Morphine-induced μ-opioid receptor isoform MOR-1X amplifies dependence-related signaling

Clinically used opioids, such as morphine, primarily act through the μ-opioid receptor (MOR), encoded by the OPRM1 gene, which undergoes extensive alternative splicing. More than 20 OPRM1 isoforms have been identified, yet functional characterization has largely focused on the canonical MOR-1 variant. With the emergence of three-dimensional human cerebral organoids (hCOs) derived from induced pluripotent stem cells (iPSCs), it is now possible to model human-specific neuronal responses to opioids more accurately. In this study, we established hCOs as a functional platform to investigate the impact of morphine on OPRM1 pre-mRNA splicing and opioid signaling. We generated iPSC-derived hCOs and neurons, after which they were treated with or without morphine and screened using cellular and molecular/biochemical assays. Our results revealed that morphine exposure selectively induced the MOR-1X isoform in hCOs and iPSC-derived neurons in a dose-dependent manner as revealed by RT-PCR and RT-qPCR. We utilized CRE/CREB overexpression plasmid and lentiviral constructs to measure effects of morphine on cyclic AMP (cAMP) signaling. Upon morphine withdrawal, cells expressing MOR-1X exhibited markedly enhanced cAMP superactivation, a molecular hallmark of opioid dependence, compared with MOR-1. Furthermore, isoform-specific knockdown of MOR-1X by a short hairpin RNA (shRNA) effectively abolished this cAMP overshoot in iPSC-derived neurons. Collectively, these findings identify MOR-1X as a morphine-inducible isoform with a potential key role in the molecular mechanisms underlying opioid signaling, adaptation, and dependence in the brain.