Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, ß- and γ-secretases is a determining factor in Alzheimer's disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aß production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aß production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.

SorCS2 is required for BDNF-dependent plasticity in the hippocampus.

SorCS2 is a member of the Vps10p-domain receptor gene family receptors with critical roles in the control of neuronal viability and function. Several genetic studies have suggested SORCS2 to confer risk of bipolar disorder, schizophrenia and attention deficit-hyperactivity disorder. Here we report that hippocampal N-methyl-d-aspartate receptor-dependent synaptic plasticity is eliminated in SorCS2-deficient mice. This defect was traced to the ability of SorCS2 to form complexes with the neurotrophin receptor p75NTR, required for pro-brain-derived neurotrophic factor (BDNF) to induce long-term depression, and with the BDNF receptor tyrosine kinase TrkB to elicit long-term potentiation. Although the interaction with p75NTR was static, SorCS2 bound to TrkB in an activity-dependent manner to facilitate its translocation to postsynaptic densities for synaptic tagging and maintenance of synaptic potentiation. Neurons lacking SorCS2 failed to respond to BDNF by TrkB autophosphorylation, and activation of downstream signaling cascades, impacting neurite outgrowth and spine formation. Accordingly, Sorcs2-/- mice displayed impaired formation of long-term memory, increased risk taking and stimulus seeking behavior, enhanced susceptibility to stress and impaired prepulse inhibition. Our results identify SorCS2 as an indispensable coreceptor for p75NTR and TrkB in hippocampal neurons and suggest SORCS2 as the link between proBDNF/BDNF signaling and mental disorders.

Lipoprotein receptors: new roles for ancient proteins.

Lipoprotein receptors used to be viewed simply as the means by which cells were supplied with lipids for energy production and membrane synthesis. This perception has now changed dramatically. Megalin, a member of the low density lipoprotein receptor gene family, turns out to mediate the endocytic uptake of retinoids and steroids, thus helping to regulate their biological function. Other members of this receptor family interact with cytosolic signalling proteins, giving this evolutionarily ancient family of receptors an entirely unexpected new role as transducers of extracellular signals.

Remnant lipoproteins inhibit malaria sporozoite invasion of hepatocytes.

Remnants of lipoproteins, intestinal chylomicrons, and very low density lipoprotein (VLDL), are rapidly cleared from plasma and enter hepatocytes. It has been suggested that remnant lipoproteins are initially captured in the space of Disse by heparan sulfate proteoglycans (HSPGs), and that their subsequent internalization into hepatocytes is mediated by members of the LDL-receptor gene family. Similarly to lipoprotein remnants, malaria sporozoites are removed from the blood circulation by the liver within minutes after injection by Anopheles mosquitoes. The sporozoite's surface is covered by the circumsporozoite protein (CS), and its region II-plus has been implicated in the binding of the parasites to glycosaminoglycan chains of hepatocyte HSPGs. Lactoferrin, a protein with antibacterial properties found in breast milk and neutrophil granules, is also rapidly cleared from the circulation by hepatocytes, and can inhibit the hepatic uptake of lipoprotein remnants. Here we provide evidence that sporozoites, lactoferrin, and remnant lipoproteins are cleared from the blood by similar mechanisms. CS, lactoferrin, and remnant lipoproteins compete in vitro and in vivo for binding sites on liver cells. The relevance of this binding event for sporozoite infectivity is highlighted by our demonstration that apoliprotein E-enriched beta-VLDI and lactoferrin inhibit sporozoite invasion of HepG2 cells. In addition, malaria sporozoites are less infective in LDL-receptor knockout (LDLR -/-) mice maintained on a high fat diet, as compared with littermates maintained on a normal diet. We conclude that the clearance of lipoprotein remnants and sporozoites from the blood is mediated by the same set of highly sulfated HSPGs on the hepatocyte plasma membrane.

Interaction of the apolipoprotein E receptors low density lipoprotein receptor-related protein and sorLA/LR11.

In this study, we examined protein-protein interactions between two neuronal receptors, low density lipoprotein receptor-related protein (LRP) and sorLA/LR11, and found that these receptors interact, as indicated by three independent lines of evidence: co-immunoprecipitation experiments on mouse brain extracts and mouse neuronal cells, surface plasmon resonance analysis with purified human LRP and sorLA, and fluorescence lifetime imaging microscopy (FLIM) on rat primary cortical neurons. Immunocytochemistry experiments revealed widespread co-localization of LRP and sorLA within perinuclear compartments of rat primary neurons, while FLIM analysis showed that LRP-sorLA interactions take place within a subset of these compartments.

Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist.

The low density lipoprotein receptor-related protein (LRP) has been proposed to mediate in concert with the LDL receptor (LDLR) the uptake of dietary lipoproteins into the hepatocytes. This hypothesis was tested by transient inactivation of LRP in vivo. Receptor-associated protein (RAP), a dominant negative regulator of LRP function, was transferred by an adenoviral vector to the livers of mice lacking LDLR (LDLR-/-). The inactivation of LRP by RAP was associated with a marked accumulation of chylomicron remnants in LDLR-/- mice and to a lesser degree in normal mice, suggesting that both LDLR and LRP are involved in remnant clearance.

A novel renal carbonic anhydrase type III plays a role in proximal tubule dysfunction.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.

Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show that previously identified low-molecular-weight renal albumin binding proteins are fragments of cubilin. In addition, we find that mice lacking the endocytic receptor megalin show altered urinary excretion, and reduced tubular reabsorption, of albumin. Because cubilin has been shown to colocalize and interact with megalin, we propose a mechanism of albumin reabsorption mediated by both of these proteins. This process may prove important for understanding interstitial renal inflammation and fibrosis caused by proximal tubule uptake of an increased load of filtered albumin.

Sustained somatic gene inactivation by viral transfer of Cre recombinase.

Transgenic and knockout mice have proven invaluable tools for analyzing physiologically relavant functions of numerous genes. In some cases, however, pleiotropic effects that result from a variable requirement for a particular gene in different tissues, cell types, or stages of embryonic development may complicate the analysis due to a complex phenotype or embryonic lethality. The loxP/Cre-mediated recombination system, which allows tissue-specific gene targeting in the mouse, can be used to overcome these problems. A limitation of current methods is that a mouse carrying a loxP-tagged gene must be crossed with a transgenic mouse expressing the Cre recombinase in an appropriate tissue to obtain the desired gene rearrangement. We have used recombinant adenovirus carrying the Cre recombinase to induce virtually quantitative somatic cell gene disruption in the liver. The targeted gene was the multifunctional low-density lipoprotein receptor-related protein (LRP), a cell surface receptor for alpha 2-macroglobulin and other ligands. Transient expression of Cre following adenoviral infection produced the predicted gene rearrangement, functionally inactivating LRP in the liver. Rearrangement occurred within 6 days after infection and remained stable for at least 28 days. The results demonstrate the suitability of adenoviral Cre gene transfer to induce long-term, quantitative, and temporally controlled gene disruption in the mouse.

The low-density lipoprotein receptor gene family: multiple roles in lipid metabolism.

The low-density lipoprotein receptor gene family encompasses a class of endocytic receptors that exhibit structural similarities to the low-density lipoprotein receptor. Members of this gene family are present in both vertebrate and nonvertebrate species. The identification of naturally occurring mutations and the application of gene targeting to inactivate receptor genes enabled us to develop animal models to investigate the consequences of individual receptor defects in vivo. Analysis of these animal models revealed exciting new functions of lipoprotein receptors not only in systemic clearance of lipoproteins but also in other important biological processes including reproduction, brain development. and adipositas.

Animal models for disorders of hepatic lipoprotein metabolism.

The application of methods to create transgenic mice in which a gene of interest is either overexpressed or genetically inactivated has provided us with an ever-growing number of animal models to study complex physiological processes in vivo. Analysis of these mouse models has increased our knowledge about basic mechanisms that control biological systems and the pathological processes in human genetic disorders. This review focuses on the analysis of mouse models in which individual components of the hepatic clearance pathway for plasma lipoproteins have been inactivated. These studies have demonstrated that two hepatic lipoprotein receptors, the low-density lipoprotein receptor and the low-density lipoprotein receptor-related protein operate jointly in the uptake of dietary lipoproteins from the circulation. These findings have important implications for our understanding of pathophysiological processes resulting in hyperlipoproteinemia and atherosclerosis in patients.

Gene transfer and disruption strategies to elucidate hepatic lipoprotein receptor functions.

Recent technological advances have enabled us to manipulate specific genes in laboratory animals in a specific predetermined manner. This has opened new areas of research on physiological processes not previously accessible to such precise experimental manipulation. Over-expression of genes by traditional transgenic techniques has recently been complemented by methods that allow the efficient transfer of exogenous genes into various somatic tissues of adult animals. The development of homologous recombination technology in embryonic stem cells (ESC) and the application of this technology to specifically disrupt a given gene of interest in the germline of a mouse has been particularly useful to determine the physiologically relevant processes in which these genes participate in vivo. Rather than introducing random mutations into the genome by chemical mutagenesis or by retroviral insertion, techniques that have been employed in the past, gene targeting not only allows us to disrupt any cloned gene, but also to specifically introduce single nucleotide changes into its genomic sequence. The past few years have witnessed an explosion of research reports in all areas of biological research that have employed these ground-breaking tools of modern genetics to study the physiological roles of a plethora of different genes in neurobiology immunology, endocrinology, development, etc. Our laboratory has also extensively used these new approaches to study the function of several genes that are involved in the metabolism of lipoproteins on the systemic as well as on the cellular level. In this article, we will review the various approaches we have used to define the roles of the low density lipoprotein (LDL) receptor, the LDL receptor-related protein (LRP) and the receptor-associated protein (RAP) in hepatic lipoprotein metabolism.

Holoprosencephaly and low molecular weight proteinuria: the human homologue of murine megalin deficiency.

We encountered a child with holoprosencephaly, pulmonary insufficiency, absent circulating vitamin D metabolites, mild albuminuria, and urinary excretion of vitamin D-binding protein. The child displayed a phenotype highly reminiscent of that observed in mice genetically deficient for megalin, a member of the low-density lipoprotein receptor superfamily. Only the Guthrie card was available from the child; the DNA sufficed for a limited haplotype analysis. We were not able to implicate the megalin gene locus directly; however, the possibility of a functional megalin defect in this child remains. To the best of our knowledge, this patient represents the first report that pathologic abnormalities consistent with megalin deficiency are present in humans.

Mutually dependent localization of megalin and Dab2 in the renal proximal tubule.

Disabled-2 (Dab2) is a cytoplasmic adaptor protein that binds to the cytoplasmic tail of the multiligand endocytic receptor megalin, abundantly expressed in renal proximal tubules. Deletion of Dab2 induces a urinary increase in specific plasma proteins such as vitamin D binding protein and retinol binding protein (Morris SM, Tallquist MD, Rock CO, and Cooper JA. EMBO J 21: 1555-1564, 2002). However, the subcellular localization of Dab2 in the renal proximal tubule and its function have not been fully elucidated yet. Here, we report the characterization of Dab2 in the renal proximal tubule. Immunohistocytochemistry revealed colocalization with megalin in coated pits and vesicles but not in dense apical tubules and the brush border. Kidney-specific megalin knockout almost abolished Dab2 staining, indicating that Dab2 subcellular localization requires megalin in the proximal tubule. Reciprocally, knockout of Dab2 led to a redistribution of megalin from endosomes to microvilli. In addition, there was an overall decrease in levels of megalin protein observed by immunoblotting but no decrease in clathrin or alpha-adaptin protein levels or in megalin mRNA. In rat yolk sac epithelial BN16 cells, Dab2 was present apically and colocalized with megalin. Introduction of anti-Dab2 antibody into BN16 cells decreased the internalization of 125I-labeled receptor-associated protein, substantiating the role of Dab2 in megalin-mediated endocytosis. The present study shows that Dab2 is localized in the apical endocytic apparatus of the renal proximal tubule and that this localization requires megalin. Furthermore, the study suggests that the urinary loss of megalin ligands observed in Dab2 knockout mice is caused by suboptimal trafficking of megalin, leading to decreased megalin levels.

Mechanisms of hepatic chylomicron remnant clearance.

Experimental evidence suggests that chylomicron remnants, the carriers of dietary lipids, are highly atherogenic and that their postprandial plasma concentration is directly correlated with the progression of coronary artery disease. Much interest has therefore focused on elucidation of the various steps involved in uptake of these lipoprotein particles from the circulation. Recently, gene targeting has been applied to generate mouse models with deficiencies in lipoprotein metabolism and to dissect the chylomicron remnant clearance pathway in vivo. These experiments, together with studies performed in cultured cells, have demonstrated that plasma clearance of chylomicron remnants is a two-step process. The lipoprotein particles are rapidly sequestered in the liver by binding to the heparan sulphate proteoglycan surface (HSPG) of hepatocytes. This constitutes a high-capacity reservoir for adsorbing large amounts of lipoproteins in the postprandial state. Binding to HSPG is facilitated by apolipoprotein E (apo E) molecules present on the surface of the remnant particles. Once bound to the hepatocellular surface, the remnants are further enriched with apo E secreted by the hepatocytes. They are then internalized by two lipoprotein receptors: the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP). Characterization of this distinct clearance pathway for chylomicron remnants helps to direct further research towards developing an understanding of pathological abnormalities in postprandial lipoprotein metabolism.

Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors.

A number of cellular control mechanisms have evolved that facilitate and evaluate post-translational steps in protein biosynthesis. Chaperones or escort proteins are an important part of these cellular control mechanisms. They associate with newly synthesized proteins and assure correct folding and post-translational modification including disulfide bridge formation, glycosylation and complex formation. The receptor-associated protein (RAP) is a novel type of chaperone recently identified that is especially designed to assist in the biosynthesis and intracellular transport of endocytic receptors. Experimental evidence suggests that RAP acts as a receptor antagonist and prevents association of newly synthesized receptors with their ligands during transport to the cell surface. This mechanism seems to be required in cell types that express both receptor and ligand because premature receptor-ligand interaction in the secretory pathway interferes with proper export of the receptors to the cell surface. This review describes studies that have uncovered this unique protein biosynthesis mechanism.