THE FOLLOWING POWERPOINT HAS BEEN APPROVED FOR ALL

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

T 8 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

Normal Brain Aging 20 -40 yrs. 40 -60 yrs. Images from Med. Expert. net 80 + yrs.

Abnormal Alzheimer’s Brain

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

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

Why is Alzheimer’s Important? Most common form of dementia Affects all areas of the brain starting in the hippocampus l 2005: 4. 5 million Americans suffer 2025: estimated 14 million l Risk increases with age l 10% of patients older than 65 l 50% over 85 l Monetary cost l $174, 000 / person’s lifetime l $100 billion annually in U. S. l l

Hallmarks of AD: Plaques and Tangles Courtesy American Health Assistance Foundation • Cellular signs of Alzheimer's: Ø neurofibrillary tangles Ø amyloid plaques P: GSS 2005T 8PRESENTATIONvideo 2. asx

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

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

Different Types of Glial cells include astrocytes, oligodendrocytes, and microglial cells l We worked with the BV-2 cell line: immortalized murine microglial cells l

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

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

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

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

What are Statins? • 3 -hydroxyl-3 -methylglutaryl coenzyme A reductase inhibitor (HMG-Co A reductase inhibitor) • Used for hypercholesterolemia treatments • Observed to reduce NO levels in neurons Lovastatin C 24 H 36 O 5 MW 404. 5454 Mevastatin C 23 H 34 O 5 MW 390. 5186 Simvastatin C 25 H 38 O 5 MW 418. 5722 Pravastatin C 23 H 36 O 7 MW 424. 5332

Statins: Their role in AD

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

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

Methods: LPS Treatment Cell cultures immersed in serumfree culture l Used various statins: Ø Mevastatin Ø Lovastatin Ø Pravastatin Ø Simvastatin - With or without LPS activation l Plate #1 Control LPS Prava + LPS Simva + LPS Lova +LPS Plate #2 Control LPS Meva + LPS

Methods: Nitrite/Griess Assay l Inside the cell: unstable nitric oxide is metabolized to nitrite (NO 2) l Nitrite is stable product and easy to measure l The Nitrite/Griess Assay is used to measure the nitrite concentration in various cell media exposed to different statin treatments

Nitrite Assay: Serial dilutions Na. NO 3 + DMEM standard solutions

Nitrite Assay: 96 -well plate

Nitrite Assay: Microplate template

Nitrite Assay: Microplate Reader

Graphs for Statins: Griess Assay * * *

More Statins Data: Griess Assay

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

Immunocytochemistry: How it works Primary antibody Mouseαi. NOS Y Y FITC Secondary antibody RabbitαMouse Ig. G with FITC i. NOS

Immunocytochemistry Data: i. NOS

Mitochondrial staining l Mitochondrial staining- mitochondrial distribution under AD simulated stress

Methods: Fluorescence Staining l l 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

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

Mitochondrial Stains CONTROL GLUTAMATE

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

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

Discussion Conclusion This experiment demonstrated that statins do have ameliorative effects in reducing the production of NO in microglial cells. l Future experiments l l The effect of statins on neurons using another cell line Evaluate expression levels of i. NOS 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.

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.