Heather Ali Fayetteville High School Fayetteville, AR

1. The Effects of MPP+ on Gene Expression and Dopamine in PC-12 Cells:An In-Vitro Model of Parkinson’s Disease Heather Ali Fayetteville High School Fayetteville, AR

2. Introduction • Parkinson disease (PD) is the most common neurodegenerative disease after Alzheimer’s (National Parkinson Foundation). • It is called Parkinson’s disease because it was described in 1817 by Dr. James Parkinson, a British physician. • PD occurs when certain nerve cells (neurons) in a part of the brain called the substantia nigra die or become impaired. • It is characterized by four major features: 1. Rest tremors of limbs 2. Slowness of movement 3. Rigidity 4. Poor balance

3. Social Impacts of Parkinson’s Disease • Each year Parkinson’s disease affects 50,000 Americans, adding to the 1 million already living with the disease. • This creates a burden on the healthcare system in the United States with spending of approximately $5.6 billion per year (Society for Neuroscience).

4. Many famous people have been diagnosed with Parkinson’s Disease The pictures above were taken from the website of Online NewsHour under the article Testifying for Parkinson’s athttp://www.pbs.org/newshour/bb/health/july-dec99/parkinson_9-28.html.

5. Normal Human Brain The picture above was taken from the Neurobiological Lab at the University Rostock website at http://www.med.univ-rennes1.fr/resped/cours/pharmaco/media/nigra.jpg.

6. Brain Region Involved in Parkinson’s Disease The picture above was taken from American Accreditation HealthCare Commission (www.urac.org).

7. Normal Dopamine Neuron in the Brain The picture above was taken from Pfizer Inc (http://www.pfizer.com/brain/dlgame.html)

8. Dopamine Neurons in Normal Brain and Parkinson's Disease Brain The picture above was taken from NCERx LLC(http://www.about-dementia.com/parkinsons/pd-causes.php)

9. Problem Does MPP+ produce dopaminergic neurotoxicity similar to Parkinson’s disease in PC-12 cells by altering the concentration of the neurotransmitter dopamine, RNA, altering selective gene expression and morphological changes?

10. Hypothesis MPP+ will produce dopaminergic neurotoxicity similar to Parkinson’s disease in PC-12 cells by decreasing the concentration of the neurotransmitter dopamine, RNA, altering selective gene expression and causing major morphological changes

11. Specific Aims • Effects of MPP+ on PC-12 Cells: • DA, DOPAC and HVA concentration by HPLC/EC • Total RNA content by Nano Labchip Kits and Agilant Bioanalyser • Gene expression by Real-time-PCR • Morphological changes by Hoechst 33258 Staining

12. MPP+ Background • MPTP discovered by mistake in the illegal formation of Heroin. • MPTP has been shown to produce Parkinson’s-like symptoms in both humans and animal models such as mouse and monkeys. • MPP+ is the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrdine (MPTP). • This in-vitro cell model of Parkinson’s disease was created by treating PC-12 cells in culture with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+).

13. PC-12 Cultured Cells • Pheochromocytoma (PC-12) cell line established by Greene and Tischler in 1976 • PC-12 cultured cells, derived from a transplantable rat adrenal • The cells are used to study brain biochemistry because they have the ability to synthesize and release the neurotransmitter dopamine, as do dopaminergic brain cells • Their ATCC (American Type Culture Collection) number is CRL-1721

14. Materials and Methods • Growing/Splitting PC-12 Cells • Dosing PC-12 Cells • Performing HPLC/EC on PC-12 Cells • Performing RNA Extraction on PC-12 Cells • RNA Integrity Number Standardization of RNA Quality Control of PC-12 cells • Gene Expression of PC-12 cells • Nuclear Staining of PC-12 cells

15. Growing/Splitting PC-12 Cells

16. Dosing PC-12 Cells • Cells dosed with 500uM of MPP+ at 4 hours, 8 hours, and 24 hours • Control had no exposure to MPP+

17. Performing HPLC on PC-12 Cells to Measure Dopamine

18. Dopamine Concentration in PC-12 Cells Exposed to MPP+

19. Performing RNA Extraction on PC-12 Cells • RNA was extracted using Qiagen RNAeasy mini kit • After extraction the quantity of RNA was measured using NanoDrop ND-1000 UV-Vis spectrophotometer

20. Total RNA Content in PC -12 Cells after Exposure to MPP+

21. RNA Integrity Number Standardization of RNA Quality Control of PC-12 cells • This method utilized the Agilent 2100 bioanalyzer and associated RNA 6000 Nano Assay (Nano LabChip kit) to access the integrity of the RNA

22. Gene Expression of PC-12 cells • Real-Time Polymerase Chain Reaction (RT-PCR) method was used to analyze the gene expression

23. Alpha-Synuclein Gene Expression in PC-12 Cells Exposed to MPP+

24. DAT Gene Expression in PC-12 Cells Exposed to MPP+

25. Parkin Gene Expression in PC-12 Cells Exposed to MPP+

26. Nuclear Staining of PC-12 cells • Cells were grown and dosed on a cover slip inside the wells • Olympus Fluorescence microscope at an excitation wavelength of 365 nm used to view the staining

27. Hoechst 33258 Staining Top two photomicrographs are control 24 hours cells and bottom two photomicrographs are cells that have been exposed to 500 micromolars of MPP+ for 24 hours.

28. Summary • MPP+ produced a time-dependent effect on dopamine levels in PC-12 cells. As the time of incubation was increased more significant effects resulted as shown the dopamine concentration graph. • MPP+ produced a time-dependent decrease in the total RNA in PC-12 cells, as shown in the total RNA graph. • MPP+ effected the expression of both alpha-synuclein and DAT genes which were shown to be significantly different after 24 hours of exposure. Although there were changes in other genes such as Parkin, these changes did not reach statistical significance. • Fluorescent microscopy with Hoechst 33258 staining showed that MPP+ produced an increase in nuclear fragmentation and condensation in PC-12 cells, as shown in the Hoechst photomicrographs.

29. Conclusion • The data collected in this project summarized that MPP+ produces neurotoxic effects in PC-12 cells. • It can then be extrapolated that the exposure of MPP+ on PC-12 cells would be similar to the effects on human brain cells in Parkinson’s disease. • Therefore, this will be a good in vitro model of Parkinson’s disease and can be used to screen new drugs to treat this neurodegenerative disease.

30. Acknowledgments • This work was performed at the National Center for Toxicological Research under the direction of Dr. Tucker Patterson. I would like to thank Ms. Helen Duhart and Dr. Tucker Patterson for providing the facilities to perform this work. • I would like to thank Dr. Syed Ali for taking the photographs during the course of this research project. • I would like to thank Dr. Pat Briney for critical review of my project. • Thanks to Mr. Rosser, my Biology teacher at Fayetteville High School to encourage and allow me to do this project.

31. References • Bergman H, Deuschl G. Patholphysiology. Lancet 337 (2002): 1321-1324. • Bradbury, Jane. “Immunotherapy for Parkinson’s disease: a developing therapeutic strategy.” News and Comment 10 (2005): 1075-1076. • Kim, S., et al. “Alpha-synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson’s disease.” FASEB J 18 (2004): 1615-1617. • Landrigan, Philip J., et al. “Early Environmental Origins of Neurodegenerative Disease in Later Life.” Environmental Health Perspectives 113 (2005): 1230-1233. • Lehmensiek, V., et al. “Expression of mutant alpha-synuclein enhances dopamine transporter-mediated MPP+ toxicity in vitro.” Neuroreport 13 (2002): 1279-1283. • Logroscino, Giancarlo. “The Role of Early Life Environmental Risk Factors in Parkinson Disease: What is the Evidence?” Environmental Health Perspectives 113 (2005): 1234-1238. • Miller, RM., & Federoff, HJ. “Altered gene expression profiles reveal similarities and differences between Parkinson disease and model systems.” Neuroscientist 11 (2005): 539-549.

32. Morris, Richard and Marianne Fillenz, ed. Neuroscience Science of the Brain: An Introduction for Young Students. Liverpool: British Neuroscience Association, 2003. • National Parkinson Foundation. 2004. Parkinson Primer. Di Minno, Mariann and Michael J. Aminoff: Parkinson’s Disease Clinic and Research Center. Available: http://www.parkinson.org/site/pp.asp?c=9dJFJLPwB&b=71354 [accessed 15 January 2006]. • Olds, M.E., et al. “Behavioral and Anatomical Effects of Quinolinic Acid in the Striatum of the Hemiparkinsoninan Rat.” Synapse 55 (2005): 26-36. • Sawada, H., et al. “Methylphenylpyridium ion (MPP+) enhances glutamate-induced cytotoxicity against dopaminergic neurons in cultured rat mesencephalon.” Neuroscience Research 43 (1996): 55-62. • Society for Neuroscience. 2004. Brain Research Success Stories: Parkinson’s Disease. Available: http://apu.sfn.org/content/Publications/BrainResearchSuccessStories/BRSS_Parkinsons_Disease.pdf [accessed 16 January 2006]. • Weiner, William J., et al. Parkinson’s Disease: A Complete Guide for Patients and Families. Baltimore: John Hopkins University Press, 2001. • Xu, Z., et al. “Selective Alterations of Gene Expression in Mice Induced by MPTP.” Synapse 55 (2005): 45-51. • Xu, Z., et al. “Selective alterations of transcription factors in MPP+ induced neurotoxicity in PC12 cells.” Neurotoxicology 26 (2005): 729-737.