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. Picture of oligo- precursor cells stained with RIP (only oligodendrocytes stained)

Untreated

H2O2

3

hours

H2O2

24

hours

5%

EtOH

3

hours

5%

EtOH

24

hours

15%

EtOH

3

hours

15%

EtOH24

hours

Fig 4. Gel of PKA

Fig 5. Gel of beta actin PKA

Fig 6. Graph of PKA signaling

Fig 7. Gel of CREB

Fig 8. Gel of beta actin CREB

Fig 9. Graph of CREB signaling

Fig 10. Picture of cells treated with 5% ethanol, stained with Hoechst (blue) and PI (red)

Fig 11. Picture of cells treated with 15% ethanol stained with Hoechst (blue) and PI (red)

Fig 12. Graph of cell death assay

References

1. Baud O., Greene A., Li J., Wang H. , Volpe J, and Rosenberg P. (2004) Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. The Journal of Neuroscience. 24(7). 1531-1540.

2. Belayev L, Khoutorova L, Zhang Y, Belayev A, Zhao W, Busto R and Ginsberg M. (2004) Caffeinol confers cortical but not subcortical neuroprotection after transient focal cerebral ischemia in rats. Brain Research. 1008. 278-283.

3. Bracken MB. (2001) Methylprednisolone and acute spinal cord injury: an update of the randomized evidence. Spine. 26 (24). 47-54.

4. Chen Y, Balasubramaniyan V, Tallquist M, Li J and Richard Lu Q. (2007) Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nature Protocols 2. 1044 – 1051.

5. Chiarugi P. (2003) Reactive oxygen species as mediators of cell adhesion. Ital J Biochem. 52. 28–32.

6. Conti A., Maas J., Moulder K., Jiang X., Dave B., Mennerick S., and Muglia L. (2009) Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment. Plos One. 4(5).

7. Dohrman D, Diamond I and Gordon A. (1996) Ethanol causes translocation of cAMP- dependent protein kinase catalytic subunit to the nucleus. The Proceedings of the National Academy of Sciences Online (U.S.). 93. 10217- 10221.

8. Fee J. R, Knapp D. J., Sparta D. R., Breese G. R., Picker M. J., and Theele T. E. (2006) Involvement of protein Kinase a in ethanol-induced locomotor activity and sensitization. Neuroscience. 140 (1). 21-31.

9. Halliwell B, Gutteridge JMC (1999) Antioxidant defence enzymes: the glutathione peroxidase family. In: Free radicals in biology and medicine, Ed 3, pp 140-146, 170-172. Oxford, UK: Oxford UP.

10. Haynes RL, Folkerth RD, Keefe RJ, Sung I, Swzeda LI, Rosenberg PA, Volpe JJ, Kinney HC (2003) Nitrosative and oxidative injury to premyelinating OLs in periventricular leukomalacia. J Neuropathol Exp Neurol .62. 441-450

11. Hoek JB and Pastorino JG. (2002) Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol. 27 (1). 63-8

12. Pearse D, Pereira F, Marcillo A, Bates M, Berrocal Y, Filbin M and Bartlett M. (2004) cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nature Medicine. 1-7

13. Qun W, Sun A, Simonyi A, Kalogeris T, Miller D, Sun G and Korthuis R. (2007) Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: Role of NADPH oxidase-derived ROS. Free Radic Biol Med. 43 (7). 1048- 1060.

14. Ryu H., Lee J.,Impey S., Ratan R., and Ferrante R. (2005) Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons. Proc Natl Acad Sci U S A. 102(39). 13915–13920.

15. Wang Q, Sun A, Simonyi A, Kalogeris T, Miller D, Sun G and Korthuis R. (2007) Ethanol preconditioning protects against ischemia/ reperfusion- induced brain damage: Role of NADPH oxiddase- derived ROS. Free Radic Biol Med.43 (7). 1048- 1060.

16. Wang L, Renault G., Garreau H and Jacquet M. (2004) Stress induces depletion of Cdc25p and decreases the cAMP producing capability in Saccharomyces cerevisiae.

Microbiology. 150. 3383-3391.