Transcript Nitric Oxide

Beta Amyloid and Nitric Oxide :
Putative Links
Umesh Chaudhary
Contents
• Introduction
• Nitric oxide and neurological functioning
• Molecular basis of Alzheimer’s disease
• B-Amyloid stimulation of iNOS :
TNF alpha and NF-kB dependent iNOS expression.
• Current prospects in Alzheimer therapy
Nitric Oxide
A class of inter- and intra-cellular messenger molecules
– Small molecular weight, Highly diffusible, Gaseous,
Highly reactive stable free radical
Second messenger role requires
Cytotoxic effects due to a combination of
•low concentrations of NO (<2mM)
•Elevated concentration (>10mM)
•occurs usually via guanylate cyclase. •Formation of highly reactive peroxynitrite
Direct Effects of Nitric Oxide
Chemistry of Indirect Effects
Free Radical Biology & Medicine, Vol. 25, Nos. 4/5, pp. 434–456, 1998
Properties of NOS Isozymes
Type I
Type II
Type III
Tissue in which
first described
Cerebellum
Tissue based
terminology
nNOS
iNOS
eNOS
Expression
Constitutive
Inducible
Constitutive
Intracellular free
Calcium
Regulates
No Effect
Regulates
Size
161KDa
131KDa
!33KDa
Location of
Gene
Chrom-12
Chrom-17
Chrom-7
Neuronal
Immunologically
Vascular
activated
Endothelial Cells
Macrophages
Nitric Oxide Biosynthesis
Overall reaction Catalyzed and cofactors of NOS
Biochem. J. (2001) 357, 593-615
Nitric Oxide and Nervous System
• Extensive distribution of NOS positive neuron
• Role in long term potentiation
• Learning and Memory
• Pain perception
• Neuromodulatory functions
• Host defense against pathogens
• Neurotoxicity
Alzheimer’s Disease
Epidemiology
• Most common Neurodegenerative disorder & dementia
• Currently affecting nearly 5 million individuals in USA alone.
Symptoms

Progressive cognitive decline affecting memory, learning,
emotions and behaviour
Pathology
• Neurofibrillary Tangles, Senile Plaques and Synapse Loss
• Mutation in one of the genes that codes for three transmembrane proteinsAPP, PS1 and PS2
• APOe4 allele is higher
3
Neuropathological Hallmarks
Central Role of Amyloid b in Neurotoxicity
VARADARAJAN ET AL.
Journal of Structural Biology
130, 184–208 (2000)
Amyloid Precursor Protein Processing
•
APP is 770AA Transmembrane protein,
Gene located on Chromosome 21
•
Cleavage by a, b and g Secretase
yields Amyloid b ( 1-40, 42)
•
Conversion of soluble forms to
insoluble fibrils accounts for its toxicity
•
Amyloid b binding to RAGE leads to
formation of plaque
•
Amyloid can directly produce
Hydrogen peroxide through metal ion
reduction
VARADARAJAN ET AL.
Journal of Structural Biology
130, 184–208 (2000)
Mutations in Presenilin genes
Presenilin 1 (PS1) on chromo 14 encodes 467 residue polypeptide &
Presenilin 2 (PS2) on chromo 1 encodes for 448 residue polypeptide
 PS1 and PS2 are found in nuclear membrane ,ER and Golgi body
 Necessary for Neurogenesis and Neuronal survival
 50 mutations of PS1 and 2 mutations of PS2 found in AD families

Apolipoprotein E
A plasma protein Involved in transport and metabolism of
triglyceride and cholesterol
 e2, e3 and e4 allelic variants
ApoE e4 allelic variant is high and linked with Alzheimer

All mutations enhances production of Fibrillar A b 42 and Tau
Hyper-phosphorylation leading to NFT’s
Neurofibrillary Tangles

Intra neuronal lesions in degenerating neurones

Composed of hyper-phosphorylated tau-protein
organised in paired helical filaments
Tau is a soluble, microtubule-associated phospho-protein
involved in neuronal stabilisation
p
p
ppp p
pp p
P sites
-C
NMicrotubule binding
domain (MBD)

In AD Tau becomes hyper-phosphorylated
NO Neurotoxicity and Neuroprotection in AD
Law et al . / Brain Research Reviews 35 (2001) 73– 96
b -Amyloid Stimulation of Inducible Nitric-oxide
Synthase in Astrocytes Is Interleukin-1b- and Tumor
Necrosis Factor-a (TNFa)-dependent, and Involves a
TNFa Receptor-associated Factor- and NFkBinducing Kinase-dependent Signaling Mechanism
(Keith T. Akama and Linda J. Van Eldik)
From the ‡Department of Cell and Molecular Biology and §Northwestern Drug
Discovery Program, NorthwesternUniversity Medical School, Chicago, Illinois
60611
The Journal of Biological Chemistry, Vol-275. No. 11, March 17, pp –7918-7924
A- b stimulated Cytokine production occurs before iNOS Production
Protein synthesis inhibitor blocks A b stimulated iNOS mRNA levels
IL-1 b Receptor antagonist Decreases the levels of
Amyloid b-stimulated i-NOS and Nitrite
Production
Dominant Negative TRAF Proteins can
Block NF-kB Activation in Astrocytes
Dominant Negative TRAF6 Inhibits
iNOS Promoter Activation by A b 42
IL-1 b Localizes to Microglia and iNOS Localizes to
Astrocytes in A b 42 Stimulated Glial Cultures
SUMMARY
• Amyloid b activates Microglia to produce Pro-Inflammatory
Cytokines such as IL-1 b and TNF- a
• These Cytokines in turn activate surrounding Astrocytes, which
exacerbate the inflammation with the production of Neurotoxic
Mediators such as iNOS.
• The resultant NO and Peroxynitrite ultimately damages local
neurons and contributes to the Neurodegeneration observed in AD
Evidence for NF-kB and Cytokine dependent iNOS induction
Other Signaling Cascade Activated by Amyloid b
• Binding to Neurotrophin receptor leads to
Apoptotic cell death
• Induction of Transcription of c-JUN mRNA
and stimulates JUN Amino Terminal Kinase
Neuroscience Letters 312 (2001) 177–179
Amyloid can Directly activate Caspase 3
and induce Apoptosis in Neurons
Science Vol 293, 21 September 2001 P- 219294
Potential Novel Therapeutic Approach
Focusing on b -Amyloid plaques
Enhance alpha-Secretase activity or inhibit beta or gamma-Secretase activity
 Correcting APP or presenilin mutations
Antioxidants such as Vitamin E and Extracts from roots of Ginko biloba
 Oestrogen modulate APP processing
and reduce Amyloid- b 42
Vaccination with Amyloid Beta

Focusing on Apolipoprotein E
Replace ApoE e4 alleles with e2/3 alleles
 Cholesterol lowering drugs ( Lipitor )

Tau protein based therapies

Prevent t-protein phosphorylation
Gene Therapy - Nerve Growth Factor, Propentophylline
22
Conclusions
• Oxidative stress plays a vital role in Alzheimer Pathology
• Amyloid-b can initiate a variety of signalling cascades
leading to Neuronal cell death in AD
• Amyloid-b can stimulate Pro inflammatory cytokines and
ultimately contribute to Oxidative and Nitrosative Stress
induced cell death and Apoptosis
• Further Detailed Studies needed to have a deeper Insight
into Signal transduction pathways involved in Alzheimer
Disease
TIMES - SUNDAY, FEBRUARY 24, 2002
Twelve Alzheimer's patients injected with an
experimental vaccine are suffering serious brain
inflammation.
The vaccine's manufacturer halted the experiment
last month when it discovered that the first four
patients, all from France, were suffering the
encephalitis-like reaction.
Since then, doctors have discovered eight more
people with the apparent side effect, which can be
hard to distinguish from worsening Alzheimer's.