Characteristic pathological landmarks in the AD brain include the presence of activated microglial, reactive astrocytes and degenerating neurons with dystrophic neurites in brain regions around beta amyloid plaques. Due to the complex cellular organization of the brain, studies using suitable cell models and culture conditions are needed to stimulate the environment in the AD brain and to explore temporal events leading to the deleterious cellular effects induced by Abeta peptides. The overall objective of the Cell Culture Core (G. Weisman, CL) will be to provide PPG investigators with a reliable source of cell cultures from a centralized cell culture facility. In addition, the Cell Culture Core will oversee preparation and characterization of Abeta peptides and lipoproteins to be used by all PPG projects.
Specific Aims are: To prepare primary cortical and hippocampal neurons, cortical astrocytes and microglial cells from neonatal and adult mouse brain. To provide cultures of immortalized astrocytes (DITNC cells), microglial (BV-2) cells and other cell lines relevant to research in the PPG . To provide cultures of human astrocytes and neurons. To characterize and assess cell homogeneity and viability. To standardize methodology for preparation of aggregated Abeta (1-40, 1-42 and the inactive peptide 42-1) to be used by PPG investigators. To prepare and characterize native and oxidized lipoproteins simulating those in human brain. To prepare characterize native and oxidized lipoproteins simulating those in human brain. An advantage of the cell model approach is the ability to generate multiple conditions that mimic those found in the AD brain. Cell culture studies using neurons and glial cells from the C57BL/6J mouse strain will be useful for comparison with studies in which cells are derived from transgenic animals. The Cell Culture Core will be responsible for maintenance of transgenic mice colonies: APP Tg 2576 transgenic mice, human apoE transgenic mice and cPLA2 deficient mice. Furthermore, models are available for co-culture of neurons and glial cells and for obtaining conditioned media from one cell type to test responses in another cell type. Studies using cell models will provide information on the effects of Abeta on lipid peroxidation, lipoprotein oxidation, altered cholesterol homeostasis and receptor signaling processes that affect neurons and glial. The facilities in the Cell Culture Core will enable investigators to study biochemical and biophysical mechanisms using single or multiple brain cell types to understand the pathogenesis of AD.
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