Toward the therapy of AUNA1 auditory neuropathy – Diaphanous

To decipher the mechanism responsible of the AUNA1 deafness, we will take advantage of two transgenic mouse lines, which carry the mouse Diap3 gene (homologue to the human DIAPH3) under the control of the cytomegalovirus promoter and therefore overexpress the Diaph3 protein (collaboration with Dr. Marci Lesperance, University of Michigan).

Our preliminary result shows that the two transgenic lines overexpressing Diaph3 protein, display progressive threshold shift with preserved outer hair cells (OHCs) function, mimicking therefore the AUNA1 human auditory neuropathy. Our preliminary data indicate an aberrant shape of the apical part of the inner hair cells (IHCs), i.e., swelling of the cuticular plate, which is a dense structure composed of an actin gel, suggesting that IHCs are not able to encode sound stimulation. Altogether, these preliminary results suggest that mutant mice that overexpress Diaph3 replicate AUNA1 and pinpoint a defect in IHCs.

Up to now, the therapeutic strategies for hearing deficits remain limited. Because auditory neuropathy degrades the signal transfer from the cochlea to the brain and leave the cochlear amplification unaffected, hearing aids are no help against auditory neuropathies. Identifying the signaling pathway in AUNA1 should target attractive candidate for futur therapies.

Hearing impairments are major health and socio-economical issues in industrialized country. During the two last decades, a novel form of deafness called auditory neuropathy has been identified. The auditory neuropathy arises from a defect of the sensory inner hair cells (IHCs) of the cochlea and/or in the afferent fibers along the ascending auditory pathway. Because auditory neuropathy degrades the signal transfer from the cochlea to the brain, unraveling the molecular mechanism of auditory neuropathy is of prime interest to understand poor speech intelligibility.

The Diaphanous homolog 3 (DIAPH3) gene is responsible for the autosomal dominant nonsyndromic auditory neuropathy 1 (AUNA1). DIAPH3 belongs to the formin related family and is involved in the actin nucleation. Strikingly, the DIAPH3 gene mutation leads to the overexpression of the DIAPH3 protein. Here, we propose to decipher the mechanism responsible for AUNA1 from the molecular signaling pathways to the system level in order to envision future therapies. Our project therefore consists in i) phenotyping the AUNA1 mouse model ii) unraveling the signaling pathways of AUNA1 at the molecular level and iii) developing rescue strategies for the AUNA1 mouse model.

Because the point mutation in the 5’untranslated region of the human DIAPH3 gene, responsible for AUNA1, leads to DIAPH3 protein overexpression, the phenotype investigation will be carried-out on transgenic mouse lines overexpressing the Diaph3 protein. Our preliminary result shows that the two existing transgenic lines display progressive threshold shift with preserved outer hair cells (OHCs) function, mimicking therefore AUNA1 deafness. In addition, scanning electron microscopy shows aberrant shape of the apical part of the IHCs (i.e., swelling of the cuticular plate and fused stereocilia), suggesting that IHCs are not able to encode sound stimulation. Altogether, these preliminary results demonstrate that mice overexpressing Diaph3 replicate AUNA1 and pinpoint a defect in the IHCs. Next experiments will consist in examining the anatomy of the cuticular plate and its associated area using electron and confocal microscopy and in probing the IHCs biophysical activity using the patch-clamp technique.

To decipher the Diaph3 signaling pathway, temporal and spatial Diaph3 expression will be assessed using immunohistochemistry and in situ hybridization. Interacting partners will be identified using mass spectrometry or yeast two-hybrid screen. Alternatively, a differential cDNA library can be constructed using subtractive hybridization between IHCs and OHCs, leading to specific IHCs proteins isolation with potential interactions with Diaph3. The interacting partners and signaling cascade will then be identified in situ in the wild-type and Diaph3 mutant mice using immunohistochemistry and western-blot.

The last goal consists in developing novel therapeutic strategies for AUNA1. Because Diaph3 overexpression in mice leads to a deafness mimicking AUNA1, one attractive strategy is to down-regulate Diaph3 expression or to reduce Diaph3 activity. To do so, siRNA, peptide competitor will directly be infused into the cochlea through trans-tympanic delivery. If successful, these experiments will constitute the necessary proof-of-concept for translational research as they demonstrate the backbone for AUNA1 patient’s treatment.