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Thomas E Willnow

Publications and source records attributed to Thomas E Willnow.

At least 19 recordsLinked to original sources
PubMed2007

The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.

The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid beta peptide (Abeta) in Alzheimer disease. We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset Alzheimer disease. These variants, which occur in at least two different clusters of intronic sequences within the SORL1 gene (also known as LR11 or SORLA) may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways and that when SORL1 is underexpressed, APP is sorted into Abeta-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing Alzheimer disease.

Age of Onset↗
PubMed2006

Lipoprotein receptors in Alzheimer's disease.

Lipoprotein receptors have important roles in pathological processes that lead to Alzheimer's disease (AD). Previously, they were believed to act mainly by modulating the neuronal metabolism of cholesterol and apolipoprotein E, major risk factors for spontaneous AD. However, recent findings point towards an unexpected new function for lipoprotein receptors in regulation of intracellular transport and processing of the amyloid precursor protein (APP) to give amyloid-beta peptide, the principal component of senile plaques. Here, we will discuss how lipoprotein receptors might modulate distinct steps in neuronal trafficking of APP, and how an intricate balance between opposing receptor activities might be a crucial determinant of APP processing, and of onset and progression of neurodegeneration.

Alzheimer Disease↗
PubMed2006

Elucidation of megalin/LRP2-dependent endocytic transport processes in the larval zebrafish pronephros.

Megalin/LRP2 is an endocytic receptor in the proximal tubules of the mammalian kidney that plays a central role in the clearance of metabolites from the glomerular filtrate. To establish a genetic model system for elucidation of molecular components of this retrieval pathway, we characterized orthologous transport processes in the zebrafish. We show that expression of megalin/LRP2 and its co-receptor cubilin is conserved in the larval zebrafish pronephros and demarcates a segment of the pronephric duct that is active in clearance of tracer from the ultrafiltrate. Knock-down of megalin/LRP2 causes lack of Rab4-positive endosomes in the proximal pronephric duct epithelium and abrogates apical endocytosis. Similarly, knock-down of the megalin/LRP2 adaptor Disabled 2 also blocks renal clearance processes. These results demonstrate the conservation of the megalin/LRP2 retrieval pathway between the larval zebrafish pronephros and the mammalian kidney and set the stage for dissection of the renal endocytic machinery in a simple model organism. Using this model system, we provide first genetic evidence that renal tubular endocytosis and formation of endosomes is a ligand-induced process that crucially depends on megalin/LRP2 activity.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11.

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.

Amyloid beta-Protein Precursor↗
PubMed2006

Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme.

sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.

Amyloid Precursor Protein Secretases↗
PubMed2006

Hyporesponsiveness to glucocorticoids in mice genetically deficient for the corticosteroid binding globulin.

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.

Adrenocorticotropic Hormone↗
PubMed2005

Role of endocytosis in cellular uptake of sex steroids.

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.

Androgens↗
PubMed2005

Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.

sorLA (Sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid beta-peptide (Abeta), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to Abeta, whereas ablation of sorLA expression in knockout mice results in increased levels of Abeta in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into Abeta and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase Abeta production and plaque formation and promote spontaneous AD.

Alzheimer Disease↗
PubMed2005

p75NTR--live or let die.

During neuronal development, neurotrophins are essential factors that promote survival, differentiation and myelination of neurons. The trophic signals are relayed to the cells via binding to Trk receptor tyrosine kinases and the p75 neurotrophin receptor. Paradoxically, the p75 neurotrophin receptor also ensures rapid and appropriate apoptosis of neonatal neurons not reaching their proper targets and transmits death signals to injured neurons. Until recently, the mechanisms by which the p75 neurotrophin receptor governs these opposing functions have remained elusive. By the identification of new ligands and cytosolic interacting partners, receptor cleavage products and coreceptors, some of these mechanisms are now being unraveled. Here, we review recent progress in delineating the molecular networks that enable p75(NTR) to dictate life and death.

Animals↗
PubMed2005

Megalin-mediated reuptake of retinol in the kidneys of mice is essential for vitamin A homeostasis.

The reuptake of retinol (ROH) and retinol-binding protein (RBP) in the kidneys is mediated by the endocytic receptor megalin, suggesting an important role for this receptor in vitamin A (VA) metabolism. We examined the extent to which megalin deficiency may affect urinary ROH excretion, levels of ROH and RBP in plasma, as well as storage of VA in liver and kidney. For this purpose, mice with a kidney-specific megalin gene defect (megalin(lox/lox); apoE(Cre)) and control mice (megalin(lox/lox)) were fed either a basal diet containing 4500 retinol equivalents (RE)/kg diet or a diet without VA during experimental periods of 42 and 84 d. Urinary ROH excretion was observed only in megalin(lox/lox); apoE(Cre) mice (P < 0.0001, 2-way ANOVA) and not in the controls. Plasma ROH and RBP differed only by diet (P < 0.05), but not genotype (P = 0.615). A major effect of megalin deficiency, however, was evident in retinyl ester levels in the liver (P < 0.05), which were approximately 37% lower than those in megalin(lox/lox) controls (P < 0.05, Student's t test) during the 84-d period of dietary VA deprivation. Kidney levels of VA were not affected by the receptor gene defect. The findings demonstrate that urinary ROH excretion caused by megalin deficiency requires accelerated mobilization of hepatic VA stores to maintain normal plasma ROH levels, which suggests that megalin plays an essential role in systemic VA homeostasis.

Animals↗
PubMed2005

LRP2/megalin is required for patterning of the ventral telencephalon.

Megalin is a low-density lipoprotein receptor-related protein (LRP2) expressed in the neuroepithelium and the yolk sac of the early embryo. Absence of megalin expression in knockout mice results in holoprosencephaly, indicating an essential yet unidentified function in forebrain development. We used mice with complete or conditional megalin gene inactivation in the embryo to demonstrate that expression of megalin in the neuroepithelium but not in the yolk sac is crucial for brain development. During early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression and signaling in the rostral dorsal neuroepithelium, and a subsequent loss of sonic hedgehog (Shh) expression in the ventral forebrain. As a consequence of absent SHH activity, ventrally derived oligodendroglial and interneuronal cell populations are lost in the forebrain of megalin-/- embryos. Similar defects are seen in models with enhanced signaling through BMPs, central regulators of neural tube patterning. Because megalin mediates endocytic uptake and degradation of BMP4, these findings indicate a role for megalin in neural tube specification, possibly by acting as BMP4 clearance receptor in the neuroepithelium.

Animals↗
PubMed2005

Megalin is essential for renal proximal tubule reabsorption of (111)In-DTPA-octreotide.

UNLABELLED: Radiolabeled somatostatin analogs have been shown to be important radiopharmaceuticals for tumor diagnosis and radionuclide therapy. The kidney has appeared to be the critical organ during radionuclide therapy because of peptide reabsorption and retention in the proximal tubules after glomerular filtration. The molecular mechanism of renal reabsorption of these analogs has not been clarified. A possible receptor candidate is megalin, a multiligand scavenger receptor in the renal proximal tubules. The objective of this study was to investigate the role of megalin in tubular reabsorption of radiolabeled somatostatin analogs by using kidney-specific megalin-deficient mice versus mice with normal renal megalin expression. [(111)In-Diethylenetriaminepentaacetic acid (DTPA)]octreotide was used as a practical model of peptide. METHODS: Renal uptake of [(111)In-DTPA]octreotide was determined by animal SPECT scintigraphy at different time points after injection of the tracer and by measurement of radioactivity after isolation of the organs. Furthermore, ex vivo autoradiography of renal sections revealed the zonal distribution of radioactivity in the megalin-deficient and megalin-expressing kidneys. RESULTS: SPECT scintigraphy of [(111)In-DTPA]octreotide at 3 and 24 h after injection clearly showed lower renal radioactivity in megalin-deficient kidneys than in megalin-expressing kidneys, both in male and in female mice, in accordance with counts obtained after isolation of the organ (70%-85% reduction of uptake in the megalin-deficient kidneys, P < 0.001). Renal uptake of [(111)In-DTPA]octreotide was significantly higher in female than in male kidneys (P < 0.001). Ex vivo autoradiograms clearly showed that renal radioactivity was not homogeneously distributed in the megalin-expressing kidneys but localized in the renal cortex. Quantification of the autoradiogram data confirmed the reduced radioactivity in the renal cortex of megalin-deficient kidneys. CONCLUSION: This study revealed the molecular mechanism of [(111)In-DTPA]octreotide uptake in renal proximal tubules involving the receptor megalin. Identification of megalin may be crucial for further research into strategies to reduce renal uptake.

Absorption↗
PubMed2004

Low-density lipoprotein receptor-related protein interacts with MafB, a regulator of hindbrain development.

The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development.

Amino Acid Sequence↗
PubMed2004

Sortilin is essential for proNGF-induced neuronal cell death.

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Kidney-specific inactivation of the megalin gene impairs trafficking of renal inorganic sodium phosphate cotransporter (NaPi-IIa).

Renal reabsorption of inorganic phosphate is mediated by the type IIa sodium phosphate cotransporter (NaPi-IIa) of the proximal tubule. Changes in renal phosphate handling are mainly attributable to altered NaPi-IIa brush border membrane (BBM) expression. Parathyroid hormone (PTH) induces inactivation of NaPi-IIa by endocytic membrane retrieval and degradation. The key elements triggering this process are not clear to date. Megalin serves as a receptor for the endocytosis of multiple ligands and is coexpressed with NaPi-IIa in the proximal tubule. Investigated was the role of megalin in the regulation of NaPi-IIa in steady state and during inactivation. Kidneys and tubular BBM fractions from mice with a renal-specific megalin gene defect and from controls were analyzed by light and electron microscopic histochemical techniques and Western blot test. Steady-state levels of NaPi-IIa in BBM were significantly enhanced, mRNA levels preserved, and phosphaturia reduced in the absence of megalin. Fluid-phase endocytosis was prevented and the apical endocytic apparatus markedly reduced. Systemic administration of PTH resulted in a defective retrieval and impaired degradation of NaPi-IIa. In vitro, the application of various stimuli of the PTH-induced signaling cascade had no effect either. Adequate steady-state expression of NaPi-IIa and the capacity of the proximal tubule cell to react on PTH-driven inactivation of NaPi-IIa by endocytosis and intracellular translocation require the presence of megalin.

Animals↗
PubMed2003

Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: a control mechanism for thyroid hormone release.

Hormone secretion by thyrocytes occurs by fluid phase uptake and lysosomal degradation of the prohormone thyroglobulin (Tg). However, some Tg internalized by megalin bypasses lysosomes and is transcytosed across cells and released into the bloodstream. Because the hormone content of Tg is variable, we investigated whether this affects transcytosis. We found that rat Tg with a low hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is transcytosed by megalin across thyroid FRTL-5 cells to a greater extent than rat Tg with a high hormone content [hormonogenic rat Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg sequence Arg-2489-Lys-2503 (required for binding to megalin and heparan sulfate proteoglycans) was found to be more exposed in low-horm-rTg, which accounted for its preferential transcytosis. Thus, removal of surface heparan sulfate proteoglycans from FRTL-5 cells or blocking of 2489-2503 reduced transcytosis of low-horm-rTg to a greater extent than that of horm-rTg. Preferential transcytosis of low-horm-rTg affected hormone release. Thus, the increase in hormone release from horm-rTg in FRTL-5 cells determined by megalin blocking (due to reduced transcytosis and enhanced Tg degradation) was rescued by low-horm-rTg, suggesting that megalin is required for effective hormone release. This finding was confirmed in a small number of megalin-deficient mice, which had serological features resembling mild hypothyroidism. Reduced hormone formation within Tg in vivo, due to treatment of rats with aminotriazole or of patients with Graves' disease with methimazole, resulted in increased Tg transcytosis via megalin, in confirmation of results with FRTL-5 cells. Our study points to a major role of megalin in thyroid homeostasis with possible implications in thyroid diseases.

Adult↗
PubMed2003

Differential binding of ligands to the apolipoprotein E receptor 2.

Apolipoprotein E receptor 2 (apoER2) is an important participant in the Reelin signaling pathway that directs cell positioning during embryogenesis. ApoER2 is a cell surface molecule that elicits intracellular signal transduction through binding of Reelin. The structural requirements for Reelin binding to apoER2 and the receptor domains involved in this process are unclear at present. Using a series of receptor mutants, we characterized the interaction of apoER2 with Reelin and compared this interaction to that of apoER2 with the receptor-associated protein (RAP), an apoER2 ligand that does not induce signaling. By surface plasmon resonance we demonstrate that apoER2 exhibits 6-fold higher affinity for Reelin than the very low density lipoprotein receptor (VLDLR), which also functions as a Reelin receptor (K(D) 0.2 nM versus K(D) 1.2 nM). Acidic amino acid residues in complement-type repeat domains 1 and 3 of apoER2 are required for Reelin binding. The same regions of the receptor are also bound by RAP with a 25-fold lower affinity (K(D) 5 nM). Whereas RAP binds to apoER2 with a 1:1 stoichiometry, experimental evidence suggests that Reelin associates with two or more receptor molecules simultaneously to achieve high-affinity interaction. This finding indicates that aggregation of apoER2 by multivalent ligands such as Reelin may be the structural basis for signal transduction.

Animals↗