Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood–Brain Barrier In Vitro System
Abstract
:1. Introduction
2. Results
2.1. Brain Capillary Endothelial Cells
2.2. PrP 106-126 Alters cAMP and Nitric Oxide Levels in Brain Endothelial Cells
2.3. PrP 106-126 Modulates the Activation State of the Small GTPases Rac1 and RhoA
2.4. Stress Fibers Formation and Cortactin Reduction in Brain Endothelial Cells Exposed to PrP 106-126
2.5. PrP 106-126 Increases Cytoskeleton-Related Proteins Expression in Brain Endothelial Cells
3. Discussion
4. Materials and Methods
4.1. Ethics
4.2. Materials
4.3. Media
4.4. Peptides
4.5. Cells
4.6. In-Vitro BBB Model and Transendothelial Electrical Resistance Measurements
4.7. cAMP Determination
4.8. Nitrite Measurements
4.9. eNOS, iNOS and Cortactin Expression
4.10. Rac1 and RhoA Activation Assay
4.11. Immunocytochemistry
4.12. Liquid Chromatography-Mass Spectrometry (LC-MS)
4.12.1. Proteomic Analysis
4.12.2. Shotgun Proteomics
4.12.3. Targeted Proteomics—Selective Reaction Monitoring
4.13. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AJ | adherence junctions |
BBB | blood-brain barrier |
BCA | bicinchoninic acid |
BEC | brain endothelial cells |
GTPases | small guanosine triphosphatases |
iNOS and eNOS | inducible and endothelial nitric oxide synthase |
LC-MS | liquid chromatography-mass spectrometry |
LPS | lipopolysaccharide |
NO | nitric oxide |
TJ | tight junction |
PBS | phosphate-buffered saline |
PrPC | cellular human prion protein |
ROS | reactive oxygen species |
TEER | trans-endothelial electrical resistance |
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Cooper, I.; Cohen-Kashi Malina, K.; Levin, Y.; Gabashvili, A.; Mohar, B.; Cagnotto, A.; Salmona, M.; Teichberg, V.I. Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood–Brain Barrier In Vitro System. Int. J. Mol. Sci. 2022, 23, 12126. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232012126
Cooper I, Cohen-Kashi Malina K, Levin Y, Gabashvili A, Mohar B, Cagnotto A, Salmona M, Teichberg VI. Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood–Brain Barrier In Vitro System. International Journal of Molecular Sciences. 2022; 23(20):12126. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232012126
Chicago/Turabian StyleCooper, Itzik, Katayun Cohen-Kashi Malina, Yishai Levin, Alexandra Gabashvili, Boaz Mohar, Alfredo Cagnotto, Mario Salmona, and Vivian I. Teichberg. 2022. "Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood–Brain Barrier In Vitro System" International Journal of Molecular Sciences 23, no. 20: 12126. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232012126