Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.

Hearing loss is one of the most prevalent sensory deficits worldwide and can result from the death of mechanosensory hair cells that transduce auditory signals in the cochlea. The mammalian cochlea lacks the capacity to regenerate these hair cells once damaged, and currently there are no biological therapies for hearing loss. Understanding the signaling pathways responsible for hair cell development can inform regenerative strategies and identify targets for treating hearing loss. The canonical Wnt and Notch pathways are critical for cochlear development; they converge on several key molecules, such as Atoh1, to regulate prosensory specification, proliferation, hair cell differentiation, and cellular organization. Much work has focused on Wnt and Notch modulation in the neonatal mouse cochlea, where they can promote hair cell regeneration. However, this regenerative response is limited in the adult cochlea and this might be attributed to age-dependent epigenetic modifications. Indeed, the epigenetic status at key gene loci undergoes dynamic changes during cochlear development, maturation, and aging. Therefore, strategies to improve regenerative success in the adult cochlea might require the modulation of Wnt, Notch, or other pathways, as well as targeted epigenetic modifications to alter the activity of key genes critical for supporting cell proliferation or transdifferentiation.

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.