Effect of Homocysteine on Autophagy in Rat Primary Neuronal Culture

Hyperhomocysteinemia is a well-established risk factor for neurodevelopmental disorders, but its impact on autophagy in the developing brain remains insufficiently understood. This study evaluated autophagic vacuole formation in homocysteine-exposed primary cultures of immature neurons derived from the embryonic cortex of Wistar rats. Cells were incubated with homocysteine (7.5–200 µM) for 3 h, 24 h, or 5 days, as well as with methionine (0.1–5 mg/mL) and modulators of autophagy (rapamycin, chloroquine, bafilomycin A1). Bafilomycin and chloroquine increased the number of cells containing autophagic vacuoles (up to 29.0 and 46.5%, respectively). Rapamycin did not induce autophagic vacuole formation or alter p-mTOR and p-rpS6 levels, suggesting distinct regulatory features of autophagy in neuronal cells. Homocysteine (up to 200 µM) did not affect autophagic vacuole formation, levels of Beclin-1, LC3B-II, LAMP-2, or mTOR signaling proteins under either 24 h or 5 days exposure, despite a dose-dependent decrease in cell viability. Methionine also had no effect on autophagic vacuole formation. Prolonged (5 days), but not short-term (24 h), homocysteine combined with chloroquine increased LC3B-II levels versus chloroquine alone. These findings indicate that homocysteine at hyperhomocysteinemia-relevant concentrations does not substantially affect the mTOR-autophagy axis and autophagic vacuole formation in immature cortical neurons.