Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder affecting millions of patients globally. Despite significant efforts from researchers in recent decades, there are still many unanswered questions about AD pathogenesis. AD patient brains manifest changes in extracellular vesicles (EVs) secreted from diseased neurons, and the effect of this phenomenon remains poorly understood. EVs contain a variety of biomolecules and play a critical role in cell-to-cell communication in all eukaryotic organisms. Here, we report a thorough characterization of small EVs purified from cultures of human cerebrocortical organoids. These organoids are differentiated from human patient-derived stem cells that bear a familial AD mutation in the presenilin 1 (PSEN1) gene, or from an isogenic wildtype (WT) control. The organoid conditioned media was aspirated from cultures and processed for EV enrichment using a non-invasive technique that requires no cellular disruption. EVs purified from AD organoid conditioned media have a wider size distribution and show differential expression of tetraspanins CD63, CD9, and CD81 when compared to WT organoid-derived EVs. AD organoid-derived EVs can have single, double, and even triple membranes and display luminal fibrillar material. A deep proteomic profiling of the EVs reveals several statistically significant differences, including evidence for modifications in secretory autophagy. EV isolates from both WT and AD organoids show strong binding to amyloid detecting dyes, both in bulk fluorescence and fluorescence microscopy assays. After a 1-week co-culture of AD organoids with WT organoids, there is evidence of endosomal membrane transfer between the isogenic cultures with an increase in amyloid-β peptides in the WT organoids. These observations support the notion that non-cell-autonomous spread of amyloid-containing EVs in human AD brains can be modeled in a cerebral organoid system. ### Competing Interest Statement J.W.K. discloses that he receives royalties for Tafamidis sales as an inventor and has received additional payments from Pfizer. J.W.K is a founder and major shareholder of Protego, which is developing immunoglobulin light chain kinetic stabilizers and other stabilizers for misfolding diseases; he serves on its Board of Directors and Scientific Advisory Board and acts as a consultant. J.W.K. is a member of the Scientific Advisory Boards of 3D BioAnalytix, Inc., Attralus (focused on antibodies to transthyretin), Curebound (cancer funding), and Exokeryx, Inc. (focused on Parkinson's research). He serves as a consultant for the Dominantly Inherited Alzheimer Network Trial Unit in reviewing drug candidates. S.A.L. discloses that he is an inventor on worldwide patents for the use of memantine, NitroSynapsin, related compounds, and NRF2 activators for neurodegenerative and neurodevelopmental disorders. Per Harvard University guidelines, he participates in a royalty-sharing agreement with his former institution, Boston Children's Hospital/Harvard Medical School, which licensed the FDA-approved drug memantine (Namenda) to Forest Laboratories, Inc./Actavis/Allergan/AbbVie. S.A.L. is also a scientific founder of Adamas Pharmaceuticals, Inc. (now owned by Supernus Pharmaceuticals, Inc.), which developed or comarketed FDA-approved forms of memantine- or amantadine-containing drugs (NamendaXR, Namzaric, and GoCovri), and of EuMentis Therapeutics, Inc., which has licensed NitroSynapsin and related aminoadamantane nitrates from S.A.L. He also serves on the Scientific Advisory Board of Point6 Bio. National Institute on Aging, RF1AG061846, 5R01AG075862, 5R01AG077046, R01AG073418 National Institute on Alcohol Abuse and Alcoholism, T32AA007456 National Institute on Aging, https://ror.org/049v75w11, U01AG088679, R01AG056259, R35AG071734