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.

Fig 1. The LMNA mutations cause perturbations of the A-type lamin Ig-fold tertiary structure.

(A) The ¹⁵N/¹H HSQC NMR spectrum of each mutant Ig-fold (left column) was superimposed onto that of the wild type Ig-fold in green (middle column) PDB 1IVT was used to generate a ribbon plot of the wild type Ig-fold showing the perturbed amino acids (yellow, Δδ_ppm ≥ 0.15 ppm) and the unperturbed backbone atoms (blue, Δδ_ppm ≤ 0.15 ppm) backbone (right column). Perturbation was determined by calculating the chemical shift difference for each backbone amide cross peak using the equation Δδ_ppm = ([Δδ(¹H_ppm)]² + [0.1 • Δδ(¹⁵N_ppm)]²) ^1/2. Since the ¹⁵N/¹H HSQC spectrum of the wild type was assigned (S3B Fig) and the spectra of the mutants were not assigned, the Δδ_ppm was calculated by comparing each backbone amide cross peak in the wild type spectrum (assigned) to all cross peaks present in the mutant spectrum. The smallest Δδ_ppm from this calculation was taken as the chemical shift perturbation value for that residue. In the ribbon plots, the mutated residues are shown in stick mode with their C atoms indicated by spheres. (B and C) Surface display of the Ig-fold domain showing common residues perturbed by G449V and W514R (B) and N456I and L489P (C). Amino acids not perturbed are shown in blue. Amino acid residues altered by the LMNA mutations are colored by black (G449V), purple (W514R), magenta (N456I) and red (L489P) and indicated by an arrow. Note that L489P is buried and not visible from the surface.

Fig 2. Mutant lamins mislocalize in Drosophila larval body wall muscles and have minimal dominant effects on nuclear stiffness.

(A) Immunohistochemical staining of Drosophila body wall muscles from transgenic larvae expressing full length wild type (WT) and mutant Lamin C. Phalloidin staining is represented by magenta, Lamin C by green, and DAPI by blue. (B) Normalized nuclear strain values for nuclei in Drosophila larval body wall muscle. Eleven nuclei from three different larvae were analyzed per genotype. Error bars represent standard error of the mean. Increased normalized nuclear strain values correspond to decreased nuclear stiffness. The ** indicates p ≤ 0.01 compared to the value obtained for muscles expressing wild type Lamin C.

Fig 3. Mutant lamins cause changes in Drosophila muscle gene expression and reductive stress.

(A) Venn diagram of the changes in gene expression caused by ΔN and G489V versus wild type Lamin C. (B) Alternative pathways known to cause activation of cellular detoxification genes. (C) Quantitation of oxidized glutathione (GSSG), reduced glutathione (GSH) and NADPH in Drosophila body wall muscles of larvae expressing wild type and mutant Lamin C. Analysis was performed on three independent biological samples. Error bars represent standard error of the mean. Statistical significance is indicated by * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001 when compared to values obtained for muscles expressing wild type Lamin C.

Fig 4. Mutant lamins cause enrichment of CncC/Nrf2 in myonuclei.

(A) Drosophila larval body wall muscles from transgenic larvae stained with phalloidin (magenta), DAPI (blue) and CncC (green). Increased intensity of staining was observed for all mutants, relative to wild type. (B) Human muscle biopsy tissues were stained with phalloidin (magenta), DAPI (blue) and antibodies to Nrf2 (green). Arrows indicate myonuclei enriched for staining with Nrf2 antibodies. Boxed areas are those shown enlarged below.

Fig 5. 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).