Introduction: Understanding the role of exosomes in the brain is a fundamental scientific objective with clinical relevance. Realization of this goal however has been hampered by an inability to isolate genuine exosomes from the brain. Relative to the routine isolation from extracellular fluids, many technical issues must be overcome to successfully isolate exosomes from solid tissue. Exosomes share many physical and molecular properties with other vesicles imposing important limitations. Cell integrity needs to be maintained to minimize co-isolation of particles masking as exosomes and rigorous characterisation needs to be undertaken to confirm enrichment of exosomes relative to exosome mimetics. Here we have taken a critical approach to the enrichment and characterisation of exosomes from human frontal cortex and mouse tissue.
Methods: Vesicles were isolated from human (frontal cortex; Alzheimer’s disease or neurological control) or mouse (whole) brain tissues (n=50 human/n=30 mouse) and systematically assessed for morphology, density, size distribution and proteomic and genomic content to validate the approach.
Results: Immunoblot, electron microscopy, proteomics, size distribution, RNA and density gradient analysis confirmed successful isolation of endosome derived exosomes (enriched for syntenin, tsg101 and CD81) from brain tissue. Upon comparing extracellular vesicles from Alzheimer’s disease subjects versus aged matched controls we discovered a previously unidentified pool of the disease associated proteins in the frontal cortex of Alzheimer’s disease subjects.
Conclusion: Progression in understanding the role of extracellular vesicles in the nervous system has been hindered by a lack of appropriate methodology to isolate genuine exosomes, as defined by a minimal set of experimental requirements, from tissue. Our innovative methods have enabled us to isolate genuine brain exosomes and discover a new pool of neurodegenerative disease associated protein.