Although cardiomyopathy and muscular dystrophy were the first phenotypes found in human subjects with LMNA
mutations, the pathogenic mechanisms underlying these phenotypes have been obscure. To approach this issue, Muchir et al. (100
) used microarrays to identify genes and pathways that were perturbed in hearts of LmnaH222P/H222P
mice (a mouse model of autosomal EDMD in which a single missense LMNA
mutation [H222P] known to cause autosomal EDMD in humans is introduced into Lmna
). They detected activation of the ERK and JNK branches of the MAPK signaling cascade, and these changes occurred prior to the onset of histopathologic abnormalities in the heart. Furthermore, expression of mutant forms of lamin A in cultured cells led to activation of ERK and JNK signaling. These findings were consistent with the known alterations in MAPK signaling in cardiomyopathy (101
). Muchir et al. (102
) further analyzed gene-expression profiles in hearts of Emd
-knockout mice, a model of X-linked EDMD, and found a similar molecular signature — activation of ERK and its downstream targets. Thus, MAPK signaling seems relevant to the pathogenesis of heart disease in both X-linked and autosomal dominant EDMD. However, it remains unclear how abnormalities in the nuclear envelope lead to activation of ERK and/or JNK.
Pharmacological inhibitors of MEK (the MAPK kinase that activates ERK) can be administered systemically, and some have been tested as anticancer agents in early-stage human clinical trials. The availability of these agents led Muchir et al. (103
) to hypothesize that systemic administration of a MEK inhibitor would prevent dilated cardiomyopathy in LmnaH222P/H222P
mice. Beginning at 8 weeks of age, prior to the onset of detectable cardiomyopathy, LmnaH222P/H222P
mice were treated with a MEK inhibitor. At 16 weeks of age, nontreated and placebo-treated LmnaH222P/H222P
mice manifested increased left ventricular end-systolic and end-diastolic diameters as well as a 30% reduction in ejection fraction. In contrast, hearts of LmnaH222P/H222P
mice treated with the MEK inhibitor were indistinguishable from those of wild-type mice. Treatment with a MEK inhibitor also prevented upregulation of genes encoding atrial natriuretic peptides. These mouse studies suggest that ERK inhibition might hold promise for treating cardiomyopathy in human subjects with LMNA
mutations (Figure ).
Studies from LmnaH222P/H222P knockin mice and Emd-knockout mice suggest that activation of ERK and/or JNK underlies the development of cardiomyopathy.
Although no single mechanism has emerged, several studies point to defects in genome maintenance (e.g., defective DNA repair, accumulation of DNA damage, and altered gene expression) as underlying causes of the progeroid syndromes (104
). This also seems to be important in progerias associated with ZMPSTE24 deficiency and specific LMNA
mutations. Liu et al. (105
) reported that ZMPSTE24-deficient mouse fibroblasts were more sensitive to DNA-damaging agents and were slow to repair DNA and proposed that defective DNA repair was linked to genome instability and cell senescence. DNA damage responses are also abnormal in cells from subjects with HGPS, but treatment with a protein FTase inhibitor (FTI), which blocks prenylation of progerin, did not reduce markers of DNA damage (106
). Varela et al. (107
) used microarray analysis to identify a possible link between ZMPSTE24 deficiency in mice and p53 activation. Also, there was a suggestion that disease phenotypes were less severe in Zmpste24–/–p53–/–
), which would be consistent with the notion that p53 overexpression promotes aging (108
). Shumaker et al. (109
) showed that histone methylation patterns are altered in HGPS, providing evidence that the lamina interacts with chromatin to modulate heterochromatin and presumably gene transcription. These changes preceded the appearance of abnormally shaped nuclei.
Others have proposed that abnormal nuclear mechanics are an important cellular defect in HGPS. Dahl et al. (110
) investigated the mechanical properties of the lamina in HGPS cells and found that the nuclei of HGPS cells exhibited reduced deformability when aspirated with a micropipette. They also found that HGPS nuclei were more resistant to disruption by mechanical pressure than the nuclei from wild-type cells. Verstraeten et al. (111
) showed that cultured dermal fibroblasts from patients with HGPS developed progressively stiffer nuclei with increasing passage number and exhibited decreased viability under repetitive mechanical strain as well as attenuated wound healing. FTIs reversed the nuclear stiffness phenotype and accelerated the wound-healing response in fibroblasts from subjects with HGPS and healthy controls but did not restore sensitivity to mechanical strain. While these findings are clearly intriguing, there were no obvious mechanisms connecting the defects in nuclear mechanics to other cellular phenotypes or disease phenotypes.
Signaling pathways required for maintaining normal stem cell function appear to be perturbed in cells expressing progerin or high levels of unprocessed prelamin A. Scaffidi and Misteli (112
) showed that the expression of progerin activates downstream effectors of the Notch signaling pathway and alters the differentiation potential of mesenchymal stem cells. Espada et al. (113
) showed that ZMPSTE24 deficiency caused an alteration in the number and proliferative capacity of epidermal stem cells, with alterations in molecular signaling pathways implicated in the regulation of stem cells, such as Wnt and microphthalmia transcription factor. These studies demonstrate a potential link between stem cell dysfunction and progeria, but the precise contribution of stem cells to the pathophysiology of progeria remains to be established.
In considering the absence of disease phenotypes in Zmpste24–/–Lmna+/–
mice, Fong et al. (95
) hypothesized that the farnesylated
form of prelamin A might be the molecular culprit in progeria. They further reasoned that blocking protein farnesylation might interfere with progerin targeting to the nuclear periphery, potentially reducing its toxic effects. In support of this hypothesis, Yang et al. (72
) showed that FTI treatment mislocalized progerin away from the nuclear periphery and reduced the frequency of misshapen nuclei in LmnaHG/+
fibroblasts (i.e., fibroblasts from mice in which a mutant Lmna
, encodes progerin exclusively). Shortly thereafter, Toth et al. (114
) showed that an FTI reduced the frequency of misshapen nuclei in cultured fibroblasts from humans with HGPS and ZMPSTE24 deficiency as well as in fibroblasts from Zmpste24–/–
). Several other laboratories reported the same basic findings, some using complementary approaches and different systems (115
). In the studies by Yang et al. (72
) and Toth et al. (114
), blockade of protein farnesylation was substantial, as farnesylation of human DnaJ homolog-2 (HDJ-2) (an unrelated CaaX protein) was largely blocked and nonfarnesylated prelamin A accumulated in cells. FTI treatment also reduced levels of lamin A and prelamin A (114
), suggesting that the blockade of protein farnesylation might reduce the stability of prelamin A.
The next step was to examine whether an FTI might ameliorate disease in mouse models of progeria. Fong and coworkers (96
) found that systemic administration of an FTI improved body weight curves in both male and female Zmpste24–/–
mice, although the drug also led to weight loss in wild-type mice. FTI administration also improved survival, improved grip strength performance, and reduced the number of rib fractures. However, FTI-treated Zmpste24–/–
mice still had profound disease phenotypes and succumbed to the progeroid disease. In these studies, only 10%–50% of the HDJ-2 in tail extracts was nonfarnesylated (similar to levels observed in the testing of FTIs as anticancer agents). The FTI also led to the appearance of nonfarnesylated prelamin A in tissue extracts but to a lower extent than in cell culture experiments (96
mice treated with an FTI also exhibited improvements in body weight curves, weights of fat depots, bone fractures, and bone mineralization (97
). However, as in Zmpste24–/–
mice, improvements in disease phenotypes fell far short of a cure. In these studies, more than 50% of HDJ-2 in livers of FTI-treated mice was nonfarnesylated, and small amounts of nonfarnesylated prelamin A accumulated in tissues (97
). Increasing FTI doses led to greater effects on farnesylation, but survival was adversely affected, presumably because of drug toxicity (99
The efficacy of FTIs in reducing the number of cells with misshapen nuclei (72
) and in ameliorating disease phenotypes in mouse models of progeria (96
) prompted an open-label trial of an FTI (lonafarnib) in children with HGPS (119
). The trial has been ongoing for more than a year, but neither clinical outcomes nor evidence regarding the in vivo blockade of protein farnesylation are available. In the human trial, lonafarnib is being administered to children with advanced disease phenotypes, a substantial difference from the mouse experiments, where the drug therapy was initiated prior to development of significant disease.
The improvements in nuclear shape in FTI-treated HGPS fibroblasts have in some cases been quite striking (72
). However, the benefits of FTIs in LmnaHG/+
mice, although highly statistically significant, have been less dramatic. One possible explanation is that the degree of inhibition of protein FTase was less than complete in the mice. Another possibility is that prelamin A can be alternately prenylated by geranylgeranyltransferase-I (GGTase-I) when FTase is blocked. The latter possibility is very plausible, as lamins terminate with methionine and other CaaX proteins terminating with methionine can be geranylgeranylated when FTase is blocked (120
). Varela et al. (98
) used mass spectrometry (MALDI-TOF) to examine prelamin A and progerin structure in FTI-treated cells and uncovered strong evidence that these proteins are geranylgeranylated in FTI-treated fibroblasts. Whether alternate geranylgeranylation of prelamin A also occurred in FTI-treated mice was never investigated. Varela et al. (98
) also reported that a combination of a GGTase-I inhibitor and an FTI increased prelamin A accumulation in cultured fibroblasts, again consistent with alternate prenylation. On the other hand, another group found that an FTI retards the electrophoretic migration of prelamin A, raising the possibility that the extent of alternate prenylation may be limited (or that the FTIs that were used are effective in blocking protein geranylgeranylation) (114
). More studies are clearly required to define the extent of alternate prenylation in vivo in the setting of FTI therapy and to explore its potential relevance to the pathogenesis of progeria.
Another possible explanation for the incomplete therapeutic response with an FTI in LmnaHG/+
mice is that it leads to accumulation of another abnormal lamin—nonfarnesylated progerin. If the nonfarnesylated progerin were itself toxic to cells, the benefits of FTI therapy would obviously be limited. To explore the possibility that nonfarnesylated progerin is toxic and capable of eliciting disease, Yang et al. (121
) created knockin mice expressing nonfarnesylated progerin (LmnanHG/+
mice are genetically identical to LmnaHG/+
mice, except that the cysteine in the CaaX motif of progerin was changed to serine, eliminating all protein prenylation. LmnanHG/+
mice exhibited the same phenotypes as LmnaHG/+
mice, but they were slightly milder. In addition, fewer LmnanHG/+
fibroblasts contained misshapen nuclei (121
). A likely explanation for the milder phenotypes in LmnanHG/+
mice was that steady-state cellular levels of progerin were lower in LmnanHG/+
). The milder phenotypes in LmnanHG/+
mice (compared with LmnaHG/+
mice) are consistent with the results of FTI treatment studies (97
) and support the idea that inhibiting protein farnesylation could be beneficial. On the other hand, finding that nonfarnesylated progerin caused substantial disease suggested that there could be significant limitations in the FTI treatment strategy for HGPS.
Toth et al. (114
) suggested that bisphosphonates, frequently prescribed for osteoporosis, might be useful for treating bone disease in progeria. Nitrogen-containing bisphosphonates bind avidly to bone and block farnesyl diphosphate synthase, an enzyme that produces farnesyl diphosphate (122
). Inhibiting synthesis of farnesyl diphosphate blocks protein farnesylation, protein geranylgeranylation, and cholesterol synthesis, and these effects are thought to promote apoptosis in osteoclasts, leading to improved bone density. It is not clear that increased activity of osteoclasts underlies bone disease of progeria (25
), and it seems somewhat more probable that dysfunctional osteoblasts are more important for the pathogenesis of disease (24
). In any case, Toth et al. (114
) showed that one of the nitrogen-containing bisphosphonates, alendronate, inhibited prelamin A processing in wild-type and HGPS fibroblasts, although the blockade of lamin A biogenesis was less than with an FTI. The impact of these drugs on prelamin A processing in vivo (in mouse or humans) is not yet clear. A potential advantage of bisphosphonates is that they would interfere with geranylgeranylation by GGTase-I, if indeed this enzyme were active in the posttranslational modification of prelamin A in vivo. Another advantage of bisphosphonates is that these drugs are concentrated in bone, a tissue that is severely affected in progeria. On the other hand, it seems unlikely that these drugs would be helpful for treating disease phenotypes unrelated to bone, such as lipodystrophy and vascular disease.
Varela et al. (98
) tested a combination of a potent nitrogen-containing bisphosphonate and a statin in Zmpste24–/–
mice and documented improved survival and improvements in bone abnormalities. The rationale for this drug combination was to inhibit prelamin A farnesylation in a synergistic manner and also to block any alternate geranylgeranylation that might occur in the tissues of mice. Although it is clear that the mice treated with the drug combination exhibited an improvement in disease phenotypes, they did not report whether the drug combination actually affected the prenylation of prelamin A (or any other protein) in the tissues of mice; thus, the mechanism for the observed improvements in disease phenotypes is not yet entirely clear. Varela et al. (98
) did not find a beneficial effect of a statin alone or a bisphosphonate alone on the survival of Zmpste24–/–
mice, although the number of mice in the latter experiments was small.
Blocking protein prenylation with an FTI represents a “blunt instrument” for the treatment of HGPS, in that FTIs block the posttranslational processing of many CaaX proteins, including the B-type lamins. Similarly, the bisphosphonate/statin combination would inhibit the processing of both farnesylated and geranylgeranylated proteins, at least in bone. A more specific approach would be to identify therapies that interfere with the proximal cause of HGPS — the utilization of the alternate splice donor site in exon 11 of LMNA
. Scaffidi and Misteli (123
) transfected HGPS fibroblasts with a morpholino oligonucleotide directed against the abnormal splice donor site and found reduced levels of progerin in cells, reduced frequency of misshapen nuclei in fibroblasts, and normalized expression of aberrantly expressed genes. Huang et al. (124
) tested an RNA interference approach to reduce levels of progerin in HGPS cells. They identified a short hairpin RNA directed against sequences unique to progerin that reduced progerin transcripts and protein levels by approximately 25%. Despite this modest effect, the frequency of misshapen nuclei was reduced and cell proliferation rates increased. Another strategy was suggested by the absence of disease in mice carrying two Lmna
alleles that exclusively produce lamin C (LmnaLCO/LCO
mice, so called lamin C–only mice) (125
). If lamin A and prelamin A are dispensable, it might make sense to treat HGPS by eliminating all prelamin A transcripts (both for progerin and wild-type prelamin A) with antisense oligonucleotides. Fong et al. (125
) identified an antisense oligonucleotide that potently reduced prelamin A transcripts and, when tested in fibroblasts from mice lacking ZMPSTE24, reduced both prelamin A levels and the frequency of misshapen nuclei. Thus far, no laboratory has tested oligonucleotide therapeutics in animal models.