The recent observations that both DNA adducts and oxidative base damage are increased in the brains from Alzheimer s and Parkinson s patients and in spinal cord tissue of patients with amyotrophic lateral sclerosis (ALS) support the idea that oxidative DNA damage may contribute to the observed loss of neurons in these neurological disorders (8,9). Therefore, understanding how oxidative DNA damage is repaired (or not) is critical for understanding the pathology underlying these diseases. The overall hypothesis of this proposal is that oxidative stress induced DNA damage in neuronal cell lines leading to cellular malfunction and/or death and DNA repair enzymes are critical for the protection of neurons against oxidative stress. The following specific Aims will address this hypothesis.
Specific Aim 1 : Functional and biochemical characterization of the repair enzymes Ogg1, NTH, MTH, and MYH in the neuroblastoma cell lines SHSY5Y and Neuro-2A and in primary cultures of hippocampal neurons. We will measure the steady- state level of mRNA of the four repair enzymes, cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2. Experiments will also be performed in post-mitotic SHSY5Y cells.
Specific Aim 2 : Determine the effects of over-expressing these oxidative DNA repair genes on extotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG following exposure to L-Dopa, dopamine, menadione, or H2O2. Both mitotic and post-mitotic SHSY5Y cells and the Neuro-2A cells will be analyzed.
Specific Aim 3 : Determine the effects of antisence gene expression specific for the four oxidative DNA repair genes on cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2 in SHSY5Y mitotic and post-mitotic cells. If increasing the levels of DNA repair enzymes protects cells against oxidative stress, then this could provide a novel therapeutic basis for the treatment of neurological disorders such as ALS, aging, Alzheimer s and Parkinson s diseases.
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