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.

Impaired aldosterone responsiveness in corticosteroid binding globulin deficient mice.

AIM: Corticosteroid binding globulin (CBG) is the high affinity plasma carrier protein for cortisol. It keeps the steroids inactive, prevents them from degradation and defines the amount of free hormone acting on target tissues. Previous findings have shown insufficient responsiveness of corticosterone in peripheral tissues in CBG⁻(/)⁻ mice despite elevated free plasma corticosterone. In the large intestine, glucocorticoids synergistically enhance the pro-absorptive effects of aldosterone. We therefore hypothesized that CBG⁻(/)⁻ mice have reduced responsiveness to aldosterone. METHODS: We used CBG⁻(/)⁻ and CBG(+/+) mice to investigate distal colonic electrogenic Na(+) absorption. An Ussing chamber was used to quantify amiloride-sensitive Na(+) transport in distal colonic mucosa (ΔI(sc) (amil)) as a measure of the physiological effect of aldosterone. RESULTS: No differences were observed in ΔI(sc) (amil) or aldosterone levels in animals on control diet. When Na(+) restricted, CBG(+/+) mice responded with a marked up-regulation of ΔI(sc) (amil) (25-fold). In CBG⁻(/)⁻ mice this up-regulation was greatly attenuated as seen in a markedly reduced amiloride-sensitive short circuit current (reduced by ∼50%), a reduced ability to lower faecal Na(+) excretion and a significantly attenuated up-regulation of the ENaC channel γ-subunit. Diet-induced increases of total plasma aldosterone were similar in both genotypes, but CBG⁻(/)⁻ mice had an increased free plasma aldosterone fraction. SUMMARY: This study defines the functional hyporesponsiveness and aldosterone resistance in distal colon of CBG⁻(/)⁻ mice. This resistance occurs despite sufficient free corticosterone plasma level. Thus, steroid actions require an intrinsic but unknown function of CBG, which allows the sufficient supply of the hormone/s to the target tissue.

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.

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.

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.

Efficient eukaryotic expression system for authentic human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) is the main carrier for androgens and oestrogens in humans. It mediates the transport of steroid hormones in the circulation and testicular fluid, and regulates their bioavailability to steroid-responsive tissues. In addition, the protein interacts with membrane receptors expressed in target tissues. Binding to the receptors is suspected to facilitate the uptake of steroid hormones and/or elicit cellular signal transduction. The identity of the SHBG receptor has not yet been resolved, in part due to a lack of sufficient quantities of authentic SHBG for receptor purification and molecular characterization. We have successfully addressed this problem by establishing an episomal expression system in human embryonic kidney cells that produces 5 mg of fully active human SHBG per litre. The recombinant protein resembles native SHBG in terms of structure, glycosylation pattern and steroid-binding activity. Moreover, the protein interacts with plasma membranes in steroid target tissues, an activity not observed with SHBG from other recombinant expression systems. Thus our studies have removed an important obstacle to the further elucidation of the role SHBG plays in steroid hormone action.

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3).

Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D(3) complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D(3) in the urine and develop bone disease. Here, we report that cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid-carrier complexes on the cellular surface before megalin-mediated internalization of the cubilin-bound ligand. Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D(3). This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.

Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia.

Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B(12) complex in the intestine and as a receptor for apolipoprotein A1 and albumin reabsorption in the kidney proximal tubules and the yolk sac. In the present study, we report the identification of cubilin as a novel transferrin (Tf) receptor involved in catabolism of Tf. Consistent with a cubilin-mediated endocytosis of Tf in the kidney, lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf, whereas no Tf is detectable in the endocytic apparatus of the renal tubule epithelium of dogs with deficient surface expression of cubilin. As a consequence, these dogs excrete increased amounts of Tf in the urine. Mice with deficient synthesis of megalin, the putative coreceptor colocalizing with cubilin, also excrete high amounts of Tf and fail to internalize Tf in their proximal tubules. However, in contrast to the dogs with the defective cubilin expression, the megalin-deficient mice accumulate Tf on the luminal cubilin-expressing surface of the proximal tubule epithelium. This observation indicates that megalin deficiency causes failure in internalization of the cubilin-ligand complex. The megalin-dependent, cubilin-mediated endocytosis of Tf and the potential of the receptors thereby to facilitate iron uptake were further confirmed by analyzing the uptake of (125)I- and (59)Fe-labeled Tf in cultured yolk sac cells.

A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney.

Clara cell secretory protein (CCSP) is a transport protein for lipophilic substances in bronchio-alveolar fluid, plasma, and uterine secretion. It acts as a carrier for steroid hormones and polychlorinated biphenyl metabolites. Previously, the existence of receptors for uptake of CCSP.ligand complexes into the renal proximal tubules had been suggested. Using surface plasmon resonance analysis, we demonstrate that CCSP binds to cubilin, a peripheral membrane protein on the surface of proximal tubular cells. Binding to cubilin results in uptake and lysosomal degradation of CCSP in cultured cells. Surprisingly, internalization of CCSP is blocked not only by cubilin antagonists but also by antibodies directed against megalin, an endocytic receptor that does not bind CCSP but associates with cubilin. Consistent with a role of both receptors in renal uptake of CCSP in vivo, patients deficient for cubilin or mice lacking megalin exhibit a defect in tubular uptake of the protein and excrete CCSP into the urine. These findings identify a cellular pathway consisting of a CCSP-binding protein (cubilin) and an endocytic coreceptor (megalin) responsible for tissue-specific uptake of CCSP and associated ligands.

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.

Evidence for the role of megalin in renal uptake of transthyretin.

The kidney is a major organ for uptake of the thyroid hormone thyroxine (T(4)) and its conversion to the active form, triiodothyronine. In the plasma, one of the T(4) carriers is transthyretin (TTR). In the present study we observed that TTR, the transporter of both T(4) and retinol-binding protein, binds to megalin, the multiligand receptor expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney, megalin plays an important role in tubular uptake of macromolecules filtered through the glomerulus. To evaluate the importance of megalin for renal uptake of TTR, we performed binding/uptake assays using immortalized rat yolk sac cells with high expression levels of megalin. Radiolabeled TTR, free as well as in complex with thyroxine or retinol-binding protein, was rapidly taken up by the cells, and the uptake was strongly inhibited by a polyclonal megalin antibody and by the receptor-associated protein, a chaperone-like protein inhibiting ligand binding to megalin. In cell culture, different TTR mutations presented different levels of cell association and degradation, suggesting that the structure of TTR is important for megalin recognition. Both the apo form and the T(4)-bound form were taken up by the cells. Analysis of urine from patients with Dent's disease, a renal tubular disorder that alters receptor-mediated endocytic reabsorption of proteins, identified TTR as an abundant excreted protein. Furthermore, analysis of kidney sections of megalin-deficient mice revealed no immunohistochemical TTR labeling in intracellular vesicles in the proximal tubule cells when compared with wild type control littermates. Taken together, the present data indicate that TTR represents a novel megalin ligand of importance in the thyroid hormone homeostasis.

Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor.

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.

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.

Cellular signalling by lipoprotein receptors.

Lipoprotein receptors are commonly thought merely to mediate the internalization of lipoprotein particles or the exchange of lipids at the cell surface. Recent findings have now implicated these multifunctional receptors in cellular signalling mechanisms that extend beyond simple ligand endocytosis. By mediating the cellular uptake of lipophilic vitamins and hormones, megalin, a member of the LDL receptor gene family, regulates critical hormonal and metabolic processes. Other members of the LDL receptor family interact with cytoplasmic adaptor and scaffold proteins, which allows them to transmit signals directly across the plasma membrane of the target cell. This sheds a new light on the emerging roles of lipoprotein receptors in pathologic disease processes such as Alzheimer's disease.

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.

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Evidence for an essential role of megalin in transepithelial transport of retinol.

Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.