Ultrasound-mediated augmented exosome release from astrocytes alleviates amyloid-β-induced neurotoxicity

Background: Extracellular vesicles, including exosomes, are secreted by a variety of cell types in the central nervous system. Exosomes play a role in removing intracellular materials from the endosomal system. Alzheimer's disease (AD) is caused by an overproduction or reduced amyloid-beta (Aβ) peptide clearance. Increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway. Therapeutic ultrasound stimulation demonstrated its potential for promoting Aβ degradation efficiency in clinical trials. However, the underlying mechanism of ultrasound stimulation is still unclear. Methods: In this study, astrocytes, the most abundant glial cells in the brain, were used for exosome production. Post insonation, exosomes from ultrasound-stimulated HA cells (US-HA-Exo) were collected, nanoparticle tracking analysis and protein analysis were used to measure and characterize exosomes. Neuroprotective effect of US-HA-Exo in oligomeric Aβ₄₂ toxicated SH-SY5Y cells was tested. Cellular uptake and distribution of exosomes were observed by flow cytometry and confocal laser scanning microscopy. Focused ultrasound (FUS) with microbubbles was employed for blood-brain-barrier opening to achieve brain-targeted exosome delivery. After US-HA-Exo/FUS treatment, amyloid-β plaque in APP/PS1 mice were evaluated by Aβ immunostaining and thioflavin-S staining. Results: We showed that ultrasound resulted in an almost 5-fold increase in the exosome release from human astrocytes. Exosomes were rapidly internalized in SH-SY5Y cells, and colocalized with FITC-Aβ₄₂, causing a decreased uptake of FITC-Aβ₄₂. CCk-8 test results showed that US-HA-Exo could mitigate Aβ toxicity to neurons in vitro. The therapeutic potential of US-HA-Exo/FUS delivery was demonstrated by a decrease in thioflavin-S-positive amyloid plaques and Aβ immuno-staining, a therapeutic target for AD in APP/PS1 transgenic mice. The iTRAQ-based proteomic quantification was performed to gain mechanistic insight into the ultrasound effect on astrocyte-derived exosomes and their ability to alleviate Aβ neurotoxicity. Conclusion: Our results imply that US-HA-Exo have the potential to provide neuroprotective effects to reverse oligomeric amyloid-β-induced cytotoxicity in vitro and, when combined with FUS-induced BBB opening, enable the clearance of amyloid-β plaques in vivo.