Diseases of the Nuclear Envelope

In the past decade, a wide range of fascinating monogenic diseases have been linked to mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. These diseases include dilated cardiomyopathy with variable muscular dystrophy, Dunnigan-type familial partial lipodystrophy, a Charcot-Marie-Tooth type 2 disease, mandibuloacral dysplasia, and Hutchinson-Gilford progeria syndrome. Several diseases are also caused by mutations in genes encoding B-type lamins and proteins that associate with the nuclear lamina. Studies of these so-called laminopathies or nuclear envelopathies, some of which phenocopy common human disorders, are providing clues about functions of the nuclear envelope and insights into disease pathogenesis and human aging.Mutations in LMNA encoding the A-type lamins cause a group of human disorders often collectively called laminopathies. The major A-type lamins, lamin A and lamin C, arise by alternative splicing of the LMNA pre-mRNA and are expressed in virtually all differentiated somatic cells. Although the A-type lamins are widely expressed, LMNA mutations are responsible for at least a dozen different clinically defined disorders with tissue-selective abnormalities. Mutations in genes encoding B-type lamins and lamin-associated proteins, most of which are similarly expressed in almost all somatic cells, also cause tissue-selective diseases.Research on the laminopathies has provided novel clues about nuclear envelope function. Recent studies have begun to shed light on how alterations in the nuclear envelope could explain disease pathogenesis. Along with basic research on nuclear structure, the nuclear lamins, and lamina-associated proteins, clinical research on the laminopathies will contribute to a complete understanding of the functions of the nuclear envelope in normal physiology and in human pathology.     

A Highlights from MBoC Selection: Systematic identification of pathological lamin A interactors

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.     

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.     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.     

Disheveled Hair and Ear (Dhe), a Spontaneous Mouse Lmna Mutation Modeling Human Laminopathies

Background

Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe), which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10.

Findings

Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling.

Conclusion

Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.     

Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin

A‐lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A‐lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease‐associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson‐Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose‐ and E3 ligase‐dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic–lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A‐lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2–lamin A axis in age‐related diseases such as cancer.     

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

Background

Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways.

Methodology and Principal Findings

We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna^+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna^+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna^+/− embryonic stem cells.

Conclusions

We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development.     

Lamin A/C mutations alter differentiation potential of mesenchymal stem cells

Mutations in the lamin A/C gene (LMNA) lead to severe disorders collectively called laminopathies. The mechanisms by which lamin mutations cause the diseases are not clear. Since the mesenchymal lineages, adipose tissue in particular, are mostly affected in laminopathies, the aim of the study was to estimate the effect of LMNA mutations on differentiation of mesenchymal stem cells, adipose tissue stromal cells (ATSCs), into adipose lineages. ATSCs transduced with lentiviral vectors carrying LMNA gene mutations associated with various syndromes (myodystrophy, cardiomyopathy, lipodystrophy, progeroid syndrome) were induced to adipose differentiate. It was found that introduction of genetic constructions with LMNA gene point mutations G465D, R482L, and R527C promote adipogenic differentiation compared to wild-type lamin gene; mutation R471C reduced the differentiation. Introduction of R471C or R527C lamin mutations profoundly increased the expression of adipogenesis markers PPARG, SREBP1, and adipsin. Mutations in A/C lamin gene strongly and variously affect the differentiation of mesenchymal stem cells that probably underlie the pathogenic changes in patients with laminopathies.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Lamin A/C is a risk biomarker in colorectal cancer

Background

A-type lamins are type V intermediate filament proteins encoded by the gene LMNA. Mutations in LMNA give rise to diverse degenerative diseases related to premature ageing. A-type lamins also influence the activity of the Retinoblastoma protein (pRb) and oncogenes such a β-catenin. Consequently, it has been speculated that expression of A-type lamins may also influence tumour progression.

Methodology/Principal Findings

An archive of colorectal cancer (CRC) and normal colon tissue was screened for expression of A-type lamins. We used the Cox proportional hazard ratio (HR) method to investigate patient survival. Using CRC cell lines we investigated the effects of lamin A expression on other genes by RT-PCR; on cell growth by FACS analysis; and on invasiveness by cell migration assays and siRNA knockdown of targeted genes. We found that lamin A is expressed in colonic stem cells and that patients with A-type lamin-expressing tumours have significantly worse prognosis than patients with A-type lamin negative tumours (HR = 1.85, p = 0.005). To understand this finding, we established a model system based upon expression of GFP-lamin A in CRC cells. We found that expression of GFP-lamin A in these cells did not affect cell proliferation but did promote greatly increased cell motility and invasiveness. The reason for this increased invasiveness was that expression of lamin A promoted up-regulation of the actin bundling protein T-plastin, leading to down regulation of the cell adhesion molecule E-cadherin.

Conclusions

Expression of A-type lamins increases the risk of death from CRC because its presence gives rise to increased invasiveness and potentially a more stem cell-like phenotype. This report directly links A-type lamin expression to tumour progression and raises the profile of LMNA from one implicated in multiple but rare genetic conditions to a gene involved in one of the commonest diseases in the Western World.     

Progeroide Laminopathien zwischen Hutchinson-Gilford-Syndrom, restriktiver Dermopathie und mandibuloakraler Dysplasie

There is a group of primary and secondary laminopathies that show progeroid phenotypes. They are associated with pleiotropic mutations in the LMNA or ZMPSTE24 genes and are clinically associated with Hutchinson-Gilford progeria syndrome, restrictive dermopathy and mandibuloacral dysplasia. Besides these clinically distinct entities, there is also an increasing number of atypical progeroid laminopathies??also known as atypical Werner syndrome??that cannot be categorized clinically as they show features of several laminopathies. As an example, two atypical primary and secondary progeroid laminopathies that show overlapping features of Hutchinson-Gilford progeria syndrome, restrictive dermopathy, dysplasia and even muscular laminopathies are presented.     

Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation

Background

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.

Methods/Findings

To investigate the hypothesis that mutant lamin A/C changes the lamina''s ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.

Conclusions

These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.     

Mechanisms of genome instability in Hutchinson-Gilford progeria

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder. It arises from a single point mutation in the LMNA gene. This mutation stimulates an aberrant splicing event and produces progerin, an isoform of the lamin A protein. Accumulation of progerin disrupts numerous physiological pathways and induces defects in nuclear architecture, gene expression, histone modification, cell cycle regulation, mitochondrial functionality, genome integrity and much more.

Objective

Among these phenotypes, genomic instability is tightly associated with physiological aging and considered a main contributor to the premature aging phenotypes. However, our understanding of the underlying molecular mechanisms of progerin-caused genome instability is far from clear.

Results and Conclusion

In this review, we summarize some of the recent findings and discuss potential mechanisms through which, progerin affects DNA damage repair and leads to genome integrity.

     

Low Levels of the Reverse Transactivator Fail to Induce Target Transgene Expression in Vascular Smooth Muscle Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.     

Cell-Extrinsic Defective Lymphocyte Development in Lmna-/- Mice

Background

Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna^-/- mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna^-/- mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development.

Principal Findings

Lmna^-/- mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna^-/- bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4⁺ and CD8⁺ T cells. Transplantation of Lmna^-/- neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development.

Conclusions

Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna^-/- mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna ^-/- mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.