Methadone alters transcriptional programs associated with synapse formation in human cortical organoids

Opioid use disorder (OUD) among pregnant women has become an epidemic in the United States. Pharmacological interventions for maternal OUD most commonly involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms and behaviors linked with drug addiction. However, evidence of methadone's ability to readily accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on prenatal brain development. We utilized human cortical organoid (hCO) technology to probe how this drug impacts the earliest mechanisms of cortico-genesis. Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1 μM methadone for 50 days revealed a robust transcriptional response to methadone associated with functional components of the synapse, the underlying extracellular matrix (ECM), and cilia. Co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis of growth factors, developmental signaling pathways, and matricellular proteins (MCPs). TGFβ1 was identified as an upstream regulator of this network and appeared as part of a highly interconnected cluster of MCPs, of which thrombospondin 1 (TSP1) was most prominently downregulated and exhibited dose-dependent reductions in protein levels. These results demonstrate that methadone exposure during early cortical development alters transcriptional programs associated with synaptogenesis, and that these changes arise by functionally modulating extra-synaptic molecular mechanisms in the ECM and cilia. Our findings provide novel insight into the molecular underpinnings of methadone's putative effect on cognitive and behavioral development and a basis for improving interventions for maternal opioid addiction.