Neuroplastin genetically interacts with Cadherin 23 and the encoded isoform Np55 is sufficient for cochlear hair cell function and hearing

Abstract

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin (Nptn) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn-knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn-knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of Neuroplastin in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn-knockout mice exhibiting hearing loss through genetic interaction with the Cdh23ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing.

Absence of Embigin accelerates hearing loss and causes sub-viability, brain and heart defects in C57BL/6N mice due to interaction with Cdh23

“…Cdh23 c.753A>G ), we recently demonstrated that the closely related gene Neuroplastin genetically interacts with the Cdh23 ahl allele potentiating the auditory deficit exhibited by Neuroplastin mutant mice. 7 Here, we demonstrate that Embigin also genetically interacts with the Cdh23 ahl allele, causing high-frequency hearing deficits from as early as 1-month of age. However, unlike in the case of Neuroplastin , the cause of hearing impairment in the Embigin mutant is not due to a physical interaction with plasma membrane calcium ATPase (PMCA) proteins.…”

“…Furthermore, histological sections prepared from 24-week old Emb +/+ and Emb tm1b/tm1b mice maintained on a C57BL/6N background showed normal cochlear morphology in the Emb tm1b/tm1b cochleae, with no evidence for loss or degeneration of: hair cells; spiral ganglion neurons; stria vascularis or supporting cells ( Figure 4 A). Given that we have recently reported significant IHC synaptic deficits in the Embigin-related Neuroplastin null mice, 7 synaptic coupling between IHCs and afferent terminals of spiral ganglion neurons in Emb tm1b mice maintained on a C57BL/6N background was assessed by whole-mount immuno-labeling. 5 , 7 Co-labeling with anti-Ribeye (pre-synaptic ribbon marker) and anti-GluR2 (post-synaptic density marker) revealed no evidence of a synaptic deficit with similar numbers of matched, unmatched, and total number of puncta per IHC observed in Emb tm1b/tm1b mice as compared to wild type littermates at 6-week of age ( Figures 4 B and 4C).…”

“…Given that we have recently reported significant IHC synaptic deficits in the Embigin-related Neuroplastin null mice, 7 synaptic coupling between IHCs and afferent terminals of spiral ganglion neurons in Emb tm1b mice maintained on a C57BL/6N background was assessed by whole-mount immuno-labeling. 5 , 7 Co-labeling with anti-Ribeye (pre-synaptic ribbon marker) and anti-GluR2 (post-synaptic density marker) revealed no evidence of a synaptic deficit with similar numbers of matched, unmatched, and total number of puncta per IHC observed in Emb tm1b/tm1b mice as compared to wild type littermates at 6-week of age ( Figures 4 B and 4C). Importantly, this time point was chosen for the analysis of the stereocilia bundle and synapse morphology as it precedes any effects attributable to the hypomorphic Cdh23 ahl allele.…”

“…The Embigin gene ( Emb ) encodes a highly glycosylated protein belonging to a small subgroup of immunoglobulin neural cell adhesion molecules alongside Basigin ( Bsg , also known as CD147) and Neuroplastin ( Nptn ), the latter of which has recently been linked to early onset hearing loss in mice. 5 , 6 , 7 Embigin was first described as a protein expressed at high levels during the development of several different tissue types. 8 Thus, it is thought to play a role in the early stages of embryogenesis.…”

Presynaptic Nrxn3 is essential for ribbon-synapse assembly in hair cells

“…CAMs act to bridge pre- and post-synaptic domains to initiate, maintain and specify synapses (reviewed in: (Südhof, 2021)). Work in mouse auditory inner hair cells (IHCs) has implicated the CAM Neuroplastin (Np55/Np65) and the neuronal CAM (NrCAM) in the development of IHC ribbon synapses (Carrott et al, 2016; Harley et al, 2018; Newton et al, 2022). More recent work in mouse found that a well-studied family of postsynaptic CAMs, neuroligins (Nlgns), are important for ribbon-synapse assembly (Ramirez et al, 2022).…”