Farnesyltransferase inhibition in HGPS.

The ultra-rare, pediatric premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS) is caused by mutation of LMNA, encoding the nuclear architectural protein lamin A. Patients develop atherosclerosis and typically die of heart failure in their teens. FDA-approved Zokinvy prevents farnesylation of lamin A, reduces vascular stiffness, and extends survival in HGPS patients. To view this Bench to Bedside, open or download the PDF.

Abnormal nuclear morphology is independent of longevity in a zmpste24-deficient fish model of Hutchinson-Gilford progeria syndrome (HGPS).

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

Chemical inhibition of NAT10 corrects defects of laminopathic cells.

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule "Remodelin" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C-depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.

Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model.

A de novo G608G mutation in LMNA gene leads to Hutchinson-Gilford progeria syndrome. Mice lacking the prelamin A-processing metalloprotease, Zmpste24, recapitulate many of the progeroid features of Hutchinson-Gilford progeria syndrome. Here we show that A-type lamins interact with SUV39H1, and prelamin A/progerin exhibits enhanced binding capacity to SUV39H1, protecting it from proteasomal degradation and, consequently, increasing H3K9me3 levels. Depletion of Suv39h1 reduces H3K9me3 levels, restores DNA repair capacity and delays senescence in progeroid cells. Remarkably, loss of Suv39h1 in Zmpste24(-/-) mice delays body weight loss, increases bone mineral density and extends lifespan by ∼60%. Thus, increased H3K9me3 levels, possibly mediated by enhanced Suv39h1 stability in the presence of prelamin A/progerin, compromise genome maintenance, which in turn contributes to accelerated senescence in laminopathy-based premature aging. Our study provides an explanation for epigenetic alterations in Hutchinson-Gilford progeria syndrome and a potential strategy for intervention by targeting SUV39H1-mediated heterochromatin remodelling.

Targeting protein prenylation in progeria.

A clinical trial of a protein farnesyltransferase inhibitor (lonafarnib) for the treatment of Hutchinson-Gilford progeria syndrome (HGPS) was recently completed. Here, we discuss the mutation that causes HGPS, the rationale for inhibiting protein farnesyltransferase, the potential limitations of this therapeutic approach, and new potential strategies for treating the disease.

p38 (MAPK) stress signalling in replicative senescence in fibroblasts from progeroid and genomic instability syndromes.

Werner Syndrome (WS) is a human segmental progeria resulting from mutations in a DNA helicase. WS fibroblasts have a shortened replicative capacity, an aged appearance, and activated p38 MAPK, features that can be modulated by inhibition of the p38 pathway. Loss of the WRNp RecQ helicase has been shown to result in replicative stress, suggesting that a link between faulty DNA repair and stress-induced premature cellular senescence may lead to premature ageing in WS. Other progeroid syndromes that share overlapping pathophysiological features with WS also show defects in DNA processing, raising the possibility that faulty DNA repair, leading to replicative stress and premature cellular senescence, might be a more widespread feature of premature ageing syndromes. We therefore analysed replicative capacity, cellular morphology and p38 activation, and the effects of p38 inhibition, in fibroblasts from a range of progeroid syndromes. In general, populations of young fibroblasts from non-WS progeroid syndromes do not have a high level of cells with an enlarged morphology and F-actin stress fibres, unlike young WS cells, although this varies between strains. p38 activation and phosphorylated HSP27 levels generally correlate well with cellular morphology, and treatment with the p38 inhibitor SB203580 effects cellular morphology only in strains with enlarged cells and phosphorylated HSP27. For some syndromes fibroblast replicative capacity was within the normal range, whereas for others it was significantly shorter (e.g. HGPS and DKC). However, although in most cases SB203580 extended replicative capacity, with the exception of WS and DKC the magnitude of the effect was not significantly different from normal dermal fibroblasts. This suggests that stress-induced premature cellular senescence via p38 activation is restricted to a small subset of progeroid syndromes.

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

Lonafarnib for cancer and progeria.

INTRODUCTION: Lonafarnib is a non-peptidomimetic inhibitor of farnesyl transferase, an enzyme responsible for the post-translational lipid modification of a wide variety of cellular proteins that are involved in the pathogenic pathways of various diseases including cancer and progeria. Although extensive clinical research indicates limited activity of lonafarnib in solid tumors, there is recent interest in combinations of farnesyl transferase inhibitors with imatinib or bortezomib in hematological malignancies and to investigate the role of lonafarnib in progeria. AREAS COVERED: This review examines the in vitro and in vivo pharmacology of lonafarnib and the available clinical data for lonafarnib monotherapy and combination therapy in the treatment of solid and hematological malignancies as well as progeria, using studies identified from the PubMed database supplemented by computerized search of relevant abstracts from major cancer and hematology conferences. EXPERT OPINION: There is no evidence to support the use of lonafarnib in solid tumors. There is ongoing interest to explore lonafarnib for progeria and to investigate other farnesyl transferase inhibitors for chronic and acute leukemias.

Defective transcription initiation causes postnatal growth failure in a mouse model of nucleotide excision repair (NER) progeria.

Nucleotide excision repair (NER) defects are associated with cancer, developmental disorders and neurodegeneration. However, with the exception of cancer, the links between defects in NER and developmental abnormalities are not well understood. Here, we show that the ERCC1-XPF NER endonuclease assembles on active promoters in vivo and facilitates chromatin modifications for transcription during mammalian development. We find that Ercc1(-/-) mice demonstrate striking physiological, metabolic and gene expression parallels with Taf10(-/-) animals carrying a liver-specific transcription factor II D (TFIID) defect in transcription initiation. Promoter occupancy studies combined with expression profiling in the liver and in vitro differentiation cell assays reveal that ERCC1-XPF interacts with TFIID and assembles with POL II and the basal transcription machinery on promoters in vivo. Whereas ERCC1-XPF is required for the initial activation of genes associated with growth, it is dispensable for ongoing transcription. Recruitment of ERCC1-XPF on promoters is accompanied by promoter-proximal DNA demethylation and histone marks associated with active hepatic transcription. Collectively, the data unveil a role of ERCC1/XPF endonuclease in transcription initiation establishing its causal contribution to NER developmental disorders.

Age-dependent loss of MMP-3 in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, progressive segmental premature aging disease that includes scleroderma-like skin, progressive joint contracture, and atherosclerosis. Affected individuals die prematurely of heart attacks or strokes. Extracellular matrix dysregulation is implicated as a factor in disease progression. We analyzed messenger RNA and protein levels for matrix metalloproteinases (MMPs)-2,-3, and -9 in HGPS primary human dermal fibroblasts using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. MMP-3 messenger RNA and protein levels decreased significantly with increasing donor age in HGPS fibroblasts but not in controls. MMP-2 messenger RNA also showed a donor age-dependent decrease in HGPS fibroblasts, but levels of secreted protein were unchanged. MMP-9 was similar in HGPS and control cultures. The decreased MMP-3 may represent a shift in the inherent extracellular matrix-degrading proteolytic balance in favor of matrix deposition in HGPS. This metalloproteinase has the potential to serve as a biomarker of therapeutic efficacy when assessing treatments for HGPS.

Premature aging-related peripheral neuropathy in a mouse model of progeria.

Peripheral neuropathy is a common aging-related degenerative disorder that interferes with daily activities and leads to increased risk of falls and injury in the elderly. The etiology of most aging-related peripheral neuropathy is unknown. Inherited defects in several genome maintenance mechanisms cause tissue-specific accelerated aging, including neurodegeneration. We tested the hypothesis that a murine model of XFE progeroid syndrome, caused by reduced expression of ERCC1-XPF DNA repair endonuclease, develops peripheral neuropathy. Nerve conduction studies revealed normal nerve function in young adult (8 week) Ercc1(-/Δ) mice, but significant abnormalities in 20 week-old animals. Morphologic and ultrastructural analysis of the sciatic nerve from mutant mice revealed significant alterations at 20 but not 8 weeks of age. We conclude that Ercc1(-/Δ) mice have accelerated spontaneous peripheral neurodegeneration that mimics aging-related disease. This provides strong evidence that DNA damage can drive peripheral neuropathy and offers a rapid and novel model to test therapies.

3-Ketoacyl thiolase delays aging of Caenorhabditis elegans and is required for lifespan extension mediated by sir-2.1.

Studies of long-lived Caenorhabditis elegans mutants have identified several genes that function to limit lifespan, i.e., loss-of-function mutations in these genes promote longevity. By contrast, little is known about genes that normally act to delay aging and that when mutated cause premature aging (progeria). To seek such genes, we performed a genetic screen for C. elegans mutants that age prematurely. We found that loss-of-function mutations of the ketoacyl thiolase gene kat-1 result in an increased accumulation of the lipofuscin-like fluorescent aging pigment, shortened lifespan, early behavioral decline, and other abnormalities characteristic of premature aging. These findings suggest that kat-1 acts to delay C. elegans aging. kat-1 encodes a conserved metabolic enzyme that catalyzes the last step of fatty acid oxidation and was previously shown to regulate fat accumulation in worms. We observed that kat-1 is required for the extension of lifespan and enhanced thermotolerance mediated by extra copies of the deacetylase gene sir-2.1. kat-1 acts independently of other known pathways that affect longevity. Our findings suggest that defects in fatty acid oxidation can limit lifespan and accelerate aging in C. elegans and that kat-1-mediated fatty acid oxidation is crucial for overexpressed sir-2.1 to delay aging.

Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria.

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 (Lmna(HG/+)). 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 (Lmna(nHG/+)) develop progeria-like disease phenotypes. The fact that Lmna(nHG/+) mice manifest disease raised the possibility that the beneficial effects of an FTI in Lmna(HG/+) 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 Lmna(HG/+) and Lmna(nHG/+) mice. In Lmna(HG/+) mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in Lmna(nHG/+) mice. The failure of the FTI to ameliorate disease in Lmna(nHG/+) mice supports the idea that the beneficial effects of an FTI in Lmna(HG/+) mice are due to the effect of drug on the farnesylation of progerin.

Accelerated features of age-related bone loss in zmpste24 metalloproteinase-deficient mice.

Age-related bone loss is associated with changes in bone cellularity, which include marrow fat infiltration and decreasing levels of osteoblastogenesis. The mechanisms that explain these changes remain unclear. Although nuclear lamina alterations occur in premature aging syndromes that include changes in body fat and severe osteoporosis, the role of proteins of the nuclear lamina in age-related bone loss remains unknown. Using the Zmpste24-null progeroid mice (Zmpste24(-/-)), which exhibit nuclear lamina defects and accumulate unprocessed prelamin A, we identified several alterations in bone cellularity in vivo. We found that defective prelamin A processing induced accelerated features of age-related bone loss including lower osteoblast and osteocyte numbers and higher levels of marrow adipogenesis. In summary, processing of prelamin A could become a new approach to regulate osteoblastogenesis and bone turnover and thus for the prevention and treatment of senile osteoporosis.

A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging.

Although DNA damage is considered a driving force for aging, the nature of the damage that arises endogenously remains unclear. Replicative stress, a source of endogenous DNA damage, is prevented primarily by the ATR kinase. We have developed a mouse model of Seckel syndrome characterized by a severe deficiency in ATR. Seckel mice show high levels of replicative stress during embryogenesis, when proliferation is widespread, but this is reduced to marginal amounts in postnatal life. In spite of this decrease, adult Seckel mice show accelerated aging, which is further aggravated in the absence of p53. Together, these results support a model whereby replicative stress, particularly in utero, contributes to the onset of aging in postnatal life, and this is balanced by the replicative stress-limiting role of the checkpoint proteins ATR and p53.

Treatment with a farnesyltransferase inhibitor improves survival in mice with a Hutchinson-Gilford progeria syndrome mutation.

Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by multiple aging-like disease phenotypes. We recently reported that a protein farnesyltransferase inhibitor (FTI) improved several disease phenotypes in mice with a HGPS mutation (Lmna(HG/+)). Here, we investigated the impact of an FTI on the survival of Lmna(HG/+) mice. The FTI significantly improved the survival of both male and female Lmna(HG/+) mice. Treatment with the FTI also improved body weight curves and reduced the number of spontaneous rib fractures. This study provides further evidence for a beneficial effect of an FTI in HGPS.

Farnesylated lamins, progeroid syndromes and farnesyl transferase inhibitors.

Three mammalian nuclear lamin proteins, lamin B(1), lamin B(2) and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility that farnesyl transferase inhibitors can represent effective treatment for HGPS. However, there are concerns about their use for this purpose given the potential for alternative prenylation pathways.

Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.

Defects in the biogenesis of lamin A from its farnesylated precursor, prelamin A, lead to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes. A deficiency in ZMPSTE24, a protease involved in prelamin A processing, leads to prelamin A accumulation, an absence of mature lamin A, misshapen nuclei, and a lethal perinatal progeroid syndrome: restrictive dermopathy (RD). Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot be processed to lamin A. The hallmark cellular abnormality in RD and HGPS is misshapen nuclei. We hypothesized that the farnesylation of prelamin A is important for its targeting to the nuclear envelope in RD and HGPS and that blocking farnesylation would ameliorate the nuclear shape abnormalities. Indeed, when RD fibroblasts were treated with a farnesyltransferase inhibitor (FTI), prelamin A was partially mislocalized away from the nuclear envelope, and the frequency of nuclear shape abnormalities was reduced (P < 0.0001). A FTI also mislocalized prelamin A and improved nuclear shape in Zmpste24-deficient mouse embryonic fibroblasts (P < 0.0001) and improved nuclear shape in human HGPS fibroblasts (P < 0.0001). Most remarkably, a FTI significantly improved nuclear shape in two fibroblast cell lines from atypical progeria patients with lamin A missense mutations in the absence of prelamin A accumulation (P = 0.0003 and P < 0.0001). These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes and suggest a potential strategy for treating these diseases.

Lysyl oxidase in development, aging and pathologies of the skin.

Lysyl oxidase (LOX) is a copper- and lysyl-tyrosyl cofactor containing amine oxidase that has been known to play a critical role in the catalysis of lysine-derived crosslinks in extracellular matrix (ECM) proteins in the dermis. Changes in the composition and crosslinked state of the ECM and alterations in LOX synthesis and activity are known to be associated with aging and a range of acquired and heritable skin disorders. It has been assumed until recently that the LOX-related changes in the skin are mediated through the catalytic activity of LOX. However, work by several laboratories over the last few years has shown that LOX is a multifunctional protein. In this review we discuss the regulation of expression, localization and activation of LOX in the normal developing and adult skin, and alterations in LOX expression and activity associated with skin aging and senescence, and in pathological conditions, including wound healing, fibrosis, hypertrophic scarring, keloids, scleroderma, and diabetic skin. We further evaluate the role of LOX in skin ECM changes associated with the normal aging process and with these pathological states. In addition to collagen and elastin cross-linkages, regulatory and activation mechanisms and cell type specific LOX interactions may contribute to a range of novel intra- and extracellular LOX functions that appear critical determinants of the cellular microenvironment in the normal skin and in these skin disorders.