Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic human syndrome of premature aging. Children with this rare condition are normal at birth, but by age 2 they have stopped growing, lost their hair, and shown skin changes and loss of subcutaneous tissue that resemble the ravages of old age. Untreated, they rarely live past adolescence, dying almost always of advanced cardiovascular disease (heart attack and stroke). The classic syndrome has never been observed to recur in families. Our laboratory discovered that nearly all cases of HGPS harbor a de novo point mutation in codon 608 of the LMNA gene. This mutation, denoted G608G, causes disease by creating an abnormal splice donor, generating an mRNA with an internal deletion of 150 nt. This is translated into a mutant form of the lamin A protein (referred to as progerin) that lacks 50 amino acids near the C-terminus. Normally lamin A is post-translationally processed to add a farnesyl group at the C-terminus, and then the last 18 amino acids are cleaved off by the enzyme Zmpste24 to produce mature lamin A. Progerin lacks the recognition site for this final cleavage, and so remains permanently farnesylated. We have shown that this abnormal protein acts as a dominant negative to disrupt the structure of the membrane scaffold. Data from our group has also demonstrated that progerin interferes with proper chromosome segregation during mitosis, and alters the distribution of various histone chromatin marks. Our lab has developed a mouse model for HGPS, by inserting into the germline a 164 kb bacterial artificial chromosome (BAC) containing the human LMNA gene, engineered to carry the G608G mutation. Recent work has demonstrated the complex anatomy of the transgene insert, but an intact copy of LMNA is included. Animals carrying the G608G mutation show progressive loss of smooth muscle cells in the media of large vessels. Thus, the mouse model nicely replicates the cardiovascular phenotype of HGPS. We have tested the use of farnesyl transferase inhibitors (FTIs), to see if these drugs could provide benefit in HGPS by reducing the amount of the toxic progerin protein. Treatment of HGPS fibroblasts growing in cell culture demonstrates that FTIs are capable of reversing the dramatic nuclear blebbing that is the hallmark of the disease. A trial of FTIs in the HGPS mouse model has demonstrated that this drug treatment is capable of preventing and even reversing the cardiovascular phenotype. An open label clinical trial of FTIs in 30 children with the disease was initiated in May 2007. Results were published in the fall of 2012, demonstrating benefit to the vascular system, with reduction in peripheral vascular resistance in most of the 28 children treated. But this is not a cure, and so the search for other therapeutic options continues. Homozygotes for the mouse BAC transgenic have also now been bred, and show a considerably more severe phenotype. Those animals are now being used to test the effect of RAD001 (everolimus), a rapamycin analog, alone or in combination with FTIs. Rapamycin has shown to expand lifespan in mice, and its use on HGPS fibroblasts causes an improvement in phenotype with reduced nuclear blebbing and increased proliferative ability. We have shown that in cell culture, rapamycin acts by increasing turnover of progerin aggregates by activating autophagy. We are currently studying whether RAD001 has an effect on fibroblasts from patients with other LMNA or Zmpste24 mutations. Our hypothesis is that RAD001 will increase autophagy and lead to an improved phenotype in these cells. While progerin has a dramatic effect on nuclear structure and mitosis, it also disrupts the connections between the nuclear scaffold and chromatin. The consequences include disregulation of gene expression and epigenetic modification. To explore this in detail, we analyzed passage-matched normal and HGPS fibroblasts, using gene expression microarray analysis and chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) and the Hi-C method that reveals the 3-D structure of chromatin in the interphase nucleus. We found that HGPS fibroblasts have an alteration in the distribution of H3K27me3, a histone mark associated with facultative heterochromatin, causing disruption of the association between the nuclear lamina and heterochromatin. This change may cause of the loss of spatial chromosome compartmentalization we observe in HGPS cells and thus cause gene expression disregulation. Of considerable relevance to the study of normal human aging, we have also shown that progerin is made in small amounts in normal individuals, and appears to increase in quantity as cells approach senescence. Recent data points to an interesting connection between shortening of telomeres and activation of alternative splicing of dozens of genes, including production of progerin from a normal LMNA gene. In this way, senescence apparently proceeds by a positive feedback loop, once a cell has reached its maximum life span. We are in the process of creating a mouse knockin model that should be unable to make any progerin, effectively short circuiting this positive feedback loop. Cell culture and whole animal studies of this mouse model could reveal what role progerin plays in natural aging.

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Support Year
11
Fiscal Year
2013
Total Cost
$1,059,945
Indirect Cost
Name
National Human Genome Research Institute
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Sagelius, Hanna; Rosengardten, Ylva; Hanif, Mubashir et al. (2008) Targeted transgenic expression of the mutation causing Hutchinson-Gilford progeria syndrome leads to proliferative and degenerative epidermal disease. J Cell Sci 121:969-78
Merideth, Melissa A; Gordon, Leslie B; Clauss, Sarah et al. (2008) Phenotype and course of Hutchinson-Gilford progeria syndrome. N Engl J Med 358:592-604
Capell, Brian C; Olive, Michelle; Erdos, Michael R et al. (2008) A farnesyltransferase inhibitor prevents both the onset and late progression of cardiovascular disease in a progeria mouse model. Proc Natl Acad Sci U S A 105:15902-7