Human amniotic mesenchymal stem cell-derived exosomes alleviate cerebral Palsy–Associated neuronal damage via TSC1/mTOR/ULK1 autophagy suppression and PINK1/Parkin mitophagy restoration

Abstrakts

Background Cerebral palsy (CP) from perinatal hypoxic-ischaemic brain injury lacks effective therapies. Human amniotic mesenchymal stem cell-derived exosomes (hAMSC-exos) show neuroprotective potential, but their mechanisms remain unclear. This study investigated the effects and molecular pathways of hAMSC-exos in a neonatal rat CP model and an oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal model. Methods hAMSC-exos were isolated and characterised. Severe CP was induced in postnatal day 21 rats by carotid ligation and hypoxia; hAMSC-exos or vehicle were given via tail vein 24 h later. Brain histology, ultrastructure, TSC1/mTOR/ULK1 pathway proteins, and apoptosis were assessed. In vitro , cortical neurons underwent OGD/R with or without exosomes; mitochondrial function (ATP, ROS, membrane potential), morphology, dynamics proteins (DRP1, FIS1, MFN1, MFN2), and PINK1/Parkin-mediated mitophagy were evaluated. Results hAMSC-exos significantly ameliorated cortical neuronal degeneration, Nissl loss, and mitochondrial ultrastructural damage, and reduced autophagosome accumulation. They reversed CP-induced TSC1 upregulation, restored mTOR phosphorylation, and decreased ULK1 phosphorylation, correlating with fewer apoptotic neurons. In vitro , exosomes restored ATP content, reduced mitochondrial ROS, and normalised membrane potential. OGD/R caused excessive mitochondrial elongation via MFN1 upregulation; exosomes selectively downregulated MFN1 without affecting fission proteins (DRP1, FIS1). Moreover, exosomes restored impaired PINK1/Parkin-mediated mitophagy, increasing co-localisation of phospho-PINK1, phospho-Parkin, and LAMP1 with mitochondria, and rescued their phosphorylated protein levels. Conclusion hAMSC-exos protect against hypoxic-ischaemic injury through coordinated regulation of mitochondrial quality control, involving suppression of excessive TSC1/mTOR/ULK1-mediated autophagy ( in vivo ) and restoration of PINK1/Parkin-mediated mitophagy ( in vitro ), collectively preserving mitochondrial integrity and reducing neuronal apoptosis. These findings support hAMSC-exos as a promising cell-free therapeutic strategy for CP.