Endocytosis of Liposomes Containing Sulfo-Cerebrosides by an Astrocytomal Cell Line: Is it a Charge Mediated Process or Does it Involve a Specific Receptor?

Abstract

Sulfo-cerebrosides (SCB) are glycosphingolipids found in high concentrations in the central nervous system (CNS), particularly in myelin where they play an important structural role. In addition, these lipids play an important role in various biological processes such as establishing a connection between the neuronal axon and myelin, and regulating cell growth and neuronal plasticity. Moreover, recent in-vitro studies from our group have shown that astrocytes avidly endocyte liposomes containing SCB without inducing a pro-inflammatory response. This indicates that astrocytes are involved in the homeostasis of the SCB in the CNS. In this work, we explore the specificity of the process of endocytosis of liposomes containing SCB, using an astrocytoma cell line as a model. Our hypothesis is that this process is receptor-mediated and not due to an electrostatic interaction with the cell surface. We show that (PG enriched) negatively charged liposomes do enhance endocytosis, but at a significantly lower level compared to SCB. We also show that specific uptake can be inhibited by screening the surface with polyethylene glycol. Additionally, we show through the use of endocytotic inhibitors that the SCB endocytotic route is a clatherin dependent route, which differs from non-specific liposome uptake routes. In these experiments, cells were cultured with liposomes labeled with the membrane fluorescent probe (Texas Red) to measure internalization, and a liposome content marker (calcein) to measure release. Measurements were carried out by flow cytometry and the results confirmed by fluorescence microscopy. Since low density lipoproteins (LDL) have been suggested to participate in SCB transport, we propose that the endocytotic pathway involves the LDL receptor (LDLr). Preliminary results show that SCB endocytosis is inhibited by LDLr specific antibodies.