© TND 2005

Oxidative Stress and Inflammation in Neurodegenerative Diseases

George Perry and Mark A. Smith

Case Western Reserve University

Cleveland, Ohio

© TND 2005

I. Alzheimer’s Disease

Background© TND 2004

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Alzheimer’s Disease

4 million Americans have Alzheimer’s Disease; by 2050, 14 million will have AD.1

AD is one of the top 10 leading causes of death in Americans over 65 years of age.2

AD is the third most costly disease after heart disease and cancer.1

Federal funding for AD is 4 to 7 times lower than for heart disease, cancer or AIDS.1

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PET (positron emission tomography) scans show differences in brain activity between a normal brain and a brain affected by Alzheimer’s disease. Blue and black in the images above denote inactive areas.

Normal Alzheimer

Metabolism is the primary source of oxidants.

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Brain Inflammation in Alzheimer’s Disease

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Proposed Etiologies of Alzheimer Disease

• Aluminum or other toxins

• Infectious agents

• Synaptic abnormalities

• Vascular/immune abnormalities

• Lack of growth factor

• Lipoproteins

• Protein abnormalities

• Oxidative stress

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0

10

20

30

40

50

60

65-69 70-74 75-79 80-84 85-89 90-94 95+

Age

Per

cent

Pre

vale

nce

A leading hypothesis of the biological basis of aging is oxidative stress.

The prevalence of AD is strictly age-dependent

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Proposed Chronology of Changes in Proposed Chronology of Changes in ADAD

Tauist and BAPtistTauist and BAPtist

Amyloid- Deposition[Senile Plaque]

Tau Phosphorylation[Neurofibrillary Tangles]

Neu

ronal d

eath/D

ysfun

ction:

Dem

entia

Tau Phosphorylation[Neurofibrillary Tangles]

Amyloid- Deposition[Senile Plaque]

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Proposed Chronology of Changes in ADProposed Chronology of Changes in ADTauist and BAPtistTauist and BAPtist

Amyloid- Deposition[Senile Plaque]

Tau Phosphorylation[Neurofibrillary tangles]

Neuro

nal d

eath

:D

em

entia

Tau phosphorylation[Neurofibrillary tangles]

Amyloid-b deposition[Senile plaque]

Causes Consequences

Age-RelatedNeuronal dysfunction

Senile plaques&

neurofibrillary tangles

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II. Oxidative Stress

Role in AD

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Oxidative Stress

1. Classic definition: The production of reactive oxygen in excess of antioxidant mechanisms

2. Modern definition: Altered homeostatic balance resulting from oxidant insult.

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Oxidative Modifications Affect All Cellular Macromolecules

ControlAlzheimer

Lipid Peroxidation/Protein Adduction(4-HNE)

Protein Oxidation (Free Carbonyl Groups)

Nucleic Acids (8-OH-Guanosine)

Alzheimer

Alzheimer

Control

Control

Alzheimer Control

Glycoxidation (Carboxymethyllysine)

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Is oxidative stress an early event in AD? In the proposed sequence of degenerative events, it occurs earlier than cytoskeletal alterations.

? .....

.

Glycation

NormalNeuron

? Pre-NFT I-NFT E-NFT

FREE CARBONYLS

80HG

HNE

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Causes of Reactive Oxygen Species Generation in Alzheimer’s Disease

Active microglia

Redox active metalsAmyloid-

Advanced glycation endproducts

Mitochondria

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Partial reduction of oxygen generates ROS

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Consequences

Oxidation Inflammation

Superoxide dismutase

NSAIDS confer protection

Complement pathway activation

Microglial activation and association with amyloid plaques

Heme-oxygenase- 1 induction

Induction of “adaptive” gene responses and repair enzymes

Apoptosis pathway

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III. Mitochondrial and microtubule abnormalities are found in Alzheimer’s Disease.

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Mitochondrial DNA is increased in Alzheimer’s pyramidal neurons.

*

*

*

*

Deleted

Wild type

Alzheimer Control

mtDNA

Wild type probe

Chimera probe

4977 bp deletion

Deleted mtDNANormal mtDNA

8482 10897

8454 10941

1347513475

8454

8482/1346013460

ACACAAACTACCACCTACCTCCCTCACCATTGGCAGCCTA GCATT

CAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCT

16,569 bp 11,592 bp

OHOH

4977 bp deletion contains ATPase subunit 8, subunit 6, cytochrome-c oxidase subunit III, and NADH-coenzyme Q oxidoreductase subunit 3, 4 and 5.

In situ hybridization of mtDNA

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mtDNA

8OHG

Nitrotyrosine

The distribution of increased neuronal levels of mtDNA (A), 8OHG (B) and nitrotryosine (C) in Alzheimer’s Disease completely overlaps.

The same neurons in adjacent serial sections are numbered.

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Mitochondria components are in autophagosomes.

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Normal

Alzheimer

Could the mitochondrial problem be related to microtubules?

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Study of biopsy samples shows microtubules are reduced specifically in AD pyramidal neurons.

Pyramidal neurons

Non-pyramidal neurons

p=0.000004

p=0.90

R2 = 0.9841p=0.016

0

2

4

6

8

10

12

60 65 70 75 80

Age (yr)

Num

ber

or m

icro

tubu

les/

m²

Numbers of microtubules decrease

with normal aging

Microtubule Density

0

1

2

3

4

No

. o

f p

yra

mid

al n

eu

ron

s

0

1

2

3

4

No. of microtubules/mm²

No

. o

f n

on

-pyra

mid

al n

eu

ron

s

0

1

2

3

4

No. o

f pyr

amid

al n

euro

ns

0

1

2

3

4

Length (mm x 10²) of microtubules/mm²N

o. o

f n

on

-pyra

mid

al n

eu

ron

s

Microtubule Density Microtubule Length Control AD -PHF AD +PHF

Control AD -PHF AD +PHF

Control AD -PHF AD +PHF

Control AD -PHF AD +PHF

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Microtubules (arrowheads) remain intact even in close proximity to paired helical filaments (*).

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IV. Interplay of pathological lesions and oxidative stress

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Accumulation is Associated with a Reduction in Oxidative Stress

/80HG HO-1

Heme Oxygenase Alz50

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Overexpression of HO-1 Induces tausuppression

1 2 3 4

HO-1

HO-2

Tau

Ferritin

1 Cep2 Antisense3 HO-14 HO-1 + inhibitor

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Increased amyloid - (brown) correlates with decreasedoxidative damage (8OHG, blue).

Down Syndrome

17 yr.

61 yr.

31 yr.

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How do lesions protect?

• Oxidatively damaged

• Metal binding sites

• Reduce oxidative stress

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Is amyloid- protective against a cauldron of oxidative stressors in Alzheimer’s Disease?

Are there signs of established antioxidant responses in AD?

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V. Phosphorylation of Cytoskeletal Proteins Drives Oxidative Modifications.

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p-ERK p-JNK/SAPK p-p38

Stress response kinases are induced

tau assembly induced by HNE is dependent on phosphorylation

Effect of HNE on tau assembly.

Phospho-tau polymers following 1mM HNE.

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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 500 1000 1500 2000

HNE (mM)

Rela

tiv

e d

en

sit

y

B Unt

C -PO4

0 30 125 500 2000

HNE (mM)

A

NFH

NFH-PO4

In vitro modification of NFH and NFM by HNE is dependent upon

lysine residues…P h o s p h o r y l a t e d N F H

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

0 5 1 0 1 5 2 0 2 5 3 0 3 5

A g e i n m o n t h s

Relativ

e Dens

ity

C 5 7 B L 6 J

B 6 C 3 F 1

H N E - M i c h a e l

01 02 03 04 05 06 07 0

0 5 1 0 1 5 2 0 2 5 3 0 3 5

A g e i n m o n t h s

Relativ

e Dens

ity

H N E - P y r r o l e

05

1 01 52 02 53 03 54 0

0 5 1 0 1 5 2 0 2 5 3 0 3 5

A g e i n m o n t h s

Relativ

e Dens

ity

A

B

C

Levels of HNE do not accumulate with age in the

mouse’s sciatic nerve

…and phosphorylation state.

NFHNFM

A B C D

Antibody recognition of NFH and NFM (A) is abolished by HNE-lysine (B) but not cyteine (C) or histidine(D).

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VI. Therapeutics

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Progression/Incidence

EstrogenAcetylsalicylicacid (Aspirin) (-) Deprenyl

(selegiline)

Ibuprofen

Dapsone

Acetyl-L-Carnitine(ALCAR)

Vitamin E

Tenilsetam

Antioxidants and anti-inflammatories are protective

DietLipoic Acid

Fruits and vegetables

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Nutrients per 1000 kilocalories Alzheimer Disease Cases Controls p Value n=104 n=223

Vitamin A (RE) 855 983 0.001 Carotene (mcg) 294 389 0.001 Carotene (mcg) 1921 2370 0.003Pro-A Carotene (mcg) 2231 2809 0.001Lutein (mcg) 972 1214 0.015Lycopene (mcg) 666 927 0.001Vitamin C (mg) 74.6 86.7 0.007Vitamin E (a TE) 5.6 5.9 NS

Servings per dayYellow/green vegetables 2.0 2.3 0.022Vitamin C fruits, vegetables 2.4 2.6 NS

Antioxidant diet is protective

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VII. Summary

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Primary etiology

Cellular response

Oxidative stress and inflammation

Aand

AD phenotype

Current therapeutic

targets

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MitochondriaNitric Oxide

Mutated genes (SOD, PP, synuclein)

Phospholipid metabolismProteolysis

Redox Active Metals Advanced Glycation

EndproductsMicroglia

ProteinsLipids

Nucleic AcidApoptosis

Alzheimer DiseaseParkinson Disease

ALSStroke

Multiple Sclerosis

Vitamin ELipoic Acid

Metal Chelation

STOP

Amyloid Neurofilament

proteintau

GlutathioneHemeOxygenase

-1Superoxide dismutase

Classic Cellular Protein-based

STOP

STOP

STOP

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Conclusions

• Metal catalyzed oxidative damage to all categories of macromolecules is increased.

• Antioxidant pathways and inflammatory responses are induced.

• “Pathological changes” may be compensations that are critical to maintaining oxidative homeostasis.