apoE Isoforms Research Articles

Lipoprotein(a) concentrations and cardiovascular disease in patients with chronic kidney disease: Results from the German Chronic Kidney Disease study.

Lipoprotein(a) (Lp(a)) is a causal, genetically determined risk factor for cardiovascular disease (CVD) in the general population. Patients with chronic kidney disease (CKD) have an increased CVD risk and elevated Lp(a) concentrations. Only a few studies on Lp(a) were performed in persons with mild-to-moderate CKD; none of them used genetic variants to explore potential causal associations. This study aims to investigate the association of measured and genetically predicted Lp(a) concentrations on prevalent and incident CVD events in the German Chronic Kidney Disease (GCKD) study. The study included 5043 participants of European ancestry with an estimated glomerular filtration rate (eGFR) between 30 and 60mL/min/1.73m2 or an eGFR >60mL/min/1.73m2 in the presence of overt albuminuria with a follow-up of 6.5 years. With each 10mg/dL higher Lp(a) concentration, odds for prevalent CVD (1290 events) increased 1.065-fold (95%CI: 1.042-1.088, p<0.001). The risk was significantly higher in patients with Lp(a) ≥50mg/dL but most pronounced in Lp(a) ≥70mg/dL (odds ratio=1.775 [1.409-2.231], p<0.001) compared to Lp(a) <30mg/dL. Each 10mg/dL higher Lp(a) concentration and Lp(a) ≥70mg/dL increased the risk for incident 3-point major adverse cardiovascular events (MACEs) (474 events): hazard ratio [HR]=1.037 [1.009-1.067], p=0.009 and HR=1.335 [1.001-1.781], p=0.050), respectively. Similar results were obtained for 4-point MACE (653 events). Analyses based on apo(a) isoforms and genetically predicted Lp(a) concentrations led to even stronger associations. In patients with mild-to-severe CKD, elevated Lp(a) concentrations and genetic determinants of Lp(a) concentrations are significantly associated with CVD at baseline and during follow-up, independent of traditional risk factors.

APOE from astrocytes restores Alzheimer's Aβ-pathology and DAM-like responses in APOE deficient microglia.

The major genetic risk factor for Alzheimer's disease (AD), APOE4, accelerates beta-amyloid (Aβ) plaque formation, but whether this is caused by APOE expressed in microglia or astrocytes is debated. We express here the human APOE isoforms in astrocytes in an Apoe-deficient AD mouse model. This is not only sufficient to restore the amyloid plaque pathology but also induces the characteristic transcriptional pathological responses in Apoe-deficient microglia surrounding the plaques. We find that both APOE4 and the protective APOE2 from astrocytes increase fibrillar plaque deposition, but differentially affect soluble Aβ aggregates. Microglia and astrocytes show specific alterations in function of APOE genotype expressed in astrocytes. Our experiments indicate a central role of the astrocytes in APOE mediated amyloid plaque pathology and in the induction of associated microglia responses.

Therapeutic Challenges Derived from the Interaction Among Apolipoprotein E, Cholesterol, and Amyloid in Alzheimer’s Disease

The isoform E4 of the Apolipoprotein E (ApoE) represents one of the strongest genetic risk factors for late-onset Alzheimer’s disease (AD). ApoE has key roles in cholesterol transport and amyloid-β (Aβ) metabolism, which are both central to AD pathogenesis. The E4 isoform has been implicated in reduced cholesterol homeostasis, increased Aβ aggregation, and heightened tau phosphorylation, contributing to amyloid plaques and neurodegeneration. This manuscript examines the complex interactions among ApoE isoforms, cholesterol metabolism, and amyloid pathology. Moreover, the therapeutic challenges associated with lipid-lowering agents (e.g., statins, PCSK9 inhibitors), anti-amyloid immunotherapies, and anticoagulants are described, focusing on ApoE4 carriers. Decision-making challenges are discussed by analyzing the pros and cons of these therapies.

Analyzing the Role of Specific Damage-Associated Molecular Patterns-Related Genes in Osteoarthritis and Investigating the Association between β-Amyloid and Apolipoprotein E Isoforms.

Osteoarthritis (OA) is a prevalent chronic joint disorder. It is characterized by an immune response that maintains a low level of inflammation throughout its progression. During OA, cartilage degradation leads to the release of damage-associated molecular patterns (DAMPs), which intensify the inflammatory response. β-Amyloid is a well-recognized DAMP in OA, can interact with APOE isoforms. This study identified DAMPs-related genes in OA using bioinformatics techniques. Additionally, we examined the expression levels of β-amyloid and apolipoprotein E (ApoE) isoforms by enzyme-linked immunosorbent assay. We identified 10 key genes by machine learning techniques. Immune infiltration analysis revealed upregulation of various immune cell types in OA cartilage, underscoring the critical role of inflammation in OA pathogenesis. In the validation study, elevated serum levels of β-amyloid in knee osteoarthritis (KOA) patients were confirmed, showing positive correlations with ApoE2 and ApoE4. Notably, ApoE3 was identified as an independent protective factor against KOA. In this bioinformatics analysis, we identified the DAMPs-related genes of KOA and explored their potential functions and regulatory networks. The high expression of β-amyloid in KOA was confirmed by experiments, and the correlation between β-amyloid and ApoE2, ApoE4 in KOA was revealed for the first time, this provides a new way to explore the pathogenesis of KOA and to study the therapeutic targets of KOA.

Lipoprotein glomerulopathy: what is known? Literature review

The review is devoted to the important problem of contemporary nephrology — lipoprotein glomerulopathy (LPG), a rare glomerular disorder leading to nephrotic syndrome and/or chronic kidney failure (CKF). The article highlights the primary genetic defect in LPG associated with heterozygous mutations of the apolipoprotein E (ApoE) gene in the low‑density lipoprotein (LDL) binding site with the corresponding receptor, which increases the affinity of mutant ApoE‑containing lipoproteins to the glomerular capillary wall. The article presents data from population‑based studies on the prevalence of LPG and current understanding of its pathogenesis, in particular, the main pathogenetic link — damage to the structure and function of ApoE due to heterozygous mutations of the ApoE gene. The participation of ApoE‑containing lipoproteins, which include ApoE isoforms, in the formation of glomerular deposits, lipoprotein thrombi in the lumen of glomerular capillaries, as well as the role of macrophages in the pathogenesis of renal lipidosis is considered. Histological picture typical for LPG, intraglomerular and extrarenal factors of its development, and features of serum lipid spectrum accompanying the clinical course of LPG are described. Diagnostic criteria of LPG, clinical manifestations of this glomerular disorder, prognosis in LPG, its complications and some co-morbid states associated with LPG are given. Special attention is paid for the treatment of LPG, without which the disease is progressing to a terminal CKF. Some possibilities of contemporary lipid lowering agents (fibrates, nicotinic acid derivates, statins, monoclonal antibodies to apolipoprotein CIII) in correction of lipid disorders associated with LPG are discussed. Some perspective agents that could be applied in patients with LPG (inhibitors of natrium-dependent glucose co-transporter 2, antioxidants, anticonvulsant topiramate) after special trials are enumerated. Some attention is paid to other treatment measures in LPG, such as LDL-apheresis with heparin‑induced extracorporal precipitation, staphylococcal protein-A immunoadsorption therapy, and renal transplantation. Necessity is emphasized of further experimental and clinical studies that will allow to enlarge updated ideas about the etiology and pathogenesis of LPG, improve its diagnostics and treatment efficacy.

Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

As a component of circulating lipoproteins, APOE binds to cell surface receptors mediating lipoprotein metabolism and cholesterol transport. A growing body of evidence, including the identification of a broad variety of cellular proteins interacting with APOE, suggests additional independent functions. Investigating cellular localization and protein-protein interactions in cultured human hepatocytes, we aimed to contribute to the elucidation of hitherto unnoted cellular functions of APOE. We observed a strong accumulation of APOE in MAMs, equally evident for the two major isoforms APOE3 and APOE4. Using mass spectrometry proteome analyses, novel and previously noted APOE interactors were identified, including the mitochondrial proteins TOMM40, LONP1 and VDAC1. All three interactors were present in MAM fractions, which we think initially facilitates interactions with APOE. LONP1 is a protease with chaperone activity, which migrated to MAMs in response to ER stress, displaying a reinforced interaction with APOE. We therefore hypothesize that APOE may help in the unfolded protein response (UPR) by acting as a co-chaperone in cooperation with LONP1 at the interface of mitochondria and ER membranes. The interaction of APOE with the integral proteins TOMM40 and VDAC1 may point to the formation of bridging complexes connecting mitochondria with other organelles.

Analysis of early effects of human APOE isoforms on Alzheimer’s disease and type III hyperlipoproteinemia pathways using knock-in rat models with humanized APP and APOE

APOE is a major genetic factor in late-onset Alzheimer’s disease (LOAD), with APOE4 increasing risk, APOE3 acting as neutral, and APOE2 offering protection. APOE also plays key role in lipid metabolism, affecting both peripheral and central systems, particularly in lipoprotein metabolism in triglyceride and cholesterol regulation. APOE2 is linked to Hyperlipoproteinemia type III (HLP), characterized by mixed hypercholesterolemia and hypertriglyceridemia due to impaired binding to Low-Density Lipoproteins receptors. To explore the impact of human APOE isoforms on LOAD and lipid metabolism, we developed Long-Evans rats with human APOE2, APOE3, or APOE4 in place of rat Apoe. These rats were crossed with those carrying a humanized App allele to express human Aβ, which is more aggregation-prone than rodent Aβ, enabling the study of human APOE-human Aβ interactions. In this study, we focused on 80-day-old adolescent rats to analyze early changes that may be associated with the later development of LOAD. We found that APOE2hAβ rats had the highest levels of APOE in serum and brain, with no significant transcriptional differences among isoforms, suggesting variations in protein translation or stability. Aβ43 levels were significantly higher in male APOE4hAβ rats compared to APOE2hAβ rats. However, no differences in Tau or phosphorylated Tau levels were observed across the APOE isoforms. Neuroinflammation analysis revealed lower levels of IL13, IL4 and IL5 in APOE2hAβ males compared to APOE4hAβ males. Neuronal transmission and plasticity tests using field Input-Output (I/O) and long-term potentiation (LTP) recordings showed increased excitability in all APOE-carrying rats, with LTP deficits in APOE2hAβand APOE4hAβ rats compared to ApoehAβ and APOE3hAβ rats. Additionally, a lipidomic analysis of 222 lipid molecular species in serum samples showed that APOE2hAβ rats displayed elevated triglycerides and cholesterol, making them a valuable model for studying HLP. These rats also exhibited elevated levels of phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine. Minimal differences in lipid profiles between APOE3hAβ and APOE4hAβ rats reflect findings from mouse models. Future studies will include comprehensive lipidomic analyses in various CNS regions and at older ages to further validate these models and explore the effects of APOE isoforms on lipid metabolism in relation to AD pathology.

The Effect of FTY720 on Sphingolipid Imbalance and Cognitive Decline in Aged EFAD Mice.

During Alzheimer's disease (AD) progression, there is a decline in the bioactive sphingolipid sphingosine-1-phosphate (S1P). Previous research showed that FTY720, an S1P mimetic, prevented cognitive decline and reduced ceramide levels in transgenic mice with familial AD carrying the human APOE4 gene (E4FAD) at 6-7 months of age. The objective of this study is to explore the protective effects of FTY720 at late-stage AD. Male mice aged 9.5 to 10.5 months were orally administered FTY720 (0.1 mg/kg) via oral gavage for 6 weeks. A pre-test of water maze was used for evaluating the pathological status. After 4 weeks of administration, memory, locomotion, and anxiety were assessed. Cortex samples were analyzed for amyloid-β (Aβ) and sphingolipid levels. Compared with APOE3 mice, APOE4, E3FAD and E4FAD mice exhibited significant memory deficits. After 6 weeks administration, FTY720 did not alleviate memory deficits in EFAD mice. Lipid analysis revealed that S1P was significantly reduced in EFAD mice (E3FAD or E4FAD) compared to controls (APOE3 and APOE4). Ceramide level alterations were predominantly dependent on APOE isoforms rather than AD transgenes. Interestingly, Cer (d18 : 1/22 : 1) was elevated in APOE4 mice compared to APOE3, and FTY720 reduced it. E4FAD and APOE4 mice exhibited significant spatial memory deficits and higher ceramide concentrations compared to APOE3 mice. FTY720 did not reverse memory deficits in E4FAD and APOE4 mice but reduced specific ceramide species. This study provides insights into the association between sphingolipids and APOE4 in advanced AD stages, exploring potential therapeutic targeting of sphingolipid metabolism.

The influences of ApoE isoforms on endothelial adherens junctions and actin cytoskeleton responding to mCRP.

Apolipoprotein E4 (ApoE4) plays an important role responding to monomeric C-reactive protein (mCRP) via binding to CD31 leading to cerebrovascular damage and Alzheimer's disease (AD). Using phosphor-proteomic profiling, we found altered cytoskeleton proteins in the microvasculature of AD brains, including increased levels of hyperphosphorylated tau (pTau) and the actin-related protein, LIMA1. To address the hypothesis that cytoskeletal changes serve as early pathological signatures linked with CD31 in brain endothelia in ApoE4 carriers, ApoE4 knock-in mice intraperitoneal injected with mCRP revealed that mCRP increased the expressions of phosphorylated CD31 (pCD31) and LIMA1, and facilitate the binding of pCD31 to LIMA1. mCRP combined with recombinant APOE4 protein decreased interaction of CD31 and VE-Cadherin at adherens junctions (AJs), along with altered the expression of various actin cytoskeleton proteins, causing microvasculature damage. Notably, the APOE2 protein attenuated these changes. Overall, our study demonstrates that ApoE4 responds to mCRP to disrupt the endothelial AJs which link with the actin cytoskeleton and this pathway could play a key role in the barrier dysfunction leading to AD risk.

Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain.

Apolipoprotein E (ApoE) polymorphisms modify the risk of neurodegenerative disease with the ApoE4 isoform increasing and ApoE2 isoform decreasing risk relative to the 'wild-type control' ApoE3 isoform. To elucidate how ApoE isoforms alter the proteome, we measured relative protein abundance and turnover in transgenic mice expressing a human ApoE gene (isoform 2, 3, or 4). This data provides insight into how ApoE isoforms affect the in vivo synthesis and degradation of a wide variety of proteins. We identified 4849 proteins and tested for ApoE isoform-dependent changes in the homeostatic regulation of ~2700 ontologies. In the brain, we found that ApoE4 and ApoE2 both lead to modified regulation of mitochondrial membrane proteins relative to the wild-type control ApoE3. In ApoE4 mice, this regulation is not cohesive suggesting that aerobic respiration is impacted by proteasomal and autophagic dysregulation. ApoE2 mice exhibited a matching change in mitochondrial matrix proteins and the membrane which suggests coordinated maintenance of the entire organelle. In the liver, we did not observe these changes suggesting that the ApoE-effect on proteostasis is amplified in the brain relative to other tissues. Our findings underscore the utility of combining protein abundance and turnover rates to decipher proteome regulatory mechanisms and their potential role in biology.

Human apolipoprotein E glycosylation and sialylation: from structure to function.

Human apolipoprotein E (ApoE) was first identified as a polymorphic gene in the 1970s; however, the genetic association of ApoE genotypes with late-onset sporadic Alzheimer's disease (sAD) was only discovered 20 years later. Since then, intensive research has been undertaken to understand the molecular effects of ApoE in the development of sAD. Despite three decades' worth of effort and over 10,000 papers published, the greatest mystery in the ApoE field remains: human ApoE isoforms differ by only one or two amino acid residues; what is responsible for their significantly distinct roles in the etiology of sAD, with ApoE4 conferring the greatest genetic risk for sAD whereas ApoE2 providing exceptional neuroprotection against sAD. Emerging research starts to point to a novel and compelling hypothesis that the sialoglycans posttranslationally appended to human ApoE may serve as a critical structural modifier that alters the biology of ApoE, leading to the opposing impacts of ApoE isoforms on sAD and likely in the peripheral systems as well. ApoE has been shown to be posttranslationally glycosylated in a species-, tissue-, and cell-specific manner. Human ApoE, particularly in brain tissue and cerebrospinal fluid (CSF), is highly glycosylated, and the glycan chains are exclusively attached via an O-linkage to serine or threonine residues. Moreover, studies have indicated that human ApoE glycans undergo sialic acid modification or sialylation, a structural alteration found to be more prominent in ApoE derived from the brain and CSF than plasma. However, whether the sialylation modification of human ApoE has a biological role is largely unexplored. Our group recently first reported that the three major isoforms of human ApoE in the brain undergo varying degrees of sialylation, with ApoE2 exhibiting the most abundant sialic acid modification, whereas ApoE4 is the least sialylated. Our findings further indicate that the sialic acid moiety on human ApoE glycans may serve as a critical modulator of the interaction of ApoE with amyloid β (Aβ) and downstream Aβ pathogenesis, a prominent pathologic feature in AD. In this review, we seek to provide a comprehensive summary of this exciting and rapidly evolving area of ApoE research, including the current state of knowledge and opportunities for future exploration.

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer’s disease (AD) pathology. We used long-read sequencing in combination with a novel bioinformatics tool (FICLE) to profile transcript diversity in the entorhinal cortex of female transgenic (TG) mice harboring a mutant form of human tau. Our analyses revealed hundreds of novel isoforms and identified differentially expressed transcripts – including specific isoforms of Apoe, App, Cd33, Clu, Fyn and Trem2 – associated with the development of tau pathology in TG mice. Subsequent profiling of the human cortex from AD individuals and controls revealed similar patterns of transcript diversity, including the upregulation of the dominant TREM2 isoform in AD paralleling the increased expression of the homologous transcript in TG mice. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of AD neuropathology.

The paired immunoglobulin-like type 2 receptor alpha (PILRA) gene polymorphism rs1859788 reduces risk of Alzheimer's Disease in men homozygous for the ApoE ε4 allele.

The APOE gene has long been associated with Alzheimer Disease (AD) risk. Emerging research indicates that other genetic loci, including the paired immunoglobulin-like type 2 receptor alpha (PILRA) gene, may play a crucial role. In the current study we used UK Biobank data to assess the relationship between PILRA and AD. We examined the PILRA polymorphism rs1859788, a single nucleotide missense variant, G > A, minor allele frequency 0.3. Single nucleotide polymorphism (SNP) data for rs429358 and rs7412 determined APOE isoform. We used PheWeb to perform a phenome wide association study (phewas) of rs1859788 and identify other conditions that might be related to both AD and rs1859788. In male subjects homozygous for ApoE isoform ε4/ε4, of the men without AD, 9.7% had AA genotype; of the men with AD, 1.8% had AA genotype. This difference was significant (p = 0.006, two tail Fisher exact test). In female subjects homozygous for ApoE isoform ε4/ε4, of the women without AD, 10.4% had AA genotype; of the women with AD 7.9% had AA genotype. This difference was not significant (p = 0.481). In subjects not homozygous for ApoE isoform ε4/ε4, the effect of PILRA genotype was not significant. A phewas of rs1859788 found an association with megaloblastic anemia. We have confirmed the previously noted PILRA snp rs1859788 risk reduction of AD, as well as a PILRA link to the ApoE ε4 isoform that has been previously described. We are uncertain why the significant association is only with men who are homozygous for the ε4/ε4 isoform. A phewas indicated that PILRA SNP rs1859788 is associated with megaloblastic anemia, which may explain an observed association between AD and anemia. The identification of PILRA as a potential risk gene for Alzheimer's disease underscores the complexity of the genetic landscape contributing to AD. Alongside APOE, PILRA may play a significant role in modulating key pathological processes such as neuroinflammation and amyloid-beta accumulation.

Advancements in APOE and dementia research: Highlights from the 2023 AAIC Advancements: APOE conference.

The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimer's disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research. In 2023, the Alzheimer's Association convened multidisciplinary researchers at the "AAIC Advancements: APOE" conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function. This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field. Understanding apoE's multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine. APOE is a central player in the pathogenesis of Alzheimer's disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE.

APOE4 Increases Energy Metabolism in APOE-Isogenic iPSC-Derived Neurons.

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

A Polynesian-specific SLC22A3 variant associates with low plasma lipoprotein(a) concentrations independent of apo(a) isoform size in males.

Lipoprotein(a) (Lp(a)) is a low-density lipoprotein (LDL)-like particle in which the apolipoprotein B component is covalently linked to apolipoprotein(a) (apo(a)). Lp(a) is a well-established independent risk factor for cardiovascular diseases. Plasma Lp(a) concentrations vary enormously between individuals and ethnic groups. Several nucleotide polymorphisms in the SLC22A3 gene associate with Lp(a) concentration in people of different ethnicities. We investigated the association of a Polynesian-specific (Māori and Pacific peoples) SLC22A3 gene coding variant p.Thr44Met) with the plasma concentration of Lp(a) in a cohort of 302 healthy Polynesian males. An apo(a)-size independent assay assessed plasma Lp(a) concentrations; all other lipid and apolipoprotein concentrations were measured using standard laboratory techniques. Quantitative real-time polymerase chain reaction was used to determine apo(a) isoforms. The range of metabolic (HbA1c, blood pressure, and blood lipids) and blood lipid variables were similar between the non-carriers and carriers in age, ethnicity and BMI adjusted models. However, rs8187715 SLC22A3 variant was significantly associated with lower Lp(a) concentrations. Median Lp(a) concentration was 10.60 nmol/L (IQR: 5.40-41.00) in non-carrier group, and was 7.60 nmol/L (IQR: 5.50-12.10) in variant carrier group (P<0.05). Lp(a) concentration inversely correlated with apo(a) isoform size. After correction for apo(a) isoform size, metabolic parameters and ethnicity, the association between the SLC22A3 variant and plasma Lp(a) concentration remained. The present study is the first to identify the association of this gene variant and low plasma Lp(a) concentrations. This provides evidence for better guidance on ethnic specific cut-offs when defining 'elevated' and 'normal' plasma Lp(a) concentrations in clinical applications.

Synergism of ApoE4 and systemic infectious burden is mediated by the APOE-NLRP3 axis in Alzheimer's disease.

Systemic infections are associated with the development of AD, especially in individuals carrying the APOE4 genotype. However, the detailed mechanism through which APOE4 affects microglia inflammatory response remains unclear. We obtained human snRNA-seq data from the Synapse AD Knowledge Portal and assessed the DEGs between APOE3 and APOE4 isoforms in microglia. To verify the interaction between ApoE and infectious products, we used ApoE to stimulate in vitro and in vivo models in the presence or absence of LPS (or ATP). The NLRP3 gene knockout experiment was performed to demonstrate whether the APOE-NLRP3 axis was indispensable for microglia to regulate inflammation and mitochondrial autophagy. Results were evaluated by biochemical analyses and fluorescence imaging. Compared with APOE3, up-regulated genes in APOE4 gene carriers were involved in pro-inflammatory responses. ApoE4-stimulation significantly increased the levels of NLRP3 inflammasomes and ROS in microglia. Moreover, compared with ApoE4 alone, the co-incubation of ApoE4 with LPS (or ATP) markedly promoted pyroptosis. Both NF-κB activation and mitochondrial autophagy dysfunction were contributed by the increased level of NLRP3 inflammasomes induced by ApoE4. Furthermore, the pathological impairment induced by ApoE4 could be reversed by NLRP3 KO. Our study highlights the importance of NLRP3 inflammasomes in linking ApoE4 with microglia innate immune function. These findings not only provide a molecular basis for APOE4-mediated neuroinflammatory but also reveal the potential reason for the increased risk of AD in APOE4 gene carriers after contracting infectious diseases.

Effect of Apolipoprotein E isoforms on the Abundance and Function of P-glycoprotein in Human Brain Microvascular Endothelial Cells

BackgroundIndividuals with Alzheimer’s disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model.MethodsThis study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 – 10 µg/mL over 24 – 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms.ResultsP-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively.ConclusionsDifferent apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner.

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer’s disease

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Abstract 116: Apolipoprotein E Registry In Discoidal Lipoproteins Influences Uptake

Introduction: Apolipoprotein E (apoE) is of great interest due to its role as a risk factor for Alzheimer's disease and atherosclerosis. ApoE is secreted by astrocytes in the central nervous system as HDL-like lipoparticles. Currently there is no high-resolution structural model of lipidated apoE, resulting in a limited mechanistic understanding of how apoE functions in health and disease. Research Questions: The goal of this research was to utilize cryoEM to characterize the structure of apoE in discoidal lipoproteins. Methods: Astrocyte secreted apoE was immunopurified from culture media and characterized by TEM and cryoEM. Recombinant apoE (rapoE) was lipidated with DMPC to model astrocyte secreted lipoproteins. Results: TEM and cryoEM imaging of astrocyte apoE and rapoE found that apoE forms a dimer on discoidal lipoproteins. These results provide support for the “double belt” model of two apoE in an anti-parallel conformation. This conformation was independent of apoE isoform, suggesting it is a general feature of apoE in discoidal lipoproteins. ApoE isoforms differ by the addition or loss of a Cys residue. ApoE2 has 2 Cys residues (Cys112, Cys158), apoE3 has 1 Cys residue (Cys112), and apoE4 has no Cys residues. Accordingly, apoE4 cannot form disulfide bonds, apoE3 can form intermolecular disulfide bonds, and apoE2 can form both intramolecular and intermolecular disulfide bonds. Due to the presence of two apoE on discoidal lipoproteins, apoE3 can form a disulfide bridge between the two apoE protein. Disulfide bridge formation and experimental reduction of these disulfide bridges revealed that disulfide-linked apoE3 was uptaken significantly more by Neuro2a cells (P ≤ 0.0001) than nondisulfide-linked apoE3. Conclusion: All isoforms of apoE adopt an antiparallel dimer in discoidal lipoprotein. Different registries, regulated through the formation of disulfide bonds between the anti-parallel apoE polypeptides, leads to differential uptake of apoE by Neuro2a cells. Since the different isoforms of apoE have inherently different capacity to generate disulfide bridges, we conclude that this property affecting receptor uptake may strongly impact differential biological properties of the different isoforms.