Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells

Abstract

The detailed mechanisms by which the transferrin receptor (TfR) and associated ligands traffic across brain capillary endothelial cells (BECs) of the CNS-protective blood–brain barrier constitute an important knowledge gap within maintenance and regulation of brain iron homeostasis. This knowledge gap also presents a major obstacle in research aiming to develop strategies for efficient receptor-mediated drug delivery to the brain. While TfR-mediated trafficking from blood to brain have been widely studied, investigation of TfR-mediated trafficking from brain to blood has been limited. In this study we investigated TfR distribution on the apical and basal plasma membranes of BECs using expansion microscopy, enabling sufficient resolution to separate the cellular plasma membranes of these morphological flat cells, and verifying both apical and basal TfR membrane domain localization. Using immunofluorescence-based transcellular transport studies, we delineated endosomal sorting of TfR endocytosed from the apical and basal membrane, respectively, as well as bi-directional TfR transcellular transport capability. The findings indicate different intracellular sorting mechanisms of TfR, depending on the apicobasal trafficking direction across the BBB, with the highest transcytosis capacity in the brain-to-blood direction. These results are of high importance for the current understanding of brain iron homeostasis. Also, the high level of TfR trafficking from the basal to apical membrane of BECs potentially explains the low transcytosis which are observed for the TfR-targeted therapeutics to the brain parenchyma.

Modelling a Human Blood-Brain Barrier Co-Culture Using an Ultrathin Silicon Nitride Membrane-Based Microfluidic Device

“…The cell thickness of iBMECs can be advantageous as it enables the use of conventional confocal microscopy to study transcellular trafficking. Such studies are limited in in vitro models based on primary brain endothelial cells (BECs) as the thickness of these cells are below the axial resolution of the confocal microscope [ 41 ]. Higher resolutions with confocal microscopy can be achieved by the use of Zeiss’ Airyscan unit or Leica’s SP8 confocal microscope, or alternatively, the cells can be expanded after fixation (expansion microscopy [ 42 ]) and super-resolution can be achieved [ 41 ].…”

“…Such studies are limited in in vitro models based on primary brain endothelial cells (BECs) as the thickness of these cells are below the axial resolution of the confocal microscope [ 41 ]. Higher resolutions with confocal microscopy can be achieved by the use of Zeiss’ Airyscan unit or Leica’s SP8 confocal microscope, or alternatively, the cells can be expanded after fixation (expansion microscopy [ 42 ]) and super-resolution can be achieved [ 41 ]. Moreover, these iBMECs expressed the major junctional complexes and endothelial markers, including efflux MDR-1 and influx transporter GLUT-1, ( Figure 1 and Figure 3 B), and formed a very tight barrier.…”

Subcellular trafficking and transcytosis efficacy of different receptor types for therapeutic antibody delivery at the blood‒brain barrier

“…Although TfR has become the favourite cargo receptor for transcytosis across the BBB, a recent study showed bidirectional trafficking and endocytic capacity at both luminal and abluminal surfaces in BECs [ 26 ]. The study further shows transcytosis of cargo antibodies in both directions, thus limiting focused transcytosis in one direction [ 26 ]. The recycling capacity of TfR is a major determinant of its function in transporting transferrin.…”