Characterisation of pitch: an early onset model of sensorineural deafness

At MRC Harwell n-ethyl-n-nitrosourea (ENU) mutagenesis coupled with auditory phenotyping is utilised to generate new mouse models of hearing loss. This approach identified pitch, an autosomal recessive model of early-onset, sensorineural deafness (+90dB). Mapping studies identified a 1.6Mb critical region comprising a novel deafness locus on chromosome 9 and prioritised candidate gene sequencing identified a coding mutation in Neuroplastin (Nptn). This thesis details experiments to investigate the role of Neuroplastin in the auditory pathway. Complementation studies using a second nonsense allele (NptnY219X ) confirmed Neuroplastin as the causative gene in pitch. Modelling of Nptnpitch in silico suggested that the pitch mutation is likely harmful to protein function and in vitro studies using Nptn-tagged constructs revealed that the pitch mutation affected protein processing, and was thus likely a loss-of-function mutation. Expression studies identified that the 65kDa Nptn isoform, Np65, is expressed in the inner hair cells (IHCs) and outer hair cells (OHCs) of the cochlea in wild type mice. However, Np65 is absent in the cochleae of Nptnpitch/pitch animals. Inner ear morphology appears grossly normal in Nptnpitch/pitch mice at postnatal day (P)16, even though a severe auditory deficit is already present at this age. Electrophysiological studies suggest that whilst the IHCs of Nptnpitch/pitch mice begin to mature normally, their maturation arrests shortly after the onset of hearing. Immunolabelling of the auditory ribbon synapse identified an increased number of mismatched pre-synaptic ribbons (RIBEYE) and post-synaptic densities (GluR2) in Nptnpitch/pitch compared to wild type controls. In conclusion, my studies demonstrate that Np65 regulates the physiological and morphological differentiation of cochlear inner hair cells at the onset of hearing and as such coordinates one of the most distinctive functional refinements of the mammalian auditory system. These data also confirm that Nptn is a new deafness-related gene.