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1.  Lipoprotein Receptor–Related Protein-6 Protects the Brain From Ischemic Injury 
Background and Purpose
Loss-of-function mutations of the lipoprotein receptor–related protein-6 (LRP6), a coreceptor in the Wingless-related integration site-β–catenin prosurvival pathway, have been implicated in myocardial ischemia and neurodegeneration. However, it remains to be established whether LRP6 is also involved in ischemic brain injury. We used LRP6+/− mice to examine the role of this receptor in the mechanisms of focal cerebral ischemia.
Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery. Motor deficits and infarct volume were assessed 3 days later. Glycogen-synthase-kinase-3β (GSK-3β) phosphorylation was examined by Western blotting with phosphospecific antibodies, and the mitochondrial membrane potential changes induced by Ca2+ were also assessed.
LRP6+/− mice have larger stroke and more severe motor deficits, effects that were independent of intraischemic cerebral blood flow, vascular factors, or cytosolic β-catenin levels. Rather, LRP6 haploinsufficiency increased the activating phosphorylation and decreased the inhibitory phosphorylation of GSK-3β, a kinase involved in proinflammatory signaling and mitochondrial dysfunction. Accordingly, postischemic inflammatory gene expression was enhanced in LRP6+/− mice. Furthermore, the association of mitochondria with activated GSK-3β was increased in LRP6+/− mice, resulting in a reduction in the Ca2+ handling ability of mitochondria. The mitochondrial dysfunction was reversed by pharmacological inhibition of GSK-3β.
LRP6 activates an endogenous neuroprotective pathway that acts independently of β-catenin by controlling GSK-3β activity and preventing its deleterious mitochondrial and proinflammatory effects. The findings raise the possibility that emerging treatment strategies for diseases attributable to LRP6 loss-of-function mutations could also lead to new therapeutic avenues for ischemic stroke.
PMCID: PMC3933931  PMID: 23743975
cerebral ischemia; glycogen-synthase-kinase-3; mitochondria; stroke; Wnt signaling pathway
2.  A shed form of LDL receptor–related protein–1 regulates peripheral nerve injury and neuropathic pain in rodents 
Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor–related protein–1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact α-chain (sLRP-α), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-α into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-α and IL-1β locally. sLRP-α also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-α inhibited TNF-α–induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-α did not involve TNF-α binding, but rather glial cell preconditioning, so that the subsequent response to TNF-α was inhibited. Our results show that sLRP-α is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.
PMCID: PMC2104478  PMID: 18060043
3.  LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer’s disease 
The Aβ peptide that accumulates in Alzheimer’s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by β- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aβ production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aβ production.
In this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aβ, while knockdown of LRP10 expression increases Aβ production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP β-cleavage into Aβ. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.
The present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer’s disease.
PMCID: PMC3520120  PMID: 22734645
LDLR-related protein 10 (LRP10); Amyloid precursor protein (APP); Amyloid beta (Aβ); Intracellular trafficking; Alzheimer’s disease; Endosome; Trans-Golgi network (TGN); Low density lipoprotein receptor (LDLR)
4.  Analysis of multiple bone responses to graded strains above functional levels, and to disuse, in mice in vivo show that the human Lrp5 G171V High Bone Mass mutation increases the osteogenic response to loading but that lack of Lrp5 activity reduces it 
Bone  2011;49(2):184-193.
To investigate the role of the low-density lipoprotein receptor-related protein 5 (Lrp5) in bones' responses to loading, we analysed changes in multiple measures of bone architecture in tibias subjected to loading or disuse in male and female mice with the Lrp5 loss of function mutation (Lrp5−/−) or heterozygous for the Lrp5 G171V High Bone Mass (HBM) mutation (Lrp5HBM+).
Materials and methods
The right tibias of these 17 week old male and female mice and their Wild Type (WT) littermates were subjected to short periods of loading three days a week for two weeks. Each tibia was loaded for 40 cycles, to produce peak strains at the midshaft within the low, medium or high physiological range (~ 1500, 2400 and 3000 microstrain, respectively). In similar groups of mice the right sciatic nerve was severed causing disuse of the right tibia for 3 weeks. Data from microCT of loaded, neurectomised and contra-lateral control tibias were analysed to quantify changes in the cortical and cancellous regions of the bone in the absence of functional strains and in response to graded strains in addition to those derived from function.
Results and conclusion
Male WT+/+ controls showed significant strain:response curves for cortical area and trabecular thickness, but Lrp5−/− mice showed no detectable strain:response in those same outcomes. Female mice of either WT+/+ or Lrp5−/− genotype did not show significant strain:response curves for cortical or trabecular parameters, the one exception being Tb.Th in Lrp5−/− mice. Since female WT+/+ mice did not respond to loading in a significant dose:responsive manner, the similar lack of responsiveness of the Lrp5−/− females could not be ascribed to their Lrp5 status. Cortical bone loss associated with disuse showed no differences between Lrp5−/− mice and WT+/+ controls, but in cancellous bone of both male and females of these mice, there was a greater loss than in WT+/+ controls. In contrast, the tibias of male and female mice heterozygous for the Lrp5 G171V HBM mutation showed greater osteogenic responsiveness to loading and less bone loss associated with disuse than their WTHBM− controls. These data indicate that the presence of the Lrp5 G171V HBM mutation is associated with an increased osteogenic response to loading but support only a marginal gender-related role for normal Lrp5 function in this loading-related response.
Research highlights
► Lrp5−/− increased cancellous but not cortical bone loss with disuse. ► Lrp5−/− decreases load-induced new bone formation in males but not in females. ► Lrp5 G171V High Bone Mass mutation protected against bone loss with disuse but increased bone response to loading.
PMCID: PMC3121951  PMID: 21419885
Lrp5; Bone; Mechanical loading; Disuse; MicroCT
5.  Primary phagocytosis of viable neurons by microglia activated with LPS or Aβ is dependent on calreticulin/LRP phagocytic signalling 
Microglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called ‘primary phagocytosis’ or ‘phagoptosis’. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia.
We used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with lipopolysaccharide (LPS) or nanomolar concentrations of amyloid-β peptide1-42 (Aβ). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells.
We found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT-blocking antibody or LRP-blocking protein (receptor-associated protein: RAP). Activation of primary rat microglia with LPS or Aβ resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss.
CRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.
PMCID: PMC3481398  PMID: 22889139
Phagocytosis; Neuron; Microglia; Calreticulin; LRP; Inflammation; Amyloid; Neurodegeneration; Cell death; Phagoptosis
6.  Lipopolysaccharide impairs amyloid beta efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier 
Defects in the low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein (Pgp) clearance of amyloid beta (Aβ) from brain are thought to contribute to Alzheimer’s disease (AD). We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS) results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood–brain barrier.
CD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV)) or into the jugular vein (intravenous (IV)) was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF). Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine.
We found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain endothelial cells.
These results suggest that LRP-1 undergoes complex functional regulation following systemic inflammation which may depend on cell type, subcellular location, and post-translational modifications. Our findings that systemic inflammation causes deficits in both Aβ transport and bulk flow like those observed in AD indicate that inflammation could induce and promote the disease.
PMCID: PMC3410805  PMID: 22747709
Alzheimer’s disease; amyloid beta; blood–brain barrier; inflammation; lipopolysaccharide; LRP1; Pgp; ABCB1; MDR1; cerebrospinal fluid
7.  The hemopexin domain of matrix metalloproteinase-9 activates cell-signaling and promotes migration of Schwann cells by binding to low density lipoprotein receptor-related protein 
Low density lipoprotein receptor-related protein (LRP-1) is an endocytic receptor for diverse proteins, including matrix metalloproteinase-9 (MMP-9), and a cell-signaling receptor. In the peripheral nervous system (PNS), LRP-1 is robustly expressed by Schwann cells only after injury. Herein, we demonstrate that MMP-9 activates extracellular-signal regulated kinase (ERK1/2) and Akt in Schwann cells in culture. MMP-9 also promotes Schwann cell migration. These activities require LRP-1. MMP-9-induced cell-signaling and migration were blocked by inhibiting MMP-9-binding to LRP-1 with receptor-associated protein (RAP) or by LRP-1 gene-silencing. The effects of MMP-9 on Schwann cell migration also were inhibited by blocking the cell-signaling response. An antibody targeting the hemopexin domain of MMP-9, which mediates the interaction with LRP-1, blocked MMP-9-induced cell signaling and migration. Furthermore, a novel GST-fusion protein (MMP-9-PEX), which includes only the hemopexin domain of MMP-9, replicated the activities of intact MMP-9, activating Schwann cell-signaling and migration by an LRP-1-dependent pathway. Constitutively-active MEK1 promoted Schwann cell migration; in these cells, MMP-9-PEX had no further effect, indicating that ERK1/2 activation is sufficient to explain the effects of MMP-9-PEX on Schwann cell migration. Injection of MMP-9-PEX into sciatic nerves, 24 h after crush injury, robustly increased phosphorylation of ERK1/2 and Akt. This response was inhibited by RAP. MMP-9-PEX failed to activate cell-signaling in uninjured nerves, consistent with the observation that Schwann cells express LRP-1 at significant levels only after nerve injury. These results establish LRP-1 as a cell-signaling receptor for MMP-9, which may be significant in regulating Schwann cell migration and physiology in PNS injury.
PMCID: PMC3837707  PMID: 18987193
Schwann cell; peripheral nerve; proteinase; cell migration; hemopexin; phosphatidylinositol 3-kinase; ERK/MAP kinase
8.  The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1 
Molecular Biology of the Cell  2009;20(15):3552-3560.
The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.
PMCID: PMC2719573  PMID: 19477926
9.  Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning 
Neuroscience  2007;151(4):1099-1103.
Remote ischemic preconditioning is an emerging concept for stroke treatment, but its protection against focal stroke has not been established. We tested whether remote preconditioning, performed in the ipsilateral hind limb, protects against focal stroke and explored its protective parameters. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery (MCA) combined with a 30 minute occlusion of the bilateral common carotid arteries (CCA) in male rats. Limb preconditioning was generated by 5 or 15 minute occlusion followed with the same period of reperfusion of the left hind femoral artery, and repeated for 2 or 3 cycles. Infarct was measured 2 days later. The results showed that rapid preconditioning with 3 cycles of 15 minutes performed immediately before stroke reduced infarct size from 47.7±7.6% of control ischemia to 9.8±8.6%; at 2 cycles of 15 minutes, infarct was reduced to 24.7±7.3%; at 2 cycles of 5 minutes, infarct was not reduced. Delayed preconditioning with 3 cycles of 15 minutes conducted 2 days before stroke also reduced infarct to 23.0 ±10.9%, but with 2 cycles of 15 minutes it offered no protection. The protective effects at these two therapeutic time windows of remote preconditioning are consistent with those of conventional preconditioning, in which the preconditioning ischemia is induced in the brain itself. Unexpectedly, intermediate preconditioning with 3 cycles of 15 minutes performed 12 hours before stroke also reduced infarct to 24.7±4.7%, which contradicts the current dogma for therapeutic time windows for the conventional preconditioning that has no protection at this time point. In conclusion, remote preconditioning performed in one limb protected against ischemic damage after focal cerebral ischemia.
PMCID: PMC2696348  PMID: 18201834
preconditioning; remote preconditioning; limb preconditioning; cerebral ischemia; focal ischemia
10.  Low Density Lipoprotein Receptor-Related Protein (LRP) is Decreased in Optic Neuropathy of Alzheimer’s Disease 
Alzheimer’s disease (AD) is associated with optic nerve degeneration yet the underlying pathophysiology of this disease and the optic nerve disorder remains poorly understood. Low density lipoprotein receptor-related protein (LRP) is implicated in the pathogenesis of AD by mediating the transport of amyloid-β (Aβ) out of the brain into the systemic circulation. As a key player in the reaction to central nervous system (CNS) injury, astrocytes associate with LRP in AD. This study investigates the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy.
To investigate the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy, we conducted immunohistochemical (IHC) studies on postmortem optic nerves in AD patients (n = 11) and age-matched controls (n = 10) to examine the presence of LRP. Quantitative analyses using imaging software were used to document the extent of LRP in neural tissues. Axonal integrity was assessed by performing IHC on the subjects’ optic nerves with an antibody to neurofilament (NF) protein. Double-immunofluorescence labeling was performed to investigate whether LRP colocalized with astrocytes expressing glial fibrillary acidic protein (GFAP).
LRP expression was decreased in AD optic nerves compared to controls (p < 0.001). LRP immunoreactivity was observed in the microvasculature and perivascularly in close proximity to astrocytic processes. Colocalization of LRP in astrocytes of optic nerves was also demonstrated. The presence of optic neuropathy was confirmed in the AD optic nerves by demonstrating greatly reduced immunostaining for NF protein as compared to controls.
The reduction of LRP in the AD degenerative optic nerves supports the hypothesis that LRP may play a role in the pathophysiology of AD optic neuropathy.
PMCID: PMC3106991  PMID: 21593627
Alzheimer’s disease; Alzheimer’s optic neuropathy; astrocytes; low density lipoprotein receptor-related protein; microvasculature; optic nerve
11.  Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis 
Oncogene  2011;31(26):3202-3212.
The aspartic protease cathepsin-D (cath-D) is a marker of poor prognosis in breast cancer that is overexpressed and hypersecreted by human breast cancer cells. Secreted pro-cath-D binds to the extracellular domain of the β chain of the LDL receptor-related protein-1 (LRP1) in fibroblasts. The LRP1 receptor has an 85-kDa transmembrane β chain and a non-covalently attached 515-kDa extracellular α chain. LRP1 acts by (1) internalizing many ligands via its α chain, (2) activating signaling pathways by phosphorylating the LRP1β chain tyrosine, and (3) modulating gene transcription by regulated intramembrane proteolysis (RIP) of its β chain. LRP1 RIP involves two cleavages: the first liberates the LRP1 ectodomain to give a membrane-associated form LRP1β-CTF and the second generates the LRP1β intracellular domain, LRP1β-ICD, that modulates gene transcription. Here, we investigated the endocytosis of pro-cath-D by LRP1 and the effect of the pro-cath-D/LRP1β interaction on LRP1β tyrosine phosphorylation and/or LRP1β RIP. Our results indicate that pro-cath-D was partially endocytosed by LRP1 in fibroblasts. However, pro-cath-D and ectopic cath-D did not stimulate phosphorylation of the LRP1β chain tyrosine. Interestingly, ectopic cath-D and its catalytically-inactive D231Ncath-D, and pro-D231Ncath-D all significantly inhibited LRP1 RIP by preventing LRP1β-CTF production. Thus cath-D inhibits LRP1 RIP independently of its catalytic activity by blocking the first cleavage. Since cath-D triggers fibroblast outgrowth via LRP1, we propose that cath-D modulates the growth of fibroblasts by inhibiting LRP1 RIP in the breast tumor micro-environment.
PMCID: PMC3579766  PMID: 22081071
Animals; Breast Neoplasms; metabolism; pathology; COS Cells; Cathepsin D; metabolism; Cell Line, Tumor; Cell Membrane; metabolism; Cell Proliferation; Cercopithecus aethiops; Endocytosis; Enzyme Precursors; metabolism; Fibroblasts; cytology; enzymology; metabolism; pathology; Humans; Low Density Lipoprotein Receptor-Related Protein-1; chemistry; metabolism; Mammary Glands, Human; cytology; pathology; Neoplasm Invasiveness; Protein Structure, Tertiary; Proteolysis; Tumor Microenvironment; cancer; cathepsin D; LRP1; RIP; endocytosis; tyrosine phosphorylation
12.  Near-membrane ensemble elongation in the proline-rich LRP6 intracellular domain may explain the mysterious initiation of the Wnt signaling pathway 
BMC Bioinformatics  2011;12(Suppl 13):S13.
LRP6 is a membrane protein crucial in the initiation of canonical Wnt/β-catenin signalling. Its function is dependent on its proline-serine rich intracellular domain. LRP6 has five PPP(S/T)P motifs that are phosphorylated during activation, starting with the site closest to the membrane. Like all long proline rich regions, there is no stable 3D structure for this isolated, contiguous region.
In our study, we use a computational simulation tool to sample the conformational space of the LRP6 intracellular domain, under the spatial constraints imposed by (a) the membrane and (b) the close approach of the neighboring intracellular molecular complex, which is assembled on Frizzled when Wnt binds to both LRP6 and Frizzled on the opposite side of the membrane. We observe that an elongated form dominates in the LRP6 intracellular domain structure ensemble. This elongation could relieve conformational auto-inhibition of the PPP(S/T)PX(S/T) motif binding sites and allow GSK3 and CK1 to approach their phosphorylation sites, thereby activating LRP6 and the downstream pathway.
We propose a model in which the conformation of the LRP6 intracellular domain is elongated before activation. This is based on the intrusion of the Frizzled complex into the ensemble space of the proline rich region of LRP6, which alters the shape of its available ensemble space. To test whether this observed ensemble conformational change is sequence dependent, we did a control simulation with a hypothetical sequence with 50% proline and 50% serine in alternating residues. We confirm that this ensemble neighbourhood-based conformational change is independent of sequence and conclude that it is likely found in all proline rich sequences. These observations help us understand the nature of proline rich regions which are both unstructured and which seem to evolve at a higher rate of mutation, while maintaining sequence composition.
PMCID: PMC3278829  PMID: 22372892
13.  Enhanced Brain Amyloid-β Clearance by Rifampicin and Caffeine as a Possible Protective Mechanism Against Alzheimer’s Disease 
Rifampicin and caffeine are widely used drugs with reported protective effect against Alzheimer’s disease (AD). However, the mechanism underlying this effect is incompletely understood. In this study, we have hypothesized that enhanced amyloid-β (Aβ) clearance from the brain across the blood-brain barrier (BBB) of wild-type mice treated with rifampicin or caffeine is caused by both drugs potential to upregulate low-density lipoprotein receptor related protein-1 (LRP1) and/or P-glycoprotein (P-gp) at the BBB. Expression studies of LRP1 and P-gp in brain endothelial cells and isolated mice brain microvessels following treatment with rifampicin or caffeine demonstrated both drugs as P-gp inducers, and only rifampicin as an LRP1 inducer. Also, brain efflux index (BEI%) studies conducted on C57BL/6 mice treated with either drug to study alterations in Aβ clearance demonstrated the BEI% of Aβ in rifampicin (82.4 ± 4.3%) and caffeine (80.4 ± 4.8%) treated mice were significantly higher than those of control mice (62.4 ±6.1%, p <0.01). LRP1 and P-gp inhibition studies confirmed the importance of both proteins to the clearance of Aβ, and that enhanced clearance following drugs treatment was caused by LRP1 and/or P-gp upregulation at the mouse BBB. Furthermore, our results provided evidence for the presence of a yet to be identified transporter/receptor that plays significant role in Aβ clearance and is upregulated by caffeine and rifampicin. In conclusion, our results demonstrated the upregulation of LRP1 and P-gp at the BBB by rifampicin and caffeine enhanced brain Aβ clearance, and this effect could explain, at least in part, the protective effect of rifampicin and caffeine against AD.
PMCID: PMC3902015  PMID: 22504320
Alzheimer’s disease; amyloid-β; caffeine; LRP1; P-gp; rifampicin
14.  Synaptic plasticity and cognitive function are disrupted in the absence of Lrp4 
eLife  null;3:e04287.
Lrp4, the muscle receptor for neuronal Agrin, is expressed in the hippocampus and areas involved in cognition. The function of Lrp4 in the brain, however, is unknown, as Lrp4−/− mice fail to form neuromuscular synapses and die at birth. Lrp4−/− mice, rescued for Lrp4 expression selectively in muscle, survive into adulthood and showed profound deficits in cognitive tasks that assess learning and memory. To learn whether synapses form and function aberrantly, we used electrophysiological and anatomical methods to study hippocampal CA3–CA1 synapses. In the absence of Lrp4, the organization of the hippocampus appeared normal, but the frequency of spontaneous release events and spine density on primary apical dendrites were reduced. CA3 input was unable to adequately depolarize CA1 neurons to induce long-term potentiation. Our studies demonstrate a role for Lrp4 in hippocampal function and suggest that patients with mutations in Lrp4 or auto-antibodies to Lrp4 should be evaluated for neurological deficits.
eLife digest
LRP4 is a muscle protein that is found in the hippocampus, a region of the brain that controls cognitive processes such as learning and memory. However, we know very little about what exactly LRP4 does in the hippocampus, and how it affects learning and memory.
A standard way to figure out what a protein does is to study mice that have been genetically modified so that they cannot produce that protein. However, deleting the gene for LRP4 leads to muscle problems that kill these mutant mice at birth.
To get around this problem, Gomez et al. have developed a method to restore the production of LRP4 in the muscles of mutant mice but not in their brains. These mutant mice were then subjected to a battery of tests to measure their ability to learn and recall new memories. These tests showed that LRP4 must be present in the brain, otherwise learning and memory are impaired.
Gomez et al. also explored a process known as long-term potentiation. This process, which involves strengthening the functional connections between neurons, is believed to be essential for learning and other cognitive process. Gomez et al. demonstrated that long-term potentiation was disrupted by the lack of LRP4.
Further experiments are needed to work out how LRP4 controls the learning process in the hippocampus and to explore the connection between LRP4 and various neuromuscular and neurological diseases.
PMCID: PMC4270049  PMID: 25407677
synapse; low-density lipoprotein-related receptor; behavior; learning; hippocampus; long-term potentiation; mouse
15.  Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6 
PLoS ONE  2011;6(8):e23537.
Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening “gap4” region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.
PMCID: PMC3160902  PMID: 21887268
16.  Repetitive hypoxic preconditioning induces an immunosuppressed B cell phenotype during endogenous protection from stroke 
Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset.
Adult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student’s t-test or one-way analysis of variance determined significance (P < 0.05).
CXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, were upregulated.
Collectively, our data characterize an endogenous neuroprotective phenotype that utilizes adaptive immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B cells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediate an adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.
PMCID: PMC3926678  PMID: 24485041
Hypoxic preconditioning; B cells; CXCL13; Stroke; Neuroprotection; B10
17.  Sclerostin Inhibition Reverses Skeletal Fragility in an Lrp5-Deficient Mouse Model of OPPG Syndrome 
Science translational medicine  2013;5(211):211ra158.
Osteoporosis pseudoglioma syndrome (OPPG) is a rare genetic disease that produces debilitating effects in the skeleton. OPPG is caused by mutations in LRP5, a WNT co-receptor that mediates osteoblast activity. WNT signaling through LRP5, and also through the closely related receptor LRP6, is inhibited by the protein sclerostin (SOST). It is unclear whether OPPG patients might benefit from the anabolic action of sclerostin neutralization therapy (an approach currently being pursued in clinical trials for postmenopausal osteoporosis) in light of their LRP5 deficiency and consequent osteoblast impairment. To assess whether loss of sclerostin is anabolic in OPPG, we measured bone properties in a mouse model of OPPG (Lrp5−/−), a mouse model of sclerosteosis (Sost−/−), and in mice with both genes knocked out (Lrp5−/−;Sost−/−). Lrp5−/−;Sost−/− mice have larger, denser, and stronger bones than do Lrp5−/− mice, indicating that SOST deficiency can improve bone properties via pathways that do not require LRP5. Next, we determined whether the anabolic effects of sclerostin depletion in Lrp5−/− mice are retained in adult mice by treating 17-week-old Lrp5−/− mice with a sclerostin antibody for 3 weeks. Lrp5+/+ and Lrp5−/− mice each exhibited osteoanabolic responses to antibody therapy, as indicated by increased bone mineral density, content, and formation rates. Collectively, our data show that inhibiting sclerostin can improve bone mass whether LRP5 is present or not. In the absence of LRP5, the anabolic effects of SOST depletion can occur via other receptors (such as LRP4/6). Regardless of the mechanism, our results suggest that humans with OPPG might benefit from sclerostin neutralization therapies.
PMCID: PMC3964772  PMID: 24225945
18.  LRP1 shedding in human brain: roles of ADAM10 and ADAM17 
The low-density lipoprotein receptor-related protein 1 (LRP1) plays critical roles in lipid metabolism, cell survival, and the clearance of amyloid-β (Aβ) peptide. Functional soluble LRP1 (sLRP1) has been detected in circulating human placenta; however, whether sLRP1 is also present in the central nervous system is unclear.
Here we show that abundant sLRP1 capable of binding its ligands is present in human brain tissue and cerebral spinal fluid (CSF). Interestingly, the levels of sLRP1 in CSF are significantly increased in older individuals, suggesting that either LRP1 shedding is increased or sLRP1 clearance is decreased during aging. To examine potential effects of pathological ligands on LRP1 shedding, we treated MEF cells with Aβ peptide and found that LRP1 shedding was increased. ADAM10 and ADAM17 are key members of the ADAM family that process membrane-associated proteins including amyloid precursor protein and Notch. We found that LRP1 shedding was significantly decreased in MEF cells lacking ADAM10 and/or ADAM17. Furthermore, forced expression of ADAM10 increased LRP1 shedding, which was inhibited by ADAM-specific inhibitor TIMP-3.
Our results demonstrate that LRP1 is shed by ADAM10 and ADAM17 and functional sLRP1 is abundantly present in human brain and CSF. Dysregulated LRP1 shedding during aging could alter its function and may contribute to the pathogenesis of AD.
PMCID: PMC2672942  PMID: 19371428
19.  Lrp4, a Novel Receptor for Dickkopf 1 and Sclerostin, Is Expressed by Osteoblasts and Regulates Bone Growth and Turnover In Vivo 
PLoS ONE  2009;4(11):e7930.
Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.
PMCID: PMC2775917  PMID: 19936252
20.  Low-density lipoprotein receptor-related protein 1: a physiological Aβ homeostatic mechanism with multiple therapeutic opportunities 
Pharmacology & therapeutics  2012;136(1):94-105.
Low-density lipoprotein receptor-related protein-1 (LRP1) is the main cell surface receptor involved in brain and systemic clearance of the Alzheimer's disease (AD) toxin amyloid-beta (Aβ). In plasma, a soluble form of LRP1 (sLRP1) is the major transport protein for peripheral Aβ. LRP1 in brain endothelium and mural cells mediates Aβ efflux from brain by providing a transport mechanism for A across the blood-brain barrier (BBB). sLRP1 maintains a plasma ‘sink’ activity for Aβ through binding of peripheral Aβ which in turn inhibits re-entry of free plasma Aβ into the brain. LRP1 in the liver mediates systemic clearance of Aβ. In AD, LRP1 expression at the BBB is reduced and Aβ binding to circulating sLRP1 is compromised by oxidation. Cell surface LRP1 and circulating sLRP1 represent druggable targets which can be therapeutically modified to restore the physiological mechanisms of brain Aβ homeostasis. In this review, we discuss how increasing LRP1 expression at the BBB and liver with lifestyle changes, statins, plant-based active principles and/or gene therapy on one hand, and how replacing dysfunctional plasma sLRP1 on the other regulate Aβ clearance from brain ultimately controlling the onset and/or progression of AD.
PMCID: PMC3432694  PMID: 22820095
Alzheimer's disease; Amyloid β-peptide; LRP1; soluble LRP1 (sLRP1); sLRP1 fragments
21.  Phosphorylation of PPP(S/T)P Motif of the Free LRP6 Intracellular Domain Is Not Required to Activate the Wnt/β-Catenin Pathway and Attenuate GSK3β Activity 
Journal of cellular biochemistry  2009;108(4):886-895.
The canonical Wnt/β-catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co-receptor for Wnt/β-catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane-anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6-ICD) can activate the Wnt/β-catenin pathway in a β-catenin and TCF/LEF-1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6-ICD to attenuate GSK3β activity and modulate activation of the Wnt/β-catenin pathway requires phosphorylation of the LRP6-ICD PPP(S/T)P motifs, in a manner similar to the membrane-anchored LRP6 intracellular domain. Here we provide evidence that the LRP6-ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β-catenin resulting in activation of TCF/LEF-1 and the Wnt/β-catenin pathway. LRP6-ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β-mediated phosphorylation of β-catenin and tau to the same extent. These data indicate that the LRP6-ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6-ICD may be distinct from membrane-anchored LRP6, and that release of the LRP6-ICD may provide a complimentary signaling cascade capable of modulating Wnt-dependent gene expression.
PMCID: PMC2906752  PMID: 19711366
22.  Overexpression of lung resistance-related protein and P-glycoprotein and response to induction chemotherapy in acute myelogenous leukemia 
Hematology Reports  2012;4(3):e18.
Lung resistance-related protein (LRP) and P-glycoprotein (P-gp) are associated with multidrug resistance. P-gp overexpression reduces intracellular anticancer drug concentrations and is correlated with low remission rates. However, whether the presence of LRP influences the response to induction chemotherapy remains controversial. Therefore, we investigated the relationship of LRP and P-gp overexpression with the response to induction chemotherapy. Univariate analysis revealed that there was a significant difference between complete remission rates for acute myelogenous leukemia patients depending on their blast cell expressions, between LRP positive versus negative, P-gp positive versus negative, and LRP/P-gp double positive versus other groups. Crude odds ratios (ORs) for complete remission were 0.390, 0.360, and 0.307 for LRP positive, for P-gp positive, and LRP/P-gp double positive patients, respectively. After controlling the confounding variables by stepwise multivariate logistical regression analysis, the presence of LRP/P-gp double positivity and P-gp positivity were found to be independent prognostic factors; adjusted ORs were 0.233 and 0.393, respectively. Furthermore, the monoclonal antibody against LRP significantly increased daunorubicin acumulation (P=0.004) in the nuclei of leukemic blast cells with LRP positivity in more than 10% of the cells. An LRP reversing agent, PAK-104P, was found to increase the daunorubicin content with marginal significance (P=0.060). The present results suggest that not only the presence of P-gp, but also LRP in leukemic blast cells is a risk factor for resistance to induction chemotherapy. Inhibiting LRP function, similar to the inhibition of P-gp function, will be necessary to improve the effectiveness of induction chemotherapy.
PMCID: PMC3475940  PMID: 23087807
lung resistance-related protein; P-glycoprotein; reversing agent; acute myelogenous leukemia.
23.  Clinical Application of Preconditioning and Postconditioning to Achieve Neuroprotection 
Translational stroke research  2013;4(1):19-24.
Ischemic conditioning is a form of endogenous protection induced by transient, subcritical ischemia in a tissue. Organs with high sensitivity to ischemia, such as the heart, the brain, and spinal cord represent the most critical and potentially promising targets for potential therapeutic applications of ischemic conditioning. Numerous preclinical investigations have systematically studied the molecular pathways and potential benefits of both pre- and post-conditioning with promising results. The purpose of this review is to summarize the present knowledge on cerebral pre-and post-conditioning, with an emphasis in the clinical application of these forms of neuroprotection.
A systematic Medline search for the terms preconditioning and postconditioning was performed. Publications related to the nervous system and to human applications were selected and analyzed.
Pre-and post-conditioning appear to provide similar levels of neuroprotection. The preconditioning window of benefit can be subdivided into early and late effects, depending on whether the effect appears immediately after the sublethal stress or with a delay of days. In general early effects have been associated post-translational modification of critical proteins (membrane receptors, mitochondrial respiratory chain) while late effects are the result of gene up-or down-regulation. Transient ischemic attacks appear to represent a form of clinically relevant preconditioning by inducing ischemic tolerance in the brain and reducing the severity of subsequent strokes. Remote forms of ischemic pre- and post-conditioning have been more commonly used in clinical studies, as the remote application reduces the risk of injuring the target tissue for which protection is pursued. Limb transient ischemia is the preferred method of induction of remote conditioning with evidence supporting its safety. Clinical studies in a variety of populations at risk of central nervous damage including carotid disease, cervical myelopathy and subarachnoid hemorrhage have shown improvement in surrogate markers of injury.
Promising preclinical and early clinical studies noting improvement in surrogate markers of central nervous injury after the use of remote pre- and post-conditioning treatments demand follow-up systematic investigations to address effectiveness. Challenges in the application of these techniques to pressing clinical cerebrovascular disease ought to be overcome through careful, well-designed, translational investigations.
PMCID: PMC4224593  PMID: 24323188
Preconditioning; Postconditioning; Ischemia; Reperfusion Injury; Neuroprotection; Brain injury
24.  LRP1: Role in the regulation of vascular integrity 
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 (vWf). Since vWf blocks binding of fVIII to LRP1, questions remain regarding the molecule mechanisms by which LRP1 removes fVIII from the circulation. LRP1 also regulates cell surface levels of tissue factor (TF), a component of the extrinsic blood coagulation pathway. This occurs when tissue factor pathway inhibitor (TFPI) bridges the fVII/TF complex to LRP1, resulting in rapid LRP1-mediated internalization and down regulation 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 (HtrA1), MMP2, MMP9 and MT1-MMP. 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.
PMCID: PMC4304649  PMID: 24504736
25.  Urokinase Plasminogen Activator Impairs SNP and PGE2 Cerebrovasodilation after Brain Injury through Activation of LRP and ERK MAPK 
Journal of Neurotrauma  2008;25(11):1375-1381.
Pial artery dilation in response to prostaglandin (PG)E2 and the nitric oxide (NO) releaser sodium nitroprus-side (SNP) are blunted after fluid percussion brain injury (FPI), whereas responses to papaverine are unchanged. Urokinase plasminogen activator (uPA) and ERK mitogen-activated protein kinase (MAPK) are upregulated and contribute to the impairment of cerebrohemodynamics seen after FPI. PA vascular activity is mediated through the low-density lipoprotein receptor (LRP). Therefore, we investigated the role of uPA, LRP, and ERK MAPK in the impaired cerebrovasodilation response to PGE2 and SNP after FPI. Lateral FPI (2 atm) was induced in anesthetized piglets equipped with a closed cranial window. Cerebrospinal fluid (CSF) ERK MAPK was quantified by enzyme-linked immunosorbent assay (ELISA). Pretreatment with soluble uPA receptor (su-PAR), which antagonizes the vascular action of uPA, blunted the impairment of SNP and PGE2-mediated dilation seen after FPI. Pretreatment with the LRP antagonist RAP, a monoclonal antibody against LRP (Mab ag LRP) and the ERK MAPK antagonist, U 0126, all provided similar protection, whereas control immunoglobulin G (IgG) had no effect. Responses to papaverine were unchanged after FPI. Upregulation of ERK MAPK phosphorylation in CSF after FPI was blunted in animals pretreated with suPAR, RAP, MAb ag LRP, or U 0126, whereas control IgG had no effect. These data indicate that uPA contributes to the impairment of SNP and PGE2-mediated cerebrovasodilation seen after brain injury through activation of LRP and ERK MAPK.
PMCID: PMC2948470  PMID: 19061381
cerebral circulation; newborn; plasminogen activators; signal transduction

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