TAT peptide at treatment-level concentrations crossed brain endothelial cell monolayer independent of receptor-mediated endocytosis or peptide-inflicted barrier disruption

• Published in: PloS one 2023-10, Vol.18 (10), p.e0292681-e0292681
• Main Authors: Wu, Meng-Chih, Wang, Eric Yuhsiang, Lai, Ted Weita
• Format: Article
• Language: English
• Subjects: Amino acids; Analysis; Antibodies; Biology and Life Sciences; Blood-brain barrier; Brain research; Cell culture; Covalence; Disruption; Endocytosis; Endothelial cells; Endothelium; Ethylenediaminetetraacetic acid; Experiments; Fluorescein isothiocyanate; Fluorescent indicators; Health aspects; Health services; HIV; Human immunodeficiency virus; Hypothermia; Macromolecules; Medical research; Medicine and Health Sciences; Medicine, Experimental; Membrane permeability; Membranes; Metabolism; Metabolites; Monolayers; Peptides; Permeability; Physical Sciences; Proteins; Receptors; Research and Analysis Methods; Tat protein; Temperature dependence
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0292681

The peptide domain extending from residues 49 to 57 of the HIV-1 Tat protein (TAT) has been widely shown to facilitate cell entry of and blood-brain barrier (BBB) permeability to covalently bound macromolecules; therefore, TAT-linked therapeutic peptides trafficked through peripheral routes have been used to treat brain diseases in preclinical and clinical studies. Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an in vitro model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the "in vitro BBB", a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells. Our results show that although TAT crossed the monolayer of brain endothelial cells in a temperature-dependent manner, in contrast to the reported mechanism of cell entry, it did not require receptor-mediated endocytosis. Furthermore, we revisited the hypothesis that TAT facilitates brain delivery of covalently bound macromolecules by causing BBB disruption. Our results demonstrated that the dose of TAT commonly used in preclinical and clinical studies did not exert an effect on BBB permeability in vitro or in vivo; however, an extremely high TAT concentration caused BBB disruption in vitro. In conclusion, the BBB permeability to TAT is temperature-dependent, but at treatment-level concentrations, it does not involve receptor-mediated endocytosis or BBB disruption.