Phosphatidylserine signaling in neuronal refinement of the cochlea

ABSTRACT Hearing loss is one of the most common disabilities in the United States and understanding how the inner ear develops and establishes proper communication with the brain will provide invaluable knowledge that can help us generate interventions for human hearing loss. Two types of hair cells are responsible for unique cochlear functions: inner hair cells (IHCs) receive 95% of the afferent innervation by spiral ganglion neurons (SGNs), whereas outer hair cells (OHCs) are involved in cochlear amplification and also establish limited afferent connections with type II SGNs. As in general nervous system development, during cochlear development, a notable phase involves the initial abundance and subsequent pruning of spiral ganglion neurons (SGNs) and synapses before the onset of hearing. The pathways that mediate this pruning process are a topic of active investigation. We propose that after excessive innervation, SGNs with functional synaptic connections are labeled as “active” and retained, while SGNs with weak synaptic connections are labeled “inactive” and marked for removal. Phosphatidylserine (PS) has been robustly identified throughout the literature as a prime “eat me” signal in the context of apoptosis and increasingly within synaptic pruning and neuronal remodeling. Therefore, the central hypothesis of this proposal is that the presentation of PS marks inactive OHC-SGN connections and labels them for removal by a yet unknown phagocytic mechanism. This hypothesis is supported by multiple lines of evidence generated in my preliminary studies: I first showed that ATP8A2, a lipid flippase known to clear PS from the exterior leaflet of the cell membrane, is specifically localized to OHCs and type II SGNs during the pruning period. Furthermore, in a mouse line that expresses a hypofunctional variant of ATP8A2, which was de novo generated and characterized in my preliminary studies, the synaptic connection between OHCs and type II SGNs are impaired. Based on these findings, my specific hypothesis is that active OHC-SGN axes express ATP8A2 and thus clear any presentation of PS on the outer leaflet of the cell membrane which ultimately allows ATP8A2-expressing cells to survive. This hypothesis will be tested in two specific aims (SAs). In SA1, I will test the hypothesis that ATP8A2 is necessary for proper pruning of type II SGNs, and that ATP8A2 localizes to SGNs in an activity-dependent manner to prevent PS externalization. In SA2 I will identify the cell types that recognize PS and explore the mechanism by which they facilitate pruning. This work will help elucidate the cellular and molecular mediators of cochlear pruning.