( neomarkers ; fremont , ca ) and a 1:5000 dilution of an hrp - labeled anti - mouse igg ( ge healthcare ) . for both prelamin
a and hdj-2 western blots , antibody binding was detected with the ecl plus chemiluminescence system ( ge healthcare ) and exposure to x - ray film . to assess protein farnesylation in fibroblasts
, the cells were incubated for 48 h with an analog of farnesol , 8-anilinogeraniol ( ag ) ( 100 m in dmso ) ( 19 ) in the presence and absence of abt-100 ( 1 m ) .
ag is incorporated by cells into ag pyrophosphate , a farnesyldiphosphate analog , which is used by protein farnesyltransferase as a substrate to farnesylate caax proteins .
ag incorporation into cellular proteins was detected by western blotting with a mouse monoclonal antibody specific for ag , diluted 1:5000 ( 19 ) .
male and female lmna , lmna , and lmna mice were bred as described ( 12 , 15 ) .
genotyping of mice was performed by pcr with genomic dna from tail biopsies ( 12 , 15 ) .
mice were fed a chow diet and housed in a virus - free barrier facility with a 12 h light - dark cycle .
an fti , abt-100 ( 18 ) , was administered to groups of male and female lmna , lmna , and lmna mice ( n = 12 mice / group ) .
abt-100 was mixed in drinking water containing 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol at a concentration of 0.4 mg / ml , so as to deliver an approximate dose of 52 mg / kg / day .
vehicle - treated mice were given drinking water with 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol .
the fti was initiated at 4 weeks of age and was continued for up to 38 weeks of age ( at that time point , any mouse that had not yet succumbed to the disease was euthanized ) .
body weights were assessed weekly , and body weight curves were compared with repeated - measures anova and the log rank test . the number of surviving mice was recorded weekly and expressed as a percentage of the total number of mice .
body fat depots ( reproductive , inguinal , and mesenteric ) were measured when each mouse died or was euthanized .
the number of spontaneous rib fractures in lmna and lmna mice was documented when each mouse died .
after opening the thoracic cavity and removing the heart and lungs , the interior of the thorax was photographed and rib fractures were counted ( 12 , 15 ) .
numbers of rib fractures in fti- and vehicle - treated lmna and lmna mice were compared with a two - tailed student 's t - test .
mean bone density and bone cortical thickness in the ribs were measured with computer - assisted tomographic scans .
procedures for preparing liver extracts and western blotting techniques have been described previously ( 12 , 14 , 15 ) . to detect lamins a and c and progerin
, we used a 1:400 dilution of a goat anti - lamin a / c igg ( santa cruz biotechnology , santa cruz , ca ) and a 1:5000 dilution for an irdye800-conjugated donkey anti - goat igg ( li - cor ; lincoln , ne ) .
to detect prelamin a , we used a 1:2000 dilution of a rabbit anti - prelamin a antiserum ( 6 , 8 , 13 ) and a 1:5000 dilution of an hrp  labeled anti - rabbit igg ( ge healthcare , piscataway , nj ) . to detect hdj-2
, we used a 1:500 dilution of a mouse monoclonal antibody against hdj-2 ( neomarkers ; fremont , ca ) and a 1:5000 dilution of an hrp - labeled anti - mouse igg ( ge healthcare ) . for both prelamin a and hdj-2 western blots , antibody binding was detected with the ecl plus chemiluminescence system ( ge healthcare ) and exposure to x - ray film . to assess protein farnesylation in fibroblasts
, the cells were incubated for 48 h with an analog of farnesol , 8-anilinogeraniol ( ag ) ( 100 m in dmso ) ( 19 ) in the presence and absence of abt-100 ( 1 m ) .
ag is incorporated by cells into ag pyrophosphate , a farnesyldiphosphate analog , which is used by protein farnesyltransferase as a substrate to farnesylate caax proteins .
ag incorporation into cellular proteins was detected by western blotting with a mouse monoclonal antibody specific for ag , diluted 1:5000 ( 19 ) .
we administered an fti , abt-100 ( 52 mg / kg / day ) , or vehicle alone to groups of 12 male and female lmna , lmna , and lmna mice , starting at 4 weeks of age .
our goal was to determine if the fti would ameliorate progeria - like disease phenotypes in lmna mice in addition to lmna mice .
the plasma levels of abt-100 in treated mice were similar to those in earlier studies ( 1214 ) , ranging from 0.3 to 0.7 g / ml . when we incubated lmna and lmna fibroblasts with the same concentration of abt-100 that we achieved in mice ( 0.5 g / ml ) , the farnesylation of b - type lamins and progerin was inhibited , as judged by metabolic labeling experiments with a farnesol analog ( 8-anilinogeraniol ) ( fig .
1 ) . also , we observed , as expected , an accumulation of prelamin a and nonfarnesylated hdj-2 in liver extracts from fti - treated mice ( fig .
interestingly , there were lower levels of mature lamin a , relative to actin , in liver extracts from fti - treated mice ( fig .
long - term treatment of lmna and lmna fibroblasts with abt-100 also lowered levels of mature lamin a , consistent with the findings in fti - treated mice ( fig .
western blot testing the effect of abt-100 on the farnesylation of the b - type lamins and progerin in lmna and lmna fibroblasts .
for these studies , we used the same concentration of abt-100 that we achieved in the plasma of mice ( 0.5 g / ml ) .
the top panel shows the merged images of western blots with an antibody specific for lamin a / c ( red ) and an antibody specific for the farnesol analog ag ( green ) ; the middle panel shows the signal for the antibody against ag ; the bottom panel shows a loading control ( actin ) . in this experiment ,
lmna and lmna fibroblasts were incubated with ag ( 100 m ) in the presence ( + ) or absence (  ) of abt-100 .
the electrophoretic mobility of progerin , lamin b1 , and lamin b2 are virtually identical , and all are farnesylated ; hence , the antibody against ag detected a farnesylated protein in both cell lines ( in the absence of an fti ) .
when progerin and lamin a / c were immunoprecipitated with an antibody against lamin a / c , the progerin in lmna fibroblasts , but not lmna fibroblasts , stained with the antibody against ag .
treatment with an fti lowers steady - state levels of mature lamin a. a : western blots of liver extracts from lmna , lmna , and lmna mice that had been treated with a protein farnesyltransferase inhibitor ( fti ) or vehicle ( veh ) alone .
western blots were performed with antibodies against lamin a / c and prelamin a. lmna fibroblasts , treated with either the fti or the vehicle , were used as controls .
b : quantification of lamin a levels in liver extracts of lmna , lmna , and lmna mice ( n = 3 mice / group ; each sample was analyzed on two independent western blots ) .
lamin a / actin ratios in liver extracts of fti - treated mice were expressed relative to those in vehicle - treated mice . in the livers of lmna , lmna , and lmna mice , the lamin a / actin ratio in fti - treated mice was lower than in vehicle - treated mice ( p < 0.0001 ) .
long - term treatment of fibroblasts with abt-100 lowers steady - state levels of mature lamin a , relative to lamin c or actin .
lmna and lmna fibroblasts were treated with either fti ( abt-100 , 10 m ) or dmso ( as control ) for 2 days or 14 days .
( 12 , 14 ) , the fti improved body weight curves in lmna mice ( p < 0.0001 for both males and females when compared with lmna mice treated with vehicle alone ) ( figs .
in contrast , the body weight curves of fti- and vehicle - treated lmna mice were not different .
this was the case for both male and female lmna mice ( p = 0.27 and 0.54 , respectively ) .
also , there were no differences in the body weight curves of fti- or vehicle - treated lmna mice ( p = 0.36 for males and 0.52 for females ) .
an fti improves body weight curves and survival in lmna mice but not in lmna mice
. a and b : effect of the fti treatment on body weight curves in male ( a ) and female ( b ) mice .
lmna ( squares ) , lmna ( circles ) , and lmna ( triangles ) mice were given the fti ( closed symbols ) or vehicle alone ( open symbols ) , beginning at 4 weeks of age .
body weight curves for the fti - treated lmna mice were improved ( p < 0.0001 for both males and females ) .
the fti treatment did not improve the body weight curves in lmna or lmna mice .
c : kaplan - meier survival plots for lmna , lmna , and lmna mice treated with an fti or vehicle alone ( n = 24 mice / group ) .
fti treatment significantly improved survival in lmna mice ( p < 0.0001 ) . the fti had no significant effect on survival in lmna mice ( p = 0.45 ) .
kaplan - meier survival curves revealed that the fti improved survival in lmna mice ( p < 0.0001 ) , extending survival by 68 weeks ( fig .
in contrast , the fti had no effect on survival of lmna mice ( p = 0.45 ) ( fig .
consistent with the improvement in body weight curves in fti - treated lmna mice , the fti significantly increased body fat stores in lmna mice , compared with vehicle - treated mice ( p = 0.002 ) ( fig . 5 ) .
in contrast , the fti had no effect on fat stores in lmna mice ( fig . 5 ) .
body fat weights in lmna and lmna mice treated with the fti or the vehicle alone .
the fti significantly increased the weight of body fat in lmna mice ( p = 0.002 ) , but not in lmna mice ( p = 0.21 , n = 24 mice / group ) .
we assessed the impact of the fti treatment on spontaneous rib fractures in both male and female mice .
the fti clearly reduced the number of rib fractures in lmna mice ( p < 0.0001 ) ( fig .
in contrast , the drug had no significant effect on the number of rib fractures in lmna mice ( fig .
the fti improved mean bone density and bone cortical thickness in lmna mice ( p < 0.0001 for both males and females ) ( fig .
in contrast , the fti had no effect on these bone phenotypes in lmna mice ( fig .
a : numbers of rib fractures in fti - treated ( closed circles ) and vehicle - treated ( open circles ) lmna and lmna mice at the time of death . all mice that survived to 38 weeks of age were euthanized at that time point .
the fti - treated male and female lmna mice had fewer rib fractures ( p < 0.0001 ) than vehicle - treated lmna mice .
the numbers of rib fractures in fti- and vehicle - treated mice were similar ( n = 24 mice / group ) .
fti treatment improved the bone density ( b ) and cortical thickness ( c ) in the ribs of male and female lmna mice ( n = 24 mice / group , p < 0.0001 ) but not in lmna ( wt ) and lmna mice ( n = 24 mice / group ) .
( 12 , 13 ) showed that an fti ameliorates disease phenotypes in a mouse model of hgps . although the results were significant and reproducible ,
putting the findings into perspective is challenging , particularly with the discovery that lmna mice develop disease ( 15 ) .
lmna have all of the same progeria - like disease phenotypes as lmna mice , albeit somewhat milder , and they invariably succumb to the disease .
the discovery that nonfarnesylated progerin elicits disease prompted us to consider the possibility that the beneficial effects of the fti in lmna mice might have little to do with a direct effect on the farnesylation of progerin .
instead , we wondered whether the effect of the fti might be more indirect , perhaps secondary to inhibiting the farnesylation of other cellular proteins .
if the effects of the fti were indirect , one would predict the fti might be equally efficacious in lmna and lmna mice .
fti treatment had no effect on body weight , survival , the number of rib fractures , fat stores , bone cortical thickness , or bone density of lmna mice .
in contrast , the fti improved all of these phenotypes in lmna mice , and did so in a highly significant fashion .
the fact that the fti improved disease in lmna but not lmna mice suggests that the beneficial effect of the fti in lmna mice is likely a consequence of inhibiting the prenylation of progerin , rather than an indirect effect of the drug on other cellular proteins .
the beneficial effects of the fti on disease phenotypes in lmna mice were very apparent , and most of the differences were significant at a p < 0.0001 level . in contrast , the fti had no significant effects in lmna mice .
one could argue that there may have been a slight trend toward improved body weights and lower numbers of rib fractures in fti - treated lmna mice , but these differences did not achieve statistical significance .
however , if one were forced to suggest a potential explanation for the trend , one could point to the lower levels of mature lamin a in the setting of fti treatment .
genetic studies have suggested that lower levels of lamin a synthesis reduce the disease phenotypes elicited by a lmna allele ( 20 ) .
the finding of lower lamin a levels in association with fti treatment was also observed in earlier studies ( 8 , 14 , 15 ) .
the mechanism for the fall in lamin a levels is unclear but we suspect that sustained blockade of prelamin a farnesylation leads to the eventual turnover of nonfarnesylated prelamin a , reducing the production of mature lamin a. the current study reinforces the idea that an fti can ameliorate disease in lmna mice , but important questions remain about the utility of ftis in the treatment of humans with hgps .
indeed , questions were raised about whether an fti is the most appropriate drug for inhibiting the prenylation of progerin ( 21 ) .
21 ) reported data suggesting that progerin can be geranylgeranylated in the setting of an fti , and they proposed that a combination of a statin and a bisphosphonate ( which would theoretically inhibit both farnesylation and geranylgeranylation ) might be more useful for treating hgps .
they went on to show that a statin / bisphosphonate combination improved progeria - like disease phenotypes in zmpste24-deficient mice ; however , they provided no evidence that the combination actually inhibited the prenylation of prelamin a ( or any other protein ) in mice .
until such evidence is in hand , doubts will remain about the rationale for the statin / bisphosphonate combination , and in particular , whether the combination ameliorates disease indirectly ( perhaps secondary to the bone - strengthening properties of bisphosphonates ) or more directly by inhibiting the prenylation of prelamin a. for those interested in investigating the utility of a bisphosphonate / statin combination , the approach outlined in the current study should be of interest . if a statin / bisphosphonate combination were to improve disease phenotypes in lmna mice , that would suggest that the mechanism for the combination was likely indirect , perhaps due to the bone - strengthening properties of the bisphosphonates , and not to a specific effect on the prenylation of prelamin a. in summary , an fti ameliorates disease phenotypes in lmna mice , but not lmna mice .
the failure of the fti to improve disease in lmna mice suggests that the beneficial effects of the drug in lmna mice are likely due to blocking the farnesylation of progerin .
 hutchinson - gilford progeria syndrome ( hgps ) is caused by the accumulation of a farnesylated form of prelamin a ( progerin ) . 
 previously , we showed that blocking protein farnesylation with a farnesyltransferase inhibitor ( fti ) ameliorates the disease phenotypes in mouse model of hgps ( lmnahg/+ ) . 
 however , the interpretation of the fti treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin ( lmnanhg/+ ) develop progeria - like disease phenotypes . the fact that lmnanhg/+ mice manifest disease raised the possibility that the beneficial effects of an fti in lmnahg/+ mice were not due to the effects of the drug on the farnesylation of progerin , but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins . to address this issue , we compared the ability of an fti to improve progeria - like disease phenotypes in both lmnahg/+ and lmnanhg/+ mice . in lmnahg/+ mice , 
 the fti reduced disease phenotypes in a highly significant manner , but the drug had no effect in lmnanhg/+ mice . 
 the failure of the fti to ameliorate disease in lmnanhg/+ mice supports the idea that the beneficial effects of an fti in lmnahg/+ mice are due to the effect of drug on the farnesylation of progerin . 
MATERIALS AND METHODS
Knock in mice expressing progerin
Treatment with a protein farnesyltransferase inhibitor
Analysis of disease phenotypes
Western blots and metabolic labeling studies
RESULTS
DISCUSSION