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Background The aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson’s disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood. Methods We established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection. Results We found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates. Conclusions We identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Tumor cells face constant stress of ischemic (nutrient paucity and hypoxia) environment when they migrate and invade too fast to outgrow the nearest blood vessels. During the temporary loss of support from circulation, the tumor cells must act self-sufficient to survive and then to migrate to re-connect with the nearest blood supply or die. We have previously reported that ablation of the low-density lipoprotein receptor-related protein 1 (LRP-1) completely nullified the ability of tumour cells to migrate and invade under serum-free conditions in vitro and to form tumours in vivo. The mechanism behind the important function by cell surface LRP-1 was not fully understood. Herein we show that LRP-1 orchestrates two parallel cell surface signalling pathways to support the full constitutive tumour cell migration. First, LRP-1 stabilizes activated epidermal growth factor receptor (EGFR) to contribute half of the pro-motility signalling. Second, LRP-1 mediates secreted Hsp90α autocrine signalling to bring the other half of pro-motility signalling. Only combined inhibitions of the EGFR signalling and the eHsp90α autocrine signalling led to the full blockade of the tumour cell migration as the LRP-1 depletion did. This finding uncovers a novel mechanism by which certain breast cancer cells use LRP-1 to engage parallel signalling pathways to move when they lose contact with blood support.

In addition to exerting several essential house-keeping activities in the cell, heat shock proteins (HSPs) are crucial players in a well-structured molecular program activated in response to stressful challenges. Among the different activities carried out by HSPs during emergency, they reach the extracellular milieu, from where they scout the surroundings, regulate extracellular protein activity and send autocrine and paracrine signals. Cancer cells permanently experience stress conditions due to their altered equilibrium and behaviour, and constantly secrete heat shock proteins as a result. Other than supporting anti-tumour immunity, extracellular heat shock proteins (eHSPs), can also exacerbate cancer cell growth and malignancy by sustaining different cancer hallmarks. eHSPs are implicated in extracellular matrix remodelling, resistance to apoptosis, promotion of cell migration and invasion, induction of epithelial to mesenchymal transition, angiogenesis and activation of stromal cells, supporting ultimately, metastasis dissemination. A broader understanding of eHSP activity and contribution to tumour development and progression is leading to new opportunities in the diagnosis and treatment of cancer.

In the cartilage matrix, complex interactions occur between angiogenic and anti-angiogenic components, growth factors, and environmental stressors to maintain a proper cartilage phenotype that allows for effective load bearing and force distribution. However, as seen in both degenerative disease and tissue engineering, cartilage can lose its vascular resistance. This vascularization then leads to matrix breakdown, chondrocyte apoptosis, and ossification. Research has shown that articular cartilage inflammation leads to compromised joint function and decreased clinical potential for regeneration. Unfortunately, few articles comprehensively summarize what we have learned from previous investigations. In this review, we summarize our current understanding of the factors that stabilize chondrocytes to prevent terminal differentiation and applications of these factors to rescue the cartilage phenotype during cartilage engineering and osteoarthritis treatment. Inhibiting vascularization will allow for enhanced phenotypic stability so that we are able to develop more stable implants for cartilage repair and regeneration.

Background Bladder cancer (BC) is one of the most common malignancies and has a relatively poor outcome worldwide. In this study, we attempted to construct a novel metabolism-related gene (MRG) signature for predicting the survival probability of BC patients. Methods First, differentially expressed MRGs between BC and normal samples were identified and used to construct a protein-protein interaction (PPI) network and perform mutation analysis. Next, univariate Cox regression analysis was utilized to select prognostic genes, and multivariate Cox regression analysis was applied to establish an MRG signature for predicting the survival probability of BC patients. Moreover, Kaplan-Meier (KM) survival analysis and receiver operating characteristic (ROC) analysis were performed to evaluate the predictive capability of the MRG signature. Finally, a nomogram based on the MRG signature was established to better predict the survival of BC. Results In the present study, 27 differentially expressed MRGs were identified, most of which presented mutations in BC patients, and LRP1 showed the highest mutation rate. Next, an MRG signature, including MAOB, FASN and LRP1, was established by using univariate and multivariate Cox regression analysis. Furthermore, survival analysis indicated that BC patients in the high-risk group had a dramatically lower survival probability than those in the low-risk group. Finally, Cox regression analysis showed that the risk score was an independent prognostic factor, and a nomogram integrating age, pathological tumor stage and risk score was established and presented good predictive ability. Conclusion We successfully constructed a novel MRG signature to predict the prognosis of BC patients, which might contribute to the clinical treatment of BC.

The vascular endothelium is present within metabolic organs and actively regulates energy metabolism. Here we show osteocalcin, recognized as a bone-secreted metabolic hormone, is expressed in mouse primary endothelial cells isolated from heart, lung and liver. In human osteocalcin promoter-driven green fluorescent protein transgenic mice, green fluorescent protein signals are enriched in endothelial cells lining aorta, small vessels and capillaries and abundant in aorta, skeletal muscle and eye of adult mice. The depletion of lipoprotein receptor-related protein 1 induces osteocalcin through a Forkhead box O -dependent pathway in endothelial cells. Whereas depletion of osteocalcin abolishes the glucose-lowering effect of low-density lipoprotein receptor-related protein 1 depletion, osteocalcin treatment normalizes hyperglycemia in multiple mouse models. Mechanistically, osteocalcin receptor-G protein-coupled receptor family C group 6 member A and insulin-like-growth-factor-1 receptor are in the same complex with osteocalcin and required for osteocalcin-promoted insulin signaling pathway. Therefore, our results reveal an endocrine/paracrine role of endothelial cells in regulating insulin sensitivity, which may have therapeutic implications in treating diabetes and insulin resistance through manipulating vascular endothelium., The vascular endothelium contributes to metabolic regulation, however, the underlying mechanisms are not fully understood. Here the authors show that endothelial low-density lipoprotein receptor-related protein 1 regulates glucose homeostasis via osteocalcin expression.

Proteolysis is a crucial process in life, tightly controlled by numerous natural protease inhibitors. In human blood, alpha-2-macroglobulin is an emergency protease inhibitor preventing coagulation and damage to endothelia and leukocytes. With the use of a unique protease trapping mechanism, alpha-2-macroglobulin lures active proteases into its snap-trap, shields these from potential substrates and 'flags' their complex for elimination by receptor-mediated endocytosis. Matrix metalloprotease-9/gelatinase B is a secreted protease increased in blood of patients with inflammations, vascular disorders and cancers. Matrix metalloprotease-9 occurs as monomers and stable homotrimers, but the reason for their co-existence remains obscure. We discovered that matrix metalloprotease-9 homotrimers undergo reduced anti-proteolytic regulation by alpha-2-macroglobulin and are able to travel as a proteolytically active hitchhiker on alpha-2-macroglobulin. As a comparison, we revealed that monomeric active matrix metalloprotease-9 is efficiently trapped by human plasma alpha-2-macroglobulin and this masks the detection of activated matrix metalloprotease-9 with standard analysis techniques. In addition, we show that alpha-2-macroglobulin/trimer complexes escape clearance through the receptor low-density lipoprotein receptor-related protein 1, also known as the alpha-2-macroglobulin receptor. Thus, the biochemistry and biology of matrix metalloprotease-9 monomers and trimers are completely different as multimerization enables active matrix metalloprotease-9 to partially avoid alpha-2-macroglobulin regulation both by direct protease inhibition and by removal from the extracellular space by receptor-mediated endocytosis. Finally, for the biomarker field, the analysis of alpha-2-macroglobulin/protease complexes with upgraded technology is advocated as a quotum for protease activation in human plasma samples. ispartof: CELLULAR AND MOLECULAR LIFE SCIENCES vol:77 issue:15 pages:3013-3026 ispartof: location:Switzerland status: published

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

Oculopharyngodistal myopathy (OPDM) is a rare hereditary muscle disease characterized by progressive distal limb weakness, ptosis, ophthalmoplegia, bulbar muscle weakness and rimmed vacuoles on muscle biopsy. Recently, CGG repeat expansions in the noncoding regions of two genes, LRP12 and GIPC1, have been reported to be causative for OPDM. Furthermore, neuronal intranuclear inclusion disease (NIID) has been recently reported to be caused by CGG repeat expansions in NOTCH2NLC. We aimed to identify and to clinicopathologically characterize patients with OPDM who have CGG repeat expansions in NOTCH2NLC (OPDM_NOTCH2NLC). Note that 211 patients from 201 families, who were clinically or clinicopathologically diagnosed with OPDM or oculopharyngeal muscular dystrophy, were screened for CGG expansions in NOTCH2NLC by repeat primed-PCR. Clinical information and muscle pathology slides of identified patients with OPDM_NOTCH2NLC were re-reviewed. Intra-myonuclear inclusions were evaluated using immunohistochemistry and electron microscopy (EM). Seven Japanese OPDM patients had CGG repeat expansions in NOTCH2NLC. All seven patients clinically demonstrated ptosis, ophthalmoplegia, dysarthria and muscle weakness; they myopathologically had intra-myonuclear inclusions stained with anti-poly-ubiquitinated proteins, anti-SUMO1 and anti-p62 antibodies, which were diagnostic of NIID (typically on skin biopsy), in addition to rimmed vacuoles. The sample for EM was available only from one patient, which demonstrated intranuclear inclusions of 12.6 ± 1.6 nm in diameter. We identified seven patients with OPDM_NOTCH2NLC. Our patients had various additional central and/or peripheral nervous system involvement, although all were clinicopathologically compatible; thus, they were diagnosed as having OPDM and expanding a phenotype of the neuromyodegenerative disease caused by CGG repeat expansions in NOTCH2NLC.

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role., Lin et al. find that mutation in endocytic trafficking protein Lrp1 causes congenital heart defects in mice due to a requirement for Lrp1 in the neural crest lineage, where it regulates outflow tract lengthening. This study provides insights into how Lrp1 and the neural crest contribute to heart development.

Fibronectin (FN) plays a major role in the stability and organization of the extracellular matrix (ECM). We have previously demonstrated that FN interacts directly with Hsp90, as well as showing that the Hsp90 inhibitor novobiocin results in FN turnover via a receptor mediated process. However, the receptor involved has not been previously identified. LRP1 is a ubiquitous receptor responsible for the internalisation of numerous ligands that binds both Hsp90 and FN, and therefore we investigated whether LRP1 was involved in novobiocin-mediated FN turnover. FN, LRP1 and Hsp90 could be isolated in a common complex, and inhibition of Hsp90 by novobiocin increased the colocalisation of FN and LRP1. Novobiocin induced an increase (at low concentrations) followed by a loss of FN that was primarily derived from extracellular matrix-associated FN and led to a concomitant increase in intracellular FN. The effect of novobiocin was specific to LRP1-expressing cells and could be recapitulated by an LRP1 blocking antibody and the allosteric C-terminal Hsp90 inhibitor SM253, but not the N-terminal inhibitor geldanamycin. Together these data suggest that LRP1 is required for FN turnover in response to Hsp90 inhibition by novobiocin, which may have unintended physiological consequences in contexts where C-terminal Hsp90 inhibition is to be used therapeutically.

In Alzheimer's disease (AD), the impaired clearance of β-amyloid peptide (Aβ) due to disrupted tight junction and transporter proteins is the prominent cause of disease progression. Somatostatin (SST) blocks the aggregation of Aβ and inflammation whereas reduction of SST levels in the CSF and brain tissue is associated with impaired cognitive function and memory loss. However, the role of SST in preservation of blood-brain barrier (BBB) integrity and functionality in Aβ-induced toxicity is not known. In the present study using human CMEC/D3 cells, we demonstrate that SST prevents Aβ-induced BBB permeability by regulating LRP1 and RAGE expression and improving the disrupted tight junction proteins. Furthermore, SST abrogates Aβ-induced JNK phosphorylation and expression of MMP2. Taken together, results presented here suggest that SST might serve as a therapeutic intervention in AD via targeting multiple pathways responsible for neurotoxicity, impaired BBB function, and disease progression.

Treatment with doxorubicin (dox) and emodin, separately and together, under normoxic and hypoxia-like conditions induced by CoCl2, led to greater intracellular compound accumulation over 10 h post-addition in the presence of CoCl2 in lung adenocarcinoma (A549) and colorectal carcinoma (HCT-15) cell lines. Confocal microscopy revealed that emodin, by itself, showed high cytosolic distribution in both cell lines, at 40 min post-addition but had entered the nuclei by 2 h, while dox entered the nuclei by 40 min. Both compounds modulated the expression of the efflux transporters (PgP, ABCG2, or MRP1-4) and the endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), to different extents under the study conditions. Efflux transporter upregulation was linked to lower intracellular compound levels due to greater efflux. Increased dox accumulation was accompanied by unaltered expression or upregulation of LRP1 in A549 cells. In both cell lines, increased accumulation of dox and emodin was observed whenever LRP1 and the efflux transporters known to transport dox and emodin were all up- or downregulated concomitantly. Increased growth inhibition was linked to co-treatment with dox and emodin and with increased ligand accumulation. The results presented in this study raise the hypothesis that higher production of LRP1 protein may be associated with higher endocytosis of upregulated transporter proteins at the cell surface, and hence, increased dox and emodin accumulation and growth inhibition. If so, elevation of LRP1 expression may be a useful target for interventions to promote the efficacy of these and other anticancer drugs.

Poor Mg status is a risk factor for Alzheimer’s disease (AD), and the underlying mechanisms remain elusive. Here, we provided the first evidence that elevated Mg levels significantly reduced the blood-brain barrier (BBB) permeability and regulated its function in vitro. Transient receptor potential melastatin 7 (TRPM7) and magnesium transporter subtype 1 (MagT1) were two major cellular receptors mediating entry of extracellular Mg2+ into the cells. Elevated Mg levels also induced an accelerated clearance of amyloid-β peptide (Aβ) from the brain to the blood side via BBB transcytosis through low-density lipoprotein receptor-related protein (LRP) and phosphatidylinositol binding clathrin assembly protein (PICALM), while reduced the influx of Aβ from the blood to the brain side involving receptor for advanced glycation end products (RAGE) and caveolae. Mg enhanced BBB barrier properties and overall expression of LRP1 and PICALM whereas reduced that of RAGE and caveolin-1. Apical-to-basolateral and vice versa steady-state Aβ flux achieved an equilibrium of 18 and 0.27 fmol/min/cm2, respectively, about 30 min after the initial addition of physiological levels of free Aβ. Knockdown of caveolin-1 or disruption of caveolae membrane microdomains reduced RAGE-mediated influx significantly, but not LRP1-mediated efflux of Aβ. Stimulating endothelial cells with vascular endothelial growth factor (VEGF) enhanced caveolin-1 phosphorylation and RAGE expression. Co-immunoprecipitation demonstrated that RAGE, but not LRP1, was physically associated with caveolin-1. Thus, Mg can reduce BBB permeability and promote BBB clearance of Aβ from the brain by increasing the expression of LRP1 and PICALM while reducing the level of RAGE and caveolin-1.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, β- 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 Aβ 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 Aβ 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.

Previous studies have demonstrated that the low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer’s disease (AD) pathogenesis, clearing β-amyloid (Aβ) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that coexpression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aβ 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), coexpression of LRP1-CT C4408R decreases Aβ and increases sAPPα and α-CTF compared with coexpression 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 amyoloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.

Switches in sphingolipid metabolism have recently been associated with oncogenic transformation, and a role for the low-density lipoprotein receptor-related protein 1 (LRP1) in sphingosine-1-phosphate (S1P) proangiogenic signaling inferred. S1P signaling crosstalk with LRP1 in brain microvascular endothelial cells (HBMEC) is however unclear. Transient in vitro siLRP1 gene silencing was compared to stable shLRP1 knockdown. We observed decreased expression of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor for which multiple binding sites are found within the promoter sequences of all five S1P receptor members, upon stable but not transient LRP1 repression. Chemotactic migration of brain EC isolated from Lrp1(EC)-/- mice and of stable shLRP1 HBMEC became unresponsive to S1P, partly due to altered ERK and p38 MAPK pathways, whereas chemotactism remained unaltered following transient in vitro siLRP1 repression. Diminished S1P1, S1P3, and S1P5 expression were observed in stable shLRP1 HBMEC and in brain EC isolated from Lrp1(EC)-/- mice. Overexpression of LRP1 cluster IV rescued S1P-mediated cell migration through increased S1P3 transcription in shLRP1 HBMEC. Our study highlights an adaptive signaling crosstalk between LRP1 and specific S1P receptors which may regulate the angiogenic response of brain EC and be targeted at the blood-brain barrier in future therapeutic strategies.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in several neurodegenerative diseases, including stroke. Low-density lipoprotein receptor-related protein 1 (LRP1) plays pivotal roles in endocytosis and signaling in the cell. Immature LRP1 is processed by furin in the trans-Golgi network (TGN) and transported to the cell surface as its mature form. Activation of mature LRP1 exerts a protective effect against glutamate-induced degeneration of the rat retinal ganglion cells, as was shown in our previous study. However, the roles of LRP1 in the pathogenesis of excitotoxic neuronal injuries remain to be determined. The aim of this present study was to achieve further insight into the pathophysiologic roles of LRP1 after excitotoxic neuronal injuries. Our findings are the first to demonstrate that LRP1 was significantly cleaved by furin after cerebral ischemia in rats as well as after exposure of cultured cortical neurons to NMDA. It was noteworthy that the intracellular domain (ICD) of LRP1 was co-localized with TGN and furin. Furthermore, a furin inhibitor inhibited the cleavage of LRP1 and co-localization of LRP1-ICD with TGN or furin. Our findings suggest that furin-mediated cleavage of LRP1 and changes in the localization of LRP1-ICD were involved in the excitotoxic neuronal injury.

Aedes aegypti is the primary vector of a number of viruses pathogenic to humans including dengue virus (DENV). DENV infection leads to widespread transcriptomic and proteomic alterations in mosquito cells. Here we identified alterations to the mosquito cell secretome during DENV infection by performing liquid chromatography tandem mass spectrometry. We found that an extracellular fragment of low-density lipoprotein receptor-related protein 1 (LRP-1) was present during infection. Previous literature suggests that LRP-1 regulates cholesterol homeostasis. Therefore, we hypothesized that DENV modifies LRP-1 protein expression to maintain host-derived intracellular cholesterol, which would facilitate virus replication within membrane-associated replication compartments. Accordingly, stimuli that are present during flavivirus infection reduced LRP-1 protein expression. We also found that dsRNA knockdown of LRP-1 increased intracellular cholesterol and DENV viral RNA. Further, depletion of intracellular lipids reduced infection. Together, these data suggest that DENV reduces LRP-1 protein expression, possibly through regulated intramembrane proteolysis (RIP), to increase intracellular cholesterol and facilitate replication in Ae. aegypti.

Amyloid-β (Aβ) accumulation in the brain is a pathological feature of Alzheimer’s disease (AD) and enhancing Aβ clearance is a potential therapeutic strategy. Pioglitazone is a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist and is widely used to treat type 2 diabetes. We previously reported that low-dose pioglitazone increased the expression of low-density lipoprotein receptor-related protein 1 (LRP1), which upregulates the clearance of Aβ, using human brain microvascular endothelial cells. We investigated whether low-dose pioglitazone can rescue the pathological phenotype and memory impairment in senescence-accelerated mouse prone-8 (SAMP8) mice by increasing LRP1 levels. SAMP8 mice were treated with vehicle or pioglitazone in dosages of 2 or 5 mg/kg/day for 7 weeks. In the water maze test, 2 mg/kg/day of pioglitazone significantly attenuated the increased escape latency in SAMP8 mice (p = 0.026), while 5 mg/kg/day of treatment did not. Compared with vehicle treatment, the hippocampi of SAMP8 mice with 2 mg/kg/day of pioglitazone exhibited fewer Aβ deposits and reduced Aβ1–40 levels, along with elevated LRP1 expression (p = 0.005). Collectively, our results proposed that a new therapeutic application of the PPAR-γ agonist for AD treatment should be considered at a lower dose than the conventional dose used to treat diabetes.

BACKGROUND: Gut microbiota composition and function are symbiotically linked with host health and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognised mechanisms exist by which the microbiome influences the gut-brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesised interactions between circulating gut-derived microbial metabolites, and the blood-brain barrier (BBB) also contribute to the gut-brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed. RESULTS: After demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 μM) on BBB properties in vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signalling. CONCLUSIONS: Together, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut-brain axis that warrants further attention.

BackgroundGut microbiota composition and function are symbiotically linked with host health, and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognized mechanisms exist by which the microbiome influences the gut-brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesized interactions between circulating gut–derived microbial metabolites and the blood–brain barrier (BBB) also contribute to the gut–brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed.ResultsAfter demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 μM) on BBB propertiesin vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signaling.ConclusionsTogether, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut–brain axis that warrants further attention.

A growing body of evidence suggests that β-amyloid peptides (Aβ) are unlikely to be the only factor involved in Alzheimer's disease (AD) aetiology. In fact, a strong correlation has been established between AD patients and patients with type 2 diabetes and/or cholesterol metabolism alterations. In addition, a link between adipose tissue metabolism, leptin signalling in particular, and AD has also been demonstrated. In the present study we analyzed the expression of molecules related to metabolism, with the main focus on leptin and prolactin signalling pathways in an APPswe/PS1dE9 (APP/PS1) transgenic mice model, at 3 and 6 months of age, compared to wild-type controls. We have chosen to study 3 months-old APP/PS1 animals at an age when neither the cognitive deficits nor significant Aβ plaques in the brain are present, and to compare them to the 6 months-old mice, which exhibit elevated levels of Aβ in the hippocampus and memory loss. A significant reduction in both mRNA and protein levels of the prolactin receptor (PRL-R) was detected in the hippocampi of 3 months old APP/PS1 mice, with a decrease in the levels of the leptin receptor (OB-R) first becoming evident at 6 months of age. We proceeded to study the expression of the intracellular signalling molecules downstream of these receptors, including stat (1-5), sos1, kras and socs (1-3). Our data suggest a downregulation in some of these molecules such as stat-5b and socs (1-3), in 3 months-old APP/PS1 brains. Likewise, at the same age, we detected a significant reduction in mRNA levels of lrp1 and cyp46a1, both of which are involved in cholesterol homeostasis. Taken together, these results demonstrate a significative impairment in adipokine receptors signalling and cholesterol regulation pathways in the hippocampus of APP/PS1 mice at an early age, prior to the Aβ plaque formation.

Low-density lipoprotein receptor-related protein 1 (LRP1) regulates lipid and glucose metabolism in liver and adipose tissue. It is also involved in central nervous system regulation of food intake and leptin signalling. Here we demonstrate that endothelial Lrp1 regulates systemic energy homeostasis. Mice with endothelial-specific Lrp1 deletion display improved glucose sensitivity and lipid profiles combined with increased oxygen consumption during high-fat-diet-induced obesity. We show that the intracellular domain of Lrp1 interacts with the nuclear receptor Pparγ, a central regulator of lipid and glucose metabolism, acting as its transcriptional co-activator in endothelial cells. Therefore, Lrp1 not only acts as an endocytic receptor but also directly participates in gene transcription. Our findings indicate an underappreciated functional role of endothelium in maintaining systemic energy homeostasis., LDL receptor-related protein 1 (LRP1) is an endocytic receptor involved in cell signalling and energy homeostasis. Here Mao et al. demonstrate that endothelial Lrp1 modulates lipid and glucose metabolism by binding the nuclear receptor Pparγ and promoting its transcriptional activity.

Cytotoxic effect of doxorubicin in human multiple myeloma cell cultures RPMI 1640, RPMI 8226, and IM-9 was studied. The obtained data were compared with mRNA expression of MDR1, MRP1, BCRP, LRP genes responsible for the development of multiple drug resistance. IM-9 cells that differed from the other two cultures by the expression of surface differentiation markers and by mRNA expression of MDR1, BCRP, and LRP were most sensitive to doxorubicin. All cells expressed mRNA for only A-isoform of insulin receptor (IRA), while B-isoform (IRB) was not expressed. Insulin in a concentration of 5 μg/ml had no effect on the cytotoxic effect of doxorubicin in the studied cells.

Background Transport across the blood–brain barrier (BBB) is an important mediator of beta-amyloid (Aβ) 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 Aβ 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 Aβ 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 Aβ 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 Aβbrain accumulation. Results In the current studies, inhibition of ADAM10 reduced LRP1 shedding in brain endothelial cultures and increased Aβ42 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 Aβ 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 Aβ40 levels in the plasma, indicating enhanced Aβ transit from the brain to the periphery. Furthermore, both soluble and insoluble Aβ40 and Aβ42 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 Aβ across the BBB, which may provide a novel strategy for attenuating Aβ accumulation in the AD brain.

Migraine without aura is the most common form of migraine, characterized by recurrent disabling headache and associated autonomic symptoms. To identify common genetic variants associated with this migraine type, we analyzed genome-wide association data of 2,326 clinic-based German and Dutch individuals with migraine without aura and 4,580 population-matched controls. We selected SNPs from 12 loci with 2 or more SNPs associated with P values of

Background: ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood–brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells. Methods: Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines. Results: Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions. Conclusion: ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.

For over a hundred years, it has been established that tumor-specific immune responses can frequently be detected in the tumor bearing host. Whether or not these immune responses are capable of controlling the growth of the tumor is influenced by many factors. However, the mechanism by which the immune responses are initiated in the first place has remained a dilemma. In this chapter, we present evidence that heat shock protein-peptide complexes released by tumor cells are the entity responsible for initiating the immune responses. Interaction of the extracellular HSP with its receptor CD91 is necessary for priming the immune response. We propose that the disruption of the HSP-CD91 interaction may be an active mechanism by which tumors prevent the generation of immune responses against it.

Several common polymorphisms in the ApoE, ACE, MTHFR and LDLR genes have been implicated in the pathogenesis of common complex diseases across world populations. This study investigates the prevalence of five known and clinically important common polymorphisms in Angami and Lotha Naga populations. A total of 112 unrelated healthy volunteers (52 Lotha Nagas and 60 Angami Nagas) participated in the study. All the five genes were found to be polymorphic in the studied populations. The Lotha Nagas displayed higher mutant allele frequencies than the Angami Nagas except for the T allele frequency of the AvaII polymorphism of the LDLR gene, though chi square did not reveal any significant population differences by genotypes. In view of the relatively high mutant allele frequencies in both the populations, they are likely to be at a high risk of developing various complex diseases as they shift from an active and rigorous lifestyle to a more sedentary one.

The Wnt/beta-catenin signaling pathway plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Wnts transduce signals via transmembrane receptors of the Frizzled (Fzd/Fz) family and the low density lipoprotein receptor-related protein 5/6 (Lrp5/6). A key mechanism in their signal transduction is that Wnts induce Lrp6 signalosomes, which become phosphorylated at multiple conserved sites, notably at PPSPXS motifs. Lrp6 phosphorylation is crucial to beta-catenin stabilization and pathway activation by promoting Axin and Gsk3 recruitment to phosphorylated sites. Here, we summarize how proline-directed kinases (Gsk3, PKA, Pftk1, Grk5/6) and non-proline-directed kinases (CK1 family) act upon Lrp6, how the phosphorylation is regulated by ligand binding and mitosis, and how Lrp6 phosphorylation leads to beta-catenin stabilization.

The vast majority of Alzheimer's disease (AD) cases are late-onset and their development is probably influenced by both genetic and environmental risk factors. A strong genetic risk factor for late-onset AD is the presence of the epsilon4 allele of the apolipoprotein E (APOE) gene, which encodes a protein with crucial roles in cholesterol metabolism. There is mounting evidence that APOE4 contributes to AD pathogenesis by modulating the metabolism and aggregation of amyloid-beta peptide and by directly regulating brain lipid metabolism and synaptic functions through APOE receptors. Emerging knowledge of the contribution of APOE to the pathophysiology of AD presents new opportunities for AD therapy.

Alzheimer's disease (AD), characterized by accumulation of amyloid-beta protein (Abeta) in brain parenchyma, is closely associated with brain ischemia. Decreased clearance of Abeta from brain is the main cause of Abeta accumulation in sporadic AD. However, the mechanisms underlying ischemia-mediated AD pathogenesis remain unclear. The receptor for advanced end glycation products (RAGE) and low-density lipoprotein receptor-related protein-1 (LRP-1) expressed at blood brain barrier (BBB) are actively involved in Abeta clearance. RAGE is thought to be a primary transporter of Abeta across BBB into the brain from the systemic circulation, while LRP-1 mediates the transport of Abeta out of the brain. Ginkgo biloba extract EGb761, a traditional Chinese medicine, has been widely used in the treatment of AD. To investigate the effects of EGb761 on the expression of RAGE and LRP-1 in endothelial cells in response to ischemic injury, we cultured bEnd.3 cells, an immortalized mouse cerebral microvessel endothelial cell line, under a chronic hypoxic and hypoglycemic condition (CHH) to mimic ischemic injury of BBB, and then treated with EGb 761. We found that EGb 761 markedly ameliorated the damage (evaluated by MTT assay) from CHH. Moreover, we demonstrated that CHH led to a significant increase in the expression of RAGE both at the mRNA and protein levels at all times (24, 36, and 48 h), conversely; CHH induced a dramatic decrease in LRP-1 mRNA and protein expression at both 36 and 48 h. The results indicated that CHH has differential effects on the expression of RAGE and LRP-1. Furthermore, EGb761 significantly reversed CHH-induced upregulation of RAGE expression and downregulation of LRP-1 expression. Our findings suggest that EGb761 favor clearance of Abeta via regulating the expression of RAGE and LRP-1 during brain ischemia. This may provide a new insight into a possible molecular mechanism underlying brain ischemia-mediated AD pathogenesis, and potential therapeutic application of EGb 761 in treatment of AD.

LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.

Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEDelta553) was expressed. A permanent cell line (termed CY301) was established whereby PEDelta553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEDelta553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEDelta553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEDelta553 appears to be a valuable probe of LRP1 maturation and trafficking.

The beta-amyloid precursor protein (APP) shares intracellular and extracellular-binding partners with the family of receptors for apolipoprotein E (apoE). Binding of APP and apoE receptors to specific extracellular matrix proteins (F-spondin and Reelin) promotes their presence on the cell surface and influences whether they will interact with specific cytoplasmic adaptor proteins. Cleavage of APP and apoE receptors at the cell surface occurs by alpha-secretase activities; thus, the processing of these proteins can be regulated by their trafficking either to or from the cell surface. Their cleavages can also be regulated by tissue inhibitor of metalloproteinase-3 (TIMP-3), a metalloprotease inhibitor in the extracellular matrix. ApoE receptors have functions in neuronal migration during development and in proper synaptic function in the adult. Thus, the functions of apoE receptors and by analogy of APP will be modified by the various extracellular and intracellular interactions reviewed in this paper.

We developed a new method for evaluation of the purity of cell population in primary cultures of hepatocellular carcinoma. Expression of potential surface molecular markers on primary cultures of renal tumors was assayed by the method of flow cytofluorometry. CD24 and CD70 were identified as differential markers, which allowed us to distinguish cancer cells from tumor stromal cells in vitro. A negative marker CD90 was expressed on fibroblasts of the tumor stroma, but not on cancer cells. Combined use of these three markers allows evaluation of the severity of tumor culture contamination with stromal cells.

Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting.

Background The purpose of the study was to evaluate the effects of high dietary cholesterol in ovariectomized (Ovx) rats on several key markers of hepatic cholesterol and bile acid metabolism. Method Ovx and sham operated (Sham) rats were given either a standard diet (SD), a SD diet supplemented with 0.25 % cholesterol (SD + Chol), or a high fat diet supplemented with 0.25 % cholesterol (HF + Chol) for 5 weeks. Results Ovx was associated with higher (P

Increased factor VIII (FVIII) levels are a prevalent and independent risk factor for venous thromboembolism (VTE). The low density lipoprotein receptor-related protein 1 (LRP1) has been associated with FVIII catabolism. After a median of 10 years of the first thrombotic episode, we evaluated FVIII activity levels in 75 patients with VTE and high FVIII levels and in 74 healthy controls. Subsequently, we evaluated the regions of F8 and LRP1 genes coding sites of affinity between these proteins, with the objective of determining genetic alterations associated with plasma FVIII levels. After a median time of 10 years after the VTE episode, FVIII levels were significantly higher in patients when compared to controls (158.6 IU/dL vs. 125.8 IU/dL; P ≤ 0.001]. Despite the fact that we found 14 genetic variations in F8 and LRP1 genes, no relationship was found between FVIII levels with these variations. We demonstrated a persistent increase of FVIII levels in patients with VTE, but in a much lower magnitude after 10 years when compared to 3-years after the episode. Moreover, we observed no relationship of genetic variations in the gene regions coding affinity sites between LRP1 and FVIII with FVIII levels.

The receptor for advanced glycation end products (RAGE) is thought to be a primary transporter of beta-amyloid across the blood-brain barrier (BBB) into the brain from the systemic circulation, while the low-density lipoprotein receptor-related protein (LRP)-1 mediates transport of beta-amyloid out of the brain. To determine whether there are Alzheimer's disease (AD)-related changes in these BBB-associated beta-amyloid receptors, we studied RAGE, LRP-1, and beta-amyloid in human elderly control and AD hippocampi. In control hippocampi, there was robust RAGE immunoreactivity in neurons, whereas microvascular staining was barely detectable. LRP-1 staining, in contrast, was clearly evident within microvessels but only weakly stained neurons. In AD cases, neuronal RAGE immunoreactivity was significantly decreased. An unexpected finding was the strongly positive microvascular RAGE immunoreactivity. No evidence for colocalization of RAGE and beta-amyloid was seen within either microvessels or senile plaques. A reversed pattern was evident for LRP-1 in AD. There was very strong staining for LRP-1 in neurons, with minimal microvascular staining. Unlike RAGE, colocalization of LRP-1 and beta-amyloid was clearly present within senile plaques but not microvessels. Western blot analysis revealed a much higher concentration of RAGE protein in AD hippocampi as compared with controls. Concentration of LRP-1 was increased in AD hippocampi, likely secondary to its colocalization with senile plaques. These data confirm that AD is associated with changes in the relative distribution of RAGE and LRP-1 receptors in human hippocampus. They also suggest that the proportion of amyloid within the brains of AD patients that is derived from the systemic circulation may be significant.

The growth factor midkine (MK) is a cytokine that inhibits HIV infection in cell cultures in an autocrine and paracrine manner by blocking the attachment of HIV particles to permissive cells. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-gamma and activation of T lymphocytes by PHA or through the engagement of the CD28 antigen. The binding of MK to cells occurs specifically at a high and a low affinity binding site. This low affinity-binding site is the cell-surface expressed nucleolin, which is implicated in the mechanism of the initial attachment of HIV particles to cells. Accordingly, the nucleolin-binding HB-19 pseudopeptide has no effect on the MK binding to the high affinity binding site, whereas it prevents the binding of MK to the low affinity binding site, thus suggesting the low affinity receptor of MK is the cell-surface-expressed nucleolin. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicates that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, as the domain that binds MK. The specific binding of MK to the surface nucleolin is independent of heparan sulfate and chondroitin sulfate proteoglycans. After binding to cells, MK enters cells by an active process in which nucleolin and lipid rafts appear to be implicated. The potent and the distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli, point out that MK is a cytokine that could be involved in HIV pathogenesis.

We have previously reported that apolipoprotein E (apoE), a protein component of very-low-density lipoproteins (VLDL) and high-density lipoproteins and a potent plasma-borne atheroprotective factor, exerts anti-inflammatory activity in macrophages by switching the activation profile from M1 ("classic") to M2 ("alternative") in a process involving signaling via low-density lipoprotein receptor (LDLR) family members including the VLDL receptor (VLDLR) or apoE receptor-2 (apoER2). The present study was undertaken to investigate whether LDLR-related protein 1 (LRP-1), another member of the LDLR family and a ubiquitously expressed multifunctional cell surface receptor, modulates M1→M2 conversion in murine macrophages. We investigate bone marrow or peritoneal macrophages isolated from wild-type C57/Bl6 mice or mice with conditional inactivation of the LRP-1 gene in the myeloid lineage for the expression of polarization markers. Our results suggest that the deficiency of LRP-1 down-regulates M2 marker expression in macrophages, while enhancing the macrophage response to M1 stimuli. To our knowledge, this is the first demonstration that LRP-1 affects macrophage polarization and promotes the development of an anti-inflammatory M2 functional phenotype.

Chromosome 8q amplification is a common event observed in cancer. In this study, we used high-resolution array comparative genomic hybridization to resolve two neighboring regions on 8q that are both amplified in oral cancer. One region (at 8q24) contains the MYC oncogene, which is frequently overexpressed in many cancers, while the other region (at 8q22) represents a novel amplicon. The alignment of array comparative genomic hybridization profiles of 20 microdissected oral squamous cell carcinomas (OSCCs) revealed a approximately 5 Mbp region of frequent copy number alteration. This region harbors 16 known genes. Gene expression analysis comparing 15 microdissected OSCC with 16 normal epithelium samples revealed overexpression specific to LRP12 but not the neighboring genes, dihydropyrimidinase and FOG2, suggesting that LRP12 may function as an oncogene in oral tumors.

We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the beta chain linked at the amino-terminus to a Myc epitope (Myc-LRPbeta). When expressed together with v-Src, Myc-LRPbeta becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.

Exosome-mediated signal transportation plays a variety of critical roles in cancer progression and metastasis. From the aspect of cancer diagnosis, circulating exosomes are ideal resources of biomarkers because molecular features of tumor cells are transcribed on them. However, isolating pure exosomes from body fluids is time-consuming and still major challenge to be addressed for comprehensive profiling of exosomal proteins and miRNAs. Here we constructed anti-CD9 antibody-coupled highly porous monolithic silica microtips which allowed automated rapid and reproducible exosome extraction from multiple clinical samples. We applied these tips to explore lung cancer biomarker proteins on exosomes by analyzing 46 serum samples. The mass spectrometric quantification of 1,369 exosomal proteins identified CD91 as a lung adenocarcinoma specific antigen on exosomes, which was further validated with CD9-CD91 exosome sandwich ELISA measuring 212 samples. Our simple device can promote not only biomarker discovery studies but also wide range of omics researches about exosomes.

The uptake and removal of necrotic or lysed cells involves inflammation and an immune response, due in part to processes that involve members of the collectin family, surface calreticulin and CD91. Clearance of apoptotic cells, by contrast, does not induce either inflammation or immunity. Could the phosphatidylserine receptor be the molecular switch that determines what the outcome will be?

The low density lipoprotein receptor-related protein gene (LRP) is a good candidate gene for Alzheimer's Disease (AD). Its protein is involved in the physiopathology of AD and has been found in senile plaques; on the other hand, LRP is located in 12q, a region in which genetic linkage to AD was reported. Two common polymorphisms, a tetranucleotide repeat in the 5' untranslated region and a single nucleotide polymorphism at position 766 in exon 3, were found to be associated with AD, but contradictory results were obtained in subsequent association studies. In the absence of clear hypotheses concerning the association of these polymorphisms with AD and their functional role, our objective was to test the association between AD and the two LRP polymorphisms in a large French case-control sample (274 Caucasian AD patients and 290 matched controls) using haplotype analysis. First, the separate study of each polymorphism showed no significant difference in genotype and allele frequencies between AD cases and controls. Second, strong linkage disequilibrium was found between alleles of the two polymorphisms in controls and in cases and the linkage disequilibrium between the 91 bp and C alleles were opposite in cases and in controls. Third, we found that the frequency of the 91-C haplotype was higher in cases than in controls, but the type I error was 0.061, slightly higher than the conventional one of 5%. The haplotype frequencies did not vary significantly as a function of age and APOE epsilon4 status. One interest in this study is the use of the haplotype analysis, which can be used to combine information from several polymorphisms, taking into account their dependence.

Background: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar. Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR. Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients. Methods: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry. The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry. Results: LRP expression was detected in 8 of 10 Tenon specimens. Positive staining for MRP was obtained in 5 of 10 specimens. Negative controls with non-immune mouse IgG did not display any specific staining. RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil. Conclusion: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts. Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

Low-density lipoprotein receptor-related protein-1 (LRP) on brain capillaries clears amyloid beta-peptide (Abeta) from brain. Here, we show that soluble circulating LRP (sLRP) provides key endogenous peripheral 'sink' activity for Abeta in humans. Recombinant LRP cluster IV (LRP-IV) bound Abeta in plasma in mice and Alzheimer's disease-affected humans with compromised sLRP-mediated Abeta binding, and reduced Abeta-related pathology and dysfunction in a mouse model of Alzheimer disease, suggesting that LRP-IV can effectively replace native sLRP and clear Abeta.