PROJECT SUMMARY Exosomes are small extracellular vesicles that mediate intercellular signaling in the brain without requiring direct contact between cells. These vesicles are enriched with miRNAs, proteins, and lipids. Current evidence for exosome signaling in the brain points toward their role in translational regulation, neurogenesis, synaptic plasticity, and neuroinflammation, all of which have been implicated in several neurodevelopmental diseases. However, changes in exosome signaling in mental disorders have yet to be explored. Exosomes have been isolated from nearly all mammalian cell types, including cells in the central nervous system such as neurons, astrocytes, oligodendrocytes, and microglia. Neurons and glia release exosomes in vitro and in vivo. Trophic support from glial cells to neurons is thought to be conveyed by exosomes. Exosome-mediated intercellular signaling has been implicated in neurodegenerative diseases such as ALS, Parkinson’s disease, multiple sclerosis, and Alzheimer’s Disease as well as neurodevelopmental disorders. More specifically, altered neuronal exosome signaling has been implicated in Rett syndrome, a form of Autism Spectrum Disorder (ASD). However, the role of exosome signaling in ASD and other mental disorders remains to be fully understood. The goal of this application is to explore whether exosome content and numbers are altered in ASD. We have isolated exosomes from healthy control and patient brain organoid cultures by differential ultracentrifugation and evaluated purified exosomes using transmission electron microscopy, western blotting for biochemical markers, and nanoparticle characterization system. In purified exosome fractions, we will first assess exosome proteomes and nucleic acid profiles that will be compared between control and patient groups. In addition to content, there is also evidence suggesting that exosome quantity is altered in disease states. To explore the role of neural exosomes in ASD, we propose to determine 1) whether the proteome of exosomes isolated from ASD patient organoids differ from exosomes derived from healthy control organoids (e.g., by comparing protein profiles with Tandem mass tag [TMT] liquid chromatography [LC] mass spectrometry), 2) whether nucleic acid content in exosomes purified from ASD patient organoids is altered in comparison to exosomes from control (e.g., by comparing RNA/miRNA profiles with next-generation sequencing followed by bioinformatics), and 3) whether the quantity of exosomes released from ASD brain cells is altered compared to control cells (e.g., by assessing the quantity of exosomes in 2D neuronal or glial culture media using nanosight tracking analysis). Exosome analysis from organoids and 2D cultures will serve as parallel strategies to delineate neural exosome content in health and disease. Our studies have the potential to provide novel insights into the etiology of ASD. Identifying alterations in amount or content of brain exosomes in ASD will reveal underappreciated modifications in cellular communication during mental disease states.

PROJECT NARRATIVE Exosomes are membrane-enclosed nanovesicles that are increasingly being recognized as important cell communication components transmitting proteins and RNAs. However, changes in exosome signaling in mental disorders have yet to be explored. This study aims to determine whether vesicular cargo and exosome quantity are altered in human stem cell models of ASD.