Assistant Professor, Stanford University, School of Medicine, Stanford, CA, USA
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
Assistant Professor, Otolaryngology - Head & Neck Surgery Divisions
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.
Osmotic stabilization prevents cochlear synaptopathy after blast trauma
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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
Mechanosensory hair cells express two molecularly distinct mechanotransduction channels
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
Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function.
journal of neuroscience
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
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
Using injectoporation to deliver genes to mechanosensory hair cells.
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
TMIE Is an Essential Component of the Mechanotransduction Machinery of Cochlear Hair Cells.
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
TMHS Is an Integral Component of the Mechanotransduction Machinery of Cochlear Hair Cells
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
Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain
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 2010; 5 (1): 9–12
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
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
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
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
Regulator of G Protein Signaling-4 Controls Fatty Acid and Glucose Homeostasis
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
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
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
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
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
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