Low-density Lipoprotein Receptor-related Protein 1 Knockdown Research Articles

Loss of LRP1 Promotes Hepatocellular Carcinoma Progression via UFL1-Mediated Activation of NF-κB Signaling.

Low-density lipoprotein receptor-related protein-1 (LRP1) is thought to be correlated with hepatocellular carcinoma (HCC) invasion and metastasis. However, the precise mechanism through which LRP1 contributes to HCC progression remains unclear. Here, lower LRP1 levels are associated with malignant progression, and poor prognosis in patients with HCC is shown. LRP1 knockdown enhances the tumorigenicity of HCC cells in vitro and in vivo, whereas overexpression of either LRP1 or its β-chain has the opposite effect. Mechanistically, LRP1 knockdown promotes the binding of ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) to OGA and accelerates ubiquitin-mediated OGA degradation, leading to increased O-GlcNAcylation of nuclear factor-kappa B (NF-κB) and subsequent inhibition of pro-apoptotic gene expression. Conversely, exogenously expressed truncated β-chain (β∆) stabilizes OGA by disrupting the association between UFL1 and OGA, consequently abolishing the anti-apoptotic effects of O-GlcNAcylated NF-κB. The findings identify LRP1, particularly its β-chain, as a novel upstream control factor that facilitates the stabilization of the OGA protein, thereby suppressing NF-κB signaling and attenuating HCC progression, thus suggesting a novel therapeutic strategy for HCC.

LRP1 induces anti-PD-1 resistance by modulating the DLL4-NOTCH2-CCL2 axis and redirecting M2-like macrophage polarisation in bladder cancer

The tumour microenvironment (TME) drives bladder cancer (BLCA) progression. Targeting the TME has emerged as a promising strategy for BLCA treatment in recent years. Furthermore, checkpoint blockade therapies are only beneficial for a minority of patients with BLCA, and drug resistance is a barrier to achieving significant clinical effects of anti-programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) therapy. In this study, higher low-density lipoprotein receptor-related protein 1 (LRP1) levels were related to a poorer prognosis for patients with various cancers, including those with higher grades and later stages of BLCA. Enrichment analysis demonstrated that LRP1 plays a role in the epithelial–mesenchymal transition (EMT), NOTCH signalling pathway, and ubiquitination. LRP1 knockdown in BLCA cells delayed BLCA progression both in vivo and in vitro. Furthermore, LRP1 knockdown suppressed EMT, reduced DLL4-NOTCH2 signalling activity, and downregulated M2-like macrophage polarisation. Patients with BLCA and higher LRP1 levels responded weakly to anti-PD-1 therapy in the IMvigor210 cohort. Moreover, LRP1 knockdown enhanced the therapeutic effects of anti-PD-1 in mice. Taken together, our findings suggest that LRP1 is a potential target for improving the efficacy of anti-PD-1/PD-L1 therapy by preventing EMT and M2-like macrophage polarisation by blocking the DLL4-NOTCH2 axis.

Donepezil attenuates inflammation and apoptosis in ulcerative colitis via regulating LRP1/AMPK/NF-κB signaling.

This article focuses on the specific effects and mechanisms of donepezil (DNPZ) hydrochloride on inflammation and apoptosis in ulcerative colitis (UC). In vivo and in vitro models of UC were established using dextran sodium sulfate (DSS)-induced mice and NCM460 cells, respectively. Following oral administration of DNPZ, body weight, disease activity index (DAI) scores and colon lengths of mice were recorded. Histopathological damage was detected employing hematoxylin and eosin (H&E) staining. Inflammatory factors were tested using enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction, respectively. Apoptosis was estimated utilizing terminal deoxynucleotidyl transferase dUTP nick-end labeling and western blot. Low-density lipoprotein receptor-related protein 1 (LRP1)/AMP activated protein kinase (AMPK)/nuclear factor-κB (NF- κB) signaling proteins were detected utilizing western blot. NCM460 cell viability was assessed by cell counting kit (CCK)-8. We found that DNPZ partially restored body weight, reduced DAI scores and attenuated intestinal pathological damage in DSS-induced mice. Additionally, inflammatory factors decreased significantly after DNPZ treatment, accompanied by reduced apoptosis level. Phosphorylation (p)-AMPK increased and p-p65 decreased after DNPZ treatment, whereas LRP1 knockdown showed the opposite effect. Moreover, DNPZ treatment greatly restored NCM460 cell viability after DSS stimulation. DNPZ attenuated DSS-induced inflammation and apoptosis in NCM460 cells, which was reversed by LRP1 knockdown. In summary, DNPZ hydrochloride attenuates inflammation and apoptosis in UC via LRP1/AMPK/NF-κB signaling.

Jun-APOE-LRP1 axis promotes tumor metastasis in colorectal cancer.

Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains largely unexplored. This study aimed to investigate the role of apoE in CRC metastasis and to identify the transcription factor and receptor of apoE involved in regulation of CRC metastasis. Bioinformatic analyses were conducted to examine the expression pattern and prognosis of apolipoproteins. APOE-overexpressing cell lines were utilized to explore the effects of apoE on proliferation, migration and invasion of CRC cells. Additionally, the transcription factor and receptor of apoE were screened via bioinformatics, and further validated through knockdown experiments. We discovered that the mRNA levels of APOC1, APOC2, APOD and APOE were higher in lymphatic invasion group, and a higher apoE level indicated poorer overall survival and progression-free interval. In vitro studies demonstrated that APOE-overexpression did not affect proliferation but promoted the migration and invasion of CRC cells. We also reported that APOE-expression was modulated by the transcription factor Jun by activating the proximal promoter region of APOE, and APOE-overexpression reversed the metastasis suppression of JUN knockdown. Furthermore, bioinformatics analysis suggested an interaction between apoE and low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 was highly expressed in both the lymphatic invasion group and the APOEHigh group. Additionally, we found that APOE-overexpression upregulated LRP1 protein levels, and LRP1 knockdown attenuated the metastasis-promoting function of APOE. Overall, our study suggests that the Jun-APOE-LRP1 axis contributes to tumor metastasis in CRC.

Serum Exosomes From Epithelial Ovarian Cancer Patients Contain LRP1, Which Promotes the Migration of Epithelial Ovarian Cancer Cell

Ovarian cancer is a gynecological tumor with extremely high mortality and poor prognosis. Exosomes derived from tumor cells contain abundant proteins that may influence tumor metastasis. The purpose of our study was to explore the proteomic profile of serum exosomes from epithelial ovarian cancer (EOC) patients and to find potential diagnostic markers for EOC. We obtained purified exosomes from serum using ultracentrifugation. Migration assay was used to evaluate the effects of exosomes on the migration of EOC cells. Proteomic profile of serum exosomes was analyzed by liquid chromatogram-tandem mass spectrometry. The levels of low-density lipoprotein receptor-related protein 1 (LRP1) in serum and serum exosomes were determined by enzyme-linked immunosorbent assay. Western blot and Immunohistochemistry were used to determine the level of LRP1 in tissues. Moreover, we performed small-interfering RNA-mediated knockdown of LRP1 in EOC cells to obtain SI-LRP1-Exos and SI-NC-Exos. The detailed mechanisms by which exosomal LRP1 affected the migration of EOC cells in vitro and in vivo were also explored. We found that serum exosomes from EOC patients contributed to the migration of EOC cells. The level of serum exosomal LRP1 of EOC patients was significantly upregulated compared with that of healthy volunteers, which was consistent with the result of enzyme-linked immunosorbent assay. We found that exosomal LRP1 regulated the expression of MMP2 and MMP9 through ERK signaling pathway and affected the migration of EOC cells in vitro and in vivo. Therefore, we propose that exosomal LRP1 contributes to the migration of EOC and may act as an important diagnostic and prognostic biomarker of EOC.

The protective role of hippocampal LRP1 knockdown involves synaptic plasticity through the promoting microtubule dynamics and activation of Akt/GSK-3β pathway in depressive rats

BackgroundThe mechanism by which synaptic plasticity mediates the occurrence of depression is unknown. Low-density lipoprotein receptor-related protein 1 (LRP1) affects axon growth and neurogenesis in the brain, but its role in depressive-like behaviors is poorly understood. MethodsAdeno-associated virus-mediated small interfering RNA was injected into the bilateral hippocampus 14 days before chronic unpredicted mild stress (CUMS). Behavior performance was assessed for depressive-like behaviors. Western blot was conducted to detect levels of LRP1, neurogenesis-related proteins, synaptic markers, microtubule system molecules and Akt/GSK-3β signaling-related proteins. Immunohistochemical staining was performed for LRP1 protein, immunofluorescence staining was conducted to determine the Sox2 protein, Nissl's staining and transmission electron microscope staining were used to observe hippocampal morphological features. ResultsThe expression of hippocampal LRP1 was positively correlated with depressive-like behaviors. Treatment with iAAV-LRP1 exerted protective effects on depressive-like behaviors. LRP1 Knockdown relieved the inhibition of synaptic plasticity induced by CUMS. Expression of Sox2, GluR2 and SYP was significantly increased in iAAV-LRP1 CUMS rats. LRP1 knockdown reduced the p-tau (Ser262 and Thr404) and Acet-tubule levels in depressed rats. Finally, we found that LRP1 knockdown activated the PI3K/Akt pathway and inhibited GSK-3β signal transduction. LimitationsMore neurogenesis markers would be considered, and stereotactic injection into hippocampal DG region could be performed to investigate the effects of LRP1. ConclusionsThese findings indicated that hippocampal LRP1 deficiency in stressed rats plays an important protective role in depressive-like behavior by increasing synaptic plasticity mediated by microtubule dynamic and activating Akt/GSK-3β signaling pathway. Therefore, LRP1 may represent a potential therapeutic target for depression.

Exosomes from Adipose Stem Cells Promote Diabetic Wound Healing through the eHSP90/LRP1/AKT Axis.

Oxidative damage is a critical cause of diabetic wounds. Exosomes from various stem cells could promote wound repair. Here, we investigated the potential mechanism by which exosomes from adipose-derived stem cells (ADSC-EXOs) promote diabetic wound healing through the modulation of oxidative stress. We found that ADSC-EXOs could promote proliferation, migration, and angiogenesis in keratinocytes, fibroblasts, and endothelial cells. Furthermore, ADSC-EXOs reduced the reactive oxygen species (ROS) levels in these cells and protected them against hypoxic and oxidative stress damage. Finally, the local injection of ADSC-EXOs at wound sites significantly increased collagen deposition and neovascularization while reducing ROS levels and cell death; thus, it led to accelerated diabetic wound closure. The mechanism underlying ADSC-EXO functions involved heat-shock protein 90 (HSP90) expressed on the cell surface; these functions could be inhibited by an anti-HSP90 antibody. Exosomal HSP90 could bind to the low-density lipoprotein receptor-related protein 1 (LRP1) receptor on the recipient cell membrane, leading to activation of the downstream AKT signaling pathway. Knockdown of LRP1 and inhibition of the AKT signaling pathway by LY294002 in fibroblasts was sufficient to impair the beneficial effect of ADSC-EXOs. In summary, ADSC-EXOs significantly accelerated diabetic wound closure through an exosomal HSP90/LRP1/AKT signaling pathway.

Heparan Sulfate Proteoglycans (HSPGs) Serve as the Mediator Between Monomeric Tau and Its Subsequent Intracellular ERK1/2 Pathway Activation.

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1—a key enzyme in HSPG synthesis—with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1β. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1β expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12031-021-01943-2.

Endocytosis of Peptidase Inhibitor SerpinE2 promotes Myocardial Fibrosis through activating ERK1/2 and β-catenin Signaling Pathways.

Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and β-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, β-catenin signaling pathways, consequently promoting collagen production.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for apolipoprotein A4 (APOA4) in adipose tissue

Apolipoprotein A4 (APOA4) is one of the most abundant and versatile apolipoproteins facilitating lipid transport and metabolism. APOA4 is synthesized in the small intestine, packaged onto chylomicrons, secreted into intestinal lymph and transported via circulation to several tissues, including adipose. Since its discovery nearly 4 decades ago, to date, only platelet integrin αIIbβ3 has been identified as APOA4 receptor in the plasma. Using co-immunoprecipitation coupled with mass spectrometry, we probed the APOA4 interactome in mouse gonadal fat tissue, where ApoA4 gene is not transcribed but APOA4 protein is abundant. We demonstrate that lipoprotein receptor-related protein 1 (LRP1) is the cognate receptor for APOA4 in adipose tissue. LRP1 colocalized with APOA4 in adipocytes; it interacted with APOA4 under fasting condition and their interaction was enhanced during lipid feeding concomitant with increased APOA4 levels in plasma. In 3T3-L1 mature adipocytes, APOA4 promoted glucose uptake both in absence and presence of insulin in a dose-dependent manner. Knockdown of LRP1 abrogated APOA4-induced glucose uptake as well as activation of phosphatidylinositol 3 kinase (PI3K)-mediated protein kinase B (AKT). Taken together, we identified LRP1 as a novel receptor for APOA4 in promoting glucose uptake. Considering both APOA4 and LRP1 are multifunctional players in lipid and glucose metabolism, our finding opens up a door to better understand the molecular mechanisms along APOA4-LRP1 axis, whose dysregulation leads to obesity, cardiovascular disease, and diabetes.

LRP1 is a master regulator of tau uptake and spread

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer's disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation. 1 Goedert, M., Eisenberg, D. S. & Crowther, R. A. Propagation of Tau Aggregates and Neurodegeneration. Annu Rev Neurosci 40, 189-210, doi:10.1146/annurev-neuro-072116-031153 (2017). 2 Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82, 239-259, doi:10.1007/bf00308809 (1991). 3 de Calignon, A. et al. Propagation of tau pathology in a model of early Alzheimer's disease. Neuron 73, 685-697, doi:10.1016/j.neuron.2011.11.033 (2012). 4 Guo, J. L. et al. Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice. J Exp Med 213, 2635-2654, doi:10.1084/jem.20160833 (2016).

Involvement of the lipoprotein receptor LRP1 in AMP-IBP5-mediated migration and proliferation of human keratinocytes and fibroblasts

BackgroundAntimicrobial peptide derived from insulin-like growth factor binding protein-5 (AMP-IBP5) is a potent antimicrobial agent that possesses various immunomodulatory activities. The parent protein of AMP-IBP5, IGFBP-5, has been shown to exert its effects via an insulin-like growth factor-1 receptor-independent mechanism, including binding to multifunctional low-density lipoprotein receptor-related protein 1 (LRP1), which contributes to several biological processes involved in skin wound healing. ObjectivesTo investigate whether LRP1 is involved in AMP-IBP5-induced migration and proliferation of human epidermal keratinocytes and dermal fibroblasts. MethodsThe mRNA expression of LRP1 and IGFBP-5 was assessed by quantitative real-time PCR, whereas Western blotting was used to evaluate the protein expression. Production of cytokines was determined by ELISA. Cell migration was measured by the scratch wound assay, whereas cell proliferation was analyzed using the BrdU labeling assay. MAPK activation was determined by Western blotting. ResultsWe found that AMP-IBP5 markedly induced the migration and proliferation of keratinocytes and fibroblasts, and this effect was reversed by specific siRNA and neutralizing antibody targeting the LRP1 receptor. In addition, LRP1 was upregulated by lipopolysaccharide, flagellin and AMP-IBP5 in keratinocytes and fibroblasts. LRP1 knockdown also inhibited MAPK pathway activation, which was required for AMP-IBP5-mediated cell migration and proliferation, as evidenced by the specific inhibitors for extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38. ConclusionsOur results suggest that LRP1 expressed in human epidermal keratinocytes and dermal fibroblasts contributes to AMP-IBP5-mediated cell migration and proliferation, supporting its crucial role in cutaneous wound healing process.

LRP1 knockdown aggravates Aβ1–42-stimulated microglial and astrocytic neuroinflammatory responses by modulating TLR4/NF-κB/MAPKs signaling pathways

Neuroinflammation is an important pathological feature and an early event in the pathogenesis of Alzheimer's disease (AD), which is characterized by activation of microglia and astrocytes. Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor that is abundantly expressed in neurons, microglia, and astrocytes, and plays a critical role in AD pathogenesis. There is increasing evidence to show that LRP1 regulates inflammatory responses by modulating the release of pro-inflammatory cytokines and phagocytosis. However, the effects of LRP1 on β-amyloid protein (Aβ)-induced microglial and astrocytic neuroinflammatory responses and its underlying mechanisms have not been studied in detail. In the present study, knockdown of LRP1 significantly enhanced Aβ1–42-stimulated neuroinflammation by increasing the production of pro-inflammatory cytokines in both BV2 microglial cells and mouse primary astrocytes. Furthermore, it is revealed that LRP1 knockdown further led to the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways. The phosphorylation of IκBα, p38, and JNK was significantly up-regulated in LRP1 knockdown BV2 microglial cells and primary astrocytes. Meanwhile, LRP1 knockdown increased expression of the NF-κB p65 subunit in the nucleus while decreased its expression in the cytoplasm. Besides, the upstream signaling adaptor molecules such as toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were also further increased. Moreover, blockade of NF-κB, p38, and JNK inhibited the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) induced by the knockdown of LRP1. Taken together, these findings indicated that LRP1 as an effective therapeutic target against AD and other neuroinflammation related diseases.

4035 Astrocyte LDLR-Related Protein 1 Increases Cytokine Sensitivity – The Role of Glia in Recovery after Brain Damage

OBJECTIVES/GOALS: The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long-term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long-term outcome. METHODS/STUDY POPULATION: Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low-density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. RESULTS/ANTICIPATED RESULTS: Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke. CONFLICT OF INTEREST DESCRIPTION: NA.

LRP1 is a master regulator of tau uptake and spread.

SummaryThe spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation.

Dengue virus reduces expression of low-density lipoprotein receptor-related protein 1 to facilitate replication in Aedes aegypti

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.

LDLR‐Related Protein 1 Increases Cytokine Sensitivity – Implications for Recovery after Brain Damage

The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long‐term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long‐term outcome.Expression of Apolipoprotein E4 predisposes stroke patients to poor long‐term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low‐density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes.LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell‐surface TNF receptor (TNFR1) in non‐astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke.We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral‐mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice.These studies will elucidate how astrocyte‐LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long‐term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke.Support or Funding InformationInstitutional National Research Service Award, T32 NIH/NCATS, TL1TR002647, funded by National Institutes of Health/National Center for Advancing Translational SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

BackgroundApolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation.MethodsUsing U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation.ResultsA specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype.ConclusionsThese findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Pericyte implantation in the brain enhances cerebral blood flow and reduces amyloid-β pathology in amyloid model mice

Pericytes are a major component of cerebrovasculature playing a key role in maintaining cerebrovascular homeostasis. These cells have also been suggested to regulate brain metabolism of amyloid-β (Aβ), disturbances of which are believed to contribute to the pathogenesis of Alzheimer's disease (AD). To examine the effects of pericytes on brain Aβ metabolism, C3H/10T1/2 mouse mesenchymal stem cells were differentiated into pericytes and stereotaxically injected into the brains of amyloid AD model APP/PS1 mice at the age of 18 to 20months. Consistent with a role of pericytes in modulating cerebrovascular function, brain microcirculation in the pericyte-injected hemisphere of the mice was increased 3weeks after implantation compared to the contralateral hemisphere when measured by laser speckle contrast analysis technology. Importantly, enzyme-linked immunosorbent assay revealed that the levels of insoluble Aβ40 and Aβ42 were significantly lower in the hippocampus of the pericyte-injected hemisphere of the APP/PS1 mice than that of the contralateral side. Consistently, immunohistochemical analysis demonstrated that the pericyte implantation reduced Aβ deposition in the hippocampus. When brain slices from the APP/PS1 mice were incubated with C3H/10T1/2 cell-derived pericytes, Aβ42 levels were significantly reduced in a manner that depends on the expression of a major Aβ endocytic receptor, the low-density lipoprotein receptor-related protein 1 (LRP1). While LRP1 mediated the cellular uptake of Aβ in the pericytes, the amounts of major Aβ-degrading enzymes were not affected by LRP1 knockdown. Together, our findings indicate that mesenchymal stem cell–derived pericytes have the capacity to reduce brain Aβ and related pathology, and suggest that cell-based therapy through transplantation of pericytes may be a promising approach to prevent and/or treat AD.

Astrocytic LRP1 Mediates Brain Aβ Clearance and Impacts Amyloid Deposition.

Accumulation and deposition of amyloid-β (Aβ) in the brain represent an early and perhaps necessary step in the pathogenesis of Alzheimer's disease (AD). Aβ accumulation leads to the formation of Aβ aggregates, which may directly and indirectly lead to eventual neurodegeneration. While Aβ production is accelerated in many familial forms of early-onset AD, increasing evidence indicates that impaired clearance of Aβ is more evident in late-onset AD. To uncover the mechanisms underlying impaired Aβ clearance in AD, we examined the role of low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. Although LRP1 has been shown to play critical roles in brain Aβ metabolism in neurons and vascular mural cells, its role in astrocytes, the most abundant cell type in the brain responsible for maintaining neuronal homeostasis, remains unclear. Here, we show that astrocytic LRP1 plays a critical role in brain Aβ clearance. LRP1 knockdown in primary astrocytes resulted in decreased cellular Aβ uptake and degradation. In addition, silencing of LRP1 in astrocytes led to downregulation of several major Aβ-degrading enzymes, including matrix metalloproteases MMP2, MMP9, and insulin-degrading enzyme. More important, conditional knock-out of the Lrp1 gene in astrocytes in the background of APP/PS1 mice impaired brain Aβ clearance, exacerbated Aβ accumulation, and accelerated amyloid plaque deposition without affecting its production. Together, our results demonstrate that astrocytic LRP1 plays an important role in Aβ metabolism and that restoring LRP1 expression and function in the brain could be an effective strategy to facilitate Aβ clearance and counter amyloid pathology in AD.SIGNIFICANCE STATEMENT Astrocytes represent a major cell type regulating brain homeostasis; however, their roles in brain clearance of amyloid-β (Aβ) and underlying mechanism are not clear. In this study, we used both cellular models and conditional knock-out mouse models to address the role of a critical Aβ receptor, the low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. We found that LRP1 in astrocytes plays a critical role in brain Aβ clearance by modulating several Aβ-degrading enzymes and cellular degradation pathways. Our results establish a critical role of astrocytic LRP1 in brain Aβ clearance and shed light on specific Aβ clearance pathways that may help to establish new targets for AD prevention and therapy.