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FEATURES SOME NEURONAL CELL DIVISION AND DEATHFEATURES SOME NEURONAL CELL DIVISION AND DEATH

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

Normal Brain AgingImages from MedExpert.net

20-40 yrs. 40-60 yrs. 80 + yrs.

AD as a Function of Age

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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 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

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

suffer2025: 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.

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

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 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

Different Types of Glia

Glial cells include astrocytes, oligodendrocytes, and microglial cells

We worked with the BV-2 cell line:

immortalized murine microglial cells

Microglial Immune Response

Activated by A-Beta

Become phagocytotic

Engulf apoptotic cell (dying)

• Aβ activates glial cells

• Cytokines

• Transcription factors

• Produce iNOS

• Produce nitric oxide (NO)

• NO + O2- ONOO-

Aβ role in cell death

Plaque-associated protein

Degenerating Axon

Mature Plaque

Degenerating Dendrite

Aβ activates microglia

Produce glutamate

Overstimulates neuronal NMDA

Ca2+ influx excitotoxicity

An Even Closer Look at Plaques

Calcium-induced NO production

Ca2+ influx

• activates NOS

• Produces NO

• Enters mitochondria

• Radicals and caspases

• Activate NOS

• NO production

•apoptosis

What are Statins?

Lovastatin Mevastatin Simvastatin Pravastatin

• 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor (HMG-Co A reductase inhibitor)• Used for hypercholesterolemia treatments• Observed to reduce NO levels in neurons

C23H34O5 MW 390.5186

C24H36O5

MW 404.5454 C25H38O5

MW 418.5722 C23H36O7

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

Immunocytochemistry – where statins work in NO pathway (iNOS)

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

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

Methods: Nitrite/Griess Assay

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

Nitrite Assay: Serial dilutions

NaNO3 + DMEM standard solutions

Nitrite Assay: 96-well plate

Nitrite Assay: Microplate template

Nitrite Assay: Microplate Reader

Statins Versus LPS

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Graphs for Statins: Griess Assay

Statins Versus Control

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More Statins Data: Griess Assay

Percent Difference of Statins From LPS

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Conclusions for Statins

Simvastatin and pravastatin- statistical significant reduction in NO level

Simvastatin- most quantitatively significant

Immunocytochemistry: How it works

iNOSYYFITC

Primary antibody

MouseαiNOS

Secondary antibody

RabbitαMouse IgG with FITC

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Relative Fluorescence

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Immunocytochemistry Data: iNOS

Mitochondrial staining

Mitochondrial staining- mitochondrial distribution under AD simulated stress

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-1Chemical used to stain for mitochondria Indicates presence concentration of mitochondriaFluoresces after binding to mitochondria

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

Mitochondrial Stains

CONTROL GLUTAMATE

Graph for Mitochondrial Distribution

Mitochondria actually seem to shift toward the soma with stress as opposed to towards the periphery.

Mitochondiral Distribution (by Percent)

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Control 1 Control 2 Glutamate A-Beta A-Beta &Glutamate

Treatments

Percent of Mitochondria

Neurites

Soma

Graph for Neurite Mitochondrial Density

As stress increases, mitochondria in the neurites decreases.

Neurite Mitochondrial Density

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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.

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 StoneCarl WF Bird

The corporation for public broadcastingAnd

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