Prestin and electromotility may serve multiple roles in cochlear outer hair cells.

Outer hair cells (OHCs) are innervated by both medial olivocochlear (MOC) efferents and type II afferents, which also innervate supporting cells to form a local neural network. It has also been demonstrated that prestin provides the molecular basis for OHC somatic electromotility, amplifying movements within the organ of Corti. Although not anticipated, early-onset OHC loss was found in two prestin transgenic mouse models that either lack prestin protein or lack electromotility. To uncover the molecular pathways that evoke OHC death, we profiled the coding transcriptome of OHCs from wildtype (WT), prestin-knockout (KO), and 499-knockin (KI) mice using single-cell RNA sequencing (scRNA-seq). scRNA-Seq transcriptomics and pathway analyses did not reveal common pathways associated with OHC loss observed in prestin-KO and 499-KI mice. Clustering enrichment analysis showed that increased gene expression in OHCs from prestin-KO mice was associated with lipid metabolic processes and cell death pathways. These mRNA profiles likely contribute to the OHC loss observed in prestin-KO mice and support the notion that prestin is also a structural protein, important for the normal plasma membrane compartmentalization that is essential to establish MOC efferent synapses. In contrast, the mRNA profile of OHCs from 499-KI mice did not provide a rational explanation of the early-onset OHC loss in this mutant. OHCs from 499-KI mice have normal plasma membrane compartmentalization and normal OHC-MOC contacts. However, 499 prestin lacks electromotility and appears to change the local neural network around OHCs, as more synaptic markers were found near neighboring supporting cells when compared to WT and prestin-KO mice. Thus, OHCs in prestin-KOs (no prestin protein, no electromotility) and 499-KIs (prestin protein present, no electromotility) may influence local neuronal networks in different ways. Collectively, our data suggest that prestin and its motile properties are important for OHC survival and the maintenance of local afferent/efferent circuits, as well as for its role in cochlear amplification. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.