Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

Sungsu Lee,Yehoash Raphael,Donald L Swiderski,Hsin-I Jen,Diane M Prieskorn,Lisa A Beyer,Andrew K Groves,Jae-Jun Song,Melih Acar

doi:10.1038/s41598-020-78167-8

Abstract

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Highlights

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits
We evaluated the combinatorial effect of Atoh[1] and Gfi[1], both in neonatal cochlear explants and in the adult cochlea of the P ou4f3DTR mouse model, where HCs can be completely ablated by the addition of diphtheria toxin (DT)[40,41]
Atoh1-treated explants gave rise to ectopic HC-like cells (HCLCs) located in the greater epithelial ridge region, medial to the inner HCs (Fig. 1a)

Summary

Introduction

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used overexpression of the HC-specific transcription factor Atoh[1], but to date this approach has yielded low and variable efficiency of HC production. We evaluated the combinatorial effects of Atoh[1] and Gfi[1] over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Combinatorial Atoh[1] and Gfi[1] induction is a promising strategy to promote HC regeneration in the mature mammalian cochlea. In contrast to non-mammalian vertebrates, auditory HCs in the organ of Corti (OC) of mature mammals do not spontaneously regenerate once lost, and hearing loss is permanent. Notch inhibition leads to Atoh[1] activation and HC regeneration in neonatal mice, and to some extent in the mature e ar[20,21,22], once again, the ability of Notch inhibition to promote HC formation declines precipitously with a ge[23]

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Conclusion