Global Analysis of mRNA Alternative Splicing in the Trigeminal Ganglion at Different Stages of Trigeminal Neuropathic Pain in Mice.

INTRODUCTION

Trigeminal neuropathic pain (TNP) is a chronic pain disorder with incompletely understood molecular mechanisms. Alternative splicing (AS), a key post-transcriptional regulatory process, has emerged as an important modulator of neuronal excitability and synaptic plasticity. However, the temporal dynamics of AS in the trigeminal ganglion (TG) during TNP progression remain poorly defined.

METHODS

Poly(A)-enriched RNA sequencing was performed on TG tissues from a partial infraorbital nerve transection (pIONT) mouse model at day 3 and day 10 after surgery, representing the onset and maintenance phases of TNP, respectively. TG tissues from ten mice under the same condition were pooled to generate one biological sample, and two pooled biological replicates were analyzed for each condition at each time point. ASGs and RBP genes harboring differential AS events were identified from RNA-seq-based analyses, DEPs were identified by TMT-based quantitative proteomic analysis, and their cellular distribution was further characterized by single-cell RNA-seq-based cell-type mapping.

RESULTS

Exon skipping (SE) was the predominant AS event at both time points and increased markedly at day 10, indicating greater splicing complexity during the maintenance phase. ASGs at day 10 were enriched in pathways related to synaptic remodeling, neuronal signaling, and MAPK signaling. SE events showed notable clustering on chromosomes 4 and 7. Integrative analyses identified several pain-related candidates, including ASGs such as Trpv1 and Dlg3 and DEPs such as DNM1 and GAL, that were potentially associated with synaptic transmission and neuronal excitability. These RBP-associated AS changes further suggested a role for post-transcriptional regulatory networks in stage-specific splicing alterations during TNP.

DISCUSSION: These findings reveal dynamic and stage-specific AS changes in the TG during TNP progression, with more prominent splicing alterations during the maintenance phase. Our results support an association between AS, synaptic remodeling, and pain-related molecular pathways, and provide a transcriptomic, proteomic, and cell-type-resolved framework for future studies of splicing regulation in trigeminal neuropathic pain.