Reprint of: Fibrinolytic and Non-fibrinolytic Roles of Tissue-type Plasminogen Activator in the Ischemic Brain.

The neurovascular unit (NVU) is assembled by endothelial cells (ECs) and pericytes, and encased by a basement membrane (BM) surveilled by microglia and surrounded by perivascular astrocytes (PVA), which in turn are in contact with synapses. Cerebral ischemia induces the rapid release of the serine proteinase tissue-type plasminogen activator (tPA) from endothelial cells, perivascular astrocytes, microglia and neurons. Owning to its ability to catalyze the conversion of plasminogen into plasmin, in the intravascular space tPA functions as a fibrinolytic enzyme. In contrast, the release of astrocytic, microglial and neuronal tPA have a plethora of effects that not always require the generation of plasmin. In the ischemic brain tPA increases the permeability of the NVU, induces microglial activation, participates in the recycling of glutamate, and has various effects on neuronal survival. These effects are mediated by different receptors, notably subunits of the N-methyl-D-aspartate receptor (NMDAR) and the low-density lipoprotein receptor-related protein-1 (LRP-1). Here we review data on the role of tPA in the NVU under non-ischemic and ischemic conditions, and analyze how this knowledge may lead to the development of potential strategies for the treatment of acute ischemic stroke patients.

Fibrinolytic and Non-fibrinolytic Roles of Tissue-type Plasminogen Activator in the Ischemic Brain.

The neurovascular unit (NVU) is assembled by endothelial cells (ECs) and pericytes, and encased by a basement membrane (BM) surveilled by microglia and surrounded by perivascular astrocytes (PVA), which in turn are in contact with synapses. Cerebral ischemia induces the rapid release of the serine proteinase tissue-type plasminogen activator (tPA) from endothelial cells, perivascular astrocytes, microglia and neurons. Owning to its ability to catalyze the conversion of plasminogen into plasmin, in the intravascular space tPA functions as a fibrinolytic enzyme. In contrast, the release of astrocytic, microglial and neuronal tPA have a plethora of effects that not always require the generation of plasmin. In the ischemic brain tPA increases the permeability of the NVU, induces microglial activation, participates in the recycling of glutamate, and has various effects on neuronal survival. These effects are mediated by different receptors, notably subunits of the N-methyl-D-aspartate receptor (NMDAR) and the low-density lipoprotein receptor-related protein-1 (LRP-1). Here we review data on the role of tPA in the NVU under non-ischemic and ischemic conditions, and analyze how this knowledge may lead to the development of potential strategies for the treatment of acute ischemic stroke patients.

Low-density lipoprotein receptor-related protein-1 (LRP1) in the glial lineage modulates neuronal excitability.

The low-density lipoprotein related protein receptor 1 (LRP1), also known as CD91 or α-Macroglobulin-receptor, is a transmembrane receptor that interacts with more than 40 known ligands. It plays an important biological role as receptor of morphogens, extracellular matrix molecules, cytokines, proteases, protease inhibitors and pathogens. In the CNS, it has primarily been studied as a receptor and clearance agent of pathogenic factors such as Aß-peptide and, lately, Tau protein that is relevant for tissue homeostasis and protection against neurodegenerative processes. Recently, it was found that LRP1 expresses the Lewis-X (Lex) carbohydrate motif and is expressed in the neural stem cell compartment. The removal of Lrp1 from the cortical radial glia compartment generates a strong phenotype with severe motor deficits, seizures and a reduced life span. The present review discusses approaches that have been taken to address the neurodevelopmental significance of LRP1 by creating novel, lineage-specific constitutive or conditional knockout mouse lines. Deficits in the stem cell compartment may be at the root of severe CNS pathologies.

Antibody-mediated inhibition of tissue-type plasminogen activator binding to the low-density lipoprotein receptor-related protein 1 as a potential beneficial modulator for stroke therapy.

The acute ischemic stroke therapy of choice is the application of Alteplase, a drug containing the enzyme tissue-type plasminogen activator (tPa) which rapidly destabilizes blood clots. A central hallmark of stroke pathology is blood-brain barrier (BBB) breakdown associated with tight junction (TJ) protein degradation, which seems to be significantly more severe under therapeutic conditions. The exact mechanisms how tPa facilitates BBB breakdown are not entirely understood. There is evidence that an interaction with the lipoprotein receptor-related protein 1 (LRP1), allowing tPa transport across the BBB into the central nervous system, is necessary for this therapeutic side effect. Whether tPa-mediated disruption of BBB integrity is initiated directly on microvascular endothelial cells or other brain cell types is still elusive. In this study we could not observe any changes of barrier properties in microvascular endothelial cells after tPa incubation. However, we present evidence that tPa causes changes in microglial activation and BBB breakdown after LRP1-mediated transport across the BBB. Using a monoclonal antibody targeting the tPa binding sites of LRP1 decreased tPa transport across an endothelial barrier. Our results indicate that limiting tPa transport from the vascular system into the brain by coapplication of a LRP1-blocking monoclonal antibody might be a novel approach to minimize tPa-related BBB damage during acute stroke therapy.

High affinity of ß-amyloid proteins to cerebral capillaries: implications in chronic lead exposure-induced neurotoxicity in rats.

Lead (Pb) is a known environmental risk factor in the etiology of Alzheimer's disease (AD). The existing reports suggest that Pb exposure increases beta-amyloid (Aß) levels in brain tissues and cerebrospinal fluid (CSF) and facilitates the formation of amyloid plaques, which is a pathological hallmark for AD. Pb exposure has long been associated with cerebral vasculature injury. Yet it remained unclear if Pb exposure caused excessive Ab buildup in cerebral vasculature, which may damage the blood-brain barrier and cause abnormal Ab accumulation. This study was designed to investigate the impact of chronic Pb exposure on Aß accumulation in cerebral capillary and the expression of low-density lipoprotein receptor protein-1 (LRP1), a critical Aß transporter, in brain capillary and parenchyma. Sprague-Dawley rats received daily oral gavage at doses of 0, 14 (low-dose), and 27 (high-dose) mg Pb/kg as Pb acetate, 5 d/wk, for 4 or 8 wks. At the end of Pb exposure, a solution containing Aß40 was infused into the brain via the cannulated internal carotid artery. Data by ELISA showed a strikingly high affinity of Ab to cerebral vasculature, which was approximately 7-14 times higher than that to the parenchymal fractions collected from control brains. Pb exposure further aggravated the Aß accumulation in cerebral vasculature in a dose-dependent manner. Western blot analyses revealed that Pb exposure decreased LRP1 expression in cortical capillaries and hippocampal parenchyma. Immunohistochemistry (IHC) studies further revealed a disrupted distribution of LRP1 alongside hippocampal vasculature accompanied with a decreased expression in hippocampal neurons by Pb exposure. Taken together, the current study demonstrated that the cerebral vasculature naturally possessed a high affinity to Aß present in circulating blood. Pb exposure significantly increased Aß accumulation in cerebral vasculature; such an increased Aß accumulation was due partly to the diminished expression of LRP1 in response to Pb in tested brain regions. Perceivably, Pb-facilitated Ab aggravation in cerebral vasculature may contribute to Pb-associated amyloid alterations.

FUT2 inhibits the EMT and metastasis of colorectal cancer by increasing LRP1 fucosylation.

BACKGROUND: Fucosyltransferase 2(FUT2) and its induced α-1,2 fucosylation is associated with cancer metastasis. However, the role of FUT2 in colorectal cancer (CRC) metastasis remains unclear. METHODS: The expression levels and clinical analyses of FUT2 were assessed in CRC samples. Migration and invasion assays, EMT detection, nude mice peritoneal dissemination models and intestinal specific FUT2 knockout mice (FUT2△IEC mice) were used to investigate the effect of FUT2 on metastasis in colorectal cancer. Quantitative proteomics study of glycosylated protein, UEA enrichment, Co-immunoprecipitation identified the mediator of the invasive-inhibiting effects of FUT2. RESULTS: FUT2 is downregulated in CRC tissues and is positively correlated with the survival of CRC patients. FUT2 is an inhibitor of colorectal cancer metastasis which, when overexpressed, suppresses invasion and tumor dissemination in vitro and in vivo. FUT2 knock-out mice (FUT2△IEC mice) develop AMO and DSS-induced tumors and promote EMT in colorectal cancers. FUT2-induced α-1,2 fucosylation impacts the ability of low-density lipoprotein receptor-related protein 1(LRP1) to suppress colorectal cancer invasion. CONCLUSIONS: Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer. Video Abstract.

Targeting Alzheimer's Disease: The Critical Crosstalk between the Liver and Brain.

Alzheimer's disease (AD), an age-related neurodegenerative disorder, is currently incurable. Imbalanced amyloid-beta (Aß) generation and clearance are thought to play a pivotal role in the pathogenesis of AD. Historically, strategies targeting Aß clearance have typically focused on central clearance, but with limited clinical success. Recently, the contribution of peripheral systems, particularly the liver, to Aß clearance has sparked an increased interest. In addition, AD presents pathological features similar to those of metabolic syndrome, and the critical involvement of brain energy metabolic disturbances in this disease has been recognized. More importantly, the liver may be a key regulator in these abnormalities, far beyond our past understanding. Here, we review recent animal and clinical findings indicating that liver dysfunction represents an early event in AD pathophysiology. We further propose that compromised peripheral Aß clearance by the liver and aberrant hepatic physiological processes may contribute to AD neurodegeneration. The role of a hepatic synthesis product, fibroblast growth factor 21 (FGF21), in the management of AD is also discussed. A deeper understanding of the communication between the liver and brain may lead to new opportunities for the early diagnosis and treatment of AD.

The low-density lipoprotein receptor-related protein 1 (LRP1) interactome in the human cornea.

The human cornea is responsible for approximately 70% of the eye's optical power and, together with the lens, constitutes the only transparent tissue in the human body. Low-density lipoprotein receptor-related protein 1 (LRP1), a large, multitalented endocytic receptor, is expressed throughout the human cornea, yet its role in the cornea remains unknown. More than 30 years ago, LRP1 was purified by exploiting its affinity for the activated form of the protease inhibitor alpha-2-macroblulin (A2M), and the original purification protocol is generally referred to in studies involving full-length LRP1. Here, we provide a novel and simplified LRP1 purification protocol based on LRP1's affinity for receptor-related protein (RAP) that produces significantly higher yields of authentic LRP1. Purified LRP1 was used to map its unknown interactome in the human cornea. Corneal proteins extracted under physiologically relevant conditions were subjected to LRP1 affinity pull-down, and LRP1 ligand candidates were identified by LC-MS/MS. A total of 28 LRP1 ligand candidates were found, including 22 novel ligands. The LRP1 corneal interactome suggests a novel role for LRP1 as a regulator of the corneal immune response, structure, and ultimately corneal transparency.

Liuwei Dihuangwan Regulate Brain Microvessels in SAMP8 Mice Through RAGE/LRP1 Receptors / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Liuwei Dihuangwan on neurovascular injury in SAMP8 mice. MethodThe Alzheimer's disease （AD） model with insufficiency of kidney essence was induced in 75 SAMP8 mice aging 6 months. The model mice were divided into model group， positive control group （donepezil hydrochloride， 0.747 mg·kg-1·d-1）， and high-， medium-， and low-dose Liuwei Dihuangwan groups （2.700， 1.350， 0.675 g·kg-1·d-1）， with 15 mice in each group. Fifteen SAMR1 mice were assigned to a normal control group. All mice were administered continuously for 2 months. The spatial memory of mice was tested by the Morris water maze. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in the hippocampus and cortex of brain tissues. The immunohistochemical method （IHC） was used to detect the deposition of amyloid β-protein （Aβ） and the expression of von Willebrand factor （vWF） and CD34 in the hippocampus and cortex of brain tissues. Electron microscopy was used to observe the ultrastructural changes in cerebral microvessels. Western blot was used to detect the protein expression levels of the receptor of advanced glycation endproduct （RAGE）， low-density lipoprotein receptor-related protein 1 （LRP1）， vascular endothelial growth factor A （VEGF-A）， and P-selection in the hippocampus and cortex of brain tissues. ResultCompared with the normal control group， the model group showed prolonged escape latency and swimming distance （P<0.01）， increased number of glial cells， decreased number of nerve cells， blurred tight junctions or enlarged gap of the brain microvascular endothelial cells， severely injured membrane structure， swollen mitochondria of endothelial cells， ruptured membrane， massive dissolution in cristae， increased protein expression of Aβ and vWF in the hippocampus and cortex （P<0.01）， reduced protein expression of CD34 （P<0.05）， elevated protein expression of RAGE and P-selection in the cortex （P<0.01）， and decreased protein expression level of LRP1 and VEGF-A （P<0.01）. Compared with the model group， the Liuwei Dihuangwan groups showed shortened escape latency and swimming distance （P<0.05）， reduced number of glial cells in the cortex and hippocampus， increased number of microvessels in the cortex， clear double-layer membrane structure in tight junctions between the microvascular endothelial cells， increased number of mitochondria with intact membrane and recovered mitochondrial cristae， decreased protein expression of Aβ， vWF， RAGE， and P-selection in the hippocampus and cortex （P<0.05）， and increased protein expression of CD34， LRP1， and VEGF-A （P<0.05）. ConclusionLiuwei Dihuangwan can regulate Aβ metabolism through the RAGE/LRP1 receptor system and promote cerebral microvascular angiogenesis by inhibiting vWF expression and increasing VEGF-A and CD34， thereby improving cerebral microvascular injury in SAMP8 mice.

Brain endothelial LRP1 maintains blood-brain barrier integrity.

The entry of blood-borne molecules into the brain is restricted by the blood-brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor-related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreERT2 specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity.

Imaging epileptic foci in mouse models via a low-density lipoprotein receptor-related protein-1 targeting strategy.

BACKGROUND: In the setting of drug-resistant epilepsy (DRE), the success of surgery depends on the ability to accurately locate the epileptic foci to be resected or disconnected. However, the epileptic foci in a considerable percentage of the DRE patients cannot be adequately localised. This warrants the need for a reliable imaging strategy to identify the "concealed" epileptic regions. METHODS: Brain specimens from DRE patients and kainate-induced epileptic mouse models were immuno-stained to evaluate the integrity of the blood-brain barrier (BBB). The expression of low-density lipoprotein receptor-related protein-1 (LRP1) in the epileptic region of DRE patients and kainate models was studied by immunofluorescence. A micellar-based LRP1-targeted paramagnetic probe (Gd3+-LP) was developed and its ability to define the epileptic foci was investigated by magnetic resonance imaging (MRI). FINDINGS: The integrity of the BBB in the epileptic region of DRE patients and kainate mouse models were demonstrated. LRP1 expression levels in the epileptic foci of DRE patients and kainate models were 1.70-2.38 and 2.32-3.97 folds higher than in the control brain tissues, respectively. In vivo MRI demonstrated that Gd3+-LP offered 1.68 times higher (P < 0.05) T1-weighted intensity enhancement in the ipsilateral hippocampus of chronic kainite models than the control probe without LRP1 specificity. INTERPRETATION: The expression of LRP1 is up-regulated in vascular endothelium, activated glia in both DRE patients and kainate models. LRP1-targeted imaging strategy may provide an alternative strategy to define the "concealed" epileptic foci by overcoming the intact BBB. FUNDING: This work was supported by the National Natural Science Foundation, Shanghai Science and Technology Committee, Shanghai Municipal Science and Technology, Shanghai Municipal Health and Family Planning Commission and the National Postdoctoral Program for Innovative Talents.

A novel nanoparticle-based theranostic agent targeting LRP-1 enhances the efficacy of neoadjuvant radiotherapy in colorectal cancer.

Neoadjuvant radiotherapy has become an important therapeutic option for colorectal cancer (CRC) patients, whereas complete tumor response is observed only in 20-30% patients. Therefore, the development of diagnostic probe for radio-resistance is important to decide an optimal treatment timing and strategy for radiotherapy-resistant CRC patients. In this study, using the patient-derived xenograft (PDX) mouse model established with a radio-resistant CRC tumor tissue, we found low-density lipoprotein receptor-related protein-1 (LRP-1) as a radio-resistant marker protein induced by initial-dose radiation in radio-resistant CRC tumors. Simultaneously, we discovered a LRP-1 targeting peptide in a radio-resistant CRC PDX through in vivo peptide screening. We next engineered the theranostic agent made of human serum albumin nanoparticles (HSA NPs) containing 5-FU for chemo-radiotherapy and decorating LRP-1-targeting peptide for tumor localization, Cy7 fluorophore for diagnostic imaging. The nanoparticle-based theranostic agent accurately targeted the tumor designated by LRP-1 responding radiation and showed dramatically improved therapeutic efficacy in the radio-resistant PDX model. In conclusion, we have identified LRP-1 as a signature protein of radio-resistant CRC and successfully developed LRP-1-targeting HSA-NP containing 5-FU that is a novel theranostic tool for both diagnostic imaging and neoadjuvant therapy of CRC patients. This approach is clinically applicable to improve the effectiveness of neo-adjuvant radiotherapy and increase the ratio of complete tumor response in radio-resistant CRC.

Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-ß42 by LRP1-dependent apolipoprotein E isoform-specific mechanism.

BACKGROUND: Clearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer's amyloid-ß (Aß) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aß clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aß from brain capillaries, and whether removal of Aß aggregates by pericytes requires apoE, and if so, is Aß clearance on pericytes apoE isoform-specific. METHODS: We performed immunostaining for Aß and pericyte biomarkers on brain capillaries (< 6 µm in diameter) on tissue sections derived from AD patients and age-matched controls, and APPSwe/0 mice and littermate controls. Human Cy3-Aß42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrplox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aß42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student's t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis. RESULTS: First, we found that 35% and 60% of brain capillary pericytes accumulate Aß in AD patients and 8.5-month-old APPSw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aß42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aß42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aß42 clearance by mouse pericytes with silenced mouse apoE. CONCLUSIONS: Our data suggest that BBB-associated pericytes clear Aß aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aß clearance in AD.

The concerted amyloid-beta clearance of LRP1 and ABCB1/P-gp across the blood-brain barrier is linked by PICALM.

The accumulation of neurotoxic amyloid-beta (Aß) in the brain is a characteristic hallmark of Alzheimer's disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aß. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aß efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aß through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aß transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aß from the brain.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) promote neurorepair in the ischemic brain.

Despite the fact that ischemic stroke has been considered a leading cause of mortality in the world, recent advances in our understanding of the pathophysiological mechanisms underlying the ischemic injury and the treatment of acute ischemic stroke patients have led to a sharp decrease in the number of stroke deaths. However, this decrease in stroke mortality has also led to an increase in the number of patients that survive the acute ischemic injury with different degrees of disability. Unfortunately, to this date we do not have an effective therapeutic strategy to promote neurological recovery in these growing population of stroke survivors. Cerebral ischemia not only causes the destruction of a large number of axons and synapses but also activates endogenous mechanisms that promote the recovery of those neurons that survive its harmful effects. Here we review experimental evidence indicating that one of these mechanisms of repair is the binding of the serine proteinase urokinase-type plasminogen activator (uPA) to its receptor (uPAR) in the growth cones of injured axons. Indeed, the binding of uPA to uPAR in the periphery of growth cones of injured axons induces the recruitment of ß1-integrin to the plasma membrane, ß1-integrin-mediated activation of the small Rho GTPase Rac1, and Rac1-induced axonal regeneration. Furthermore, we found that this process is modulated by the low density lipoprotein receptor-related protein (LRP1). The data reviewed here indicate that the uPA-uPAR-LRP1 system is a potential target for the development of therapeutic strategies to promote neurological recovery in acute ischemic stroke patients.

Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) C4408R Mutant Promotes Amyloid Precursor Protein (APP) α-Cleavage in Vitro.

Previous studies have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer's disease (AD) pathogenesis, clearing ß-amyloid (Aß) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that co-expression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aß production, while also increasing sAPPα and APP α-C-terminal fragment (α-CTF), compared with CHO cells expressing APPswe alone. Surprisingly, the location of this mutation on LRP1 corresponded with the α-secretase cleavage site of APP. Further experimentation confirmed that in CHO cells expressing APPswe or wild-type APP (APPwt), co-expression of LRP1-CT C4408R decreases Aß and increases sAPPα and α-CTF compared with co-expression of wild-type LRP1-CT. In addition, LRP1-CT C4408R enhanced the unglycosylated form of LRP1-CT and reduced APP endocytosis as determined by flow cytometry. This finding identifies a point mutation in LRP1 which slows LRP1-CT-mediated APP endocytosis and amyloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aß secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin ß and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin ß, with the latter exhibiting a preference for APP in its dimeric state.

Inhibition of ADAM10 promotes the clearance of Aß across the BBB by reducing LRP1 ectodomain shedding.

BACKGROUND: Transport across the blood-brain barrier (BBB) is an important mediator of beta-amyloid (Aß) accumulation in the brain and a contributing factor in the pathogenesis of Alzheimer's disease (AD). One of the receptors responsible for the transport of Aß in the BBB is the low density lipoprotein receptor-related protein 1 (LRP1). LRP1 is susceptible to proteolytic shedding at the cell surface, which prevents endocytic transport of ligands. Previously, we reported a strong inverse correlation between LRP1 shedding in the brain and Aß transit across the BBB. Several proteases contribute to the ectodomain shedding of LRP1 including the α-secretase, a desintegrin and metalloproteinase domain containing protein 10 (ADAM10). METHODS: The role of ADAM10 in the shedding of LRP1 and Aß BBB clearance was assessed through pharmacological inhibition of ADAM10 in an in vitro model of the BBB and through the use of ADAM10 endothelial specific knock-out mice. In addition, an acute treatment paradigm with an ADAM10 inhibitor was also tested in an AD mouse model to assess the effect of ADAM10 inhibition on LRP1 shedding and Aßbrain accumulation. RESULTS: In the current studies, inhibition of ADAM10 reduced LRP1 shedding in brain endothelial cultures and increased Aß42 transit across an in vitro model of the BBB. Similarly, transgenic ADAM10 endothelial knockout mice displayed lower LRP1 shedding in the brain and significantly enhanced Aß clearance across the BBB compared to wild-type animals. Acute treatment with the ADAM10-selective inhibitor GI254023X in an AD mouse model substantially reduced brain LRP1 shedding and increased Aß40 levels in the plasma, indicating enhanced Aß transit from the brain to the periphery. Furthermore, both soluble and insoluble Aß40 and Aß42 brain levels were decreased following GI254023X treatment, but these effects lacked statistical significance. CONCLUSIONS: These studies demonstrate a role for ADAM10 in the ectodomain shedding of LRP1 in the brain and the clearance of Aß across the BBB, which may provide a novel strategy for attenuating Aß accumulation in the AD brain.

New Insights into VacA Intoxication Mediated through Its Cell Surface Receptors.

Helicobacter pylori (H. pylori), a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPß, and LRP1) that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin) have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage.

LRP-1 polymorphism is associated with global and regional amyloid load in Alzheimer's Disease in humans in-vivo.

OBJECTIVE: Impaired amyloid clearance has been proposed to contribute to ß-amyloid deposition in sporadic late-onset Alzheimer's disease (AD). Low density lipoprotein receptor-related protein 1 (LRP-1) is involved in the active outward transport of ß-amyloid across the blood-brain barrier (BBB). The C667T polymorphism (rs1799986) of the LRP-1 gene has been inconsistently associated with AD in genetic studies. We aimed to elucidate the association of this polymorphism with in-vivo brain amyloid load of AD patients using amyloid PET with [(11)C]PiB. MATERIALS AND METHODS: 72 patients with very mild to moderate AD were examined with amyloid PET and C667T polymorphism was obtained using TaqMan PCR assays. The association of C667T polymorphism with global and regional amyloid load was calculated using linear regression and voxel based analysis, respectively. The effect of the previously identified modulator of amyloid uptake, the apolipoprotein E genotype, on this association was also determined. RESULTS: The regression analysis between amyloid load and C667T polymorphism was statistically significant (p = 0.046, ß = 0.236). In an additional analysis ApoE genotype and gender were identified to explain further variability of amyloid load. Voxel based analysis revealed a significant (p < 0.05) association between C667T polymorphism and amyloid uptake in the temporo-parietal cortex bilaterally. ApoE did not interact significantly with the LRP-1 polymorphism. DISCUSSION: In conclusion, C667T polymorphism of LRP-1 is moderately but significantly associated with global and regional amyloid deposition in AD. The relationship appears to be independent of the ApoE genotype. This finding is compatible with the hypothesis that impaired amyloid clearance contributes to amyloid deposition in late-onset sporadic AD.