Theses Doctoral

Brain macrophage and extracellular vesicle response to focused ultrasound neuroimmunotherapy

Kline-Schoder, Alina R.

In addition to protecting the brain from circulating pathogens and neurotoxins, the blood-brain barrier (BBB) limits both the delivery of drugs to the brain and the migration of neurological disease biomarkers from the brain into the blood. Focused-ultrasound blood-brain barrier opening (FUS-BBBO) addresses both of these transport limitations by transiently and noninvasively opening the BBB. Although originally designed as a drug delivery method, FUS-BBBO has also been shown to be an effective neuroimmunotherapy and method of improving liquid biopsy specificity for neurological disease. Prior to the work presented herein, the mechanism of FUS-BBBO neuroimmunotherapy remained poorly characterized and FUS-BBBO liquid biopsy remained poorly optimized.

Initially, we present the temporal response of brain macrophages to FUS-BBBO. Due totheir role as the main phagocyte in the brain and the well-documented association between their dysfunction and neurodegenerative disease progression, we hypothesized that FUS-BBBO affects brain macrophage population composition and phenotype. Utilizing temporal single-cell RNA sequencing, we establish that treatment remodels the immune landscape via a number of processes including microglia proliferation, disease-associated microglia population size increase, and central-nervous-system associated macrophage recruitment. To further elucidate the functional role of the brain macrophage response to FUS-BBBO, we find that their depletion is associated with significantly decelerated BBB restoration.

Secondly, we compare FUS-BBBO with two other methods of focused ultrasound neuroimmunotherapy, focused ultrasound neuromodulation (FUS-N) and focused ultrasound with microbubbles without BBBO (FUS+MB). FUS-N utilizes FUS parameters that alter neuronal connectivity via a combination of mechanosensitive receptor interactions and transient hypothermia without the injection of microbubbles (MB). FUS+MB is the combination of MB and FUS below the pressure threshold for BBBO (FUS+MB). FUS+MB has been shown to trigger morphological activation of brain macrophages and has proven efficacious as a method of immunotherapy within the peripheral nervous system. Due to the findings of brain macrophage modulation in response to FUS-BBBO, we compare brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s Disease (AD) pathology. We identify FUS-BBBO as the paradigm which maximizes brain macrophage modulation including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between FUS cavitation dose and brain macrophage phagocytosis.

Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to characterize the effect of FUS-BBBO on brain macrophage distribution in both wild-type and Alzheimer’s disease animals. Given their relevance within neurodegeneration and perturbation response, we emphasize the distribution of three brain macrophage populations - disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find a genotype-specific redistribution of each population, with an overall trend towards increased interaction with the brain-cerebrospinal fluid barrier after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology.

Finally, we investigate the role of extracellular vesicles (EVs) in both the mechanism ofFUS-BBBO neuroimmunotherapy and as a method of improving FUS-BBBO liquid biopsy. EVs are lipid vesicles that are responsible for the transport and exchange of diverse cargo between cells and have been reported to modulate the immune system. Isolation of EVs has emerged as a method of improving biomarker detection. Prior to this study, the effect of FUS-BBBO neuroimmunotherapy on EV concentration and content remained unexplored. We investigate the concentration and content of isolated EVs from the serum of mice and Alzheimer’s Disease patients prior to and after treatment with FUS-BBBO. We illustrate a 100% increase in EV concentration one hour after treatment in both mice and patients.

Furthermore, we illustrate an increase in murine EV RNA that is associated with the previously reported neuroimmunotherapeutic responses to FUS-BBBO including synaptic remodeling and neurogenesis. Finally, we illustrate an increase in AD biomarker concentration within the patient EVs three days after treatment that is proportional to the volume of blood-brain barrier opening. Overall, we establish that FUS-BBBO drug-free neuroimmunotherapy triggers complex brain macrophage modulation in a manner incomparable by other FUS neuroimmunotherapy paradigms. Furthermore, we illustrate the effect of FUS-BBBO on EV concentration and content in both preclinical and clinical experiments, indicating the role of EVs in FUS-BBBO neuroimmunotherapy and their utility as a method of improving liquid biopsy specificity. The results presented herein support the potential of FUS-BBBO as both a method of neuroimmunotherapy and a method of amplifying liquid biopsy specificity in Alzheimer’s Disease.

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More About This Work

Academic Units
Biomedical Engineering
Thesis Advisors
Konofagou, Elisa E.
Degree
Ph.D., Columbia University
Published Here
October 4, 2023