In Alzheimer's disease (AD), pathological forms of tau are transferred from cell to cell and “seed” aggregation of cytoplasmic tau. Phosphorylation of tau plays a key role in neurodegenerative ...tauopathies. In addition, apolipoprotein E (apoE), a major component of lipoproteins in the brain, is a genetic risk determinant for AD. The identification of the apoE receptor, low-density lipoprotein receptor–related protein 1 (LRP1), as an endocytic receptor for tau raises several questions about the role of LRP1 in tauopathies: is internalized tau, like other LRP1 ligands, delivered to lysosomes for degradation, and does LRP1 internalize pathological tau leading to cytosolic seeding? We found that LRP1 rapidly internalizes 125I-labeled tau, which is then efficiently degraded in lysosomal compartments. Surface plasmon resonance experiments confirm high affinity binding of tau and the tau microtubule-binding domain to LRP1. Interestingly, phosphorylated forms of recombinant tau bind weakly to LRP1 and are less efficiently internalized by LRP1. LRP1-mediated uptake of tau is inhibited by apoE, with the apoE4 isoform being the most potent inhibitor, likely because of its higher affinity for LRP1. Employing post-translationally–modified tau derived from brain lysates of human AD brain tissue, we found that LRP1-expressing cells, but not LRP1-deficient cells, promote cytosolic tau seeding in a process enhanced by apoE. These studies identify LRP1 as an endocytic receptor that binds and processes monomeric forms of tau leading to its degradation and promotes seeding by pathological forms of tau. The balance of these processes may be fundamental to the spread of neuropathology across the brain in AD.
Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide ...range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.
•ProMMP-13 is constitutively produced and endocytosed by chondrocytes.•LRP1 is a key modulator of extracellular levels of proMMP-13 and MMP-13.•ProMMP-13 and MMP-13 directly bind to LRP1 via the hemopexin domain.•Unique sites on LRP1 for MMP-13 binding have been mapped.•Co-endocytosis of proMMP-13 with ADAMTS-4, -5 and TIMP-3.
Center for Vascular and Inflammatory Diseases and Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland; Department of Pathology, Duke University ...Medical Center, Durham, North Carolina; and Department of Pathology, Medical Scientist Training Program, and The BioMatrix Engineering and Regenerative Medicine Center, University of Alabama at Birmingham, Birmingham, Alabama
The LDL receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes, and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, macrophages, and adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: 1 ) its ability to recognize more than 30 distinct ligands, 2 ) its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner, and 3 ) its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases.
Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an ...increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood–brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1
−/−
) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1
−/−
mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b
+
cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1
−/−
mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.
Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil ...extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.
Thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is markedly limited owing to concerns about hemorrhagic complications and the requirement that tPA be administered ...within 3 h of symptoms. Here we report that tPA activation of latent platelet-derived growth factor-CC (PDGF-CC) may explain these limitations. Intraventricular injection of tPA or active PDGF-CC, in the absence of ischemia, leads to significant increases in cerebrovascular permeability. In contrast, co-injection of neutralizing antibodies to PDGF-CC with tPA blocks this increased permeability, indicating that PDGF-CC is a downstream substrate of tPA within the neurovascular unit. These effects are mediated through activation of PDGF-alpha receptors (PDGFR-alpha) on perivascular astrocytes, and treatment of mice with the PDGFR-alpha antagonist imatinib after ischemic stroke reduces both cerebrovascular permeability and hemorrhagic complications associated with late administration of thrombolytic tPA. These data demonstrate that PDGF signaling regulates blood-brain barrier permeability and suggest potential new strategies for stroke treatment.
Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the ...clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa, and E nterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.
According to the current view, binding of fibrin degradation product E
fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and ...fibrin-derived β15-42 peptide reduces leukocyte transmigration by competing with E
for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of β15-42 to VE-cadherin raised a question about its ability to inhibit E
-VE-cadherin interaction. Further, our previous study revealed that fibrin promotes leukocyte transmigration through the very-low-density lipoprotein (VLDL) receptor (VLDLR)-dependent pathway and suggested a possible link between the inhibitory properties of β15-42 and this pathway. To test such a link and the proposed inhibitory mechanisms for β15-42, we performed in vitro experiments using surface plasmon resonance, enzyme-linked immunosorbent assay, and leukocyte transendothelial migration assay, and in vivo studies with wild-type and VLDLR-deficient mice using mouse model of peritonitis. The experiments revealed that β15-42 cannot inhibit E
-VE-cadherin interaction at the concentrations used in the previous in vivo studies leaving the proposed Fyn-dependent signaling mechanism as a viable explanation for the inhibitory effect of β15-42. While testing this mechanism, we confirmed that Fyn plays a critical role in controlling fibrin-induced transendothelial migration of leukocytes and found that signaling through the VLDLR-dependent pathway results in inhibition of Fyn, thereby increasing leukocyte transmigration. Furthermore, our in vivo experiments revealed that β15-42 inhibits this pathway, thereby preventing inhibition of Fyn and reducing leukocyte transmigration. Thus, this study clarifies the molecular mechanism underlying the VLDLR-dependent pathway of leukocyte transmigration and reveals that this pathway is a target for β15-42.
Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling receptor that is widely expressed. In the liver, LRP1 plays an important role in regulating the plasma ...levels of blood coagulation factor VIII (fVIII) by mediating its uptake and subsequent degradation. fVIII is a key plasma protein that is deficient in hemophilia A and circulates in complex with von Willebrand factor. Because von Willebrand factor blocks binding of fVIII to LRP1, questions remain on the molecular mechanisms by which LRP1 removes fVIII from the circulation. LRP1 also regulates cell surface levels of tissue factor, a component of the extrinsic blood coagulation pathway. This occurs when tissue factor pathway inhibitor bridges the fVII/tissue factor complex to LRP1, resulting in rapid LRP1-mediated internalization and downregulation of coagulant activity. In the vasculature LRP1 also plays protective role from the development of aneurysms. Mice in which the lrp1 gene is selectively deleted in vascular smooth muscle cells develop a phenotype similar to the progression of aneurysm formation in human patient, revealing that these mice are ideal for investigating molecular mechanisms associated with aneurysm formation. Studies suggest that LRP1 protects against elastin fiber fragmentation by reducing excess protease activity in the vessel wall. These proteases include high-temperature requirement factor A1, matrix metalloproteinase 2, matrix metalloproteinase-9, and membrane associated type 1-matrix metalloproteinase. In addition, LRP1 regulates matrix deposition, in part, by modulating levels of connective tissue growth factor. Defining pathways modulated by LRP1 that lead to aneurysm formation and defining its role in thrombosis may allow for more effective intervention in patients.
Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The ...low-density lipoprotein receptor–related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 SNPs are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 KO mice revealed a critical, nonredundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these “hidden” specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.
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