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Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling.

ABSTRACT: Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

SUBMITTER: Zwerger M

PROVIDER: S-EPMC3658163 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling.

Zwerger Monika M Jaalouk Diana E DE Lombardi Maria L ML Isermann Philipp P Mauermann Monika M Dialynas George G Herrmann Harald H Wallrath Lori L LL Lammerding Jan J

Human molecular genetics 20130219 12

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, ...[more]

PMID: 23427149

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Project description:Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis.

Project description:Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus and regulate gene expression. Drosophila melanogaster models of skeletal muscle laminopathies were developed to investigate the pathological defects caused by mutant lamins and identify potential therapeutic targets. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization and up-regulation of the autophagy cargo receptor p62. We hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMP-activated protein kinase (AMPK)/4E-binding protein 1 (4E-BP1)/autophagy/proteostatic pathways. Ribosomal protein S6K (S6K) messenger RNA (mRNA) levels were increased and AMPKα and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKα and down-stream targets 4E-BP, Forkhead box transcription factors O (Foxo) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy.

Dataset Information

Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling.

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Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling.

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