Assistant Professor, Stanford University, School of Medicine, Stanford, CA, USA
Otolaryngology department

Senior Research Associate, The Scripps Research Institute, La Jolla, CA, USA
Department of Cell Biology and Dorris Neuroscience Center
Advisor: Ulrich Müller

Research Associate, The Scripps Research Institute, La Jolla, CA, USA
Department of Cell Biology and Dorris Neuroscience Center
Advisor: Ulrich Müller

Ph.D. Student, The Institute for Developmental Biology of Marseilles, France
then moved to the Ecole Normale Supérieure, Paris, France
CNRS/ENS “Development and Evolution of the Nervous System”
Advisor: Jean-François Brunet

Graduate Student, The Institute for Developmental Biology of Marseilles, France
INSERM “Development and Pathology of Spinal Motoneuron”
Advisors: Christopher E Henderson, Keith Dudley

Academic Appointments

Current Research and Scholarly Interests

Genetics of Hearing and Vestibular Impairment

The inner ear contains the sensory cells that detect sound and head motion, the hair cells. In mammals, these cells are generated during the mid-gestation and will never be replaced during the entire life. The hair cells are in constant activity and their dysfunction is a major cause of deafness and peripheral vestibular disorders: they are both the core and the Achilles’ heel of the system.
Hearing loss can result from exposure to excessive noise, chemicals and certain medications. However, susceptibility to deafness is generally dictated by genetic transmission. To this date, 136 human loci have been linked to hearing loss, but we know the corresponding affected genes for only 85 of them. These genes are very often required, directly or indirectly, for the proper hair cell function.

We want to identify the comprehensive list of genes required for hearing and head motion detection, and ultimately characterize the function of these genes at the molecular level.

Function of Hair Cells and other Inner Ear Cells

Differently from the sense of Vision, still little is known about Hearing and Balancing at their molecular level. This is due to the technical challenges associated with this organ: the paucity of the inner ear sensory cells, their inaccessibility and their fragility.
The inner ear is composed of two functional parts: the cochlea, which is the auditory organ, and the vestibule, organs responsible for head motion and balancing. In both parts, the sensory epithelia are composed by the sensory hair cells, always surrounded by supporting cells.
We want to characterize down to the molecular level the function of the cells that compose the inner ear, particularly the hair cells.

The hair cells have different functions: 1) to detect the mechanical stimuli induced by sound, and 2), to transmit this information to the central nervous system through their synapses.

2023-24 Courses

Graduate and Fellowship Programs

All Publications

  • LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission. Research square Wang, P., Miller, K. K., He, E., Dhawan, S. S., Cunningham, C. L., Grillet, N. 2024


    Hearing is initiated in hair cells by the mechanical activation of ion channels in the hair bundle. The hair bundle is formed by stereocilia organized into rows of increasing heights interconnected by tip links, which convey sound-induced forces to stereocilia tips. The auditory mechanosensitive channels are complexes containing at least four protein-subunits - TMC1/2, TMIE, CIB2, and LHFPL51-16 - and are located at the tips of shorter stereocilia at a yet-undetermined distance from the lower tip link insertion point17. While multiple auditory channel subunits appear to interact with the tip link, it remains unknown whether their combined interaction alone can resist the high-frequency mechanical stimulations owing to sound. Here we show that an unanticipated additional element, LOXHD1, is indispensable for maintaining the TMC1 pore-forming channel subunits coupled to the tip link. We demonstrate that LOXHD1 is a unique element of the auditory mechanotransduction complex that selectively affects the localization of TMC1, but not its close developmental paralogue TMC2. Taking advantage of our novel immunogold scanning electron microscopy method for submembranous epitopes (SUB-immunogold-SEM), we demonstrate that TMC1 normally concentrates within 100-nm of the tip link insertion point. In LOXHD1's absence, TMC1 is instead mislocalized away from this force transmission site. Supporting this finding, we found that LOXHD1 interacts selectively in vitro with TMC1 but not with TMC2 while also binding to channel subunits CIB2 and LHFPL5 and tip-link protein PCDH15. SUB-immunogold-SEM additionally demonstrates that LOXHD1 and TMC1 are physically connected to the lower tip-link complex in situ. Our results show that the TMC1-driven mature channels require LOXHD1 to stay coupled to the tip link and remain functional, but the TMC2-driven developmental channels do not. As both tip links and TMC1 remain present in hair bundles lacking LOXHD1, it opens the possibility to reconnect them and restore hearing for this form of genetic deafness.

    View details for DOI 10.21203/

    View details for PubMedID 38260480

    View details for PubMedCentralID PMC10802736

  • High-resolution immunofluorescence imaging of mouse cochlear hair bundles. STAR protocols Miller, K. K., Wang, P., Grillet, N. 2022; 3 (2): 101431


    High-resolution immunofluorescence imaging of cochlear hair bundles faces many challenges due to the hair bundle's small dimensions, fragile nature, and complex organization. Here, we describe an optimized protocol for hair-bundle protein immunostaining and localization. We detail the steps and solutions for extracting and fixing the mouse inner ear and for dissecting the organ of Corti. We further emphasize the optimal permeabilization, blocking, staining, and mounting conditions as well as the parameters for high-resolution microscopy imaging. For complete details on the use and execution of this protocol, please refer to Trouillet etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101431

    View details for PubMedID 35669049

  • Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1. Cell reports Deng, Q., Natesan, R., Cidre-Aranaz, F., Arif, S., Liu, Y., Rasool, R. U., Wang, P., Mitchell-Velasquez, E., Das, C. K., Vinca, E., Cramer, Z., Grohar, P. J., Chou, M., Kumar-Sinha, C., Weber, K., Eisinger-Mathason, T. S., Grillet, N., Grünewald, T., Asangani, I. A. 2022; 39 (11): 110971


    Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

    View details for DOI 10.1016/j.celrep.2022.110971

    View details for PubMedID 35705030

  • High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy. STAR protocols Grillet, N. 2022; 3 (1): 101213


    Scanning electron microscopy (SEM) allows cell surface imaging at a sub-nanometric resolution. However, the sample requires a specific preparation to sustain the high vacuum of the SEM and be electrically conductive. The sample preparation consists of dissection, fixation, dehydration, metal coating, and tissue mounting. Here we provide a comprehensive protocol to perform SEM on the mouse's inner ear, and image the hair bundles at high resolution. Hair bundles are the force-sensitive organelles located at the apical surface of hair cells. For complete details on the use and execution of this protocol, please refer to Trouillet etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101213

    View details for PubMedID 35257116

  • Loxhd1 mutations cause mechanotransduction defects in cochlear hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Trouillet, A., Miller, K. K., George, S. S., Wang, P., Ali, N., Ricci, A., Grillet, N. 2021


    Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase alpha-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type (WT) levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the MET phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphological defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT:Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multi-repeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.

    View details for DOI 10.1523/JNEUROSCI.0975-20.2021

    View details for PubMedID 33707295

  • Dimensions of a Living Cochlear Hair Bundle Front Cell Dev Biol Miller, K. K., Atkinson, P., Mendoza, K., Ó Maoiléidigh, D., Grillet, N. 2021; 9: 742529


    The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle's mechanical properties - overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.

    View details for DOI 10.3389/fcell.2021.742529

    View details for PubMedCentralID PMC8657763

  • Mechanosensory hair cells express two molecularly distinct mechanotransduction channels NATURE NEUROSCIENCE Wu, Z., Grillet, N., Zhao, B., Cunningham, C., Harkins-Perry, S., Coste, B., Ranade, S., Zebarjadi, N., Beurg, M., Fettiplace, R., Patapoutian, A., Muller, U. 2017; 20 (1): 24-33


    Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. We report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair cell stereocilia, and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS, TMIE, TMC1 and TMC2. We show here that the second ion channel is expressed at the apical surface of hair cells and that it contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca(2+) concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distributions.

    View details for DOI 10.1038/nn.4449

    View details for Web of Science ID 000391085500009

    View details for PubMedID 27893727

    View details for PubMedCentralID PMC5191906

  • TMIE Is an Essential Component of the Mechanotransduction Machinery of Cochlear Hair Cells. Neuron Zhao, B., Wu, Z., Grillet, N., Yan, L., Xiong, W., Harkins-Perry, S., Müller, U. 2014; 84 (5): 954-67


    Hair cells are the mechanosensory cells of the inner ear. Mechanotransduction channels in hair cells are gated by tip links. The molecules that connect tip links to transduction channels are not known. Here we show that the transmembrane protein TMIE forms a ternary complex with the tip-link component PCDH15 and its binding partner TMHS/LHFPL5. Alternative splicing of the PCDH15 cytoplasmic domain regulates formation of this ternary complex. Transducer currents are abolished by a homozygous Tmie-null mutation, and subtle Tmie mutations that disrupt interactions between TMIE and tip links affect transduction, suggesting that TMIE is an essential component of the hair cell's mechanotransduction machinery that functionally couples the tip link to the transduction channel. The multisubunit composition of the transduction complex and the regulation of complex assembly by alternative splicing is likely critical for regulating channel properties in different hair cells and along the cochlea's tonotopic axis.

    View details for DOI 10.1016/j.neuron.2014.10.041

    View details for PubMedID 25467981

    View details for PubMedCentralID PMC4258123

  • Using injectoporation to deliver genes to mechanosensory hair cells. Nature protocols Xiong, W., Wagner, T., Yan, L., Grillet, N., Müller, U. 2014; 9 (10): 2438-49


    Mechanosensation, the transduction of mechanical force into electrochemical signals, allows organisms to detect touch and sound, to register movement and gravity, and to sense changes in cell volume and shape. The hair cells of the mammalian inner ear are the mechanosensors for the detection of sound and head movement. The analysis of gene function in hair cells has been hampered by the lack of an efficient gene transfer method. Here we describe a method termed injectoporation that combines tissue microinjection with electroporation to express cDNAs and shRNAs in mouse cochlear hair cells. Injectoporation allows for gene transfer into dozens of hair cells, and it is compatible with the analysis of hair cell function using imaging approaches and electrophysiology. Tissue dissection and injectoporation can be carried out within a few hours, and the tissue can be cultured for days for subsequent functional analyses.

    View details for DOI 10.1038/nprot.2014.168

    View details for PubMedID 25232939

    View details for PubMedCentralID PMC4241755

  • TMHS Is an Integral Component of the Mechanotransduction Machinery of Cochlear Hair Cells CELL Xiong, W., Grillet, N., Elledge, H. M., Wagner, T. F., Zhao, B., Johnson, K. R., Kazmierczak, P., Mueller, U. 2012; 151 (6): 1283-1295


    Hair cells are mechanosensors for the perception of sound, acceleration, and fluid motion. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity. The molecular constituents of the mechanotransduction channels of hair cells are not known. Here, we show that mechanotransduction is impaired in mice lacking the tetraspan TMHS. TMHS binds to the tip-link component PCDH15 and regulates tip-link assembly, a process that is disrupted by deafness-causing Tmhs mutations. TMHS also regulates transducer channel conductance and is required for fast channel adaptation. TMHS therefore resembles other ion channel regulatory subunits such as the transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits. We conclude that TMHS is an integral component of the hair cell's mechanotransduction machinery that functionally couples PCDH15 to the transduction channel.

    View details for DOI 10.1016/j.cell.2012.10.041

    View details for Web of Science ID 000311999900017

    View details for PubMedID 23217710

  • Mutations in LOXHD1, an Evolutionarily Conserved Stereociliary Protein, Disrupt Hair Cell Function in Mice and Cause Progressive Hearing Loss in Humans AMERICAN JOURNAL OF HUMAN GENETICS Grillet, N., Schwander, M., Hildebrand, M. S., Sczaniecka, A., Kolatkar, A., Velasco, J., Webster, J. A., Kahrizi, K., Najmabadi, H., Kimberling, W. J., Stephan, D., Bahlo, M., Wiltshire, T., Tarantino, L. M., Kuhn, P., Smith, R. J., Mueller, U. 2009; 85 (3): 328-337


    Hearing loss is the most common form of sensory impairment in humans and is frequently progressive in nature. Here we link a previously uncharacterized gene to hearing impairment in mice and humans. We show that hearing loss in the ethylnitrosourea (ENU)-induced samba mouse line is caused by a mutation in Loxhd1. LOXHD1 consists entirely of PLAT (polycystin/lipoxygenase/alpha-toxin) domains and is expressed along the membrane of mature hair cell stereocilia. Stereociliary development is unaffected in samba mice, but hair cell function is perturbed and hair cells eventually degenerate. Based on the studies in mice, we screened DNA from human families segregating deafness and identified a mutation in LOXHD1, which causes DFNB77, a progressive form of autosomal-recessive nonsyndromic hearing loss (ARNSHL). LOXHD1, MYO3a, and PJVK are the only human genes to date linked to progressive ARNSHL. These three genes are required for hair cell function, suggesting that age-dependent hair cell failure is a common mechanism for progressive ARNSHL.

    View details for DOI 10.1016/j.ajhg.2009.07.017

    View details for Web of Science ID 000270104500003

    View details for PubMedID 19732867

  • The Mechanotransduction Machinery of Hair Cells SCIENCE SIGNALING Grillet, N., Kazmierczak, P., Xiong, W., Schwander, M., Reynolds, A., Sakaguchi, H., Tokita, J., Kachar, B., Mueller, U. 2009; 2 (85)


    Mechanotransduction, the conversion of mechanical force into an electrochemical signal, allows living organisms to detect touch, hear, register movement and gravity, and sense changes in cell volume and shape. Hair cells in the vertebrate inner ear are mechanoreceptor cells specialized for the detection of sound and head movement. Each hair cell contains, at the apical surface, rows of stereocilia that are connected by extracellular filaments to form an exquisitely organized bundle. Mechanotransduction channels, localized near the tips of the stereocilia, are gated by the gating spring, an elastic element that is stretched upon stereocilia deflection and mediates rapid channel opening. Components of the mechanotransduction machinery in hair cells have been identified and several are encoded by genes linked to deafness in humans, which indicates that defects in the mechanotransduction machinery are the underlying cause of some forms of hearing impairment.

    View details for DOI 10.1126/scisignal.285pt5

    View details for Web of Science ID 000275602200003

    View details for PubMedID 19706872

  • Harmonin Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells NEURON Grillet, N., Xiong, W., Reynolds, A., Kazmierczak, P., Sato, T., Lillo, C., Dumont, R. A., Hintermann, E., Sczaniecka, A., Schwander, M., Williams, D., Kachar, B., Gillespie, P. G., Mueller, U. 2009; 62 (3): 375-387


    In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.

    View details for DOI 10.1016/j.neuron.2009.04.006

    View details for Web of Science ID 000266146100009

    View details for PubMedID 19447093

  • Selection Criteria Optimal for Recovery of Inner Ear Tissues from Deceased Organ Donors. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology Aaron, K. A., Hosseini, D. K., Vaisbuch, Y., Scheibinger, M., Grillet, N., Heller, S., Wang, T., Cheng, A. G. 2022


    To identify optimal conditions for recovering viable inner ear tissues from deceased organ donors.Tertiary recovery hospitals and Donor Network West Organ Recovery Center.Recovering bilateral inner ear tissues and immunohistological analysis.Immunohistochemical analysis of utricles from human organ donors after brain death (DBD) or donors after cardiac death (DCD).Vestibular tissues from 21 organ donors (39 ears) were recovered. Of these, 18 donors (33 utricles) were examined by immunofluorescence. The sensory epithelium was present in seven utricles (two from DBD and five from DCD). Relative to DBD utricles, DCD organs more commonly displayed dense populations of hair cells and supporting cells. Relative to DBD, DCD had significantly shorter postmortem interval time to tissue recovery (<48 h). Compared to donors with no sensory epithelium, donors with intact and viable sensory epithelium (both DCD and DBD) had significantly shorter lag time to resuscitation prior to hospital admission (6.4 ± 9.2 vs 35.6 ± 23.7 min, respectively) as well as a shorter time between pronouncements of death to organ recovery (22.6 ± 30.4 vs 64.8 ± 22.8 h, respectively).Organ donors are a novel resource for bilateral inner ear organs. Selecting tissue donors within defined parameters can optimize the quality of recovered inner ear tissues, thereby facilitating future research investigating sensory and nonsensory cells.

    View details for DOI 10.1097/MAO.0000000000003496

    View details for PubMedID 35120078

  • Oncofusion driven de novo enhancer assembly promotes malignancy in Ewing sarcomavia aberrant expression of the stereociliary protein LOXHD1. Deng, Q., Natesan, R., Cidre-Aranz, F., Arif, S., Liu, Y., Rasool, R., Wang, P., Crammer, Z., Chou, M., Kumar, C., Weber, K., Eisinger, K., Grillet, N., Gruenewald, T., Asangani, I. AMER ASSOC CANCER RESEARCH. 2021
  • Discovery and characterization of LOXHD1 as a highly specific EWS-FLI1 driven oncogene in Ewing sarcoma. Deng, Q., Natesan, R., Arif, S., Rasool, R., Liu, Y., Wang, P., Crammer, Z., Mercadante, M., Gades, T., Cho, M., Eisengher, K., Grillet, N., Asangani, I. A. AMER ASSOC CANCER RESEARCH. 2020: 54–55
  • A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58. Human molecular genetics Lezirovitz, K., Vieira-Silva, G. A., Batissoco, A. C., Levy, D., Kitajima, J. P., Trouillet, A., Ouyang, E., Zebajardi, N., Sampaio-Silva, J., Pedroso-Campos, V., Nascimento, L. R., Sonoda, C. Y., Borges, V. M., Vasconcelos, L. G., Beck, R. M., Grasel, S. S., Jagger, D. J., Grillet, N., Bento, R. F., Mingroni-Netto, R. C., Oiticica, J. 2020


    Here we define a~200Kb genomic duplication in 2p14 as the genetic signature that segregates with post-lingual progressive sensorineural autosomal dominant hearing loss in 20 affected individuals from the DFNA58 family, first reported in 2009. The duplication includes two entire genes, PLEK and CNRIP1, and the first exon of PPP3R1 (protein-coding), in addition to four uncharacterized long noncoding (lnc) RNA genes and part of a novel protein-coding gene. Quantitative analysis of mRNA expression in blood samples revealed selective overexpression of CNRIP1 and of two lncRNA genes (LOC107985892 and LOC102724389) in all affected members tested, but not in unaffected ones. Qualitative analysis of mRNA expression identified also fusion transcripts involving parts of PPP3R1, CNRIP1 and an intergenic region between PLEK and CNRIP1, in the blood of all carriers of the duplication, but were heterogeneous in nature. By in situ hybridization and immunofluorescence, we showed that Cnrip1, Plek and Ppp3r1 genes are all expressed in the adult mouse cochlea including the spiral ganglion neurons, suggesting changes in expression levels of these genes in the hearing organ could underlie the DFNA58 form of deafness. Our study highlights the value of studying rare genomic events leading to hearing loss such as copy number variations. Further studies will be required to determine which of these genes, either coding proteins or non-coding RNAs, is or are responsible for DFNA58 hearing loss.

    View details for DOI 10.1093/hmg/ddaa075

    View details for PubMedID 32337552

  • Dual regulation of planar polarization by secreted Wnts and Vangl2 in the developing mouse cochlea. Development (Cambridge, England) Huarcaya Najarro, E., Huang, J., Jacobo, A., Quiruz, L. A., Grillet, N., Cheng, A. G. 2020


    Planar cell polarity (PCP) proteins localize asymmetrically to instruct cell polarity within the tissue plane, with defects leading to deformities of the limbs, neural tube, and inner ear. Wnt proteins are evolutionarily conserved polarity cues, yet Wnt mutants display variable PCP defects, thus how Wnts regulate PCP remains unresolved. Here, we used the developing cochlea as a model system to show that secreted Wnts regulate PCP through polarizing a specific subset of PCP proteins. Conditional deletion of Wntless or Porcupine, both essential for secretion of Wnts, caused misrotated sensory cells and shortened cochlea-both hallmarks of PCP defects. Wntless-deficient cochleae lacked the polarized PCP components Dishevelled1/2 and Frizzled3/6, while other PCP proteins (Vangl1/2, Celsr1, Dishevelled3) remained localized. We identified seven Wnt paralogues, including the major PCP regulator Wnt5a, which was surprisingly dispensable for planar polarization in the cochlea. Finally, Vangl2 haploinsufficiency markedly accentuated sensory cell polarization defects in Wntless-deficient cochlea. Together, our study indicates that secreted Wnts and Vangl2 coordinate to ensure proper tissue polarization during development.

    View details for DOI 10.1242/dev.191981

    View details for PubMedID 34004910

  • Osmotic stabilization prevents cochlear synaptopathy after blast trauma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J., Xia, A., Grillet, N., Applegate, B. E., Oghalai, J. S. 2018; 115 (21): E4853–E4860


    Traumatic noise causes hearing loss by damaging sensory hair cells and their auditory synapses. There are no treatments. Here, we investigated mice exposed to a blast wave approximating a roadside bomb. In vivo cochlear imaging revealed an increase in the volume of endolymph, the fluid within scala media, termed endolymphatic hydrops. Endolymphatic hydrops, hair cell loss, and cochlear synaptopathy were initiated by trauma to the mechanosensitive hair cell stereocilia and were K+-dependent. Increasing the osmolality of the adjacent perilymph treated endolymphatic hydrops and prevented synaptopathy, but did not prevent hair cell loss. Conversely, inducing endolymphatic hydrops in control mice by lowering perilymph osmolality caused cochlear synaptopathy that was glutamate-dependent, but did not cause hair cell loss. Thus, endolymphatic hydrops is a surrogate marker for synaptic bouton swelling after hair cells release excitotoxic levels of glutamate. Because osmotic stabilization prevents neural damage, it is a potential treatment to reduce hearing loss after noise exposure.

    View details for PubMedID 29735658

  • Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function. journal of neuroscience Zeng, W., Grillet, N., Dewey, J. B., Trouillet, A., Krey, J. F., Barr-Gillespie, P. G., Oghalai, J. S., Müller, U. 2016; 36 (35): 9201-9216


    Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function.Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice.

    View details for DOI 10.1523/JNEUROSCI.0093-16.2016

    View details for PubMedID 27581460

  • Two-Dimensional Cochlear Micromechanics Measured In Vivo Demonstrate Radial Tuning within the Mouse Organ of Corti. journal of neuroscience Lee, H. Y., Raphael, P. D., Xia, A., Kim, J., Grillet, N., Applegate, B. E., Ellerbee Bowden, A. K., Oghalai, J. S. 2016; 36 (31): 8160-8173


    The exquisite sensitivity and frequency discrimination of mammalian hearing underlie the ability to understand complex speech in noise. This requires force generation by cochlear outer hair cells (OHCs) to amplify the basilar membrane traveling wave; however, it is unclear how amplification is achieved with sharp frequency tuning. Here we investigated the origin of tuning by measuring sound-induced 2-D vibrations within the mouse organ of Corti in vivo Our goal was to determine the transfer function relating the radial shear between the structures that deflect the OHC bundle, the tectorial membrane and reticular lamina, to the transverse motion of the basilar membrane. We found that, after normalizing their responses to the vibration of the basilar membrane, the radial vibrations of the tectorial membrane and reticular lamina were tuned. The radial tuning peaked at a higher frequency than transverse basilar membrane tuning in the passive, postmortem condition. The radial tuning was similar in dead mice, indicating that this reflected passive, not active, mechanics. These findings were exaggerated in Tecta(C1509G/C1509G) mice, where the tectorial membrane is detached from OHC stereocilia, arguing that the tuning of radial vibrations within the hair cell epithelium is distinct from tectorial membrane tuning. Together, these results reveal a passive, frequency-dependent contribution to cochlear filtering that is independent of basilar membrane filtering. These data argue that passive mechanics within the organ of Corti sharpen frequency selectivity by defining which OHCs enhance the vibration of the basilar membrane, thereby tuning the gain of cochlear amplification.Outer hair cells amplify the traveling wave within the mammalian cochlea. The resultant gain and frequency sharpening are necessary for speech discrimination, particularly in the presence of background noise. Here we measured the 2-D motion of the organ of Corti in mice and found that the structures that stimulate the outer hair cell stereocilia, the tectorial membrane and reticular lamina, were sharply tuned in the radial direction. Radial tuning was similar in dead mice and in mice lacking a tectorial membrane. This suggests that radial tuning comes from passive mechanics within the hair cell epithelium, and that these mechanics, at least in part, may tune the gain of cochlear amplification.

    View details for DOI 10.1523/JNEUROSCI.1157-16.2016

    View details for PubMedID 27488636

  • Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain DEVELOPMENT Webb, S. W., Grillet, N., Andrade, L. R., Xiong, W., Swarthout, L., Della Santina, C. C., Kachar, B., Mueller, U. 2011; 138 (8): 1607-1617


    Protocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly, mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.

    View details for DOI 10.1242/dev.060061

    View details for Web of Science ID 000288649400016

    View details for PubMedID 21427143

    View details for PubMedCentralID PMC3062428

  • The genetics of progressive hearing loss: a link between hearing impairment and dysfunction of mechanosensory hair cells. Future neurology Müller, U., Grillet, N. 2010; 5 (1): 9-12

    View details for DOI 10.2217/fnl.09.68

    View details for PubMedID 24436636

    View details for PubMedCentralID PMC3891795

  • Regulator of G Protein Signaling-4 Controls Fatty Acid and Glucose Homeostasis ENDOCRINOLOGY Iankova, I., Chavey, C., Clape, C., Colomer, C., Guerineau, N. C., Grillet, N., Brunet, J., Annicotte, J., Fajas, L. 2008; 149 (11): 5706-5712


    Circulating free fatty acids are a reflection of the balance between lipogenesis and lipolysis that takes place mainly in adipose tissue. We found that mice deficient for regulator of G protein signaling (RGS)-4 have increased circulating catecholamines, and increased free fatty acids. Consequently, RGS4-/- mice have increased concentration of circulating free fatty acids; abnormally accumulate fatty acids in liver, resulting in liver steatosis; and show a higher degree of glucose intolerance and decreased insulin secretion in pancreas. We show in this study that RGS4 controls adipose tissue lipolysis through regulation of the secretion of catecholamines by adrenal glands. RGS4 controls the balance between adipose tissue lipolysis and lipogenesis, secondary to its role in the regulation of catecholamine secretion by adrenal glands. RGS4 therefore could be a good target for the treatment of metabolic diseases.

    View details for DOI 10.1210/en.2008-0717

    View details for Web of Science ID 000260194000043

    View details for PubMedID 18635652

  • A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function JOURNAL OF NEUROSCIENCE Schwander, M., Sczaniecka, A., Grillet, N., Bailey, J. S., Avenarius, M., Najmabadi, H., Steffy, B. M., Federe, G. C., Lagler, E. A., Banan, R., Hice, R., Grabowski-Boase, L., Keithley, E. M., Ryan, A. F., Housley, G. D., Wiltshire, T., Smith, R. J., Tarantino, L. M., Mueller, U. 2007; 27 (9): 2163-2175


    Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

    View details for DOI 10.1523/JNEUROSCI.4975-06.2007

    View details for Web of Science ID 000244758500004

    View details for PubMedID 17329413

  • Sphingosine 1-phosphate (S1P) signaling is required for maintenance of hair cells mainly via activation of S1P(2) JOURNAL OF NEUROSCIENCE Herr, D. R., Grillet, N., Schwander, M., Rivera, R., Mueller, U., Chun, J. 2007; 27 (6): 1474-1478


    Hearing requires the transduction of vibrational forces by specialized epithelial cells in the cochlea known as hair cells. The human ear contains a finite number of terminally differentiated hair cells that, once lost by noise-induced damage or toxic insult, can never be regenerated. We report here that sphingosine 1-phosphate (S1P) signaling, mainly via activation of its cognate receptor S1P2, is required for the maintenance of vestibular and cochlear hair cells in vivo. Two S1P receptors, S1P2 and S1P3, were found to be expressed in the cochlea by reverse transcription-PCR and in situ hybridization. Mice that are null for both these receptors uniformly display progressive cochlear and vestibular defects with hair cell loss, resulting in complete deafness by 4 weeks of age and, with complete penetrance, balance defects of increasing severity. This study reveals the previously unknown role of S1P signaling in the maintenance of cochlear and vestibular integrity and suggests a means for therapeutic intervention in degenerative hearing loss.

    View details for DOI 10.1523/JNEUROSCI.4245-06.2007

    View details for Web of Science ID 000244070000028

    View details for PubMedID 17287522

  • Generation and characterization of Rgs4 mutant mice MOLECULAR AND CELLULAR BIOLOGY Grillet, N., Pattyn, A., Contet, C., Kieffer, B. L., GORIDIS, C., Brunet, J. F. 2005; 25 (10): 4221-4228


    RGS proteins are negative regulators of signaling through heterotrimeric G protein-coupled receptors and, as such, are in a position to regulate a plethora of biological phenomena. However, those have just begun to be explored in vivo. Here, we describe a mouse line deficient for Rgs4, a gene normally expressed early on in discrete populations of differentiating neurons and later on at multiple sites of the central nervous system, the cortex in particular, where it is one of the most highly transcribed Rgs genes. Rgs4(lacZ/lacZ) mice had normal neural development and were viable and fertile. Behavioral testing on mutant adults revealed subtle sensorimotor deficits but, so far, supported neither the proposed status of Rgs4 as a schizophrenia susceptibility gene (by showing intact prepulse inhibition in the mutants) nor (unlike another member of the Rgs family, Rgs9) a role of Rgs4 in the acute or chronic response to opioids.

    View details for DOI 10.1128/MCB.25.10.4221-4228.2005

    View details for Web of Science ID 000228888100032

    View details for PubMedID 15870291

  • Dynamic expression of RGS4 in the developing nervous system and regulation by the neural type-specific transcription factor Phox2b JOURNAL OF NEUROSCIENCE Grillet, N., Dubreuil, R., Dufour, H. D., Brunet, J. F. 2003; 23 (33): 10613-10621


    Previous studies have shown that members of the family of regulators of G-protein signaling (RGS), including RGS4, have a discrete expression pattern in the adult brain (Gold et al., 1997). Here, we describe for RGS4 a distinct, mostly transient phase of neuronal expression, during embryonic development: transcription of RGS4 occurs in a highly dynamic manner in a small set of peripheral and central neuronal precursors. This expression pattern overlaps extensively with that of the paired-like homeodomain protein Phox2b, a determinant of neuronal identity. In embryos deficient for Phox2b, RGS4 expression is downregulated in the locus coeruleus, sympathetic ganglia, and cranial motor and sensory neurons. Moreover, Phox2b cooperates with the basic helix-loop-helix protein Mash1 to transiently switch on RGS4 after ectopic expression in the chicken spinal cord. Intriguingly, we also identify a heterotrimeric G-protein alpha-subunit, gustducin, as coexpressed with RGS4 in developing facial motor neurons, also under the control of Phox2b. Altogether, these data identify components of the heterotrimeric G-protein signaling pathway as part of the type-specific program of neuronal differentiation.

    View details for Web of Science ID 000186680700017

    View details for PubMedID 14627646

  • Responsiveness to neurturin of subpopulations of embryonic rat spinal motoneuron does not correlate with expression of GFR alpha 1 or GFR alpha 2 DEVELOPMENTAL DYNAMICS Garces, A., Livet, J., Grillet, N., Henderson, C. E., deLapeyriere, O. 2001; 220 (3): 189-197


    Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFR alpha 1 (considered to be the favored coreceptor for GDNF) or GFR alpha 2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2.

    View details for Web of Science ID 000167131400001

    View details for PubMedID 11241828