Mapping Receptor Antibody Endocytosis and Trafficking in Brain Endothelial Cells

Holst, Mikkel R.; Nielsen, Simone S. E.; Nielsen, Mogens

doi:10.1007/7651_2020_312

Cited by 5 publications

(7 citation statements)

References 8 publications

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“…Colocalization score was analyzed and measured as described previously using IMARIS software (Bitplane) for spot segmentation [ 32 ]. Image analysis of the 3D STED microscopy images was carried out in MATLAB 2020b.…”

Section: Methodsmentioning

confidence: 99%

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers

Alam

Holst

Lauritsen

et al. 2022

Fluids Barriers CNS

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Parkinson’s disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson’s disease and related synucleinopathies.

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Section: Methodsmentioning

confidence: 99%

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers

Alam

Holst

Lauritsen

et al. 2022

Fluids Barriers CNS

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“…Using an immunofluorescence based, pulse-chase methodology for mapping receptor endocytosis and trafficking as previously described [ 39 ], we found the bi-directional endocytosis of TfR to significantly differientiate in co-localization with the intracellular markers EEA1 and Rab5 (Fig. 6 ).…”

Section: Resultsmentioning

confidence: 92%

“…Following establishment and validation of the BBB model, a pulse-chase study of receptor endocytosis was applied as previously described in detail [ 39 ]. Briefly, cells were cooled to 4 °C for 30 min in order to pause cellular activity, then primary anti-TfR antibody was applied to the apical or basal compartment, respectively, and the cells kept at 4 °C for further 90 min.…”

Section: Methodsmentioning

confidence: 99%

Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells

Nielsen

Holst

Langthaler

et al. 2023

Fluids Barriers CNS

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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.

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“…In the first 20 min of endocytosis, αvβ6-A20FMDV2 is significantly colocalised with early endosome marker EEA1 ( Figure 5 ), a Rab5 effector protein ( Wilson et al, 2000 ), and an antigen ubiquitously used to identify early endosomes ( Lee et al, 2020 ; Vandesande et al, 2020 ; Eapen et al, 2021 ; Holst et al, 2021 ; Wang et al, 2021 ). A significant shift in colocalization of A20FMDV2 (Cy5-(GS) 5 -bioA20FMDV2) with EEA1 endosomes from 1 to 2 h ( p < 0.001) was also observed.…”

Section: Discussionmentioning

confidence: 99%

Ligand-bound integrin αvβ6 internalisation and trafficking

Meecham

Cutmore²,

Protopapa³

et al. 2022

Front. Cell Dev. Biol.

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The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.

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Mapping Receptor Antibody Endocytosis and Trafficking in Brain Endothelial Cells

Cited by 5 publications

References 8 publications

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers

Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells

Ligand-bound integrin αvβ6 internalisation and trafficking

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