TITLE: Modeling Neurovascular Communication in Alzheimer’s Disease via Extracellular Vesicles in a 3D In Vitro Model of Blood-Brain Barrier

AUTHORS: Vishal Singh Guleria¹, Violaine delorme-walker², Cecilia Marcondes², Charisse Winston¹

¹Department of Physiology and Neuroscience, Epstein Family Alzheimer’s Therapeutic Research Institute, Keck School of Medicine at USC, San Diego, CA, USA.    

²San Diego Biomedical Research Institute, San Diego, CA, USA.

INTRODUCTION:

HYPOTHESIS: EVs from AD individuals exhibit enhanced translocation across the BBB, thereby increasing BBB permeability and contributing to neurovascular dysfunction.

METHODS: 

Figure 1: (A) Plasma EVs were isolated and Neuronal EVs (NEVs) were enriched using magnetic immunocapture targeting L1CAM. (B) Brain tissue-derived EVs (BDEVs) were isolated via differential ultracentrifugation.

Figure 2: 3D BBB in vitro model composed of endothelial cells, astrocytes and pericytes was used to study the translocation of isolated EVs.

RESULTS:

Figure 3: (A-C) Plasma NEVs and (D-F) BDEVs were characterized using (A, D) nanoparticle tracking analysis, (B, E) ONI super-resolution imaging, and (C, F) western blotting. Both EV populations showed expected small EV size profiles and expressed canonical EV markers, including CD9, CD63, CD81, and TSG101.

Figure 4: (A-C) Compared to plasma Non-AD Control-NEVs, AD-NEVs showed decreased transendothelial electrical resistance (TEER), increased BBB permeability and showed higher post-barrier migration as measured by fluorescence intensity. (D-F) BDEVs exhibit permeability trends similar to plasma NEVs. Data represent mean ± SEM; Plasma NEVs, n=3 per group, BDEV, n=6 per group, *p<0.05.

Figure 5: (A) Representative fluorescence images showing BDEV uptake by endothelial cells in 3D BBB in vitro model exposed to BDEVs. Actin, blue; EVs, green. Images of basolateral chamber with pericytes not exposed to BDEVs, showing intact actin cytoskeletons but no BDEV signal. (B) Orthogonal projections (XZ) of images.

Figure 6: (A) 3D BBB immunofluorescence images showing VE-cadherin at endothelial junctions. VE-cadherin, red; BDEVs, green; nuclei, blue. (B) AD-BDEVs significantly reduce junctional VE-cadherin mean fluorescence intensity compared with control BDEVs. (C) AD-BDEVs significantly lower the barrier integrity index, indicating compromised BBB integrity. n=30 per group,  ***p < 0.001.

CONCLUSIONS AND FUTURE DIRECTIONS:

CONTACT: Vishal Singh Guleria, M.S, Ph.D (guleria@usc.edu)

Epstein Family Alzheimer’s Therapeutic Research Institute (ATRI), Keck School of Medicine at USC, San Diego, CA, USA