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Background <p>Hirschsprung disease (HSCR) is a congenital condition featuring aganglionosis in the distal colon, causing functional obstruction. While EGF and bFGF are well-characterized neurogenic factors, the precise mechanistic role of GDNF in modulating enteric glial cell plasticity remains incompletely understood.</p> Methods <p>EGCs were identified via proteomic profiling and immunofluorescence in Ednrb⁻/⁻ mice modeling HSCR. EGC/PK060399egfr and primary EGCs were induced with neural stem cell–inducing medium (NSC-Med). Morphological changes, EdU assay, immunofluorescence, RT‒qPCR, and Western blotting were employed to assess the expression of stemness- and neuron-associated markers. Metabolomic and transcriptomic analyses were performed to evaluate metabolic remodeling and signaling pathways.</p> Results <p>Following treatment with NSC-Med, immunofluorescence analysis revealed that neurospheres expressed high proportions of Nestin-positive (97.09%), Sox2-positive (50.11%), and p75<sup>NTR</sup>-positive (77.87%) cells. Metabolomic profiling revealed a significant enhancement of the Warburg effect in the NSC-Med group. Western blot analysis further revealed elevated expression of PKM2, along with significant increases in both extracellular and intracellular lactate levels following NSC-Med treatment. NSC-Med treatment significantly enhanced proliferation, as demonstrated by a 2.3-fold increase in EdU incorporation (<i>P</i> &lt; 0.05). Transcriptomic analysis revealed the activation of the calcium signaling pathway in the GDNF group. Western blotting revealed a significant increase in CaMKII phosphorylation, and treatment with the calcium chelator BAPTA-AM attenuated GDNF-induced NeuroD1 upregulation.</p> Conclusion <p>NSC-Med promotes stem cell-associated features and gene expression in enteric glial cells. GDNF—a key component of NSC-Med—activates a neurogenic cascade via the calcium signaling pathway (CaMKII-NeuroD1 axis), which offers a potential targeted molecular strategy for HSCR therapy.</p>

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Dual roles of GDNF in enteric glial cell plasticity: direct transdifferentiation via the CaMKII/NeuroD1 pathway and cooperative regulation in a neural stem cell-inducing medium

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月11日

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  • Wanying Jia,
  • Hanlei Yan,
  • Jingjing Huang,
  • Wei Liu,
  • Zihao Fu,
  • Donghao Tian,
  • Wenyao Xu,
  • Xinlin Chen,
  • Ya Gao,
  • Hui Yu

摘要

Background

Hirschsprung disease (HSCR) is a congenital condition featuring aganglionosis in the distal colon, causing functional obstruction. While EGF and bFGF are well-characterized neurogenic factors, the precise mechanistic role of GDNF in modulating enteric glial cell plasticity remains incompletely understood.

Methods

EGCs were identified via proteomic profiling and immunofluorescence in Ednrb⁻/⁻ mice modeling HSCR. EGC/PK060399egfr and primary EGCs were induced with neural stem cell–inducing medium (NSC-Med). Morphological changes, EdU assay, immunofluorescence, RT‒qPCR, and Western blotting were employed to assess the expression of stemness- and neuron-associated markers. Metabolomic and transcriptomic analyses were performed to evaluate metabolic remodeling and signaling pathways.

Results

Following treatment with NSC-Med, immunofluorescence analysis revealed that neurospheres expressed high proportions of Nestin-positive (97.09%), Sox2-positive (50.11%), and p75NTR-positive (77.87%) cells. Metabolomic profiling revealed a significant enhancement of the Warburg effect in the NSC-Med group. Western blot analysis further revealed elevated expression of PKM2, along with significant increases in both extracellular and intracellular lactate levels following NSC-Med treatment. NSC-Med treatment significantly enhanced proliferation, as demonstrated by a 2.3-fold increase in EdU incorporation (P < 0.05). Transcriptomic analysis revealed the activation of the calcium signaling pathway in the GDNF group. Western blotting revealed a significant increase in CaMKII phosphorylation, and treatment with the calcium chelator BAPTA-AM attenuated GDNF-induced NeuroD1 upregulation.

Conclusion

NSC-Med promotes stem cell-associated features and gene expression in enteric glial cells. GDNF—a key component of NSC-Med—activates a neurogenic cascade via the calcium signaling pathway (CaMKII-NeuroD1 axis), which offers a potential targeted molecular strategy for HSCR therapy.