Nuriel, Tal

Abstract Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimer’s disease (AD), particularly in individuals carrying the APOE-ε4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (Aβ42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-ε4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 ( FN1 ) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-ε4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-ε4 carriers. Here, we demonstrate that APOE-ε4 , Aβ42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-ε4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract Accessibility text This image illustrates the effects of different APOE isoforms (ApoE-ε3 and ApoE-ε4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-ε4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-ε3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-ε4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. Highlights APOE-ε4 drives fibronectin deposition in Alzheimer’s, disrupting astrocyte-endothelia interactions. APOE-ε4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-ε4 pathology.

AbstractBackgroundAPOEε4 significantly increases the risk of developing Alzheimer’s disease (AD). Cognitively healthy APOEε4‐carriers exist, suggesting potential protective mechanisms against APOEε4. We hypothesized that some APOEε4‐carriers may have genetic variations protecting them from developing APOEε4‐mediated AD pathology. We aim to identify these protective genetic variants.MethodsWhole genome sequencing (WGS) and cerebrospinal fluid (CSF) proteomics were performed from human cohorts to identify potential protective variants segregating exclusively among APOEε4 carriers. Bioinformatic analyses were performed to select candidate target genes. Immunohistochemistry on postmortem human brain tissues and mouse models expressing human APOE variants were performed along with in‐vivo functional studies in adult zebrafish AD model.ResultWGS analyses revealed 510 potential gene variants segregating exclusively among APOEε4 carriers, which included rare and loss‐of‐function (LOF) variants. Pathway analysis of these genes showed significant enrichment in extracellular matrix (ECM)‐related processes, suggesting protective effects of LOF in ECM proteins. This was further validated by CSF proteome profiling and subsequent analyses in APOEε4 carriers and non‐carriers. Fibronectin‐1 (FN1) and Collagen‐6A2 (COL6A2) were prioritized as candidate target genes for postmortem validation and in‐vivo functional studies. FN1 protein was increased in APOEε4 carriers resulting in thickened ECM at the basement membrane around the blood vessels, potentially impairing pathology‐induced responses such as clearance and immune system activity. This observation is validated in human brains, mouse models and zebrafish model; therefore, the pathological association of FN1 to AD is evolutionarily conserved. Supporting this hypothesis, in‐vivo functional study in zebrafish model with LOF mutations in fn1b revealed that fibronectin LOF enhanced gliovascular remodeling and microglial activation while reducing astrogliosis, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF.ConclusionThe vascular deposition of the ECM components FN1 and COL6A2 are increased in APOEε4 carriers. Rare variant in FN1 protect against APOEε4‐mediated pathogenesis in AD. We propose a new disease mechanisms and potential therapeutic intervention targets for vascular contribution to dementia to mitigate the risk of developing AD.