The long term goal is to elucidate the molecular basis by which activated glia (astrocytes and microglia) and glial derived molecules contribute to neuropathology, and to gain sufficient insight into molecular mechanisms for better treatment of neurodegenerrative diseases, such as Alzheimer's disease (AD). The focus of this AG13039 renewal application is on molecular mechanisms of glial activation induced by beta amyloid (Abeta)1-42 and inflammatory cytokine such as IL-1Beta and tumor necrosis factor (TNF). Key glial signal transduction pathways and gene expression changes mediated by AB and cytokines have been elucidated and novel mechanisms of modulation of these pathways have been defined. Abeta can stimulate astrocytes to express inducible nitric oxide synthase (iNOS), quantitatively the most important source of over expression of NO in the brain, and increased NO production can lead to neuronal death. Abeta stimulated iNOS expression is mediated through an induction of IL-1Beta and TNF alpha in microglia, which then activates astrocytes through an NFKbeta dependent mechanism to produce iNOS and NO. However, there remains a major gap in knowledge of the quantitative contribution of this cytokine induced NFKB-dependent, signaling pathways in relationship to other molecular pathways which mediates Abeta induced iNOS in astrocytes. Also, there is a lack of knowledge regarding the more upstream events by which Abeta induces the pro-inflammatory cytokines in miocroglia. In addition little is known about how modulation of specific molecular pathways are activated, affecting neuronal responses. The studies proposed here will fill these voids in knowledge and provide insight into how Abeta and cytokines are involved in progressive and chronic glial activation processes that compromise brain function.
The specific aims address the following questions: 1) What are the molecular mechanisms by which AB induces inflammatory cytokines and iNOS in activated glia? 2) What are the consequences to the neuron of modulation of glial signal transduction pathways leading to iNOS induction? Aim 1A will examine a recently discovered new pathway involving calmodulin -dependenmt kinase (CaMK) by determining if CaMKIV is involved in iNOS induction through activation of CREB-linked pathways.
Aim 1 B, will elucidate the mechanisms by which inflammatory cytokines are induced by ABeta by determining if p38 stress kinase are important for IL-1beta induction in activated microglia.
Aim2 will determine the contribution of specific signal transduction pathways leading to iNOS induction on neuronal dysfunction by utilizing a defined co-culture system of primary astrocytes and neurons where NO-dependent neuronal death occurs. Pathways will be modulated by selective pharmacological agents or molecular constructs and effects on neuronal viability will be determined. Astrocytes from genetically engineered mice with over expression of KO and specific components of glial signaling pathways will also be used to ascertain how an in vivo alteration in a specific pathway affects neuronal function. Elucidation of these molecular mechanisms and how they can be modulated as well as defining the effects on the neuron and how genotype affects the responses, will provide new insight into glial neuronal interactions and the knowledge base necessary for pursuit of novel strategies to block glial activation and its neurotoxic consequences.
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