A Neuron‑Like Cellular Model for Severe Tinnitus Associated with Rare Variations in the ANK2 Gene

Abstract

Tinnitus is the perception of sound without an external source, often associated with changes in the auditory pathway and different brain regions. Recent research revealed an overload of missense variants in the ANK2 gene in individuals with severe tinnitus. ANK2, encoding ankyrin-B, regulates axon branching and inhibits microtubule invasion. Missense mutations in ANK2 may promote excessive axonal branching and the formation of excitatory synapses. This study aims to generate a patient-derived iPSC model from an individual with severe tinnitus and to differentiate these cells into otic-neural progenitors and inner ear neurons. We successfully generated a severe tinnitus cellular model through cell reprogramming. Using a two-stage neural differentiation protocol, we differentiated these cells into otic-neural progenitors and neuron-like cells. We confirmed the expression of genes, proteins, and cellular markers, including ANK2, otic-neural progenitors, and neuron-like cells through qPCR and immunostaining. Our analysis revealed higher ANK2 expression in the control cell line compared to the patient cell line. Although both lines formed multipolar neurons, the patient cell line displayed a unique pattern of closely grouped neurons with increased neuronal projections and dendrites compared to the control. This cellular model provides a valuable tool for studying the cellular and molecular changes associated with the ANK2 gene. It holds great promise for the development of novel drug and gene-based therapies for severe tinnitus.