THE FOLLOWING POWERPOINT HAS BEEN APPROVED FOR ALL AUDIENCES THESE SLIDES HAVE BEEN RATED

1. THE FOLLOWING POWERPOINT HAS BEEN APPROVED FOR ALL AUDIENCES THESE SLIDES HAVE BEEN RATED NJGSS-A NEW JERSEY GOVERNOR SCHOOL SCIENCE AUDIENCES Some material may be inappropriate for non-scientists FEATURES SOME NEURONAL CELL DIVISION AND DEATH

3. 0 T8 Experiments on Alzheimer’s Disease Jash Bansal, Jonathan Cantalino, Morgan Greenfield, Jennifer Lee, Christina Long, Darius Rackus, Karen Sun, Sam Tarakajian, Roxinne Templonuevo, Christopher Thompson, Kathy Zhou Under the direction of:Diane Chuback, Ph.D.Carl W.F. Bird B.A.and Jonathan Stone

4. Normal Brain Aging 0 Images from MedExpert.net 20-40 yrs. 40-60 yrs. 80 + yrs.

5. Abnormal Alzheimer’s Brain 0

6. 0 “. . . aging always affects the human brain.” -Victoroff The task we face is to reduce theimpact of AD-related neurodegeneration.

7. Our Experiments and Goals 0 • The Ameliorative properties of HMG-CoA (Statins) on the Nitric Oxide production by Microglial cells: an Alzheimer’s Model • The use of NMDA receptor antagonists to help neuronal cells

8. Why is Alzheimer’s Important? 0 • Most common form of dementia • Affects all areas of the brain starting in the hippocampus • 2005: 4.5 million Americans suffer 2025: estimated 14 million • Risk increases with age • 10% of patients older than 65 • 50% over 85 • Monetary cost • $174,000 / person’s lifetime • $100 billion annually in U.S.

9. Hallmarks of AD: Plaques and Tangles 0 • Courtesy American Health Assistance Foundation • Cellular signs of Alzheimer's: • neurofibrillary tangles • amyloid plaques P:\GSS2005\T8\PRESENTATION\video2.asx

10. Goals 0 • The Ameliorative properties of Statins on the Nitric Oxide production by Microglial cells: an Alzheimer’s Model

11. Inside the Central Nervous System 0 • Neural tissue consists of both neurons and glial cells • Glia support nerve cells in the CNS by • maintaining homeostasis • buffering pH • providing insulation • regulating the concentration of neurotransmitters • eliminating foreign bodies from the brain

12. Different Types of Glia 0 • Glial cells include astrocytes, oligodendrocytes, and microglial cells • We worked with the BV-2 cell line: immortalized murine microglial cells

13. Microglial Immune Response 0 • Activated by A-Beta • Become phagocytotic • Engulf apoptotic cell (dying)

14. 0 Aβ role in cell death • Aβ activates glial cells • Cytokines • Transcription factors • Produce iNOS • Produce nitric oxide (NO) • NO + O2-  ONOO- Plaque-associated protein Mature Plaque Degenerating Axon Degenerating Dendrite

15. An Even Closer Look at Plaques 0 • Aβ activates microglia • Produce glutamate • Overstimulates neuronal NMDA • Ca2+ influx  excitotoxicity

16. Calcium-induced NO production 0 • Ca2+ influx • Enters mitochondria • Radicals and caspases • Activate NOS • NO production • activates NOS • Produces NO • apoptosis

17. Lovastatin Pravastatin Mevastatin Simvastatin What are Statins? 0 • 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor (HMG-Co A reductase inhibitor) • Used for hypercholesterolemia treatments • Observed to reduce NO levels in neurons C24H36O5MW 404.5454 C23H34O5MW 390.5186 C25H38O5MW 418.5722 C23H36O7MW 424.5332

18. Statins: Their role in AD 0

19. So what exactly did we do? 0 • Griess Assay- effect of statins on [NO] in BV-2 microglial cells • Immunocytochemistry – where statins work in NO pathway (iNOS)

20. BV2 Methods: BV2 Microglial Cell Cultures 0 • Incubated BV-2 cells • Applied a supplement media (DMEM) with 10% FBS • Seeded in 24-well plates • Incubated at 37°C for 24 hours BV2 cells were grown in T-25 flasks as shown above. 37°C incubator

21. Plate #1 Control LPS Prava Prava Simva Simva + LPS + LPS Plate #2 Control LPS Meva Meva Lova Lova + LPS +LPS Methods: LPS Treatment 0 • Cell cultures immersed in serum-free culture • Used various statins: • Mevastatin • Lovastatin • Pravastatin • Simvastatin - With or without LPS activation

22. Methods: Nitrite/Griess Assay 0 • Inside the cell: unstable nitric oxide is metabolized to nitrite (NO2) • Nitrite is stable product and easy to measure • The Nitrite/Griess Assay is used to measure the nitrite concentration in various cell media exposed to different statin treatments

23. Nitrite Assay: Serial dilutions 0 NaNO3 + DMEM standard solutions

24. Nitrite Assay: 96-well plate 0

25. Nitrite Assay: Microplate template 0

26. Nitrite Assay: Microplate Reader 0

27. * * * Graphs for Statins: Griess Assay 0

28. 0 More Statins Data: Griess Assay

29. Conclusions for Statins 0 • Simvastatin and pravastatin- statistical significant reduction in NO level • Simvastatin- most quantitatively significant

30. Y FITC FITC FITC FITC FITC Y Y Y Y Y Immunocytochemistry: How it works 0 Y Primary antibody MouseαiNOS Secondary antibody RabbitαMouse IgG with FITC Y iNOS

31. 0 Immunocytochemistry Data: iNOS

32. Mitochondrial staining 0 • Mitochondrial staining- mitochondrial distribution under AD simulated stress

33. Methods: Fluorescence Staining 0 • Culture media was taken out of the wells • New culture media with JC-1 staining agent added • Cells were incubated for 1 hour • Cells were examined under Nikon inverted microscope with rhodamine (red) filter • JC-1 • Chemical used to stain for mitochondria • Indicates presence concentration of mitochondria • Fluoresces after binding to mitochondria

34. Differentiated HCN-1A cells 0 Glial phenotype of HCN-1A cells Control group HCN-1A cells Neuronal phenotype of HCN-1A cells HCN-1A cells treated with A-beta and glutamate

35. Mitochondrial Stains 0 CONTROL GLUTAMATE

36. Graph for Mitochondrial Distribution 0 Mitochondria actually seem to shift toward the soma with stress as opposed to towards the periphery.

37. Graph for Neurite Mitochondrial Density 0 As stress increases, mitochondria in the neurites decreases.

38. Discussion 0 • Future experiments • The effect of statins on neurons using another cell line • Evaluate expression levels of iNOS after treatment of statins • Ethanol kills microglia and therefore decreases the amount of NO produced. So, find other solvents to dissolve statins. • Testing ameliorative effects of memantine in cytoxicity. Conclusion This experiment demonstrated that statins do have ameliorative effects in reducing the production of NO in microglial cells.

39. 0 Special thanks go to: The State of New Jersey RICHIE Cody Robert Weisbuch (President Bobby W.) Canada (FOR DARIUS) (AND FRANKIE)(AND SALLY) Ninja David Miyamoto, Ph.D. Tortured Ballerina Paul Quinn, Ph.D. Godfather: Steve Surace Ph.D. Dr. Diane J-Koziereski-Chuback Ph.D Jonathan Stone Carl WF Bird The corporation for public broadcasting And Viewers Like You.

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