Miller School of Medicine The involvement of cyclic AMP signaling in ethanol mediated oligoprotection following injury Lydia E. Cortés Betancourt Leadership Alliance Dr. Damien Pearse Miami Project to Cure Paralysis Abstract The treatment of injured organisms with ethanol can increase motor function. It has been demonstrated that short term exposure to ethanol increases cAMP (Cyclic adenosine monophosphate) levels. When cAMP levels increase, the catalytic subunit of PKA (cAMP dependent protein kinase) is released from the regulatory subunit. The purpose of this investigation is to prove if ethanol’s protective capacity works against oxidative stress present in injured organisms or if it has to do with another pathogenesis of the injury. The hypothesis is that ethanol protects the cells from oxidative stress. Cells that receive treatment with 15% ethanol should show more PKA and CREB expression than the cells who receive 5% ethanol, hydrogen peroxide, and neither. To test this hypothesis we obtained tissue from the spinal cord of injured animals, and then cultured them. Added ethanol 5% and 15% with hydrogen peroxide for three and twenty four hour, hydrogen peroxide for half an hour, three and twenty four hours, or neither to this media. Then, analyzed the tissue by Western Blot with antiPKA and antiCREB. According to our results, cells with 5% ethanol showed more PKA than cells with 15% ethanol, hydrogen peroxide and untreated. In the case of CREB signaling, the cells treated with 5% ethanol for twenty four hours showed more CREB, and the ones treated with hydrogen peroxide for three hours were the ones that showed less. In conclusion, a functional treatment for spinal cord injury can be the combination of 5% ethanol with another pharmacological agent. Introduction Injury to the human spinal cord produces a complex pathology, including disruption of its cellular integrity. This leads to the loss of various motor and sensory functions. Pearse et al. studied the ability of Schwann cell to repopulate and replace damaged tissue. It has been analyzed whether the combination of these cells with molecular or pharmacologic strategies will ensure the cell’s maximum efficacy for restoration. The pharmacological strategy that is being studied the most is ethanol. Studies by Yao L. et al. have demonstrated that short- term exposure to ethanol increases extracellular adenosine, which activates adenosine A2 receptors and increases cAMP levels. When cAMP levels increase, the catalytic subunit of PKA is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. There is evidence that oxidative stress plays a key role in the pathogenesis of the white-matter injury10. Oxidative stress by definition, is when there is an imbalance between the formation of reactive oxygen species (ROS) and removal of oxyradicals by antioxidants. Increase in ROS production has been directly linked to the oxidation of cellular macromolecules, which may cause direct cellular injury or induce a variety of cellular responses through the generation of secondary metabolic reactive species. Peroxides, including hydrogen peroxide (H2O2), are one of the main reactive oxygen species (ROS) leading to oxidative stress9. H2O2 is continuously generated by several enzymes (including superoxide dismutase, glucose oxidase, and monoamine oxidase) and must be degraded to prevent oxidative damage2. According to Hoek JB, et al., ethanol promotes oxidative stress, both by increased formation of ROS and by depletion of oxidative defenses in the cell. Ryu H. et al. proved that antioxidants increase the quantity of PKA, so increasing oxidative stress should decrease the quantity of PKA. In the neurons, the results of Wang, Q. et al. suggest the possibility that preconditioning ethanol with other pharmacological agents that induce a mild oxidative stress may suppress stroke-mediated damage in the brain. According to their results, moderate ethanol ingestion causes a mild oxidative stress and at the same time initiates a protective response in the brain by an oxidant-dependent mechanism. cAMP (cyclic AMP) is a second messenger that regulates the passage of Ca2+ through ion channels. Research has suggested that cyclic AMP (cyclic adenosine monophosphate) can turn on growth factor genes in nerve cells, stimulating growth and helping to overcome signals that normally inhibit regeneration. It has been shown that activation of the cAMP pathway can increase axon growth in cell implants and improve function of the cells. PKA and Creb are molecules involved in the expression of genes, and are closely related to cAMP. PKA (cAMP dependent protein kinase) is a tetrameric protein composed of two subunits that bind cAMP, and two subunits that catalyze the transfer of a phosphoryl group from ATP to a target enzyme. Results of Fee J. et al. suggest that PKA protects against ethanol-induced locomotor activity and behavioral sensitization. CREB (cAMP response element binding) is a protein that binds to DNA sequences called cAMP response elements (CRE) and affects the expression of certain genes. In the reaction chain of cAMP, cAMP first reacts with PKA and then PKA reacts with CREB. The cells that are going to be studied in this investigation are the oligodendrocytes. Oligodendrocytes play a crucial role in facilitating the rapid conduction of neuronal action potentials and supporting axonal survival. The hypothesis is that the oligodendrocytes treated with ethanol will have more PKA in comparison to the oligodendrocytes treated with hydrogen peroxide only or neither. This is because according to my hypothesis ethanol protects the oligodendrocytes from the oxidative stress. Cells treated with 15% ethanol should show more PKA and CREB expression than cells treated with 5% ethanol. At the same time, cells treated for twenty four hours with ethanol should show more than the cells treated for three hours. Ryu H et al. results proved that oxidative stress decreases the quantity of PKA. So cells treated with hydrogen peroxide only for half an hour should show more PKA than the cells treated for three and twenty four hours. Methods: Tissue obtaining Spinal cord and brain’s cells were obtained from rats. Thoracic spinal cord contusion to the T8 was performed in rats. Rats were then anesthetized with isoflourine, then injected with ketamine and xylezine. They were then decapitated. The brain and spinal cord of the rats were dissected. Adult rats were housed according to NIH and The Guide for the Care and Use of Animals. The Institutional Animal Care and Use Committee of the University of Miami approved all animal procedures. Cell Culture Oligodendrocytes were prepared and isolated from rat brains according to a method derived from Chen Y et al. First, four rats were subjected to a contusion in T8. After three days, the brain and spinal cord of the rats were extracted. The tissue was put in ethanol 70% and then in HBSS. Cortical tissues of the brain and spinal cord were diced with a sterilized razor blade into 1.5 mm3 chunks. The cells were cleaned under the dissection microscope and put in collagenase/ dispase enzyme mix for two hours. Triturated the cells with the pipette. Then centrifuged the cells and ran them in the strainer. Centrifuged the cells again, and washed the red blood cells. Let sit in ice for 10 minutes. Spin the cells and took out the supernatant. Added DMEM20S. Let grow the cells for 10 days. Staining with Hoechst and RIP To see if the cells needed for this investigation were growing in the cultures, before doing the procedure they were stained. First the cells were fixed, with 4% paraformaldehyde. The paraformaldehyde was removed and added DPBS to wash. For Permeabilization, 0.1% Triton x-100 in PBS was added to the slides for no more than 10 minutes. The Triton was removed and the slides were washed twice with DPBS. The 10% NGS (H1 normal goat serum) was added to the slides for one hour to create the blocking. Then they were washed once with DPBS. Was added the primary antibodies GFAP polyclonal (1:1000) and RIP monoclonal (1:200) overnight. The primary antibodies were removed and the secondary antibodies, GAM 594 and GAR 488, were added overnight. Removed the secondary antibody and washed with DPBS once. Added Hoechst and washed with DPBS. Adding ethanol and hydrogen peroxide Added 1micromolar hydrogen peroxide to each dish or slide. Waited half an hour. Treated the cells with 5% or 15% ethanol, depending on the condition. Waited for half an hour, three hours or twenty four hours. For use in western blot, the cells were first taken from the Petri dish and centrifuged. The supernatant was taken out, the pellet was resuspended and the solution kept in -80 degrees Celsius. For the slides, each slide was treated with the specified conditions and kept for later procedure. The conditions used in this investigation are presented in Fig 1. Staining with PI and Hoechst Cells were stained with PI for ten minutes. Then the cells were fixed, with 4% paraformaldehyde. The paraformaldehyde was removed and added DPBS to wash. For Permeabilization, 0.1% Triton x-100 in PBS was added to the slides for no more than 10 minutes. The Triton was removed and the slides were washed twice with DPBS. The 10% NGS (H1 normal goat serum) was added to the slides for one hour to create the blocking. Then they were washed once with DPBS. Was added the primary antibodies GFAP polyclonal (1:1000) and RIP monoclonal (1:200) overnight. The primary antibodies were removed and the secondary antibodies, GAM 594 and Gar 488, were added overnight. Removed the secondary antibody and washed with DPBS once. Western Blot Analysis First, the gel for Western Blot was prepared. Proteins were transferred to nitrocellulose membranes and probed using antiPKA and antiCREB. Each tissue was incubated with the antibody, washed and then exposed to autoradiographic film. Results Results in figure 6 prove that the cells who received treatment with 5% ethanol for twenty four hours showed more PKA. Cells treated with 5% ethanol showed more PKA than cells treated with hydrogen peroxide only. At the same time, cells treated with hydrogen peroxide showed more than the cells treated with 15% ethanol, and this cells more than untreated. In the case of the cells treated with ethanol, the order from the cell culture that showed most PKA to the one that showed less is: twenty four and three hours. In the cells treated with hydrogen peroxide only, the order was the opposite, being the cells treated for three hours the ones that showed more PKA, then the ones treated for twenty four hours. The difference in the quantity of PKA between the cells treated with ethanol or with hydrogen peroxide for different time intervals was small. Figure 9 shows the graph of the proteins probed with antiCREB. According to our results, the cells treated with 5% ethanol for twenty four hours showed more CREB than the cells treated with 15% ethanol for three hours, and this more than the ones with 15% ethanol for twenty four hours. The cells untreated and treated with hydrogen peroxide for three hours showed similar quantities of CREB, as the cells treated with hydrogen peroxide for twenty four hours and 5% ethanol for three hours. Discussion Results in figure 6 prove that cells treated with 5% ethanol for twenty four hours produced more PKA than the rest of the cultures studied in this investigation. The results obtained were different to the expected since the cells treated with extra oxidative stress presented more PKA than the cells treated with 15% ethanol. The explanation to this could be that a 15% ethanol treatment is too strong and kills the cells. This analysis is based too in the graph of cell death assay (figure 12). In this graph, even though the cells treated with 5% ethanol had more PI staining (stains dead cell), in the cell cultures treated with 15% ethanol was found less quantity of cells. This may had happened because when the cells die, they don’t attach to the dish, so they can be lost during the procedure. The results obtained in the analysis of CREB signaling were similar to the ones recovered in PKA, only that in the CREB analysis 15% ethanol treatment for three hours showed more CREB signaling than the hydrogen peroxide treatment. After this can be concluded that a treatment with a strong ethanol concentration as 15% ethanol is functional, but in short term. Since CREB is produced during stress stages of a cell life, is important to point that the quantity of CREB signaling in the cells untreated and the cells treated with hydrogen peroxide for three hours was nearly equal. This can be explained after the fact that the untreated cells already presented oxidative stress, this is because they were recovered from a spinal cord injured animal, and oxidative stress is one of the principal characteristics present in the pathogenesis of the injured animal’s cell. The same happened to the cells treated with hydrogen peroxide for twenty four hours and 5% ethanol for three hours. After this result can be concluded that ethanol treatment does cause oxidative stress, and this may be important to understand how it protects the cell from apoptosis. The graph of cell death assay, figure 2, presents that a treatment with H2O2 causes more cell death than a treatment with 15% ethanol, and this more than a treatment with 5% ethanol. This is because in the cell cultures treated with H2O2 was found the less quantity of cells, so the rest must have died, didn’t attach to the slide and were lost during the procedure. The cells treated with 15% ethanol showed more PI than the cells treated with H2O2, so it is possible that this concentration of ethanol does protect the cells (they died but did not come out of the slide), but not as much as ethanol 5%. The results obtained in the death assay of the cells treated with 5% ethanol were great, since nearly 50% of the cells survived. According to the results obtained in this investigation, ethanol increases cAMP, which at the same time increases PKA, but not necessarily increases CREB. One remaining issue is to identify by which mechanism ethanol protects against oxidative stress. The results obtained in this investigation show that a treatment of 5% ethanol is functional. This is because it increases PKA. By increasing PKA can be concluded that cAMP was increase too. Researchers are looking forward to increase cAMP in injured organisms because it increases at the same time neuronal regeneration. It was proven in this investigation too that ethanol protects against oxidative stress, one of the principal causes of oligodendrocyte’s apoptosis. Ethanol in combination with other treatments should increase motor function in injured humans. Tables and figures Fig 1. Conditions used to treat the cell cultures Fig 2. Picture of oligo- precursor cells stained with Hoechst (nuclei stained) Fig 3. 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