Alexander Laatsch

Impaired Recycling of Apolipoprotein E4 Is Associated with Intracellular Cholesterol Accumulation

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.

The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.

Insulin stimulates hepatic low density lipoprotein receptor-related protein 1 (LRP1) to increase postprandial lipoprotein clearance

BACKGROUND While the role of insulin in glucose uptake and its aberration in diabetes are well established, the effect of insulin on lipoprotein clearance in the postprandial phase is not yet fully understood. The dietary lipids are carried in chylomicron remnants (CR) which are taken up into the liver mainly via LDLR-related protein 1 (LRP1). In this study, the effect of insulin on LRP1-mediated hepatic CR uptake was investigated. METHODS The study was based on determining the subcellular localisation of LRP1 by subcellular fractionation and immunofluorescence microscopy and correlating those findings with the hepatic uptake of fluorescently or radioactively labelled LRP1-specific ligands and CR in hepatoma cells, primary hepatocytes and mouse models. RESULTS AND CONCLUSION In vitro and in vivo, insulin stimulated the translocation of hepatic LRP1 from intracellular vesicles to the plasma membrane, which correlates with an increased uptake of LRP1-specific ligands. In wild-type mice, a glucose-induced insulin response increased the hepatic uptake of LRP1 ligands while in leptin-deficient obese mice (ob/ob), which are characterised by hepatic insulin resistance, insulin-inducible LRP1 ligand uptake was abolished. Finally, upon hepatic LRP1 knockdown, insulin no longer significantly enhanced CR uptake into the liver. The insulin-induced LRP1-mediated CR uptake, as demonstrated here, suggests that impaired hepatic LRP1 translocation can contribute to the postprandial lipaemia in insulin resistance.

Knockdown of Hepatic ABCA1 by RNA Interference Decreases Plasma HDL Cholesterol Levels and Influences Postprandial Lipemia in Mice

Objective—To investigate the impact of hepatic ABCA1 on systemic lipoprotein metabolism in vivo by an adenovirus-mediated RNA interference approach. Methods and Results—Efficiency of plasmid-based small interference RNA (siRNA)-induced knockdown of cotransfected murine ATP binding cassette transporter A1 (mABCA1) in HEK-293 cells was judged by RT-polymerase chain reaction, immunofluorescence, and Western blot analysis. The most effective plasmid was used to generate a recombinant adenovirus as a tool to selectively downregulate ABCA1 expression in mouse liver (C57BL/6). In comparison to controls, Western blot analysis from liver membrane proteins of Ad-anti-ABCA1 infected mice resulted in an ≈50% reduction of endogenous ABCA1 and a clear upregulation of apolipoprotein E. Fast protein liquid chromatography analysis of plasma revealed that hepatic ABCA1 protein reduction was associated with an ≈40% decrease of HDL cholesterol and a reduction of HDL-associated apolipoprotein A-I and E. In the fasted state, other lipoprotein classes were not affected. To analyze the influence of ABCA1 downregulation on postprandial lipemia, infected mice were given a gastric load of radiolabeled trioleate in olive oil. In Ad-anti-ABCA1 infected mice, the postprandial increase of chylomicrons and chylomicron-associated apolipoproteins B and E was significantly reduced as compared with controls. Conclusion—Hepatic ABCA1 contributes to HDL plasma levels and influences postprandial lipemia.

Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

Background Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. Principal Findings Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. Conclusion We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.