Low-density Lipoprotein Receptor-related Protein Research Articles

Selection of LRP1 ligand phage-displayed single domain antibody that transmigrates BBB

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood–brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Renal Proximal Tubule Cell-Specific Megalin Deletion Does Not Affect Atherosclerosis But Induces Tubulointerstitial Nephritis in Mice Fed a Western Diet.

Pharmacological inhibition of megalin (also known as LRP2 [low-density lipoprotein receptor-related protein-2]) attenuates atherosclerosis in hypercholesterolemic mice. Since megalin is abundant in renal proximal tubule cells (PTCs), the purpose of this study was to determine whether PTC-specific deletion of megalin reduces hypercholesterolemia-induced atherosclerosis in mice. Female Lrp2 f/f mice were bred with male Ndrg1-Cre ERT2 +/0 mice to develop PTC-LRP2 +/+ and PTC-LRP2 -/- littermates. To study atherosclerosis, all mice were bred to an LDL (low-density lipoprotein) receptor -/- background and fed a Western diet to induce atherosclerosis. PTC-specific megalin deletion did not attenuate atherosclerosis in LDL receptor -/- mice in either sex. Serendipitously, we discovered that PTC-specific megalin deletion led to interstitial infiltration of CD68+ cells and tubular atrophy. The pathology was only evident in male PTC-LRP2 -/- mice fed a Western diet but not in mice fed a normal laboratory diet. Renal pathologies were also observed in male PTC-LRP2 -/- mice in an LDL receptor +/+ background fed the same Western diet, demonstrating that the renal pathologies were dependent on diet and not on hypercholesterolemia. In contrast, female PTC-LRP2 -/- mice had no apparent renal pathologies. In vivo multiphoton microscopy demonstrated that PTC-specific megalin deletion dramatically diminished ALB (albumin) accumulation in PTCs within 10 days of Western diet feeding. RNA-sequencing analyses demonstrated the upregulation of inflammation-related pathways in the kidney. PTC-specific megalin deletion does not affect atherosclerosis but leads to tubulointerstitial nephritis in mice fed a Western diet, with severe pathologies in male mice.

The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue- specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine-knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

The role of neutrophils in tPA thrombolysis after stroke: a malicious troublemaker.

Acute ischemic stroke represents a critical, life-threatening condition affecting the central nervous system. Intravenous thrombolysis with tissue plasminogen activator (tPA) remains a cornerstone for achieving vascular recanalization in such patients; however, its therapeutic utility is limited, with only approximately 10% of patients benefiting due to the narrow therapeutic window and significant risk of hemorrhagic transformation. Enhancing the efficacy of tPA thrombolysis is therefore imperative. Neutrophils have been identified as key modulators of thrombolytic outcomes, interacting with tPA post-stroke to influence treatment effectiveness. The binding of tPA to low-density lipoprotein receptor-related protein 1 (LRP-1) on neutrophil surfaces induces degranulation and formation of neutrophil extracellular traps (NETs). Conversely, neutrophils impede the thrombolytic action of tPA by obstructing its interaction with fibrin and activating platelets. These findings suggest that targeting neutrophils may hold promise for improving thrombolysis outcomes. This review explores the role of neutrophils in tPA-mediated thrombolysis following acute ischemic stroke, examines neutrophil-associated biomarkers, and outlines potential strategies for enhancing tPA efficacy.

Systemic Lipid Metabolism Dysregulation as a Possible Driving Force of Fracture Non-Unions?

Non-unions are fractures that do not heal properly, resulting in a false joint formation at the fracture site. This condition leads to major health issues and imposes a burden on national healthcare systems. The etiology of non-unions is still not fully understood; therefore, we aimed to identify potential systemic factors that may contribute to their formation. We conducted a cross-sectional concomitant proteomic and metabolomic pilot study of blood plasma in patients with non-unions (N = 11) and compared them with patients with bone fracture in the normal active healing phase (N = 12). We found five significantly upregulated proteins in the non-union group: immunoglobulin heavy variable 3-74, immunoglobulin lambda variable 2-18, low-density lipoprotein receptor-related protein 4, zinc-alpha-2-glycoprotein, and serum amyloid A-1 protein; and we found one downregulated protein: cystatin-C. The metabolomic study found differences in alanine, aspartate and glutamate metabolism pathways between two groups. The combined results of proteomic and metabolomic analyses suggest that the dysregulation of lipid metabolism may contribute to non-union formation.

ApoE Receptor-2 R952Q Variant in Macrophages Elevates Soluble LRP1 to Potentiate Hyperlipidemia and Accelerate Atherosclerosis in Mice.

apoER2 (apolipoprotein E receptor-2) is a transmembrane receptor in the low-density lipoprotein receptor (LDLR) family with unique tissue expression. A single-nucleotide polymorphism that encodes the R952Q sequence variant has been associated with elevated plasma cholesterol levels and increased myocardial infarction risk in humans. The objective of this study was to delineate the mechanism underlying the association between the apoER2 R952Q variant and increased atherosclerosis risk. An apoER2 R952Q mouse model was generated using a CRISPR/Cas9 strategy, intercrossed with LDLR knockout mice, followed by feeding a Western-type high-fat high-cholesterol diet for 16 weeks. Atherosclerosis was investigated by immunohistology. Plasma lipids and lipid distributions among the various lipoprotein classes were analyzed by colorimetric assay. Tissue-specific effects of the R952Q sequence variant on atherosclerosis were analyzed by bone marrow transplant studies. sLRP1 (soluble low-density lipoprotein receptor-related protein 1) was measured in plasma and conditioned media from bone marrow-derived macrophages by ELISA and GST-RAP (glutathione S-transferase-receptor-associated protein) pull-down, respectively. P38 MAPK (mitogen-activated protein kinase) phosphorylation in VLDL (very-low-density lipoprotein)-treated macrophages was determined by Western blot analysis. Consistent with observations in humans with this sequence variant, the apoER2 R952Q mutation exacerbated diet-induced hypercholesterolemia, via impediment of plasma triglyceride-rich lipoprotein clearance, to accelerate atherosclerosis in Western diet-fed LDLR knockout mice. Reciprocal bone marrow transplant experiments revealed that the apoER2 R952Q mutation in bone marrow-derived cells instead of non-bone marrow-derived cells was responsible for the increase in hypercholesterolemia and atherosclerosis. Additional data showed that the apoER2 R952Q mutation in macrophages promotes VLDL-induced LRP1 (low-density lipoprotein receptor-related protein 1) shedding in a p38 MAPK-dependent manner. The apoER2 R952Q mouse model recapitulates characteristics observed in human disease. The underlying mechanism is that the apoER2 R952Q mutation in macrophages exacerbates VLDL-stimulated sLRP1 production in a p38 MAPK-dependent manner, resulting in its competition with cell surface LRP1 to impede triglyceride-rich lipoprotein clearance, thereby resulting in increased hypercholesterolemia and accelerated atherosclerosis.

Lrp1 facilitates infection of neurons by Jamestown Canyon virus.

Jamestown Canyon virus (JCV) is a bunyavirus and arbovirus responsible for neuroinvasive disease in the United States. Little is known about JCV pathogenesis, and no host factors required for cellular infection have been identified. Recently, we identified low-density lipoprotein receptor related protein 1 (Lrp1) as a host entry factor for two other bunyaviruses Rift Valley fever virus (RVFV) and Oropouche virus (OROV). Here, we assessed the role of Lrp1 in mediating JCV cellular infection of neurons. Both neuronal and non-neuronal immortalized cell lines deficient for Lrp1 displayed reduction in infection with JCV, and early stages of infection such as binding and internalization were impacted by lack of Lrp1. In primary rat neurons, Lrp1 was highly expressed, and the neurons were highly permissive for JCV infection. Treatment of primary neurons with recombinant receptor-associated protein (RAP), a high affinity ligand for Lrp1, resulted in reduced infectivity with JCV. In addition, pretreatment of cells with RVFV Gn inhibited JCV infection, suggesting that the two viruses may share overlapping binding sites. These results provide compelling evidence that Lrp1 is an important cellular factor for efficient infection by JCV, and thus multiple bunyaviruses with varying clinical manifestations and tissue tropism are facilitated by the host cell Lrp1. Reliance of multiple bunyaviruses on Lrp1 makes it a promising target for pan-bunyaviral antivirals and therapeutics.

A phase I dose escalation study of the LRP5 antagonist BI 905681 in patients with advanced and metastatic solid tumors

The Wnt pathway is involved in proliferation and tissue homeostasis. Aberrant activation promotes cancer cell proliferation and survival. Inhibition of the low-density lipoprotein receptor-related protein 5/6 (LRP5/6) coreceptors that regulate Wnt signaling could prevent cancer cell proliferation. BI 905681 is a novel LRP5 antagonist that has demonstrated potent invivo antitumor activity. This was a phase I, dose escalation study (NCT04147247) evaluating BI 905681 in patients with advanced solid tumors over two dosing schedules (schedule A: every 3 weeks, 3-week cycles and schedule B: every 2 weeks, 4-week cycles). The primary endpoint was the maximum tolerated dose (MTD) of BI 905681 and the number of patients experiencing adverse events (AEs). Other endpoints were pharmacokinetics, pharmacodynamics, and efficacy. As a result of difficulties enrolling patients, the trial was terminated early and the MTD for schedule A could not be determined. Twenty-one patients received BI 905681 over five dose cohorts (schedule A: 1.0, 2.5, 5.0, 7.0, and 8.5 mg/kg). No patients received schedule B. No dose-limiting toxicities (DLTs) were reported during the MTD evaluation period. However, during the entire treatment period, two patients (9.5%) experienced a DLT of grade 1 C-telopeptide increase in the 5.0 and 8.5 mg/kg dose cohorts. The most frequent treatment-related AEs were diarrhea (23.8%), vomiting (23.8%), nausea (19.0%), and infusion-related reactions (IRRs; 14.3%). Despite premedication to mitigate IRRs, one patient experienced a grade 2 IRR. The pharmacokinetic profiles of BI 905681 were biphasic, with a rapid distribution phase in the beginning followed by a slower elimination phase. The objective response rate was 0%; 5 (23.8%) and 14 patients (66.7%) had a best overall response of stable disease and progressive disease, respectively. BI 905681 has minimal efficacy in an unselected patient population and was generally well tolerated.

Serum megalin levels in type-2 diabetes mellitus with and without cardiovascular disease

ABSTRACT Background: Low-density lipoprotein receptor-related protein-2 (LRP2), also called megalin, is a multi-ligand receptor of the LDL receptor gene family mediating reabsorption of ligands like Apo-A1. Type 2 diabetes mellitus (T2DM) may possibly disrupt megalin functions as it is found to be regulated by insulin. This might cause cardiovascular complications due to derangement in lipoprotein metabolism. The current study was carried out to assess the serum megalin levels among T2DM individuals with cardiovascular complications in the Indian population. Methods: This was a cross-sectional study involving 80 patients with T2DM. 40 T2DM patients with known cardiovascular disease were selected as cases and 40 of those without evidence of cardiovascular disease were selected as controls. Demographic details, DM duration and class of oral hypoglycemic agents (OHA) used were collected from medical records while details of lipid profile, fasting glucose, serum creatinine and urea were collected from laboratory information system. Serum megalin levels were estimated using left-over samples by ELISA. Results: The study groups showed no statistical significance in baseline laboratory parameters except for serum creatinine and low density lipoprotein cholesterol (LDL-c). Mean serum megalin levels were statistically insignificant between cases and controls (0.91 ± 0.78 ng/mL vs. 0.85 ± 0.69 ng/mL, P 0.74).The subgroup analysis of serum megalin levels based on OHA consumption among cases was also statistically insignificant (P 0.056). Pearson’s correlational analysis was statistically insignificant between serum megalin and lipid profile parameters among cases (P > 0.05). Conclusion: Serum megalin alone may not serve as a biomarker for cardiovascular disease.

Myelin debris phagocytosis in demyelinating disease.

Demyelinating diseases are often caused by a variety of triggers, including immune responses, viral infections, malnutrition, hypoxia, or genetic factors, all of which result in the loss of myelin in the nervous system. The accumulation of myelin debris at the lesion site leads to neuroinflammation and inhibits remyelination; therefore, it is crucial to promptly remove the myelin debris. Initially, Fc and complement receptors on cellular surfaces were the primary clearance receptors responsible for removing myelin debris. However, subsequent studies have unveiled the involvement of additional receptors, including Mac-2, TAM receptors, and the low-density lipoprotein receptor-related protein 1, in facilitating the removal process. In addition to microglia and macrophages, which serve as the primary effector cells in the disease phase, a variety of other cell types such as astrocytes, Schwann cells, and vascular endothelial cells have been demonstrated to engage in the phagocytosis of myelin debris. Furthermore, we have concluded that oligodendrocyte precursor cells, as myelination precursor cells, also exhibit this phagocytic capability. Moreover, our research group has innovatively identified the low-density lipoprotein receptor as a potential phagocytic receptor for myelin debris. In this article, we discuss the functional processes of various phagocytes in demyelinating diseases. We also highlight the alterations in signaling pathways triggered by phagocytosis, and provide a comprehensive overview of the various phagocytic receptors involved. Such insights are invaluable for pinpointing potential therapeutic strategies for the treatment of demyelinating diseases by targeting phagocytosis.

The Low-Density Lipoprotein Receptor-Related Protein-1 Is Essential for Dengue Virus Infection.

Dengue virus (DENV) causes the most prevalent and rapidly spreading arboviral disease of humans. It enters human cells by receptor-mediated endocytosis. Numerous cell-surface proteins were proposed as DENV entry factors. Among these, the phosphatidylserine receptor TIM-1 is the only one known to mediate virus internalization. However, several cellular models lacking TIM-1 are permissive to DENV infection, suggesting that other receptors exist. Here, we show that the low-density lipoprotein receptor-related protein-1 (LRP1) binds DENV virions by interacting with the DIII of the viral envelope glycoprotein. DENV infection is effectively inhibited by the purified receptor at 5 × 10-8 mol/L, and the interaction of the envelope protein with LRP1 is also blocked by a natural ligand of LRP1. The depletion of LRP1 causes 100-fold lower production of infectious virus than controls. Our results indicate that LRP1 is another DENV receptor, thus becoming an attractive target to evaluate for the development of effective antiviral drugs against DENV.

Protective effects of electroacupuncture on senile osteoporosis in rats.

The objectives were to explore the protective effects of electroacupuncture (EA) on senile osteoporosis in aged rats and investigate the underlying mechanisms. This study included aged (24-month-old; n = 16) and young (3-month-old; n = 8) male Sprague-Dawley rats. Aged rats were further randomized 1:1 to an aged control group (Aged; n = 8) and an EA treatment group (EA; n = 8). The 3-month-old rats served as young controls (Young). EA rats received EA at ST36, SP6, GB34 and SP10 bilaterally for 30 min a day, 5 days a week, for 8 weeks. EA significantly increased serum markers of bone formation in Aged rats. There were no significant differences in serum markers of bone resorption between EA and Aged rats. Deterioration of bone mineral density (BMD) and trabecular bone architecture was observed in the Aged group, while EA significantly increased BMD of the left femur and L5 vertebral body in aged rats. Aging-induced deterioration of trabecular bone architecture was partially reversed in EA rats. Runx2 and Osterix mRNA and protein levels were significantly increased and peroxisome proliferator-activated receptor (PPAR)γ was significantly decreased in bone marrow cells in EA compared with Aged groups. The mRNA and protein levels of core constituents of the Wnt/β-catenin signaling pathway (Wnt3a, low-density lipoprotein receptor-related protein (LRP)5 and β-catenin) were significantly increased and Dickkopf 1 was significantly decreased in bone marrow cells in EA compared with Aged groups. EA may prevent bone loss and deterioration in aged rats by promoting osteogenesis via a mechanism that may involve activation of the Wnt/β-catenin signaling pathway. EA may represent a therapeutic option for senile osteoporosis.

Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

The interaction of sclerostin (Scl) with the low-density lipoprotein receptor-related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/β-catenin pathway. To characterize the Scl-LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single-mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4-a reduction that was not a result of changes in the variants' secondary structure or stability. We found that Scl positions K75 (loop 1) and V136 (loop 3) are critical hotspots for binding to LRP4. Our findings establish the foundation for targeting these hotspots for developing novel therapeutic strategies to promote bone formation.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

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.

Low to moderate ethanol exposure reduces astrocyte-induced neuroinflammatory signaling and cognitive decline in presymptomatic APP/PS1 mice

Alcohol use disorder has been associated with the development of neurodegenerative diseases, including Alzheimer’s disease (AD). However, recent studies demonstrate that moderate alcohol consumption may be protective against dementia and cognitive decline. We examined astrocyte function, low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), and the NF-κB p65 and IKK-α/β signaling pathways in modulating neuroinflammation and amyloid beta (Aβ) deposition. We assessed apolipoprotein E (ApoE) in the brain of APP/PS1 mice using IHC and ELISA in response to low to moderate ethanol exposure (MEE). First, to confirm the intracerebral distribution of ApoE, we co-stained with GFAP, a marker for astrocytes that biosynthesize ApoE. We sought to investigate whether the ethanol-induced upregulation of LRP1 could potentially inhibit the activity of IL-1β and TNF-α induced IKK-α/β towards NF-κB p65, resulting in a reduction of pro-inflammatory cytokines. To evaluate the actual Aβ load in the brains of APP/PS1 mice, we performed with a specific antibody Aβ (Thioflavin S) on both air- and ethanol-exposed groups, subsequently analyzing Aβ levels. We also measured glucose uptake using 18F- fluorodeoxyglucose (FDG)-positron emission tomography (PET). Finally, we investigated whether MEE induced cognitive and memory changes using the Y maze, noble object recognition test, and Morris water maze. Our findings demonstrate that MEE reduced astrocytic glial fibrillary acidic protein (GFAP) and ApoE levels in the cortex and hippocampus in presymptomatic APP/PS1 mice. Interestingly, increased LRP1 protein expression was accompanied by dampening the IKK-α/β-NF-κB p65 pathway, resulting in decreased IL-1β and TNF-α levels in male mice. Notably, female mice show reduced levels of anti-inflammatory cytokines IL-4, and IL-10 without altering IL-1β and TNF-α concentrations. In both males and females, Aβ plaques, a hallmark of AD, were reduced in the cortex and hippocampus of APP/PS1 mice exposed to ethanol starting at pre-symptomatic stage. Consistently, MEE increased FDG-PET-based brain activities and normalized cognitive and memory deficits in the APP/PS1 mice. Our findings suggest that MEE may benefit AD pathology via modulating LRP1 expression, potentially reducing neuroinflammation and attenuating Aβ deposition. Our study implies that reduced astrocyte-derived ApoE and LDL cholesterol levels are critical for attenuating AD pathology.

Regulatory role of Heparan sulfate in leptin signaling

Leptin, a hormone mainly secreted by adipocytes, has attracted significant attention since its discovery in 1994. Initially known for its role in appetite suppression and energy regulation, leptin is now recognized for its influence on various physiological processes, including immune response, bone formation, and reproduction. It exerts its effects by binding to receptors and initiating an intracellular signaling cascade. Heparan sulfate (HS) is known to regulate the intracellular signaling of various ligands. HS is present as the glycan portion of HSPGs on cell surfaces and in intercellular spaces, with diverse structures due to extensive sulfation and epimerization. Although HS chains on HSPGs are involved in many physiological processes, the detailed effects of HS chains on leptin signaling are not well understood.This study examined the role of HS chains on HSPGs in leptin signaling using Neuro2A cells expressing the full-length leptin receptor (LepR). We showed that cell surface HS was essential for efficient leptin signaling. Enzymatic degradation of HS significantly reduced leptin-induced phosphorylation of downstream molecules, such as signal transducer and activator of transcription 3 and p44/p42 Mitogen-activated protein kinase. In addition, HS regulated LepR expression and internalization, as treatment with HS-degrading enzymes decreased cell surface LepR. HS was also found to exhibit a weak interaction with LepR. Enzymatic removal of HS enhanced the interaction between LepR and low-density lipoprotein receptor-related protein 1, suggesting that HS negatively regulates this interaction. In conclusion, HS plays a significant role in modulating LepR availability on the cell surface, thereby influencing leptin signaling. These findings provide new insights into the complex regulation of leptin signaling and highlight potential therapeutic targets for metabolic disorders and obesity.

LRP1 Repression by SNAIL Results in ECM Remodeling in Genetic Risk for Vascular Diseases.

Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry. Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix and connective tissue development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3 (Mothers against decapentaplegic homolog 2/3). Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61 (cystein rich angiogenic protein 61), a known LRP1 ligand involved in vascular integrity and TIMP3 (Metalloproteinase inhibitor 3), implicated in extracellular matrix maintenance and also known to interact with LRP1. Our findings support allele-specific LRP1 expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

PPARs (Peroxisome Proliferator-activated Receptors) and Their Agonists in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease leading to dementia because of complex phathomechanisms like amyloid β (Aβ) aggregation, tau aggregates, and neurofibrillary tangles. Peroxisome proliferator-activated receptor (PPAR) agonists have been reported recently with neuroprotective and anti-inflammatory properties. PPARs belong to the superfamily of nuclear hormone receptors and function as ligand-activated transcription factors. These have emerged as crucial players in the pathogenesis of AD. This review presented the potential of PPARs and their agonists in treating neurodegenerative diseases like AD. <P> </P> PPARs regulate the expression of specific genes vital for synaptic function and neurotransmitter release. PPAR agonists play a critical role in increasing the clearance of Aβ peptides by lowdensity lipoprotein receptor-related protein 1 (LRP1) in the microvascular endothelial cells of the human brain. Studies have shown that PPAR agonists reduce the level of APoE-mRNA, contributing to the accumulation of Aβ plaques and up-regulation of PPAR. A knockout of miR-128 has been found to inhibit AD-like cognitive decline, amyloid precursor protein (APP) amyloidogenic processing, and inflammatory responses in AD. <P> </P> PPARs are involved in the pathomechanism of AD, and therefore, PPAR agonists could be viable options for controlling the neurodegenerative symptoms and may be useful in treating AD.