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). 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. 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. 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: It is characterized by four major features: 1. Rest tremors of limbs 1. Rest tremors of limbs 2. Slowness of movement 2. Slowness of movement 3. Rigidity 3. Rigidity 4. Poor balance 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. 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). 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 at http://www.pbs.org/newshour/bb/health/july- dec99/parkinson_9-28.html. http://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. http://www.med.univ- rennes1.fr/resped/cours/pharmaco/media/nigra.jpghttp://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) The picture above was taken from NCERx LLC (http://www.about- dementia.com/parkinsons/pd-causes.php )http://www.about- dementia.com/parkinsons/pd-causes.phphttp://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: 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 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. 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). 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 + ). 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 Materials and Methods Growing/Splitting PC-12 Cells Growing/Splitting PC-12 Cells Dosing PC-12 Cells Dosing PC-12 Cells Performing HPLC/EC on PC-12 Cells Performing HPLC/EC on PC-12 Cells Performing RNA Extraction on PC-12 Cells Performing RNA Extraction on PC-12 Cells RNA Integrity Number Standardization of RNA Quality Control of PC-12 cells RNA Integrity Number Standardization of RNA Quality Control of PC-12 cells Gene Expression of PC-12 cells Gene Expression of PC-12 cells Nuclear Staining 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 RNA was extracted using Qiagen RNAeasy mini kit After extraction the quantity of RNA was measured using NanoDrop ND-1000 UV-Vis spectrophotometer 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 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 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 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 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 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 + 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. 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. 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. 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. 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. 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. 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. 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. 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. Thanks to Mr. Rosser, my Biology teacher at Fayetteville High School to encourage and allow me to do this project.

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