Engineering the blood-brain barrier: molecular approaches for the modulation of tight junctions

The blood-brain barrier (BBB) is a multicellular structure comprising pericytes, astrocytes, and endothelial cells, which together safeguard the brain parenchyma and preserve its homeostasis. Endothelial cells are the walls of brain capillaries, acting as the first line of defence against the uncontrolled passive diffusion of solutes from the bloodstream. The selective transcellular transport of molecules is facilitated by specific transmembrane transporters, while the paracellular space between adjacent cells is tightly regulated by protein complexes known as tight junctions (TJs). Claudin5 (CLDN5) proteins are the primary components of TJs in the BBB, forming multimeric strands along the lateral membranes of neighbouring cells to effectively block the passage of nearly all solutes through the paracellular pathway. Although essential for brain protection, the BBB often represents an obstacle for brain-directed treatments. Due to its protective function, BBB indeed prevents most drugs from reaching the brain. Thus, one of the actual major challenges of the pharmaceutical industry is the development of drugs able to reach the brain and exert their functions. Moreover, the malfunction of some transporters can lead to severe impairments as for Glucose Transporter 1 Deficiency Syndrome (GLUT1-DS), since the transport cannot rely on the paracellular route. In this context, the possibility of regulating the BBB permeability in a transient and reversible manner is an intriguing opportunity for the pharmacological treatment of brain diseases. In this thesis, a robust workflow of structural modelling and in vitro validation techniques was designed to select and test short CLDN5-binding peptides (about 15 amino acids), derived from the CLDN5 extracellular domains and the non-toxic C-terminal domain of Clostridium perfringens enterotoxin, to transiently modulate BBB permeability and favour paracellular transport, especially of glucose, as a potential therapy for GLUT1-DS. The effective workflow successfully identified a novel CLDN5-derived peptide, called f1-C5C2, based on its solubility in biological media and the efficient binding to CLDN5. This selected peptide, tested in multiple in vitro BBB models, promoted a transient increase of size-selective permeability. The peptide effects on brain endothelial cells and TJs physiology were investigated in both the short- and long-term, suggesting that the proven alteration of TJs integrity can be reverted by shorter incubation time, still allowing BBB modulation. Despite a long way to go, taken together, the results presented in this thesis provide a strong foundation for advancing the promising field of TJ modulation with potential beneficial impact from a clinical perspective.