Transcript Document

Nitric Oxide Synthase
in Mouse Brain Tissue that
Exhibits Alzheimer’s Disease
http://www.nsrl.ttu.edu/tmot1/mus_musc.htm
Patrick McCarthy 2003-04
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.
NOS-catalyzed production of NO
via L-arginine to L-citrulline pathway
NOS
N
N
O
N
O
N
O
N
N
L-arginine
N
L-citrulline
+
NO
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 nNOScontaining 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. 18.0
NOS Turnover (Rate/Second)
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
Tg. Negative
Tg. Positive
Mouse Type
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. Neuronal Nitric Oxide Synthase
(mg/mL)
0.00120
0.00100
0.00080
0.00060
0.00040
0.00020
0.00000
Tg. Negative
Tg. Positive
M ouse Type
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