Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-β-cyclodextrin (MβCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MβCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor–related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer’s disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER^T2 Lrp1^fl/fl mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [¹²⁵I] Aβ_1–42. Additionally, in the 5xFAD mouse model of AD, brain endothelial–specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD.