Fernando G. Osorio

Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging

Several human progerias, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by the accumulation at the nuclear envelope of farnesylated forms of truncated prelamin A, a protein that is also altered during normal aging. Previous studies in cells from individuals with HGPS have shown that farnesyltransferase inhibitors (FTIs) improve nuclear abnormalities associated with prelamin A accumulation, suggesting that these compounds could represent a therapeutic approach for this devastating progeroid syndrome. We show herein that both prelamin A and its truncated form progerin/LAΔ50 undergo alternative prenylation by geranylgeranyltransferase in the setting of farnesyltransferase inhibition, which could explain the low efficiency of FTIs in ameliorating the phenotypes of progeroid mouse models. We also show that a combination of statins and aminobisphosphonates efficiently inhibits both farnesylation and geranylgeranylation of progerin and prelamin A and markedly improves the aging-like phenotypes of mice deficient in the metalloproteinase Zmpste24, including growth retardation, loss of weight, lipodystrophy, hair loss and bone defects. Likewise, the longevity of these mice is substantially extended. These findings open a new therapeutic approach for human progeroid syndromes associated with nuclear-envelope abnormalities.

Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging

Antisense oligonucleotides reverse premature aging and extend life span in mutant mice that mimic aberrant splicing in progeria patients. Countering Careless Cutting Carpenters warn that one should “measure twice, cut once” to avoid unfixable assaults on building materials. Indeed, careless cutting lies at the heart of Hutchinson-Gilford progeria syndrome (HGPS). This premature aging disease is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site in LMNA RNA; aberrant cutting and splicing results in the production of an mRNA that encodes progerin, a truncated form of the lamin A protein that is also produced in small amounts during normal aging. Until now, no model system has recapitulated the pathogenic LMNA splicing that occurs in HGPS patients. Here, Osorio et al. characterize such HGPS mutant mice mimics—called LmnaG609G/G609G mice—and show that antisense oligonucleotide–based therapy reverses various premature aging phenotypes and extends life span. Encoded by the LMNA gene, lamin A is a nuclear envelope protein that is important for nuclear stability, chromatin structure, and regulation of gene expression. Osorio et al. showed that the LmnaG609G/G609G mice produced reduced amounts of intact lamin A, accumulated progerin, displayed the nuclear abnormalities and transcriptional alterations seen in other progeroid models, and sported the key clinical features of human HGPS, such as a shortened life span, reduced size, disrupted metabolism, and enhanced bone and cardiovascular maladies relative to wild-type animals. The authors then used their newly characterized HGPS animal model to test the effects of antisense morpholino oligonucleotides that bound to and blocked the aberrant splice donor site in Lmna RNA. These reagents reduced progerin accumulation and corrected the nuclear abnormalities in both cultured mutant mouse and human HGPS fibroblasts. Furthermore, LmnaG609G/G609G mice that were treated with a combination of two antisense oligonucleotides that blocked aberrant splicing displayed reduced amounts of accumulated progerin, enhanced life expectancy, and a reversal of the phenotypical and molecular alterations associated with HGPS, including the righting of gene expression aberrations and normalization of blood glucose levels. Together, these findings provide preclinical proof of concept for the use of antisense oligonucleotide–based therapies in the treatment of HGPS. Furthermore, because progerin also accumulates during normal aging, the LmnaG609G/G609G mutant mice may be useful for preclinical testing of therapies designed to slow the human aging process and prevent age-related diseases. As the poet Ralph Waldo Emerson noted, “All diseases run into one—old age.” Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino–based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide–based therapies for treating human diseases of accelerated aging.

Nuclear lamina defects cause ATM-dependent NF-κB activation and link accelerated aging to a systemic inflammatory response

Alterations in the architecture and dynamics of the nuclear lamina have a causal role in normal and accelerated aging through both cell-autonomous and systemic mechanisms. However, the precise nature of the molecular cues involved in this process remains incompletely defined. Here we report that the accumulation of prelamin A isoforms at the nuclear lamina triggers an ATM- and NEMO-dependent signaling pathway that leads to NF-κB activation and secretion of high levels of proinflammatory cytokines in two different mouse models of accelerated aging (Zmpste24(-/-) and Lmna(G609G/G609G) mice). Causal involvement of NF-κB in accelerated aging was demonstrated by the fact that both genetic and pharmacological inhibition of NF-κB signaling prevents age-associated features in these animal models, significantly extending their longevity. Our findings provide in vivo proof of principle for the feasibility of pharmacological modulation of the NF-κB pathway to slow down the progression of physiological and pathological aging.

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome.

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.

Defective Extracellular Pyrophosphate Metabolism Promotes Vascular Calcification in a Mouse Model of Hutchinson-Gilford Progeria Syndrome That Is Ameliorated on Pyrophosphate Treatment

Background— Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS. Methods and Results— We performed expression and functional studies in wild-type mice and knock-in LmnaG609G/+ mice expressing progerin, which mimic the main clinical manifestations of HGPS. LmnaG609G/+ mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, LmnaG609G/+ vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of LmnaG609G/+ mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice. Conclusions— Excessive vascular calcification in LmnaG609G mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Insulin-like growth factor 1 treatment extends longevity in a mouse model of human premature aging by restoring somatotroph axis function

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A, an essential component of the nuclear envelope. Zmpste24-deficient mice exhibit multiple defects that phenocopy human accelerated aging processes such as Hutchinson–Gilford progeria syndrome. In this work, we report that progeroid Zmpste24-/− mice present profound transcriptional alterations in genes that regulate the somatotroph axis, together with extremely high circulating levels of growth hormone (GH) and a drastic reduction in plasma insulin-like growth factor 1 (IGF-1). We also show that recombinant IGF-1 treatment restores the proper balance between IGF-1 and GH in Zmpste24-/− mice, delays the onset of many progeroid features, and significantly extends the lifespan of these progeroid animals. Our findings highlight the importance of IGF/GH balance in longevity and may be of therapeutic interest for devastating human progeroid syndromes associated with nuclear envelope abnormalities.

NF-κB activation impairs somatic cell reprogramming in ageing.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor–Guillermo progeria syndrome and Hutchinson–Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Identification of mitochondrial dysfunction in Hutchinson-Gilford progeria syndrome through use of stable isotope labeling with amino acids in cell culture.

UNLABELLED Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental premature aging disorder that recapitulates some biological and physical aspects of physiological aging. The disease is caused by a sporadic dominant mutation in the LMNA gene that leads to the expression of progerin, a mutant form of lamin A that lacks 50 amino acids and retains a toxic farnesyl modification in its carboxy-terminus. However, the mechanisms underlying cellular damage and senescence and accelerated aging in HGPS are incompletely understood. Here, we analyzed fibroblasts from healthy subjects and HGPS patients using SILAC (stable isotope labeling with amino acids in cell culture). We found in HGPS cells a marked downregulation of mitochondrial oxidative phosphorylation proteins accompanied by mitochondrial dysfunction, a process thought to provoke broad organ decline during normal aging. We also found mitochondrial dysfunction in fibroblasts from adult progeroid mice expressing progerin (Lmna(G609G/G609G) knock-in mice) or prelamin A (Zmpste24-null mice). Analysis of tissues from these mouse models revealed that the damaging effect of these proteins on mitochondrial function is time- and dose-dependent. Mitochondrial alterations were not observed in the brain, a tissue with extremely low progerin expression that seems to be unaffected in HGPS. Remarkably, mitochondrial function was restored in progeroid mouse fibroblasts treated with the isoprenylation inhibitors FTI-277 or pravastatin plus zoledronate, which are being tested in HGPS clinical trials. Our results suggest that mitochondrial dysfunction contributes to premature organ decline and aging in HGPS. Beyond its effects on progeria, prelamin A and progerin may also contribute to mitochondrial dysfunction and organ damage during normal aging, since these proteins are expressed in cells and tissues from non-HGPS individuals, most prominently at advanced ages. BIOLOGICAL SIGNIFICANCE Mutations in LMNA or defective processing of prelamin A causes premature aging disorders, including Hutchinson-Gilford progeria syndrome (HGPS). Most HGPS patients carry in heterozygosis a de-novo point mutation (c.1824C>T: GGC>GGT; p.G608G) which causes the expression of the lamin A mutant protein called progerin. Despite the importance of progerin and prelamin A in accelerated aging, the underlying molecular mechanisms remain largely unknown. To tackle this question, we compared the proteome of skin-derived dermal fibroblast from HGPS patients and age-matched controls using quantitative stable isotope labeling with amino acids in cell culture (SILAC). Our results show a pronounced down-regulation of several components of the mitochondrial ATPase complex accompanied by up-regulation of some glycolytic enzymes. Accordingly, functional studies demonstrated mitochondrial dysfunction in HGPS fibroblasts. Moreover, our expression and functional studies using cellular and animal models confirmed that mitochondrial dysfunction is a feature of progeria which develops in a time- and dose-dependent manner. Finally, we demonstrate improved mitochondrial function in progeroid mouse cells treated with a combination of statins and aminobisphosphonates, two drugs that are being evaluated in ongoing HGPS clinical trials. Although further studies are needed to unravel the mechanisms through which progerin and prelamin A provoke mitochondrial abnormalities, our findings may pave the way to improved treatments of HGPS. These studies may also improve our knowledge of the mechanisms leading to mitochondrial dysfunction during normal aging, since both progerin and prelamin A have been found to accumulate during normal aging.

Nuclear envelope alterations generate an aging-like epigenetic pattern in mice deficient in Zmpste24 metalloprotease

Mutations in the nuclear envelope protein lamin A or in its processing protease ZMPSTE24 cause human accelerated aging syndromes, including Hutchinson–Gilford progeria syndrome. Similarly, Zmpste24‐deficient mice accumulate unprocessed prelamin A and develop multiple progeroid symptoms, thus representing a valuable animal model for the study of these syndromes. Zmpste24‐deficient mice also show marked transcriptional alterations associated with chromatin disorganization, but the molecular links between both processes are unknown. We report herein that Zmpste24‐deficient mice show a hypermethylation of rDNA that reduces the transcription of ribosomal genes, being this reduction reversible upon treatment with DNA methyltransferase inhibitors. This alteration has been previously described during physiological aging in rodents, suggesting its potential role in the development of the progeroid phenotypes. We also show that Zmpste24‐deficient mice present global hypoacetylation of histones H2B and H4. By using a combination of RNA sequencing and chromatin immunoprecipitation assays, we demonstrate that these histone modifications are associated with changes in the expression of several genes involved in the control of cell proliferation and metabolic processes, which may contribute to the plethora of progeroid symptoms exhibited by Zmpste24‐deficient mice. The identification of these altered genes may help to clarify the molecular mechanisms underlying aging and progeroid syndromes as well as to define new targets for the treatment of these dramatic diseases.

Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion

Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A accumulating) and Zmpste24-proficient (mature lamin A containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.