Microplastic exposure as a potential APOE-dependent risk factor for human blood-brain barrier dysfunction

Gavin Spillard^1,2, Ruslan Rust^1,2

1 Department of Physiology and Neuroscience, University of Southern California, Los Angeles, CA 90033, USA
2 Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

1. Abstract

Plastic particles at the micro- and nano-scale (microplastics and nanoplastics, MNPs) are now detected in the human brain, where they accumulate around blood vessels and reach higher levels in individuals with dementia. Whether they damage the blood-brain barrier (BBB), the protective interface between blood and brain, remains unclear. Pericytes are contractile cells that wrap brain capillaries and maintain the BBB. Their early loss is a hallmark of Alzheimer’s disease, and carriers of APOE4 (a major genetic risk factor) show pericyte injury and BBB dysfunction before cognitive symptoms appear. We propose to test whether MNP exposure damages human pericytes and whether APOE4 makes them more vulnerable. Using isogenic APOE3 and APOE4 iPSC-derived brain pericytes (iPSC-PC), which we recently showed closely resemble primary human brain pericytes and can repair leaky vessels in vivo, we will (i) expose them to fluorescently labeled polystyrene and polyethylene MNPs and image uptake, (ii) profile the transcriptomic response by RNA-seq, and (iii) assess function in a pericyte-endothelial co-culture BBB model and by vessel-homing in acute brain slices from pericyte-deficient mice. We will then use the Connectivity Map (CMAP) to predict drugs that reverse the APOE-specific MNP signature and test top candidates in the same assays. The project will run for 6 months at USC under existing ethics approvals. Data will be deposited in public repositories.

2. Introduction

Plastic pollution is a rising public health concern. Recent post-mortem studies show that MNPs accumulate in the human brain, with polyethylene (PE) as the dominant polymer and concentrations rising steadily between 2016 and 2024¹. Brain MNPs are enriched in cerebrovascular walls and immune cells, and reach higher levels in individuals with dementia¹. Independent analyses detect MNPs in the human olfactory bulb² and cerebrospinal fluid, with CSF levels that correlate with BBB leakiness³. Animal studies further show that polystyrene (PS) nanoplastics cross the BBB, disrupt tight junctions, and trigger neuroinflammation^4,5.

However, the cellular mechanisms by which MNPs damage the human BBB remain elusive. Pericytes are the primary mural cells of brain capillaries and are required for BBB formation and maintenance⁶. Pericyte degeneration is an early feature of Alzheimer's disease, and APOE4, the strongest genetic risk factor for late-onset Alzheimer's disease, causes BBB breakdown that precedes cognitive decline in human carriers and mouse models^7,8,9. Whether MNPs damage human pericytes, and whether this injury is modulated by APOE genotype, has not been tested.

To address these questions, we will use isogenic APOE3 and APOE4 iPSC-PC recently established in the Rust lab. These cells share 96% of the proteome and 98% of phosphopeptide sites with primary human brain pericytes, home to host brain vessels in pericyte-deficient mice, and repair BBB leaks in vivo¹⁰. We will (i) quantify uptake of fluorescently labeled PS and PE MNPs by live imaging, (ii) profile the transcriptomic response by RNA-seq, and (iii) assess function in a pericyte-endothelial co-culture BBB model and by vessel-homing in acute brain slices from pericyte-deficient mice. We will then use the Connectivity Map (CMAP) to query the APOE-specific MNP-response signatures against compound-induced transcriptome libraries, predict drugs that reverse the MNP phenotype, and test top candidates in the same assays. This approach will reveal whether APOE4 sensitizes human pericytes to MNP exposure and will nominate drug candidates for follow-up studies.

Hypotheses

H1 (primary). Microplastic exposure alters pericyte transcriptomic state and function, with APOE4 iPSC-PC showing larger effects than isogenic APOE3 iPSC-PC.

H2 (secondary). CMAP analysis of the APOE3- and APOE4-specific MNP response signatures will identify compounds predicted to reverse the phenotype; top candidates will rescue pericyte transcriptional state, BBB integrity, and vessel homing in the same assays.

Primary outcome: differential RNA-seq signature (APOE4 vs APOE3 after MNP exposure), BBB permeability in pericyte-EC co-culture, and vessel-homing index in the acute-slice assay.

Secondary outcomes: MNP uptake rate per cell, cell viability, tight junction protein expression, and extent of rescue by top CMAP-predicted compounds.

3. Methods

Participants/Sample

Cell lines. We will use isogenic APOE3/E3 and APOE4/E4 iPSC lines generated from a CRISPR-Cas9-edited KOLF2.1J iPSC line from JAX¹¹ and differentiate them into brain pericytes (iPSC-PC) using our published protocol^10,12. We will validate these findings in independent, non-isogenic iPSC-PC lines if follow-up funding is secured.

Animals. Acute brain slices will be prepared from pericyte-deficient Pdgfrb^F7/F7 mice at 6-8 months, males and females, n = 5 per group. Sample size was estimated by power analysis assuming an effect size of Cohen’s d = 0.8 for vessel-homing based on Rust et al.^10,12, alpha = 0.05, power = 0.8.

Inclusion criteria. Passage-matched iPSC-PC with ≥90% PDGFRβ+/CD13+ purity. Exclusion: lines with karyotype abnormalities or failed differentiation (<70% pericyte markers).

Resources: all cell lines and animals are already established in the Rust lab or at USC core facilities.

Materials and Procedures

Microplastics. Fluorescently labeled polystyrene (PS) and polyethylene (PE) MNPs of 200-9900 nm sizes from Polysciences / Cospheric. Particles will be characterized by dynamic light scattering and TEM before each experiment to confirm size and dispersion. Size and polymer selection reflects the dominant species recovered from human brain tissue¹.

Exposure. iPSC-PC will be seeded at 34,000 cells/cm² on Poly-L-Lysine-coated dishes and exposed to MNPs at 1, 10, 25 μg/mL for 24 h, the time point that gives the most robust transcriptional response in our hands. Each polymer (PS and PE) will be paired with a matched PBS vehicle control at every concentration. The concentration range is anchored to MNP loads reported in human brain tissue¹.

Uptake imaging. Live-cell confocal imaging at 24 h to quantify fluorescent MP internalization per cell, using co-staining with CellMask and Hoechst. Image segmentation with CellProfiler / Fiji. Uptake will be reported as particles/cell and integrated fluorescence/cell.

Bulk RNA-seq. Total RNA from iPSC-PC (APOE3 and APOE4; PBS vehicle vs PS- or PE-exposed; n = 3 biological replicates per condition) will be extracted, libraries prepared with Illumina TruSeq Stranded mRNA and sequenced on an Illumina NovaSeq 6000 to 10M reads/sample.

Acute brain slice homing assay. 250 μm coronal sections from pericyte-deficient mice will be live-cultured on membrane inserts. GFP-labeled iPSC-PC (APOE3 or APOE4; PBS vehicle, PS- or PE-exposed) will be applied to the slice surface, cultured for 24 h, and imaged by confocal microscopy to quantify vessel association (IB4/CD31+) as in Rust et al.¹⁰.

BBB integrity readout (EC co-culture). To test whether MNP-exposed pericytes still support a functional BBB, we will run two complementary assays. (i) Conditioned-medium transfer: supernatant from PBS-vehicle, PS- or PE-exposed iPSC-PC (APOE3 or APOE4) is applied to human brain endothelial cells (hCMEC/D3), and tight-junction expression (claudin-5, occludin, ZO-1) and permeability are measured, as we have previously done¹⁰. (ii) Transwell pericyte-EC co-culture: ECs on the apical side and iPSC-PC on the basolateral side of 0.4 μm pore inserts, with TEER measurements and FITC-dextran permeability over 24, 48, and 72 h.

CMAP-based drug prediction and rescue. MNP-induced differential expression signatures (APOE3 and APOE4, analyzed separately) will be queried against the Connectivity Map (CMAP) / LINCS L1000 compound-induced transcriptome library via clue.io to rank small molecules whose signatures are negatively correlated with, and therefore predicted to reverse, the MNP phenotype. Top N = 5 per genotype candidates, prioritized by BBB penetrance and clinical stage, will be re-tested in iPSC-PC for rescue of MNP-induced transcriptional changes (RNA-seq), BBB permeability (Transwell and conditioned-medium assays), and vessel homing in acute slices. This will provide a path to actionable therapeutic candidates for APOE-stratified environmental exposure.

Planned Analyses

Uptake. Particles per cell compared by 2-way ANOVA (genotype × concentration) with Tukey post hoc tests.

RNA-seq. Reads will be aligned with STAR, quantified with featureCounts, and differential expression called with DESeq2 (FDR < 0.05, |log₂FC| > 1). Pathway enrichment with Enrichr and GSEA. We will specifically test enrichment of inflammation, oxidative stress, tight-junction, and ECM-remodeling pathways in APOE4 vs APOE3.

Homing. Vessel-associated iPSC-PC will be scored as the ratio of GFP+ cells within 5 μm of an IB4+ vessel to total GFP+ cells, compared by 2-way ANOVA (genotype × exposure).

BBB integrity. TEER and FITC-dextran permeability will be analyzed by mixed-effects models with repeated measures over time. Tight-junction protein expression (claudin-5, occludin, ZO-1) will be quantified by Western blot and immunocytochemistry, compared by 2-way ANOVA (genotype × exposure).

CMAP analysis. Ranked DEG lists (top 150 up / 150 down genes by fold change and FDR) from APOE3 and APOE4 MNP-exposed iPSC-PC will be uploaded to clue.io to generate connectivity scores against the LINCS L1000 library. Compounds with negative connectivity scores will be prioritized. Rescue experiments will be analyzed by 2-way ANOVA (compound × exposure) within each genotype.

Regardless of outcome, results will be interpreted in terms of APOE-dependent susceptibility to environmental plastic exposure and will inform follow-up in vivo studies. Deviations from the planned sample (e.g., differentiation failures) will be handled by replacing failed lines with additional rounds of differentiation, and final n reported per group.

Ethics and Data Management

iPSC work and animal procedures are covered under USC IRB/IACUC protocols. Raw and processed RNA-seq data will be deposited in GEO. Imaging datasets will be shared via BioImage Archive. Protocols and analysis code will be released via the Rust lab GitHub and protocols.io upon publication.

4. Pilot Data (Optional)

We have established and characterized several iPSC-PC lines from healthy donors^10,12. By quantitative LC/MS, we showed that iPSC-PC closely resemble primary human brain pericytes, sharing 96% of 8,344 quantified proteins and 98% of 20,572 phosphopeptides (Fig. 1 a-b), and express the pericyte marker PDGFRβ in nearly all cells (Fig. 1c). In acute brain slices from pericyte-deficient Pdgfrb^F7/F7 mice, labeled iPSC-PC home to lectin⁺ host capillaries within 24 h, with the majority extending processes along the vessel wall (Fig. 1 d-f). After intracerebral transplantation, engrafted iPSC-PC wrap around host brain vessels in vivo (Fig. 1 g-h). Isogenic APOE3/E3 and APOE4/E4 iPSC-PC lines have also been generated using this protocol and will be used in this study.

Figure 1. iPSC-derived brain pericytes closely resemble primary human brain pericytes and home to host brain vasculature. (a) Workflow for quantitative LC/MS comparison of iPSC-PC and primary human brain pericytes (PC). (b) iPSC-PC closely resemble PC, sharing 96% of 8,344 quantified proteins and 98% of 20,572 phosphopeptides. (c) iPSC-PC express the pericyte marker PDGFRβ (magenta); nuclei, DAPI (blue). (d-f) Ex vivo homing assay. GFP-labeled iPSC-PC applied to acute brain slices from pericyte-deficient Pdgfrb F7/F7 mice (d) home to lectin+ host capillaries within 24 h (e), with the majority extending processes along the vessel wall (f). (g-h) Intracerebral transplantation into Pdgfrb F7/F7 mice (g) shows engrafted iPSC-PC (green) ensheathing host brain vessels (magenta) in 3D reconstructions (h). Parts of this figure are adapted from Rust et al., bioRxiv 2024 (PMID: 40291694).¹⁰

5. Budget

An itemized estimate is provided below. Values are not binding and will be refined based on pilot data.

Item	Estimated cost (USD)
Consumables (microplastics, fluorophores, iPSC culture media, antibodies)	500
RNA-seq (library prep and sequencing)	950
Animal costs (pericyte-deficient mouse line)	400
CMAP hits (drug procurement, Transwell inserts, TEER electrodes)	0 (covered by other grants)
Personnel (research assistant)	0 (covered by other grants)
Publication fees (open access)	0 (covered by other grants)
Total	1850

6. References

1. Nihart, A. J. et al. Bioaccumulation of microplastics in decedent human brains. Nat. Med. 31, 1114-1119 (2025).

2. Amato-Lourenço, L. F. et al. Microplastics in the olfactory bulb of the human brain. JAMA Netw. Open 7, e2440018 (2024).

3. Xie, J. et al. Blood-brain barrier damage accelerates the accumulation of micro- and nanoplastics in the human central nervous system. J. Hazard. Mater. 480, 136028 (2024).

4. Shan, S. et al. Polystyrene nanoplastics penetrate across the blood-brain barrier and induce activation of microglia in the brain of mice. Chemosphere 298, 134261 (2022).

5. Jin, H. et al. Evaluation of neurotoxicity in BALB/c mice following chronic exposure to polystyrene microplastics. Environ. Health Perspect. 130, 107002 (2022).

6. Daneman, R., Zhou, L., Kebede, A. A. & Barres, B. A. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562-566 (2010).

7. Montagne, A. et al. APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature 581, 71-76 (2020).

8. Barisano, G. et al. A multi-omics analysis of blood-brain barrier and synaptic dysfunction in APOE4 mice. J. Exp. Med. 219, e20221137 (2022).

9. Montagne, A. et al. APOE4 accelerates advanced-stage vascular and neurodegenerative disorder in old Alzheimer's mice via cyclophilin A independently of amyloid-β. Nat. Aging 1, 506-520 (2021).

10. Rust, R. et al. Molecular signature and functional properties of human pluripotent stem cell-derived brain pericytes. bioRxiv (2025). doi:10.1101/2023.06.26.546577.

11. Pantazis CB, Yang A, Lara E, et al. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell. 2022;29(12):1685-1702.e22. doi:10.1016/j.stem.2022.11.004

12. Zhang M, Kim Y, Bosworth A, Tcw J, Nih LR, Kisler K, Sagare AP, Rust R. Brain pericytes derived from human pluripotent stem cells retain vascular and phagocytic functions under hypoxia. Stem Cells. 2025 Oct 16;43(11):sxaf055. doi: 10.1093/stmcls/sxaf055. PMID: 40737487; PMCID: PMC12573254.

Microplastic exposure as a potential APOE-dependent risk factor for human blood-brain barrier dysfunction

Microplastic exposure as a potential APOE-dependent risk factor for human blood-brain barrier dysfunction

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