Myopathic Lamin Mutations Cause Reductive Stress and Activate the Nrf2/Keap-1 Pathway

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.

Increasing autophagy and blocking Nrf2 suppress laminopathy‐induced age‐dependent cardiac dysfunction and shortened lifespan

“…Mutations in LMNA that give rise to Lamin A/C R190W are associated with progressive cardiac defect (including conduction defects) and reduced cardiac performance (Arbustini et al., 2002; Heller et al., 2017; Hermida‐Prieto et al., 2004). Mutations in LMNA that give rise to Lamin A/C G449V cause congenital muscular dystrophy, which is characterized by skeletal muscle defects in childhood and age‐dependent dilated cardiomyopathy (Dialynas et al., 2015). Cardiac‐specific expression of Drosophila LamC was obtained using the Gal4/UAS system with a Hand‐Gal4 driver (Han & Olson, 2005; Melkani et al., 2013) .…”

“…Nrf2 and its cytoplasmic binding partner Keap1 function in cellular detoxification and are conserved in Drosophila (Deng & Kerppola, 2013, 2014). In a Drosophila model of noncardiac muscle laminopathies, Cap‐and‐collar C (CncC) [the orthologue of mammalian nuclear erythroid 2‐related factor 2 (Nrf2)] redox transcriptional regulator (Deng & Kerppola, 2013, 2014) accumulated in myonuclei and activated cellular detoxification genes (Dialynas et al., 2015). To determine whether CncC accumulated in this cardiac model of laminopathies, hearts expressing wild‐type and mutant LamC were stained with antibody to CncC (Deng & Kerppola, 2013, 2014).…”

“…Translocation of Nrf2 (CncC) into the nucleus in the Drosophila model of noncardiac muscle laminopathies was driven by cytoplasmic protein aggregation (Dialynas et al., 2015). Abundant protein aggregates increased levels of the mammalian autophagy cargo receptor p62 and the Drosophila orthologue Ref(2)P (Nezis et al., 2008).…”

“…Abundant protein aggregates increased levels of the mammalian autophagy cargo receptor p62 and the Drosophila orthologue Ref(2)P (Nezis et al., 2008). The p62 [Ref(2)P] protein binds Keap1, sequestering it from Nrf2 (CncC) in the cytoplasm, thereby allowing Nrf2 (CncC) to translocate into the nucleus (Dialynas et al., 2015; Jain et al., 2010). Antibodies showed elevated levels of Ref(2)P in protein extracts from hearts expressing mutant LamC via western and antibody dot blots (Figure 4b–d) as well as in immunostained tissues relative to hearts expressing wild‐type LamC (Figure 4e, red foci) Thus, cardiac‐specific expression of mutant LamC elevated levels of Ref(2)P, promoting CncC nuclear translocation.…”

“…Furthermore, studies on the mouse models demonstrated that the cardiac defects and shortened lifespan can be partially reversed by treatment with rapamycin and temsirolimus (a derivative of rapamycin) (Choi et al., 2012; Ramos et al., 2012). Studies in Drosophila demonstrated that mutant lamins, modeled after those that cause human disease, lead to cytoplasmic aggregation of nuclear envelope proteins and loss of redox homeostasis in larval body wall muscles (Dialynas et al., 2012, 2015). Consistent with these findings, human muscle biopsy tissues showed both cytoplasmic aggregation of nuclear envelope (NE) proteins and activation of the Nrf2/Keap‐1 signaling pathway.…”