The Effects of MPPThe Effects of MPP++ on Gene on Gene Expression and Dopamine in PC-12 Expression and Dopamine in PC-12

Cells:Cells:An An In-VitroIn-Vitro Model of Parkinson’s Model of Parkinson’s

DiseaseDisease

Heather AliHeather AliFayetteville High SchoolFayetteville High School

Fayetteville, ARFayetteville, AR

IntroductionIntroduction Parkinson disease (PD) is the most common Parkinson disease (PD) is the most common

neurodegenerative disease after Alzheimer’s neurodegenerative disease after Alzheimer’s (National Parkinson Foundation).(National Parkinson Foundation).

It is called Parkinson’s disease because it was It is called Parkinson’s disease because it was described in 1817 by Dr. James Parkinson, a British described in 1817 by Dr. James Parkinson, a British physician.physician.

PD occurs when certain nerve cells (neurons) in a PD occurs when certain nerve cells (neurons) in a part of the brain called the substantia nigra die or part of the brain called the substantia nigra die or become impaired. 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. Rigidity3. Rigidity

4. Poor balance4. Poor balance

Social Impacts of Parkinson’s Social Impacts of Parkinson’s DiseaseDisease

Each year Parkinson’s disease affects Each year Parkinson’s disease affects 50,000 Americans, adding to the 1 50,000 Americans, adding to the 1 million already living with the million already living with the disease. disease.

This creates a burden on the This creates a burden on the healthcare system in the United healthcare system in the United States with spending of States with spending of approximately $5.6 billion per year approximately $5.6 billion per year (Society for Neuroscience).(Society for Neuroscience).

Many famous people have been Many famous people have been

diagnosed with Parkinson’s Diseasediagnosed 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

.

Normal Human BrainNormal Human Brain

The picture above was taken from the Neurobiological Lab at the The picture above was taken from the Neurobiological Lab at the University Rostock website at University Rostock website at

http://www.med.univ-rennes1.fr/resped/cours/pharmaco/media/nigra.jphttp://www.med.univ-rennes1.fr/resped/cours/pharmaco/media/nigra.jpgg

. .

Brain Region Involved in Parkinson’s Brain Region Involved in Parkinson’s DiseaseDisease

The picture above was taken from The picture above was taken from American American Accreditation HealthCare Commission (www.urac.org).Accreditation HealthCare Commission (www.urac.org).

Normal Dopamine Neuron in the Normal Dopamine Neuron in the BrainBrain

The picture above was taken from Pfizer Inc The picture above was taken from Pfizer Inc (http://www.pfizer.com/brain/dlgame.html)(http://www.pfizer.com/brain/dlgame.html)

Dopamine Neurons in Normal Brain Dopamine Neurons in Normal Brain and Parkinson's Disease Brainand Parkinson's Disease Brain

The picture above was taken from NCERx LLCThe picture above was taken from NCERx LLC ((http://www.about-dementia.com/parkinsons/pd-causes.php ))

ProblemProblem

Does MPPDoes MPP++ produce dopaminergic produce dopaminergic neurotoxicity similar to neurotoxicity similar to

Parkinson’s disease in PC-12 Parkinson’s disease in PC-12 cells by altering the cells by altering the concentration of the concentration of the

neurotransmitter dopamine, neurotransmitter dopamine, RNA, altering selective gene RNA, altering selective gene expression and morphological expression and morphological

changes?changes?

HypothesisHypothesis

MPPMPP++ will produce dopaminergic will produce dopaminergic neurotoxicity similar to neurotoxicity similar to

Parkinson’s disease in PC-12 Parkinson’s disease in PC-12 cells by decreasing the cells by decreasing the

concentration of the concentration of the neurotransmitter dopamine, neurotransmitter dopamine,

RNA, altering selective gene RNA, altering selective gene expression and causing major expression and causing major

morphological changesmorphological changes

Specific AimsSpecific Aims

Effects of MPPEffects of MPP++ on PC-12 Cells: on PC-12 Cells: DA, DOPAC and HVA concentration by DA, DOPAC and HVA concentration by

HPLC/EC HPLC/EC Total RNA content by Nano Labchip Total RNA content by Nano Labchip

Kits and Agilant BioanalyserKits and Agilant Bioanalyser Gene expression by Real-time-PCRGene expression by Real-time-PCR Morphological changes by Hoechst Morphological changes by Hoechst

33258 Staining33258 Staining

MPPMPP++ Background Background MPTP discovered by mistake in the illegal MPTP discovered by mistake in the illegal

formation of Heroin.formation of Heroin. MPTP has been shown to produce MPTP has been shown to produce

Parkinson’s-like symptoms in both humans Parkinson’s-like symptoms in both humans and animal models such as mouse and and animal models such as mouse and monkeys.monkeys.

MPPMPP++ is the active metabolite of 1-methyl-4- is the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrdine (MPTP).phenyl-1,2,3,6-tetrahydropyrdine (MPTP).

This This in-vitroin-vitro cell model of Parkinson’s cell model of Parkinson’s disease was created by treating PC-12 cells disease was created by treating PC-12 cells in culture with the neurotoxin 1-methyl-4-in culture with the neurotoxin 1-methyl-4-phenylpyridinium (MPPphenylpyridinium (MPP++).).

PC-12 Cultured CellsPC-12 Cultured Cells Pheochromocytoma (PC-12) cell line Pheochromocytoma (PC-12) cell line

established by Greene and Tischler in 1976 established by Greene and Tischler in 1976

PC-12 cultured cells, derived from a PC-12 cultured cells, derived from a transplantable rat adrenaltransplantable rat adrenal

The cells are used to study brain The cells are used to study brain biochemistry because they have the ability biochemistry because they have the ability to synthesize and release the to synthesize and release the neurotransmitter dopamine, as do neurotransmitter dopamine, as do dopaminergic brain cells dopaminergic brain cells

Their ATCC (American Type Culture Their ATCC (American Type Culture Collection) number is CRL-1721Collection) number is CRL-1721

Materials and MethodsMaterials and Methods

Growing/Splitting PC-12 CellsGrowing/Splitting PC-12 Cells Dosing PC-12 CellsDosing PC-12 Cells Performing HPLC/EC on PC-12 CellsPerforming HPLC/EC on PC-12 Cells Performing RNA Extraction on PC-12 CellsPerforming RNA Extraction on PC-12 Cells RNA Integrity Number Standardization of RNA Integrity Number Standardization of

RNA Quality Control of PC-12 cellsRNA Quality Control of PC-12 cells Gene Expression of PC-12 cellsGene Expression of PC-12 cells Nuclear Staining of PC-12 cellsNuclear Staining of PC-12 cells

Growing/Splitting PC-12 Growing/Splitting PC-12 CellsCells

Dosing PC-12 CellsDosing PC-12 Cells

Cells dosed with 500uM of MPPCells dosed with 500uM of MPP++ at 4 at 4 hours, 8 hours, and 24 hours hours, 8 hours, and 24 hours

Control had no exposure to MPPControl had no exposure to MPP++

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

Dopamine Concentration in PC-12 Dopamine Concentration in PC-12 Cells Exposed to MPPCells Exposed to MPP++

control 4 hrs 8hrs 24hrs0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Exposure Time to MPP+

Ave

rag

e D

op

amin

eC

on

cen

trat

ion

(n

M/w

ell)

**

*

* p<0.001 Significantly Different from Control* Control means no exposure to MPP+

Performing RNA Extraction Performing RNA Extraction on PC-12 Cellson PC-12 Cells

RNA was extracted RNA was extracted using Qiagen using Qiagen RNAeasy mini kitRNAeasy mini kit

After extraction After extraction the quantity of the quantity of RNA was measured RNA was measured using NanoDrop using NanoDrop ND-1000 UV-Vis ND-1000 UV-Vis spectrophotometerspectrophotometer

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

control 4 hrs 8hrs 24hrs0

40

80

120

160

200

Exposure Time to MPP+

Ave

rage

Am

ount

of

Tota

l RN

A (u

g)

* p<0.001 Signif icantly Different from Control

*

* Control means no exposure to MPP+

RNA Integrity Number RNA Integrity Number Standardization of RNA Quality Standardization of RNA Quality

Control of PC-12 cellsControl of PC-12 cells

This method This method utilized the Agilent utilized the Agilent 2100 bioanalyzer 2100 bioanalyzer and associated and associated RNA 6000 Nano RNA 6000 Nano Assay (Nano Assay (Nano LabChip kit) to LabChip kit) to access the access the integrity of the integrity of the RNA RNA

Gene Expression of PC-12 Gene Expression of PC-12 cellscells

Real-Time Real-Time Polymerase Chain Polymerase Chain Reaction (RT-PCR) Reaction (RT-PCR) method was used method was used to analyze the to analyze the gene expressiongene expression

Alpha-Synuclein Gene Alpha-Synuclein Gene Expression in PC-12 Cells Expression in PC-12 Cells

Exposed to MPPExposed to MPP++

0

1

2

3

4

5

6

7

8

4 hrs 8 hrs 24 hrs

Exposure Time to MPP+

Ch

ang

es i

n C

ycle

sF

rom

Co

ntr

ol

*p<0.001 Signif icantlyDifferent from Control

*

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

00.5

11.5

22.5

33.5

44.5

5

4 hrs 8 hrs 24 hrs

Exposure Time to MPP+

Ch

ang

es in

Cyc

les

Fro

m C

on

tro

l

*p<0.001 Signif icantlyDifferent from Control

*

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

0

0.5

1

1.5

2

2.5

3

3.5

4

4 hrs 8 hrs 24 hrs

Exposure Time to MPP+

Ch

ang

es i

n C

ycle

sF

rom

Co

ntr

ol

Nuclear Staining of PC-12 Nuclear Staining of PC-12 cellscells

Cells were grown and dosed on a cover slip inside Cells were grown and dosed on a cover slip inside the wellsthe wells

Olympus Fluorescence microscope at an excitation Olympus Fluorescence microscope at an excitation wavelength of 365 nm used to view the stainingwavelength of 365 nm used to view the staining

Hoechst 33258 StainingHoechst 33258 Staining Top two photomicrographs are control 24 hours cells and Top two photomicrographs are control 24 hours cells and

bottom two photomicrographs are cells that have been bottom two photomicrographs are cells that have been exposed to 500 micromolars of MPPexposed to 500 micromolars of MPP++ for 24 hours. for 24 hours.

SummarySummary MPPMPP++ produced a time-dependent effect on dopamine produced a time-dependent effect on dopamine

levels in PC-12 cells. As the time of incubation was levels in PC-12 cells. As the time of incubation was increased more significant effects resulted as shown the increased more significant effects resulted as shown the dopamine concentration graph. dopamine concentration graph.

MPPMPP++ produced a time-dependent decrease in the total produced a time-dependent decrease in the total RNA in PC-12 cells, as shown in the total RNA graph. RNA in PC-12 cells, as shown in the total RNA graph.

MPPMPP++ effected the expression of both alpha-synuclein and effected the expression of both alpha-synuclein and DAT genes which were shown to be significantly different DAT genes which were shown to be significantly different after 24 hours of exposure. Although there were changes after 24 hours of exposure. Although there were changes in other genes such as Parkin, these changes did not in other genes such as Parkin, these changes did not reach statistical significance.reach statistical significance.

Fluorescent microscopy with Hoechst 33258 staining Fluorescent microscopy with Hoechst 33258 staining showed that MPP+ produced an increase in nuclear showed that MPP+ produced an increase in nuclear fragmentation and condensation in PC-12 cells, as shown fragmentation and condensation in PC-12 cells, as shown in the Hoechst photomicrographs.in the Hoechst photomicrographs.

ConclusionConclusion The data collected in this project The data collected in this project

summarized that MPPsummarized that MPP++ produces neurotoxic produces neurotoxic effects in PC-12 cells.effects in PC-12 cells.

It can then be extrapolated that the It can then be extrapolated that the exposure of MPPexposure of MPP++ on PC-12 cells would be on PC-12 cells would be similar to the effects on human brain cells similar to the effects on human brain cells in Parkinson’s disease.in Parkinson’s disease.

Therefore, this will be a good Therefore, this will be a good in vitroin vitro model model of Parkinson’s disease and can be used to of Parkinson’s disease and can be used to screen new drugs to treat this screen new drugs to treat this neurodegenerative disease. neurodegenerative disease.

AcknowledgmentsAcknowledgments This work was performed at the National Center for This work was performed at the National Center for

Toxicological Research under the direction of Dr. Toxicological Research under the direction of Dr. Tucker Patterson. I would like to thank Ms. Helen Tucker Patterson. I would like to thank Ms. Helen Duhart and Dr. Tucker Patterson for providing the Duhart and Dr. Tucker Patterson for providing the facilities to perform this work.facilities to perform this work.

I would like to thank Dr. Syed Ali for taking the I would like to thank Dr. Syed Ali for taking the photographs during the course of this research photographs during the course of this research project.project.

I would like to thank Dr. Pat Briney for critical review I would like to thank Dr. Pat Briney for critical review of my project.of my project.

Thanks to Mr. Rosser, my Biology teacher at Thanks to Mr. Rosser, my Biology teacher at Fayetteville High School to encourage and allow me Fayetteville High School to encourage and allow me to do this project. to do this project.

ReferencesReferences Bergman H, Deuschl G. Patholphysiology. Lancet 337 (2002): Bergman H, Deuschl G. Patholphysiology. Lancet 337 (2002):

1321-1324.1321-1324. Bradbury, Jane. “Immunotherapy for Parkinson’s disease: a Bradbury, Jane. “Immunotherapy for Parkinson’s disease: a

developing therapeutic strategy.” News and Comment 10 (2005): developing therapeutic strategy.” News and Comment 10 (2005): 1075-1076.1075-1076.

Kim, S., et al. “Alpha-synuclein induces apoptosis by altered Kim, S., et al. “Alpha-synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson’s expression in human peripheral lymphocyte in Parkinson’s disease.” FASEB J 18 (2004): 1615-1617.disease.” FASEB J 18 (2004): 1615-1617.

Landrigan, Philip J., et al. “Early Environmental Origins of Landrigan, Philip J., et al. “Early Environmental Origins of Neurodegenerative Disease in Later Life.” Environmental Health Neurodegenerative Disease in Later Life.” Environmental Health Perspectives 113 (2005): 1230-1233.Perspectives 113 (2005): 1230-1233.

Lehmensiek, V., et al. “Expression of mutant alpha-synuclein Lehmensiek, V., et al. “Expression of mutant alpha-synuclein enhances dopamine transporter-mediated MPP+ toxicity in vitro.” enhances dopamine transporter-mediated MPP+ toxicity in vitro.” Neuroreport 13 (2002): 1279-1283.Neuroreport 13 (2002): 1279-1283.

Logroscino, Giancarlo. “The Role of Early Life Environmental Risk Logroscino, Giancarlo. “The Role of Early Life Environmental Risk Factors in Parkinson Disease: What is the Evidence?” Factors in Parkinson Disease: What is the Evidence?” Environmental Health Perspectives 113 (2005): 1234-1238.Environmental Health Perspectives 113 (2005): 1234-1238.

Miller, RM., & Federoff, HJ. “Altered gene expression profiles Miller, RM., & Federoff, HJ. “Altered gene expression profiles reveal similarities and differences between Parkinson disease and reveal similarities and differences between Parkinson disease and model systems.” Neuroscientist 11 (2005): 539-549.model systems.” Neuroscientist 11 (2005): 539-549.

Morris, Richard and Marianne Fillenz, ed. Neuroscience Science of Morris, Richard and Marianne Fillenz, ed. Neuroscience Science of the Brain: An Introduction for Young Students. Liverpool: British the Brain: An Introduction for Young Students. Liverpool: British Neuroscience Association, 2003.Neuroscience Association, 2003.

National Parkinson Foundation. 2004. Parkinson Primer. Di Minno, National Parkinson Foundation. 2004. Parkinson Primer. Di Minno, Mariann and Michael J. Aminoff: Parkinson’s Disease Clinic and Mariann and Michael J. Aminoff: Parkinson’s Disease Clinic and Research Center. Available: Research Center. Available: http://http://www.parkinson.org/site/pp.asp?cwww.parkinson.org/site/pp.asp?c=9dJFJLPwB&b=71354=9dJFJLPwB&b=71354 [accessed 15 January 2006]. [accessed 15 January 2006].

Olds, M.E., et al. “Behavioral and Anatomical Effects of Quinolinic Olds, M.E., et al. “Behavioral and Anatomical Effects of Quinolinic Acid in the Striatum of the Hemiparkinsoninan Rat.” Synapse 55 Acid in the Striatum of the Hemiparkinsoninan Rat.” Synapse 55 (2005): 26-36.(2005): 26-36.

Sawada, H., et al. “Methylphenylpyridium ion (MPP+) enhances Sawada, H., et al. “Methylphenylpyridium ion (MPP+) enhances glutamate-induced cytotoxicity against dopaminergic neurons in glutamate-induced cytotoxicity against dopaminergic neurons in cultured rat mesencephalon.” Neuroscience Research 43 (1996): cultured rat mesencephalon.” Neuroscience Research 43 (1996): 55-62.55-62.

Society for Neuroscience. 2004. Brain Research Success Stories: Society for Neuroscience. 2004. Brain Research Success Stories: Parkinson’s Disease. Available: Parkinson’s Disease. Available: http://apu.sfn.org/content/Publications/BrainResearchSuccessStories/BRSS_http://apu.sfn.org/content/Publications/BrainResearchSuccessStories/BRSS_Parkinsons_Disease.pdfParkinsons_Disease.pdf [accessed 16 January 2006]. [accessed 16 January 2006].

Weiner, William J., et al. Parkinson’s Disease: A Complete Guide Weiner, William J., et al. Parkinson’s Disease: A Complete Guide for Patients and Families. Baltimore: John Hopkins University for Patients and Families. Baltimore: John Hopkins University Press, 2001.Press, 2001.

Xu, Z., et al. “Selective Alterations of Gene Expression in Mice Xu, Z., et al. “Selective Alterations of Gene Expression in Mice Induced by MPTP.” Synapse 55 (2005): 45-51.Induced by MPTP.” Synapse 55 (2005): 45-51.

Xu, Z., et al. “Selective alterations of transcription factors in Xu, Z., et al. “Selective alterations of transcription factors in MPP+ induced neurotoxicity in PC12 cells.” Neurotoxicology 26 MPP+ induced neurotoxicity in PC12 cells.” Neurotoxicology 26 (2005): 729-737.(2005): 729-737.