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• W2962912382 abstract "Article19 July 2019free access Proteostasis is essential during cochlear development for neuron survival and hair cell polarity Stephen Freeman Corresponding Author [email protected] orcid.org/0000-0003-2151-2550 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Susana Mateo Sánchez GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Ronald Pouyo GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Pierre-Bernard Van Lerberghe GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Kevin Hanon GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Nicolas Thelen GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Marc Thiry GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Giovanni Morelli GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium UHasselt, BIOMED, Hasselt, Belgium Search for more papers by this author Laura Van Hees GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Sophie Laguesse GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Alain Chariot GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium GIGA-Molecular Biology of Diseases, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wavre, Belgium Search for more papers by this author Laurent Nguyen GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Laurence Delacroix Corresponding Author [email protected] orcid.org/0000-0003-3440-5359 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Brigitte Malgrange Corresponding Author [email protected] orcid.org/0000-0002-8957-2528 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Stephen Freeman Corresponding Author [email protected] orcid.org/0000-0003-2151-2550 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Susana Mateo Sánchez GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Ronald Pouyo GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Pierre-Bernard Van Lerberghe GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Kevin Hanon GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Nicolas Thelen GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Marc Thiry GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Giovanni Morelli GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium UHasselt, BIOMED, Hasselt, Belgium Search for more papers by this author Laura Van Hees GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Sophie Laguesse GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Alain Chariot GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium GIGA-Molecular Biology of Diseases, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wavre, Belgium Search for more papers by this author Laurent Nguyen GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Laurence Delacroix Corresponding Author [email protected] orcid.org/0000-0003-3440-5359 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Brigitte Malgrange Corresponding Author [email protected] orcid.org/0000-0002-8957-2528 GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium Search for more papers by this author Author Information Stephen Freeman *,1,‡, Susana Mateo Sánchez1,‡, Ronald Pouyo1, Pierre-Bernard Van Lerberghe1, Kevin Hanon1, Nicolas Thelen1, Marc Thiry1, Giovanni Morelli1,2, Laura Van Hees1, Sophie Laguesse1, Alain Chariot1,3,4, Laurent Nguyen1, Laurence Delacroix *,1,‡ and Brigitte Malgrange *,1,‡ 1GIGA-Neurosciences, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium 2UHasselt, BIOMED, Hasselt, Belgium 3GIGA-Molecular Biology of Diseases, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), C.H.U. Sart Tilman, University of Liège, Liège, Belgium 4Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wavre, Belgium ‡These authors contributed equally to this work as first, second authors ‡These authors contributed equally to this work as last authors *Corresponding author. Tel: +32 4 3662178; E-mail: [email protected] *Corresponding author. Tel: +32 4 3662178; E-mail: [email protected] *Corresponding author. Tel: +32 4 3665905; E-mail: [email protected] EMBO Rep (2019)20:e47097https://doi.org/10.15252/embr.201847097 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components. Synopsis Loss of the Elongator subunit Elp3 in the inner ear results in enhanced protein misfolding and aggregation. Impaired proteostasis negatively affects neuron survival and sensory cell polarity and results in deafness. Aggresome-like structures are found in Elongator-deficient cells. Embryonic spiral ganglion neurons (SGNs) depend on Elongator and proteostasis for survival. Cochlear hair cells (HCs) depend on Elongator and proteostasis for appropriate localization of the kinocilium and stereociliary bundle. Accumulation of misfolded protein aggregates in HEK293T and cochlear HCs reduces microtubular transport and affects the distribution of LGN polarity protein. Introduction Sensorineural hearing loss, affecting millions of people worldwide, results from damage to the cochlear mechanosensory cells—i.e. the so-called hair cells (HCs)—or to their innervating spiral ganglion neurons (SGNs). There is increasing evidence that protein misfolding and aggregation are involved in hearing loss caused by environmental factors such as exposure to noise and ototoxic drugs 1. Misfolded proteins are detrimental to cellular homeostasis because of their propensity to self-aggregate and aberrantly interact with other cellular components, disrupting normal cellular processes. Protein homeostasis (herein referred to as proteostasis) is reliant upon chaperones that assist protein folding or re-folding, and on their principal degradation systems, i.e. the ubiquitin–proteasome system and autophagy 2. Polyubiquitinated proteins that fail to be degraded by the proteasome are transported along microtubules towards the microtubule-organizing centre (MTOC), where they gather to form a cytoprotective structure called an aggresome. Under pathological stress, these protein quality control systems can be overwhelmed and the accumulation of misfolded proteins can lead to cell death 3. The ability to maintain proteostasis declines with age, and numerous age-related diseases have been linked to proteostatic disruption, including age-related hearing loss 4. However, little is known about the importance of managing the proteome during the development of the auditory portion of the inner ear. Most of the cells composing the cochlea derive from the otic placode, which consists of a thickened region of neuroectoderm. The SGNs are born early during development and start extending their peripheral processes towards the sensory epithelium (the organ of Corti, OC) before their target HCs have differentiated 5. As the differentiation of HCs proceeds, these cells acquire dual levels of planar polarity, both of which are crucial for auditory perception. Each HC develops a highly polarized mechanosensitive organelle at its apical surface to detect sound signals. This organelle is a v-shaped bundle of actin-rich microvilli (named stereocilia) that emerges in close association with the HC's specialized primary cilium (named the kinocilium). In addition to this cell-intrinsic planar polarity, all HCs are co-ordinately oriented in the plane of the epithelium—along the medio-lateral axis of the OC. This is referred to as “tissue planar polarity” and is most obviously indicated by the vertex of the stereociliary bundles pointing towards the lateral side of the epithelium. Both levels of polarity are dependent upon the asymmetric distribution of specific proteins in opposing medio-lateral domains 6. Tissue planar polarity is governed by the so-called core planar cell polarity (PCP) proteins, which include Van Gogh-like (Vangl1 and Vangl2), Frizzled (Fz3 and Fz6) and Dishevelled 7-9. Cell-intrinsic planar polarity is controlled by the asymmetric enrichment of GTP-binding protein alpha-I subunit 3 (Gαi3), G-protein-signalling modulator 2 (GPSM2 or LGN), mammalian inscuteable (mInsc) and the atypical protein kinase C zeta (aPKC). These proteins have been shown to regulate mitotic spindle orientation in various tissues and organisms and are of crucial importance to centriole positioning from flies to mammals 10, 11. In the cochlear epithelium, these polarity proteins act to define the position of the kinocilium and the shape of the stereociliary bundle 12, which are crucial for normal audition 13. Elongator is a highly conserved complex composed of duplicate copies of six subunits (Elp1-Elp6). Elp3 is the enzymatic core containing a lysine acetyltransferase motif and a radical S-adenosylmethionine (SAM) domain 14, but all subunits are required to serve the major function of the complex, which is to control translational efficiency via its regulation of tRNA modifications 15. Elongator is the primary component of an enzymatic cascade that facilitates the addition of 5-methoxycarbonylmethyl and 5-carbamoylmethyl (mcm5 and ncm5, respectively) groups to the wobble uridine (U34) in the anticodon of 11 different tRNAs 16. These chemical modifications are essential to normal proteostasis, as they ensure high fidelity and speed of protein translation, and thus minimize the occurrence of protein misfolding 17, 18. In humans, mutations or genetic variations of genes encoding the various Elongator subunits have been linked to neurological disorders 19. In the present study, we investigated the importance of proteostasis during cochlear development using conditional Elp3 knockout mice (Elp3cKO) and chemical modulation of proteostasis. We found that protein misfolding and aggregation result in severe hearing loss by inducing apoptosis of SGNs and affecting the establishment of HC intrinsic planar polarity. We show that the accumulation of protein aggregates at the apical surface of HCs interferes with vesicular transport along microtubules and alters the distribution of the proteins that control HC intrinsic planar polarity. Reducing the load of aggregates, through chemical chaperone treatment, restores HC intrinsic planar polarity although it is not sufficient to improve SGN survival. These results shed light on a new role for proteostasis during cochlear development, which is essential for normal hearing. Results Elp3 expression in the cochlea is essential for hearing We analysed the temporal and spatial cochlear expression pattern of Elp3 during embryonic and neonatal development using in situ hybridization and immunohistochemistry. Elp3 mRNA is detected in the developing inner ear at embryonic day 12.5 (E12.5) in a ventro-medial region of the cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG; Fig 1A). Later, we found Elp3 transcripts are strongly expressed in the SGNs and in nascent HCs (Fig 1A, E16.5) and remain present in differentiated HCs in early postnatal animals (Fig 1A, P1). Immunofluorescent labelling of Elp3 protein at P1 confirmed this expression pattern (Fig 1B). Other proteins of the Elongator complex, such as Elp1, Elp5 and Elp6, are similarly expressed in the neonatal cochlea (Appendix Fig S1). To study the function of Elongator in inner ear development, we used a conditional knockout mouse line (Elp3lox/lox) 15, which when bred with the FoxG1:Cre transgenic line 20, resulted in the genetic ablation of Elp3 in the otic vesicle from E8.5 onwards (conditional knockout mice are referred to as Elp3cKO from herein). The loss of Elp3 expression in the cochlea of Elp3cKO mice was confirmed by in situ hybridization and Western blot (Fig 1C and D). Figure 1. Elp3 expression in the inner ear is essential for hearing A. In situ hybridizations for Elp3 transcripts in the developing inner ear. Transcripts are present at E12.5 in a ventro-medial region of the developing cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG). At E16.5, transcripts are present in the spiral ganglion neurons (SGNs) and in the newly formed hair cells (HCs) at the base of the cochlea, where they remain strongly expressed at P1. Scale bar = 50 μm; inset = 25 μm. B. Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar = 10 μm. C, D. Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (C) At P1, Elp3 mRNA is absent from Elp3cKO. Scale bars = 50 μm. In situ hybridizations were repeated four times. (D) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level (cochleae from three different animals are pooled per sample). E. Representative ABR recording from P19 WT and Elp3cKO animals reveals an absence of sound-evoked potentials in Elp3cKO animals, indicating that they suffer from severe hearing loss. F. Elp3cKO animals present increased ABR thresholds (WT/Elp3cKO n = 5/3. Unpaired two-tailed t-test, **P = 0.0017, t = 5.369, DF = 6, mean ± SD). Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: organ of Corti. Download figure Download PowerPoint We initially noticed a potential hearing deficit in Elp3cKO mice when observing the Preyer's reflex in P19 animals, as none of them elicited a rapid movement of the body in response to a sharp handclap. We then recorded auditory brainstem responses (ABR) and confirmed this observation (Fig 1E and F). Sound-evoked potentials were observed from 35 to 40 decibels (dB) upwards in wild-type (WT) mice, whereas no neuronal activity could be recorded in Elp3cKO animals at all amplitudes tested, indicating profound hearing impairment (Fig 1E). Thus, conditional deletion of Elp3 in the inner ear leads to deafness in mice. Elp3 is crucial to spiral ganglion development To investigate the possible causes of deafness upon Elp3 depletion in the inner ear, we analysed semi-thin sections from P15 WT and Elp3cKO cochleae. While the organs of Corti showed no gross abnormality, Elp3-depleted spiral ganglia were drastically reduced in size and SGN loss was obvious in all cochlear turns (Fig 2A). To assess if the remaining SGNs in Elp3cKO cochleae were still innervating their target cells at this stage, we performed specific labellings of the pre- and post-synaptic markers of the ribbon synapses, Ctbp2 and glutamate receptor GluR2/3, respectively (Fig 2B). In the absence of Elp3, Ctbp2 and GluR2/3 were still in close apposition in inner HCs (IHCs), suggesting the synapses may be functional. However, the number of these synapses was decreased by 65% when compared to control IHCs (Fig 2C), thus confirming massive SGN loss upon Elp3 depletion. In order to evaluate the temporal window of neuronal loss, we counted Gata3 or Mafb-labelled SGN nuclei on WT and Elp3cKO cochlear sections at different stages of embryonic development (Fig 2D and E). While the mean number of neurons was similar at E13.5 for Elp3cKO and controls, it was significantly reduced upon Elp3 invalidation at all later stages. At birth, the SGN population in Elp3cKO cochleae was decreased by 59% compared to controls (Fig 2E). These results suggest that SGNs are correctly specified but subsequently die during their differentiation. Therefore, we next checked for markers of apoptosis by performing cleaved caspase-3 labelling on cochlear sections. We observed a significant increase in the number of apoptotic SGNs in Elp3cKO cochleae at E13.5 and E14.5 (Fig 2F and G). Altogether, these results show that Elp3 ensures SGN survival at early stages of development. Figure 2. Elp3cKO SGNs are lost before birth as a result of early apoptosis Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar = 100 μm, OC: organ of Corti. Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar = 20 μm. The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n = 6 animals per genotype; unpaired two-tailed t-test, ***P < 0.001, t = 13.48, DF = 10, mean ± SD). NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar = 100 μm. SGN quantifications on cochlear sections from different embryonic stages reveal a loss of SGNs from E14.5 onwards (n = 3–6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***P < 0.001; *P < 0.05; F = 20.43; DF = 7; mean ± SD). Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar = 100 μm. The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n = 3–4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***P < 0.001; **P < 0.01; F = 23.74; DF = 7; mean ± SD). Download figure Download PowerPoint Elp3 deficiency is associated with the presence of aggresome-like structures in the developing SGNs and HCs Loss of Elp3 is known to cause inefficient decoding by tRNAs lacking U34 modification and to increase the frequency of protein misfolding and aggregation 18. Since neurons are particularly vulnerable to proteotoxic stress 21, we reasoned that SGN apoptosis in Elp3cKO could be a consequence of unresolved protein aggregation. Thus, we checked for the presence of aggregates in SGNs at E13.5, immediately before SGN number begins to fall in Elp3cKO cochleae. Using the PROTEOSTAT® aggresome detection kit, we observed an intense labelling in SGNs of E13.5 Elp3cKO cochleae (Fig 3A). To confirm this finding, we performed transmission electron microscopy (TEM) and identified cells harbouring large electron dense structures that distort the shape of the cell nucleus (Fig 3B). We observed that these structures are not enclosed by a membrane, further confirming that they present typical aggresome characteristics 22. These results demonstrate that proteostasis is heavily compromised in Elp3-deficient SGNs, as misfolded proteins have accumulated to form large juxtanuclear aggresome-like structures. Figure 3. Aggresome-like structures are formed in SGNs and HCs from Elp3cKO animals A. Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar = 100 μm. B. Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structure typical of aggresomes (dashed circle). Scale bars = 10 and 2 μm (last panel). C. Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n = 8/6 for WT/KO; unpaired t-test two-tailed; P = 0.0001/0.0015/0.0010; t = 9.535/4.090/4.307; DF = 12/12/12 for Elp3, Chop and Chac1 respectively; mean ± SEM presented), indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs. D, E. Proteostat staining of whole-mounted P0 organs of Corti reveals the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars = 5 μm. #IHC; *OHC1; **OHC2; ***OHC3. F. Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption. G. The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n = 3 animals per genotype; unpaired t-test two-tailed; P = 0.0003/0.4296/0.056; t = 11.62/0.8778/2.662; DF = 4/4/4 for Elp3, Chop and Chac1 respectively; mean ± SEM presented), suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis. Download figure Download PowerPoint Abnormal amounts of misfolded proteins are known to activate the unfolded protein response (UPR). This adaptive response induces cellular chaperones, increases proteasomal and autophagy capacities and reduces the rate of protein synthesis. However, if these programmes are not sufficient to alleviate the load of misfolded proteins, UPR can induce apoptosis by upregulating Chop transcription factor 3 and its target gene Chac1 23. Hence, we performed RT–qPCR analysis on E14.5 control and Elp3cKO cochleae and found a significant increase in Chop and Chac1 transcripts in the absence of Elp3 (Fig 3C), suggesting that the pro-apoptotic arm of UPR is induced in response to compromised protein homeostasis. Collectively, these results demonstrate that SGNs survival during embryonic stages of cochlear development is reliant on Elp3 to ensure proteome integrity. As Elp3 is expressed in the auditory HCs (Fig 1B), we next investigated whether aggresome-like structures would also be present in HCs of Elp3cKO animals. By performing PROTEOSTAT® staining, we detected protein aggregates that accumulate predominantly in the most apical portion of P0 HCs (Fig 3D and E, orthog). In addition, we found that the level of polyubiquitinated conjugates is drastically increased in neonatal Elp3cKO cochlea (Fig 3F). This confirms that the extent of misfolded and aggregated proteins exceeds the capacity of the cells to degrade them. Interestingly, proteostasis disruption in Elp3cKO HCs was not sufficient to activate the pro-apoptotic arm of UPR, as Chop and Chac1 transcript levels in neonatal OC were similar than those of controls (Fig 3G). Altogether, our results indicate that Elp3 is crucial to proteome integrity in the developing cochlea but that SGNs and HCs respond differently to protein homeostasis impairment. Elp3cKO HCs exhibit defective polarity and ciliogenesis Scanning electron microscopy (SEM) of P19 cochleae revealed that HCs are able to survive to proteostasis disruption in Elp3cKOs, as none of them are missing (Fig 4A). However, the polarity of the auditory epithelium appeared to be affected by the absence of Elp3 as some stereociliary bundles protruding from the apical surface of HCs displayed modest alterations in morphology and orientation (Fig 4A, indicated by arrowheads and arrows, respectively) when compared to controls. Actin stainings of P1 cochleae revealed that the polarity defects were more pronounced at birth, as some actin-rich bundles in Elp3cKO were misoriented along the medio-lateral axis and many of them appear flatter than those of WT (Fig 4B). Figure 4. Elp3 is necessary for embryonic establishment of HC polarity and ciliogenesis A. Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity, as misaligned HCs and dysmorphic stereociliary bundles are visible (red arrows and white arrowheads, respectively). Scale bars = 5 μm. B. Surface view of P1 actin-stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio-lateral axis (M-L) or misshaped. Scale bars = 5 μm. C. Elp3cKOs exhibit a broader distribution of hair cell orientation. The angles of deviation between the stereociliary bundle and the medio-lateral axis are plotted in rose diagrams, for each OHC row. Whereas the majority of WT bundles are pointing to the most lateral region of the tissue (within a range of 10°), many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation (WT/Elp3cKO OHC1: n = 99/99, P > 0.1, DF = 99; OHC2: n = 99/100, P < 0.001, DF = 99; OHC3: n = 99/98, P < 0.001, DF = 98; pooled data from six animals per genotype; Watson U2 test). D–G. Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (D) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae (scale bar = 2 μm) illustrating the misshaped stereociliary bundles and the shorter kinocilium (false coloured in pink) of Elp3cKO HCs. (E) Graph presents the mean angle of HC bundle (n = 6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***P < 0.001, F = 13.69, DF = 7; mean ± SD). (F) Elp3cKO HCs possess a significantly larger bare zone (WT/KO IHC: n = 23/24 cells; OHC1: n = 27/27; OHC2: n = 27/29; OHC3: n = 30/29; cumulative data collected from three animals per genotype; one-way ANOVA, Tukey's multiple comparisons test; ***P < 0.001, *P < 0.05, F = 16.72, DF = 7; mean ± SD). (G) Elp3cKO HCs possess a significantly smaller medial zone (WT/KO IHC: n = 23/24 cells; OHC1: n = 27/27; OHC2: n = 27/29; OHC3: n = 27/29; cumulat" @default.
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• W2962912382 title "Proteostasis is essential during cochlear development for neuron survival and hair cell polarity" @default.
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