Project Summary The main way in which non-mammalian vertebrates, such as fish, restore sensory hair cells is through proliferation and differentiation of the residual population of supporting cells. In contrast, supporting cells lose the capacity to proliferate postnatally in mammals, and the molecular machinery preventing cell cycle reentry remains poorly understood. Our work has established that Hippo signaling serves as a major repressive mechanism that blocks supporting cell proliferation and plasticity in the mammalian inner ear. In three Aims, we will identify the molecular mechanism by which Hippo inhibition promotes mitotic sensory receptor regeneration in the adult utricle explants (Aim 1); assess whether reversible pharmacologic inactivation of Hippo signaling stimulates bona fide vestibular hair cell regeneration to support functional recovery in vivo (Aim 2); and assess the pathway’s interaction with the cell cycle inhibitor p27Kip1, specific to the organ of Corti, in the adult inner ear in vivo (Aim 3). The long-term goal of this proposal is to identify therapeutic strategies for hearing and balance restoration through controlled manipulation of the Hippo pathway. Due to its relatively recent discovery, study of the Hippo pathway in the inner ear is innovative in itself. Furthermore, our group has pioneered this field and developed several specialized research tools to aid the study of the pathway in the inner ear. Most notably, we identified the first small-molecule inhibitor of Lats kinases – the core enzymes in Hippo signaling – that we show to potently induce supporting cell proliferation and the initial stages of hair cell regeneration in vitro and in vivo. We also optimized posterior semicircular canal approach for LKI delivery into the inner ear and utilize several cutting-edge genetic and epigenetic techniques (e.g multiome sequencing, CUT&RUN). The proposed basic research is significant because understanding the molecular machinery blocking cell cycle reentry in the inner ear may determine new therapeutic targets for induction of hair cell regeneration. Remarkably, we demonstrate that brief pharmacologic inhibition of Lats kinases induces supporting cell proliferation in the adult utricle, allowing progeny to re-exit the cell cycle and spontaneously upregulate sensory receptor genes upon drug withdrawal. Collectively our data show that temporal inactivation of Hippo signaling is sufficient to promote the initial stages of hair cell regeneration through supporting cell division – a process thought to be permanently suppressed in the adult mammalian inner ear.

Project Narrative The progressive loss of hair cells, the sensory receptors within the cochlea and vestibular system, results in irreversible deterioration of the auditory and vestibular functions. Our work has established that Hippo signaling serves as a major repressive mechanism preventing supporting cell-mediated hair cell regeneration in the mammalian inner ear. The pathway, therefore, represents a new therapeutic target for induction of hair cell regeneration, and the proposed work is directly relevant to the NIH’s mission to uncover new knowledge that will lead to better health for everyone.