Amyloid-Beta Peptides 40 and 42 Employ Distinct Molecular Pathways for Cell Entry and Intracellular Transit at the Blood-Brain Barrier Endothelium.

The blood-brain barrier (BBB) plays a critical role in maintaining the equilibrium between amyloid beta (A) levels in blood and the brain by regulating A transport. Our previous publications demonstrated that BBB trafficking of A42 and A40 is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle A40 and A42 have not been clearly elucidated. In this study, we identified mechanisms of A endocytosis in polarized human cerebral microvascular endothelial cell monolayers. Our studies demonstrated that A peptides with fluorescent label (F-A) were internalized by BBB endothelial cells via energy, dynamin, and actin-dependent endocytosis. Interestingly, endocytosis of F-A40 but not F-A42 was substantially reduced by clathrin inhibition, whereas F-A42 but not F-A40 endocytosis was reduced by half after inhibiting the caveolae-mediated pathway. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. Although A42 was shown to accumulate more in the lysosomes, which could lead to its higher degradation and/or aggregation at lower lysosomal pH, A40 demonstrated robust accumulation in recycling endosomes, which may facilitate its exocytosis by the endothelial cells. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport A40 versus A42. This knowledge contributes to the understanding of molecular pathways underlying A accumulation in the BBB endothelium and associated BBB dysfunction. Moreover, it allows us to establish mechanistic rationale for altered A40:A42 ratios and anomalous amyloid deposition in the cerebral vasculature as well as brain parenchyma during Alzheimer's disease progression. SIGNIFICANCE STATEMENT: Differential interaction of Aβ40 and Aβ42 isoforms with the blood-brain barrier (BBB) endothelium may contribute to perturbation in Aβ42:Aβ40 ratio, which is associated with Alzheimer's disease (AD) progression and severity. The current study identified distinct molecular pathways by which Aβ40 and Aβ42 are trafficked at the BBB, which regulates equilibrium between blood and brain Aβ levels. These findings provide molecular insights into mechanisms that engender BBB dysfunction and promote Aβ accumulation in AD brain.