Sensorineural hearing loss and vestibular disorders are permanent impairments for many people. They often stem from the loss of sensory hair cells in the inner ear, a population of cells that cannot be replaced when they die. Recent reports have shown that inner ear supporting cells, which neighbor sensory hair cells, can be coaxed into directly transdifferentiating into sensory hair cells in injured tissue. However, to achieve tue regeneration, inner ear supporting cells must also be guided into proliferation. We hypothesize that signaling through the ErbB2 receptor is sufficient to drive inner ear supporting cells to proliferate. We will test this hypothesis by over-expressing a constitutively activated (CA) ErbB2 receptor in auditory and balance supporting cells in the inner ear, and assessing subsequent proliferation. We present preliminary data with two independent CA-ErbB2 expression systems. Moreover, we show that both neonatal cochlear supporting cells and adult utricular supporting cells proliferate in response to CA-ErbB2 signaling in organ culture. Our first system uses two custom-built adenoviruses to over-express two mutated versions of the ErbB2 protein: one that is constitutively active, and one that is inert.
In Aim 1, we will infect neonatal cochlear supportng cells with these viruses in vitro. We show preliminary data demonstrating that only the CA-ErbB2 virus activates phosphoinositol 3-kinase's (PI3K) regulatory subunit. Moreover, we show that the CA-ErbB2 virus drives proliferation among neonatal cochlear supporting cells in vitro. Surprisingly, CA-ErbB2 exerts some of its effects indirectly, as cells neighboring infected cells are also stimulated to divide. This finding highlights the power of using overexpression to investigate receptor function. We will quantify these results and probe the downstream effectors of the CA-ErbB2 receptor with reporter assays and chemical inhibitors. These experiments will define ErbB2's role in proliferation in neonatal mouse cochlear supporting cells. Our second system uses transgenic Tet-On technology to over-express a mutated, constitutively active ErbB2 protein in mouse supporting cells at different stages. We show preliminary data demonstrating that this system also activates PI3K. We also show that transgenic CA-ErbB2 signaling drives proliferation in neonatal cochlear supporting cells in vitro. Because Tet-On technology allows us to control both the timing and duration of the CA-ErbB2 signal, we will deliver a pulse of signaling and assess first if supporting cells divide, and second, if they trans differentiate into hair cells.
In Aim 2, we propose to use this system to activate CA-ErbB2 signaling in cochlear supporting cells at different stages, with and without hair cell injury, to determine if ErbB2 signaling is sufficient to promote proliferation in vivo.
In Aim 3, we will use both the CA-ErbB2 virus and the transgenic system to drive proliferation in adult utricular supporting cells in vitro, after hair cell injury. These experiments will illuminate how proliferaton in mammalian cochlear supporting cells can be regulated and significantly advance our understanding of inner ear regeneration.

Public Health Relevance

Our goal is to drive cell replacement in the injured inner ear by promoting supporting cell proliferation. We will test if activation of the ErbB signaling pathwa is sufficient to promote such proliferation in the auditory and vestibular system, at all ages, in he mouse.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC014261-05
Application #
9632803
Study Section
Auditory System Study Section (AUD)
Program Officer
Freeman, Nancy
Project Start
2015-03-06
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Rochester
Department
Neurosciences
Type
School of Medicine & Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Zhang, Jingyuan; Wang, Quan; Abdul-Aziz, Dunia et al. (2018) ERBB2 signaling drives supporting cell proliferation in vitro and apparent supernumerary hair cell formation in vivo in the neonatal mouse cochlea. Eur J Neurosci 48:3299-3316
Paquette, Stephen T; Dawes, Ryan P; Sundar, Isaac K et al. (2018) Chronic cigarette smoke exposure drives spiral ganglion neuron loss in mice. Sci Rep 8:5746
Gilels, Felicia; Paquette, Stephen T; Beaulac, Holly J et al. (2017) Severe hearing loss and outer hair cell death in homozygous Foxo3 knockout mice after moderate noise exposure. Sci Rep 7:1054
White, Patricia M (2016) The Role of the Transcription Factor Foxo3 in Hearing Maintenance: Informed Speculation on a New Player in the Cochlea. Biomed Res Int 2016:1870675
Paquette, Stephen T; Gilels, Felicia; White, Patricia M (2016) Noise exposure modulates cochlear inner hair cell ribbon volumes, correlating with changes in auditory measures in the FVB/nJ mouse. Sci Rep 6:25056
Gilels, Felicia; Paquette, Stephen T; Zhang, Jingyuan et al. (2013) Mutation of Foxo3 causes adult onset auditory neuropathy and alters cochlear synapse architecture in mice. J Neurosci 33:18409-24