(J-368) The role of RhoA/ROCK Signaling Pathway in Oligomeric Aβ and Tau-induced Inflammatory Responses and Cellular Mechanical Alterations in Cerebral Endothelial Cells
Professor University of Illinois at Chicago, United States
Introduction:: Introduction: Dysfunction of Cerebral Endothelial Cells (CECs) has been implicated in the pathology of Alzheimer’s disease (AD). Despite evidence showing the toxicity of oligomeric amyloid-β (oAβ) and Tau (oTau) peptides in cells in the central nervous system (CNS), the effects of these peptides on CECs are not yet fully understood.
Materials and Methods:: Materials and Methods: Primary mouse CECs were treated with oAβ, oTau, and their combination to study the effects of these peptides on CECs. To demonstrate the involvement of RhoA/ROCK pathway, cells were pre-treated with Fasudil, a RhoA/ROCK signaling inhibitor. Atomic force microscopy (AFM) with cantilevers biofunctionalized by Sialyl-LewisX (sLex) was employed to measure cell adhesion mechanics. P-selectin expression at the cell surface and actin polymerization within cells were quantified using immunofluorescence microscopy (QIM). Expressions of IL-1β, phosphorylated (p)-NF-kB p65, p-RhoA, GTP-RhoA, and p-ROCK2 were assayed by Western blot analysis, and TNF-α by Enzyme-linked Immunosorbent Assay (ELISA).
Results, Conclusions, and Discussions:: Results: Exposing CECs to oAβ and oTau increased cell stiffness, membrane tethering force and adhesion probability through the p-selectin/sLeX bonding at the cell surface. These cellular mechanical alterations were further significantly enhanced by the combined treatment of oAβ and oTau. Consistent with the cellular mechanical alterations, these treatments also increased actin polymerization, and p-selectin expression at the cell surface. In addition, treatments with oAβ and oTau also triggered inflammatory responses through activation of the RhoA/ROCK pathway, as indicated by significant upregulations of p-NF-kB p65, IL1β, TNFα, p-RhoA, GTP-RhoA, and p-ROCK2. While these cellular mechanical alterations, and biochemical processes were further enhanced by the combined treatment (oAβ+oTau), they were significantly suppressed by the RhoA/ROCK inhibitor, Fasudil.
Conclusion: In sum, our data suggests that oAβ, oTau and their combined treatment triggered inflammatory responses, and cellular mechanical alterations in CECs through the RhoA/ROCK pathway.
Acknowledgements (Optional): : This work was supported by National Institutes of Health R01AG044404 (JCL).
