tRF‐M2–Regulated Dopamine Receptor D2 Expression Attenuates Methamphetamine Reinstatement Behavior in Rats

Methamphetamine addiction is a chronic disorder characterized by compulsive drug-seeking and high relapse rates, driven by dopamine-mediated neuroadaptations in reward circuits that induce persistent behavioral and synaptic changes that endure despite abstinence, making treatment challenging. In this study, we demonstrated significant downregulation of the tRNA-derived small RNA (tsRNA) tRF-M2 (tRF-1:32-Gly-GCC-2-M2) in the nucleus accumbens (NAc) of rats exposed to methamphetamine self-administration (METH SA). Through bioinformatic prediction and experimental validation via dual-luciferase reporter assays, we identified that dopamine receptor D2 (Drd2) mRNA is a direct molecular target of tRF-M2. We found that NAc-specific tRF-M2 delivery attenuated reinstatement behaviors in METH SA rats, potentially mediated through downstream Akt-Gsk3β signaling cascade and the transcriptional regulator CREB. The antireinstatement effect mirrored the behavioral outcomes observed following Drd2 knockdown in the NAc, which similarly showed concomitant changes in phosphorylated Akt (p-Akt) and Gsk3β (p-Gsk3β). These findings show that tRF-M2 regulates Drd2 expression via Akt-Gsk3β-CREB signaling, thereby inhibiting drug-seeking behavior in addiction models. These results provide valuable mechanistic insights into neuroepigenetic regulation of psychostimulant addiction and identify promising candidates for therapeutic intervention against methamphetamine relapse.