The most common malignant primary brain tumor, gliomas usually derive from glial cells including oligodendrocytes and astrocytes. These tumors are characterized by high rates of proliferation and aberrant migration which make them notoriously difficult to treat using standard treatment paradigms such as chemotherapy and radiation. In order for glioma cells to migrate into the surrounding brain tissue, they must undergo rapid and dynamic volume changes. Previous work published by the Sontheimer laboratory and others indicates glioma utilize the flux of ions across the cell membrane to aid in volume changes associate with cell migration. In this dissertation, I show the Sodium-Potassium- Chloride Cotransporter Isoform-1 (NKCC1) plays an important role in glioma biology, providing the ionic gradients required for volume regulation. Emphasizing its role in migration and invasion, I demonstrate that NKCC1 contributes to glioma cell migration using pharmacological or genetic inhibition of NKCC1. In addition, a genetic knockdown of NKCC1 abolishes Regulatory Volume Increase (RVI) in human glioma cells, suggesting that RVI in gliomas is solely dependent on NKCC1. Furthermore, NKCC1 inhibition reduced the invasion distance of glioma cells implanted into the brains of immunocompromised mice. Volume changes must be tightly coordinated to support successful cell invasion. One known regulator of NKCC1 in other cells is With-No-Lysine kinase 3 (WNK3). Here, I show that WNK3 is expressed in glioma cell lines and appears to have increased expression in patient biopsies of gliomas. I show hitherto unrecognized proii tein-protein interaction occurring between WNK3 and NKCC1 under hyperosmotic con-ditions. WNK3 inhibition through genetic knockdown reduces glioma cell migration and abolishes the cells’ ability to undergo RVI to a similar extent as a loss of NKCC1. This data suggests that WNK3 is the exclusive regulator of NKCC1 in glioma cells and that targeting NKCC1 and its regulation would provide innovative approaches to treating ma-lignant gliomas.

1 online resource (xii, 109 p.) : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

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According to the Central Brain Tumor Registry of the United States, the most common primary brain tumors are gliomas, tumors composed of cells of glial origin, most commonly astrocytes and oligodendrocytes. The most aggressive of these tumors are characterized by hyperproliferation, marked cellular and nuclear atypia, extensive infiltration into surrounding normal brain tissue, and large areas of cell and tissue death. Previous data published by our lab and others suggest that these biological processes may involve regulated cell volume changes. Using cell volume regulation in the presence of an anisosmotic challenge as a model for cell swelling and shrinkage, cell volume changes have been shown to involve the movement of molecules, or osmolytes, across the plasma membrane through channels and transporters. Water is osmotically obliged to follow the net movement of molecules, resulting in a net flux of water across the plasma membrane and an overall change in cell volume. As the most abundant anion in biological systems, chloride has been shown to be involved in the volume regulation of several cell types. However, chloride-mediated volume changes in human glioma cells have not been extensively studied. It is the primary goal of this dissertation to examine the mechanisms employed by human glioma cells to dynamically regulate their cell volume at rest, in the presence of anisosmotic conditions, and importantly during the biological processes of apoptosis and migration. We confirm the expression of the voltage gated chloride channels ClC-2, 3, and 5 in human glioma cell lines and patient biopsies. In addition, we demonstrate the expression of the cation chloride cotransporters, KCC1, KCC3, and NKCC1. Using a variety of techniques, including electrophysiology, Coulter Counter, and chloride-sensitive fluorescent dyes, we establish that the resting intracellular chloride concentration and cell volume are maintained by the basal activity of chloride cotransporters. While pharmacological inhibition of these cotransporters suggests that they are also involved in cell volume regulation during an aniosomotic challenge, chloride efflux through channels plays a more significant role in post-hyposmotic volume decrease. Similarly, inhibition of chloride channels, but not chloride cation cotransporters, inhibits the cell condensation that occurs in the presence of apoptotic stimuli. Cells treated with chloride channel inhibitors also demonstrated limited caspase 3 activity and DNA fragmentation, suggesting that the volume decrease is necessary for apoptosis. Finally, transwell migration of human glioma cells was blocked in the presence of chloride channel and transport inhibitors, suggesting that the mechanisms involved in cell shrinkage are necessary in glioma cell migration.

xi, 176 p. : ill., digital, PDF file

Neurobiology

Joint Health Sciences

glioma astrocytoma chloride channels migration apoptosis volume regulation

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Congenital HCMV infection of the developing brain is the leading viral cause of mental retardation and sensorineural hearing loss. To elucidate the pathogenesis of congenital HCMV CNS infections, we developed a small animal model of CMV infection where newborn Balb/c mice are peripherally inoculated with murine cytomegalovirus. In this model we observed transient deficits in cerebellar/hindbrain development as well as the recruitment of peripheral immune effector cells to the CNS parenchyma. CD8+ T-lymphocytes were the predominant mononuclear cellular infiltrates in the brain and immune-depletion of CD8+ cells resulted in increased viral genome copy numbers in the CNS. CD8+ T-lymphocytes exhibited an activated phenotype and were primarily specific against the IE1-168 epitope of MCMV. Ex-vivo stimulation of brain T-lymphocytes with IE1-168 peptide resulted in increased IFN-[gamma] and TNF-[alpha] production as well as degranulation as shown by increased surface staining with CD107a. Adoptively transferred brain mononuclear cells from neonatally infected animals were able to control MCMV replication in immune incompetent, MCMV infected adult mice. A substantial fraction of CD8+ T-lymphocytes in the CNS expressed CCR5 early in infection. To determine if CCR5 is critical for mononuclear cell infiltration into the MCMV infected neonatal brain, CCR5-/- mice were infected with MCMV. Infection was completely lethal to CCR5-/- mice by PN day 13. There was an overall decrease in leukocyte infiltration to the CNS in CCR5-/- animals. However, there was an unexpected increase in CD8+ T-lymphocyte frequency and magnitude in the CNS of MCMV infected CCR5-/- mice. These CD8+ T-lymphocytes primarily displayed a partially activated phenotype and displayed deficits in IFN-[gamma] and TNF-[alpha] production following peptide stimulation. Loss of CCR5 did not yield differences in CNS viral genome copy number between wild type and CCR5-/- animals. Together, these results suggest a role for CD8+ T-lymphocytes in the control of MCMV growth in the newborn CNS. Additionally, our results suggest that CCR5 does not play a role in T-lymphocyte recruitment but it could potentially promote CD8+ T-lymphocyte maturation in MCMV infected animals.

xi, 133 p. : ill., digital, PDF file.

Microbiology

Joint Health Sciences

MCMV CD8 T-cells CNS CCR5 newborn

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The recombinant fifth kringle domain of plasminogen (rK5) has been shown to induce apoptosis of dermal microvessel endothelial cells (MvEC), and this pro-apoptotic effect required rK5 binding to cell surface glucose-regulated protein 78 (GRP78). GRP78 is a member of the heat shock protein family and under certain conditions is expressed on the cell surface. I am interested in identifying new anti-angiogenic therapy for glioblastoma tumors. The efficacy of certain anti-angiogenic therapy can be improved when combined with radiation, and radiation is a standard therapy for glioblastoma tumors; therefore, I investigated the pro-apoptotic effect of rK5 combined with radiation on primary human brain MvEC. I found that treatment of brain MvEC with rK5 induced apoptosis in a dose- and time-dependent manner, and that prior irradiation significantly sensitized (500-fold) the cells to the pro-apoptotic effect of rK5. In both the unirradiated and irradiated MvEC, the rK5-induced apoptosis required the expression of GRP78 and the low density lipoprotein receptor-related protein 1 (LRP1), a scavenger receptor. This was determined by blocking studies with an antibody directed toward GRP78 and with a competitive inhibitor of ligand binding to LRP1, as well as by downregulation studies with small interfering RNA. Also, I found p38 MAP kinase to be a necessary downstream effector of rK5-induced apoptosis, in contrast to Erk and JNK. These data suggest that irradiation sensitizes brain MvEC to rK5-induced apoptosis and that this signal requires LRP1 internalization of GRP78 and the activation of p38 MAP kinase. The physiologic relevance of these findings are supported by my observation that expression of GRP78 protein is upregulated on the brain MvEC in glioblastoma tumor biopsies as compared to the normal brain, suggesting a potential tumor-specific effect of rK5. In addition, in an orthotopic intracerebral xenograft mouse model of malignant glioma, I found that treatment with rK5 significantly decreased tumor volume. Taken together, these in vitro and in vivo data potentially present an important new therapeutic role for rK5 in the treatment of malignant gliomas.

xvi, 124 p. : ill., digital, PDF file

Neurobiology

Joint Health Sciences

Glioma Angiogenesis GRP78 LRP1 Radiation Kringle 5

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The hypothesis that cell volume and the progression of the cell cycle are interdependent has surfaced off and on in the cell cycle literature for the past 30 years. However, a conclusion as to how cell volume is mechanistically involved in cell division has not been reached in mammalian cells. The aim of this dissertation was to establish how volume changes modulate cell cycle progression. Most of the studies addressing this question have examined mass content yet, more recently, focus has been placed on intracellular water, which is determined by the balance between mechanical and osmotic forces. As a result, ion channels and transporters which regulate intracellular osmotic content are integral to the maintenance of cell volume. In this dissertation, I show that a large, rapid and regulated volume decrease occurs as glioma cells progress through mitosis. I refer to this process as pre-mitotic condensation (PMC). This process is functionally linked to DNA condensation prior to cell division as the two events occur simultaneously, and inhibition of PMC results in a prolongation of DNA condensation. Further, my data demonstrates that glioma cells actively accumulate chloride, which acts as the primary energetic driving for cell volume changes in these cells. During the process of PMC, this gradient drives the efflux of chloride through ClC3 channels, which mediates water loss and the volume decrease. Interestingly, chloride accumulation to similar levels can be observed in immature astrocytes and neurons, suggesting that glioma cells recapitulate the biology of immature proliferating cells in the brain. This also suggests that my findings may have broader applicability to cell division in both neural cells and cancer.

xii, 132 p. : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

Voltage Gated Chloride Channels Pre-miotic Condensation DND Condensation C1C3 Mitosis Volume Regulation

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Na-Coupled Bicarbonate Transporters (NCBTs) are members of the bicarbonate transporter superfamily that play important roles in regulating intracellular pH (pHi) and extracellular pH (pHo) in the central nervous system. Electrogenic Na/bicarbonate Cotransporter 1 (NBCe1) is an NCBT that is expressed in different mammalian tissues including the brain. NBCe1 has three splice variants – NBCe1-A, -B and -C – that differ in the amino and carboxy termini. We have first performed a systematic characterization of the localization profiles of the three NBCe1 splice variants at mRNA and protein levels in rat brain. In these studies, we have used anti-sense probes and C-terminal antibodies that distinguish between the different NBCe1 splice variants. Using in situ hybridization technique, we have found that NBCe1-A is absent in rat brain whereas NBCe1-B and NBCe1-C are the brain-specific variants. Our results from immunohistochemistry were confirmed by those from immunoelectron microscopy demonstrating intracellular expression of NBCe1-A/B in neurons, and plasma membrane expression of NBCe1-C in glial cells more so than the surrounding neurons. Therefore, NBCe1 splice variants have different expression profiles in rat brain. Next, we examined whether the electrogenic NBC expressed in neurons is functional. We performed electrophysiology experiments in cultured rat hippocampal neurons bathed in either 5% CO2/24mM HCO3 – or 20% CO2/96mM HCO3 – using two different assays in whole-cell and perforated-patch modes. Based on the results from both assays, we found a population of rat hippocampal neurons that exhibited functional electrogenic NBC activity. At the immunohistochemistry level, using C-terminal antibodies to NBCe1-A/B and -C we found that all neurons in culture expressed NBCe1-A/B and -C variants. Also, performing double labeling studies on neurons using antibodies to NBCe1-A/B and -C and an antibody to glutamic acid decarboxylase 67 (marker of inhibitory neurons), we found that NBCe1-A/B and -C were expressed in both excitatory and inhibitory neurons. Therefore, the heterogeneity in NBC function was not due to differences in NBCe1 expression.

1 online resource (xi, 145 p. : ill., digital, PDF file)

Physiology and Biophysics

Joint Health Sciences;

expression excitatory functional nervous system protein variants

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Gliomas, primary brain tumors derived from glial cells, constitute the majority of malignant tumors within the central nervous system. The most malignant of these tumors, grade IV Glioblastoma multiforme, are characterized by extensive proliferation, cellular and nuclear atypia, angiogenesis, areas of necrosis, and widespread invasion into the brain parenchyma. Data from our lab and others have implicated ion channels in the invasion and proliferation of glioma cells. Moreover, calcium signaling in gliomas and other cells has been implicated in both migration and proliferation. The aim of this dissertation was to investigate Transient Receptor Potential Canonical (TRPC) channels role in glioma cell biology. TRPC channels are non-selective cation channels whose activation is downstream of the phospholipase C cascade and their role in glioma biology was previously unknown. In this dissertation, I show expression of TRPC channel subunits within glioma cell lines and glioblastoma multiforme patient biopsy lysates. Further, I demonstrate that these subunits form functional channels on the plasma membrane. Whole-cell patch clamp electrophysiology shows that TRPC channel inhibitors block small, linear currents and affect glioma calcium signaling by decreasing store-operated calcium entry. These pharmacological inhibitors additionally chronically impair glioma proliferation. To assess whether TRPC channels specifically impact glioma calcium signaling and proliferation, shRNA plasmids directed against TRPC1 were utilized. These plasmids specifically decrease TRPC1 subunit expression as shown by western blot analysis and I show through whole-cell recordings and calcium imaging that TRPC1 channels are functionally impaired. Furthermore, TRPC1 shRNA plasmids decrease glioma proliferation. With TRPC1 channel inhibition, glioma cells become larger and, often, multinucleated indicating a role for TRPC1 channels in cytokinesis. In this dissertation, I also show that TRPC1 channels localize to lipid raft fractions of the plasma membrane and propose that the localization and timing of TRPC channel activation plays a crucial role in glioma cell biology.

Neurobiology;

Joint Health Sciences;

TRPC proliferation multinucleation calcium

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Astrocytes, a type of glial cell in the central nervous system, are recognized for their support roles to neurons. They supply neurons with metabolites, maintain ion homeostasis and clear the synaptic space of neurotransmitters. However, it has been found that some astrocytes have receptors for neurotransmitter and neuroligands, exhibit Ca2+ excitability when stimulated via these receptors, and secrete gliotransmitters as an output of this Ca2+ excitability. In the Ca2+-dependent release of glutamate, it has been shown that the endoplasmic reticulum is the predominant source and the extracellular space is the auxiliary source of free Ca2+ necessary for triggering exocytosis. Because astrocytes possess mitochondria and plasma membrane Ca2+ transport systems that play roles in Ca2+ signaling in other cell types including neurons, I hypothesized that these elements also regulate Ca2+ signaling in astrocytes, and can modulate glutamate release. Employing cultured astroctyes from the visual cortex of newborn rats, I measured changes in cytosolic Ca2+ in stimulated astrocytes, and in parallel experiments measured glutamate release through a glutamate dehydrogenase assay. I took a pharmacological approach to manipulate the Ca2+ handling of mitochondria and the plasma membrane Ca2+ transport systems. I showed that regulation of Ca2+ signaling by mitochondria and the plasma membrane Ca2+ transport systems correlates with modulation of glutamate release where an increase in cytosolic Ca2+ resulted in increase glutamate release. These studies expand what is known about Ca2+ signaling and exocytotic transmitter release in astrocytes. These studies may have medical relevance since the importance of Ca2+ signaling in astrocytes has been implicated in models of various neurological diseases.

1 online resource (x, 141 p. : ill., digital, PDF file)

Neurobiology;

Joint Health Sciences;

astrocytes Ca2+ mitochondria plasma membrane exocytosis glutamate

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Aquaporins (AQP) constitute the primary pathway for water movement across cellular membrances. As a result, their expression and function are important for regulating cell volume. Both gliomas and astrocytes have highly effective volume regulatory mechanisms capable of rapidly moving ions and water into and out of cells following edemic episodes. In this dissertation, we examined the expression of AQPs in both gliomas and astrocytes in order to understand their function role as well as their regulation. We have shown that primary gliomas highly express AQP1 and AQP4 whereas cell lines express no AQP4 and variable AQP1. We reintroduced AQP1 and AQP4 into D54MG cell line lacking AQPs and examined their function. AQP1 enhanced tumor swelling and migration while AQP4 enhanced cell swelling and adhesion. PKC activation is known to regulate AQP function. Using PKC modulators, we found that while PKC did not regulate AQP1, AQP4 function was highly dependent on PKC activity. Specifically, phorbol 12-myristate 13-acetate, PKC activator, reduced cell swelling in AQP4-expressing tumors as well as migration. Chelerythrine, PKC inhibitor, enhanced both cell swelling and migration. Additionally, knocking out the PKC phosphorylation site at S180 eliminated the regulation of AQP4 water permeability and tumor migration. We verified this data by implanting AQP1, AQP4 and S180-AQP4 intracranially into mice and examined glioma invasion. We found that AQP1 and S180-AQP4 invaded on average significantly further than either AQP4 or control tumor cells lacking AQPs. AQP1 and S180-AQP4 invaded much further distances (>1500 μm) suggesting that AQP1 expressing tumors invade into tissue to set up satellite colonies characteristic of gliomas whereas AQP4 mediated invasion is regulated by PKC activity. Finally, we wanted to understand the regulation of AQP1 expression in astrocytes following injury. AQP1 is upregulated following cortical stab injury, and we could mimic this upregulation using an in vitro wound assay. We found that pMEK1/2 and pERK1/2 was increased up to 60 min post-injury but then subsequently decreased. AQP1 expression was increased by 16hrs. MEK1/2 and ERK1/2 activity as well as AQP1 expression was completely inhibited by U0126, a MAPK inhibitor. Astrocyte upregulation following injury is regulated by MAPK signaling.

1 online resource (x, 127 p. : ill., digital, PDF file)

Neurobiology

Joint Health Sciences

volume regulation tumor edema migration invasion cell signaling

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Cytoskeletal actin filaments underlie dendritic spine plasticity, critical for several forms of learning and memory. Therefore, understanding the mechanisms that regulate actin dynamics is essential to elucidate memory formation pathways. Myosins, a superfamily of actin binding proteins, have emerged as candidates for regulation of actin dynamics in the brain. Several myosin class II isoforms have been identified in brain, but their individual contribution to synaptic activity is still unknown. Based on the finding that myosin IIB regulates actin polymerization in the growth cone of developing neurons and that it is necessary for maintenance of dendritic spine structure, I hypothesized that in dendritic spines myosin IIB regulates actin dynamics, affecting synaptic transmission. I show that in addition to basal transmission, myosin IIB affects the maintenance of long term potentiation through a mechanism leading to reorganization and de novo synthesis of actin filaments. I further show that myosin IIB activity on actin filaments is critical for long-term memory consolidation. In addition to advancing our understanding of myosin IIB function in neurons, I was also able to characterize and identify a distinct muscle isoform of myosin II, MyH7B, in brain. The effects on dendritic spine morphology and synaptic strength of this isoform are different from those of myosin IIB. MyH7B acts as a structural myosin that maintains synaptic morphology and regulates trafficking of AMPA receptors to the synaptic surface through its activity on actin filament dynamics. Through my research I was able to identify a novel function for two distinct myosin II isoforms in brain, finding them critical for synaptic activity.

1 online resource (viii, 205 p. : ill., digital, PDF file)

Neurobiology;

Joint Health Sciences;

actin myosin dendritic spine synaptic transmission AMPA receptors plasticity

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Changes in the glioma microenvironment including oxygen (O2) levels, supply of amino acid such as L-glutamate and L-cystine and glutathione (GSH) concentrations play a critical role in glioma biology. Previous data from our laboratory and others have implicated the L-cystine/L-glutamate exchanger, system xc - in the invasion and proliferation of cancers including glioma. The central aim of this dissertation was to characterize the contribution of L-cystine uptake, GSH synthesis and L-glutamate release to migration and proliferation of glioma cells. In my first study, I examined the role of system xc - mediated L-glutamate release on glioma migration. I show that activation of Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-Rs) induces intracellular Ca2+ oscillations which promote migration. These findings suggest that a primary role of L-glutamate release in glioma cells is to promote migration through the autocrine and paracrine activation of Ca2+ permeable AMPA-Rs. In my second study, I examined how hypoxia affects the dependency of glioma growth on L-cystine and GSH. I show that glioma cells respond to hypoxia with increases in free radical production. When D54-MG cells were propagated under hypoxic conditions, we observed an increase in surface expression of xCT alone. I also show that hypoxia increases system xc - sensitive L-cystine uptake and consumption rates of GSH. In my last study, I examined the expression profile of xCT across high grade human glioma cell lines and in biopsied glioma samples from over 30 patients. The results show a heterogeneous expression of xCT ranging from no expression to abundant expression. Interestingly, glioma cells with minimal xCT expression no longer depend on L-cystine or GSH for growth. These studies show multiple important roles for the system xc - transporter in glioma biology.

1 online resource (xiii, 132 p. : ill., digital, PDF file)

Neurobiology;

Joint Health Sciences;

System xc- Glutamate Cystine Glioma Migration Proliferation

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The most common and most malignant gliomas are the Grade IV Glioblastoma Multiforme (GBM), characterized by a highly proliferative tumor mass and extremely invasive phenotype that allows for profuse dispersal of tumor cells throughout the brain. GBM cells must specifically regulate their cell volume to thrive within the edematous tumor mass and infiltrate throughout the tortuous extracellular spaces of the brain. These rapid and directed volume changes are governed by the controlled flux of potassium and chloride ions across the cell membrane, which move osmotically obliged water. The goal of this dissertation was to investigate the role of calcium-activated potassium channels (KCa channels) in regulating the potassium efflux pathways during volume change. The KCa channels are the major contributors to potassium conductance in GBM cells and are ideal for translating calcium changes into potassium efflux and volume decrease. In this dissertation, I report the activity of the IK channel, previously unknown to be expressed in GBM cells, in addition to the well characterized BK channel. Both KCa channels are biochemically isolated in lipid-raft domains together with the ClC-3 chloride channel and co-localize in lipid-raft domains in situ, suggesting that these channels are coordinated to specific areas of the plasma membrane for directed movement of KCl. During apoptosis cells undergo apoptotic volume decrease (AVD) that requires volume decrease under normotonic conditions. I demonstrate that the intrinsic pathway, induced by staurosporine, initiates AVD through IK channels in a manner that is dependent on global calcium increase. Blockade of IK channels with clotrimazole or TRAM-34 significantly reduced AVD and impaired downstream caspase-3 activation. In contrast, the extrinsic pathway, stimulated by treatment with TRAIL, induced a paxilline sensitive AVD and caused no detectable changes in calcium. Finally, during exposure to hypotonic solution, GBM cells swell and regulate their volume back to baseline using potassium efflux primarily through IK channels. This regulatory volume decrease (RVD) was blocked by clotriamazole and TRAM-34 while paxillne was ineffective at preventing RVD. These studies have revealed IK channels as an intriguing new player in GBM biology, especially in volume regulation and possibly many other processes integral for GBM malignancy.

1 online resource (ciii, 107 p.) : ill., digital, PDF file.

Neurobiology;

Joint Health Sciences;

Glioma KCa Channels IK Channel BK Channel Apoptosis Volume

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A major challenge in neuroscience is understanding how the different neural cell types work together to process information and produce a behavioral output. Glial cells of the human brain have long been thought to act as support for the fundamental cell to cell communication at the core of cognition: neuronal synaptic communication. Research over the past several decades measuring glial activity and experimentally controlling glial cells in rodent model systems has shown that the two macroglia sub-types of glial cells (astrocytes and oligodendrocytes) have active roles in establishment, maintenance, and modulation of synaptic communication in the mammalian brain. Much of this research has utilized cell-type specific promoters to identify and genetically manipulate mammalian glial cells to better understand their role in intercellular communication in the brain. My research is aimed at examining the glia of C. elegans: a model organism chosen for a reductionist approach to research on how gene products and gene regulation work in development and function of a simple nervous system. I chose to focus on a subset of glia in this nematode, the sheath glia of the cephalic sensilla, due to cellular characteristics unique to this invertebrate and their morphological similarity to mammalian astrocytes and oligodendrocytes. I hypothesized that as the most evolutionarily ancient proto-astrocytes with some oligodendrocytes characteristics, these glia of C. elegans would exhibit a hallmark of mammalian glia: robust calcium dynamics upon stimulation. To test this hypothesis, I determined experimental applications for the hlh-17 gene promoter that would avoid confounding effects on behavior and used it to examine cultured C. elegans glia for the first time. I then adapted a genetically encoded calcium sensor to show that cultured glial cephalic sheath cells respond to membrane depolarization with increases in cytoplasmic calcium. I show that voltage-gated calcium channels underlie this response, indicating that glia of C. elegans have taken on a functional profile less like that known for mature mammalian glial cells but with some remaining commonalities. This establishes C. elegans as a model organism that can be used to study glia in a simple nervous system through contrast and comparison with macroglia of mammalian model organisms with similarities possibly representing ancient roles of glia and differences possibly representing roles taken on to meet demands imposed by a more complex nervous system.

1 online resource (xvi, 162 p.) : ill., digital, PDF file.

Neurobiology;

Joint Health Sciences;

glia Caenorhabditis elegans model organism astrocyte oligodendrocyte

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Malignant gliomas, including glioblastomas, are the main primary adult brain tumor. Even with current therapies, the median survival time for patients diagnosed with glioblastomas is only about 12 months. Therefore, it is imperative to identify pathways that can induce glioma cell death. This dissertation provides evidence defining how Endoplasmic Reticulum Stress Response (ERSR) induction promotes cell death selectively in malignant glioma cells (MGCs). I present data showing a correlation between ER Ca2+ storage abnormalities, ER ribosome-translocon expression and activity, ERSR intensity and cell death in MGCs. These data show ERSR induction with thapsigargin (THAP) results in a larger loss of ER Ca2+, an enhancement of ERSR, ER-associated caspases (4/12) and caspase 3 activation and death in MGCs to a much higher degree than in primary astrocytes. MGCs have been reported to have enhanced levels of protein synthesis. I confirm that increased expression of the translocon, a part of the ribosomal complex responsible for ER protein translocation, is a phenomenon associated with ER Ca2+ loss. Pharmacologically inhibiting translocon Ca2+ permeability prevented the THAP-induced ER Ca2+ loss, ERSR deployment, and MGCs death. Conversely, stimulating translocon Ca2+ permeability caused ER Ca2+ loss, ERSR deployment, and MGCs death. Based on these findings, we set out to identify novel small molecules that could mimic THAP actions. We engineered a high throughput assay and queried a library of 1200 FDA approved drugs for agents able to activate ERSR. Spiperone (SPIP), a small molecule identified in our screening, causes ERSR induction and marked cytotoxicity in MGCs. Post screening validation indicates that SPIP induced GRP78 expression and activated ER associated caspases and caspase 3 in MGCs. SPIP also causes ER Ca2+ release from the THAP sensitive store in MGCs. In silico molecular docking analysis indicates that SPIP could bind to SERCA at the cyclopiazonic acid binding site but not to the inositol 1, 4, 5 triphosphate receptor. Future plans include in vivo studies to determine the effect of SPIP on tumor regression. In conclusion, these findings reveal a weakness of MGCs that could be exploited to identify and develop novel therapeutic strategies to treat incurable glial malignancies.

PhD

1 online resource (xi, 118 pages) :illustrations

Ph.D.University of Alabama at Birmingham2012.

Neurobiology

Joint Health Sciences

calcium cytotoxicity endoplasmic reticulum stress response glioma cells spiperone translocons

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Acid Sensing Ion Channel 1 is one of the many proteins in the Epithelial Sodium Channel/Degenerin family. The proteins in this family interact to form cation channels with unique biophysical properties and can all be inhibited by the small molecule amiloride. Their expression in many different cell types underlies their involvement in a large variety of physiological and pathophysiological processes. ASIC-1 containing channels, specifically, are an important therapeutic target for ischemic stroke, nociception, the invasiveness of glioblastoma cells, and many other processes including anxiety and memory formation. Of the members of the ENaC/Deg family, chicken ASIC-1 is the only protein for which crystal structures have been solved. Using homology modeling, structures of the human proteins in the ENaC/Degenerin family were deduced. Further computational methods were applied to examine the interaction of hASIC-1 with known small molecule inhibitors such as amiloride and Psalmotoxin-1, a highly potent peptide toxin. Experimental verification of these computational results was performed using whole-cell and single-channel electrophysiology. Amiloride analogs not known to interact with ASIC-1 were identified by computational studies and verified functionally, showing the strength of this technique for discovering new ASIC ligands. Fully understanding the interactions of these inhibitors with ASIC-1 should permit their manipulation for the benefit of those afflicted with glioblastomas, stroke, anxiety, or pain.

1 online resource (xi, 135 p.) : ill., digital, PDF file.

Physiology and Biophysics

Joint Health Sciences

Acid Sensing Ion Channel Epithelial Sodium Channel Pslamotoxin-1 Homlogy Modelling Protein Docking Drug Discovery

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Drug-seeking behavior following chronic drug use lasts for many months or even years. Short-term withdrawal experiments have suggested that the neuroadaptations thought to underlie learning and memory may also contribute to addictive behavior. However, there is little information about the physiological mechanisms that participate in craving and relapse following long-term withdrawal. Here I show in hippocampal slices from rats treated with nicotine for 1 week that there is a change in the excitability of CA1 pyramidal cells that persists for up to 9 months following the cessation of drug treatment. The expression of this enhanced excitability is dependent on different mechanisms immediately after cessation of the drug compared to following longer withdrawal. After short-term withdrawal (1 day), the enhanced excitability is dependent on spontaneous activity upstream of CA1 that results in a depolarization of the CA1 pyramidal cells. Following longer withdrawal ([greater than] 4 weeks) the enhanced excitability is localized to the CA1 region and mediated via an increase in the intrinsic excitability of the CA1 pyramidal cells. Re-exposure to nicotine in vitro restores hippocampal function to normal after 1 day of withdrawal, but not at withdrawal times later than 4 weeks suggesting a homeostatic induction mechanism followed by long-lasting neuroadaptations downstream of nicotinic receptors that may contribute to the expression of nicotine craving and relapse.

xii, 133 p. : ill., digital, PDF file

Neurobiology

Joint Health Sciences

nicotine hippocampus plasticity addiction

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Neuroinflammation and Fragile X syndrome (FXS) are two particularly devastating neurologic conditions for which no adequate treatment exists and much is still unknown about the underlying cellular and molecular processes. Neuroinflammation contributes to the pathogenesis of many neurologic and psychiatric disorders, thus treatment of the underlying inflammation has broad implications. FXS is the most common cause of inherited intellectual and developmental delay and one of the few known genetic causes of autism. Neuroinflammation and FXS have potential links with glycogen synthase kinase-3 (GSK3) and its inhibitor, lithium. Therefore, the overall goal of this research was to examine the role of GSK3 in neuroinflammation and FXS, as well as the potential therapeutic value of GSK3 inhibitors in these neurologic conditions. Most neuroinflammatory processes result in activation of microglia, the resident immune cells of the brain. Microglial responses include migration to an injury site and the release of pro- and anti-inflammatory factors, which ultimately can affect neuronal survival. Treatment with GSK3 inhibitors attenuated several microglial responses in vitro and in situ after lipopolysaccharide (LPS)-induced activation of microglia, including migration, activation, and production of pro-inflammatory molecules. GSK3 inhibitors also provided protection from inflammation-induced neuronal toxicity. Thus, the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia and provides neuroprotection after an inflammatory insult. FXS results from an expansion-mutation upstream of the fragile X mental retardation 1 (Fmr1) gene causing loss of the gene product. Together with previous results, it was discovered that impaired inhibitory serine-phosphorylation of GSK3 is a robust phenotype in the brains of Fmr1 knockout mice and two potential therapeutics for FXS, a metabotropic glutamate receptor antagonist and lithium, converge to inhibit GSK3. Chronic lithium treatment reversed the hyperactive GSK3 in the brains of Fmr1 knockout mice and improved many of the tested FXS-associated behaviors. Therefore, impaired inhibitory regulation of GSK3 plays a prominent role in the pathogenesis of FXS and supports GSK3 as a potential therapeutic target. Overall, this work provides novel insight regarding the function of GSK3 in two pathologic processes in the central nervous system, and further supports GSK3 inhibitors as viable therapeutics for both neuroinflammation and FXS.

vii, 141 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Neuroinflammation Microglia Glycogen Synthase Kinase 3 Lithium Neurodevelopmental Disorders Fragile X Syndrome

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Cholinergic innervation to visual cortex is essential for spatial learning and memory as well as normal visual function. Long-term depression (LTD) or potentiation (LTP) dependent upon muscarinic acetylcholine receptor (mAChR) activation (mLTD and mLTP), has been previously described, however only mLTD in hippocampus has been fully characterized. It is possible that mLTD and mLTP underlie the cholinergic requirement of normal functioning of the visual system. First, examination of intracellular mechanisms mediating mLTD in rodent visual cortex revealed that inhibiting m1 mAChRs, as well as Src and ERK kinases, prevented mLTD. Furthermore, mLTD does not require NMDARs but utilizes voltage-gated Ca2+ channels for induction and requires protein synthesis for long-term maintenance. Cholinergic denervation is a hallmark of many neurodegenerative diseases, including Alzheimer’s disease. Following cholinergic denervation in both humans and animals, there is an adrenergic sympathetic sprouting originating from superior cervical ganglion. Previously, in hippocampus we have shown that mLTD is rescued by cholinergic rennervation accompanying sympathetic sprouting. I examined the effects of cholinergic denervation and sympathetic sprouting on mLTD induction in visual cortex. mLTD is lost following cholinergic denervation and is rescued by sympathetic sprouting; low frequency stimulation-induced LTD (LFS-LTD) is also affected by these lesions. mLTD induction in sham lesioned animals requires both m1 and m3 receptor as well as ERK activity. In lesioned animals with intact ganglia (to allow for sprouting), mLTD no longer requires m1 receptor activity. We examined if tree shrews exhibited muscarinic dependent plasticity. Visual cortex is segregated into two regions where ocular inputs originate from either one eye (monocular) or both eyes (binocular), and in tree shrew this segregation is highly defined. In the monocular region, mLTD is induced in an m3, ERK dependent fashion, while in the binocular region, mLTP occurs and its induction requires m1 receptors and PLC activation. Collectively, these data characterize intracellular signaling events mediating mLTD/mLTP and demonstrate that mLTD is affected by cholinergic denervation and sympathetic sprouting occurring in visual cortex. Furthermore, higher order species exhibit forms of muscarinic dependent plasticity. Taken together, these data support the hypothesis that mLTD/mLTP are mechanisms by which the cholinergic system modulates visual cortical function.

1 online resource (ix, 134 p. : ill., digital, PDF file)

Neurobiology

Joint Health Sciences

LTD ERK mAChR LTP PLC

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Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. While previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1’s contribution to normal brain function. In the central nervous system (CNS), PAR1 is expressed in glial cells in the hippocampus, a brain region critical for memory formation. I am particularly interested in PAR1 because its activation enhances the function of N-methyl-D-aspartate receptors (NMDARs), which are required for some forms of behavioral learning and synaptic plasticity. Thus, the work in this dissertation is driven by the central hypothesis that PAR1 function is a regulator of NMDAR-dependent memory formation and synaptic function. In this dissertation, I explore the consequences of loss of PAR1 function on long-term memory formation and long-term synaptic plasticity in the PAR1 -/- mouse. I demonstrate that whereas baseline behavioral measures were largely unaffected by PAR1 removal, PAR1-/- mice showed deficits in hippocampus-dependent memory tasks. I also show that while PAR1 -/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1synapses, they exhibit severe deficits in NMDAR-dependent long-term potentiation (LTP). Mounting evidence indicates that PAR1 is expressed predominantly in astrocytes in the hippocampus, and that activation of PAR1 leads to glutamate release from astrocytes and potentiation of NMDAR responses in CA1 pyramidal cells. Taken together, these data suggest an important role for PAR1 function in astrocyte-neuron interactions subserving memory formation and synaptic plasticity.

1 online resource (xiii, 175 p. : ill., digital, PDF file)

Neurobiology

Joint Health Sciences

serine protease PAR hippocampus behavior synaptic plasticity long-term potentiation

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Spinal cord injury (SCI) is a devastating condition resulting in loss of motor function as well as sensory abnormalities. Insight into the pathophysiology of SCI progression has been gained through use of pre-clinical animal models, however these have not been successful in yielding pharmacological interventions for clinical management of SCI. One proposed reason for this discrepancy may be the use of SCI models which are not fully clinically relevant and do not assess the contribution of gray matter pathology to SCI functional outcomes. Post-SCI inflammation is welldocumented and may lead to downstream loss of motor function. Additionally, inflammation is thought to play a role in the development of neuropathic pain through activation of glial cell populations. The transcription factor nuclear factor kappa B (NF- κB) controls the production of multiple proinflammatory mediators and is acutely upregulated post-SCI. Thus, inhibition of NF-κB may serve as a key target to preserve downstream motor function and inhibit neuropathic pain post-SCI. The first experiment performed describes the effect of varying impact forces of cervical hemicontusion SCI on locomotor and forelimb function as well as tissue lesion characteristics. We report that histological assessments of gray and white matter tissue damage are significantly correlated with impairments in forelimb and locomotor function. These findings indicate that both gray and white matter pathology are implicated in the loss of motor function post-cervical SCI. The second experiment describes the effect of a previously described neuroprotectant, 17β-estradiol (E2), on functional recovery and histological lesion characteristics using this newly characterized cervical SCI model. We report that E2 administration post-SCI significantly increases forelimb and locomotor function with corresponding decreased gray and white matter tissue damage. The third experiment describes the effect of sulfasalazine (SSZ), an inhibitor of NF-κB, on functional recovery and neuropathic pain post-SCI. We report that SSZ administration post-SCI has no effect on functional recovery, however significantly decreases the incidence of neuropathic pain behaviors through inhibition of NF-κB acutely post-SCI. Collectively, this body of work describes the characterization of a clinically relevant model of cervical SCI and the promising effects of two novel pharmacological interventions on functional recovery and neuropathic pain.

2011

1 online resource (ix, 200 p.) : ill., digital, PDF file.

Neurobiology

Joint Health Sciences;

cervical spinal cord injury sulfasalazine neuropathic pain NF-κB gliosis

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