Hearing Restoration Mechanisms Downstream of ERBB2 Signaling in the Mouse Cochlea
Abstract
In people hearing loss from noise does not improve over time. Traumatic noise injury can kill high-frequency auditory receptor cells, called hair cells, which amplify and detect sound vibrations. It can also break cellular structures, drive synaptic loss, and promote inflammation. Birds suffer similar injuries from traumatic noise, but their cochleae are able to recover hearing function through a combination of regeneration and repair. It takes over six weeks for birds to regain auditory discrimination between sounds. Our objective is to identify mechanisms that promote long-term auditory recovery in mammals, that is, improvements to auditory thresholds that arise two or three months after injury. We use mouse genetic models to investigate candidate signaling pathways that could induce this kind of hearing recovery. We are investigating one such pathway, called ERBB2. We used genetic tools to drive expression of a protein, a variant of ERBB2 that is always activated, called CA-ERBB2. In preliminary experiments, we generated mice that could express CA-ERBB2 in supporting cells, which are adjacent to hair cells. We tested hearing in young adult mice, exposed them to traumatic noise, and re-tested their hearing. Three days after noise exposure, we treated the mice to induce CA-ERBB2 expression. While mice with CA-ERBB2 had hearing thresholds similar to their control littermates before, right after, and one month after noise damage, we saw significant, partial, low-frequency auditory recovery for CA-ERBB2 mice two and three months later. Littermate controls did not improve. CA-ERBB2 mice also had significantly better hair cell survival compared to controls. These results show that supporting cells can be induced to protect and/or repair hair cells days after injury. In this proposal, we seek to characterize how CA-ERBB2 could drive these improvements. We are proposing to characterize a pathway that we already know can improve long-term auditory recovery and cell survival in adults after noise damage. CA-ERBB2 by itself cannot be used in humans, as it has oncogenic properties. Consequently, we must identify its downstream effectors to build a usable therapy. We hypothesize that CA-ERBB2 changes how supporting cells act after noise damage by altering what proteins they make. We can test this hypothesis by sequencing mRNAs, which are copies of the DNA sequences, or genes, that cells use to make new proteins. An important consideration for our experimental design is that there are multiple kinds of supporting cells, and we do not know which one or ones promote hearing recovery. Thus, an approach that looks at changes in cells individually will provide us with more information than one that looks at changes in the tissue overall. To address this question, we propose in Aim 1 to perform single-cell mRNA sequencing. We will treat young mice to induce CA-ERBB2 in supporting cells, and wait 24 hours for its signaling to change what proteins are made in those cells. We will then collect the cochleae, dissociate them into a single-cell suspension, isolate individual CA-ERBB2+ cells, and sequence their mRNA. We can isolate the cells because the genetic system includes a green fluorescent protein (GFP), which is expressed in CA-ERBB2+ cells. Control mice express GFP in the same cell types, but have no CA-ERBB2 protein. We can compare individual CA-ERBB2+ cells to the same cells from control mice to find differences, using known markers for the different supporting cells to group them by kind. Statistical techniques will enable us to find real differences providing that we obtain enough live cells. For that reason, we propose to use younger mice for this screen, as we know from previous work that activating CA-ERBB2 in younger supporting cells has regeneration-like effects. In Aim 2, we will validate our findings and determine which changes are also seen in young adult mouse cochleae, after both noise da
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010515
Entities
People
- Patricia White
Organizations
- United States Army
- University of Rochester