Step 2. Transfection of plasmid into human neuronal cells

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Transcript Step 2. Transfection of plasmid into human neuronal cells

Increased Expression of Tryptophan Hydroxylase as a Potential
Treatment of Methylenedioxymethamphetamine Depletion of
Serotonin in Human Neuronal Cells
www.nida.nih.gov
Antony Ngondara
Figure 1. Methylenedioxymethamphetamine
Department of Biological Sciences, York College of Pennsylvania
Abstract
Research Design and Methods
Introduction
MDMA or ecstasy is an illegal, synthetic drug that has
grown in popularity since the early 1990’s
Step 1. Generate TPH expression plasmid
Isolate human tissue RNA, RT to produce cDNA
PCR using primers specific for human TPH
Figure 3. Biosynthetic Pathway of Serotonin where
tryptophan hydroxylase is the rate-limiting enzyme in serotonin
production (Wood and Russo 2001).
Review of Literature
Toxicity do to amphetamines and methamphetamines is
a result of mass reduction in brain dopamine and
serotonin levels as well as reduction of reuptake sites in
the brain (Ricaurte et al 1985).
Isolate band, cut plasmid (Fig. 5) and TPH gene with
restriction digests Xho1 and Pme 1 and purify product
Ligate and grow up in E.Coli with ampicillin as the
selectable marker
Observations by Wood and Russo found that the TPH
promoter could be amplified 20 fold by the MAP kinase
cascade of phosphorylation and suggests that regulation
of the enzyme can be manipulated (Fig. 4, 2001).
Few treatment options for MDMA-induced neuronal
damage exist; it is unknown whether damage is reversible
Step 2. Transfection of plasmid into human
neuronal cells
Transfection- the implementation of DNA into neuronal
cells allowing for changes in cell function.
Done transiently with amphiphilic liposomes
(Lipofectamine, invitrogen)
Select with neomycin
Serotonin
Serotonin
receptor
www.nida.nih.gov
Figure 2. Model of Synapse Action
200
TPH
Untreated Cells
MDMA and TPH
MDMA
150
100
50
Figure 6. Expected Results of TPH Transfection
Theoretical graph illustrating expected results from
specified treamtment groups, where untreated cells
(100%) serves as a basis for comparison.
Future Studies
Another option to confirm function of TPH as the
determinant factor would be to label Tryptophan amino
acid and document if degradation is increased with
increased enzyme activity
Investigation into an endogenous conversion mechanism
by which TPH and serotonin can be increased
Literature Cited
Ricaurte, G.A., Bryan, G., Strauss, L., Seiden, L. and
Schuster, C. 1985. Science 229: 986-989.
Ricaurte, G.A., Forno, L.S., Wilson, M.A., DeLanney, L.E.,
Irwin, I., Molliver, M.E. and Langston, J.W. 1988. Journal
of the American Medical Association 260: 51-56.
Objective
Reuptake
transporter
Expected Results
Treatments
Structure allows MDMA to have a higher affinity for
reuptake sites than serotonin itself (Ricaurte et al. 1985)
Figure 4. Activation of the TPH promoter by activated MEK1 and
repression by CGS. TPH promoter luciferase reporter plasmids were
transiently cotransfected into CA77 cells with a plasmid encoding
constitutively active MEK1 or vector control and then treated with CGS
(10 µM) or vehicle (0.0001 N HCl) for 24 h prior to harvest. Promoter
constructs from top to bottom are TPH0.045-luc and TPH0.15-luc.
Luciferase activity is expressed as relative light units per 20 µg of
protein ± S.E. Data represent at least three independent experiments
(Taken from Wood and Russo 2001).
Differences in intensity is scanned by NIH computer
software and yields score for amount of serotonin present
0
Figure 5. pcDNA 3.1 Vector contains powerful CMV
promoter, Xho 1 and Pme 1 as restriction sites
(invitrogen)
One of the dangers associated with MDMA use is
neurotoxicity by way of damage to serotonergic nerve
fibers (Ricaurte et al. 1988)
ELISA using serotonin antibody (ab8882, Abcam)
Sequence plasmid to ensure gene is present
Evidence of nerve terminal damage and serotonin loss
occurred the the same location linking these two
observations in nonhuman primates (Ricaurte et al 1988).
MDMA releases and inhibits reuptake of serotonin from
axon terminals producing heightened stimulatory effects
by saturation of serotonin receptors (Ricaurte et al. 1985)
Confirm enzyme function by screening for 5-HTP protein
using western blotting using 5-HTP antibody (ab8890,
Abcam)
Pixel Density Measure
(% control)
Ecstasy, or methylenedioxymethamphetamine (MDMA),
releases and inhibits reuptake of serotonin from axons
producing euphoria. Prolonged use of MDMA results in
serotonergic nerve damage and reduced axon function.
While use of this drug has escalated, few treatment
options exist. We hypothesize that amplification of
tryptophan hydroxylase (TPH) enzyme, which is the ratelimiting step in converting tryptophan into serotonin, will
increase serotonin levels after MDMA-induced depletion.
Human neuronal cells will be transfected with a CMV
driven, full-length human TPH gene in plasmid with
neomycin as a selectable marker. Activity of TPH will be
detected by western blot analysis of 5-HTP and serotonin
levels quantified by ELISA. We expect that cells
transfected with TPH will show increases in protein and
serotonin levels, while cells transfected with an empty
vector will show no recovery. If this study shows an
increase in neuronal function, induction of TPH may be a
potential treatment for ecstasy-induced neuron damage.
Step 4. Enzyme Function and Serotonin
Quantification
 To successfully transfect the TPH gene into
MDMA-induced, serotonin depleted neuronal cells
Step 3. Treatment Groups
To determine if transfection of TPH will increase
enzyme activity and serotonin expression
No MDMA and Transfection (positive control)
To test whether increases in serotonin can
increase neuronal function of serotonin
transmission in presence of MDMA
MDMA and Transfection (experimental group)
No MDMA and No Transfection (untreated sample)
MDMA and No Transfection (negative control)
Wood, J. L., and Russo, A.F. 2001. Journal of Biological
Chemistry 276: 21262-21271.
Acknowledgements
Dr. Kaltreider-YCP research mentor for his cellular and
molecular prowess and Dr. Thompson