From Blood to Brain: Age-Specific Exosomal Signatures modulate Neurodegeneration in a Human Brain-on-Chip Model

Introduction: : Aging systemic blood milieu profoundly influences the cellular function across tissues, acting as a key driver of age-related cognitive decline. Techniques like therapeutic plasma exchange and heterochronic parabiosis have widely explored the role of plasma on various tissues. However, the role of red blood cells (RBCs) remains largely unexplored. In addition to the role of RBCs in transporting oxygen, RBCs have also shown morphological changes in the patients with neurodegenerative diseases. Furthermore, RBC-derived EVs have been explored for their role in disease biomarker discovery. Studies on exosomes have shown to transfer nucleic acid material to tissues and alter the functionality of the cells. This suggests that there could be molecular interplay between the exosomes and their recipient cells. In this study, we investigated the impact of red blood cell (RBC) and their exosomal cargo from young (20-40yo) and old (60-80yo) donors (both male and female) on human brain cells using a brain-on-chip microphysiological model.

Materials and

Methods: : We developed a scalable, membrane-free brain-on-chip microfluidic device that incorporates integrated electrodes for trans-endothelial electrical resistance (TEER) measurements. The device consists of three interconnected microchannels. Channel 1, referred to as the vascular channel, is used to seed cerebral endothelial cells (hCMEC/D3) (Figure 1A). Adjacent to it is Channel 2, known as the brain channel, where human astrocytes (HA) are co-cultured with human microglia (HMC3) in a three-dimensional hydrogel matrix. Channel 3 serves to supply culture media to the brain channel, supporting cell viability and function. The RBCs and their exosomes from the young and old donors were circulated in the vascular channel of the brain-on-chip device. Using confocal microscopy, the impact of RBCs and RBC-EVs on the brain tissues were examined. Next, we conducted a molecular profiling of the exosomes from the RBCs of young and old donors using microRNA seq analysis and proteomics. To understand the impact of RBC-derived exosomes on the brain cells, we investigated the change in proteome expression of the brain cells (astrocytes and microglia) in response to the exosomes from the RBCs of young and old donors.

Results, Conclusions, and Discussions:: Confocal immunographs show establishment of endothelium barrier with astrocytes and microglia in proximity similar to the microenvironment of brain tissues (Figure 1B). We validated the human brain-on-chip by measuring the formation of barrier using the TEER electrodes (Figure 1C).The RBCs from young and old donors (both male and female) were circulated in the vascular channel of the brain-on-chip. In comparison to the young, the RBCs from the old donors show elevated levels of inflammatory markers and heme accumulation in the brain cells (astrocytes and microglia) (Figure 2 A-C). Molecular profiling of the RBC-derived exosomes using proteomics and microRNA seq analysis revealed age-associated differences in cargo, including downregulation of proteins linked to neuroprotective pathways and upregulation of those involved in inflammation and synaptic dysregulation. Proteomic analysis of the brain cells in response to the RBC-derived exosomes from older donors altered brain cell homeostasis by modulating key signaling pathways such as cytokine signaling, neurotrophin signaling, metabolic activity, and DNA repair (Figure 3). Sex-specific trends in the miRNA and proteomic profiles of the RBC-exosomes were also observed, although no distinct sex-specific brain cell responses were identified. Our findings suggest that RBC-derived exosomes act as carriers of aging signals capable of influencing neurobiological processes.

Conclusion: Cumulatively, our multi-omics analysis provides compelling evidence that RBC-derived exosomes from aged individuals modulate the brain microenvironment through molecular exchange of miRNA and protein cargo. These exosomal cargo have the potential to induce neuroinflammation, BBB leakage, and stress-associated cellular reprogramming. The integration of miRNA and proteomics data from exosomes and proteome expression in brain cells highlights key molecular pathways, including DNA repair, mitochondrial metabolism, synaptic regulation, and immune signaling, that might lead to age-related neurodegenerative progression. This work positions RBC-derived exosomes as a minimally invasive biomarker platform and opens new avenues to explore hematopoietic contributions to brain aging. Targeting the systemic exosomal signaling-brain axis may provide novel therapeutic strategies for delaying or reversing age-related cognitive decline.

Acknowledgements and/or References (Optional):: The authors thank UCSD school of Medicine Microscopy Core facility for the usage of Confocal microscope. The microscopy core facility is supported by a grant from NIH (NS047101). The authors would also like to thank UC San Diego Altman Clinical and Translational Research Institute (ACTRI) for granting us vouchers to perform the bioinformatics analysis. The project was partially supported by the National Institutes of Health, Grant UL1TR001442. The content is solely the responsibility of the author(s) and does not necessarily represent the official views of the NIH.