TGFβR-1/ALK5 inhibitor RepSox induces enteric glia-to- neuron transition and influences gastrointestinal mobility in adult mice

Promoting adult neurogenesis in the enteric nervous system (ENS) may be a potential therapeutic approach to cure enteric neuropathies. Enteric glial cells (EGCs) are the most abundant glial cells in the ENS. Accumulating evidence suggests that EGCs can be a complementary source to supply new neurons during adult neurogenesis in the ENS. In the brain, astrocytes have been intensively studied for their neuronal conversion properties, and small molecules have been successfully used to induce the astrocyte-to-neuron transition. However, research on glia-to-neuron conversion in the ENS is still lacking. In this study, we used GFAP-Cre:Rosa-tdTomato mice to trace glia-to-neuron transdifferentiation in the ENS in vivo and in vitro. We showed that GFAP promoter-driven tdTomato exclusively labelled EGCs and was a suitable marker to trace EGCs and their progeny cells in the ENS of adult mice. Interestingly, we discovered that RepSox or other ALK5 inhibitors alone induced efficient transdifferentiation of EGCs into neurons in vitro. Knockdown of ALK5 further confirmed that the TGFβR-1/ALK5 signalling pathway played an essential role in the transition of EGCs to neurons. RepSox-induced neurons were Calbindin- and nNOS-positive and displayed typical neuronal electrophysiological properties. Finally, we showed that administration of RepSox (3, 10 mg· kg⁻¹ ·d⁻¹, i.g.) for 2 weeks significantly promoted the conversion of EGCs to neurons in the ENS and influenced gastrointestinal motility in adult mice. This study provides a method for efficiently converting adult mouse EGCs into neurons by small-molecule compounds, which might be a promising therapeutic strategy for gastrointestinal neuropathy.