Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

Background  

The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene.     

Direct interaction between emerin and lamin A

Emerin is the protein of the inner nuclear membrane that is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. The autosomal dominant form of the disease is caused by mutations in the lamin A/C gene. Several lines of circumstantial evidence have suggested an interaction of emerin with lamins in the nuclear lamina but direct interaction between the two proteins has not yet been demonstrated. We now demonstrate direct interaction between recombinant emerin and lamin A molecules using biomolecular interaction analysis (BIA) and monoclonal antibodies. An emerin-lamin A interaction system may be related in function to the LAP2-lamin B system at the inner nuclear rim.     

Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.     

A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans

Mutations in the human LMNA gene underlie many laminopathic diseases, including Emery-Dreifuss muscular dystrophy (EDMD); however, a mechanistic link between the effect of mutations on lamin filament assembly and disease phenotypes has not been established. We studied the ΔK46 Caenorhabditis elegans lamin mutant, corresponding to EDMD-linked ΔK32 in human lamins A and C. Cryo-electron tomography of lamin ΔK46 filaments in vitro revealed alterations in the lateral assembly of dimeric head-to-tail polymers, which causes abnormal organization of tetrameric protofilaments. Green fluorescent protein (GFP):ΔK46 lamin expressed in C. elegans was found in nuclear aggregates in postembryonic stages along with LEM-2. GFP:ΔK46 also caused mislocalization of emerin away from the nuclear periphery, consistent with a decreased ability of purified emerin to associate with lamin ΔK46 filaments in vitro. GFP:ΔK46 animals had motility defects and muscle structure abnormalities. These results show that changes in lamin filament structure can translate into disease-like phenotypes via altering the localization of nuclear lamina proteins, and suggest a model for how the ΔK32 lamin mutation may cause EDMD in humans.     

Primary laminopathy fibroblasts display altered genome organization and apoptosis

A number of diseases associated with specific tissue degeneration and premature aging have mutations in the nuclear envelope proteins A-type lamins or emerin. Those diseases with A-type lamin mutation are inclusively termed laminopathies. Due to various hypothetical roles of nuclear envelope proteins in genome function we investigated whether alterations to normal genomic behaviour are apparent in cells with mutations in A-type lamins and emerin. Even though the distributions of these proteins in proliferating laminopathy fibroblasts appear normal, there is abnormal nuclear positioning of both chromosome 18 and 13 territories, from the nuclear periphery to the interior. This genomic organization mimics that found in normal nonproliferating quiescent or senescent cells. This finding is supported by distributions of modified pRb in the laminopathy cells. All laminopathy cell lines tested and an X-linked Emery-Dreifuss muscular dystrophy cell line also demonstrate increased incidences of apoptosis. The most extreme cases of apoptosis occur in cells derived from diseases with mutations in the tail region of the LMNA gene, such as Dunningan-type familial partial lipodystrophy and mandibuloacral dysplasia, and this correlates with a significant level of micronucleation in these cells.     

The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope

We have analyzed the interaction of soluble nuclear lamins with the nuclear envelope by microinjection of normal and mutated lamins into the cytoplasm of Xenopus laevis oocytes. Our results demonstrate that the conserved cysteine of the carboxy-terminal tetrapeptide Cys Ala/Ser Ile Met of lamins is essential for their association with the nuclear envelope. Removal of this sequence or replacement of the cysteine by serine resulted in Xenopus lamin L1 remaining in a soluble, non-envelope-associated state within the nucleus. Similar mutations of Xenopus lamin A resulted in only partial reduction of nuclear envelope association, indicating that lamin A contains additional signals that can partially compensate for the lack of the cysteine. Mammalian lamin C lacks this tetrapeptide and is not associated with the nuclear envelope in our experimental system. Cloning of the tetrapeptide Cys Ala Ile Met to the carboxy terminus of human lamin C resulted in lamin being found in a nuclear envelope-associated form in oocytes. Mutations at the amino terminus and in the alpha-helical region of lamin L1 revealed that the carboxy terminus mediates the association of lamins with the nuclear envelope; however, this alone is insufficient for maintenance of a stable association with the nuclear envelope.     

Aging and nuclear organization: lamins and progeria

The discoveries of at least eight human diseases arising from mutations in LMNA, which encodes the nuclear A-type lamins, have revealed the nuclear envelope as an organelle associated with a variety of fundamental cellular processes. The most recently discovered diseases associated with LMNA mutations are the premature aging disorders Hutchinson-Gilford progeria syndrome (HGPS) and atypical Werner's syndrome. The phenotypes of both HGPS patients and a mouse model of progeria suggest diverse compromised tissue functions leading to defects reminiscent of aging. Aspects of the diseases associated with disrupted nuclear envelope/lamin functions may be explained by decreased cellular proliferation, loss of tissue repair capability and a decline in the ability to maintain a differentiated state.     

Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene

Mutations in the LMNA gene encoding nuclear lamins A and C are responsible for seven inherited disorders affecting specific tissues. We have analyzed skin fibroblasts from a patient with type 1B limb-girdle muscular dystrophy and from her deceased newborn grandchild carrying, respectively, a heterozygous (+/mut) and a homozygous (mut/mut) nonsense Y259X mutation. In fibroblasts(+/mut), the presence of only 50% lamins A and C promotes no detectable abnormality, whereas in fibroblasts(mut/mut) the complete absence of lamins A and C leads to abnormally shaped nuclei with lobules in which none of the analyzed nuclear proteins were detected, i.e., B-type lamins, emerin, nesprin-1alpha, LAP2beta, and Nup153. These lobules perturb cell division as fibroblast(mut/mut) cultures with large proportions of cells with dysmorphic nuclei grow more slowly than controls and the cell proliferation normalizes when the number of these abnormally shaped nuclei declines. In all fibroblasts(mut/mut), nesprin-1alpha-like emerin exhibited aberrant localization in the endoplasmic reticulum. Transfection of wild-type lamin A or C cDNAs restored the correct localization of both emerin and nesprin-1alpha. These data demonstrate that lamin C, like lamin A, interacts in vivo directly with nesprin-1alpha and with emerin and that lamin A or C is sufficient for the correct anchorage of emerin and nesprin-1alpha at the nuclear envelope in human cells.     

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.     

Structural and physiological phenotypes of disease-linked lamin mutations in C. elegans

The nuclear lamina is a major structural element of the nucleus and is predominately composed of the intermediate filament lamin proteins. Missense mutations in the human lamins A/C cause a family of laminopathic diseases, with no known mechanistic link between the position of the mutation and the resulting disease phenotypes. The Caenorhabditis elegans lamin (Ce-lamin) is structurally and functionally homologous to human lamins, and recent advances have allowed detailed structural analysis of Ce-lamin filaments both in vitro and in vivo. Here, we studied the effect of laminopathic mutations on Ce-lamin filament assembly in vitro and the corresponding physiological phenotypes in animals. We focused on three disease-linked mutations, Q159K, T164P, and L535P, which have previously been shown to affect lamin structure and nuclear localization. Mutations prevented the proper assembly of Ce-lamin into filament and/or paracrystalline arrays. Disease-like phenotypes were observed in strains expressing low levels of these mutant lamins, including decreased fertility and motility coincident with muscle lesions. In addition, the Q159K- and T164P-expressing strains showed a reduced lifespan. Thus, different disease-linked mutations in Ce-lamin exhibit major effects in vivo and in vitro. Using C. elegans as a model system, a comprehensive analysis of the effects of specific lamin mutations from the level of in vitro filament assembly to the physiology of the organism will help uncover the mechanistic differences between these different lamin mutations.     

Structure of the globular tail of nuclear lamin

The nuclear lamins form a two-dimensional matrix that provides integrity to the cell nucleus and participates in nuclear activities. Mutations in the region of human LMNA encoding the carboxyl-terminal tail Lamin A/C are associated with forms of muscular dystrophy and familial partial lipodystrophy (FPLD). To help discriminate tissue-specific phenotypes, we have solved at 1.4-A resolution the three-dimensional crystal structure of the lamin A/C globular tail. The domain adopts a novel, all beta immunoglobulin-like fold. FPLD-associated mutations cluster within a small surface, whereas muscular dystrophy-associated mutations are distributed throughout the protein core and on its surface. These findings distinguish myopathy- and lipodystrophy-associated mutations and provide a structural framework for further testing hypotheses concerning lamin function.     

Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.     

Specific contribution of lamin A and lamin C in the development of laminopathies

Mutations in the lamin A/C gene are involved in multiple human disorders for which the pathophysiological mechanisms are partially understood. Conflicting results prevail regarding the organization of lamin A and C mutants within the nuclear envelope (NE) and on the interactions of each lamin to its counterpart. We over-expressed various lamin A and C mutants both independently and together in COS7 cells. When expressed alone, lamin A with cardiac/muscular disorder mutations forms abnormal aggregates inside the NE and not inside the nucleoplasm. Conversely, the equivalent lamin C organizes as intranucleoplasmic aggregates that never connect to the NE as opposed to wild type lamin C. Interestingly, the lamin C molecules present within these aggregates exhibit an abnormal increased mobility. When co-expressed, the complex formed by lamin A/C aggregates in the NE. Lamin A and C mutants for lipodystrophy behave similarly to the wild type. These findings reveal that lamins A and C may be differentially affected depending on the mutation. This results in multiple possible physiological consequences which likely contribute in the phenotypic variability of laminopathies. The inability of lamin C mutants to join the nuclear rim in the absence of lamin A is a potential pathophysiological mechanism for laminopathies.     

The nuclear envelope, muscular dystrophy and gene expression

Lamins and other nuclear envelope proteins organize nuclear architecture through structural attachments that vary dynamically during the cell cycle and cell differentiation. Genetic studies have now shown that people with mutations in either lamins A/C or emerin, a nuclear membrane protein, develop Emery-Dreifuss muscular dystrophy. A mouse model for this rare disease has been created by knocking out the gene that encodes lamin A/C. This article discusses these and other recent results in the wider context of nuclear envelope function, as a framework for thinking about the possible ways in which defects in nuclear envelope proteins can lead to disease.     

Expression of lamin A mutated in the carboxyl-terminal tail generates an aberrant nuclear phenotype similar to that observed in cells from patients with Dunnigan-type partial lipodystrophy and Emery-Dreifuss muscular dystrophy

Autosomal dominantly inherited missense mutations in lamins A and C cause familial partial lipodystrophy of the Dunnigan-type (FPLD), and myopathies including Emery-Dreifuss muscular dystrophy (EDMD). While mutations responsible for FPLD are restricted to the carboxyl-terminal tails, those responsible for EDMD are spread throughout the molecules. We observed here the same structural abnormalities in the nuclear envelope and chromatin of fibroblasts from patients with FPLD and EDMD, harboring missense mutations at codons 482 and 453, respectively. Similar nuclear alterations were generated in fibroblasts, myoblasts, and preadipocytes mouse cell lines overexpressing lamin A harboring either of these two mutations. A large variation in sensitivity to lamin A overexpression was observed among the three cell lines, which was correlated with their variable endogenous content in A-type lamins and emerin. The occurrence of nuclear abnormalities was reduced when lamin B1 was coexpressed with mutant lamin A, emphasizing the functional interaction of the two types of lamins. Transfected cells therefore develop similar phenotypes when expressing lamins mutated in the carboxyl-terminal tail at sites responsible for FPLD or EDMD.