The Tryptophan Hydroxylase 2 (TPH2), Phosphorylated

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

• Nisma Mujahid

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Serotonin and Alcoholism

• Various scientific studies support the hypothesis
  that reduced serotonin neurotransmission is
  linked with alcohol-dependence.

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Biosynthesis of Serotonin
The initial and rate-limiting step during the
biosynthesis of serotonin is the hydroxylation of
L-tryptophan to 5-hydroxytryptophan by the
rate-limiting enzyme tryptophan hydroxylase or
TPH.
5-hydroxytryptophan is then decarboxylated into
5-hydroxytryptamine or serotonin by the aromatic
amino acid decarboxylase.
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Tryptophan Hydroxylase

• TPH exists in two different forms in humans
• TPH1-found primarily in the pineal gland
• TPH2-found predominately in the brain

Film autoradiographic images comparing the
localization and level of expression of TPH1
versus TPH2 mRNA in the dorsal raphe nucleus and
pineal gland (insets) of a normal control subject.
Structure of TPH
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The Interaction of TPH2, Phosphorylated TPH2, and
14-3-3 eta Proteins

• TPH2 is an unstable molecule with low activity at
  its initial state.
• To higher the activity, TPH2, is phosphorylated
  by the enzyme Kinase.
• TPH2-phosphorylated is a more stable molecule and
  has higher activity than TPH2.
• Binding of the intracellular regulatory molecule
  14-3-3 eta to TPH2-P has two effects.
• It increases its hydroxylase activity.
• It also prevents dephosphorylation by altering
  its conformation or directly blocking
  phosphatases access to the phosphorylated TPH2.

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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.

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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.

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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.

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2) Load samples of proteins onto gel and run
electrophoresis
1) Denature proteins with sodium dodecyl sulfate

• 3) Transfer gel onto membrane

Block non-specific binding using milk buffer and
incubate in primary and secondary antibodies to
detect specific binding. Incubate membrane with
western lightning chemiluminescence reagent.
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TPH2, TPH2 Phosphorylated and 14-3-3 eta Proteins
Levels in Prefrontal Cortex in Alcohol Dependence
Subjects
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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.

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Acknowledgements

• Dr. Mark Austin
• Dr. Bernadeta Szewczyk
• Tarsha Harris