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Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway.

Dialynas G, Shrestha OK, Ponce JM, Zwerger M, Thiemann DA, Young GH, Moore SA, Yu L, Lammerding J, Wallrath LL - PLoS Genet. (2015)

Bottom Line: 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.Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Mutant lamins cause increased levels of p62/SQSTM1 in Drosophila muscles and human muscle biopsy tissues.(A) Drosophila larval body wall muscles from transgenic larvae were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/Ref(2)P (green). Increased intensity of staining and the number of cytoplasmic foci were greater in muscle expressing mutant lamins compared to wild type. (B) Human muscle biopsy tissues were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/SQSTM1 (green). Arrows indicate muscle fibers with enhanced staining with anti-p62/SQSTM1 antibodies. Boxed areas are those shown enlarged below. (C) Unified model to explain the activation of cellular detoxification genes due to mutant lamins. Mutant lamins have altered tertiary structures that promote cytoplasmic aggregation. In addition, other nuclear envelope proteins such as LEM domain proteins and nuclear pore proteins accumulate in the cytoplasm via unknown mechanisms [9] (green cloud structures). These aggregates cause elevated levels of the autophagy adaptor protein p62/SQSTM1 (orange circles), which in turn binds Keap-1 (grey oval). This competitive binding allows Nrf2 (light blue oval) to translocate into the nucleus, bind to a partner protein (dark blue oval), and activate target genes possessing anti-oxidant response elements (AREs).
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pgen.1005231.g005: Mutant lamins cause increased levels of p62/SQSTM1 in Drosophila muscles and human muscle biopsy tissues.(A) Drosophila larval body wall muscles from transgenic larvae were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/Ref(2)P (green). Increased intensity of staining and the number of cytoplasmic foci were greater in muscle expressing mutant lamins compared to wild type. (B) Human muscle biopsy tissues were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/SQSTM1 (green). Arrows indicate muscle fibers with enhanced staining with anti-p62/SQSTM1 antibodies. Boxed areas are those shown enlarged below. (C) Unified model to explain the activation of cellular detoxification genes due to mutant lamins. Mutant lamins have altered tertiary structures that promote cytoplasmic aggregation. In addition, other nuclear envelope proteins such as LEM domain proteins and nuclear pore proteins accumulate in the cytoplasm via unknown mechanisms [9] (green cloud structures). These aggregates cause elevated levels of the autophagy adaptor protein p62/SQSTM1 (orange circles), which in turn binds Keap-1 (grey oval). This competitive binding allows Nrf2 (light blue oval) to translocate into the nucleus, bind to a partner protein (dark blue oval), and activate target genes possessing anti-oxidant response elements (AREs).

Mentions: In mammalian systems Nrf2 and p62/SQSTM1 are co-regulated [21]. Given our findings of reductive stress and Nrf2 myonuclei enrichment, we hypothesized that levels of the autophagy cargo protein p62/SQSTM1 would be elevated in the muscles expressing mutant lamins relative to controls. To test this hypothesis, we stained Drosophila larval body wall muscles with antibodies to Drosophila p62/Ref(2)P, a homologue of human p62/SQSTM1 [26]. Muscles expressing wild type Lamin C showed hardly any staining for p62/Ref(2)P (Fig 5A). In contrast, muscles expressing mutant lamins showed increased cytoplasmic foci of staining (Fig 5A). The elevated levels of p62/Ref(2)P were validated by western analysis of protein extract from larval body wall muscles (S6 Fig). Thus, mutant lamins cause elevated levels of cytoplasmic p62/Ref(2)P, which is consistent with the increased cytoplasmic aggregation of mutant lamins (Fig 2A).


Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway.

Dialynas G, Shrestha OK, Ponce JM, Zwerger M, Thiemann DA, Young GH, Moore SA, Yu L, Lammerding J, Wallrath LL - PLoS Genet. (2015)

Mutant lamins cause increased levels of p62/SQSTM1 in Drosophila muscles and human muscle biopsy tissues.(A) Drosophila larval body wall muscles from transgenic larvae were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/Ref(2)P (green). Increased intensity of staining and the number of cytoplasmic foci were greater in muscle expressing mutant lamins compared to wild type. (B) Human muscle biopsy tissues were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/SQSTM1 (green). Arrows indicate muscle fibers with enhanced staining with anti-p62/SQSTM1 antibodies. Boxed areas are those shown enlarged below. (C) Unified model to explain the activation of cellular detoxification genes due to mutant lamins. Mutant lamins have altered tertiary structures that promote cytoplasmic aggregation. In addition, other nuclear envelope proteins such as LEM domain proteins and nuclear pore proteins accumulate in the cytoplasm via unknown mechanisms [9] (green cloud structures). These aggregates cause elevated levels of the autophagy adaptor protein p62/SQSTM1 (orange circles), which in turn binds Keap-1 (grey oval). This competitive binding allows Nrf2 (light blue oval) to translocate into the nucleus, bind to a partner protein (dark blue oval), and activate target genes possessing anti-oxidant response elements (AREs).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440730&req=5

pgen.1005231.g005: Mutant lamins cause increased levels of p62/SQSTM1 in Drosophila muscles and human muscle biopsy tissues.(A) Drosophila larval body wall muscles from transgenic larvae were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/Ref(2)P (green). Increased intensity of staining and the number of cytoplasmic foci were greater in muscle expressing mutant lamins compared to wild type. (B) Human muscle biopsy tissues were stained with phalloidin (magenta), DAPI (blue) and antibodies to p62/SQSTM1 (green). Arrows indicate muscle fibers with enhanced staining with anti-p62/SQSTM1 antibodies. Boxed areas are those shown enlarged below. (C) Unified model to explain the activation of cellular detoxification genes due to mutant lamins. Mutant lamins have altered tertiary structures that promote cytoplasmic aggregation. In addition, other nuclear envelope proteins such as LEM domain proteins and nuclear pore proteins accumulate in the cytoplasm via unknown mechanisms [9] (green cloud structures). These aggregates cause elevated levels of the autophagy adaptor protein p62/SQSTM1 (orange circles), which in turn binds Keap-1 (grey oval). This competitive binding allows Nrf2 (light blue oval) to translocate into the nucleus, bind to a partner protein (dark blue oval), and activate target genes possessing anti-oxidant response elements (AREs).
Mentions: In mammalian systems Nrf2 and p62/SQSTM1 are co-regulated [21]. Given our findings of reductive stress and Nrf2 myonuclei enrichment, we hypothesized that levels of the autophagy cargo protein p62/SQSTM1 would be elevated in the muscles expressing mutant lamins relative to controls. To test this hypothesis, we stained Drosophila larval body wall muscles with antibodies to Drosophila p62/Ref(2)P, a homologue of human p62/SQSTM1 [26]. Muscles expressing wild type Lamin C showed hardly any staining for p62/Ref(2)P (Fig 5A). In contrast, muscles expressing mutant lamins showed increased cytoplasmic foci of staining (Fig 5A). The elevated levels of p62/Ref(2)P were validated by western analysis of protein extract from larval body wall muscles (S6 Fig). Thus, mutant lamins cause elevated levels of cytoplasmic p62/Ref(2)P, which is consistent with the increased cytoplasmic aggregation of mutant lamins (Fig 2A).

Bottom Line: 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.Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus