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

3. 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 T8 Experiments on Alzheimer’s Disease

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

5. Abnormal Alzheimer’s Brain

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

7. Our Experiments and Goals 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? 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 Cellular signs of Alzheimer's:  neurofibrillary tangles  amyloid plaques Courtesy American Health Assistance Foundation P:\GSS2005\T8\PRESENTATION\video2.asx

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

11. Inside the Central Nervous System 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 Glial cells include astrocytes, oligodendrocytes, and microglial cells We worked with the BV-2 cell line: immortalized murine microglial cells

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

14. Aβ activates glial cells Cytokines Transcription factors Produce iNOS Produce nitric oxide (NO) NO + O 2 -  ONOO - Aβ role in cell death Plaque-associated protein Degenerating Axon Mature Plaque Degenerating Dendrite

15. Aβ activates microglia Produce glutamate Overstimulates neuronal NMDA Ca 2+ influx  excitotoxicity An Even Closer Look at Plaques

16. Calcium-induced NO production Ca 2+ influx activates NOS Produces NO Enters mitochondria Radicals and caspases Activate NOS NO production apoptosis

17. What are Statins? LovastatinMevastatinSimvastatinPravastatin 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor (HMG-Co A reductase inhibitor) Used for hypercholesterolemia treatments Observed to reduce NO levels in neurons C 23 H 34 O 5 MW 390.5186 C 24 H 36 O 5 MW 404.5454 C 25 H 38 O 5 MW 418.5722 C 23 H 36 O 7 MW 424.5332

18. Statins: Their role in AD

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

20. Methods: BV2 Microglial Cell Cultures 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 BV2 cells were grown in T-25 flasks as shown above. 37°C incubator

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

22. Methods: Nitrite/Griess Assay Inside the cell: unstable nitric oxide is metabolized to nitrite (NO 2 ) 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 NaNO 3 + DMEM standard solutions

24. Nitrite Assay: 96-well plate

25. Nitrite Assay: Microplate template

26. Nitrite Assay: Microplate Reader

27. * * Graphs for Statins: Griess Assay *

28. More Statins Data: Griess Assay

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

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

31. Immunocytochemistry Data: iNOS

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

33. 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 Methods: Fluorescence Staining JC-1 Chemical used to stain for mitochondria Indicates presence concentration of mitochondria Fluoresces after binding to mitochondria

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

35. Mitochondrial Stains CONTROL GLUTAMATE

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

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

38. Discussion 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. 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. Special thanks go to: