Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Despite the potential importance, very little is known about the fibrotic response in the central nervous system (CNS), and how this may impact the regenerative process. We identified persistent fibrotic scarring in the brain and spinal cord following neuroinflammation. Using lineage tracing and single-cell sequencing in the EAE mouse model of multiple sclerosis, we determined that this fibrotic scar is derived from proliferative CNS resident fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibroblasts using the fibroblast-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within inflammatory lesions and a reduction in motor disability. We further identified that interferon gamma pathway genes are enriched in CNS fibroblasts, and the fibroblast-specific deletion of Ifngr1 resulted in a reduced fibrotic scar in EAE. Additionally, we identified that platelet derived growth factor receptor signaling affects brain fibroblast development and fibroblast migration. In the retina, fibrosis occurs following retinopathies and can lead to retinal detachment and blindness, and yet little is known about the origin of this fibrosis as it has been difficult to model in mice. We generated a novel mouse model of oxygen-induced retinopathies that exhibits persistent fibrotic pathology, and used histology and single-cell sequencing to determine that the origin of the fibrotic scar in the retina is likely pericytes. These data delineate a framework for understanding CNS fibrosis.

The blood vessels in the central nervous system (CNS) have a series of unique properties, termed the blood-brain barrier (BBB), which stringently regulate the entry of molecules into the brain, thus maintaining proper brain homeostasis. Despite the dynamic nature of the brain, the BBB has largely been studied in a static context. We sought to understand if the BBB exhibited plasticity and whether neural activity could regulate BBB properties. Using both chemogenetics and a volitional behavior paradigm, we identified a core set of brain endothelial genes whose expression is regulated by neural activity. In particular, neuronal activity regulates BBB efflux transporter expression and function, which is critical for excluding many small lipophilic molecules from the brain parenchyma. Furthermore, we found that neural activity regulates the expression of circadian clock genes within brain endothelial cells, which in turn mediate the activity-dependent control of BBB efflux transport. These results have important clinical implications for efficiency of CNS drug delivery in the day vs. night, the BBB’s role in clearance of CNS waste products including Aβ in health and Alzheimer’s Disease, and understanding how neural activity can control these and other diurnal processes.

The aim of this dissertation is to investigate how dietary macronutrient composition, particularly ketogenic feeding, influences systemic metabolism, circadian rhythms, physical endurance, and blood-brain barrier function. While the ketogenic diet is employed increasingly for its neuroprotective and metabolic effects, its impact on behavioral rhythmicity and blood-brain barrier physiology remains incompletely understood. This dissertation addresses these knowledge gaps through a multi-modal approach, combining behavioral assays, circadian analyses, and molecular profiling of the blood-brain barrier.

In Chapter 2, I characterize how ketogenic feeding alters diurnal and circadian rhythms in mice, revealing a marked sex-dependent effect. Male mice fed a ketogenic diet exhibit significantly earlier onset of activity during the dark cycle, as well as a shortened endogenous circadian period under constant darkness conditions, suggesting a shift in central clock timing. In both sexes, ketogenic diet-fed mice show diminished voluntary locomotion, indicating impairments in physical stamina. Interestingly, female mice exhibit reduced locomotion without corresponding shifts in circadian or diurnal rhythmicity, highlighting a sexually dimorphic behavioral output of metabolic state.

In Chapter 3, I examine how different diets affects blood-brain barrier structure, function, and transcriptional identity. While high-fat/high-sucrose and ketogenic diet-fed mice show radically different metabolic phenotypes, neither diet induces overt changes in vesicular transport or tight junction morphology, nor grossly alters permeability to small hydrophilic solutes. However, transcriptomic profiling of isolated brain endothelial cells reveals robust ketogenic diet-induced changes in circadian gene expression, including core clock genes and circadian transcription factors, suggesting circadian modulation at the blood-brain barrier. I then show that the function of the efflux transporter P-glycoprotein displays circadian oscillation in conventionally fed animals, but loses amplitude under ketogenic diet feeding, despite an overall increase in efflux transport levels. These findings show that metabolic state can reprogram the temporal dynamics and magnitude of efflux transport across the blood-brain barrier.

Together, these findings reveal that the ketogenic diet exerts profound effects on circadian timing, locomotion, and neurovascular function. By integrating behavioral chronobiology with cellular analyses of the blood-brain barrier, this work reveals how a systemic shift in metabolic fuel utilization can influence both physiological rhythms and neurovascular transport dynamics in the brain.

The aim of this dissertation is to increase the understanding of how the cerebral vasculature (CV) becomes dysregulated in neurological disease, with a focus on the blood-brain barrier (BBB). I begin this dissertation with a literature review on the topics of the BBB in health and disease, the complex pathology of sporadic Alzheimer’s disease (AD), the importance of lipids in neuroinflammatory contexts such as AD, evidence of CV dysfunction in AD, and a brief overview of the key findings of my dissertation research establishing a connection between these topics. A large body of literature implicates CV dysfunction, including avascularity, reduced cerebral blood flow (CBF), hemorrhage, and BBB permeability, as a common feature of AD pathology; however, the molecular nature of this dysfunction had never been explored. To address this knowledge gap, I employed a multi-omic approach, leveraging both proteomics and lipidomics, to identify the molecular changes to the CV in post-mortem sporadic AD patients. This multi-omic strategy revealed a loss of sphingolipid (SL) and ceramide (CER) biosynthetic machinery at the protein level, and a distinct loss of CERs at the lipid level. To understand the functional importance of these changes to the CV, I used mouse genetics to conditionally inhibit SL biosynthesis specifically in endothelial cells (ECs), the BBB-containing cells lining the lumen of the CV. In doing so, I found that loss of EC SL biosynthesis promotes BBB permeability in healthy adult mice and inhibits angiogenesis capacity promoting hemorrhage in response to a hypoxic environment; all of which have been shown to occur in AD patients. In this process, I noticed a fatty acid (FA) elongase enzyme, Elovl7, to be downregulated in the AD CV at the protein and transcript level. Further, I found that Elovl7 is uniquely expressed in brain ECs in both humans and mice and becomes dysregulated in several mouse models of neurological disease. To clarify the role of Elovl7 in brain ECs, I used mouse genetics to conditionally delete Elovl7 from brain ECs of adult mice. In doing so, I discovered that Elovl7 generates phosphatidylcholines (PCs) and lyso-PCs (LPCs) for the CV, and its absence results in decreased levels of docosapentaenoic acid (DPA), an anti-inflammatory FA, in the brain. When confronted with a systemic inflammatory stimulus, loss of Elovl7 exacerbated hemorrhage and drove persistent reactivity in astrocytes and microglia. Together, these findings show that lipids synthesized within brain ECs are important in maintaining CV integrity and modulating neuroinflammation. This body of work reveals multiple novel targetable therapeutic avenues to fortify the brain vasculature across a broad spectrum of neurological disorders.

Throughout the body, the vasculature functions as a delivery system, supplying oxygen and nutrients crucial for cell activity and survival. Central nervous system (CNS) vasculature plays a second critical role as a barrier system, limiting brain entry of bloodborne ions, molecules, and cells. Indeed, CNS blood vessels are endowed with a series of unique properties that allow them to tightly regulate the extracellular environment of the brain, and together these properties are termed the “blood-brain barrier” (BBB). The barrier properties of CNS vasculature are critical for proper brain function; BBB dysfunction during disease or injury can lead to a cascade of consequences resulting in neuronal death. Several questions remain regarding how BBB properties are maintained in health and the mechanisms underlying their dysfunction in neuropathologies.

The brain uses an extraordinary amount of the body’s energy, and without constant blood supply from the vasculature, its functions quickly deteriorate. Indeed, neuronal activity leads to a temporary increase in local blood flow to provide neurons with sufficient energy and oxygen, and this phenomenon is known as neurovascular coupling (NVC). NVC has been shown to involve various cell types and signaling molecules, but it is still unclear to what extent endothelial cells, the cells that form the innermost walls of the vasculature, participate in NVC.

This dissertation explores brain vasculature in health and disease. Chapter I focuses on BBB dysfunction in disease, identifying PDLIM1 as a marker for BBB dysfunction and characterizing its role in CNS endothelial cells. Chapter II focuses on the neurovascular unit, specifically whether microglia play a role in maintaining barrier properties in healthy vasculature. Chapter III demonstrates the dynamic nature of neurovascular cholesterol metabolism and how this metabolic pathway influences NVC.

This dissertation opens with a review on the important players in the sphere of glutamate in the brain as well as the sphere of the blood-brain barrier to best understand their overlap and intersection. By delving into that which we know about the need for glutamate in different processes and the capabilities of the brain vasculature, we can highlight what is left to conjecture about which we do not know. Glutamate is already highly regulated by astrocytes and neurons, however, evidence of the brain vasculature highly expressing glutamate transporter, slc1a1, opens the possibility that the blood-brain barrier is participating in this very necessary glutamate regulation. Slc1a1 is also an interesting gene to further investigate because of its association with neuropsychiatric conditions, obsessive-compulsive disorder, schizophrenia, and some ambiguous, understudied cognitive symptoms in dicarboxylic aminoaciduria. When we sought to further characterize the localization of slc1a1 in the brain, we found that slc1a1 is indeed expressed by the vasculature of the brain, but in a region-dependent manner. The cerebellum and hypothalamus are the regions with the greatest amount of vascular slc1a1, and the pattern of endothelial slc1a1 levels expressed per region is inverse to the pattern of slc1a1 detected in the nonvascular parenchyma. At an ultrastructural level, slc1a1 was detected to have signal in both the luminal and abluminal membranes of the endothelial cell. These findings unveiled new questions about the slc1a1 function in the brain. By conditionally knocking out this gene in endothelial cells in mice, we further investigated slc1a1’s role in the brain vasculature. These endothelial cell-specific knockouts of slc1a1 have a sociability-associated behavioral phenotype when compared to control mice in the same contexts. At rest, we did not identify large differences in glutamate metabolite levels across the compartments of the blood-brain barrier nor in seizure susceptibility or spontaneous excitability. However, when we modeled an environment where there might be an excess of glutamate in the extracellular space using a subseizure dose of chemoconvulsant, kainic acid, we identified some differences. With subseizure kainic acid doses, the brains of the endothelial cell-specific knockouts of slc1a1 demonstrated higher excitability on electroencephalogram. Additionally, subseizure kainic acid doses elicited differences in glutamine and cystine content within the sample of enriched microvessels in the endothelial cell-specific knockouts compared to the littermate controls. This dissertation aims to seek greater understanding of the action that the blood-brain barrier takes in regulating brain behavior and glutamate-associated signaling.

The blood-brain barrier (BBB) is a set of properties belonging to endothelial cells that make up blood vessels in the central nervous system (CNS). The BBB’s function is to reduce the transport of nonspecific ions and molecules between the blood and brain and import specific molecules to maintain brain homeostasis. The cerebral vasculature is also known to be tightly coupled to neural activity and dynamically changes blood flow to satisfy the energy demand of active brain regions. Although the cerebral vascular blood flow is tightly coupled to neural activity, the question remains whether BBB properties can dynamically respond to neural activity, as well. Through manipulating glutamatergic neurons in mice, we have shown that neural activity can dynamically regulate both the expression and function of BBB genes in the cerebral vasculature, namely, ATP- binding cassette (ABC) efflux transporters. Additionally, we found that neural activity dynamically regulates circadian clock-related genes in endothelial cells. This led us to ask the question of what is the functional role of the circadian clock in endothelial cells of the cerebral vasculature? Through knocking out a core clock gene, Bmal1, specifically in endothelial cells, we found that the vascular clock regulates ABC efflux transport and animal behavior. We propose the idea that neural activity entrains the vascular-specific circadian clock, which regulates ABC transporter activity in a circadian manner, and deletion of the clock in endothelial cells causes dysregulation of ABC transport, thus disrupting the chemical microenvironment of the brain, resulting in complex behavioral phenotypes.

Wound healing is paramount for the survival of multicellular organisms, but this process can lead to scarring if left uncontrolled. Retinal scarring, in particular, occurs in many prevalent retinal diseases and can be devastating as this pathology leads to blindness. Unfortunately, little is known about retinal scarring, such as the origin of cells producing the scar and the cellular mechanisms leading to scar formation. Understanding these elements behind scar production is vital in creating therapeutic targets that can prevent potential blindness in those affected by retinal scarring. Although there are many retinal disease models in mice, a reliable mouse retinal scarring model has not been established. In this study, I develop two scarring models in mice. One is a mouse model for the disease phthisis bulbi, which is characterized as a shrunken, non-functional eye that is caused by injury, inflammation or other infections. We induce these characteristic symptoms by injecting dispase, a neutral protease, into the eyes of mice, causing degradation of retinal tissue, eye shrinkage, and the production of scars. The other scarring model is termed as Long Oxygen Induced Retinopathy (LOIR), which is a more pathologically severe model of the traditional “Smith” OIR mouse model of retinopathy of prematurity. This newly developed scar producing model is induced by prolonging the exposure of highly concentrated atmospheric oxygen from the traditional “Smith” OIR model protocol of 5 days to 14 days. With these models, I answer what cell type(s) are producing scar tissue in the retina.