Monday, May 5, 2025
5/5/2025
Single Exosome Technology for Alzheimer's Disease - PROJECT SUMMARY This project responds to the important need for improved diagnostics for Alzheimer’s disease (AD). Existing methods based on biomarkers such as Ab and tau protein ratios from cerebrospinal fluid (CSF) are useful but incomplete. Furthermore, CSF sampling requires lumbar puncture and is too expensive for broad screening. A class of extracellular vesicles (EVs), exosomes, provide an attractive target for AD diagnostics. Exosomes freely cross the blood-brain barrier and can be readily sampled in peripheral blood, enabling a blood-based liquid biopsy. Exosomes also provide rich signatures for disease detection, including both proteins and nucleic acids (mRNA, miRNA, and other non-coding RNAs). The majority of studies on EVs and AD have been performed with bulk or batch analyses. Bulk analysis has a fundamental limitation because the relatively rare exosomes specific to the central nervous system (CNS) are easily confounded (swamped) by the exosome contributions of peripheral cells. To overcome this limitation, we are developing methods for combined protein/nucleic acid analysis in single exosomes. Our innovative, high-content, high-throughput method is designed to simultaneously analyze, in one pass, up to 10 potential AD biomarker cargoes in as many as 107 individual, CNS-tagged blood exosomes. These unique capabilities provide multiple advantages over previous approaches. Our method can rapidly: 1) distinguish and separately analyze both exosomes and other EVs; 2) discriminate and simultaneously evaluate multiple CNS-specific exosome surface markers, whereas conventional approaches can only evaluate one CNS-specific surface marker at a time, significantly limiting the ability to identify exosomes of CNS origin; 3) individually interrogate each and every exosome in a sample for its cargoes, dramatically raising information content compared to conventional methods where exosome cargoes must be pooled; and 4) search for unique combinations of biomarkers within unique, CNS-specific exosomal populations, an impossibility with conventional approaches. We therefore propose the following stepwise objectives for this Phase I project. First, to optimize the combined protein/nucleic acid analysis of exosomes produced by human SH-SY5Y cells. Second, to assay exosomes in brain homogenate samples from rapid autopsies of 40 AD, 40 mild cognitive impairment (MCI), 40 non-AD neurological conditions (nADneuro) (e.g., Parkinson’s disease, amyotrophic lateral sclerosis), and 40 normal elderly control (NC) subjects. Third, to assay exosomes in rapid autopsy blood samples from AD, MCI, nADneuro, and NC subjects that provided brain samples used in Objective 2. We anticipate that our novel imaging platform has the potential to become a new research/diagnostic/ prognostic tool for the clinical management of AD or other pathologies in which EV/exosomal analysis could provide clinically useful information, such as other neurodegenerative diseases, cancer, and cardiovascular disease. This capability may even assist in developing exosome-based therapies for these pathologies.
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