ARTICLE Impairment of SLC17A8 Encoding Vesicular Glutamate Transporter-3, VGLUT3, Underlies Nonsyndromic Deafness DFNA25 and Inner Hair Cell Dysfunction in Null Mice Je ´ro ˆme Ruel, 1,2,8 Sarah Emery, 3,8 Re ´gis Nouvian, 4 Tiphaine Bersot, 1,2 Be ´ne ´dicte Amilhon, 5 Jana M. Van Rybroek, 6 Guy Rebillard, 1,2 Marc Lenoir, 1,2 Michel Eybalin, 1,2 Benjamin Delprat, 1,2 Theru A. Sivakumaran, 3 Bruno Giros, 5 Salah El Mestikawy, 5 Tobias Moser, 4,7 Richard J.H. Smith, 6 Marci M. Lesperance, 3, * and Jean-Luc Puel 1,2 Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-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 with DFNA25 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 VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emis- sions were recorded. Ca 2þ -triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse. Introduction Age-related hearing impairment, or presbycusis, is the most common sensory disorder in older individuals, with prevalence increasing with age. 1 Both genetic factors and environmental factors such as noise exposure influence the development of presbycusis. 2,3 Although a remarkable number of genes responsible for hereditary hearing impair- ment have been identified in recent years, a significant number of genes associated with mapped deafness loci still await discovery. 4 The dominant (DFNA) loci represent interesting candidates for presbycusis, because the hearing loss associated with these loci is typically delayed-onset and progressive, and with few exceptions, predominantly affects high frequencies. We previously mapped one such locus, DFNA25 (MIM 605583), to chromosome 12q21- q24. 5 Molecular genetic studies of mutant mice and of hu- mans with hereditary hearing loss have contributed to our understanding of the unique properties of the afferent synapse of cochlear inner hair cells. 6,7 Bassoon (MIM 604020) is a presynaptic scaffolding protein that is required to anchor the ribbon at the presynaptic active zone of the sensory inner hair cell (IHC) synapse and for synchronous signaling to the auditory nerve. 8 Bassoon mutant mice show reduction of the presynaptic readily releasable pool of vesicles and impaired synchronous and sound-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 2þ sensor that triggers membrane fusion at the IHC ribbon synapse. 9 Mutations in OTOF are responsible for DFNB9 (MIM 601071) deaf- ness, which is characterized by profound hearing loss with documentation of preserved outer hair cell (OHC) function as measured by otoacoustic emission (OAE) in some patients. 10,11 Very recently, Seal et al. 12 identified another key compo- nent of the IHC synapse, the vesicular glutamate trans- porter-3 (VGLUT3 [MIM 607557]), which is encoded by the SLC17A8 gene and transports the neurotransmitter into synaptic vesicles before it is released into the synaptic cleft. The genetic deletion of Slc17a8 in mice results in pro- found deafness because of the lack of glutamate release. Because SLC17A8 maps within the DFNA25 interval, we evaluated SLC17A8 as a candidate gene and studied 1 Inserm U 583, Institut des Neurosciences, Ho ˆpital Saint Eloi, 34091 Montpellier, France; 2 Universite ´ Montpellier 1, 34091 Montpellier, France; 3 Division of Pediatric Otolaryngology, Department of Otolaryngology-Head and Neck Surgery, University of Michigan Health System, Ann Arbor, MI 48109-5241, USA; 4 InnerEarLab, Department of Otolaryngology and Center for Molecular Physiology of the Brain, University of Goettingen Medical School, Goettingen 37075, 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.edu DOI 10.1016/j.ajhg.2008.07.008. ª2008 by The American Society of Human Genetics. All rights reserved. 278 The American Journal of Human Genetics 83, 278–292, August 8, 2008