Title: “Message in a Bottle”
Description: Background: The physiological and pathophysiological roles of secreted membrane-enclosed vesicles known as extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. EVs and their encapsulated molecular material including microRNAs are key mediators of intercellular communication, making EVs analogous to a message in a bottle. This discovery has fundamentally changed the study of gene regulation, and understanding the central role of EVs and their cargo in health and disease will generate new opportunities for basic biology, diagnostics, and disease treatment. EV release and the packaging of molecular material into EVs can be altered by stressors such as air pollution exposure. Exposure to air pollution is associated with significant morbidity among individuals with asthma, especially children who participate in more frequent outdoor activities and are more susceptible to exposure due to their narrower airways and higher breathing rate.

Thus, sensitive biomarkers of air pollution exposure are needed to identify children at risk of worsened symptoms and asthma exacerbations. Given their role in cell-to-cell communication, EVs also represent a plausible molecular mechanism in the etiology of disorders such as aging-related cognitive decline. Individuals with mild cognitive impairment and experiencing increased rates of cognitive decline are more likely to develop Alzheimer’s disease and other dementias, signifying the importance of identifying and treating cognitive impairment early. More precise identification of the neurobiological processes of cognitive decline in aging populations may provide critical insights into the precursors of late-life dementias and identify health interventions that can delay cognitive impairment or therapeutic targets for treatment. This dissertation evaluates the utility of EV-encapsulated microRNAs (EV-miRNAs) as biomarkers of environmental exposure (i.e., air pollution) and assesses their role in disease risk (i.e., cognitive decline) in two separate studies.

First, in Chapters 2-3, using a cohort of children with asthma in the greater Boston area, we describe saliva EVs isolated from these children using a high-throughput method and explore the potential of salivary EV-miRNAs as easy-to-measure biomarkers of exposure to fine particulate matter (PM2.5), nitrogen dioxide (NO2), and ozone (O₃). Then, in Chapter 4, we evaluate the association between EV-miRNAs and cognitive function and rates of cognitive decline in a cohort of elderly men and discuss the utility of circulating EV-miRNAs as biomarkers of risk. Furthermore, we discuss the pathways that these EV-miRNAs target if they play a causal role in cognitive decline which could have implications for development of therapeutics. Methods: Drawing from the School Inner-City Asthma Study (SICAS), we isolated salivary EVs and EV-miRNA from children with asthma for analysis in relation to ambient exposure to PM₂.₅, NO₂, and O₃. In accordance with the recommended minimal experimental requirements for the definition of EVs, in Chapter 2 we employ multiple orthogonal methods to describe the EVs that were isolated from cell-free saliva using a high-throughput polymer-based reagent (ExoQuick-TC).

In Chapter 3, we utilize EV-miRNA data generated via RNA sequencing and ambient air pollution data estimated using a validated spatiotemporal high-resolution model. We perform differential expression analyses to examine the effect of high exposure to PM₂.₅, NO₂, and O₃ on saliva EV-miRNA abundance. In Chapter 4, we leverage data from the Normative Aging Study, a longitudinal cohort of elderly men, to investigate whether circulating EV-miRNAs are associated with cognitive function and rates of cognitive decline. We used linear models to assess the relationship between plasma EV-miRNAs and cognitive function and linear mixed models to evaluate the relationship between plasma EV-miRNAs and rates of cognitive decline. We performed gene ontology functional enrichment and pathway enrichment analyses to identify the biological pathways that these EV-miRNAs would target if they play causal roles in cognitive decline. Results:In Chapter 2, we demonstrate that EVs can be isolated from human saliva using ExoQuick-TC.

The saliva EVs isolated from ExoQuick (N=180) ranged in size but were mostly ~55 nm in diameter and expressed tetraspanins CD9 and CD63, canonical markers for EVs, but did not highly express the tetraspanin CD81. In Chapter 3, in a subset of the SICAS cohort (N=69), we show that relatively high (>19.37 parts per billion) short-term ambient NO2 exposure and relatively high (>38.38 parts per million) prior-day O3 exposure are associated with down-regulation of several saliva EV-miRNAs. We did not detect differential expression of any EV-miRNAs in relation to PM₂.₅ exposure over multiple time windows of exposure. Finally, in Chapter 4, multivariable linear and linear mixed models demonstrated a relationship between several plasma EV-miRNAs and global cognitive function and rates of global cognitive decline, measured by the Mini-Mental State Examination. Functional enrichment and pathway enrichment analyses revealed that the biological pathways targeted by these miRNAs are relevant in neurodegeneration, including pathways regulating synaptic function and plasticity and neuronal death.

We found no association between EV-miRNAs and cognitive function or cognitive decline as assessed by cognitive tests measuring specific domains of cognitive function. Conclusion: This work demonstrates the opportunities that EV-miRNAs can create for advancing environmental health research. EV-miRNAs may serve as sensitive biomarkers of environmental exposures as well as biomarkers of risk and may play mechanistic roles in disease. We also make recommendations for integrating EV research into the field of environmental health. Future studies should continue to evaluate the potential of EV-miRNAs and seek to identify EV-miRNAs that can serve as mechanistic biomarkers between exposures and effect across all stages of life to (1) increase our understanding of the consequences of circulating miRNA changes and the contribution of the environment to heterogeneous disorders, (2) advance development of non-invasive diagnostics to allow for longitudinal monitoring, and (3) pave the way for new opportunities for disease prevention and treatment.