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Proteomic analyses of src +/+ and src −/− mouse brain microvessels reveals insights into the regulation of the blood brain barrier integrity
Author(s) -
Minder Petra,
Scott Michael,
Moresco James J,
Baird Andrew,
Yates John R,
Eliceiri Brian P,
Torbett Bruce E
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb455
Subject(s) - proto oncogene tyrosine protein kinase src , microbiology and biotechnology , blood–brain barrier , biology , tight junction , tyrosine kinase , vascular permeability , proteomics , signal transduction , biochemistry , neuroscience , central nervous system , gene , endocrinology
The blood brain barrier (BBB) is a highly selective permeability barrier, composed of endothelial cells with tight intercellular junctions, surrounded by a basal lamina, astrocytes, and pericytes, which regulates the entry of compounds and cells between the blood and brain. The integrity of the BBB is necessary for maintaining brain function, whereas a number of severe brain diseases, such as stroke, edema, brain tumors and multiple sclerosis, have been associated with deregulation of BBB integrity. Therefore, understanding the molecular and cellular mechanisms whereby BBB integrity is maintained or deregulated, will be essential for restoration of BBB homeostasis after injury, as well as for selective therapeutic delivery. To interrogate the cellular and molecular mechanisms that maintain BBB integrity, we utilized Multidimensional Protein Identification Technology (MudPIT) proteomics to identify proteins from normal mouse ( src +/+ ) brain microvessels and microvessels from a leakage‐resistant vascular phenotype resulting from loss of Src ( src −/− ). Src is a non‐receptor tyrosine kinase that phosphorylates specific tyrosine residues and has been demonstrated to be necessary for the maintenance of BBB integrity. src −/− mice support relatively normal vessel development and sprouting, but demonstrate a leakage‐resistant phenotype. We identified 2479 proteins from MudPIT proteomics from src +/+ and src −/− mouse brain microvessel membrane preparations (n=3 biological samples from each phenotype), of which 150 proteins were determined to be differentially expressed in src −/− as compared to src +/+ microvessels. The top five cellular compartments showing protein changes were membrane, cytoplasm, nucleus, mitochondrion, and cell junction. Comparing microvessels of src +/+ to src −/− mice, we found differential expression of 42 proteins involved in transport across the plasma membrane and 47 proteins involved in cell adhesion and cytoskeleton remodeling. GeneGo analysis software was used to generate Pathway Maps. The majority of pathways and the relevant proteins identified are involved in cell adhesion and cytoskeleton development and regulation. Moreover, the Src‐regulated gene, Cdc42, was downregulated in src −/− compared to src +/+ tissue. Furthermore, we identified altered regulation of proteins necessary for actin dynamics and junctional remodeling in src −/− vessels. Imaging of actin in src −/− brain microvessels revealed that actin filaments are altered as compared to src +/+ mice. Our findings reveal that specific proteins are necessary for regulating the actin cytoskeleton to maintain BBB integrity through precise control of specific transporters. No significant alterations in the BBB basement membrane appeared to be necessary for alterations in BBB permeability.