A pulse-labeling assay to track extracellular vesicle spreading in the brain

PROJECT SUMMARY Extracellular vesicles (EVs) are nano-sized secreted vesicles that carry biomolecules and mediate cell-cell communication. EVs are implicated in the development and progression of Alzheimer’s disease (AD) through their role in spreading pathogenic proteins (i.e. amyloid-beta, Tau) through the brain. EVs also have utility as AD biomarkers, given their ability to cross the blood-brain-barrier (BBB) and their presence in multiple bodily fluids. Further, EVs are promising drug delivery vehicles, with the ability to target specific cell types. However, despite their promise as biomarkers and therapeutics, EV biology remains poorly understood due to the lack of tools for visualizing EVs in their physiological environments. Such tools will be essential for uncovering mechanisms of EV spreading and uptake by different brain cell types, information critical for slowing AD progression and harnessing EVs as AD therapeutics. In the current proposal, we will develop a pulse-labeling assay for tracking cell type-specific EV secretion and spreading in the murine brain (Aim 1). We will then use this assay to examine how APOE4 genotype, the strongest genetic risk factor for AD, impacts EV spreading in the dentate gyrus (Aim 2). The Halotag EV pulse-chase (HEVPL) assay will have the following three components: 1) EV-enriched tetraspanin membrane proteins conjugated to the self-labeling Halotag, to enable in vivo pulse labeling with fluorescent ligands that cross the BBB and covalently attach to Halotag; 2) AAV delivery coupled with cell type- specific promoters/enhancers to express tetraspanin-Halotag proteins in specific brain cells; 3) coexpression of tetraspanin-Halotags with GFP at equimolar concentration via the 2A cleavage sequence, allowing us to distinguish GFP+ ‘donor’ cells that secrete tetraspanin-Halotag-containing EVs and GFP- ‘recipient’ cells that internalize these EVs. The assay will be developed through in vitro and in vivo experiments to optimize tetraspanin-Halotag expression and labeling, test the assay’s dynamic range, and determine the time course over which labeled EVs can be detected in the brain. In Aim 2, we will use HEVPL to visualize the impact of the APOE4 gene on EV secretion and spreading from granule cells of the dentate gyrus (DG). ApoE4 was recently shown to compromise global EV secretion in aging humans and mice, but the specific cell types and brain regions impacted remain unknown. The DG is one of the earliest brain regions affected in AD, and granule cells appear to be particularly vulnerable to ApoE4-related pathology. Using the AAV-mscRE4 enhancer to drive CD63- Halotag expression in granule cells, together with Halotag pulse-labeling and automated confocal microscopy/3D image reconstruction, we will quantify and compare EV secretion and spreading over time for humanized APOE3 vs. APOE4 mice. These experiments will provide the first information about how ApoE4 impacts EV release and spreading in an AD-vulnerable brain region, shedding light on a mechanism by which APOE4 genotype may accelerate AD progression.