Patricia M. White, Ph.D. - US grants

? DESCRIPTION (provided by applicant): 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.

A significant fraction of patients with Alzheimer's Disease (AD) also have acquired hearing loss. Why these two diseases of the aged are correlated is a matter of great debate. In some cases, both may arise from the same cause. In that case, acquired hearing loss might become a useful biomarker for early treatment of AD. Alternatively, untreated hearing loss might drive AD progression, either through increasing social isolation or by exacerbating working memory deficits during communication. In the latter case, treating hearing loss could delay the onset or progression of AD. Unfortunately, hearing aid use is not widespread among people with acquired hearing loss, prompting interest in biological cures. This parent R01 grant investigates ERBB2 signaling as a candidate for stimulating inner ear regeneration, using transgenic mouse models to over-express an constitutively active form of ERBB2 (CA- ERBB2) in supporting cells of the inner ear. We have recently published that activation of ERBB2 in the newborn mouse cochlear drives the production of ectopic hair cells. We also show preliminary data indicating that young adult mice with CA-ERBB2 significantly recover low-frequency hearing after traumatic noise exposure, compared to their control siblings. While still quite preliminary, this exciting result may also be useful in studies of the interaction of AD and acquired hearing loss. We seek supplemental funding to expand our ongoing experiments, so that we might address new basic questions about AD in the context of this new hearing restoration. We plan to employ mice harboring two transgenes, the K670N and M671L mutations in the amyloid precursor protein (APP), and the M146L mutation in presenilin 1 (PS1). We will cross such APP/PS1de9 mice into our current transgenic system, where we obtain mice that can express CA-ERBB2 under control of a Tet-ON promoter regulated by a supporting cell specific CRE recombinase, Fgfr3-iCRE and ROSA-rtTA. We will then investigate whether mice with both CA- ERBB2 and APP/PS1de9 exhibit similar recovery dynamics from damage as those with CA-ERBB2 alone. We have the advantage that we can design follow-up experiments regardless of the outcome. If AD-related transgenes prevent hearing loss recovery, then we will plan to investigate a causative role for these genes in hearing loss. If AD-related transgenes do not prevent hearing recovery, then we plan to investigate if this intervention can delay onset or progression of AD-like symptoms in mice. Thus, we predict that these experiments will further a greater understanding into this puzzling dynamic of hearing loss and AD progression.

Noise induced hearing loss (NIHL) has disabled millions of people world-wide. Individual risk for NIHL varies from person to person under similar exposure conditions, suggesting that genetic factors contribute to susceptibility. We have found that mice lacking a transcription factor called FOXO3 become severely and permanently deafened after a noise exposure that only briefly affects their wild-type littermates. FOXO3 has multiple functions in other cell types, including oxidative stress reduction, autophagy, and directly inducing apoptosis. A recent study linked human genetic variations in FOXO3 to a greater susceptibility to occupational NIHL. However, the FOXO3 alleles associated with NIHL drive increased expression of FOXO3. Thus, there is evidence to indicate that FOXO3 is important for hearing preservation, but there is also evidence that excess FOXO3 drives NIHL. In this grant, we seek to address this knowledge gap by researching the mechanisms of FOXO3 function, using translatome sequencing, cell-specific Foxo3 conditional knockouts (cKO), and CRISPR modifications to generate mouse lines that can be used to investigate the human NIHL-linked FOXO3 allele. In Foxo3-knockout (KO) mice, noise eliminates high-frequency outer hair cells (OHCs). We show that this occurs through a rapid cell death program called parthanatos, which is caspase-independent apoptosis. Parthanatos indicates that in the absence of any noise damage, Foxo3-KO OHCs are primed for death. Bulk RNA-Seq data from control Foxo3-KO and wild-type littermates show no evidence for changes in oxidative stress reducers known to be regulated by FOXO3. Instead, we see changes in actin binding genes expressed in OHCs. In Aim 1, we propose to validate this screen and identify markers of OHC distress in the Foxo3-KO through translatome sequencing. In Aim 2, we propose to make cell-specific Foxo3-cKO to identify the cells in which FOXO3 acts. Wild-type mice express FOXO3 protein in both OHCs and in the surrounding supporting cells (SCs). By using inducible DNA recombinases lines specific to either OHCs or SCs, we can ablate FOXO3 function in either cell type. We will expose such Foxo3-cKO mice to noise and determine their NIHL susceptibility. Finally, in Aim 3, we have used CRISPR genetic modification technology to create two mouse lines, one with control sequences (Foxo3-T-allele mice), as well as one homologous to the human FOXO3 allele that confers NIHL susceptibility (Foxo3-G-allele mice). We will validate that the Foxo3-G-allele mouse line has increased levels of FOXO3 in cochlear cells after noise exposure. We hypothesize that this modification promotes apoptosis from FOXO3 activation, and we will test that hypothesis by exposing Foxo3- G-allele mice to noise, measuring their hearing and analyzing potential cellular losses. In sum, through both loss-of-function and gain-of-function experiments, we will analyze FOXO3's role in hearing loss from noise.