Drug-induced stress granule formation protects sensory hair cells in mouse cochlear explants during ototoxicity

Stress granules regulate RNA translation during cellular stress, a mechanism that is generally presumed to be protective, since stress granule dysregulation caused by mutation or ageing is associated with neurodegenerative disease. Here, we investigate whether pharmacological manipulation of the stress granule pathway in the auditory organ, the cochlea, affects the survival of sensory hair cells during aminoglycoside ototoxicity, a common cause of acquired hearing loss. We show that hydroxamate (-)-9, a silvestrol analogue that inhibits eIF4A, induces stress granule formation in both an auditory cell line and ex-vivo cochlear cultures and that it prevents ototoxin-induced hair-cell death. In contrast, preventing stress granule formation using the small molecule inhibitor ISRIB increases hair-cell death. Furthermore, we provide the first evidence of stress granule formation in mammalian hair cells in-vivo triggered by aminoglycoside treatment. Our results demonstrate that pharmacological induction of stress granules enhances cell survival in native-tissue, in a clinically-relevant context. This establishes stress granules as a viable therapeutic target not only for hearing loss but also other neurodegenerative diseases.EI:595 - Action on Hearing Loss; 091092/Z/09/Z - Wellcome Trust (Wellcome); MR/N004329/1 - RCUK | Medical Research Council (MRC)Published versio

Regulation of the orphan nuclear receptor Nr2f2 by the DFNA15 deafness gene Pou4f3.

Hair cells are the mechanotransducing cells of the inner ear that are essential for hearing and balance. POU4F3--a POU-domain transcription factor selectively expressed by these cells--has been shown to be essential for hair cell differentiation and survival in mice and its mutation in humans underlies late-onset progressive hearing loss (DFNA15). The downstream targets of POU4F3 are required for hair cell differentiation and survival. We aimed to identify such targets in order to elucidate the molecular pathways involved in hair cell production and maintenance. The orphan thyroid nuclear receptor Nr2f2 was identified as a POU4F3 target using a subtractive hybridization strategy and EMSA analysis showed that POU4F3 binds to two sites in the Nr2f2 5' flanking region. These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5' flanking region reporter construct to POU4F3. Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing. NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised. These data demonstrate that Nr2f2 is a direct target of POU4F3 in vitro and that this regulatory relationship may be relevant to hair cell development and survival

Expression of NR2F2 in the embryonic rat cochlea.

<p><i>a</i>. Overview of NR2F2 expression throughout the cochlea at E18. NR2F2 is most strongly expressed in the cochlear duct. In the apical duct, strong expression is seen in the super-medial wall (<i>S-MW</i>), greater epithelial ridge (<i>GER</i>), and lesser epithelial ridge (<i>LER</i>). This expression is reduced to the lesser epithelial ridge in the basal duct (<i>arrowhead</i>). <i>b–e</i>. Apical cochlear duct; <i>f–i</i>. Middle cochlear duct; and <i>j–m</i>. Basal cochlear duct. <i>b, f & j</i>, cell nuclei stained with DAPI; <i>c, g & k</i>, NR2F2 labelling; <i>d, h & l</i>, filamentous actin labelled by Phalloidin; and <i>e, i & m,</i> Merge of DAPI (<i>blue</i>), NR2F2 (<i>green</i>) and Phalloidin (<i>red</i>), s<i>cale bars</i>: 100 µm in <i>a</i> and 20 µm in <i>e, i & m</i>.</p

Evolutionary conservation of transcription factor binding sites in the <i>Nr2f2</i> 5′ flanking region.

<p>Schematic representation of the conservation of the region surrounding the human PRE1 sequence (<i>a</i>) and the human PRE2 sequence (<i>b</i>) compared to corresponding mouse, rat and chimpanzee sequences. <i>a</i>. The well-conserved PRE1 sequence is indicated by asterisks with evolutionarily conserved binding sites for the POU domain transcription factors OCT1 and OCT_C indicated by horizontal bars. Conserved bases are represented by black boxes with mismatches in grey. <i>b</i>. PRE2 is less well conserved with eight mismatched bases and no corresponding POU domain transcription factor binding sites in the TRANSFAC database. Conserved bases are again represented by black boxes with mismatches in grey.</p

Expression of NR2F2 in the postnatal mouse inner ear.

<p>Cryotome sections of P1 and adult mouse inner ears were subjected to immunohistochemistry in order to characterise their postnatal NR2F2 expression pattern. NR2F2 labelling is seen throughout the sensory epithelia of the cochlea and cristae. In the cochlea, NR2F2 labelling is of greater intensity in the apex than the base. NR2F2 is expressed in the nuclei of apical hair cells at all ages examined. In P1 mice, NR2F2 expression is reduced in basal hair cells. In adult mice, the expression of NR2F2 in basal hair cell nuclei appears further reduced. NR2F2 is also expressed in the hair cells of the cristae of the semicircular canals and this expression is maintained into adulthood. In merged images DAPI is <i>blue</i>, NR2F2 is <i>green</i>, Phalloidin is r<i>ed</i>, a<i>rrowheads</i> indicate hair cells and s<i>cale bars</i>: 20 µm.</p

POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb <i>Nr2f2</i> 5′ flanking sequence.

<p><i>a</i>. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an <i>Nr2f2</i> 5′ flanking region fragment (<i>grey box</i>) cloned upstream of a luciferase reporter gene (<i>LUC</i>). The location of this fragment relative to the <i>Nr2f2</i> transcriptional start site is shown. <i>b</i>. Evaluation of the response of 4.2 kb-<i>Nr2f2</i>-Luc to increasing levels of POU4F3 or <i>dreidel</i> mutant POU4F3 (<i>Ddl</i>) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. <i>c</i>. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and <i>dreidel</i> expression constructs. <i>d</i>. Evaluation of the response of PRE2-Luc in similar experiments to <i>c</i>. Error bars represent the s.e.m in <i>b, c</i> and <i>d</i> (n = 6 for each data point).</p

POU4F3 recognition element mutation attenuates POU4F3 binding and activation of the <i>Nr2f2</i> 5′ flanking region.

<p><i>a</i>. EMSA analysis of POU4F3 recognition element mutations on POU4F3 binding. Probes were either incubated alone or with <i>in vitro</i> translated POU4F3. <i>Closed arrowheads</i> indicate POU4F3-specific PRE1 bandshifts and the <i>open arrowhead</i> indicates the POU4F3-specific PRE2 bandshift. The mutation of each site severely compromises the ability of POU4F3 to bind to each sequence. <i>b</i>. Luciferase reporter assay upon co-transfection of POU4F3 with the 4.2 kb-<i>Nr2f2</i>-Luc-Mut reporter construct. Error bars represent the s.e.m (n = 6 for each data point).</p

Identification and verification of POU4F3 recognition elements in the <i>Nr2f2</i> 5′ flanking region.

<p><i>a</i>. Schematic diagram of the location of two putative POU4F3 recognition elements (PREs) identified in the <i>Nr2f2</i> 5′ flanking region and used in EMSA analysis. Underlined bases correspond to POU4F3 binding sites predicted by MatInspector software with capital letters denoting matches to the core sequence. The asterisks signify matrix position conservation >60/100 (<i>TSS</i>, transcriptional start site and <i>UTR</i>, un-translated region). <i>b</i>. Sequences shown in <i>a</i> were used as radiolabelled probes in EMSA analysis with <i>in vitro</i> translated POU4F3 or a Luciferase control. Bandshifts due to protein-specific binding by POU4F3 are indicated by arrowheads. <i>c</i>. The same probes were used in EMSA analysis with UB/OC-2 cell nuclear protein extract. Reactions were incubated either alone, with UB/OC-2 cell nuclear protein extract or with the nuclear protein extract and an excess of non-radiolabelled competitor oligonucleotide as indicated. The ‘<i>P</i>’ suffix refers to non-radiolabelled POU4F3 binding sequence whereas the ‘<i>A’</i> suffix indicates a non-radiolabelled AP4 binding sequence. POU4F3-sequence-specific shifts are indicated by arrowheads.</p

Targeted deletion of the RNA-binding protein Caprin1 leads to progressive hearing loss and impairs recovery from noise exposure in mice

Cell cycle associated protein 1 (Caprin1) is an RNA-binding protein that can regulate the cellular post-transcriptional response to stress. It is a component of both stress granules and neuronal RNA granules and is implicated in neurodegenerative disease, synaptic plasticity and long-term memory formation. Our previous work suggested that Caprin1 also plays a role in the response of the cochlea to stress. Here, targeted inner ear-deletion of Caprin1 in mice leads to an early onset, progressive hearing loss. Auditory brainstem responses from Caprin1-deficient mice show reduced thresholds, with a significant reduction in wave-I amplitudes compared to wildtype. Whilst hair cell structure and numbers were normal, the inner hair cell-spiral ganglion neuron (IHC-SGN) synapse revealed abnormally large post-synaptic GluA2 receptor puncta, a defect consistent with the observed wave-I reduction. Unlike wildtype mice, mild-noise-induced hearing threshold shifts in Caprin1-deficient mice did not recover. Oxidative stress triggered TIA-1/HuR-positive stress granule formation in ex-vivo cochlear explants from Caprin1-deficient mice, showing that stress granules could still be induced. Taken together, these findings suggest that Caprin1 plays a key role in maintenance of auditory function, where it regulates the normal status of the IHC-SGN synapse