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Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.

<p dir="ltr">Neurotrauma encompasses a broad category of injuries affecting the central nervous system (CNS), which includes both the traumatic brain injury (TBI) and spinal cord injury (SCI). These injuries can result from various causes, including accidents, falls, sports-related incidents, and other traumatic events, affecting millions of individuals annually. Traumatic injuries are the leading cause of disability, and moreover are associated with elevated risk of developing cognitive impairments and neurodegenerative diseases (ND) such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). The elevated ND risk arising from neurotrauma poses significant burdens on healthcare systems and affect life quality of affected individuals, emphasizing the critical need for research aimed at understanding the underlying mechanisms conferring ND risk from the lesion center to CNS. The goal of my thesis is to understand persistent molecular changes post SCI associated with ND using a combination of a rat animal model and neuronal cultures derived from human induced pluripotent stem cells.</p><p dir="ltr">I started with Sprague-Dawley rats with T10 spinal cord contusive injury; and assessed immediate and persistent changes in transcriptomic and epigenetic markers via next generation sequencing (NGS) at primary lesion site and distal spinal cord tissue. Along with global changes in chromatin arrangements and DNA methylation, we observed significant transcriptomic changes enriched for pathways of inflammatory responses, and synaptogenesis. These changes were further verified using immunohistochemistry and super resolution microscopy. To further understand the long-term brain abnormality linked to SCI, we investigated persistent alterations in the composition and molecular profiles of both the male and female motor cortex 30 days after injury. Immunohistochemistry revealed that SCI leads to neuronal loss and changes in synaptic density and morphology; and significant alterations in the neuron-astrocyte ratio and astrocyte morphology, in male motor cortex supporting our hypothesis that SCI may increase the risk of neurodegeneration by affecting the motor cortex. Comparison of transcriptomic data collected at a sub-acute stage in male rats, namely 7 days post injury, with 30 days post injury, identified persistent and de novo changes that occur primarily after recovery of spinal cord injury, which are enriched for neuronal and synaptic function related pathways. Interestingly, neuroendocrine-related pathways were prominently implicated at the chronic stage of SCI, with Esr1 identified as a major upstream regulator offering protective effects in females that did not exhibit significant alterations in cellular composition or morphology after SCI. Collectively, our study paved the way towards understanding sexual dimorphism in brains after spinal cord injury and provides a plausible connection between spinal cord injury and neurodegeneration later in life that were further investigated using a humanized culture model.</p><p dir="ltr">We established the feasibility of using hiPSC derived neurons to examine long term neurotoxic mechanism using lead (Pb) as a model chemical with strong associations with elevated AD risks later in life. A similar culture system was then used to assess persistent neurotoxicity of acrolein, a chemical that is known to emerge in brains post traumatic injury. We found that acrolein induced alterations in neuronal network morphology, synaptic density, and excitability. Furthermore, acrolein exposure negatively impacted mitochondrial function and persistently altered neuronal resilience towards a secondary stressor of mitochondria, namely MPP+. Acrolein exposure also alters the expression of tau and tau phosphorylation which collectively result in increased cellular vulnerability toward paired helical filament (PHF-tau) seeding, a known neurotoxin associated with ND. These findings collectively provide molecular insights as to how acrolein can partake alterations in neural function and resilience to stressors; and relay ND risks in neurotrauma patients later in life.</p><p dir="ltr">In conclusion, our comprehensive investigation employing both rat and hiPSC models uncovers plausible molecular pathways connecting SCI to neurodegenerative diseases, providing insights into the enduring consequences of these injuries on affected patients.</p>

<p dir="ltr">Neurodegenerative diseases are progressive, incurable conditions that affect tens of millions of people worldwide and are characterized by the aggregation of misfolded protein in the brain. Though the precise role of these amyloid aggregates in the onset and progression of these diseases is not clear at this time, there is a pressing need to understand how they form and spread in human disease. In service to these aims, I have conducted three small projects to expand knowledge in this regard. I first investigated the use of thioflavin T, a common amyloid stain, as an affinity reagent for the general purification of amyloid filaments from <i>ex vivo </i>samples, observing strong potential using a relatively simple, inexpensive magnetic bead conjugation technique. I next analyzed the formation of filaments of a truncated recombinant amyloid-beta peptide with residues 1-35, observing a new filament type formed at low pH in the wild-type sequence of this truncated peptide. Finally, I conducted structural studies on amyloid-beta(1-42) filaments prepared under different conditions consistent with traumatic brain injury to observe their effect on amyloid folding. While I found no effect of differential conditions on filament type, the low-resolution structures solved were highly consistent with aggregates found in Alzheimer’s disease patients, presenting a promising way forward for <i>in vitro</i> modeling of amyloid filaments that are true to pathology. In sum, the work here presented advances the concepts of both how amyloid aggregates from patient brains can be best prepared for structural analysis, and the factors underpinning their aggregation at the onset of neurodegenerative disease.</p>

<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>