Pejvakin-Mediated Pexophagy Protects Auditory Hair Cells Against Noise-Induced Damage
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Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage Jean Defournya,b,c, Alain Aghaieb,c,d, Isabelle Perfettinia,b,c, Paul Avane,f, Sedigheh Delmaghania,b,c,1,2, and Christine Petita,b,c,d,g,1,2 aUnité de Génétique et Physiologie de l’Audition, Institut Pasteur, 75015 Paris, France; bUnité Mixte de Recherche en Santé (UMRS) 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), 75015 Paris, France; cComplexité du Vivant, Sorbonne Universités, Université Pierre et Marie Curie, Université Paris 06, 75005 Paris, France; dSyndrome de Usher et Autres Atteintes Rétino-Cochléaires, Institut de la Vision, 75012 Paris, France; eLaboratoire de Biophysique Sensorielle, Faculté de Médecine, Centre Jean Perrin, Université d’Auvergne, 63000 Clermont-Ferrand, France; fUMR 1107, INSERM, 63000 Clermont-Ferrand, France; and gCollège de France, 75005 Paris, France Contributed by Christine Petit, February 18, 2019 (sent for review January 3, 2019; reviewed by Karen B. Avraham and Neil Segil) Noise overexposure causes oxidative stress, leading to auditory oxidative stress in auditory hair cells and neurons. Pejvakin is a hair cell damage. Adaptive peroxisome proliferation involving pejva- member of the gasdermin protein family, which has six members kin, a peroxisome-associated protein from the gasdermin family, has in humans (gasdermins A–E and pejvakin), four of which (gas- been shown to protect against this harmful oxidative stress. How- dermins A, B, D, and E) trigger pyroptosis, a form of programmed ever, the role of pejvakin in peroxisome dynamics and homeostasis lytic cell death initiated by inflammasome activation (10). These remains unclear. Here we show that sound overstimulation induces gasdermins have an autoinhibited two-domain architecture, with an early and rapid selective autophagic degradation of peroxisomes an N-terminal pore-forming domain in the plasma membrane and (pexophagy) in auditory hair cells from wild-type, but not pejvakin- a C-terminal repressor domain, separated by a linker region − − deficient (Pjvk / ), mice. Noise overexposure triggers recruitment of (11–14). Their pyroptotic pore-forming activity results from the autophagosome-associated protein MAP1LC3B (LC3B; microtubule- inflammatory caspase cleavage at the linker domain, sepa- associated protein 1 light chain 3β) to peroxisomes in wild-type, but not rating the N-terminal pore-forming domain from the C- −/− Pjvk , mice. We also show that pejvakin–LC3B binding involves an terminal repressor domain. Pejvakin has a markedly differ- LC3-interacting region within the predicted chaperone domain of pej- ent structure than that of other gasdermins, with a divergent vakin. In transfected cells and in vivo transduced auditory hair cells, shorter C-terminal domain and no identifiable cleavable linker cysteine mutagenesis experiments demonstrated the requirement for domain (11, 15). both C328 and C343, the two cysteine residues closest to the C terminus Pejvakin is associated with peroxisomes and is required for the – of pejvakin, for reactive oxygen species-induced pejvakin LC3B interac- sound-induced proliferation of these organelles (1). However, its tion and pexophagy. The viral transduction of auditory hair cells from role in peroxisome dynamics, in peroxisome proliferation itself, or Pjvk−/− mice in vivo with both Pjvk and Lc3b cDNAs completely re- at other steps in peroxisome turnover, and the direct or indirect stored sound-induced pexophagy, fully prevented the development nature of its action, remain unknown. In this study, we addressed of oxidative stress, and resulted in normal levels of peroxisome pro- these issues. We first studied sound-induced peroxisome pro- − − liferation, whereas Pjvk cDNA alone yielded only a partial correction of liferation in both wild-type and Pjvk / mice. We then characterized the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify de- fective pexophagy as a cause of noise-induced hearing loss. They Significance suggest that pejvakin acts as a redox-activated pexophagy receptor/ adaptor, thereby identifying a previously unknown function of gas- Noise-induced hearing loss is a highly prevalent form of sen- dermin family proteins. sorineural hearing impairment affecting individuals of all ages. Noise exposure triggers a rapid increase in reactive oxygen pejvakin | noise-induced hearing loss | LC3B | pexophagy | intracochlear species (ROS) levels, causing oxidative damage to auditory hair viral transduction cells and resulting in hearing loss. Here we describe a novel mechanism of protection against noise-induced auditory hair cell damage. In response to sound exposure, pejvakin, a peroxisome- oise overexposure increases reactive oxygen species (ROS) associated protein, acts as an ROS sensor and recruits the auto- levels, causing oxidative damage to auditory hair cells and N phagy machinery to trigger the degradation of peroxisomes resulting in hearing loss (1, 2). Excessive ROS production can be (pexophagy) damaged by oxidative stress. We characterized the counteracted by the antioxidant activities of ROS-metabolizing pejvakin interactions involved in this process. We provide evi- organelles, mitochondria, and peroxisomes (3, 4). Diverse metabolic dence that pejvakin-mediated pexophagy plays a key role in pathways, including the β-oxidation of branched and very-long- controlling peroxisome proliferation and protecting auditory hair chain fatty acids and hydrogen peroxide (H O ) metabolism, oc- 2 2 cells against noise-induced damage. cur in peroxisomes. The number, size, shape, and molecular content of these organelles continually adapt to the changing needs of the Author contributions: P.A., S.D., and C.P. designed research; J.D., A.A., I.P., and S.D. per- cell and external conditions (5, 6). The balance between peroxisome formed research; J.D., A.A., and S.D. analyzed data; and J.D., S.D., and C.P. wrote biogenesis and degradation is crucial for redox cell homeostasis and the paper. may be dysregulated in peroxisomal biogenesis disorders (7). Per- Reviewers: K.B.A., Tel Aviv University; and N.S., Keck School of Medicine of the University oxisome biogenesis can occur de novo or through the proliferation of Southern California. of existing peroxisomes (5, 6), and damaged peroxisomes are de- The authors declare no conflict of interest. graded by selective autophagy (pexophagy) in yeast and plant vac- Published under the PNAS license. uoles and in mammalian lysosomes (8). 1S.D. and C.P. contributed equally to this work. We recently showed that the physiological response to noise 2To whom correspondence may be addressed. Email: [email protected] or overexposure includes peroxisome proliferation (1). We found [email protected]. that defects of pejvakin, underlying an autosomal recessive form This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of deafness in humans (DFNB59) (9), cause hypervulnerability 1073/pnas.1821844116/-/DCSupplemental. to sound in humans and mice (1) associated with high levels of Published online April 1, 2019. 8010–8017 | PNAS | April 16, 2019 | vol. 116 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1821844116 Downloaded by guest on September 29, 2021 theroleofpejvakininpexophagyandinprotectionoftheauditory 1.5 peroxisomes per IHC), 24% lower (24.8 ± 1.4 peroxisomes per system against noise-induced oxidative stress. IHC; P = 0.0002) at the end of the 1 h of exposure to loud broadband sound (5–40 kHz, 105 dB SPL), and 39% lower at 1 h Results after the end of sound exposure (20 ± 1.2 peroxisomes per IHC; Early Pexophagy Precedes the Proliferation of Peroxisomes Induced P < 0.0001) (Fig. 1A). Two hours later, the number of peroxisomes by Sound Overexposure. We studied peroxisome kinetics in response had started to increase, peaking at 48 h at a level 4.3 times that at to sound in the inner hair cells (IHCs), the genuine sensory cells of 1 h after the end of the stimulation (85.3 ± 4.2 peroxisomes per + + the cochlea, the auditory sensory organ, in wild-type (Pjvk / )and IHC; 2.6 times that before stimulation). It decreased slowly there- −/− + + Pjvk mice. Surprisingly, Pjvk / mice were found to have fewer after, reaching baseline values within 10 days (Fig. 1A). In contrast, peroxisomes per IHC than unexposed mice (mean ± SEM, 32.8 ± the number of peroxisomes remained unchanged at 1 h after the NEUROSCIENCE + + Fig. 1. Pejvakin promotes pexophagy in response to sound in auditory hair cells. (A) Changes in peroxisome number in P21 Pjvk / IHCs (n = 60; four mice per time point) after sound exposure (5–40 kHz, 105 dB SPL, 1 h). The arrows indicate the sound stimulation period (1 h). (B) Impaired early elimination and − − − − proliferation of peroxisomes in Pjvk / IHCs in response to sound (n = 60 IHCs; four mice per time point). (C) LC3B-mediated pexophagy is altered in Pjvk / IHCs. The number of coimmunolabeled LC3B/PMP70 spots increased at 1 h after sound exposure in Pjvk+/+ IHCs, but not in Pjvk−/− IHCs. (D) The number of LC3B/pejvakin PLA spots (in red) was higher at 1 h after sound exposure in Pjvk+/+ IHCs. No LC3B/pejvakin PLA spots were observed in Pjvk−/− IHCs. The bar + + − − + + − − charts quantify LC3B/PMP70 coimmunolocalization in IHCs from Pjvk / and Pjvk / mice (C) and LC3B/pejvakin PLA spots in Pjvk / and Pjvk / IHCs (D) before and at 1 h after sound exposure (n = 40 IHCs; four mice per genotype and condition). Data are mean ± SEM. ***P < 0.001; n.s., not significant, unpaired Student’s t test. (Scale bars: 5 μm.) Defourny et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 8011 Downloaded by guest on September 29, 2021 −/− end of sound exposure in Pjvk IHCs (mean ± SEM, 29.3 ± investigating whether mutations of its key residues, F215 and L218, 1.3 and 26.1 ± 1.2 peroxisomes per IHC before and after sound affected pejvakin binding to LC3B under H2O2-induced oxidative exposure, respectively; P = 0.08) and decreased gradually there- stress. After H2O2 treatment, the numbers of LC3B/pejvakin- after, by 56% (mean ± SEM, 12.8 ± 1.8; P < 0.0001)over48h coimmunolabeled spots and LC3B/pejvakin PLA spots increased (Fig.