Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and employ Mrc1a for cargo uptake

Abstract

19Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells within 20 the meningeal layer of the zebrafish brain. We previously reported that BLECs efficiently endocytose 21 extracellular cargo molecules (van Lessen et al., 2017), but how this is accomplished and controlled on 22 the molecular level remains unclear. We here compare BLECs to scavenging endothelial cells (SECs) in 23 the embryonic cardinal vein and find them to accept an identical set of substrate molecules. While there 24is redundancy in the type of scavenger receptors being used, the two cell populations rely for specific 25 substrate molecules on different cell surface receptors to mediate their physiological role: Stab2 26 appears more critical within SECs in the cardinal vein, while BLECs depend more on the Mrc1a receptor 27for internalization of cargo. Given the striking similarities to the substrate specificity of cardinal vein 28SECs, we postulate that BLECs qualify functionally as SECs of the brain. 29 30 31

Cells with Many Talents: Lymphatic Endothelial Cells in the Brain Meninges

“…Injection of Amyloid β into the CSF of larval zebrafish [ 54 , 55 ] resulted in its internalization by BLECs. Microglia have long been established as the main cells removing extracellular debris from the brain [ 56 ]; hence, identifying a cell population with high endocytic capacity immediately adjacent to the brain raises the question about ‘division of labor’ between microglia and BLECs, particularly as BLECs appear to be more efficient in direct comparison [ 54 ]. A formal analysis is required in order to decipher the differences between BLECs and microglia in terms of substrate specificity and context of action.…”

“…Recently, Campbell et al showed the presence of SECs in several zebrafish veins, including the embryonic cardinal vein, and that their clearance mechanism is highly dependent on the transmembrane receptor stabilin-2 (Stab2) [ 57 ]. Comparing embryonic SECs within the PCV and larval BLECs within the meninges in terms of scavenging exogenous substances led to the conclusions that BLECs qualify as SECs [ 54 ].…”

Differential Clearance of Aβ Species from the Brain by Brain Lymphatic Endothelial Cells in Zebrafish

“…In contrast, the co-localization of oAβ42 in BLECs was quite scarce following oAβ42 injection (Figure 3B,F: the co-localization frequency of oAβ42 in prox1a+ BLECs, 12.04%), and the endocytic vesicles in BLECs were barely positive with oAβ42 fluorescence (Figure 3F, white arrows in Figure 3F ). To validate that the injected mAβ42 was internalized into endocytic vesicles, we used pHrodo Green dextran (pHrodoGreen), which emitted a pH-sensitive fluorescence upon its internalization in the acidic environment and was used to reveal the intracellular uptake in BLECs [16,18]. After mAβ42 or oAβ42 injection, following pHrodo injection (Figure S5A), we observed the co-localization of mAβ42 labeled with HiLyte Fluor 647 (mAβ42-HiLyte Fluor 647) and pHrodo in BLECs (Figure S5B), while oAβ42 was undetected in BLECs (Figure S5C), indicating that the mAβ42 was actively internalized into the endocytic compartment of BLECs.…”

“…To validate the internalization of BLECs, an endocytic marker, pHrodoGreen dextran, 10 kDa (ThermoFischer, Waltham, MA, USA, P35368) was prepared in PBS at 2 mg/mL concentration and injected into the brain ventricle of 3 dpf zebrafish larvae [16,18] before mAβ42 or oAβ42 was introduced. In the mannan experiment, pHrodoGreen was injected after PBS or mannan administration.…”

“…Recently, these BLECs were also found in mouse and human leptomeninges, sharing morphological features and molecular markers [17]. BLECs are able to internalize macromolecules such as Aβ40 from the brain or take up lipids with a close proximity to the vasculature, suggesting their involvement in the brain clearance system [14,[16][17][18]. However, the exact role of BLECs in AD pathology and the mechanism by which BLECs clear different forms of Aβ species remain to be determined.…”