Annadora Bruce-Keller - US grants

The role of activated microglia in Alzheimers disease (AD) has been widely studied, but is minimally understood. Microglial cells are activated by amyloid beta peptide in vitro, and a halo of activated microglial cells surrounds senile plaques in AD brain, leading some investigators to speculate that microglial cells are in part responsible for neurodegeneration in and around plaques. However, evidence for this hypothesis is circumstantial, and microglial cells also secrete neurotrophins, indicating that activated microglial cells in the vacinity of amyloid deposits could serve to support neuronal survival under local conditions of stress. This proposal seeks to determine the role of activated microglal cells in models of AD-like pathology in vitro and in AD brain, and specifically tests the hypothesis that microglial activation can be neuroprotective both in vitro and in vivo. Specific aim 1 tests the hypothesis that microglial activation is neuroprotective in vitro by modulating miroroglial cell activation and determining resultant neurnal pathology and glial response following amyloid beta peptide toxicity, excitotoxicity, or hypoglycemia in organotypic hippocampal cultures. Specific aim 2 tests the role of the neuroprotective cytokine tumor necrosis factor (TNF) in microglial- mediated neuroprotection by applying the paradigms listed in specific aim 1 to cultures drawn from TNF receptor deficient mice. Specific emphasis will be placed on the role of TNF-induced MnSOD and iNOS activation, and the selective induction of apoptosis over necrosis. Specific aim 3 will probe AD and control brain tissue for TNF and the TNF signalling moieties listed in specific aim 2 to determine if these pathways are activated in either neurons or glial cells of AD brain. Particular emphasis will be placed on identification of pathways activated in neurons and glial cells surrounding diffuse, non-neuritic amyloid deposits versus those activated in cells in the vacinity of dense core senile plaques. Collectively, the above studies will help elucidate the role of neuron/glial interactions in AD pathology, and potentially highlight novel therapeutic strategies for AD research, as well as for other neurodegenerative disorders of aging such as Huntington's disease and stroke.

The nervous and immune systems of man are made up of complex networks of cells that monitor specific body signals and respond appropriately. While effects of the brain on the immune system are well characterized, modulation of brain cell function by immunocompetent cells, especially under conditions of brain injury, is not well characterized. These studies seek to identify and characterize endogenous inflammatory pathways that both increase neuronal resistance to brain injury, and increase synaptic recovery following brain injury. Additionally, pharmacological agents that could mimic these neuroprotective pathways will be evaluated. This proposal, therefore, will determine the role of microglial cells, brain resident immunologic cells, in the development of brain injury. Using a well characterized in slice culture model that preserves the physiological, three-dimensional architecture of the brain, this propose will employ well-characterized pharmacological agents to modulate microglial activation in models of excitotoxicity (i.e., seizure-induced damage), anoxia (i.e., stroke), and amyloid- dependent (i.e., Alzheimer s like pathology). Specific Aim 1 tests the general hypothesis that activation of microglial cells is neuroprotective in these models. Specific Aim 2 tests the hypothesis that microglia protect neuronal systems by channeling death pathways towards apoptosis rather than necrosis, and then isolating/removing the apoptotic cells. Specific Aim 3 tests a hypothesis that microglial activation increases both neuronal to injury and neuronal recovery following injury by selective targeting and removal of white matter (myelin) debris. It is especially important to understand the specific contributions of inflammatory mediators to brain injury because under most clinical settings the first priority is to stop immune and inflammatory processes. The possibility therefore exists that new pharmacological interventions could be devised based on studies such as these that lead to increased efficacy in the treatment and therapeutic recovery of victims of traumatic spinal or brain injury.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) afflicts almost 500,000 Americans a year, but unfortunately, existing treatments have only minimal ability to prevent secondary brain damage accompanying traumatic brain injury. Epidemiological data that suggests that women fare better then men following TBI, but the basis for this difference is not understood. It is likely that action of female sex hormones, particularly estrogen, may have significant effects on the progression of brain injury, and recent data from our laboratory suggests that estrogen has potent anti-inflammatory properties that could account for its ability to attenuate traumatic brain injury. In particular, data indicates that estrogen is able to decrease oxidative burst activity and subsequent redox-based inflammatory signaling in glial cells, thereby attenuating neurotoxic brain inflammation. Therefore, it is proposed that estrogen acts to preserve brain function following TBI by decreasing both blood-brain barrier (BBB) breech and neuronal injury, and that these distinct endpoints are mediated by a single mechanism: modulation of the glial oxidative burst. Specific Aim 1 will test the hypothesis that estrogen is able to significantly attenuate oxidative burst activity in astrocytes, microglial cells, and endothelial cells both in vitro and in vivo, and will determine the role of estrogen receptors in this process through use of estrogen receptor knockout mice. Specific Aim 2 will test the hypothesis that by directly interfering with oxidative burst activity, estrogen blocks the release of matrix metalloproteinases and thus preserves blood-brain barrier integrity in mice following traumatic brain injury. Specific Aim 3 will build upon these studies by testing the hypothesis that by decreasing oxidative burst activity and redox signaling, estrogen blocks the formation of neurotoxic inflammatory mediators (excitotoxins and cytokines), culminating in decreased injury and increased recovery following traumatic brain injury. Completion of these studies will result in a thorough understanding of the mechanisms of estrogen-mediated neuroprotection in TBI and could highlight a novel target for therapeutic intervention following brain trauma in both women and men.

Alzheimer's disease (AD) is the major age-related dementia, and is expected to take an increasing toll on society as the population ages. Histopathologically, while the diagnostic criterion for AD is the presence of amyloid-containing plaques and neurofibrilliary tangles, AD brains are typified by increased oxidative stress and focal neuronal death. While the exact relationship between beta amyloid (AP) deposition, oxidative stress, and neuronal injury is still unresolved, increasing evidence suggests that the NADPH oxidase system may be altered in AD and in response to Ap. NADPH oxidase (NOX), originally described as the main component of the leukocyte oxidative burst, produces reactive oxygen species (ROS) that are involved in both intracellular signaling and oxidative stress. NOX expression has been established in neurons, thus raising the possibility that aberrant activation of neuronal NOX by toxic forms of A(3 could contribute to ADrelated neuronal alterations. The focus of this project is to test the hypothesis that sustained activation of NOX by Ap triggers a pernicious cascade linking increases in ROS production to altered intracellular redoxbased signaling and oxidative stress, culminating in neuron degeneration and death. The specific aims to test this hypothesis are as follows: 1) To test the hypothesis that NOX activity is increased during the progression of AD;2) To test the hypothesis that NOX activity is increased in response to increasing Ap deposition in a specific APP/PS1 knock-in mouse model of amyloidogenesis;3) To test the hypothesis that toxic forms of Ap increase neuronal oxidative stress and cell death via NOX;4) To test the hypothesis that microglial NOX activation exacerbates neuronal responses to AP;and 5) To test the hypothesis that genetic or pharmacologic ablation of NOX in the APP/PS1 transgenic mice attenuates oxidative stress and Ap pathogenesis in vivo. The proposed experiments will make use of our extensive and well-characterized tissue bank of human brain specimens, as well as a novel and highly relevant mouse model of beta amyloid pathogenesis. Finally, studies will determine the specific effects of distinct physiological forms of AP(1-40/1- 42/oligomer/fibrillar) on NOX activation both in vitro and in vivo. Completion of these studies will result in a more thorough understanding of the relationship between Ap deposition and AD-related neuronal pathology, and could highlight an innovative and highly promising target for therapeutic intervention in AD.