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ARTICLE
Impairment of
SLC17A8
Encoding Vesicular GlutamateTransporter-3, VGLUT3, Underlies Nonsyndromic DeafnessDFNA25 and Inner Hair Cell Dysfunction in Null Mice
Je´roˆme Ruel,
Sarah Emery,
Re´gis Nouvian,
Tiphaine Bersot,
Be´ne´dicte Amilhon,
Jana M. Van Rybroek,
Guy Rebillard,
Marc Lenoir,
Michel Eybalin,
Benjamin Delprat,
Theru A. Sivakumaran,
Bruno Giros,
Salah El Mestikawy,
Tobias Moser,
Richard J.H. Smith,
Marci M. Lesperance,
3,
*and Jean-Luc Puel
Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, themost common sensory defect associated with aging in humans. We have identified
SLC17A8
, which encodes the vesicular glutamatetransporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyn-dromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C
/
T (p.A211V), was found to segregate withDFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant com-mon ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUTsubtypes (VGLUT1-3), suggesting animportant functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs)beforereleasingitontoreceptorsofauditory-nerveterminals.Nullmicewithatargeteddeletionof
Slc17a8
exon2lackedauditory-nerveresponsestoacousticstimuli, althoughauditory brainstem responsescouldbe elicitedbyelectricalstimuli,androbustotoacousticemis-sions were recorded. Ca
2
þ
-triggered synaptic-vesicle turnover was normal in IHCs of
Slc17a8
null mice when probed by membranecapacitancemeasurementsat2weeksofage.Later,thenumberofafferentsynapses,spiralganglionneurons,andlateralefferentendingsbelowsensoryIHCsdeclined.Ribbonsynapsesremainingby3monthsofagehadanormalultrastructuralappearance.Weconcludethatdeafness in
Slc17a8
-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential forauditory coding at the IHC synapse.
Introduction
Age-related hearing impairment, or presbycusis, is themost common sensory disorder in older individuals, withprevalence increasing with age.
Both genetic factors andenvironmental factors such as noise exposure influencethe development of presbycusis.
Although a remarkablenumberofgenesresponsibleforhereditaryhearingimpair-ment have been identified in recent years, a significantnumber of genes associatedwithmapped deafness loci stillawait discovery.
The dominant (DFNA) loci representinteresting candidates for presbycusis, because the hearingloss associated with these loci is typically delayed-onsetand progressive, and with few exceptions, predominantlyaffects high frequencies. We previously mapped one suchlocus, DFNA25 (MIM 605583), to chromosome 12q21-q24.
Molecular genetic studies of mutant mice and of hu-mans with hereditary hearing loss have contributed toour understanding of the unique properties of the afferentsynapse of cochlear inner hair cells.
Bassoon (MIM604020) is a presynaptic scaffolding protein that isrequired to anchor the ribbon at the presynaptic activezone of the sensory inner hair cell (IHC) synapse and forsynchronous signaling to the auditory nerve.
Bassoonmutant mice show reduction of the presynaptic readilyreleasable pool of vesicles and impaired synchronous andsound-evoked activation of spiral ganglion neurons. Oto-ferlin (MIM 603681), a protein encoded by the
OTOF
gene, is essential for the late step of synaptic-vesicle exocy-tosis and may act as the major Ca
2
þ
sensor that triggersmembrane fusion at the IHC ribbon synapse.
Mutationsin
OTOF
are responsible for DFNB9 (MIM 601071) deaf-ness, which is characterized by profound hearing losswith documentation of preserved outer hair cell (OHC)function as measured by otoacoustic emission (OAE) insome patients.
Very recently, Seal et al.
identified another key compo-nent of the IHC synapse, the vesicular glutamate trans-porter-3 (VGLUT3 [MIM 607557]), which is encoded bythe
SLC17A8
gene and transports the neurotransmitterinto synaptic vesicles before it is released into the synapticcleft.The geneticdeletionof
Slc17a8
inmiceresultsinpro-found deafness because of the lack of glutamate release.Because
SLC17A8
maps within the DFNA25 interval, weevaluated
SLC17A8
as a candidate gene and studied
1
InsermU583,InstitutdesNeurosciences,HoˆpitalSaintEloi,34091Montpellier,France;
2
Universite´Montpellier1,34091Montpellier,France;
3
Divisionof Pediatric Otolaryngology,Department ofOtolaryngology-HeadandNeckSurgery,UniversityofMichigan Health System,Ann Arbor,MI48109-5241,USA;
4
InnerEarLab, Department of Otolaryngology and Center for Molecular Physiology of the Brain, University of Goettingen Medical School, Goettingen37075, Germany;
5
Inserm U 513, 9 Quai Saint Bernard, 75252 Paris, France;
6
Department of Otolaryngology and Head and Neck Surgery, University of Iowa, Iowa City, IA 52242, USA;
7
Bernstein Center for Computational Neuroscience, University of Goettingen, Goettingen 37075, Germany
8
These authors contributed equally to this work *Correspondence:lesperan@med.umich.eduDOI 10.1016/j.ajhg.2008.07.008.
ª
2008 by The American Society of Human Genetics. All rights reserved.
The American Journal of Human Genetics
83
, 1–15, August 8, 2008
1
Please cite this article in press as: Ruel et al., Impairment of
SLC17A8
Encoding Vesicular Glutamate Transporter-3, VGLUT3, UnderliesNonsyndromic Deafness DFNA25 and Inner..., The American Journal of Human Genetics (2008), doi:10.1016/j.ajhg.2008.07.008
amousewithatargeteddeletionof
Slc17a8
exon2.
Here,we provide the first evidence that a glutamate-transmis-sion deficit is directly responsible for human sensorineuraldeafness, DFNA25, because of a mutation of
SLC17A8
, seg-regating in two families. Although
Slc17a8
À
/
À
mice exhibitno response to sound, electrical stimulation applied to theround-window membrane elicits clear auditory brainstemresponses.Inaddition,wedemonstratethatIHCpresynap-tic function and morphology are intact up to the stage of transmitter release after vesicle fusion, making
Slc17a8
À
/
À
mutant mice an excellent model to test therapeutic inter-ventions in the inner ear.
Subjects and Methods
Human Subjects
These studies were approved by the Institutional Review Boards of the University of Michigan (IRBMED) and the University of Iowa.
Phenotype and Mapping of DFNA25
The DFNA25 locus was mapped in an American family (family 1)of Czech descent segregating autosomal-dominant, progressive,high-frequency sensorineural hearing loss.
The DFNA25 intervalas defined by key recombination events spanned 20 cM and14.3 Mb on chromosome 12q21-q24
and includes more than60genes, including
SLC17A8
(UCSCGenomeBrowser).Penetrancewas shown to be age dependent and possibly also dependent onthe sex of the parent of origin.
Iowa Family 1490, an American family of German descent, seg-regated a similar type of high-frequency, autosomal-dominantprogressive hearing loss. A genome-wide linkage analysis wasperformed as previously described
and identified a locus on12q21.2-q24.21 defined by markers D12S326 and D12S79, span-ning 38.9 cM, with a maximum LOD score of 3.91 at
q
¼
0 forD12S78. In both families, hearing loss appeared to be nonsyn-dromic, without a history of seizure disorders or other neurologicdysfunction in affected family members.
Sequencing SLC17A8 from Human Genomic DNA Samples
DNAsequencingofall12codingexonsandintron-exonjunctionswas performed in four DNA samples from individuals in family 1(three affected and one unaffected) and one affected individualfrom family 1490. In addition, five DNA samples from individuals(three affected and two unaffected) in an unrelated family (family63) with nonsyndromic, dominant, progressive, high-frequencyhereditaryhearingimpairmentwereassayed.Primersforpolymer-ase chain reaction (PCR) were designed with the Primer 3.0 pro-gram (Table S1available online). PCR was done with Failsafe(EpicenterBiotechnologies,Madison,WI,USA)orBiolinereagents(Bioline, Taunton, MA, USA) according to the manufacturer’sinstructions. Standard thermocycling conditions were used with50
C–70
C annealing temperatures and 30 s extension for 35 cy-cles. PCR products were purified with the QiaQuick DNA extrac-tion kit (QIAGEN, Valencia, CA, USA) and sequenced by dye-ter-minator cycle sequencing on a 3730
xl
DNA analyzer (AppliedBiosystems, Weiterstadt, Germany) at the University of MichiganDNA Sequencing Core or a 3130
xl
Genetic Analyzer (AppliedBiosystems) at the Molecular Otolaryngology Research Laboratorywith the manufacturer’s recommended reagents and software. Se-quence data were compared to reference sequences NM_139319(mRNA) and NP_647480 (protein) with Lasergene (DNAStar, Mad-ison, WI, USA).
Assaying the Mutation for Segregation in the Families and Presence in Controls
The presence of the c.632T
/
C mutation was assayed by sequenc-ing exon 5 in remaining available DNA samples (17 affected, 19unaffected,9unknownstatus,and8married-inspouses)infamily1andincontrols.Atotalof267unrelatedcontrolsweresequencedof which 90 were from the DNA Polymorphism DiscoveryResource Panel, 99 were from the Caucasian North AmericanPanel, and 78 were from Human Variation Panels, consisting of Northern European (ten samples), Czech (ten samples), AfricanAmerican, Mexican, Southeast Asian, South American, MiddleEastern, South African, Ashkenazi Jewish, Chinese, and NorthAfrican panels (Coriell Cell Repositories, Camden, NJ, USA).For family 1490, the presence of the c.632T
/
C mutation wasdetermined by KpnI (New England Biolabs, Ipswich, MA, USA)restriction digest of exon 5 in remaining available DNA samples(seven affected, five unaffected, and five married-in spouses).
Conservation Analysis
Amino acid sequences representing exons 5 and 6 of select pro-teins from the VGLUT protein family in
H. sapiens
(VGLUT1[MIM 605208], VGLUT2 [MIM 607563], and VGLUT3),
R. norvegi-cus
, and
M. musculus
and homologous proteins in
C. elegans
and
D.melanogaster
wereobtainedfromtheNCBIdatabaseandalignedwith ClustalX2 software.
Identification of Previously Unreported STRs and SNPs
We identified short tandem repeats (STRs) and single-nucleotidepolymorphisms (SNPs) not previously reported by searchingsequences from bacterial artificial chromosomes mapping to theregionofthe
SLC17A8
genewithTandemRepeatsFindersoftware.Primer design and PCR for screening newly identified STRs wasdone as described above for
SLC17A8
exon primers. PCR productwas separated by polyacrylamide gel electrophoresis and detectedwith silver staining. Primer design and PCR for SNP screening wasalso done as described above for
SLC17A8
exon primers, and PCR products were analyzed by restriction digests. Restriction enzymesfor screening SNPs were used according to the manufacturer’sinstructions (New England Biolabs) and are as follows: rs79703820with BsrI; rs11110349 with AseI; rs7485480 with DdeI; rs11568544with NdeI; rs11110390 with NheI; and rs35735 with BstEII.
Haplotype Analysis
Haplotype analysis was done with STRs and single nucleotideSNPs (Table S2). Primers to amplify previously reported STRswere designed on the basis of reports (NCBI) and fluorescentlylabeled. PCR was done with AmpliTaq Gold polymerase andbuffers (Applied Biosystems) according to the manufacturer’sinstructions, and products were evaluated on a 3130
xl
GeneticAnalyzer (Applied Biosystems). Data analysis was performedwith GeneMarker software (Softgenetics, State College, PA, USA),and haplotype reconstruction was done with custom-madeGeneScreen software (Itty Bitty Computers, San Jose, CA, USA).
Animals
The care and use of animals followed the animal-welfareguidelines of the Institut National de la Sante´et de la RechercheMe´dicale (Inserm), and was approved by the Ministe`re Franc¸aisde l’Agriculture et de la Peˆche (authorization number A 3417231).Alleffortsweremadetominimizethenumberandsufferingoftheanimals used.
Generation of the Slc17a8
À
/
À
Mice
The entire procedure to generate the
Slc17a8
knockout mice wasperformed by the Institut Clinique de la souris (Illkirch, France).
2
The American Journal of Human Genetics
83
, 1–15, August 8, 2008
Please cite this article in press as: Ruel et al., Impairment of
SLC17A8
Encoding Vesicular Glutamate Transporter-3, VGLUT3, UnderliesNonsyndromic Deafness DFNA25 and Inner..., The American Journal of Human Genetics (2008), doi:10.1016/j.ajhg.2008.07.008
In brief, the null mutation in the
Slc17a8
mouse gene, located onchromosome 10, was created by homologous recombination.
Removalofexon2,afterinsertionofthetargetingconstruct,intro-duced a stop codon into exon 3 in frame with exon 1, resulting ina complete loss of allele function. Consequently, mutant mice nolonger expressed
Slc17a8
mRNA. All experiments were performedwithF2wild-typeandhomozygouslittermatesderivedfromcross-ing F1 heterozygous
Slc17a8
þ
/
À
mice (129/Sv
3
C57BL/6). Micewere identified by PCR analysis of tail DNA with three primerslocated in intron 2–3 of the
Slc17a8
gene or in the neomycincassette.
Animal Surgery
Animals were anaesthetized by an intraperitoneal injection of a mixture of Rompun 2% (3 mg/kg) and Zoletyl 50 (40 mg/kg).Both pinna were severed to facilitate the insertion and placementof the distortion product otoacoustic emissions (DPOAEs) probe.A retroauricular incision of the skin was performed on both sides,andthebullatympaniwereopenedtoexposetheroundwindows.
Distortion Product Otoacoustic Emission Recordings
DPOAEs were recorded in the external auditory canal with anER-10C S/N 2525 probe (Etymotic Research, Elk Grove Village,IL, USA) consisting of two emitters and one microphone. Thetwo primary tones were generated, and the distortion was pro-cessed by the Cubdis system HID 40133DP (Mimosa Acoustics,Champaign, IL, USA). The probe was self-calibrated for the twostimulating tones before each recording. The two tones werepresented simultaneously, sweeping f2 from 0.5 kHz to 20 kHzby quarter octave steps, and maintaining the f2/f1 ratio constantat 1.2. The primary intensities of f2 and f1 were set at 60 dB and55 dB SPL (ref. 2
3
10
À
5
Pa), respectively. For each frequency,the cubic distortion product (2f1-f2) and the neighboring noisemagnitudes were measured and expressed as a function of f2.Data are expressed as means
5
standard error of the mean (SEM).
Recording of Cochlear- and Auditory-Nerve Potentials
The acoustical stimuli were generated by an arbitrary functiongenerator (type 9100R; LeCroy Corporation, Chestnut Ridge, NY,USA), consisting of 9 ms tone bursts with a 1 ms rise and falltime delivered at a rate of 10/s. Tone bursts were passed througha programmable attenuator and delivered to the animal by a JBL075 loudspeaker (James B. Lansing Sound, Los Angeles, CA, USA)in a calibrated free field. Cochlear potentials were obtained via asilver electrode placed on the round window and then amplified(
3
2000) by a Tektronix (TM 503) differential amplifier and digita-lized(50kHzsamplingrate,witha12-bitdynamicrangeand1024samples per burst), averaged 256 times, and stored on a computer(Dell Dimensions, Austin, TX, USA). This signal was then digitallyfiltered either with a low-pass filter (cutoff frequency 3.5 kHz) todisplay the compound action potential (CAP) and the summatingpotential (SP) or with a band-pass filter centered on the frequencyof stimulation with a 4 kHz span to display the cochlear micro-phonic (CM). We obtained intensity-amplitude functions of thesepotentialsateachfrequencytested(2,4,6,8,10,12,16,20,26,32,and 64 kHz) by varying the intensities of the tone bursts from 0 to100 dB SPL, in 5 dB steps. The CAP, SP, and CM thresholds weredefined as the minimum sound intensity necessary to elicita clearly distinguishable response.
Electrical Stimulation of the Auditory Nerve
Electricalstimulationconsistedin25
m
srectangularpulsesofalter-natepolaritydeliveredatarateof20/sbyacustom-madeDCstim-ulator.Theintensityofthepulseswasgradedfrom100
m
Ato2mAby100
m
Asteps.Stimuliweredeliveredthroughapairofelectrodesplaced respectively on the round window and in the neighboringmuscles. To insure that the electrical pulses directly activatedcochlear spiral ganglion neurons, we applied a droplet of artificialperilymphcontaining10
m
MofNa
2
þ
channelblockertetrodotoxin(TTX)intotheround-windowniche.Auditorybrainstemresponses(ABRs)wererecordedbeforeand30minafterTTXapplication.Theartificial perilymph solution had the following composition: 137mM NaCl, 5 mM KCl, 2 mM CaCl
2
, 1 mM MgCl
2
, 1 mM NaHCO
3
,11mMglucose,and10mMHEPES.ThepHwas7.4andtheosmo-larity was maintained at 300 mOsm/kg H
2
O.
Brainstem-Response Recordings
Brainstem-evokedresponsestoeitheracousticalstimulationoftheear or to electrical stimulation of the cochlear nerve were differen-tially recorded by needle electrodes placed subcutaneously respec-tively at the vertex and at base of the pinna, whereas the groundelectrode was placed in the muscles of the posterior leg. The phys-iological signal was amplified 100,000 times by a Grass P5 seriesamplifier(GrassInstruments,Quincy,MA,USA),digitalized(50kHzsampling rate, with a 12-bit dynamic range and 1024 samples perburst), and averaged 1000 times (Dell Dimensions).
Patch-Clamp Recordings of Ca
2
þ
Currents and Membrane Capacitance
The animals were maintained according to the animal-welfareguidelines of the University of Goettingen and the State of LowerSaxony. After cervical dislocation of mice (postnatal day 12 [P12]to 18 [P18]), IHCs of the apical coil of freshly dissected organs of Corti were patch-clamped at their basolateral face at room tem-perature in perforated-patch configuration as described previ-ously.
The pipette solution for perforated-patch recordingscontained the following: 130 mM Cs-gluconate, 10 mM tetra-ethylammonium-Cl (TEA-Cl), 10 mM HEPES, 1 mM MgCl
2
, and10mM4-Aminopyridine(4-AP)aswellas250
m
g/mlamphotericinB. The extracellular solution contained the following: 105 mMNaCl, 35 mM TEA-Cl, 2.8 mM KCl, 1 mM MgCl
2
, 10 mM HEPES,2 mM CaCl
2
, 1 mM CsCl, and 10 mM D-glucose. Solutions wereadjusted to pH 7.2 and had osmolarities between 290 and 310mOsm/kgH
2
O.AllchemicalswereobtainedfromSigma(St.Louis,MO, USA), with the exception of CsOH (Aldrich, Milwaukee, WI,USA) and amphotericin B (Calbiochem, La Jolla, CA, USA). Ca
2
þ
current integrals were calculated from the total depolarization-evoked inward current, including Ca
2
þ
tail currents after P/10leak subtraction (i.e., from the start of the depolarization step to1.5 ms after the end of the depolarization step). Cells that dis-playedamembranecurrentexceeding
À
30pAat
À
87mVweredis-carded from the analysis. No series resistance (
R
s
) compensationwas applied; however, recordings with
R
s
>
30 M
U
were discardedfrom the analysis. Current was low-pass filtered at 4 kHz and sam-pled at 10 kHz for capacitance measurements and at 40 kHz forrecordings of Ca
2
þ
current-voltage relationships. All voltageswere corrected for the liquid junction potential between pipetteand bath (calculated at 16.9 mV).
Capacitance Measurements
Membranecapacitance(
C
m
)wasmeasuredwiththeLindau-Nehertechnique
implemented in the software-lockin module of Pulse(HEKAElektronik,Lambrecht,Germany)combinedwithcompen-sation of pipette and resting cell capacitances by the EPC-9 (HEKAElektronik)compensationcircuitries.A1kHz,70mVpeak-to-peak sinusoid was applied about the holding potential of
À
87 mV.
D
C
m
was estimated as the difference of the mean
C
m
over 400 ms afterthe end of the depolarization (the initial 250 ms was skipped) andthemeanprepulsecapacitance(400ms).Mean
D
C
m
andCa
2
þ
cur-rent estimates present grand averages calculated from the meanestimates of individual IHCs. Data analysis was performed withIgor Pro software (WaveMetrics, Lake Oswego, OR, USA).
The American Journal of Human Genetics
83
, 1–15, August 8, 2008
3
Please cite this article in press as: Ruel et al., Impairment of
SLC17A8
Encoding Vesicular Glutamate Transporter-3, VGLUT3, UnderliesNonsyndromic Deafness DFNA25 and Inner..., The American Journal of Human Genetics (2008), doi:10.1016/j.ajhg.2008.07.008
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