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Background <p>Neuropathic pain is a debilitating condition with limited effective treatments, highlighting an urgent need for non-opioid analgesic targets. Gαo, encoded by the Gnao1 gene, is an abundant G protein in the nervous system and couples to multiple inhibitory GPCRs, but its role in chronic pain remains poorly understood.</p> Methods <p>Spatial transcriptomics and bulk RNA-sequencing were performed on the spinal cord and dorsal root ganglia (DRG) of mice following spared nerve injury (SNI). Adeno-associated virus (AAV)-mediated Gnao1 overexpression or knockdown was performed in the spinal cord or DRG, followed by pain behavioral testing using von Frey filaments, acetone evaporation test, and mechanical conflict avoidance (MCA) assay. Microglial activation, neuronal injury, and signaling pathways were examined by immunohistochemistry (IHC), Western blotting (WB), and calcium imaging. In vitro studies used BV2 microglia and primary DRG neurons with lentiviral Gnao1 overexpression and LPS or db-cAMP stimulation.</p> Results <p>Spatial transcriptomics revealed that Gnao1 is enriched in the spinal dorsal horn (SDH) and significantly downregulated after SNI. Bulk RNA-seq confirmed Gnao1 downregulation in pain-processing regions (DRG and SDH). IHC showed that its cellular localization shifted from neurons to glial cells. Overexpression of Gnao1 in the spinal cord or DRG markedly alleviated SNI-induced mechanical allodynia, cold hypersensitivity, and pain-related avoidance behaviors. Mechanistically, transcriptomic profiling revealed that Gnao1 overexpression broadly suppressed pro-inflammatory and pro-nociceptive pathways. Experimental validation confirmed that Gnao1 overexpression reduced p38, ERK, and NF-κB phosphorylation, and decreased microglial activation. In primary DRG neurons, Gnao1 overexpression also lowered cAMP levels and suppressed db-cAMP-evoked Ca<sup>2+</sup> influx.</p> Conclusion <p>Gnao1 acts as a gatekeeper negative regulator of neuropathic pain by silencing distinct pro-nociceptive signaling cascades, and highlights its potential as a non-opioid analgesic target.</p> Clinical trial number <p>Not applicable.</p> Graphical Abstract <p></p>

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Gnao1 acts as a gatekeeper to alleviate neuropathic pain by silencing pro-nociceptive signaling cascades

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月23日

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  • Jieyi Cai,
  • Hui Li,
  • Lü Chen,
  • Ling Luo,
  • Xin Wu,
  • Min Yuan,
  • Maolin Han,
  • Shirong Wen,
  • Changlai Zhu,
  • Yun Gu

摘要

Background

Neuropathic pain is a debilitating condition with limited effective treatments, highlighting an urgent need for non-opioid analgesic targets. Gαo, encoded by the Gnao1 gene, is an abundant G protein in the nervous system and couples to multiple inhibitory GPCRs, but its role in chronic pain remains poorly understood.

Methods

Spatial transcriptomics and bulk RNA-sequencing were performed on the spinal cord and dorsal root ganglia (DRG) of mice following spared nerve injury (SNI). Adeno-associated virus (AAV)-mediated Gnao1 overexpression or knockdown was performed in the spinal cord or DRG, followed by pain behavioral testing using von Frey filaments, acetone evaporation test, and mechanical conflict avoidance (MCA) assay. Microglial activation, neuronal injury, and signaling pathways were examined by immunohistochemistry (IHC), Western blotting (WB), and calcium imaging. In vitro studies used BV2 microglia and primary DRG neurons with lentiviral Gnao1 overexpression and LPS or db-cAMP stimulation.

Results

Spatial transcriptomics revealed that Gnao1 is enriched in the spinal dorsal horn (SDH) and significantly downregulated after SNI. Bulk RNA-seq confirmed Gnao1 downregulation in pain-processing regions (DRG and SDH). IHC showed that its cellular localization shifted from neurons to glial cells. Overexpression of Gnao1 in the spinal cord or DRG markedly alleviated SNI-induced mechanical allodynia, cold hypersensitivity, and pain-related avoidance behaviors. Mechanistically, transcriptomic profiling revealed that Gnao1 overexpression broadly suppressed pro-inflammatory and pro-nociceptive pathways. Experimental validation confirmed that Gnao1 overexpression reduced p38, ERK, and NF-κB phosphorylation, and decreased microglial activation. In primary DRG neurons, Gnao1 overexpression also lowered cAMP levels and suppressed db-cAMP-evoked Ca2+ influx.

Conclusion

Gnao1 acts as a gatekeeper negative regulator of neuropathic pain by silencing distinct pro-nociceptive signaling cascades, and highlights its potential as a non-opioid analgesic target.

Clinical trial number

Not applicable.

Graphical Abstract