Low-density Lipoprotein Receptor-related Protein Mediates the Endocytosis of Anionic Liposomes in Neurons

Abstract

We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000 -32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-labeled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of ALCy3ONs was widespread and time-and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/ lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.Endocytosis in neurons has mainly been studied in the context of synaptic vesicle recycling (1) and the regulation of neurotransmitter receptor numbers in the post-synaptic membrane (2). Constitutive endocytosis also occurs in neurons, albeit at a slower rate than in non-polarized or mitotically active cells. Growth factors and hormones modulate the rate of constitutive endocytosis of neurotransmitter receptors as well as receptors involved in nutrient acquisition and metabolism. Neurotrophins such as nerve growth factor and bone-derived neurotrophic factor increase both the recruitment of clathrin to the plasma membrane and the rate of constitutive endocytosis of transferrin in hippocampal neurons (3).Little is known, however, about the mechanisms of internalization of exogenous macromolecules such as phospholipids and nucleic acids in neurons. Insight into the interactions between neurons and these molecules would further our understanding of lipid and DNA transport pathways and provide potential therapeutic advantages. We have recently designed and developed an anionic liposome vector (composed of the anionic phospholipid dioleoylphosphatidylglycerol and the zwitterionic phospholipid dioleoylphosphatidylcholine) for oligonucleotide delivery to neurons. Antisense oligonucleotides targeted to the p53 tumor suppressor mRNA, delivered to neurons via anionic liposomes, efficiently protected hippocampal neurons fr...

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Radiation improves gene delivery by a novel transferrin-lipoplex nanoparticle selectively in cancer cells

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