The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

Toth, Andrea E.; Siupka, Piotr; Augustine, Thomas J P; Venø, Susanne T.; Thomsen, Louiza Bohn; Moos, Torben; Lohi, Hannes; Madsen, Peder; Lykke‐Hartmann, Karin; Nielsen, Morten S.

doi:10.1007/s12035-018-0988-x

Cited by 11 publications

(14 citation statements)

References 81 publications

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“…TAMRA-Tat- N -dimer appeared to be taken up by the primary BCECs cultured on microscope slides to the same extent as TAMRA-Tat and TAMRA-Tat-NR2B9c, which contradicts the observed degree of uptake in the in vitro BBB model. Recently, Toth et al demonstrated that co-culture of BCECs with astrocytes affects the endo-lysosomal sorting system [ 42 ]. Whether the presence of the astrocytes affects the uptake of TAMRA-Tat- N -dimer in the co-culture BBB model, but not in the BCECs when cultured alone on microscope slides, requires further study before conclusions can be drawn.…”

Section: Discussionmentioning

confidence: 99%

Conjugation of Therapeutic PSD-95 Inhibitors to the Cell-Penetrating Peptide Tat Affects Blood–Brain Barrier Adherence, Uptake, and Permeation

et al. 2020

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Novel stroke therapies are needed. Inhibition of the interaction between the postsynaptic density-95 (PSD-95)/disc large/ZO-1 (PDZ) domains of PSD-95 and the N-methyl-D-aspartate (NMDA) receptor has been suggested as a strategy for relieving neuronal damage. The peptides NR2B9c and N-dimer have been designed to hinder this interaction; they are conjugated to the cell-penetrating peptide Tat to facilitate blood–brain barrier (BBB) permeation and neuronal uptake. Tat-N-dimer exhibits 1000-fold better target affinity than Tat-NR2B9c, but the same magnitude of improvement is not observed in terms of therapeutic effect. Differences in BBB permeation by Tat-NR2B9c and Tat-N-dimer may explain this difference, but studies providing a direct comparison of Tat-NR2B9c and Tat-N-dimer are lacking. The aim of the present study was therefore to compare the BBB uptake and permeation of Tat-NR2B9c and Tat-N-dimer. The peptides were conjugated to the fluorophore TAMRA and their chemical stability assessed. Endothelial membrane association and cell uptake, and transendothelial permeation were estimated using co-cultures of primary bovine brain capillary endothelial cells and rat astrocytes. In vivo BBB permeation was demonstrated in mice using two-photon microscopy imaging. Tissue distribution was evaluated in mice demonstrating brain accumulation of TAMRA-Tat (0.4% ID/g), TAMRA-Tat-NR2B9c (0.3% ID/g), and TAMRA-Tat-N-dimer (0.25% ID/g). In conclusion, we demonstrate that attachment of NR2B9c or N-dimer to Tat affects both the chemical stability and the ability of the resulting construct to interact with and permeate the BBB.

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Section: Discussionmentioning

confidence: 99%

Conjugation of Therapeutic PSD-95 Inhibitors to the Cell-Penetrating Peptide Tat Affects Blood–Brain Barrier Adherence, Uptake, and Permeation

et al. 2020

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“…We consider that to improve the current understanding of BBB transcytosis and reveal the specific mechanisms of intracellular transport within BECs, it is necessary to adopt emerging technologies from the field of molecular cell biology, such as super-resolution microscopy, unbiased image analysis, and genome editing. The value of quantitative microscopy/image analysis for the BBB is highlighted by the findings on new mechanisms of transferrin receptor sorting [48] and on the regulation of the endo-lysosomal network of porcine BECs by astrocytes [113]. However, a major limitation of these studies is that they relied on two-dimensional cellular models that do not fully recapitulate the complex cellular architecture of the NVU.…”

Section: Open Questions and Challengesmentioning

confidence: 99%

Intracellular transport and regulation of transcytosis across the blood–brain barrier

Villaseñor

Lampe

Schwaninger

et al. 2018

Cell. Mol. Life Sci.

152

107

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The blood–brain barrier is a dynamic multicellular interface that regulates the transport of molecules between the blood circulation and the brain parenchyma. Proteins and peptides required for brain homeostasis cross the blood–brain barrier via transcellular transport, but the mechanisms that control this pathway are not well characterized. Here, we highlight recent studies on intracellular transport and transcytosis across the blood–brain barrier. Endothelial cells at the blood–brain barrier possess an intricate endosomal network that allows sorting to diverse cellular destinations. Internalization from the plasma membrane, endosomal sorting, and exocytosis all contribute to the regulation of transcytosis. Transmembrane receptors and blood-borne proteins utilize different pathways and mechanisms for transport across brain endothelial cells. Alterations to intracellular transport in brain endothelial cells during diseases of the central nervous system contribute to blood–brain barrier disruption and disease progression. Harnessing the intracellular sorting mechanisms at the blood–brain barrier can help improve delivery of biotherapeutics to the brain.

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“…Assumptions on their subcellular trafficking systems are based on observations from epithelial cell lines [3]. A recent study has pointed out that subcellular trafficking in primary BECs differs in detail from that of epithelial cells and therefore needs to be specifically examined [17]. Therefore, our aim was to investigate and characterize the endo-lysosomal system in the mouse bEnd.3 and the human hCMEC/D3 cell lines, which are widely used for the investigation of receptor-mediated transcytosis in in vitro drug transport studies.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, our aim was to investigate and characterize the endo-lysosomal system in the mouse bEnd.3 and the human hCMEC/D3 cell lines, which are widely used for the investigation of receptor-mediated transcytosis in in vitro drug transport studies. In our previous study, we already described and characterized the endo-lysosomal structure of primary porcine BECs (PBEC) [17]. Consequently, we compare our observations on the cell lines to the PBEC model.…”

Section: Introductionmentioning

confidence: 99%

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells

Toth

Nielsen

Tomaka

et al. 2019

Fluids Barriers CNS

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Background Brain endothelial cell-based in vitro models are among the most versatile tools in blood–brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial cell lines of in vitro blood–brain barrier models has not been investigated in detail, our aim was to characterize this system in different models. Methods For the investigation, we have chosen two widely-used models for in vitro drug transport studies: the bEnd.3 mouse and the hCMEC/D3 human brain endothelial cell line. We compared the structures and attributes of their endo-lysosomal system to that of primary porcine brain endothelial cells. Results We detected significant differences in the vesicular network regarding number, morphology, subcellular distribution and lysosomal activity. The retromer-positive vesicles of the primary cells were distinct in many ways from those of the cell lines. However, the cell lines showed higher lysosomal degradation activity than the primary cells. Additionally, the hCMEC/D3 possessed a strikingly unique ratio of recycling endosomes to late endosomes. Conclusions Taken together our data identify differences in the trafficking network of brain endothelial cells, essentially mapping the endo-lysosomal system of in vitro blood–brain barrier models. This knowledge is valuable for planning the optimal route across the blood–brain barrier and advancing drug delivery to the brain. Electronic supplementary material The online version of this article (10.1186/s12987-019-0134-9) contains supplementary material, which is available to authorized users.

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The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

Cited by 11 publications

References 81 publications

Conjugation of Therapeutic PSD-95 Inhibitors to the Cell-Penetrating Peptide Tat Affects Blood–Brain Barrier Adherence, Uptake, and Permeation

Conjugation of Therapeutic PSD-95 Inhibitors to the Cell-Penetrating Peptide Tat Affects Blood–Brain Barrier Adherence, Uptake, and Permeation

Intracellular transport and regulation of transcytosis across the blood–brain barrier

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells

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