From lamins to lamina: a structural perspective

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Nuclear Envelope, Nuclear Lamina, and Inherited Disease

The human genome is contained within the nucleus and is separated from the cytoplasm by the nuclear envelope. Mutations in the nuclear envelope proteins emerin and lamin A cause a number of diseases including premature aging syndromes, muscular dystrophy, and cardiomyopathy. Emerin and lamin A are implicated in regulating muscle- and heart-specific gene expression and nuclear architecture. For example, lamin A regulates the expression and localization of gap junction and intercalated disc components. Additionally, emerin and lamin A are also required to maintain nuclear envelope integrity. Demonstrating the importance of maintaining nuclear integrity in heart disease, atrioventricular node cells lacking lamin A exhibit increased nuclear deformation and apoptosis. This review highlights the present understanding of lamin A and emerin function in regulating nuclear architecture, gene expression, and cell signaling and discusses putative mechanisms for how specific mutations in lamin A and emerin cause cardiac- or muscle-specific disease.

Association of lamin A/C with muscle gene-specific promoters in myoblasts

Nuclear lamins and nuclear pore complexes (NPCs) are major components of the nuclear envelope in metazoan cells. The objectives of this thesis were first to study interactions between nuclear lamins and the nuclear pore protein Nup153 (Chapters 2 and 3) and second to determine lamin assembly conditions of the Xenopus oocytes LIII in vitro (Chapter 4). Nuclear lamins are major constituents of the nuclear lamina underlying the nuclear periphery along with inner nuclear membrane proteins. The nuclear lamina provides stability and determines the nuclear architecture and spacing of the NPCs. NPCs form supramolecular assemblies that regulate nucleocytoplasmic transport. An overview of the functional aspects associated with the nuclear lamina and NPCs in health and disease is provided in Chapter 1. In depth analysis of the interaction of nuclear lamins with the nucleoporin Nup153 is revealed in Chapters 2 and 3. Using in vitro solution binding assays as well as immunoprecipitation assays, in chapter 2 we show direct associations between Nup153 and nuclear lamins. This work is explored even further in chapter 3 using binding assays and immunofluorescence microscopy as well as immunoprecipitation assays; we examined the interaction in the presence of lamin related mutations. Finally, in chapter 4 we established buffer conditions for LIII assembly in vitro, analysed by electron microscopy (EM) using glycerol spraying/low-angle rotary metal shadowing and negative staining. Our results presented in this thesis contribute to expand our current knowledge of the interactions of the NPCs with the nuclear lamins, as well as to increase our understanding of the impact of mutations in lamins that can cause laminopathies. In addition, the studies on the assembly conditions of LIII provide a vehicle for further characterisation of the influence of binding partners and the importance of lamin sub-fragments on the formation of higher order assemblies.

The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level.

Cutaneous asthenia is a hereditary connective tissue disease, primarily of dogs and cats, resembling Ehlers-Danlos syndrome in man. Collagen dysplasia results in skin hyperextensibility, skin and vessel fragility, and poor wound healing. The purpose of this study was to describe the histological findings in a dog with a collagenopathy consistent with cutaneous asthenia. An 8-month-old crossbreed female dog presented with lacerations and numerous atrophic and irregular scars. The skin was hyperextensible and easily torn by the slightest trauma. Ultrastructurally, the dermis was comprised of elaunin and oxytalan microfibrils. These are immature fibres in which the fibrillar component is increased but elastin is reduced. Collagen fibres were profoundly disorganized. The fibrils had a highly irregular outline and a corroded appearance when viewed in cross-section, and were spiralled and fragmented in a longitudinal view. Dermal fibroblasts displayed a conspicuous thickening of the nuclear lamina. Nuclear lamins form a fibrous nucleoskeletal network of intermediate-sized filaments underlying the inner nuclear membrane. Mutations in lamins or lamin-associated proteins cause a myriad of genetic diseases collectively called laminopathies. Disruption of the nuclear lamina seems to affect chromatin organization and transcriptional regulation of gene expression. A common link among all laminopathies may be a failure of stem cells to regenerate mesenchymal tissue. This could contribute to the connective tissue dysplasia seen in cutaneous asthenia.

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

Nuclear lamins are type V intermediate filament proteins that polymerize into complex filamentous meshworks at the nuclear periphery and in less structured forms throughout the nucleoplasm. Lamins interact with a wide range of nuclear proteins and are involved in numerous nuclear and cellular functions. Within the nucleus, they play roles in chromatin organization and gene regulation, nuclear shape, size, and mechanics, and the organization and anchorage of nuclear pore complexes. At the whole cell level, they are involved in the organization of the cytoskeleton, cell motility, and mechanotransduction. The expression of different lamin isoforms has been associated with developmental progression, differentiation, and tissue-specific functions. Mutations in lamins and their binding proteins result in over 15 distinct human diseases, referred to as laminopathies. The laminopathies include muscular (e.g., Emery–Dreifuss muscular dystrophy and dilated cardiomyopathy), neurological (e.g., microcephaly), and metabolic (e.g., familial partial lipodystrophy) disorders as well as premature aging diseases (e.g., Hutchinson–Gilford Progeria and Werner syndromes). How lamins contribute to the etiology of laminopathies is still unknown. In this review article, we summarize major recent findings on the structure, organization, and multiple functions of lamins in nuclear and more global cellular processes.

Premature aging syndromes: From patients to mechanism

BackgroundLamins 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/FindingsTo 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.ConclusionsThese 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.

Cardiac aging is considered as natural and age-related changes in the heart, which can lead to a decline in heart function. Cardiac aging increases the risk of heart failure, atrial fibrillation, and other cardiovascular complications. Understanding the molecular and cellular mechanisms of cardiac aging is crucial for preventing and managing age-related heart problems. Among nuclear lamins, Lamin A/C (LMNA) is the most studied protein linked to cardiac aging. LMNA protein, the alternative splice product of LMNA gene, is crucial for maintaining nuclear inner membrane structure, regulating expression of different genes, chromatin organization, and cell division. A mutation in LMNA gene causes progeria, a rare genetic disorder that causes premature aging and cardiovascular diseases. In addition to progeria, muscular dystrophy, lipodystrophy, and cardiomyopathies are among the conditions that can arise from LMNA mutations. Besides gene mutations, altered posttranslational modifications (PTMs) of this protein can disrupt their maturation and function, interfere with cellular signaling, and destabilize nuclear architecture. Several PTMs of LMNA have been implicated in cardiac aging by causing cellular dysfunction, elevated production of reactive oxygen species, and weakening of muscle cells. In this short review, we summarize the various types of PTMs such as phosphorylation, acetylation, SUMOylation, farnesylation, and palmitoylation of LMNA and explained how their modification can contribute to cardiac ageing and related cardiovascular disorders.

Dilated cardiomyopathy (DCM) is one of the most common cardiac phenotypes caused by mutations of lamin A/C (LMNA) gene in humans. Almost 50% of LMNA variants reported in ClinVar as pathogenic or likely pathogenic are due to frameshift, nonsense, or splicing variants, predictive of haploinsufficiency for LMNA protein. In contrast, synonymous variants that do not immediately suggest a haploinsufficiency mechanism are much harder to interpret. We evaluated a cohort of 57 patients who underwent heart transplant for dilated cardiomyopathy for LMNA variants and identified a novel synonymous variant c.936G>A in the last nucleotide of exon 5 of LMNA in a DCM family. Clinically, LMNA c.936G>A carriers presented with severe DCM, conduction disease and high creatine-kinase levels. Sanger sequencing showed the presence of LMNA c.936G>A in the genomic DNA but not in the cDNA derived from one family member’s heart tissue. RT-PCR using RNA from the patient’s heart tissue was performed with primers that amplified LMNA exons 4-6. DNA fragments amplified by RT-PCR were then cloned into pcDNAII vector and 40 clones were picked and sequenced. An aberrantly spliced mRNA containing the variant and a premature stop codon was identified in 1 of the 40 clones. Real-time quantitative PCR showed significantly lower LMNA mRNA levels in the patient’s heart compared to the controls. Our findings suggest that the c.936G>A LMNA variant resulted in aberrant RNA splicing, which possibly triggers nonsense-mediated RNA decay, leading to reduced mRNA and likely protein expression. These results expand the understanding on the association between synonymous LMNA variants and the molecular pathogenesis of LMNA-DCM.