This application focuses on mechanisms of amyloid beta-protein perturbation of cholesterol and apoE homeostasis within astrocytes and resulting consequences on neuronal function. There is a dynamic interaction between cholesterol and amyloid beta-protein (ABeta), a protein that is thought to be an important contributor to neurodegeneration that occurs with Alzheimer's disease. Cholesterol levels modulate amyloid precursor protein expression and ABeta1-42 production. Conversely, ABeta1-42 alters cellular cholesterol dynamics particularly cholesterol trafficking in astrocytes and neurons. The Golgi complex play an important role in protein and lipid trafficking and recent work from our laboratory has shown that ABeta1-42 modified cholesterol distribution within the Golgi complex in astrocytes, reduced cholesterol levels in the plasma membrane and increased apoE levels. These results lead us to hypothesize that: ABeta1-42 disrupts cholesterol and apoE homeostasis in astrocytes and effects are apoE isoform dependent. Mechanisms of ABeta1-42 effects involve a caveolin associated pathway between the Golgi complex and the plasma membrane, and transcriptional regulation of apoE expression that is dependent on stimulation of Beta-adrenergic receptors, cAMP formation and the transcription factor AP-2. Consequences of ABeta1-42 perturbation of astrocyte cholesterol and apoE homeostasis are alterations in neuronal cholesterol domains and the synaptophysin/synaptobrevin complex. To test this we will: 1: Evaluate effects of ABeta1-42 on caveolin levels in the cis-medial and trans- regions of the Golgi complex of astrocytes. Examine proteomics of the Golgi complex regions of astrocytes treated with ABeta1-42 Evaluate caveolae and lipid raft structure and function in astrocytes treated with ABeta1-42. Primary astrocytes from mice expressing human apoE2, apoE3, apoE4, apoE-null mice, caveolin-l-null mice and human astrocytes will be used. 2: Examine the effects of ABeta1-42 on distribution of apoE levels in astrocyte organelles and conditioned media. Evaluate apoE mRNA abundance in astrocytes incubated with ABeta1-42. Evaluate effects of ABeta1-42 on Beta-adrenergic receptors, cAMP formation, transcription factor AP-2 levels and DNA binding of AP-2. Astrocytes of mice expressing human apoE2, apoE3, and apoE4 will be used. 3: Evaluate neuronal lipid raft proteins, lipids and transbilayer cholesterol distribution using neurons of C57BL/6 mice co-cultured with astrocytes from mice expressing human apoE2, apoE3, apoE4, and apoE-null mice. Examine synaptophysin/synaptobrevin complex in neurons of C57BL/6 mice co-cultured with astrocytes from mice expressing human apoE2, apoE3, apoE4, and apoE-null mice. Determine if the neuronal apoE4 and apoE-null phenotype can be """"""""rescued"""""""" by incubation with astrocytes of apoE2, apoE3, or wildtype mice. In some experiments astrocytes will be treated with ABeta1-42.
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