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.

促进肠神经系统 (ENS) 中的成人神经发生可能是治愈肠神经病的潜在治疗方法。肠神经胶质细胞 (EGC) 是 ENS 中最丰富的神经胶质细胞。越来越多的证据表明，EGC 可以作为 ENS 成体神经发生过程中新神经元的补充来源。在大脑中，星形胶质细胞的神经元转换特性已得到深入研究，小分子已成功用于诱导星形胶质细胞到神经元的转换。然而，仍然缺乏对 ENS 中神经胶质细胞到神经元转化的研究。在这项研究中，我们使用 GFAP-Cre:Rosa-tdTomato 小鼠在体内和体外追踪 ENS 中的胶质细胞到神经元的转分化。我们发现 GFAP 启动子驱动的 tdTomato 专门标记了 EGC，并且是追踪成年小鼠 ENS 中的 EGC 及其后代细胞的合适标记。有趣的是，我们发现 RepSox 或其他 ALK5 抑制剂单独诱导 EGCs 在体外有效转分化为神经元。ALK5 的敲低进一步证实了 TGFβR-1/ALK5 信号通路在 EGC 向神经元的转变中发挥了重要作用。RepSox 诱导的神经元呈 Calbindin 和 nNOS 阳性，并显示出典型的神经元电生理特性。最后，我们证明 RepSox (3, 10 mg·kg ALK5 的敲低进一步证实了 TGFβR-1/ALK5 信号通路在 EGC 向神经元的转变中发挥了重要作用。RepSox 诱导的神经元呈 Calbindin 和 nNOS 阳性，并显示出典型的神经元电生理特性。最后，我们证明 RepSox (3, 10 mg·kg ALK5 的敲低进一步证实了 TGFβR-1/ALK5 信号通路在 EGC 向神经元的转变中发挥了重要作用。RepSox 诱导的神经元呈 Calbindin 和 nNOS 阳性，并显示出典型的神经元电生理特性。最后，我们证明 RepSox (3, 10 mg·kg^-1  ·d ^-1 , ig) 持续2 周显着促进成年小鼠中EGC 向ENS 中的神经元的转化并影响胃肠动力。该研究提供了一种通过小分子化合物将成年小鼠 EGCs 高效转化为神经元的方法，这可能是胃肠道神经病变的一种有前途的治疗策略。