An Atlas of the Quantitative Protein Expression of Anti-Epileptic-Drug Transporters, Metabolizing Enzymes and Tight Junctions at the Blood-Brain Barrier in Epileptic Patients.

The purpose of the present study was to quantitatively elucidate the levels of protein expression of anti-epileptic-drug (AED) transporters, metabolizing enzymes and tight junction molecules at the blood-brain barrier (BBB) in the focal site of epilepsy patients using accurate SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Brain capillaries were isolated from focal sites in six epilepsy patients and five normal brains; tryptic digests were produced and subjected to SWATH analysis. MDR1 and BCRP were significantly downregulated in the epilepsy group compared to the normal group. Out of 16 AED-metabolizing enzymes detected, the protein expression levels of GSTP1, GSTO1, CYP2E1, ALDH1A1, ALDH6A1, ALDH7A1, ALDH9A1 and ADH5 were significantly 2.13-, 6.23-, 2.16-, 2.80-, 1.73-, 1.67-, 2.47- and 2.23-fold greater in the brain capillaries of epileptic patients than those of normal brains, respectively. The protein expression levels of Claudin-5, ZO-1, Catenin alpha-1, beta-1 and delta-1 were significantly lower, 1.97-, 2.51-, 2.44-, 1.90- and 1.63-fold, in the brain capillaries of epileptic patients compared to those of normal brains, respectively. Consistent with these observations, leakage of blood proteins was also observed. These results provide for a better understanding of the therapeutic effect of AEDs and molecular mechanisms of AED resistance in epileptic patients.

Comparison of Absolute Protein Abundances of Transporters and Receptors among Blood-Brain Barriers at Different Cerebral Regions and the Blood-Spinal Cord Barrier in Humans and Rats.

This work was designed to clarify the absolute abundances of transporters and receptors at different cerebral regions of the blood-brain barriers (BBB) and blood-spinal cord barrier (BSCB) in humans and rats, using physiologically relevant units (pmol/g tissue and fmol/cm2); 39 and 29 proteins including tight-junction proteins and markers were quantified in human and rat capillary samples, respectively. Protein expression levels of almost all proteins were identical within a 2-fold range between BBB and BSCB in rats, while many proteins showed >2-fold smaller expression levels in BSCB than BBB in humans. Protein expression levels of transporters and receptors in humans were remarkably smaller than those in rats in both BBB and BSCB in units of pmol/g tissue and fmol/cm2. Protein expression levels (fmol/cm2) of MDR1 and BCRP at the BBB in humans were 9.88-fold and 5.23-fold smaller than those in rats, respectively. GLUT1 expression (pmol/g tissue) at cortical BBB in a human was 2.49- and 3.76-fold greater than that at white matter BBB and BSCB, respectively. INSR and LRP1 proteins were detected at cortical BBB, but not at white matter BBB or BSCB in humans. These findings throw light on regional differences and species differences in pharmacokinetics and physiological functions in the central nervous system.

Cluster of Differentiation 46 Is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).

Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).

Evaluation of Organic Anion Transporter 1A2-knock-in Mice as a Model of Human Blood-brain Barrier.

The present study aimed to establish a humanized mouse model with which to explore OATP1A2-mediated transcellular transport of drug substrates across the blood-brain barrier (BBB) and to evaluate the usefulness of the humanized mice in preclinical studies. Sulpiride, amisulpride, sultopride, and triptans were used as probes to discriminate OATP1A2 and Oatp1a4. We generated a mouse line humanized for OATP1A2 by introducing the coding region downstream of the Oatp1a4 promoter using the CRISPR/Cas9 technique. In the mice generated, OATP1A2 mRNA in the brain was increased corresponding to disappearance of Oatp1a4. OATP1A2 was localized on both the luminal and abluminal sides of the BBB. Unfortunately, study in vivo employing sulpiride, sumatriptan, and zolmitriptan as probes did not indicate any difference in their brain-to-plasma ratio between the control and humanized mice. Quantitative targeted absolute proteomic analysis of the BBB fraction from the humanized mice revealed that almost all analyzed transporters and membrane proteins were expressed at similar levels to those in control mice. The quantitative levels of OATP1A2 differed depending on the peptide quantified, which suggests that incomplete translation or posttranslational modification may occur. The blood-to-brain transport of zolmitriptan, determined by brain perfusion in situ, was 1.6-fold higher in the humanized mice than in the controls, whereas that of sulpiride was not significantly changed. To our knowledge, we established a mouse line humanized for a BBB uptake transporter for the first time. Unfortunately, because of limited impact, there is still room for improvement of the model system.