Beta Amyloid and Nitric Oxide : Putative Links

Umesh Chaudhary

Contents

• Introduction

• Current prospects in Alzheimer therapy

• B-Amyloid stimulation of iNOS :

TNF alpha and NF-kB dependent iNOS expression.

• Molecular basis of Alzheimer’s disease

• Nitric oxide and neurological functioning

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

•low concentrations of NO (<2M)

•occurs usually via guanylate cyclase.

Cytotoxic effects due to a combination of

•Elevated concentration (>10M)

•Formation of highly reactive peroxynitrite

Free Radical Biology & Medicine, Vol. 25, Nos. 4/5, pp. 434–456, 1998

Direct Effects of Nitric Oxide Chemistry of Indirect Effects

Properties of NOS Isozymes

Type I Type II Type III

Tissue in which first described

Cerebellum

Neuronal

Immunologically activated

Macrophages

Vascular Endothelial Cells

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

Overall reaction Catalyzed and cofactors of NOS

Biochem. J. (2001) 357, 593-615

Nitric Oxide Biosynthesis

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.

3

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 proteins-

APP, PS1 and PS2• APOe4 allele is higher

Neuropathological Hallmarks

Journal of Structural Biology

VARADARAJAN ET AL.

130, 184–208 (2000)

Central Role of Amyloid in Neurotoxicity

Journal of Structural Biology

VARADARAJAN ET AL.

130, 184–208 (2000)

Amyloid Precursor Protein Processing

• APP is 770AA Transmembrane protein, Gene located on Chromosome 21

• Cleavage by and Secretase yields Amyloid

• Conversion of soluble forms to insoluble fibrils accounts for its toxicity

• Amyloid binding to RAGE leads to formation of plaque

• Amyloid can directly produce Hydrogen peroxide through metal ion reduction

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

Mutations in Presenilin genes

All mutations enhances production of Fibrillar A 42 and Tau Hyper-phosphorylation leading to NFT’s

A plasma protein Involved in transport and metabolism of triglyceride and cholesterol e2, e3 and e4 allelic variantsApoE e4 allelic variant is high and linked with Alzheimer

Apolipoprotein E

N- -CMicrotubule binding domain (MBD)

P sites

In AD Tau becomes hyper-phosphorylated

p p p p p p ppp

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

Law et al . / Brain Research Reviews 35 (2001) 73– 96

NO Neurotoxicity and Neuroprotection in AD

-Amyloid Stimulation of Inducible Nitric-oxide Synthase in Astrocytes Is Interleukin-1- and Tumor Necrosis Factor- (TNF)-dependent, and Involves a TNF Receptor-associated Factor- and NFB-inducing 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- stimulated Cytokine production occurs before iNOS Production

Protein synthesis inhibitor blocks A stimulated iNOS mRNA levels

IL-1 Receptor antagonist Decreases the levels of Amyloid -stimulated i-NOS and Nitrite

Production

Dominant Negative TRAF Proteins can Block NF-B Activation in Astrocytes

Dominant Negative TRAF6 Inhibits iNOS Promoter Activation by A

IL-1 Localizes to Microglia and iNOS Localizes to Astrocytes in A 42 Stimulated Glial Cultures

SUMMARY• Amyloid activates Microglia to produce Pro-Inflammatory Cytokines such as IL-1 and TNF-

• 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

• 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

Science Vol 293, 21 September 2001 P- 2192-94

Amyloid can Directly activate Caspase 3 and induce Apoptosis in Neurons

Potential Novel Therapeutic Approach

Focusing on -Amyloid plaques Enhance alpha-Secretase activity or inhibit beta or gamma-Secretase activity Correcting APP or presenilin mutationsAntioxidants such as Vitamin E and Extracts from roots of Ginko biloba Oestrogen modulate APP processing and reduce Amyloid- 42Vaccination with Amyloid Beta

22

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

Conclusions

• Oxidative stress plays a vital role in Alzheimer Pathology

• Amyloid- can initiate a variety of signalling cascades leading to Neuronal cell death in AD

• Amyloid- 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

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.

TIMES - SUNDAY, FEBRUARY 24, 2002