No description available.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). Neuropathological studies in both human MS and the experimental autoimmune encephalomyelitis (EAE) animal model have shown that endothelial cell (EC) inflammation, associated with focal breakdown of the blood-brain barrier (BBB) and neo-angiogenesis, is prevalent in demyelinating plaques. Neo-angiogenesis and BBB damage contribute to leakage of serum components, infiltration of immune cells into the CNS, neuroinflammation, axonal demyelination, neuronal dysfunction, and disease progression. In Chapter 1, I introduce MS and its pathological hallmarks related to immune and vascular dysfunctions, the clinical course of MS progression, genetic and environmental influences, current treatments, and animal models. Next, I elaborate upon the pathways and processes involved in the development of a functioning CNS vascular system and the BBB. Finally, I discuss what is currently known about the contribution and the underlying mechanisms of neo-angiogenesis in MS and other diseases. While an increase in vessel density has been documented for both MS and EAE lesions, the origin and pathways that drive formation of new, but leaky, blood vessels in EAE are poorly understood. In Chapter 3, I address these questions by performing single-cell RNA-sequencing (scRNA-seq) of 45309 ECs isolated from the spinal cord of control, acute and chronic MOG35-55 EAE mice. Based on expression patterns of blood vessel subtype-specific markers, I identified 23 distinct EC clusters with arterial, capillary, venule, and vein identities in either control or disease states. I performed differential gene expression and gene set enrichment analyses comparing control and disease EC clusters for each vascular subtype to identify which vessels exhibited gene expression profiles indicative of neo-angiogenesis in EAE. I found that molecular signatures of neo-angiogenesis are upregulated specifically in venous ECs during acute and, to a lesser extent, chronic EAE. Consistent with these data, EC proliferation is upregulated in veins in the EAE spinal cord. RNA fluorescent in situ hybridization and immunofluorescence staining confirmed increased expression of key angiogenic markers Egfl7, Ecm1, Serpine1 and Emcn, and the tip cell marker Mcam, with a corresponding increase in vein density, in demyelinating white matter lesions of EAE spinal cords relative to controls. I also assessed changes in expression of some of these markers in human MS tissue and discovered upregulated expression of EGFL7 in cortical white matter lesions of MS patients, concomitant with increased vascular density. In Chapter 4, I examine the signaling pathways that may trigger pathogenic angiogenesis in EAE. I discovered that, in contrast to developmental angiogenesis, VEGF-A and TGF-β signaling may act as the driver of neo-angiogenesis in EAE. To test this hypothesis, I used a humanized VEGF-A blocking antibody, bevacizumab, to block VEGF signaling and found that this treatment ameliorated the MOG35-55 EAE neurological score by reducing expression of several angiogenic markers Egfl7, Ecm1, Serpine1, and Emcn, as confirmed by both in situ hybridization and computational analysis of scRNA-seq data. Immune profiling of spleens and spinal cords by flow cytometry did not show changes in immune cell activation in bevacizumab-treated mice relative to IgG controls, indicating that the protective effects of VEGF blockade are not due to defects in the initiation of the immune response. Finally, in Chapter 5, I summarize the major findings of my dissertation and propose a model for the mechanisms by which neo-angiogenesis contributes to pathology in MS/EAE. I also present several future avenues of research that can be pursued to further our understanding of the molecular and cellular changes underlying pathogenic angiogenesis and its role in MS/EAE. While most current disease-modifying MS therapies aim to reduce inflammation and infiltration of immune cells into the CNS, these findings may lead to development of additional potential therapeutics that may reduce pathogenic neo-angiogenesis in order to alleviate long-term neurological deficits in MS. Additionally, since postcapillary venules and veins are the major sites of immune cell infiltration, BBB damage and neo-angiogenesis in EAE, the findings of this study suggest that development of treatment modalities that target venous ECs with anti-angiogenic compounds may be more effective in inhibiting the growth of pathogenic neovessels than therapies directed against the entire endothelium.

This thesis is broken into four chapters. The first two chapters summarize two separate lines of investigation into the role of a putative neurotoxin in the pathogenesis of Huntington's Disease (HD). The third chapter outlines an investigation of the putative role of beta-N-methylamino-L-alanine (BMAA) in the pathogenesis of amyotrophic lateral sclerosis (ALS), while the final chapter details a post-mortem investigation of the contents of biogenic amines and amino acids in the brain of a man who died of a familial form of parkinsonism. Chapter I is a description of a chromatographic technique developed to isolate quinolinic acid (QA), an endogenous compound implicated in the pathogenesis of HD, from deproteinized human sera. A cation exchange column was used to selectively isolate QA, which was eluted with 10 mM HCl. The eluted fractions were analyzed by UV spectrometry to isolate and quantify QA. Once the fractions corresponding the elution of authentic QA were isolated, concentrated and the excess HCl removed, the fractions were added to growing fetal rat striatal explant cultures as an assay of neurotoxicity. Since HD involves the selective degeneration of GABAergic neurons in the striatum, the activity of glutamic acid decarboxylase, the final enzyme in the synthesis of GABA, was used to determine the viability of the cultures. Unfortunately, the method was confounded by the contamination of all effluents by compounds originating from the cation exchange resin, which were discovered to be neurotoxic to the striatal cultures, and as a result the investigation had to be abandoned. Chapter II describes an investigation designed to further characterize the nature of neurotoxicity observed in the sera obtained from patients with HD (Perry et al. 1987). Compounds with the capacity to selectively stimulate neurons at the N-methyl-D-aspartate (NMDA) receptor have been implicated in a variety of neurodegenerative disorders, including HD. Selective antagonists at the NMDA receptor have been shown to protect neurons from the degenerative effects of such "excitotoxins". The investigation described used MK-801, a potent noncompetitive NMDA antagonist, in an attempt to protect fetal rat striatal cultures from the neurodegenerative effects of the sera obtained from HD patients. The results obtained were equivocal. No evidence was obtained to support a role of the NMDA receptor in the mediation of the neurotoxicity, and in addition the neurodegenerative effects of HD sera were not reproduced in the present investigation. A variety of possible explanations for the apparent discrepancy are suggested. Chapter III describes an experiment intended to produce an animal model of ALS based on the observations by Spencer et al. 1987 that chronic oral administration of BMAA in monkeys produced the histological and behavioural characteristics of this disease. In the present investigation synthetic D,L-BMAA was given by gavage to mice over an eleven week period. Since BMAA is known to act at the NMDA receptor, a subset of the mice were also given MK-801 in an effort to protect them from any deleterious effects based on the action of BMAA at this receptor. The animals were sacrificed at the end of the experiment, and biochemical analyses were performed on the striata and cortices of the animals. In addition, neuropathological studies were performed on the spinal cords, basal ganglia and related structures. The results indicated no biochemical or neuropathological abnormality as a result of BMAA administration. Chapter IV describes a post-mortem investigation of a man who was a member of a well described pedigree which carries an autosomal dominant form of parkinsonism. The object of the investigation was to determine post-mortem levels of dopamine, noradrenaline, serotonin and their metabolites, in addition to amino acids in various regions of brain. Although conflicting evidence was obtained during life, neuropathological findings and the present neurochemical analyses confirm the degeneration of the nigrostriatal dopaminergic tract, characteristic of parkinsonism, in this man. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

This project consists of three distinct, but sequential studies that explore the psychological factors and neuropathological substrates of metacognition or self-awareness among older adults. Study 1 examines the premorbid, psychological characteristics associated with metamemory—the mainstay of metacognitive research—in a healthy, community-dwelling cohort of older adults. Study 2 builds on these analyses, and examines the psychological characteristics associated with metacognition, more broadly, in a neurologic cohort of older adults with Essential Tremor (ET). Study 3, which utilizes post-mortem evaluations of participants from Study 2, goes beyond premorbid characteristics and examines whether distortions in metacognition are in part attributable to an underlying disease process. Findings demonstrated that psychological characteristics were associated with metacognitive accuracy in a healthy, community-dwelling cohort of older adults, but not among individuals with ET; further, distortions in metacognition among individuals with ET were better attributable to non-ET specific pathologies, such as amyloid β, neurofibrillary tangles, and regional-specific atrophy. This project underscores the importance of employing a biopsychosocial approach to understanding the factors that influence metacognition. Ultimately, by understanding and working effectively with awareness phenomena, there is a strong potential to reduce disability and enhance well-being.

High-voltage activated calcium channels underlie many critical functions in excitable cells and their dysfunction has been implicated in a myriad of cardiovascular and neurological diseases. These channels are multimeric protein complexes composed of α1, β, and α2δ subunits; currently, all calcium channel blockers target either the pore-forming α1 or extracellular-facing α2δ auxiliary subunit. These pharmacological agents have been invaluable in delineating the individual function of each subunit within excitable cells that express multiple calcium channels. Yet, no current tool allows similar pharmacological dissection of individual cytosolic β subunits, preventing our understanding of how distinct β subunits affect the function of calcium channel complexes. Further, small-molecule calcium channel blockers are highly-valued therapeutics for certain conditions, yet their propensity for off-target effects precludes their use in other diseases. In certain applications, genetically-encoded calcium channel blockers may enable channel inhibition with greater tissue-precision and versatility than is achievable with small molecules. Previous work that found the family of RGK proteins powerfully inhibits high-voltage activated calcium channels in part via an association with the β subunit. However, the myriad functions of RGK proteins limit the utility of this approach. In this work, we circumvent this issue by isolating single-domain antibodies (nanobodies) that target the auxiliary CaVβ subunit. We then paired these nanobodies with the powerful enzymatic activity of the HECT domain E3 ubiquitin ligase Nedd4L, to selectively target the calcium channel for ubiquitination. We found this strategy effectively eliminated functional calcium channels from the surface of HEK293 cells, myocytes, and DRG neurons. This modular design permitted us to characterize a pan-β inhibitor (CaV-aβlator) in chapter 2 while refining the approach with a β1-selective channel inhibitor in chapter 3. In chapter 4 I demonstrate that it is possible to hijack the endogenous ubiquitin machinery of the cell by creating Divas: divalent nanobodies that are capable of recruiting endogenous Nedd4L to regulate the calcium channel. Finally, we demonstrate the potential for these genetically-encoded calcium inhibitors to be employed as therapeutic agents by targeting CaV-aβlator to sensory neurons in order to reduce the onset of neuropathic pain. Altogether, this work lays the foundation for nanobody-based genetically-encoded calcium channel inhibitors that have the potential to achieve superior precision in regards to molecular and tissue specificity.

Traumatic brain injury (TBI) is a prevalent condition worldwide with 1.7 million incidences in the U.S. alone. A range of clinical outcomes have been reported post TBI, including dementia, memory loss, and impaired balance and coordination. The lack FDA approved treatments for TBI drives the need for improved prevention and therapeutic strategies. Finite element (FE) models of brain injury mechanics can be used to advance these efforts. These computational models require appropriate constitutive properties in order to predict accurate brain tissue response to injury loading. Suitable experimental models need to be implemented to match the resolution and computational power of FE models. The first aim of this thesis was to characterize the mechanical properties of brain tissue. Here, human, porcine, and rat brain tissue mechanical responses to multistep indentation of increasing strains up to 30% strain were recorded. We tested whether the quasilinear theory of viscoelasticity (QLV) was required to capture the mechanical behavior of brain tissue, but observed that linear viscoelasticity was sufficient under the loading condition applied. Using this fitting model, brain tissue stiffness was found to be dependent on anatomical region, loading direction, age, sex and species to varying degrees. This analysis elucidated factors that affect brain tissue injury mechanics and can be used to improve the accuracy of FE models of brain tissue deformation to predict a biofidelic response to TBI. There is growing evidence linking TBI to pathologies leading to increased risk of neurodegeneration, like tauopathies. However better understanding of these underlying mechanisms is still needed. In our study, we utilized a custom shock tube design to induce blast TBI (bTBI). To isolate the effect of bTBI-induced tau pathology, tau was extracted from sham and shockwave exposed mice 24 hours post injury, referred to as sham and blast tau respectively. We showed that bTBI increased phosphorylation of tau and its propensity to oligomerize. Treatment with blast tau resulted in impaired behavior in mice as well as reduced long term potentiation (LTP) in acute hippocampal slices. Treatment with brain isolate from shockwave exposed tau knockout mice did not exhibit altered behavior or LTP response, eliminating the possibility that any confounding factor in the blast tau preparation was responsible for the impaired outcome. Administration of de-oligomerized blast tau prevented these cognitive impairments, suggesting that toxic effect of blast tau was attributed to its oligomeric form. Here we showed that blast injury can initiate cascades in tau pathology and exposure to this progression results in worsened neurological outcome. Tau phosphorylation is mainly regulated by protein phosphatase 2A (PP2A), whose activity can be altered by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1). We sought to leverage this mechanism by infusing LCMT-1 and PME-1 transgenic mice with sham and blast tau. LCMT-1 overexpression prevented behavior and LTP deficits induced by oligomeric blast tau. Furthermore, PME-1 overexpression worsened behavior and LTP response at subthreshold doses of oligomeric blast tau. Together, this illustrated the ability of these two enzymes to regulate the response to exposure of bTBI-induced pathogenic forms of tau. This study indicates the potential of targeting PP2A activity as a viable strategy for therapeutic intervention. In conclusion, this research expands our understanding of the complexity of brain tissue injury mechanics to inform computational models of TBI, illustrates the deleterious effect of pathogenic forms of tau induced by blast injury on cognitive function, and presents a potential target mechanism for the investigation of therapeutic strategies.

Advances in sequencing technologies have sparked the discovery of new genetic etiologies for neurological and neurodevelopmental disorders. As new disease-causing mutations are unveiled, questions into the specific mechanisms of pathogenicity and potential therapeutic approaches arise. To address these questions, in vivo and in vitro models have been generated and analyzed; but how best to utilize these models, and how well they recapitulate the human brain, is still not fully understood. Within the work discussed in this thesis, we address this problem through the transcriptomic and functional interrogation of these models in the context of neurodevelopment and disease. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing to examine the longitudinal transcriptomic profiles of neuronal network establishment and maturation in ex vivo mouse cortex- and hippocampus-derived cultures. Our data highlights unique developmental transcriptomic profiles for individual genes, disease gene subclasses, and biological processes, and discusses cell population-specific divergent transcriptomic profiles between genes associated with neurological diseases, focusing on epilepsy and autism spectrum disorder. We also compared the data from our ex vivo system to transcriptomic data collected from in vivo neonatal and adult mouse brains and human cortical organoids, highlighting the importance of the generation and consideration of system-specific transcriptomic datasets when looking into a gene, disease, or biological process of interest, and serves as a vital resource for researchers. In Chapter 3, we propose a high-throughput drug discovery paradigm utilizing the application of transcriptome reversal for neurodevelopmental disorder-associated genes that affect the transcriptome. This approach describes the idea that if gene dysregulation is causal for the pathogenicity of a disease, then correcting the transcriptional signature should have a therapeutic effect. We demonstrated that small-molecule induced gene expression changes vary between both cell lines and neural cell populations, and highlight both the importance of selecting the appropriate model of disease and creating cell population-specific signatures for compounds and disease. In Chapter 4, we focus on the utilization of multi-electrode arrays for the electrophysiological characterization of primary cortical networks derived from mouse models of epileptic encephalopathy. This technique allows for the analysis of numerous neuronal and network synchronization metrics for spontaneous longitudinal activity and responses to external stimuli in the form of electrical stimulation and compound addition. In particular, mouse models with mutations in the genes Grin2a, Gnb1, and Scn1a were analyzed. We discovered significant hyperexcitability, bursting, and synchrony phenotypes, and discuss how acute and chronic compound addition can be used to interrogate biological pathways and reverse disease activity signatures.

Paget's disease of the bone is characterized by a focal increase in the rate of bone turnover, which goes through phases of activity and quiescence. Most patients are asymptomatic. The two cardinal features are pain and deformities, and many complications may arise. Diphosphonates and calcitonin are the main therapeutic modalities.

The dysregulation of immune system function has been centrally implicated in numerous age-related and neurodegenerative disorders, including Alzheimer’s disease (AD). Genetic susceptibility studies have positioned microglia, brain-resident immune cells, as critical actors in the development and the progression of the disease. Microglia are highly plastic cells with diverse functions across many modalities, and the appropriate regulation of their activities are a prerequisite for central nervous system homeostasis and cognitive health. Aging and pathogenic contexts are posited to modify microglial behavior, inhibiting their neuroprotective function and promoting a dysfunctional state that drives disease. However, the mechanisms underlying these pathogenic alterations in microglial state and function are complex and poorly understood. This thesis identifies three elements that are altered in the AD brain and investigates how these mechanisms may serve as triggers producing microglial dysregulation in AD. Chapter 3 examines the role of expression of the transposable element LINE-1 in AD-related microglial dysfunction. Chapter 4 explores the regulation of PLCG2, which encodes a critical AD-associated signaling enzyme. Chapter 5 investigates the role of the AD-linked sorting receptor SORL1 in microglia. Together, these data expand our understanding of mechanisms driving altered microglial pathophysiology in AD and illuminate pathways of interest with potential therapeutic applications meriting deeper exploration.

The functional compartmentalization underlying neuronal polarity makes tightly regulated intracellular transport between the cell body, axons, and dendrites essential for proper development and homeostatic maintenance. Disruptions to neuronal trafficking are a major cause of neurodegenerative disease. Pathogenic variants in the microtubule motor protein KIF1A cause KIF1A Associated Neurological Disorder (KAND), a spectrum of rare neurodegenerative conditions. KAND is clinically and genetically heterogeneous, with a broad phenotypic spectrum and over a hundred pathogenic variants identified. KAND is poorly understood at both the clinical and molecular level, and there is currently no treatment. This work characterizes the natural history of KAND and describes a novel heuristic severity score. This severity score is then used to show how the location of pathogenic missense variants within the KIF1A motor domain correlates with disease severity, providing evidence the clinical phenotypic heterogeneity in KAND reflects and parallels the molecular phenotypes. Insights from the neuropathology of deceased KAND patients is used to focus a histopathologic assessment of the C3-Kif1aLgdg mouse model. C3-Kif1aLgdg/Lgdg mice have a cerebellar axonal torpedo phenotype, paralleling some of the pathological changes seen in the patients. Phenotypically, the C3-Kif1aLgdg mice were found to recapitulate some of the symptoms seen in patients including progressive spasticity and gait abnormalities associated with hind limb paralysis. To model the disease at a cellular level, iPSCs were derived from affected individuals and successfully used to generate neural stem cells and neurons. These patient-derived neurons were found to have increased markers of protein aggregates, a cellular phenotype that can be used to test potential treatments. Taken together, these studies provide foundational knowledge for future therapeutic development.