Mechanisms and applications of Bri2 BRICHOS passage across the blood-brain barrier

Neurodegenerative diseases constitute a major and growing medical and healthcare challenge to modern societies. Effective therapies remain limited, largely due to the blood-brain barrier (BBB), which restricts biological molecules from entering the central nervous system (CNS) from the bloodstream. The protein domain Bri2 BRICHOS inhibits the aggregation and toxicity of several amyloidogenic proteins. Recombinant Bri2 BRICHOS is therefore being evaluated for its therapeutic potential against neurodegenerative diseases, mainly Alzheimer's disease. During these studies it was found that Bri2 BRICHOS enters the brain when injected intravenously in mice. The overall aim of this thesis was to investigate the mechanisms underlying Bri2 BRICHOS passage from blood to brain and to evaluate its potential as a transport vehicle for biological drugs into the CNS. In paper I, we demonstrate that recombinant Bri2 BRICHOS can carry cargo proteins across a human BBB in vitro model system. We also found evidence in vivo in wild type mice that Bri2 BRICHOS can cross brain vascular endothelial cells and enter neurons. Building on these results, paper II explores the use of Bri2 BRICHOS for endothelial transcytosis of antibodies, that exhibit very limited BBB passage, which is a major hurdle for their use as CNS therapeutics. We show that coupling Bri2 BRICHOS to antibodies strongly promotes their endocytosis, endosomal routing and transcytosis in vitro in the human BBB model. In paper III we investigate the mechanisms responsible for Bri2 BRICHOS endo- and transcytosis in cerebral endothelial cells. Using proximity biotinylation, protein affinity studies, inhibitor experiments and fluorescence microscopy, we demonstrate that Bri2 BRICHOS transcytosis is bidirectional, and that the entry into endothelial cells is saturable and dynamin dependent. Our results suggest that Bri2 BRICHOS can bind transiently to ß-sheet motifs of multiple membrane proteins, perhaps in similar ways as it binds to amyloid fibrils, and thereby trigger transcytosis. Finally, in paper IV we demonstrate that recombinant Bri2 BRICHOS production is scalable in a bioreactor system. The findings presented herein expand the known functions of Bri2 BRICHOS, they suggest an unexplored mechanism by which Bri2 BRICHOS can cross the BBB and they display its potential as a transport vehicle to increase the efficacy of proteins for CNS therapies. List of scientific papers I. Bri2 BRICHOS, a molecular chaperone-like domain that can cross brain vascular endothelial cells and deliver proteins into neurons. Tigro, H., Moëlo, C., Pelcman, J., Paslawski, W., Chen, G., Poska, H., Shimmo, R., Svenningsson, P., Kronqvist, N. & Johansson, J. Journal of Controlled Release, 2025, 388, 114403. https://doi.org/10.1016/j.jconrel.2025.114403 II. Bri2 BRICHOS enables transcytosis of IgG antibodies over a human blood-brain barrier model. Pelcman, J., Moëlo, C., Kronqvist, N. & Johansson, J. [Manuscript] III. Mechanisms for transcytosis of the molecular chaperone Bri2 BRICHOS across human brain vascular endothelial cells. Pelcman, J., Kronqvist, N., Johansson, J., Moëlo, C. [Manuscript] IV. Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture. Schmuck, B., Chen, G., Pelcman, J., Kronqvist, N., Rising, A. & Johansson, J. Microbial Cell Factories, 2021, 20, 150. https://doi.org/10.1186/s12934-021-01638-8