Understanding the rules of the road: proteomic approaches to interrogate the blood brain barrier

Bruce E Torbett,Brian P Eliceiri,Andrew Baird

doi:10.3389/fnins.2015.00070

Abstract

The blood brain barrier (BBB) is often regarded as a passive barrier that protects brain parenchyma from toxic substances, circulating leukocytes, while allowing the passage of selected molecules. Recently, a combination of molecular profiling techniques have characterized the constituents of the BBB based on in vitro models using isolated endothelial cells and ex vivo models analyzing isolated blood vessels. Characterization of gene expression profiles that are specific to the endothelium of brain blood vessels, and the identification of proteins, cells and multi-cellular structure that comprise the BBB have led to a emerging consensus that the BBB is not, in and of itself, a simple barrier of specialized endothelial cells. Instead, regulation of transcytosis, permeability, and drug translocation into the central nervous system is now viewed as a collection of neurovascular units (NVUs) that, together, give the BBB its unique biological properties. We will review recent technology advancing the understanding of the molecular basis of the BBB with a focus on proteomic approaches.

Highlights

Characterization of gene expression profiles that are specific to the endothelium of brain blood vessels, and the identification of proteins, cells and multi-cellular structure that comprise the blood brain barrier (BBB) have led to a emerging consensus that the BBB is not, in and of itself, a simple barrier of specialized endothelial cells
Regulation of transcytosis, permeability, and drug translocation into the central nervous system is viewed as a collection of neurovascular units (NVUs) that, together, give the BBB its unique biological properties
We address the challenges of analyzing the protein composition of the BBB from the perspective of the endothelial cells, a compartment that has been the focus of in vitro models, and the importance of considering the BBB as a multicellular structure with extracellular matrix (ECM) and other cell types relevant in BBB formation (Chun et al, 2011; Hoshi et al, 2013; Badhwar et al, 2014)

Summary

Proteomics for Blood Brain Barrier Interrogation

Proteomics, the global interrogation of the protein population expressed by a genome, cells, or tissue types, makes use of biochemical and physical methods to determine the identity of proteins present (Yates, 1998, 2000; Banks et al, 2000; McDonald and Yates, 2000; Pandey and Mann, 2000; Yates, 2000; Mann and Pandey, 2001; Mann et al, 2001). Examples of these methods are Stable Isotope Labeling with Amino acids in cell Culture (SILAC), which relies on labeling of proteins in cellular samples by metabolic labeling with 15N where the differential in 15N/14N labeling can be used to quantify the relative abundance of identical proteins grown under different conditions (Wu et al, 2004; Lu et al, 2007; Gouw et al, 2008; Haqqani et al, 2008; Kamiie et al, 2008; Liao et al, 2008; Evans et al, 2012) This approach and other related metabolic cell labeling technologies are powerful tools where the model system, either cell culture or an animal, can be metabolically labeled to enable absolute quantification based on the relative uptake of labeled isotopes. Physiological context can be used to categorize various proteins that may be identified as a consequence of increased/decreased BBB integrity based on the presence of various specific cell types and their response to local or distal stimuli

Proteomics Approaches Based on Isolated Blood Vessels

Vessel Heterogeneity

Proteomic Analyses in Disease and Animal Models