The Tryptophan Hydroxylase 2 TPH2, Phosphorylated TPH2, and 14-3-3 eta Protein Levels in the Prefrontal Cortex of Alco

1. The Tryptophan Hydroxylase 2 (TPH2), Phosphorylated � TPH2, and 14-3-3 eta Protein Levels in the Prefrontal Cortex of Alcohol-Dependent Subjects. Nisma Mujahid

2. Serotonin and Alcoholism Various scientific studies support the hypothesis that reduced serotonin neurotransmission is linked with alcohol-dependence.

3. Biosynthesis of Serotonin

4. Tryptophan Hydroxylase TPH exists in two different forms in humans: TPH1-found primarily in the pineal gland TPH2-found predominately in the brain

5. The Interaction of TPH2, Phosphorylated TPH2, and 14-3-3 eta Proteins

6. Hypotheses Considering all the previous information, 3 hypotheses can be formulated to explain the biochemical mechanisms contributing to the pathophysiology of alcohol-dependence: 1. The concentration of TPH2 protein is increased in the prefrontal cortex (PFC) of alcohol-dependent subjects relative to control subjects. but 2. The levels of phosphorylated TPH is decreased in the PFC of alcohol-dependent subjects relative to control subjects. and 3. The biosynthesis of 14-3-3 eta is decreased in the PFC of alcohol-dependent subjects relative to control subjects.

7. Subjects 9 subjects who had a diagnosis of alcohol-dependence were matched with psychiatrically normal control subject for gender and as closely as possible for age and post-mortem interval, which is the time of death till the time of freezing the brain. Some of the pairs were also matched for race.

8. Western Blotting Prefrontal cortex samples were homogenized in Tris homogenization buffer, and protein concentrations were determined. Samples were then diluted in sample buffer. Protein samples were separated on a sodium-dodecyl-lauryl-sulfate(SDS)-polyacrylamide gel by electrophoresis. Then the gel was transferred onto a nitrocellulose membrane using an electrical current. The membrane was incubated in 5% non-fat dried milk/PBS to block non-specific binding. The membrane was incubated overnight at 4oC in primary antibody. TPH2, phosphorylated TPH2, and 14-3-3 eta proteins were labeled using rabbit polyclonal antibody. Actin was used as a control of loading and transfer. The membrane was then incubated in secondary antibody (anti-rabbit) for 2 hours to amplify the proteins. For actin, anti-mouse was used. Membrane was incubated in western lightning chemiluminescence reagent. Enzyme Horseradish peroxidase (HRP) catalyzes light emission from luminol oxidation and is conjugated with the secondary antibody. Illumination was captured on X-ray film. Band densities for each protein were analyzed using imaging software. Relative optical density values of experimental protein were normalized to values of control protein.

9. 2) Load samples of proteins onto gel and run electrophoresis 3) Transfer gel onto membrane

10. TPH2, TPH2 Phosphorylated and 14-3-3 eta Proteins Levels in Prefrontal Cortex in Alcohol Dependence Subjects

11. Conclusions The elevated protein level of TPH2 in the prefrontal cortex of alcoholics may be due to a homeostatic or compensatory response to the reduced serotonin neurotransmission detected in the brain of alcohol dependent subjects. The lack of significant changes in TPH2 phosphorylated and 14-3-3 eta proteins in the prefrontal cortex of alcoholic subjects may reflect regional brain differences in these proteins. For example, perhaps TPH2 phosphorylated and 14-3-3 eta may be altered to a greater extend in the serotonin cell body regions of the dorsal raphe of alcohol-dependent subjects as compared to the prefrontal cortex. Measuring these proteins in the dorsal raphe of alcoholic subjects is planned for future studies.

12. Acknowledgements Dr. Mark Austin Dr. Bernadeta Szewczyk Tarsha Harris