HIV protease inhibitors induce senescence and alter osteoblastic potential of human bone marrow mesenchymal stem cells: beneficial effect of pravastatin

HIV‐infected patients receiving antiretroviral therapy present an increased prevalence of age‐related comorbidities, including osteoporosis. HIV protease inhibitors (PIs) have been suspected to participate to bone loss, but the mechanisms involved are unknown. In endothelial cells, some PIs have been shown to induce the accumulation of farnesylated prelamin‐A, a biomarker of cell aging leading to cell senescence. Herein, we hypothesized that these PIs could induce premature aging of osteoblast precursors, human bone marrow mesenchymal stem cells (MSCs), and affect their capacity to differentiate into osteoblasts. Senescence was studied in proliferating human MSCs after a 30‐day exposure to atazanavir and lopinavir with or without ritonavir. When compared to untreated cells, PI‐treated MSCs had a reduced proliferative capacity that worsened with increasing passages. PI treatment led to increased oxidative stress and expression of senescence markers, including prelamin‐A. Pravastatin, which blocks prelamin‐A farnesylation, prevented PI‐induced senescence and oxidative stress, while treatment with antioxidants partly reversed these effects. Moreover, senescent MSCs presented a decreased osteoblastic potential, which was restored by pravastatin treatment. Because age‐related bone loss is associated with increased bone marrow fat, we also evaluated the capacity of PI‐treated MSCs to differentiate into adipocyte. We observed an altered adipocyte differentiation in PI‐treated MSCs that was reverted by pravastatin. We have shown that some PIs alter osteoblast formation by affecting their differentiation potential in association with altered senescence in MSCs, with a beneficial effect of statin. These data corroborate the clinical observations and allow new insight into pathophysiological mechanisms of PI‐induced bone loss in HIV‐infected patients.     

LMNA-linked lipodystrophies: from altered fat distribution to cellular alterations

Mutations in the LMNA gene, encoding the nuclear intermediate filaments the A-type lamins, result in a wide variety of diseases known as laminopathies. Some of them, such as familial partial lipodystrophy of Dunnigan and metabolic laminopathies, are characterized by lipodystrophic syndromes with altered fat distribution and severe metabolic alterations with insulin resistance and dyslipidaemia. Metabolic disturbances could be due either to the inability of adipose tissue to adequately store triacylglycerols or to other cellular alterations linked to A-type lamin mutations. Indeed, abnormal prelamin A accumulation and farnesylation, which are clearly involved in laminopathic premature aging syndromes, could play important roles in lipodystrophies. In addition, gene expression alterations, and signalling abnormalities affecting SREBP1 (sterol-regulatory-element-binding protein 1) and MAPK (mitogen-activated protein kinase) pathways, could participate in the pathophysiological mechanisms leading to LMNA (lamin A/C)-linked metabolic alterations and lipodystrophies. In the present review, we describe the clinical phenotype of LMNA-linked lipodystrophies and discuss the current physiological and biochemical hypotheses regarding the pathophysiology of these diseases.     

Lamin A, farnesylation and aging

Lamin A is a component of the nuclear envelope that is synthesized as a precursor prelamin A molecule and then processed into mature lamin A through sequential steps of posttranslational modifications and proteolytic cleavages. Remarkably, over 400 distinct point mutations have been so far identified throughout the LMNA gene, which result in the development of at least ten distinct human disorders, collectively known as laminopathies, among which is the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). The majority of HGPS cases are associated with a single point mutation in the LMNA gene that causes the production of a permanently farnesylated mutant lamin A protein termed progerin. The mechanism by which progerin leads to premature aging and the classical HGPS disease phenotype as well as the relationship between this disorder and the onset of analogous symptoms during the lifespan of a normal individual are not well understood. Yet, recent studies have provided critical insights on the cellular processes that are affected by accumulation of progerin and have suggested that cellular alterations in the lamin A processing pathway leading to the accumulation of farnesylated prelamin A intermediates may play a role in the aging process in the general population. In this review we provide a short background on lamin A and its maturation pathway and discuss the current knowledge of how progerin or alterations in the prelamin A processing pathway are thought to influence cell function and contribute to human aging.     

Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

The R527H mutation in LMNA gene causes an increased sensitivity to ionizing radiation

Mandibuloacral dysplasia type A (MADA; OMIM # 248370) is a premature ageing disease caused by the homozygous R527H mutation in the LMNA gene. At the cellular level, MADA is characterized by unprocessed prelamin A accumulation, nuclear architecture alterations, chromatin defects and increased incidence of apoptosis. In some progeroid laminopathies (e.g. HGPS) it has been demonstrated that such biochemical and morphological alterations are strongly linked with genomic instability. To test this also in MADA fibroblasts, their response to the ionising radiation- induced damage was analysed. We observed that their ability to repair the damage was significantly impaired, as demonstrated by the increased chromosome damage and the higher percentage of residual γ-H2AX foci, corresponding to unrepaired DNA-damage sites. Moreover, MADA fibroblasts showed a markedly reduced phosphorylation of p53 at Ser15(S15) and a lower induction of p53 and CDKN1A proteins after irradiation, compared to the control cell line. Upon irradiation, we also detected differences in the expression of some p53 downstream target genes. In addition, MADA cells showed partial defects in the checkpoint response, particularly in G1/S transition. Our results indicate that accumulation of the lamin A precursor protein determines a defect in DNA damage response after X-ray exposure, supporting a crucial role of lamin A in regulating DNA repair process and cell cycle control.     

Protein profiling reveals energy metabolism and cytoskeletal protein alterations in LMNA mutation carriers

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.     

Identification of FAM96B as a novel prelamin A binding partner

Prelamin A accumulation causes nuclear abnormalities, impairs nuclear functions, and eventually promotes cellular senescence. However, the underlying mechanism of how prelamin A promotes cellular senescence is still poorly understood. Here we carried out a yeast two-hybrid screen using a human skeletal muscle cDNA library to search for prelamin A binding partners, and identified FAM96B as a prelamin A binding partner. The interaction of FAM96B with prelamin A was confirmed by GST pull-down and co-immunoprecipitation experiments. Furthermore, co-localization experiments by fluorescent confocal microscopy revealed that FAM96B colocalized with prelamin A in HEK-293 cells. Taken together, our data demonstrated the physical interaction between FAM96B and prelamin A, which may provide some clues to the mechanisms of prelamin A in premature aging.     

Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging

A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging has been observed in animals and humans. These include (i) decline in mitochondrial respiratory function; (ii) increase in mitochondrial production of reactive oxygen species (ROS) and the extent of oxidative damage to DNA, proteins, and lipids; (iii) accumulation of point mutations and large-scale deletions of mtDNA; and (iv) enhanced apoptosis. Recent studies have provided abundant evidence to substantiate the importance of mitochondrial production of ROS in aging. On the other hand, somatic mtDNA mutations can cause premature aging without increasing ROS production. In this review, we focus on the roles that ROS play in the aging-associated decline of mitochondrial respiratory function, accumulation of mtDNA mutations, apoptosis, and alteration of gene expression profiles. Taking these findings together, we suggest that mitochondrial dysfunction, enhanced oxidative stress, subsequent accumulation of mtDNA mutations, altered expression of a few clusters of genes, and apoptosis are important contributors to human aging.     

Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.     

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.     

Collagen expression in fibroblasts with a novel LMNA mutation

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.     

Accelerated ageing: from mechanism to therapy through animal models

Ageing research benefits from the study of accelerated ageing syndromes such as Hutchinson-Gilford progeria syndrome (HGPS), characterized by the early appearance of symptoms normally associated with advanced age. Most HGPS cases are caused by a mutation in the gene LMNA, which leads to the synthesis of a truncated precursor of lamin A known as progerin that lacks the target sequence for the metallopotease FACE-1/ZMPSTE24 and remains constitutively farnesylated. The use of Face-1/Zmpste24-deficient mice allowed us to demonstrate that accumulation of farnesylated prelamin A causes severe abnormalities of the nuclear envelope, hyper-activation of p53 signalling, cellular senescence, stem cell dysfunction and the development of a progeroid phenotype. The reduction of prenylated prelamin A levels in genetically modified mice leads to a complete reversal of the progeroid phenotype, suggesting that inhibition of protein farnesylation could represent a therapeutic option for the treatment of progeria. However, we found that both prelamin A and its truncated form progerin can undergo either farnesylation or geranylgeranylation, revealing the need of targeting both activities for an efficient treatment of HGPS. Using Face-1/Zmpste24-deficient mice as model, we found that a combination of statins and aminobisphosphonates inhibits both types of modifications of prelamin A and progerin, improves the ageing-like symptoms of these mice and extends substantially their longevity, opening a new therapeutic possibility for human progeroid syndromes associated with nuclear-envelope defects. We discuss here the use of this and other animal models to investigate the molecular mechanisms underlying accelerated ageing and to test strategies for its treatment.     

Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.     

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.     

Prelamin A-mediated nuclear envelope dynamics in normal and laminopathic cells

Prelamin A is the precursor protein of lamin A, a major constituent of the nuclear lamina in higher eukaryotes. Increasing attention to prelamin A processing and function has been given after the discovery, from 2002 to 2004, of diseases caused by prelamin A accumulation. These diseases, belonging to the group of laminopathies and mostly featuring LMNA mutations, are characterized, at the clinical level, by different degrees of accelerated aging, and adipose tissue, skin and bone abnormalities. The outcome of studies conducted in the last few years consists of three major findings. First, prelamin A is processed at different rates under physiological conditions depending on the differentiation state of the cell. This means that, for instance, in muscle cells, prelamin A itself plays a biological role, besides production of mature lamin A. Secondly, prelamin A post-translational modifications give rise to different processing intermediates, which elicit different effects in the nucleus, mostly by modification of the chromatin arrangement. Thirdly, there is a threshold of toxicity, especially of the farnesylated form of prelamin A, whose accumulation is obviously linked to cell and organism senescence. The present review is focused on prelamin A-mediated nuclear envelope modifications that are upstream of chromatin dynamics and gene expression mechanisms regulated by the lamin A precursor.     

DNA damage, cellular senescence and organismal ageing: causal or correlative?

Cellular senescence has long been used as a cellular model for understanding mechanisms underlying the ageing process. Compelling evidence obtained in recent years demonstrate that DNA damage is a common mediator for both replicative senescence, which is triggered by telomere shortening, and premature cellular senescence induced by various stressors such as oncogenic stress and oxidative stress. Extensive observations suggest that DNA damage accumulates with age and that this may be due to an increase in production of reactive oxygen species (ROS) and a decline in DNA repair capacity with age. Mutation or disrupted expression of genes that increase DNA damage often result in premature ageing. In contrast, interventions that enhance resistance to oxidative stress and attenuate DNA damage contribute towards longevity. This evidence suggests that genomic instability plays a causative role in the ageing process. However, conflicting findings exist which indicate that ROS production and oxidative damage levels of macromolecules including DNA do not always correlate with lifespan in model animals. Here we review the recent advances in addressing the role of DNA damage in cellular senescence and organismal ageing.     

Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening

H2O2 has been the most commonly used inducer for stress-induced premature senescence (SIPS), which shares features of replicative senescence. However, there is still uncertainty whether SIPS and replicative senescence differ or utilize different pathways. 'Young' human diploid fibroblasts (HDFs), treated with prolonged low doses of hydrogen peroxide, led to irreversible cellular senescence. Cells exhibited senescent-morphological features, irreversible G1 cell cycle arrest and irreversible senescence-associated beta-galactosidase positivity. The appearance of these cellular senescence markers was accompanied by significant increases of p21, gadd45 expression and p53 binding activity, as well as a significant decline in DNA repair capability and accelerated telomere shortening. Our results suggest that multiple pathways might be involved in oxidative SIPS, including genes related to DNA-damage-and-repair and telomere shortening, and that SIPS shares the same mechanisms with replicative senescence in vivo. Our findings indicate that several aging theories can be merged together by a common mechanism of oxidative damage, and that the level of oxidative DNA-damage-and-repair capacity may be exploited as reliable markers of cell senescence.