Cellular and Molecular Mechanisms of Cochlear Innervation

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PROJECT SUMMARY Synaptic connections between spiral ganglion neurons (SGNs) and hair cells in the cochlea are critical for hearing and lost in many forms of hearing impairment. These synapses form prior to hearing onset and are activated by supporting cell-induced, intrinsically generated activity that is prominent within the developing cochlea. This periodic and spontaneous synaptic activity initiates SGN burst firing, which promotes SGN survival, SGN maturation, and development of frequency tuning in central auditory circuits. Our prior studies revealed an unexpected role for otic mesenchyme cells (OMCs) in establishing appropriate SGN-hair cell connectivity through activation of POU3F4, a transcription factor associated with X-linked deafness. POU3F4 is expressed only by OMCs in the cochlea. We found that, in OMCs adjacent to developing SGNs, POU3F4 upregulates Eph receptor-A4 (EphA4) to promote SGN fasciculation. Subsequently, we discovered that POU3F4 is also necessary for SGN survival. Recent data from Ca2+ imaging and single cell RNA sequencing (scRNAseq) experiments support a model whereby OMCs promote SGN development by regulating spontaneous activity through POU3F4, insulin-like growth factor (IGF) and Semaphorin (SEMA) signaling. Here, we will test the hypothesis that expression of these factors by OMCs promotes both prehearing spontaneous activity and the establishment of hair cell–SGN synaptic connections to enable hearing. This hypothesis will be tested in three aims. In Aim 1, we will determine the role of POU3F4 and OMCs in generating prehearing spontaneous activity. In Aim 2, we will define the mechanisms by which SEMA5A inhibits SGN spontaneous activity. In Aim 3, we will determine how POU3F4 promotes IHC innervation. These studies will incorporate a range of Ca2+ imaging, physiology, molecular profiling, and tissue culture techniques. We and others have documented mechanisms of SGN guidance and survival, but there is still limited understanding of how SGNs differentiate and form synapses with hair cells. After acoustic overexposure, SGN cell bodies can survive for long periods of time, but their peripheral processes retract away from the hair cells without easily reconnecting. At present, how to re-establish these connections is not well understood. Successful completion of these aims will define how genes expressed by OMCs control the formation of the first synapses in the auditory pathway. By understanding the mechanisms of cochlear innervation during development, we will begin to build a “toolbox” that could be used to develop molecular therapies for rewiring the damaged adult cochlea. This research will also reveal key mechanisms required for the development and regulation of cochlear spontaneous activity. Since neural activity is recognized as a crucial aspect of circuit formation, it is possible that activity could be an important consideration in cochlear rewiring.