The molecular mechanisms of human neurodegenerative diseases have so far been studied in animals models but the validity of extrapolating these studies to human in uncertain. There is a need, therefore, to study human neurons at the cellular and molecular levels of understanding their normal behavior and functioning and the changes which lead to neurodegenerative diseases. The neurodegenerative effects of BetaA or its peptide fragments in vitro have mostly been studied on rat neuronal cells or tissue slices and only in few instances on human cells cultured from cryopreserved brains. Propagation and establishment of long-term cultures of human neurons promises to be a powerful approach in achieving this goal. We propose to propagate and establish long-term cultures of human neuronal cells by utilizing the proliferative property of basic fibroblast growth factor (bFGF). Cell will be characterized according to morphology and phenotypes. Establishment of such cultures will enable us to generate large number of cells from specific areas of the brain using small amounts of scarce fetal tissues. We have already succeeded in propagating and establishing long-term neuronal cultures from rat brain tissues. We will transduce amyloid precursor protein (APP) and Beta-amyloid (BetaA) genes into human and rat neurons and study both the expression of these proteins at the molecular levels and their modulation by factors such as the state of differentiation and growth factors. In In vitro experiments BetaA peptides will be added to neuronal cultures to determine their neurodegenerative effects. The neurotoxic effects of glutamate, a neurotransmitter, on cultured neurons (control) or neuron expressing APP proteins (test) in the absence and in the presence of bFGF will be studied. Parallel cultures of human and rat neuronal cultures will be used for these studies and the results will be compared. This will allow us to determine whether rat neurons can be used as model for studies of human neurodegenerative diseases at the molecular level. The establishment of perpetual human neuronal cultures will enable the studies of their functioning at the cellular and molecular level in general and, specific to this proposal, an understanding of the modulation of BetaA by environmental effectors including neurotransmitter and growth factors.
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