Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.     

Genomic instability and innate immune responses to self-DNA in progeria

In the last decade, we have seen increasing evidence of the importance of structural nuclear proteins such as lamins in nuclear architecture and compartmentalization of genome function and in the maintenance of mechanical stability and genome integrity. With over 400 mutations identified in the LMNA gene (encoding for A-type lamins) associated with more than ten distinct degenerative disorders, the role of lamins as genome caretakers and the contribution of lamins dysfunction to disease are unarguable. However, the molecular mechanisms whereby lamins mutations cause pathologies remain less understood. Here, we review pathways and mechanisms recently identified as playing a role in the pathophysiology of laminopathies, with special emphasis in Hutchinson Gilford Progeria Syndrome (HGPS). This devastating incurable accelerated aging disease is caused by a silent mutation in the LMNA gene that generates a truncated lamin A protein “progerin” that exerts profound cellular toxicity and organismal decline. Patients usually die in their teens due to cardiovascular complications such as myocardial infarction or stroke. To date, there are no efficient therapies that ameliorate disease progression, stressing the need to understand molecularly disease mechanisms that can be targeted therapeutically. We will summarize data supporting that replication stress is a major cause of genomic instability in laminopathies, which contributes to the activation of innate immune responses to self-DNA that in turn accelerate the aging process.

     

Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.     

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively,in Hutchinson–Gilford progeria syndrome fibroblasts

Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.     

Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disease resulting from a mutation in the LMNA gene, which encodes nuclear lamins A and C. Lamin A is synthesized as a precursor (prelamin A) with a C-terminal CaaX motif that undergoes farnesylation, endoproteolytic cleavage, and carboxylmethylation. Prelamin A is subsequently internally cleaved by the zinc metalloprotease Ste24 (Zmpste24) protease, which removes the 15 C-terminal amino acids, including the CaaX modifications, to yield mature lamin A. HGPS results from a dominant mutant form of prelamin A (progerin) that has an internal deletion of 50 aa near the C terminus that includes the Zmpste24 cleavage site and blocks removal of the CaaX-modified C terminus. Fibroblasts from HGPS patients have aberrant nuclei with irregular shapes, which we hypothesize result from the abnormal persistence of the farnesyl and/or carboxylmethyl CaaX modifications on progerin. If this hypothesis is correct, inhibition of CaaX modification by mutation or pharmacological treatment should alleviate the nuclear morphology defect. Consistent with our hypothesis, we find that expression in HeLa cells of GFP-progerin or an uncleavable form of prelamin A with a Zmpste24 cleavage site mutation induces the formation of abnormal nuclei similar to those in HGPS fibroblasts. Strikingly, inhibition of farnesylation pharmacologically with the farnesyl transferase inhibitor rac-R115777 or mutationally by alteration of the CaaX motif dramatically reverses the abnormal nuclear morphology. These results suggest that farnesyl transferase inhibitors represent a possible therapeutic option for individuals with HGPS and/or other laminopathies due to Zmpste24 processing defects.     

Age-related changes of nuclear architecture in Caenorhabditis elegans

Mutations in lamins cause premature aging syndromes in humans, including the Hutchinson-Gilford Progeria Syndrome (HGPS) and Atypical Werner Syndrome. It has been shown that HGPS cells in culture undergo age-dependent progressive changes in nuclear architecture. However, it is unknown whether similar changes in nuclear architecture occur during the normal aging process. We have observed that major changes of nuclear architecture accompany Caenorhabditis elegans aging. We found that the nuclear architecture in most nonneuronal cell types undergoes progressive and stochastic age-dependent alterations, such as changes of nuclear shape and loss of peripheral heterochromatin. Furthermore, we show that the rate of these alterations is influenced by the insulin/IGF-1 like signaling pathway and that reducing the level of lamin and lamin-associated LEM domain proteins leads to shortening of lifespan. Our work not only provides evidence for changes of nuclear architecture during the normal aging process of a multicellular organism, but also suggests that HGPS is likely a result of acceleration of the normal aging process. Because the nucleus is vital for many cellular functions, our studies raise the possibility that the nucleus is a prominent focal point for regulating aging.     

Mechanical model of blebbing in nuclear lamin meshworks

Much of the structural stability of the nucleus comes from meshworks of intermediate filament proteins known as lamins forming the inner layer of the nuclear envelope called the nuclear lamina. These lamin meshworks additionally play a role in gene expression. Abnormalities in nuclear shape are associated with a variety of pathologies, including some forms of cancer and Hutchinson–Gilford Progeria Syndrome, and often include protruding structures termed nuclear blebs. These nuclear blebs are thought to be related to pathological gene expression; however, little is known about how and why blebs form. We have developed a minimal continuum elastic model of a lamin meshwork that we use to investigate which aspects of the meshwork could be responsible for bleb formation. Mammalian lamin meshworks consist of two types of lamin proteins, A type and B type, and it has been reported that nuclear blebs are enriched in A-type lamins. Our model treats each lamin type separately and thus, can assign them different properties. Nuclear blebs have been reported to be located in regions where the fibers in the lamin meshwork have a greater separation, and we find that this greater separation of fibers is an essential characteristic for generating nuclear blebs. The model produces structures with comparable morphologies and distributions of lamin types as real pathological nuclei. Thus, preventing this opening of the meshwork could be a route to prevent bleb formation, which could be used as a potential therapy for the pathologies associated with nuclear blebs.     

Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody

Hutchinson-Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC-->GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria.     

Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.     

Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins.

Four proteins of Mr approximately equal to 60,000, 65,000, 67,000, and 70,000 coisolate with the major intermediate filament (IF) structural proteins of BHK-21 cells. These proteins are keratin-like, they form distinctive paracrystals in vitro, and they are concentrated at the nuclear surface. Since these properties indicate similarities with the nuclear lamins, we have prepared conventional fractions of BHK-21 nuclei from which the same type of paracrystal is obtained. Furthermore, biochemical and immunological data demonstrate that the lamins are identical to the Mr 60,000-70,000 proteins found in IF preparations, and both sets of proteins are similar to keratin. These results suggest that an IF-like protein network is present in the nuclear lamina. We speculate that in some unknown way this network connects to the cytoplasmic IF network that courses from the juxtanuclear region to the cell surface. These proposed interconnecting networks may form part of the infrastructure of cytoskeletal-nuclear matrix-connecting links involved in signal transmission between the nuclear and cytoplasmic compartments of eukaryotic cells.     

The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.

Mutations in the lamin A (LMNA) gene are associated with the tissue-specific diseases Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD-1B), dilated cardiomyopathy with conduction system disease (DCM-CD), and Dunnigan's familial partial lipodystrophy (FPLD). Lamins A and C, the products of the LMNA gene, are nuclear intermediate filament proteins and are the major structural components of the lamina network that underlies and supports the nuclear envelope. Nuclear fragility and mislocalization of the nuclear envelope protein emerin are two defects induced by a lack of the A-type lamins. These observations reveal that organization and structural integrity of the nucleus are critical factors in the origins of certain dystrophic and cardiovascular diseases.     

Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina

Apoptosis, a physiologically critical process, is characterized by a destruction of the cell after sequential degradation of key cellular components. Here, we set out to explore the fate of the physiologically indispensable nuclear envelope (NE) in this process. The NE mediates the critical nucleocytoplasmic transport through nuclear pore complexes (NPCs). In addition, the NE is involved in gene expression and contributes significantly to the overall structure and mechanical stability of the cell nucleus through the nuclear lamina, which underlies the entire nucleoplasmic face of the NE and thereby interconnects the NPCs, the NE, and the genomic material. Using the nano-imaging and mechanical probing approach atomic force microscopy (AFM) and biochemical methods, we unveiled the fate of the NE during apoptosis. The doomed NE sustains a degradation of both the mediators of the critical selective nucleocytoplasmic transport, namely NPC cytoplasmic filaments and basket, and the nuclear lamina. These observations are paralleled by marked softening and destabilization of the NE and the detection of vesicle-like nuclear fragments. We conclude that destruction of the cell nucleus during apoptosis proceeds in a strategic fashion. Degradation of NPC cytoplasmic filaments and basket shuts down the critical selective nucleocytoplasmic cross-talk. Degradation of the nuclear lamina disrupts the pivotal connection between the NE and the chromatin, breaks up the overall nuclear architecture, and softens the NE, thereby enabling the formation of nuclear fragments at later stages of apoptosis.     

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.     

Automated image analysis of nuclear shape: what can we learn from a prematurely aged cell?

The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin, an inhibitor of the mTOR pathway, reduced nuclear blebbing in HGPS fibroblasts. However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective. Here, we show a novel, automated and high throughput nuclear shape analysis that quantitatively measures curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells. We examined HGPS fibroblast cells treated with rapamycin and RAD001 (an analog to rapamycin). Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls. In addition, we find that rapamycin treatment reduces the area of the nucleus, but leaves the eccentricity unchanged. Our nuclear shape analysis provides an unbiased, multidimensional "fingerprint" for a population of cells, which can be used to quantify treatment efficacy and analyze cellular aging.     

Lamin B1 is required for mouse development and nuclear integrity

Lamins are key structural components of the nuclear lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane. Lamins play a role in nuclear architecture, DNA replication, and gene expression. Mutations affecting A-type lamins have been associated with a variety of human diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, and progeria, but mutations in B-type lamins have never been identified in humans or in experimental animals. To investigate the in vivo function of lamin B1, the major B-type lamin, we generated mice with an insertional mutation in Lmnb1. The mutation resulted in the synthesis of a mutant lamin B1 protein lacking several key functional domains, including a portion of the rod domain, the nuclear localization signal, and the CAAX motif (the carboxyl-terminal signal for farnesylation). Homozygous Lmnb1 mutant mice survived embryonic development but died at birth with defects in lung and bone. Fibroblasts from mutant embryos grew under standard cell-culture conditions but displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence. Thus, the lamin B1 mutant mice provide evidence for a broad and nonredundant function of lamin B1 in mammalian development. These mutant mice and cell lines derived from them will be useful models for studying the role of the nuclear lamina in various cellular processes.     

Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement

Mutations in LMNA, which encodes A-type lamins, result in disparate diseases, known collectively as laminopathies, that affect distinct tissues, including striated muscle and adipose tissue. Lamins provide structural support for the nucleus and sites of attachment for chromatin, and defects in these functions may contribute to disease pathogenesis. Recent studies suggest that A-type lamins may facilitate connections between the nucleus and the cytoskeleton mediated by nuclear envelope nesprin and SUN proteins. In mammalian cells, however, interfering with A-type lamins does not affect the localization of these proteins. Here, we used centrosome orientation in fibroblasts, which requires separate nuclear and centrosome positioning pathways, as a model system to understand how LMNA mutations affect nucleus-cytoskeletal connections. We find that LMNA mutations causing striated muscle diseases block actin-dependent nuclear movement, whereas most that affect adipose tissue inhibit microtubule-dependent centrosome positioning. Genetic deletion or transient depletion of A-type lamins also blocked nuclear movement, showing that mutations affecting muscle exhibit the null phenotype. Lack of A-type lamins, or expression of variants that cause striated muscle disease, did not affect assembly of nesprin-2G and SUN2 into transmembrane actin-associated nuclear (TAN) lines that attach the nucleus to retrogradely moving actin cables. Nesprin-2G TAN lines were less stable, however, and slipped over the nucleus rather than moving with it, indicating that they were not anchored. Nesprin-2G TAN lines also slipped in SUN2-depleted cells. Our results establish A-type lamins as anchors for nesprin-2G-SUN2 TAN lines to allow productive movement and proper positioning of the nucleus by actin.     

Progeria of stem cells: stem cell exhaustion in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal genetic disorder that is characterized by segmental accelerated aging. The major causal mutation associated with HGPS triggers abnormal messenger RNA splicing of the lamin A gene leading to changes in the nuclear architecture. To date, two models have been proposed to explain how mutations in the lamin A gene could lead to HGPS, structural fragility and altered gene expression. We favor a compatible model that links HGPS to stem cell-driven tissue regeneration. In this model, nuclear fragility of lamin A-deficient cells increases apoptotic cell death to levels that exhaust tissues' ability for stem cell-driven regeneration. Tissue-specific differences in cell death or regenerative potential, or both, result in the tissue-specific segmental aging pattern seen in HGPS. We propose that the pattern of aging-related conditions present or absent in HGPS can provide insight into the genetic and environmental factors that contribute to normal aging.