Activation of the Keap1/Nrf2 stress response pathway in autophagic vacuolar myopathies.

Steve Duleh,Xianhong Wang,Marta Margeta,Allison Komirenko

doi:10.1186/s40478-016-0384-6

Steve Duleh, Xianhong Wang + Show 2 more

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https://doi.org/10.1186/s40478-016-0384-6

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Abstract

Nrf2 (nuclear factor [erythroid-derived 2]-like 2; the transcriptional master regulator of the antioxidant stress response) is regulated through interaction with its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), which under basal conditions targets Nrf2 for proteasomal degradation. Sequestosome 1 (SQSTM1)/p62–a multifunctional adapter protein that accumulates following autophagy inhibition and can serve as a diagnostic marker for human autophagic vacuolar myopathies (AVMs)–was recently shown to compete with Nrf2 for Keap1 binding, resulting in activation of the Nrf2 pathway. In this study, we used 55 human muscle biopsies divided into five groups [normal control, hydroxychloroquine- or colchicine-treated non-AVM control, hydroxychloroquine- or colchicine-induced toxic AVM, polymyositis, and inclusion body myositis (IBM)] to evaluate whether Keap1-SQSTM1 interaction led to increased Nrf2 signaling in human AVMs. In toxic AVMs and IBM, but not in control muscle groups or polymyositis, Keap1 antibody labeled sarcoplasmic protein aggregates that can be used as an alternate diagnostic marker for both AVM types; these Keap1-positive aggregates were co-labeled with the antibody against SQSTM1 but not with the antibody against autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). In human AVM muscle, sequestration of Keap1 into the SQSTM1-positive protein aggregates was accompanied by an increase in mRNA and protein levels of Nrf2 target genes; similarly, treatment of differentiated C2C12 myotubes with autophagy inhibitor chloroquine led to an increase in the nuclear Nrf2 protein level and an increase in expression of the Nrf2-regulated genes. Taken together, our findings demonstrate that Nrf2 signaling is upregulated in autophagic muscle disorders and raise the possibility that autophagy disruption in skeletal muscle leads to dysregulation of cellular redox homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0384-6) contains supplementary material, which is available to authorized users.

Highlights

Macroautophagy is a catabolic pathway that contributes to cellular homeostasis by mediating routine protein and organelle turnover through lysosomal degradation [1, 2]
Kelch-like ECH-associated protein 1 (Keap1) immunohistochemistry in toxic Autophagic vacuolar myopathy (AVM) To determine whether autophagy impairment alters Keap1 subcellular localization in the human skeletal muscle, immunohistochemistry for Keap1 was performed on Formalin-fixed paraffin-embedded (FFPE) tissue from human subjects with no detectable muscle disease, human subjects treated with colchicine or HCQ but no evidence of AVM, and human subjects with colchicine- or HCQ-induced AVM
Muscle samples from the toxic AVM group showed coarse Keap1-positive sarcoplasmic puncta that often localized to the area of myofibrillary disorganization and/or vacuolization in the fiber center (Fig. 1e-h); this coarsely punctate staining pattern was never observed in the normal muscle and was only rarely seen in the muscle from the drug-treated control subjects. [With lower antibody dilutions, the control muscle showed a checkerboard pattern of diffuse sarcoplasmic Keap1 staining, raising a possibility that Keap1 protein is expressed at different levels by fast and slow twitch muscle fibers (Additional file 2: Figure S1A); future work will be required to fully evaluate this possibility

Summary

Introduction

Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway that contributes to cellular homeostasis by mediating routine protein and organelle turnover through lysosomal degradation [1, 2]. Autophagic disruption is a hallmark of several inherited skeletal myopathies including X-linked myopathy with excessive autophagy and infantile autophagic vacuolar myopathy [5, 6]; these disorders are characterized by defects in lysosomal degradation that lead to secondary accumulation of autophagic vacuoles [5]. CQ and its derivative HCQ are thought to inhibit autophagy through lysosome alkalinization, which interferes with function of the pH-sensitive lysosomal enzymes [10]; in contrast, colchicine disrupts microtubule cytoskeleton, inhibiting autophagosome-lysosome fusion (which requires translocation of autophagosomes along microtubules) [9]. All AVMs clinically present with muscular weakness but only a minority of AVM muscle biopsies show muscle fiber degeneration (a classic feature of both inherited and toxic myopathies); the causal mechanism linking autophagy impairment with muscle weakness remains poorly understood

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