Nitric Oxide Synthasein Mouse Brain Tissue that

Exhibits Alzheimer’s Disease

Patrick McCarthy 2003-04

http://www.nsrl.ttu.edu/tmot1/mus_musc.htm

Introduction

• Alzheimer’s disease (AD)

• Future impact

• Characteristics: Beta-amyloid plaque

http://www.ahaf.org/alzdis/about/AmyloidPlaques.htm

Introduction

• Transgenic mouse model of Alzheimer’s disease using species, Mus musculus

• Two genetically engineered mouse types:- Transgenic positive (Tg. +):

with AD-expressing gene- Transgenic negative (Tg. -):

without AD-expressing gene

Introduction

• Nitric oxide synthase (NOS)

- Enzyme throughout body

• 1989, NOS in brains

- Bredt et al.

• Early 2000s, NOS and AD relation strongly showed

- de La Torre et al., Law et al.

N

O

N

N

N

N

O

N

O

N

N

O

+

NOS-catalyzed production of NO via L-arginine to L-citrulline pathway

L-arginineL-citrulline

NOS

Introduction

• Three NOS isoforms

- neuronal NOS (nNOS)

- endothelial NOS (eNOS)

- inducible NOS (iNOS)

Hypothesis (1)

• Luth et al. found iNOS and eNOS activity increased in AD

• Luth et. al and Quinn et. al found nNOS activity increased in AD

• First hypothesis: total NOS activity increased in AD brains

Hypothesis (2)

• Norris et al. found number of nNOS-containing neurons decreased

• Fewer neurons, less total concentration of nNOS

• Second hypothesis: nNOS concentrations in AD brains were lower

Procedure• Tissue preparation• Determining activity

- Spectrophotometer to assay

Results• NOS activity significantly different (p = 0.014) • Tg. + NOS activity 58 % greater than Tg. -

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

Tg. Negative Tg. Positive

Mouse Type

NOS Turnover (Rate/Second)

Procedure

• Concentrations

- Slot Blot

- Western Blot

Results

Tg. +

Tg. -

• Intensity of band corresponds to concentrations• Darker band, higher concentration • This test inconclusive

Procedure

• Concentrations

-ELISA assay

Results• nNOS concentrations significantly different (p = 0.008)• Tg. + 36% less nNOS concentration than Tg. -

0.00000

0.00020

0.00040

0.00060

0.00080

0.00100

0.00120

Tg. Negative Tg. Positive

Mouse Type

Neuronal Nitric Oxide Synthase

(mg/mL)

Conclusion

(1) Total NOS was increased in Tg. + brains

(2) Concentrations of nNOS lower in Tg. + brains

Conclusion

• Suggest neurons initially damaged or further degenerated by the toxicity of the free radical NO

• Increase in NOS activity can most likely be attributed to increase in nNOS and/or eNOS

• Support the role of NOS and nNOS in AD

Potential Future Studies

• Find concentrations of other two isoforms of NOS, iNOS and eNOS

• Use different-aged brains to study the role of NOS activity and concentration before, during, and after the onset of AD

• Further test certain areas of the brain (human and mice) to investigate role in AD

Acknowledgements

• Dr. Ian Armitage and Dr. Bruce Martin

• Abigail Tolkheim and rest of the Armitage Lab team

• Ms. Lois Fruen

• Team Research

The Role of Nitric Oxide Synthase in Alzheimer’s Disease

Patrick McCarthy 2003-04