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. This treatment 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. An application for Phase 1 trial testing of RAD001 in children with progeria is currently under consideration by the FDA. We are currently studying whether RAD001 has an effect on fibroblasts from patients with other LMNA mutations, including those that cause cardiomyopathy and Emery-Dreifuss muscular dystrophy. Intriguingly, in this cell culture model we have found evidence that activation of autophagy may also benefit these other conditions. Specifically, we have found that RAD001 decreases nuclear blebbing and eliminates senescence-associated beta galatosidase expression in most of the atypical LMNA mutation lines we have tested. In all of the cell lines tested, we found that RAD001 improves the cells proliferative ability over time. 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 have created a mouse knockin model that alters the sequence of mouse exon 11, and should be unable to make any progerin, effectively short circuiting this positive feedback loop. The knockin heterozygotes and homozygotes are viable and have no overt, unusual features so far. We hypothesize that an inability to produce progerin may result in increased lifespan in vivo and a resistance to age related phenotypes such as senescence in vitro. Cell culture and whole animal studies of this mouse model are underway, and could reveal what role progerin plays in natural aging.
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