Optimization of the Isolation of Brain-Derived Exosomes from Blood: The Development of a Blood-Based Biomarker for Neurodegenerative Disease
Chemically modified aberrant Transactive Response DNA Binding Protein 43 (TDP-43) derivatives were found to represent a major accumulating protein in neuronal cytoplasmic inclusions and in exosomes in Frontotemporal Lobar Degeneration (FTLD), and in ALS patients. Exosomes are nano-size membranous vesicles that contain several macromolecules including aberrant pathological proteins. The size and membranous structure of exosomes allows them to pass through the blood brain barrier. These features make exosomes a potential platform in which targeted biomolecules can be analyzed. Our objective is to develop a method to isolate serum/plasma-derived brain-cell originated exosomes and assess their TDP-43 content as part of a surrogate biomarker development for limbic-predominant age-related TDP-43 encephalopathy (LATE).
A heterogenous mixture of extracellular vesicles was obtained by running healthy human plasma through an Izon qEVoriginal size exclusion chromatography column with 70-1000 nm matrix size. The exosome enriched fractions were pooled, and brain-derived exosomes were isolated by antibody cross-linked high-performance immunoprecipitation (HPIP) tips in conjunction with urea elution. For the isolation of astrocyte-derived exosomes, anti-GLAST antibody was cross-linked for the HPIP.
Immunoblot analysis and transmission electron microscopy (TEM) were used to confirm the presence of exosomes and quantify the amounts of TDP-43 and its phosphorylated form (pTDP-43). Immunoblot results showed the presence of TSG101, GLAST, TDP-43 and pTDP-43 in the HPIP eluates. TEM images confirm the presence of intact exosomes in the GLAST cross-linked HPIP eluates. TDP-43 and pTDP-43 signals were elevated in LATE samples compared to controls. These results suggest that the astrocyte-derived x exosomes may be a good analytical tool to monitor longitudinal changes in TDP-43 and its derivatives in neurodegenerative diseases.