Excitotoxicity can lead to increases in intracellular Na+ and Ca2+ concentrations via the glutamatergic NMDA receptors, which can lead to cell death. Detailing the time-dependent degradation of neuronal components in response to excitotoxic challenge may help elucidate the sequence in which these signaling pathways are initiated and further, associate these pathways with topographic cellular demise. Using organotypic hippocampal slice culture technique, tissue from neonatal rat pups was exposed to NMDA, APV, or co-exposed for 24, 72 or 120 hours. Fluorescent microscopy of propidium iodide (PI) was used to evaluate neuronal membrane damage, changes in the density of mature soma (NeuN) and NMDA NR2B subunits were measured using immunohistochemical procedures. After 24 hours of exposure, the CA1 showed an increased PI signal and a decrease in NeuN marker, indicating somatic injury occurs shortly after excitotoxic challenge; these effects were blocked by co-administration of APV. Also in the CA1, loss of NR2B subunits, heavily expressed in dendritic processes, declined following 72 hours of exposure. Because somatic injury precedes loss of distal NR2B subunits, it is possible that these events involve different mechanisms, findings that may be relevant in the development of therapies to target neurodegeneration resulting from excitotoxicity.

There is evidence that the excitotoxin quinolinic acid (QUIN) synthesized through the kynurenine pathway (KP) by activated microglia may play a role in the pathogenesis of several major neuroinflammatory diseases and more particularly in Alzheimer??s disease (AD). The hypothesis of this project is QUIN affects the function and morphology of astrocytes. In this study I used human foetal astrocytes stimulated with AD associated cytokines including IFN-gamma, TNF-alpha, TGF-alpha and different concentrations of QUIN ranging from low physiological to high excitotoxic concentrations. I found that QUIN induces IL-1beta expression in human astrocytes and subsequently, contribute to the inflammatory cascade that is present in AD pathology. Glial fibrillary acid protein (GFAP) and vimentin protein expression were complementary in expression to each other after 24 hr stimulation with different QUIN doses. However, there were marked increases in GFAP levels and reduction in vimentin levels compared to controls with QUIN treatment indicating that QUIN can trigger astrogliosis in human astrocytes. Glutamine synthetase (GS) activity was used as a functional metabolic test for astrocytes and I found a dose-dependent inhibition of GS activity by QUIN. This inhibition was inversely correlated with iNOS expression whereby reduced GS activity is accompanied with an increase expression of iNOS in human astrocytes. These results suggest that reduction in GS activity can lead to accumulation of extracellular glutamate then leading to exacerbated excitotoxicity via NMDA receptor over-activation and ultimately neuronal death. PCR array results showed that at least four different pathways were activated with pathological concentration of QUIN including p38 MAPK that is associated with pro-inflammatory cytokine production, ERK/MAPK growth and differentiation that can modulate structural proteins, mitochondrial-induced apoptotic cascade and cell cycle control pathway. QUIN-induced astrogliosis and excitotoxicity could lead to glial scar formation and prevention of axonal growth thus exacerbation of neurodegeneration via synaptosomal NMDA receptor over-activation. All together, this study showed that, in the context of AD, QUIN is an important factor for astroglial activation, dysregulation and death, which can be mediated by the previously mentioned pathways.

Orientador: Lília Freira Rodrigues de Souza Li / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T19:22:58Z (GMT). No. of bitstreams: 1 Nascimento_PaulaHespanholo_M.pdf: 1475049 bytes, checksum: f1ec759ca189c1e3404667fb1ce01abc (MD5) Previous issue date: 2012 / Resumo: A esclerose hipocampal está associada à epilepsia de lobo temporal medial (ELT) e causa expressão alterada de receptores tais como o Receptor Metabotrópico de Glutamato (mGluR1). Contudo, ainda há controvérsias se sua expressão está aumentada ou diminuída em ELT. O Receptor Sensor de Cálcio (CASR), outro receptor da mesma família do mGluR1, é expresso em hipocampo, mas seu papel no cérebro ainda é desconhecido. VILIP-1 é uma proteína sensora de cálcio neuronal (NCS) expressa predominantemente no cérebro e em humanos e sua expressão foi mapeada por imunoistoquímica na subpopulação de neurônios piramidais em CA1 e CA4 de hipocampo. Sugere-se também que a ativação de mGluR possa regular a expressão de VILIP-1 durante a plasticidade hipocampal. No entanto, não há estudos associando VILIP-1 e esclerose hipocampal. Nós hipotetizamos que além do mGluR1, o CASR e o VILIP-1 estão associados a esclerose hipocampal em ELT. O objetivo deste trabalho foi analisar o padrão de expressão de VILIP-1, CASR e mGluR1, em hipocampo de pacientes com ELT submetidos a amigdalohipocampectomia. Nossos resultados demonstraram a presença de EH nos tecidos hipocampais de pacientes com ELT com redução no número de neurônios em CA1 e presença de intensa gliose. Pela análise da expressão dos transcritos VILIP-1, CASR e mGluR1 em hipocampo total utilizando PCR em tempo real não encontramos diferença na expressão dos RNAs mensageiros dos pacientes quando comparado com os controles. Entretanto, quando comparamos a expressão protéica em hipocampo de pacientes e controles, utilizando o método de imunoistoquímica, encontramos não somente redução significativa no número de neurônios presentes em CA1 de pacientes, mas também redução importante nos neurônios positivamente marcados para VILIP-1, CASR e mGluR1. Estes achados sugerem que não apenas mGluR1, mas também CASR e VILIP-1, estão associados à EH em pacientes com ELT / Abstract: Hippocampal sclerosis (HS) is associated to temporal lobe epilepsy (TLE) and cause altered expression of neurotransmitter receptors such as metabotropic glutamate receptor 1 (mGluR1). However, whether its expression level is increased or decreased in temporal lobe epilepsy is still controversial. Calcium-sensing receptor (CASR), another receptor from the same family of mGluR1, is expressed in hippocampus, but its role in brain is unknown. VILIP-1, a neuronal calcium sensing protein (NCS) is expressed predominantly in brain and in humans its expression was identified by immunohistochemistry in subpopulations of pyramidal neurons in CA1 and CA4 in hippocampus. Activation of mGluR1 is suggested that may regulates VILIP-1 expression during hippocampal plasticity. However, there are no studies associating VILIP-1 and hippocampal sclerosis. We hypothesized that not only mGluR1 but also VILIP and CASR is involved in hippocampal sclerosis in TLE patients. The objective of this study was to analyze the pattern of expression of VILIP-1, CASR and mGluR1 in hippocampal tissues from patients with TLE who underwent amygdalohippocampectomy. Our results demonstrated the presence of hippocampal sclerosis in hippocampal tissues in patients with TLE with reduction in the number of neurons in CA1 and gliosis. By the expression analysis of the transcripts of VILIP-1, CASR and mGluR1 in total hippocampus using real time PCR, we did not find differences on mRNAS expression of patients compared with controls. However, when we compared the protein expression from hippocampi from patients with controls, by immunohistochemistry, we not only found an important reduction on neuron cell number in patients, but also an important reduction on positively stained neurons for VILIP-1, CASR and mGluR1, suggesting that not only mGluR1, but also CASR and VILIP1 are associated to HS in patients with TLE / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

No description available.

Excitotoxicity is the overexcitation of neurons due to the excessive activation of excitatory amino acid receptors and is thought to be involved in many neurodegenerative states. The manner in which the neuron breaks down during excitotoxicity is still unclear. The current study used the organotypic hippocampal slice culture model to examine the time-dependent loss of the synaptic vesicular protein synaptophysin and the loss of N-methyl-D-aspartate (NMDA) receptor NR1 subunit availability following an excitotoxic insult (20 μM NMDA) to provide a better understanding of the topographical nature of neuronal death following NMDA receptor activation. Significant NMDA-induced cytotoxicity in the CA1 region of the hippocampus (as measured by propidium iodide uptake) was evident early (15 minutes after exposure) while significant loss of the NR1 subunit and synaptophysin was found at later timepoints (72 and 24 hours, respectively), suggesting delayed downregulation or degradation in axons and dendrites as compared to the soma. The addition of the competitive NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV) significantly attenuated all NMDA-induced effects. These results suggest that NR1 and synaptophysin levels as measured by immunoreactivity are not reliable indicators of early cell death.

Title: The role of nitric oxide in the pathophysiology of neurodegenerative diseases Objectives: The main objective of this thesis is to evaluate the effect of nitric oxide on the formation and development of neurodegenerative diseases. Another objective was to determinate, whether NO affects by its impact processes involved in apoptosis in the CNS. Methods: The thesis is prepared in the form of research, drawing from available relevant resources. Results: Nitric oxide is widely applied in the pathophysiology of selected neurodegenerative diseases, either directly or through other reactive nitrogen and oxygen. It also affects other factors that are involved in apoptosis in the CNS. Keywords: Nitric oxide, NMDA receptors, neurodegenerative diseases, excitotoxicity, apoptosis

Neuroinflammation is a major driver of secondary brain cell death after ischaemic stroke, seizure activity and traumatic brain injury. In a model of excitotoxic neuroinflammation, striatal injection of a toxic dose of AMPA causes cell death in the striatum after 24 hours. Co-injection of AMPA with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to additional cortical cell death. Injected alone, IL-1β leads to little or no cell death. It is hypothesised that IL-1β may exacerbate cell death by interfering with blood flow coupling. In the first study, two-dimensional optical imaging spectroscopy was used to measure early changes in the haemodynamic response in the anaesthetised rat barrel cortex before and for 6 hours after injection of vehicle, AMPA, IL-1β, or AMPA+IL-1β. After injection of IL-1β, with or without AMPA, the oxygenated blood flow response to mechanical whisker stimulation approximately halved over the course of 6h. In the second study, to determine whether the IL-1β-dependent changes in blood flow response are reflected by altered cellular activity, local field potentials, multi-unit activity and local tissue oxygenation responses to whisker stimulation were recorded simultaneously from the active barrel before and up to 6h after injection. A similar reduction in the size of the oxygenation response was seen again in the IL-1β- and AMPA+IL-1β-treated groups. Importantly, the level of gamma frequency oscillations at stimulus onset decreased within the first hours after injection of AMPA+IL-1β or IL-1β, suggesting a disruption of the fast-spiking interneuron network in the barrel cortex. These findings, along with histological observations of IL-1β-dependent markers of neuroinflammation, suggest that IL-1β may exacerbate AMPA-induced excitotoxicity by potentiating seizure activity and decoupling the neurovascular response in the cortex.

Degree awarded (2004): PhDBmS, Neuroscience Program, George Washington University / Glutamate is critically important as an excitatory neurotransmitter in the central nervous system. Increasing evidence suggests additional signaling roles for glutamate in cell proliferation and migration in normal and oncogenic states. Recently, glutamate release from glioma cells has been shown to increase tumor growth in vivo. To investigate the mechanism of glutamate enhancement of tumor growth, we investigated the effect of glutamate on tumor cell proliferation, invasion, and glioma-induced cell death. Here we demonstrate that glutamate enhances tumor growth via increasing tumor cell proliferation and inducing excitotoxic death of cells surrounding the solid tumor mass, thereby facilitating tumor expansion. The evidence that glutamate enhances tumor growth suggests that regulating extracellular levels of glutamate may restrict tumor growth. In the normal brain, extracellular glutamate levels are maintained by a family of glutamate transporters. To investigate the therapeutic potential of regulating extracellular glutamate concentrations on tumor growth, we utilized a transgenic mouse model of EAAT2 glutamate transporter overexpression. In this report, we demonstrate that increased glutamate transport limits tumor growth in vivo and provides protection against glioma-associated neuronal cell death. In addition, seizure activity, often associated with the presence of a CNS tumor, is attenuated in transgenic mice overexpressing the glutamate transporter, EAAT2. These findings suggest that glutamate transporters may provide a new therapeutic target for limiting tumor expansion and secondary epileptogenesis. / Advisory Committee: Dr. Margaret Sutherland (Chair), Dr. Steven Patierno (Chair), Dr. Tim Hales, Dr. Vincent Chiappinelli, Dr. Linda Werling, Dr. Frances Noonan

Spinal cord injury (SCI) often results in irreversible paralysis and widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate that inhibition of astroglial-NF-κB activation reduces white matter damage and improves functional recovery in a mouse model of SCI. Here we provide novel evidence demonstrating that astrocytes directly regulate oligodendrocyte fate after trauma by a glutamate-mediated AMPA receptor dependent mechanism. Following trauma, elevated expression of the SLC39a10 zinc transporter correlated with an increase in zinc uptake by astrocytes, thereby reducing extracellular zinc concentrations required for AMPA receptor inhibition. Stimulation of AMPA receptors on oligodendrocytes by glutamate induced oligodendrocyte toxicity through the activation of the NADPH oxidase enzyme within oligodendrocytes. Genetic and pharmacological inhibition of active NADPH oxidase was sufficient to attenuate oligodendrocyte death in vitro. Following SCI, NADPH oxidase inhibition reduced oligodendrocyte death by ~75%, suggesting that glutamate-mediated oligodendrocyte death is dependent on the activation of the NADPH oxidase enzyme within oligodendrocytes. Combined treatment of the NADPH oxidase inhibitor apocynin and the AMPA receptor inhibitor NBQX significantly improved hind limb locomotor behavior, reduced white matter damage and lesion volume, and significantly spared descending serotonergic fibers. These studies provide a novel mechanism of oligodendrocyte death and may lead to clinically relevant therapeutics after SCI.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Extracellular glutamate concentrations are tightly regulated to avoid over-stimulation of glutamate receptors, which leads to a cascade of deleterious processes collectively known as excitotoxicity. Excitotoxicity is common to several neurodegenerative disorders and CNS injuries, including stroke and Alzheimer’s disease (AD). The projects described in this thesis were designed to uncover novel protective pathways in excitotoxic neurodegeneration. Excessive activation of the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), is a convergence point for neuron death signaling in excitotoxic pathways. In AD, the peptide amyloid-β1-42 (Aβ1-42) is aberrantly produced, leading to excitotoxic neuron death in vitro. To investigate links between Aβ1-42 and PARP, we treated cultured cortical neurons with Aβ1-42 and determined whether PARP-1 contributes to neuron death. Increased neuron death was observed after Aβ1-42 exposure. A non-selective PARP-1/2 inhibitor significantly reduced Aβ1-42-induced death while elimination of PARP-1 alone was not neuroprotective. This suggests that PARP-2 or combined effects of PARP-1 and PARP-2 are required for Aβ1-42-induced neuron death. A hallmark of PARP over-activation is depletion of intracellular NAD+ and ATP levels, yet nearly all studies examining adenine nucleotide levels use separate biochemical samples to measure nucleotides individually. We developed two HPLC methods for simultaneous separation of NAD+, ATP, ADP and AMP. We determined that PARP-1 activation in astrocytes leads to near complete NAD+ depletion, followed by partial loss of ATP pools and total adenine nucleotide pools. Finally, we hypothesized that conjugated linoleic acid (CLA), a naturally occurring polyunsaturated fatty acid, is capable of enhancing neuron survival after an excitotoxic insult. Cultured cortical neurons were exposed to glutamate in the presence and absence of CLA. CLA levels likely achievable in human plasma and brain tissue during dietary supplementation regimens, protected neurons against glutamate excitotoxicity when given during or up to five hours after glutamate exposure. Several markers of mitochondrial damage and intrinsic apoptosis were examined. CLA stabilized mitochondrial membrane potential and permeability, shedding light on the mechanism of CLA neuroprotection. Overall, our research suggests a role for PARP in Aβ1-42 toxicity and identifies a novel role for CLA in neuroprotection following excitotoxicity.