The in vitro effects of AIT-082 on ATP levels in cortical neurons and phosphorylation levels in cortical neurons and astrocytes
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Date
2003-08-29
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Degree Level
Masters
Abstract
The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimer’s disease.The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Ying’s (Ying, 1996a) theory of the development of Alzheimer’s disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082.
Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimer’s disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.
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Keywords
Ischemia, Phosphorylation, Immunophilins, ATP, Hypoxia, Alzheimer's disease, Purines, AIT-082
Citation
Degree
Master of Science (M.Sc.)
Department
Anatomy and Cell Biology
Program
Anatomy and Cell Biology