Activation of Endothelium in Alzheimer's Brain by Soluble Amyloid-B Aggregates

1. Activation of endothelium in Alzheimer’s brain involves soluble aggregates of the amyloid-b protein F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler, J.A. Kotarek, D.D. Soto-Ortega, and M.A. Moss University of South Carolina • College of Engineering and Computing • University of South Carolina

2. Alzheimer’s Pathology Alzheimer’s Brain Amyloid Plaques Pathology Ab Fibrils Courtesy of D. Dickson, Mayo Clinic, Jacksonville. Cerebral Amyloid Angiopathy Nichols et al. (2002) Biochemistry, 41: 6115-27. http://www.adrc.wustl.edu/adrc2.html College of Engineering and Computing University of South Carolina

3. Cerebral Amyloid Angiopathy • Characterized by deposition of Ab in the cerebrovasculature • Primarily Ab1-40 • Identified in over 90% of AD patients • Ab mutations: Dutch, Italian, Iowa, and Arctic • Dutch and Italian mutations result exclusively in CAA • Associated with an increased risk of hemorrhagic stroke • Correlates with disease progression Washington University ADRC College of Engineering and Computing University of South Carolina

4. Cellular degeneration Compromised BBB integrity Increased vessel-associated monocyte cells Toxicity Increased permeability Increased monocyte adhesion, transmigration Upregulated adhesion molecule expression Ab and Endothelium Cell Culture Cerebrovasculature Washington University ADRC College of Engineering and Computing University of South Carolina

5. Research Questions… What form of Ab interacts with endothelial cells to promote activation What signaling pathways are involved monomer nucleus soluble intermediates fibril College of Engineering and Computing University of South Carolina

6. Isolation of Ab Species 2-10 mM NaCl Mixture 150 mM NaCl Fibrils College of Engineering and Computing University of South Carolina

7. A model endothelium exhibiting blood-brain barrier properties was developed Gonzalez-Velasquez and Moss (2008) J Neurochem, 104: 500-13. College of Engineering and Computing University of South Carolina

8. Endothelial Cell Assays Adhesion Transmigration Permeability % adherent cells % transmigrated cells permeability coefficient Pe College of Engineering and Computing University of South Carolina

9.  MIX  MON  SOL  FIB Soluble Ab aggregates selectively activate endothelial cells Gonzalez-Velasquez and Moss (2008) J Neurochem, 107: 466-77; Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem, 104: 500-14. College of Engineering and Computing University of South Carolina

10. Endothelial activation is more pronounced in the presence of smaller soluble Ab aggregation intermediates  Adhesion  Permeability Gonzalez-Velasquez and Moss (2008) J Neurochem, 107: 466-77; Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem, 104: 500-14. College of Engineering and Computing University of South Carolina

11. NF-kB and Ab • NF-kB is involved in the activation of endothelium • Adhesion molecule upregulation • Permeability • Ab is capable of activating NF-kB • Neuronal cells • Glial cells • Ab can activate NF-kB via binding of several receptors • Receptor for advanced glycation end products (RAGE) • Toll-like receptors (TLRs) Hayden and Ghosh (2008) Cell, 132:344-62. College of Engineering and Computing University of South Carolina

12. NF-kB nuclear translocation is selectively stimulated by soluble Ab aggregates CONT TNF MIX MON SOL FIB Gonzalez-Velasquez, Reed, et al, and Moss (2010) Curr Alzheimer Res, 8: 81-94. . College of Engineering and Computing University of South Carolina

13. NF-kB activation is observed at physiological concentrations of Ab Gonzalez-Velasquez, Reed, et al, and Moss (2011) Curr Alzheimer Res, 8: 81-94. College of Engineering and Computing University of South Carolina

14.  Control  MG 132 Inhibiting NF-kB nuclear translocation abrogates Ab-induced adhesion, transmigration, and permeability Gonzalez-Velasquez, Reed, et al, and Moss (2011) Curr Alzheimer Res, 8: 81-94. College of Engineering and Computing University of South Carolina

15. Summary • Ab is capable of activating endothelial monolayers • Monocyte adhesion • Monocyte transmigration • Permeability • Specifically, soluble Ab aggregation intermediates are responsible for endothelial activation • Smaller soluble aggregation intermediates are the most potent activators of the endothelium • NF-kB signaling is involved in Ab activation of endothelial monolayers College of Engineering and Computing University of South Carolina

16. Acknowledgements • National Science Foundation • CAREER Award (BES 0644826) • American Heart Association, Mid-Atlantic Affiliate • Beginning Grant-In-Aid (0565387U) • Alzheimer’s Association • New Investigator Research Grant (NIRG-07-60412) • USC Complementary and Alternative Medicine Center Research Associate Francisco Gonzalez-Velasquez Undergraduate Students Charlotte Cooper Tim Davis Anthony Egal McCall Rogers Emily Matherly Graduate Students Adriana Reyes Barcelo Joseph Kotarek Will Reed Deborah Soto-Ortega Chen Suo Jui-Heng Tseng Kelly Wilson College of Engineering and Computing University of South Carolina

17. Activation of endothelium in Alzheimer’s brain involves soluble aggregates of the amyloid-b protein F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler, J.A. Kotarek, D.D. Soto-Ortega, and M.A. Moss University of South Carolina • College of Engineering and Computing • University of South Carolina