Integrated miRNA and metabolomic profiling to target brain endothelial cell senescence

1. Overview

The blood-brain barrier (BBB) regulates brain homeostasis and is responsible for the exchange between blood vessels and the brain. As we age, brain endothelial cells (BECs), the main cell type of the BBB, deteriorate structurally and functionally, which compromises BBB integrity and promotes neurovascular injury and cognitive decline. But still the exact mechanisms underlying the deterioration of BECs remain unexplored. In this study, we propose to characterize senescence in mouse BECs using microRNA sequencing (miRNA sequencing). Senescence will be induced chemically by the known inducer doxorubicin (Dox) and the oxidative stress inducer hydrogen peroxide (H₂O₂). This approach enables us to identify shared miRNAs responsible for regulating BEC senescence, which will form the basis for the development of novel therapeutic targets. Thereafter, we plan to compare the obtained data with our previously obtained dataset on metabolomic changes of senescent BECs to better understand the regulatory networks that drive metabolic reprogramming during BBB senescence. We confirm that this study does not require any ethical approvals. The project will run over 9 months, RNA extraction, library preparation and sequencing (3 months), and analysis of miRNA sequencing data (3 months), integration of miRNA data with previously obtained metabolomics data (3 months). We commit to share the obtained results in a public repository to promote transparency and further use of this data.

2. Introduction

The main cell type of the blood-brain barrier (BBB) are brain endothelial cells (BECs) that line the inner lumen of blood vessels and prevent the leakage of molecules from the blood to the brain by the expression of tight junction proteins and efflux transporters [1], [2]. Aging is a phenomenon that naturally occurs in all cell types in our body. During aging, several changes to the morphology and integrity of the BBB occur, such as increased non-specific protein leakage and decreased efflux transporter systems expressed in BECs [1]. Cellular senescence, a hallmark of aging, occurs when cells continue to proliferate without undergoing cell death [3]. Senescence is influenced by various factors, including genomic damage, telomere shortening, and stress signals. Senescent cells then undergo distinct changes in gene expression, secretory patterns, and morphology, contributing to the aging process and age-related diseases. When it comes to BBB cells, an increase in the number of BECs with high levels of senescence-related gene expression has been observed in a transcriptomics study in aged mice [4]. microRNAs (miRNAs) are small, non-coding RNAs that control the expression of post-transcriptional genes that cause transcriptional repression or degradation. While previous studies have described several changes at miRNAs levels that contribute to senescence (i.e., miR-34a, promoting the repression of SIRT1) [5], but still the role of miRNAs during senescence, in particular with respect to BECs and the BBB, is limited. In this proposal, we will profile the miRNAs responsible for cellular senescence in BECs using chemical inducers of senescence, i.e., the known inducer doxorubicin (Dox) and the oxidative stress inducer hydrogen peroxide (H₂O₂). In a second step, we will compare these changes with our previously obtained metabolomic dataset in senescent BECs [6] using the same experimental setup.

Research questions:

1. Which miRNAs are expressed differently in BECs during chemically induced senescence induced by Dox and H₂O₂compared with control?

2. Which pathways are associated with identified miRNA targets for senescence?

3. How do microRNA-driven regulatory changes contribute to the metabolic reprogramming of senescent BECs, and can integrated miRNA-metabolite network analysis reveal key molecular targets for preventing BBB aging?

Hypotheses (H1, H2, etc.).

H1: Senescent BECs exhibit specific miRNAs shared among two chemical inducers of senescence.

H2: Senescence in BECs rewires metabolism via miRNA-mediated repression of enzymes and transporters; thus, deregulated miRNAs will anti-correlate with their target enzymes’ activity and associate with characteristic metabolite shifts.

Explain the study's importance with an impact statement, regardless of potential outcomes.

Aging of the BBB accelerates cognitive decline and increases vulnerability to neurodegenerative diseases, yet the molecular mechanisms linking gene regulation to metabolic dysfunction in senescent BECs remain poorly understood. By integrating miRNA sequencing with metabolomics, this project will uncover regulatory networks that drive metabolic reprogramming during BBB senescence. Identifying these miRNA-metabolite signatures will not only deepen our understanding of endothelial aging but also provide novel, mechanistically grounded targets for therapeutic interventions aimed at preserving BBB integrity and preventing age-related neurological disorders.

3. Methods

Participants/Sample

We will use the mouse brain endothelial cell line bEnd.3.

Materials and Procedures

Senescence induction: Senescence will be induced by 750 µM H₂O₂ or 100 nM Dox for 72 hours and assessed by SA-β-gal staining (microscopy) as well as p16 or P21 mRNA expression (rt-PCR) as previously described [6]. Cells will be harvested for miRNA analysis.

miRNA analysis: RNA will be extracted (E.Z.N.A. HP Total RNA Kit) from three independent experiments and samples will be processed for RNA sequencing using Illumina NovaSeqX at the Vienna BioCenter Sequencing Facility. All samples per experiment will be sequenced together in one run using multiplexing to avoid batch effects.

Planned Analyses

· QC/normalize miRNA-seq (DESeq2) and metabolomics (QC-based normalization, ComBat)

· Identify DE miRNAs and differential metabolites (FDR<0.05) per condition and overlap across Dox/H₂O₂

· Predict/validate miRNA targets; map to enzymes and pathways

· Dual enrichment: pathway analysis for miRNA targets and metabolites; intersect results

· Sample-level integration: partial correlations and network building for plausible miRNA–enzyme–metabolite triads

· Multiblock modeling (DIABLO/sCCA or MOFA+) to derive joint signatures discriminating senescence

Ethics and Data Management

Ethical Approval: Not required (no human or animal subject research).

Confidentiality: Not applicable (public dataset).

Data Sharing: public repository, e.g. figshare

4. Pilot Data (Optional)

We already established BEC senescence and performed metabolomics [6]. The same conditions will be used for the miRNA study and compared to the metabolomic signatures identified in senescent BECs.

5. Budget

miRNA sequencing NovaSeqX 10B 300 cycles Lane including Library QC and miRNA library preparation for 9 samples = 5 000 USD

6. References

[1] Banks, W. A., Reed, M. J., Logsdon, A. F., Rhea, E. M., & Erickson, M. A. (2021). Healthy aging and the blood–brain barrier. Nature Aging, 1(3), 243–254. doi.org/10.1038/s43587-021-00043-5

[2] Knox, E. G., Aburto, M. R., Clarke, G., Cryan, J. F., & O’Driscoll, C. M. (2022). The blood-brain barrier in aging and neurodegeneration. Molecular Psychiatry, 27(6), 2659–2673. doi.org/10.1038/s41380-022-01511-z

[3] López-Otín, C., et al (2023). Hallmarks of aging: An expanding universe. Cell, 186(2), 243–278.doi.org/10.1016/j.cell.2022.11.001

[4] Kiss, T., et al (2020). Single-cell RNA sequencing identifies senescent cerebro-microvascular endothelial cells in the aged mouse brain. GeroScience, 42(2), 429–444. doi.org/10.1007/s11357-020-00177-1

[5] Yamakuchi M, et al. miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci USA. 2008 Sep 9;105(36):13421-6. doi: 10.1073/pnas.0801613105.

[6] doi: https://doi.org/10.1101/2025.08.14.670255.

Funding

Funding

Integrated miRNA and metabolomic profiling to target brain endothelial cell senescence

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI

Integrated miRNA and metabolomic profiling to target brain endothelial cell senescence

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI