Abstract
Background: Autophagy dysregulation plays a crucial role in the early pathological stage of Parkinson’s disease (PD), in which dopaminergic (DAergic) neurons undergo degeneration. Transcranial direct current stimulation (tDCS) has shown promising neuroprotective effects in studies related to PD. However, the molecular basis underlying its effects remains unclear, and direct evidence regarding whether it exerts neuroprotective effects through the reactivation of autophagic homeostasis is still lacking. Objective: This study aimed to systematically evaluate the neuroprotective effects of tDCS on DAergic neurons via both in vivo and in vitro PD models and to identify potential biological pathways and regulatory targets associated with tDCS intervention through bioinformatic analysis. Furthermore, this study explored whether tDCS exerts its neuroprotective effects by restoring the activity of the Mlst8/mTOR/Ulk1 signaling pathway to regulate autophagic homeostasis, thereby providing a theoretical basis for mechanistic research and potential clinical applications of tDCS. Materials and methods: Injections of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were used to establish an in vivo model of PD in male C57BL/6J mice aged 6–8 weeks. Behavioural assays, immunohistochemistry, and western blotting were employed to evaluate the neuroprotective effects of tDCS. Transcriptomic analysis was conducted to identify the biological pathways involved in the tDCS intervention, and autophagy levels in the substantia nigra were assessed before and after the tDCS intervention via transmission electron microscopy (TEM) and immunofluorescence. Structural modeling and visualization and gene set enrichment analyses (GSEA) were employed to identify autophagy-related regulatory targets, which were subsequently validated via RT‒qPCR and western blotting. Finally, in an in vitro MPP+ -induced model, flow cytometry, immunofluorescence, laser confocal microscopy, and western blotting indicated that Target of rapamycin complex subunit LST8 (Mlst8)-mediated mTOR/Ulk1 activity may represent a key mechanism contributing to the suppression of excessive autophagy by tDCS. Results: The tDCS intervention significantly improved behavioural impairments in MPTP-induced PD mice and reduced the loss of DAergic neurons in the substantia nigra. Transcriptomic analysis revealed that the Autophagy and Lysosomal pathways were among the most significantly enriched terms, suggesting that tDCS may exert neuroprotective effects in PD by modulating the autophagy–lysosome pathway. Immunofluorescence and TEM of the substantia nigra revealed that MPTP induced abnormally activated autophagic flux, which exacerbated the degeneration of DAergic neurons. In contrast, both tDCS intervention and an autophagy inhibitor suppressed the excessive activation of autophagic flux and significantly reduced the loss of DAergic neurons. Further analysis via GSEA revealed Mlst8 as a potential negative regulator of autophagy. RT‒qPCR confirmed that its expression was significantly upregulated after tDCS intervention, which is consistent with the RNA-Seq results. Structural modeling suggested that Mlst8 may regulate autophagy initiation through the mTOR/Ulk1 signaling pathway. Western blot analysis further confirmed that tDCS increased Mlst8 expression, restored mTOR/Ulk1 activity and suppressed the excessive activation of the autophagy–lysosome pathway in the PD model. In vitro experiments revealed that the knockdown of Mlst8 abolished the suppressive effect of tDCS on excessive autophagy, suggesting that Mlst8-mediated mTOR/Ulk1 activity may serve as a potential mechanism underlying for the modulation of excessive autophagy by tDCS. Conclusion: This study suggested that tDCS exerts neuroprotective effects on DAergic neurons in both MPTP-induced PD models in vivo and MPP+ -induced PD models in vitro. For the first time, we proposed that tDCS may suppress MPTP/MPP+ -induced excessive autophagy by restoring Mlst8 expression and thereby enhancing the activity of the mTOR/Ulk1 signaling pathway in PD models. Evidence supports that Mlst8-mediated mTOR/Ulk1 activity may serves as the potential molecular mechanism through which tDCS inhibits excessive autophagy. Collectively, our findings elucidate the molecular mechanisms underlying the neuroprotective effects of tDCS, providing a theoretical foundation for its clinical translation in PD treatment. Supplementary Information: The online version contains supplementary material available at 10.1186/s12967-025-07597-7.