Methadone induces phospholipidosis and lysosomal dysfunction in astrocytes in vitro and in the developing brain

Methadone maintenance therapy is the standard treatment for opioid dependence during pregnancy, as its long half-life helps prevent relapse to short-acting abused opioids. However, the cellular mechanisms underlying methadone's neurodevelopmental toxicity remain poorly understood. Lipid dysregulation has emerged as a pathological feature in several neurological conditions and may contribute to cognitive impairment in children exposed to methadone in utero.

To determine whether methadone exposure alters lipid homeostasis in astrocytes and to assess whether such alterations are associated with cellular dysfunction and inflammatory responses.

Primary rat astrocytes were treated with methadone for three days. Lipid accumulation, lipid droplet formation, and lysosomal distribution were assessed using BODIPY-493/503, perilipin-2 immunostaining, and LysoTracker, respectively, followed by confocal microscopy and colocalization analyses. Ultrastructural changes were examined by electron microscopy. Lipid composition was quantified by mass spectrometry-based lipidomics. Findings were validated in cortical organotypic cultures and in the cortex of pups born to methadone-exposed dams using RT-qPCR, immunofluorescence, and ultrastructural analyses. Translational relevance was further assessed in primary human fetal astrocytes.

Methadone exposure induced a dose and time-dependent accumulation of lysosomal lipids in primary neonatal rat astrocytes, independent of lipid droplet formation and opioid receptor activation. Electron microscopy revealed multilamellar bodies characteristic of drug-induced phospholipidosis, a known effect of many cationic amphiphilic drugs. Lipidomics confirmed elevated levels of ceramides and triglycerides. Methadone-treated astrocytes displayed impaired lysosomal degradation capacity, indicating functional compromise. Similar lysosomal and inflammatory alterations were observed in methadone-exposed organotypic cultures and in the cortex of offspring from methadone-treated dams. Human fetal astrocytes replicated these findings, demonstrating cross-species conservation.

Methadone induces phospholipidosis and lysosomal dysfunction in astrocytes, accompanied by neuroinflammatory activation during brain development. These findings uncover an unrecognized mechanism linking methadone's amphiphilic properties to developmental neurotoxicity and emphasize the need to re-evaluate the safety of methadone use during pregnancy.