Regulation of Cochlear Nucleus Synaptogenesis by Progranulin in a Stem Cell-Based Coculture Model

The cochlear nucleus (CN) is the first central relay of auditory information, receiving input from spiral ganglion neurons (SGNs) through specialized synapses whose molecular regulation remains incompletely defined. Here, we examined the role of Progranulin (Grn) in a stem cell-based CN synaptogenesis using an in vitro coculture model. DsRed-expressing mouse embryonic stem cells were differentiated into SGN-like neurons (deSGNs) and cocultured for 4 days with primary CN neurons isolated from postnatal days 3-6 wild-type (Grn^+/+) or Grn-deficient (Grn^-/-) mice. Cocultures containing Grn^+/+ mouse CN neurons exhibited abundant neurite interactions and synaptic puncta between deSGNs and CN neurons. In contrast, neural connectivity and synaptic puncta were markedly reduced in cocultures with Grn^-/- mouse CN neurons. Supplementation with recombinant Progranulin protein significantly restored neurite connectivity and synaptic puncta formation in Grn^-/- mouse CN neuron cocultures. Dose-response analysis further demonstrated that Progranulin concentrations of 0.1-1 µM effectively rescued neurite connection and synaptic features to levels comparable to those observed in Grn^+/+ mouse CN neuron cocultures. These findings identify Grn as a critical regulator of CN synapse formation in vitro and provide mechanistic insight relevant to stem cell-based strategies aimed at reconstructing SGN-CN synapses for future auditory repair.