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.

How do mutations in lamins A and C cause disease?

Mutations in lamins A and C, nuclear intermediate-filament proteins in nearly all somatic cells, cause a variety of diseases that primarily affect striated muscle, adipocytes, or peripheral nerves or cause features of premature aging. Two new studies (see the related articles beginning on pages 357 and 370) use lamin A/C-deficient mice, which develop striated muscle disease, as a model to investigate pathogenic mechanisms. These reports provide evidence for a stepwise process in which mechanically stressed cells first develop chromatin and nuclear envelope damage and then develop secondary alterations in the transcriptional activation of genes in adaptive and protective pathways.

Expression of a mutant lamin A that causes Emery-Dreifuss muscular dystrophy inhibits in vitro differentiation of C2C12 myoblasts.

Autosomal dominantly inherited missense mutations in lamins A and C cause several tissue-specific diseases, including Emery-Dreifuss muscular dystrophy (EDMD) and Dunnigan-type familial partial lipodystrophy (FPLD). Here we analyze myoblast-to-myotube differentiation in C2C12 clones overexpressing lamin A mutated at arginine 453 (R453W), one of the most frequent mutations in EDMD. In contrast with clones expressing wild-type lamin A, these clones differentiate poorly or not at all, do not exit the cell cycle properly, and are extensively committed to apoptosis. These disorders are correlated with low levels of expression of transcription factor myogenin and with the persistence of a large pool of hyperphosphorylated retinoblastoma protein. Since clones mutated at arginine 482 (a site responsible for FPLD) differentiate normally, we conclude that C2C12 clones expressing R453W-mutated lamin A represent a good cellular model to study the pathophysiology of EDMD. Our hypothesis is that lamin A mutated at arginine 453 fails to build a functional scaffold and/or to maintain the chromatin compartmentation required for differentiation of myoblasts into myocytes.

Nuclear envelope, nuclear lamina, and inherited disease.

The nuclear envelope is composed of the nuclear membranes, nuclear lamina, and nuclear pore complexes. In recent years, mutations in nuclear-envelope proteins have been shown to cause a surprisingly wide array of inherited diseases. While the mutant proteins are generally expressed in most or all differentiated somatic cells, many mutations cause fairly tissue-specific disorders. Perhaps the most dramatic case is that of mutations in A-type lamins, intermediate filament proteins associated with the inner nuclear membrane. Different mutations in the same lamin proteins have been shown to cause striated muscle diseases, partial lipodystrophy syndromes, a peripheral neuropathy, and disorders with features of severe premature aging. In this review, we summarize fundamental aspects of nuclear envelope structure and function, the inherited diseases caused by mutations in lamins and other nuclear envelope proteins, and possible pathogenic mechanisms.

The Ig-like structure of the C-terminal domain of lamin A/C, mutated in muscular dystrophies, cardiomyopathy, and partial lipodystrophy.

Lamins are nuclear intermediate filaments that, together with lamin-associated proteins, maintain nuclear shape and provide a structural support for chromosomes and replicating DNA. We have determined the solution structure of the human lamin A/C C-terminal globular domain which contains specific mutations causing four different heritable diseases. This domain encompasses residues 430-545 and adopts an Ig-like fold of type s. We have also characterized by NMR and circular dichroism the structure and thermostability of three mutants, R453W and R482W/Q, corresponding to "hot spots" causing Emery-Dreifuss muscular dystrophy and Dunnigan-type lipodystrophy, respectively. Our structure determination and mutant analyses clearly show that the consequences of the mutations causing muscle-specific diseases or lipodystrophy are different at the molecular level.

Antinuclear antibodies specific for primary biliary cirrhosis.

The serological hallmark of primary biliary cirrhosis (PBC) is the presence of antimitochondrial antibodies. However, antinuclear antibodies (ANA) are also detectable in approximately 50% of subjects with PBC. Most clinical laboratories use indirect immunofluorescence microscopy to detect ANA and two labeling patterns that predominate in PBC are 'punctate nuclear rim' and 'multiple nuclear dots.' Work over the past several years has shown that antibodies giving these patterns most often recognize nuclear pore membrane protein gp210 and nuclear body protein sp100, respectively. These ANA are highly specific for PBC and detected in approximately 25% of patients. Less frequently, ANA apparently unique to PBC recognize other proteins of the nuclear envelope and nuclear bodies. While antibodies against gp210, sp100 and some other nuclear proteins are very specific to PBC and may therefore be useful diagnostic markers, their connection to pathogenesis remains to be elucidated.

Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway.

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.

The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers.

Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.

Autoimmune cytopenias associated with autoantibodies to nuclear envelope polypeptides.

A subset of anti-nuclear autoantibodies (ANA) are directed against nuclear envelope (NE) polypeptides and display by indirect immunofluorescence (IIF) a ring-like fluorescent pattern. We report herein 19 patients with autoimmune cytopenias associated with antibodies (Abs) to NE polypeptides. Anti-NE specificity was determined by immunoblot, using NE preparations and purified lamina fractions. Eleven sera reacted with lamin B(1), and two reacted with both lamin B(1) and an unidentified 150-kDa protein (p150). One serum reacted with only p150. Four sera reacted with lamins A and C, and one reacted with and an unidentified 52-kDa NE polypeptide (p52). Autoimmune cytopenias included hemolytic anemia (7 cases), thrombocytopenia (13 cases), and neutropenia (6 cases). Five patients had 2 (3 cases) or 3 (2 cases) different cytopenias. Antiphospholipid antibodies (APLA) were detected in 14 patients, 2 of whom experienced thromboembolic events. A liver disorder was present in 7 patients. Systemic lupus erythematosus and lupus-like syndrome were diagnosed in 11 and 2 patients, respectively. Cytopenias responded to steroids alone (13 patients), or together with intravenous immunoglobulins (2 patients), or cyclophosphamide (2 patients). Two patients did not require treatment. Our results suggest that anti-NE Abs need to be sought for in patients with peripheral cytopenias, particularly when they are associated with APLA and/or liver disorders. Their detection strongly suggests an autoimmune process. Such cytopenias are often manifestations of a lupus or lupus-like disease and are responsive to steroids.

The carboxyl-terminal region common to lamins A and C contains a DNA binding domain.

Lamins A and C are intermediate filament proteins which polymerize into the nucleus to form the nuclear lamina network. The lamina is apposed to the inner nuclear membrane and functions in tethering chromatin to the nuclear envelope and in maintaining nuclear shape. We have recently characterized a globular domain that adopts an immunoglobulin fold in the carboxyl-terminal tail common to lamins A and C. Using an electrophoretic mobility shift assay (EMSA), we show that a peptide containing this domain interacts in vitro with DNA after dimerization through a disulfide bond, but does not interact with the core particle or the dinucleosome. The covalent dimer binds a 30-40 bp DNA fragment with a micromolar affinity and no sequence specificity. Using nuclear magnetic resonance (NMR) and an EMSA, we observed that two peptide regions participate in the DNA binding: the unstructured amino-terminal part containing the nuclear localization signal and a large positively charged region centered around amino acid R482 at the surface of the immunoglobulin-like domain. Mutations R482Q and -W, which are responsible for Dunnigan-type partial lipodystrophy, lower the affinity of the peptide for DNA. We conclude that the carboxyl-terminal end of lamins A and C binds DNA and suggest that alterations in lamin-DNA interactions may play a role in the pathophysiology of some lamin-linked diseases.

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.