The Influences of APOE Isoforms on Endothelial Adherens Junctions and Actin Cytoskeleton Responding to MCRP

Geriatrics Neurology Biochemistry Cell Biology Medicine Life Sciences Immunology
APOE Monomeric C-reactive protein Endothelial cells Cytoskeleton Alzheimer's disease Cerebrovascular F-actin

Research Background

Alzheimer’s Disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloid-beta (Aβ) plaques and abnormal phosphorylation of tau protein. Recent studies have increasingly highlighted the role of cerebrovascular alterations in the pathogenesis of AD. Specifically, endothelial cell damage and inflammatory responses are considered critical components of AD pathology. Apolipoprotein E (APOE), particularly the APOE4 allele, is a major genetic risk factor for AD. APOE4 may impair endothelial cell function, leading to blood-brain barrier (BBB) disruption, neuroinflammation, and neuronal damage.

Monomeric C-Reactive Protein (mCRP), a marker of chronic inflammation, has also gained attention in AD research. mCRP binds to CD31 on endothelial cells, potentially triggering cerebrovascular inflammation and promoting AD pathology. However, the mechanisms by which mCRP affects endothelial cell junctions and the cytoskeleton, and the role of APOE genotypes in this process, remain unclear.

Research Objectives

This study aimed to investigate how APOE genotypes modulate endothelial cell junctions and the cytoskeleton in response to mCRP, thereby influencing AD pathology. Specifically, the researchers hypothesized that APOE4 disrupts endothelial cell junctions and the cytoskeleton, leading to BBB dysfunction and increased AD risk.

Research Methods

1. Human Brain Tissue Analysis

The research team obtained temporal lobe tissue samples from 8 healthy controls and 8 AD patients from the Boston University Alzheimer’s Disease Center (BU ADC). Through immunofluorescence staining and phosphoproteomic profiling, the researchers measured the expression levels of mCRP and phosphorylated CD31 (pCD31) in AD brain microvessels and analyzed changes in cytoskeletal proteins.

2. Mouse Model Experiments

The study used APOE2, APOE3, and APOE4 knock-in mice, as well as APOE knockout mice. Mice were intraperitoneally injected with mCRP for 6 weeks. Subsequently, microvessels were isolated from mouse brain tissue for proteomic analysis and immunofluorescence staining to assess the effects of mCRP on cytoskeletal and junctional proteins.

3. In Vitro Experiments

The research team isolated CD31-positive endothelial cells from mouse brain tissue and cultured them in vitro. These cells were exposed to mCRP, mCRP combined with recombinant APOE2 or APOE4 protein, and changes in cytoskeletal and junctional proteins were measured.

4. Data Analysis

Through phosphoproteomics and Gene Set Enrichment Analysis (GSEA), the researchers identified molecular pathways significantly upregulated or downregulated in AD brain microvessels. Additionally, Protein-Protein Interaction Analysis (Proximity Ligation Assay, PLA) was used to detect interactions between CD31 and LIMA1.

Research Findings

1. Cytoskeletal and Junctional Alterations in AD Brain Microvessels

The study found that mCRP and pCD31 expression were significantly higher in AD brain microvessels compared to healthy controls. Phosphoproteomic analysis revealed elevated phosphorylation levels of microtubule-associated protein tau (MAPT), particularly pTau_T217 and pTau_T231, in AD microvessels. These phosphorylated tau proteins were significantly correlated with AD pathology scores (Braak staging) and cognitive function (MMSE scores).

2. APOE4 Mice Respond to mCRP

In APOE4 knock-in mice, mCRP significantly increased pCD31 and LIMA1 expression and promoted the binding of pCD31 to LIMA1. Additionally, mCRP reduced the interaction between CD31 and VE-cadherin at cell junctions, leading to microvascular damage. In contrast, APOE2 protein attenuated these changes.

3. APOE Genotype-Dependent Effects of mCRP on Cytoskeleton and Junctions

The study found that mCRP significantly increased LIMA1 expression and promoted pCD31-LIMA1 binding in APOE4 mice, but not in APOE2 and APOE3 mice. Furthermore, mCRP significantly reduced CD31-VE-cadherin colocalization in APOE4 mice, leading to microvascular damage. APOE2 protein reversed these changes, while APOE4 protein exacerbated the toxic effects of mCRP.

4. In Vitro Validation

In cultured brain endothelial cells, the combination of mCRP and APOE4 protein significantly increased LIMA1 expression and F-actin formation, while APOE2 protein inhibited these changes. Additionally, the combination of mCRP and APOE4 protein increased the expression of ARPc4 and ITGB1, proteins closely associated with F-actin network formation.

Research Conclusions

This study is the first to reveal that APOE genotypes modulate endothelial cell cytoskeleton and junctions in response to mCRP, thereby influencing AD pathology. Specifically, APOE4 increases pCD31 and LIMA1 expression, promotes F-actin formation, and disrupts endothelial cell junctions, leading to microvascular damage and increased AD risk. In contrast, APOE2 attenuates these changes, protecting endothelial cell integrity.

Research Significance

This study provides new insights into the role of APOE genotypes in AD pathogenesis, particularly how APOE4 modulates endothelial cell cytoskeleton and junctions in response to chronic inflammation, leading to BBB dysfunction and neuroinflammation. Additionally, the study suggests that APOE2 may act as a protective factor, mitigating the toxic effects of mCRP on endothelial cells. This provides a potential theoretical basis for developing AD treatment strategies targeting APOE4 carriers.

Research Highlights

  1. First to reveal that APOE genotypes modulate endothelial cell cytoskeleton and junctions in response to mCRP, influencing AD pathology.
  2. Found that APOE4 increases pCD31 and LIMA1 expression, promotes F-actin formation, and disrupts endothelial cell junctions.
  3. APOE2 attenuates the toxic effects of mCRP on endothelial cells, protecting endothelial integrity.
  4. Provides a potential theoretical basis for developing AD treatment strategies targeting APOE4 carriers.

Additional Valuable Information

The study also found that the combination of mCRP and APOE4 protein significantly increased oxidative stress markers (e.g., OXCT1 and ROS), further exacerbating endothelial cell damage. This suggests that APOE4 may modulate mitochondrial function, affecting endothelial cell metabolism and inflammatory responses, thereby promoting AD pathology.