Periodic Reporting for period 1 - ClearPath (Amyloid-β clearance in Alzheimer's disease: Unravelling the role of endocytic pathways of endothelial cells)
Reporting period: 2023-05-01 to 2025-08-31
Alzheimer’s disease (AD) is characterized by the progressive extracellular accumulation of β-amyloid peptide (Aβ) in the brain, leading to the formation of amyloid plaques. Aβ deposition is a major risk factor for the onset and progression of AD. While research has traditionally focused on neurons, glial cells, and astrocytes, increasing evidence suggests that the brain’s vascular component plays a significant role in disease development. Notably, up to 80% of AD patients exhibit cerebral amyloid angiopathy (CAA), with Aβ accumulation in cerebral vessel walls, which disrupts vessel integrity and impairs blood-brain barrier (BBB) functionality.
The brain vascular network also plays a critical role in Aβ clearance, maintaining low levels of this toxic peptide in the brain. Endothelial cells (ECs), which form the BBB, are central to this process. Clathrin-mediated endocytosis (CME) allows ECs to internalize Aβ from the neuronal side, transport it across the cell, and release it into the blood, facilitating delivery to peripheral tissues (e.g. liver) for degradation. Impaired CME in some AD patients contributes to Aβ accumulation in the brain. Importantly, CME is only one of several endocytic pathways utilized by ECs, and the contribution of other endocytic mechanisms to Aβ clearance remains largely unexplored.
Overall Objectives
Investigate the unexplored endocytic pathways involved in brain Aβ clearance.
Identify the relationship between endocytic abnormalities and AD pathology.
Approach
To achieve these objectives, the project will:
a. Develop and characterize a novel in vitro transwell BBB model using isogenic iPSC lines derived from AD patients with APOE 4/4 genotype.
b. Utilize this model to identify distinct endocytic pathways involved in Aβ clearance.
c. Perform large-scale bioinformatics analyses of genome-wide and transcriptome datasets from AD patients to identify novel disease-associated genes and pathways implicated in endocytic routes. This will enable the development of a genetic risk score for AD onset and MCI-to-AD progression.
d. Validate in vitro findings in established AD animal models.
Using the in vitro BBB model, we will examine the effects of Aβ peptides released from iPSC-derived neurons on the endothelial monolayer, focusing on the role of distinct endocytic pathways in Aβ clearance. A multidisciplinary approach, combining advanced cellular models, bioinformatics, and in vivo validation, will provide a comprehensive understanding of the endothelial component of the neurovascular unit.
Expected Impact
Understanding the mechanisms regulating Aβ clearance will provide critical insights into AD pathogenesis and may inform rational drug design. Given the growing prevalence of AD and the associated socioeconomic burden, identifying novel targets for intervention could accelerate the development of precision therapies. While the therapeutic potential of endocytic Aβ clearance is not yet established, this research will provide foundational knowledge for novel treatment strategies for AD and related neurodegenerative diseases.
The brain vascular network also plays a critical role in Aβ clearance, maintaining low levels of this toxic peptide in the brain. Endothelial cells (ECs), which form the BBB, are central to this process. Clathrin-mediated endocytosis (CME) allows ECs to internalize Aβ from the neuronal side, transport it across the cell, and release it into the blood, facilitating delivery to peripheral tissues (e.g. liver) for degradation. Impaired CME in some AD patients contributes to Aβ accumulation in the brain. Importantly, CME is only one of several endocytic pathways utilized by ECs, and the contribution of other endocytic mechanisms to Aβ clearance remains largely unexplored.
Overall Objectives
Investigate the unexplored endocytic pathways involved in brain Aβ clearance.
Identify the relationship between endocytic abnormalities and AD pathology.
Approach
To achieve these objectives, the project will:
a. Develop and characterize a novel in vitro transwell BBB model using isogenic iPSC lines derived from AD patients with APOE 4/4 genotype.
b. Utilize this model to identify distinct endocytic pathways involved in Aβ clearance.
c. Perform large-scale bioinformatics analyses of genome-wide and transcriptome datasets from AD patients to identify novel disease-associated genes and pathways implicated in endocytic routes. This will enable the development of a genetic risk score for AD onset and MCI-to-AD progression.
d. Validate in vitro findings in established AD animal models.
Using the in vitro BBB model, we will examine the effects of Aβ peptides released from iPSC-derived neurons on the endothelial monolayer, focusing on the role of distinct endocytic pathways in Aβ clearance. A multidisciplinary approach, combining advanced cellular models, bioinformatics, and in vivo validation, will provide a comprehensive understanding of the endothelial component of the neurovascular unit.
Expected Impact
Understanding the mechanisms regulating Aβ clearance will provide critical insights into AD pathogenesis and may inform rational drug design. Given the growing prevalence of AD and the associated socioeconomic burden, identifying novel targets for intervention could accelerate the development of precision therapies. While the therapeutic potential of endocytic Aβ clearance is not yet established, this research will provide foundational knowledge for novel treatment strategies for AD and related neurodegenerative diseases.
In the ClearPath project, we successfully differentiated iPSC lines into functional brain endothelial cells, validating their barrier phenotype through tight junction marker expression and TEER measurements. This enabled the establishment of an in vitro BBB model, which was subsequently used to perform permeability assays and investigate the effect of Aβ exposure on barrier integrity.
In parallel, using endothelial cells such as HUVECs and commercially available Human Brain Microvascular Endothelial Cells (HBMVECs), we investigated Aβ uptake and transport across the endothelial layer, confirming the suitability of our model for mechanistic clearance studies.
The fellow completed a short-term secondment at Johns Hopkins School of Medicine, acquiring hands-on training in hiPSC differentiation and maintenance. This training was critical for implementing advanced stem cell methodologies and directly contributed to the successful establishment of the BBB model at BRI-FORTH.
Using the in vitro BBB models, we systematically investigated the endocytic routes of Aβ uptake in brain endothelial cells. Confocal imaging and colocalization studies demonstrated that Aβ is internalized, in part, through macropinocytosis, as indicated by its overlap with dextran-positive vesicles.
Complementary bioinformatics analyses were performed using large-scale genome-wide datasets, including single-nucleus transcriptomes from AD patients, with a focus on brain endothelial cells. A second short-term secondment at the Department of Bioinformatics, Ionian University (Corfu, Greece), provided training in the biological interpretation of large gene lists. This analysis identified potential pathogenic factors, differentially expressed genes, and novel associations between AD and specific genes. Candidate genes will be further validated using the established in vitro BBB models.
To confirm the contribution of endocytic pathways to Aβ clearance, we employed in vivo models, including C. elegans expressing pan-neuronal human Aβ1–42 (CL2355) and the 5XFAD transgenic mouse model, widely used in AD research.
Overall, the ClearPath project demonstrated the originality and strength of integrating multidisciplinary techniques, combining iPSC technology from AD patients, advanced molecular and cellular biology, bioinformatic analysis of large-scale omics data, and in vivo model organisms to address key questions regarding Aβ clearance and BBB function.
In parallel, using endothelial cells such as HUVECs and commercially available Human Brain Microvascular Endothelial Cells (HBMVECs), we investigated Aβ uptake and transport across the endothelial layer, confirming the suitability of our model for mechanistic clearance studies.
The fellow completed a short-term secondment at Johns Hopkins School of Medicine, acquiring hands-on training in hiPSC differentiation and maintenance. This training was critical for implementing advanced stem cell methodologies and directly contributed to the successful establishment of the BBB model at BRI-FORTH.
Using the in vitro BBB models, we systematically investigated the endocytic routes of Aβ uptake in brain endothelial cells. Confocal imaging and colocalization studies demonstrated that Aβ is internalized, in part, through macropinocytosis, as indicated by its overlap with dextran-positive vesicles.
Complementary bioinformatics analyses were performed using large-scale genome-wide datasets, including single-nucleus transcriptomes from AD patients, with a focus on brain endothelial cells. A second short-term secondment at the Department of Bioinformatics, Ionian University (Corfu, Greece), provided training in the biological interpretation of large gene lists. This analysis identified potential pathogenic factors, differentially expressed genes, and novel associations between AD and specific genes. Candidate genes will be further validated using the established in vitro BBB models.
To confirm the contribution of endocytic pathways to Aβ clearance, we employed in vivo models, including C. elegans expressing pan-neuronal human Aβ1–42 (CL2355) and the 5XFAD transgenic mouse model, widely used in AD research.
Overall, the ClearPath project demonstrated the originality and strength of integrating multidisciplinary techniques, combining iPSC technology from AD patients, advanced molecular and cellular biology, bioinformatic analysis of large-scale omics data, and in vivo model organisms to address key questions regarding Aβ clearance and BBB function.
Beyond the State of the Art
Accumulation of amyloid-β (Aβ) in the brain is a defining feature of Alzheimer’s disease (AD), driven in part by impaired clearance across the blood–brain barrier (BBB). The cellular mechanisms underlying Aβ transport through brain endothelium have remained poorly understood. Our work identifies macropinocytosis as a critical pathway for Aβ uptake and clearance across the BBB, complementing the previously described clathrin-mediated endocytosis (CME).
Key Results:
In vitro validation: Pharmacological inhibition revealed that EIPA, a macropinocytosis inhibitor, reduced Aβ uptake by ~65% in brain endothelial cells (BECs) and ~46% in HUVECs, while dynasore, a CME inhibitor, reduced uptake by 40% and 16%, respectively. Combined inhibition decreased Aβ internalization by ~85%, demonstrating the complementary roles of these pathways. siRNA-mediated knockdown of key macropinocytic regulators further confirmed these findings.
In vivo validation: In C. elegans (CL2355 strain), RNAi knockdown of endocytic regulators impaired locomotion, reduced Aβ clearance, and increased aggregation, as confirmed by Thioflavin S staining. In 5xFAD mice, pharmacological inhibition of macropinocytosis increased amyloid plaque burden, while inhibition of dynamin-dependent endocytosis reduced deposition. Strikingly, combined inhibition nearly abolished detectable plaques, revealing a compensatory interplay between distinct endocytic routes.
Molecular insights: Bioinformatics analyses and functional studies identified candidate genes and signaling pathways potentially involved in endocytic dysfunction and amyloid pathology.
Potential Impact:
Therapeutic potential: Macropinocytosis and CME are now established as key mechanisms for maintaining Aβ proteostasis, highlighting these pathways as potential targets for therapeutic intervention. Enhancing endocytic clearance of Aβ could slow or prevent AD progression.
Research advancement: These findings expand the understanding of BBB function beyond current models, addressing a major gap in AD research.
Translational opportunities: Candidate genes and pathways identified may guide future drug discovery, biomarker development, and precision interventions. Further research, including optimization of modulators of endocytosis and testing in human-relevant models, could facilitate uptake and translation to clinical applications.
Accumulation of amyloid-β (Aβ) in the brain is a defining feature of Alzheimer’s disease (AD), driven in part by impaired clearance across the blood–brain barrier (BBB). The cellular mechanisms underlying Aβ transport through brain endothelium have remained poorly understood. Our work identifies macropinocytosis as a critical pathway for Aβ uptake and clearance across the BBB, complementing the previously described clathrin-mediated endocytosis (CME).
Key Results:
In vitro validation: Pharmacological inhibition revealed that EIPA, a macropinocytosis inhibitor, reduced Aβ uptake by ~65% in brain endothelial cells (BECs) and ~46% in HUVECs, while dynasore, a CME inhibitor, reduced uptake by 40% and 16%, respectively. Combined inhibition decreased Aβ internalization by ~85%, demonstrating the complementary roles of these pathways. siRNA-mediated knockdown of key macropinocytic regulators further confirmed these findings.
In vivo validation: In C. elegans (CL2355 strain), RNAi knockdown of endocytic regulators impaired locomotion, reduced Aβ clearance, and increased aggregation, as confirmed by Thioflavin S staining. In 5xFAD mice, pharmacological inhibition of macropinocytosis increased amyloid plaque burden, while inhibition of dynamin-dependent endocytosis reduced deposition. Strikingly, combined inhibition nearly abolished detectable plaques, revealing a compensatory interplay between distinct endocytic routes.
Molecular insights: Bioinformatics analyses and functional studies identified candidate genes and signaling pathways potentially involved in endocytic dysfunction and amyloid pathology.
Potential Impact:
Therapeutic potential: Macropinocytosis and CME are now established as key mechanisms for maintaining Aβ proteostasis, highlighting these pathways as potential targets for therapeutic intervention. Enhancing endocytic clearance of Aβ could slow or prevent AD progression.
Research advancement: These findings expand the understanding of BBB function beyond current models, addressing a major gap in AD research.
Translational opportunities: Candidate genes and pathways identified may guide future drug discovery, biomarker development, and precision interventions. Further research, including optimization of modulators of endocytosis and testing in human-relevant models, could facilitate uptake and translation to clinical applications.