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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