Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

Abstract

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental timepoint of 72 hours post-fertilization (hpf) low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after upregulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggest a function for the transmembrane domain in liposome docking. We conclude that that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

Otoferlin requires a free intravesicular C-terminal end for synaptic vesicle docking and fusion

“…Taken together, our results suggest that tagging the C-terminal otoferlin TMD with GFP impairs the protein’s function, potentially by reducing vesicular interaction and/or tethering—an indispensable step for synaptic vesicle docking and fusion. In good agreement with the study of Manchanda et al, (2021) showing that the truncation of TMD reduces membrane docking in zebrafish hair cells, our study underlines the importance of TMD.…”

The sensory shark: high-quality morphological, genomic and transcriptomic data for the small-spotted catsharkScyliorhinus caniculareveal the molecular bases of sensory organ evolution in jawed vertebrates

“…in elasmobranch fishes, Bellono et al 2017; and in bony fish, Modrell et al 2017). Our analysis of transcriptomic data supports the identification of ribbon-synapse components in electrosensory cells of the catshark, similar to what was proposed for the paddlefish (Modrell et al 2017), with expression of glutamate transporters, L-type voltage-gated calcium channels (Ca v 1 channels) and the transmembrane protein Otoferlin (Modrell et al 2017; Manchanda et al 2021). In the paddlefish, little transcriptomic data differentiated hair cells from electrosensory cells with the exception of two potassium voltage-gated channels described to be specific to the ampullary organs (Modrell et al 2017).…”

Putative COVID-19 therapies imatinib, lopinavir, ritonavir, and ivermectin cause hair cell damage: A targeted screen in the zebrafish lateral line

“…An increase in ribbon number is unusual and could reflect increased synaptic inhibition via GABAergic signaling. GABA receptors are expressed in zebrafish lateral line hair cells and in multiple cell types within the mammalian cochlea, including outer hair cells and type I afferent neurons ( Plinkert et al, 1989 ; Arnold et al, 1998 ; Wedemeyer et al, 2013 ; Toro, 2018 ; Manchanda et al, 2021 ). GABA agonism could reduce glutamatergic signaling, initiating a compensatory mechanism that results in more excitatory synapses.…”