On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias

Abstract

The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor–related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.

The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis

“…Based on these findings, it was thus hypothesised that PACSIN-2 mediates vesicle or tubule formation for endocytosis. Interestingly, our recent study demonstrated that PACSIN-2 participates in the formation of tubular carriers for a fast transcytosis of LRP1 across BECs [7]. Furthermore, we also demonstrated that BECs employ PACSIN-2 for the clearance of amyloid-ß via LRP1 across the BBB [84].…”

“…A generally established concept is that endosomes undergo maturation, starting from the initial early endosomes (within 1-5 min, vesicles positive for Rab5 and early endosome antigen 1, EEA1), late endosomes (10-15 min, Rab7-positive vesicles) and lysosomes for degradation (after 30 min of the initiation of CME in vesicles associated with a lysosomal-associated membrane protein 1, LAMP1) [115]. However, this timing (15 to 30 min) is inconsistent with the rapid rate of transcytosis observed in vivo, which has been found to occur in <60 s in the lung epithelium [85] and even faster in BECs [7,117].…”

“…Apart from the BAR domain, BAR proteins contain other variable domains targeting distinct cellular components with different moieties to guide their cellular location [ 22 , 28 ] ( Table 1 ). Within the numerous members of BAR proteins, N-BAR (amphiphysin and endophilin) [ 18 , 29 ] and F-BAR proteins, including PACSIN-1 and PACSIN-2 [ 7 , 19 ], are expressed in the mammalian brain. These proteins have been implicated in intracellular trafficking in neurons; yet their actual role in BECs remains to be explored.…”

“…By analogy to the epithelium, it is generally established that the trafficking across the brain endothelium occurs via vesicular carriers. Nevertheless, recent findings suggest that BECs employ sorting tubules as a fast mechanism of transcytosis, in which the cargo is efficiently transported across via tubular carriers, avoiding endosomes and lysosomal degradation [ 6 , 7 ]. Interestingly, these results corroborate evidence from the 1970s and 1990s, in which it was suggested that intracellular trafficking occurs through a network of tubules at the brain endothelium [ 8 , 9 , 10 , 11 , 12 ].…”

“…Although the GC is not featured in canonical models of membrane shape regulation, GC polymers can generate forces that deform the cell membrane [ 17 ]. Even though the BAR proteins and GC are abundant in the brain endothelium [ 7 , 18 , 19 , 20 ] and intrinsically associated with the cell membrane and membrane curvature, these elements are often neglected in terms of their role in the formation of vesicular or tubular carriers for the transport and intracellular fate of the cargo in endothelial cells.…”