"Laminopathies": a wide spectrum of human diseases

Mutations in genes encoding the intermediate filament nuclear lamins and associated proteins cause a wide spectrum of diseases sometimes called "laminopathies." Diseases caused by mutations in LMNA encoding A-type lamins include autosomal dominant Emery-Dreifuss muscular dystrophy and related myopathies, Dunnigan-type familial partial lipodystrophy, Charcot-Marie-Tooth disease type 2B1 and developmental and accelerated aging disorders. Duplication in LMNB1 encoding lamin B1 causes autosomal dominant leukodystrophy and mutations in LMNB2 encoding lamin B2 are associated with acquired partial lipodystrophy. Disorders caused by mutations in genes encoding lamin-associated integral inner nuclear membrane proteins include X-linked Emery-Dreifuss muscular dystrophy, sclerosing bone dysplasias, HEM/Greenberg skeletal dysplasia and Pelger-Huet anomaly. While mutations and clinical phenotypes of "laminopathies" have been carefully described, data explaining pathogenic mechanisms are only emerging. Future investigations will likely identify new "laminopathies" and a combination of basic and clinical research will lead to a better understanding of pathophysiology and the development of therapies.     

Thematic review series: Adipocyte Biology. Lipodystrophies: windows on adipose biology and metabolism

The lipodystrophies are characterized by loss of adipose tissue in some anatomical sites, frequently with fat accumulation in nonatrophic depots and ectopic sites such as liver and muscle. Molecularly characterized forms include Dunnigan-type familial partial lipodystrophy (FPLD), partial lipodystrophy with mandibuloacral dysplasia (MAD), Berardinelli-Seip congenital generalized lipodystrophy (CGL), and some cases with Barraquer-Simons acquired partial lipodystrophy (APL). The associated mutant gene products include 1) nuclear lamin A in FPLD type 2 and MAD type A; 2) nuclear lamin B2 in APL; 3) nuclear hormone receptor peroxisome proliferator-activated receptor gamma in FPLD type 3; 4) lipid biosynthetic enzyme 1-acylglycerol-3-phosphate O-acyltransferase 2 in CGL type 1; 5) integral endoplasmic reticulum membrane protein seipin in CGL type 2; and 6) metalloproteinase ZMPSTE24 in MAD type B. An unresolved question is whether metabolic disturbances are secondary to adipose repartitioning or result from a direct effect of the mutant gene product. Careful analysis of clinical, biochemical, and imaging phenotypes, using an approach called "phenomics," reveals differences between genetically stratified subtypes that can be used to guide basic experiments and to improve our understanding of common clinical entities, such as metabolic syndrome or the partial lipodystrophy syndrome associated with human immunodeficiency virus infection.     

The nuclear lamina and inherited disease

Inherited disorders of the nuclear lamina present some of the most intriguing puzzles in cell biology. Mutations in lamin A and lamin C – nuclear intermediate filament proteins that are expressed in nearly all somatic cells – cause tissue-specific diseases that affect striated muscle, adipose tissue and peripheral nerve or skeletal development. Recent studies provide clues about how different mutations in these proteins cause either muscle disease or partial lipodystrophy. Although the precise pathogenic mechanisms are currently unknown, the involvement of lamins in several different disorders shows that research on the nuclear lamina will shed light on common human pathologies.     

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.     

A Highlights from MBoC Selection: Lamin B1 protein is required for dendrite development in primary mouse cortical neurons

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution.     

Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in HeLa cells

Patients with the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) or familial partial lipodystrophy (FPLD) have specific mutations in the lamin A gene. Three such point mutations, G465D (FPLD), R482L, (FPLD), or R527P (EDMD), were introduced by site-specific mutagenesis in the C-terminal tail domain of a FLAG-tagged full-length lamin A construct. HeLa cells were transfected with mutant and wild-type constructs. Lamin A accumulated in nuclear aggregates and the number of cells with aggregates increased with time after transfection. At 72 h post transfection 60-80% of cells transfected with the mutant lamin A constructs had aggregates, while only 35% of the cells transfected with wild-type lamin A revealed aggregates. Mutant transfected cells expressed 10-24x, and wild-type transfected cells 20x, the normal levels of lamin A. Lamins C, B1 and B2, Nup153, LAP2, and emerin were recruited into aggregates, resulting in a decrease of these proteins at the nuclear rim. Aggregates were also characterized by electron microscopy and found to be preferentially associated with the inner nuclear membrane. Aggregates from mutant constructs were larger than those formed by the wild-type constructs, both in immunofluorescence and electron microscopy. The combined results suggest that aggregate formation is in part due to overexpression, but that there are also mutant-specific effects.     

Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.     

Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.     

In silico investigation of molecular mechanism of laminopathy caused by a point mutation (R482W) in lamin A/C protein

Lamin A/C proteins are the major components of a thin proteinaceous filamentous meshwork, the lamina, that underlies the inner nuclear membrane. A few specific mutations in the lamin A/C gene cause a disease with remarkably different clinical features: FPLD, or familial partial lipodystrophy (Dunnigan-type), which mainly affects adipose tissue. Lamin A/C mutant R482W is the key variant that causes FPLD. Biomolecular interaction and molecular dynamics (MD) simulation analysis were performed to understand dynamic behavior of native and mutant structures at atomic level. Mutant lamin A/C (R482W) showed more interaction with its biological partners due to its expansion of interaction surface and flexible nature of binding residues than native lamin A/C. MD simulation clearly indicates that the flexibility of interacting residues of mutant are mainly due to less involvement in formation of inter and intramolecular hydrogen bonds. Our analysis of native and Mutant lamin A/C clearly shows that the structural and functional consequences of the mutation R482W causes FPLD. Because of the pivotal role of lamin A/C in maintaining dynamics of nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.     

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (?607–656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.     

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.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery–Dreifuss muscular dystrophy fibroblasts

Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2α was also mislocalized in X-EDMD fibroblasts.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery-Dreifuss muscular dystrophy fibroblasts

Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2alpha was also mislocalized in X-EDMD fibroblasts.     

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.     

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.     

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.