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Alzheimer’s

OH.OH.

OH. NO.NO.

Oxidative stress isthe imbalance between cellular production of reactive oxygen species and the ability of cellsto defend against them.

Reactiveoxygenspecies

Defensemechanisms

Reactive oxygen species (ROS) = any chemical speciesthat contain one or more unpaired electrons.

Act as an electron acceptor= Oxidizing agents or

Free radicals

The most common cellular free radicals-hydroxyl radical (OH●)-superoxide radical (O2

-●)-nitric monoxide (NO●)

Others (not free radicals but can lead tothe generation of free radicals)

-hydrogen peroxide (H2O2)-peroxynitrite (ONOO-)

Cellular defense mechanism against ROS�Intracellular enzymes: superoxide dismutase (SOD),

catalase, glutathione peroxidase (GSH-Px)

�Endogenous molecules: glutathione (GSH), sulfhydryl

groups, alpha lipoic acid, CoQ10, thioredoxin

�Essential nutrients: vitamin C, vitamin E, selenium,N-acetylcysteine (NAC)

�Dietary compounds: bioflavonoids, proanthocyanidins

Radiation

Cellular metabolism

Smoking

Air pollutants

Agriculturalchemicals

Additives

DetergentsActivated oxygen

Free radicals

Defense Mechanisms

Aging TraumaStrokeParkinson’s disease (PD)Huntington’s disease (HD)Alzheimer’s disease (AD)Amyotrophic lateral sclerosis (ALS)Multiple sclerosis (MS)

GI

Eye

Heart

Joints

LungMulti-organ

Vessels

Kidney Skin

OXIDATIVE STRESS

Brain

Degenerative retinal damageCataractogenesis

Renal graftGlomerulonephritis

Ischemic bowelLiver injury

VasospasmAtherosclerosis

AgingCancerDM

AsthmaHyperoxia

Rheumatoid arthritis

BurnDermatitisPsoriasis

Infarction

Illness suggested to be associatedwith oxidative stress

Illness suggested to be associated

with oxidative stress

The brighter neuron shows an increase in free radical activity that peaked three hours after neurons were deprived of a life-sustaining substance called nerve growth factor. This suggests that free radicals may play a role in the death of neurons and, possibly, neurodegenerative disorders (Eugene M. Johnson).

Free radicalactivity

Neurons

Reactions important in the production anddefense from reactive species in neurons

O2 + e- O2-. H2O2 + O2SOD

2H+

OH. + OH- + Fe3+(Fenton Reaction)

Fe2+/Cu+

R. (organic radical)

RH (organic compound)

RO2 (peroxy radical)

O2

ONOO-

NO2+

Nitration ofresiduetyrosine

O2 + OH- + OH.

(Haber-Weissreaction)

.NO2 + OH.

H2O + O2H2O + O2

GSH-Px GSH-Red

GSSG

GSH

Catalase

SOD

H2O2

NO.

Fe2+ + O2

Fe3+

GSH-Px = glutathione peroxidase; SOD=super oxide dismutase; ONOO-=peroxynitrite;GSSH = glutathione disulfide; GSH = glutathione; O2

-.=superoxide species;NO.=nitricmonoxide; GSH-red = glutathione reductase; OH.=hydroxyl species

ROS

CNS and Oxidative stress

�������������� ��������� � (PUFAs) ����������� �!"#����� oxidative stress�����!-#���.����!�� �� ���/��� �0������1��2 oxidants����� neural mitochondria 91:��#� superoxide anion �<#�� �������� Antioxidant enzymes !� extracellular space ���#�2:

-SOD !� neurons; GSH 91: GSH-Px !� astrocytes-activity ��� Catalase 91: GSH-Px �����

�Oxidative stress ���<�0H��<#I<2"12�1�� �-��-���J��� intracellular free Ca2+-��"1 �� excitatory amino acids (Glutamate)***

OH.OH.

OH. NO.NO.

Activated oxygen

Free radicals

Radiation

Cellular metabolism

Smoking

Air pollutants

Agriculturalchemicals

Additives

Detergents

IonotropicReceptors(NMDA)

~40% of all synapsesin mammalian CNS

Excitotoxic Damage

Implicated in neurodegeneration,age-associated cognitive and

memory impairments.

= harmful agentsROS

Important role assignaling molecules

Neurotransmitter releaseNeurotransmitter release

Nitric oxide (NO) as-an intercellular messenger-an atypical neurotransmitterIn neurotransmitter

release

As neurotoxin

-�����2��/# 91:N����-neuroplasticity, etc.

Long term potentiation(LTP)

Vicious cycle

(Abel&Lattel,2001;Johnston et al., 2003;Nguyen&Woo,2003)

PostSynapticMembrane

Neurotransmitter release,

Synaptic plasticity,Neuronal survival& neurogenesis

Trophicfactors

Synaptic protein,Neurite outgrowth

GrowthFactors

PPGRBRas

Raf

MEK

MAPK

RSK2

LTP=long-term potentiationPKA=Protein kinase APKC=Protein kinase C

RSK2=Ribosomal S6 kinase-2CaMK=Calcium/calmodulin kinaseCRE=c-AMP-response elementCREB=c-AMP-response element

binding protein MAPK=mitogen-activated protein kinase (ERK)

PPCREB

CBP

TICThyr

Transcription

CRE

Nucleus

Glutamate Receptors

P P

CaMKIIPCa2+

NMDA AMPA Na+

Ca2+LTP

PKC

CaMKIV

GlutamateACh

Serotonin

PLCPKC

DopamineNorepinephrine

Serotonin

AdenylateCyclase

cAMP

PKA

GlutamateGlutamate

Glutamate & Neuronal Cell Death:“Excitotoxicity”

Damaging enzymesFree radicals

Apoptosis

[Ca++]

NMDA receptor

AMPA receptor

depolarization

LipidperoxidationNuclear

damage

Membranedamage

ProteasesKinases

Nucleases

Lipases

Mitochondrialdamage

Energydeprivation

Glutamate

Voltage-dependentNa+ channel

Voltage-dependentCa++ channel

Free radicals“NO”

H2O2 (low dose) enhanced NMDA-dependent LTP in hippocampus

Synaptic plasticity

Functions of brain plasticity -Brain development-Learning & memory-Psychiatric disorders-Neurological disorders

H2O2, a membrane-permeable form of ROS,normally produced in living cells and synapses.

Hydrogen peroxide (H2O2)

(Kamsler & Segal,J Neurosci 2003)

H2O2 (high dose) inhibit EPSP

Ischemic Stroke Hemorrhagic Stroke

(From Calabresi et al.,2003)

Aging, Trauma & StrokeAging, Trauma & Stroke

Na+-K+-pump failure

Membrane depolarization

Opening of voltage-sensitive Ca2+ channels

Elevation of intracellular Ca2+ levels

Glutamate release

Activation of NMDA,AMPA& metabotropic receptors

StrokeReduction of blood flow (ischemia/hypoxia)

Depletion of energy stores

Activation of NOsynthase,lipases,proteases andendonuclease

Apoptosis Irreversible cell damageCELL DEATH

Acidosis

Reperfusion

Inflammation

Release of cytokines

Failure of Ca2+

buffering sys-tems and pumps

NO production

Free-radicalformation

Lipid peroxidation

Negative correlation between serum levels ofnitric oxide (NO) and Canadian Neurological

Scale scores (r=-0.573, p=0.000).

Negative correlation between serum levels ofMalondialdehyde (MDA) and Canadian Neurological

Scale scores (r=-0.360, p=0.002).

-Measured at 48 h after stroke in 70 patients-No correlation between GSH level and CNS scores

(J clin Neurosci 2007)

Heme and Iron Metabolism:Role in Cerebral Hemorrhage Wagner et al., J Cereb Blood Flow Met 2008

In cerebral ischemia: NO significantlyenhanced angiogenesis and neurogenesis

(Murphy&Gibson Biochem Soc Trans 2007).

Alzheimer’s DiseaseAlzheimer’s Disease

Aββββ generation

Aββββ aggre-gation

Inflam-mation

Tau hyper-phosphorylation

Cognitive& behavioralabnormalities-Neurotransmitter

deficit,-Loss ofneuroplasticity

Senile plaque withmicroglial activation

Neurofibrillarytangles

α α α α secretase

Non amyloidogenicpathway

Amyloidogenic pathwayγ γ γ γ secretase γ γ γ γ secretase

Aβββββ β β β secretase

Celldeath

(Gamblin et al., 2000)

Reactive Oxygen Species

Oxidation Excito-toxicity

HN

N

O

H2N.NH

NH.CHO

HN

N

O

H2N

NH.CHO

NH2

OH●

Hydroxylradical

HN

N

O

H2N

N

NH

Guanine

HN

N

O

H2N

.N

NH

OH

N

C8-OH-adductRadical of Guanine

HN

N

O

H2N

N

NH

OH

8-hydroxyguanine

Oxidation

HN

N

O

H2N

HN

NH

OH

N

7-hydro-8-hydroxyguanine

ReductionRingopening

ReductionRing

opening

2,6-diamino-4-hydroxy5-formamidopyrimidine

Reaction pathway for hydroxylradical attack of guanine toform 8-hydroxyguanine and2,6-diamino-4-hydroxy-5-formamidopyrimidine.

Increased inlate-stage AD

Nucleic Acids Res 2007

(Mandel et al., 2003)

Nitric oxide

Release offerritin iron

Reduction in ubiquitin-proteosome system

Proteinaggregation

Neuronaldeath

Biochemical events associated withneurodegeneration of DAneurons in PD

Glutamate excitotoxicity

neurotoxins

Impaired cellularrespiration

Iron accumulation, oxidativestress & inflammation

Parkinson’s DiseaseParkinson’s Disease

(Fang et al., PNAS 2007)

TrxR=thioredoxin reductase,Trx=thioredoxin,Srx=sulfiredoxin

Peroxi-redoxins

disulfide

Sulfinic &Sulfonic acid

Sulfenicacid

S-nitro-sylatedPrx

Oxidative Stress

Cell Death

Amyotrophic Lateral SclerosisAmyotrophic Lateral Sclerosis

(Eisen, 2000)

Free Radical Damage to Motor Neurons

Hydrogenperoxide

Oxygen radical

Environmental factors Genetic factors

ROSproduction

Macrophage

Excitotoxicity Transcription factors

Glutamate

Demyelination Gene upregulation (TNF-αααα) Axonal damage

Oligodendrocyte and neuronal loss

Sources of ROS & cellular events in MS

Multiple SclerosisMultiple Sclerosis

(Gilgun-Sherki et al.J Neurol, 2004)

Cellular Pathogenesis in HDCellular Pathogenesis in HD

(Ross & Thompson, Nat Med 2006)

(Gysin et al., PNAS 2007)

GCL=Glutamate cysteine ligase:used in GSH synthesisGCLM=modulatory unitGCLC=catalytic unit

Control Patients

Schizophrenia �complex multifactorial brain disorder with a genetic component�evidences implicated oxidative stress & glutathione (GSH)deficits in the pathogenesis of schizophrenia.

(Hayley et al., Neuroscience, 2005)

IDO=Indoleamine 2,3 dioxygenase;Kyn=Kynurenine

BDNF=Brain-Derived Neurotrophic Factor

Working model depicting potential routesby which chronic or severe stressors

could impact depressive state

GABAmodulation

CRH 5-HT

BDNF

Bcl-2

ACTH

CORT

Cytokine

Neuro-plasticity

+ +

++

-

-

-

IDO Kyn

3-OHKyn

HippocampalNMDA

Receptors+

+

+

++

-

SevereStressor

-+

++

-

Depression

Glu+

+

+

Apoptoticoxidativeexcitotoxic

Oxidative Stress & Brain disorders

Free radicals cellular defence mechanism-enzymes: SOD, Catalase-others: vit.C, vit.E etc.

Oxidative metabolism

-(PD) DA DOPACMAO-B

.OH + OH-

-Inflammation(MS)

Abnormal proteins-Alzheimer’s disease & ββββ amyloid-Prion protein &Prion disease (Mad cow)

Excitotoxicity(Trauma, Stroke, Aging)

Genetic defect:e.g. Mutation ofSOD1 & ALS,GSH & schizophrenia

Mitochondrial dysfunction& cell damage

Oxidative stress

mismatch

Energy deprivation& Cell deathBrain disorders

“Antioxidants! Antioxidants!….”

•Antioxidant drugs & vitamins

•Plant polyphenols

•Human endogenous ligands

•Female sex hormone: Estrogen

& Phytoestrogens

Antioxidant Drugs & Vitamins

Ascorbic acid(vit C)

Alpha-tocopheral(vit E)

Antioxidants Pro-oxidants

Neuroprotectants

•Both Vit C & E do notreduce risk of dementiaor PD(CNS Drugs 2003;Cummings,N Engl J Med 2004)

•Vit E but not Vit Ccould have a role in ALSprevention: clinical trials(Ascherio et al., Ann Neurol 2005)

•Potentiate extrapyramidal effects of haloperidol & NOS inhibitors (Lazzarini et al., Psychopharmacol,2005)

•Vit C: Hb denaturation in G-6-PD def. (Papandreou & Rakitzis, 1990)

•Vit E: antioxidative enzymes in erythrocytes (Eder et al., 2002)

Vitamin A & beta-carotene

(Ono et al., Exp Neurol 2004)

retinol = retinal > beta-carotene > retinoic acid.

Vitamins B2, B6, C, and E at50 and 100 µM

had no inhibitory effect

Antiamyloidogenic activity(in cell culture)

Electronmicrographof fibril extension

Control: 0 h Control: 6 h +retinol: 6 h

Rasagiline=antioxidant, MAO inhibitorLadostigil=Rasagiline+Rivastigmine=antioxidant,

selective brain MAO inhibitor andanti-cholinesterase

(Youdim&Buccafusco,J Neural Transm, 2005)

-Ginkgo biloba (EGb)-Catechins -Caffeic acid phenethyl ester(from honeybee’s propolis)

Ginkgo biloba (EGb)9�:�Q�2

Free radical scavengersFlavonoidsEgb had small but significant effect in AD patients

(Cummings, N Engl J Med 2004)

EGb=Gingko biloba extract3 wks pretreatment

Control

10 µµµµg 6-OHDA +50 mg/kg EGb

+100 mg/kg EGb +150 mg/kg EGb

(Ahmad et al., J Neurochem, 2005)

The expression of tyrosinehydroxylase (DA neuron) in

substantia nigra of rat (PD model)

•antioxidant•free radical scavenging•MAO-B inhibiting•DA-enhancing mechanisms

Rescue the DA neurons(PD model)

CatechinsCamellia sinensis

A group of flavonoids; ~30-45% ofthe solid green tea extract

(-)-epigallocatechin-3-gallate (EGCG)

(-)-epigallocatechin (EGC)(-)-epicatechin (EC)

(-)-epicatechin-3-gallate (ECG)

~10%

EGCG: modulation of cell death genein Parkinson’s model

(Mandel& Youdim, Free Rad Biol Med 2004)

EGCG=potential candidatefor the treatment of

neurodegenerative disorders

Antioxidant prop.EGCG=ECG>EGC>EC

50mg/kg EGCG i.p., after ischemia;rats were killed 72h post ischemia.

(Rahman et al., Neurosci Lett 2005)

EGCG as an interventionof cerebral ischemia

Infarc Size (mm3)

VDAC=Voltage dependent anion channel

ANT=Adenosine nucleotide translocase

PBR=Peripheral benzodiazepine receptor

CK=Creatinine kinase

CyD=Cyclophilin D

EGCG polyphenols

(Mandel & Youdim,Free Rad Biol Med 2004;Weinreb et al., J NutrBiochem 2004))

Radical scavengingIron chelation

Increasing antioxidant defense

Green Tea Polyphenols

Neurotoxin-inducedROS

PKC ANTVDAC

COMT

Apoptotic genes Aβ β β β fibrils

NEUROPROTECTION

sAPPαααα

?

?

X

Suggested potential targets of EGCG

Caffeic acid phenethyl ester(CAPE)

Active antioxidant flavonoids (45-55%)from honeybee propolis

Cultured cerebellargranule neurons (CGN)

red fluorescent=death neuron

Control

6-OHDA

6-OHDA+ CAPE

Effect of CAPE (µΜµΜµΜµΜ)))) on Ca2+-induced Cyt-C releasein rat liver mitochondria

(Noelker et al., Neurosci Lett 2005)

Propolis = neuroprotectant; a goodcandidate for in vivo models

Coenzyme Q10 (ubiquinone)

=important antioxidant in bothmitochondria and lipid membrane

Slow down functional decline in PD patients(Frucht, CNS Drugs 2005)

Protect DA neuronal death from pesticide rotenone(Moon et al., J Neurochem 2005)

360mg/day: therapeutic effect in HD patients(Korozhetz et al., Ann Neurol 1997)

Coenzyme Q10 has the potentialto be used as a therapeutic

intervention for neurodegenerativediseases.

(Somayajulu et al., Neurobiol Dis 2005)

MelatoninMelatonin

(Hardeland & Pandi-Perumal, Nutr Met 2005)

Natural compound of almost ubiquitous occurrence

AMK=Melatonin metabolite

Female sex hormone: Estrogen& Phytoestrogens

Female sex hormone: Estrogen& Phytoestrogens

(Amantea et al., Pharmacol Res, 2005)

Modulation ofgene

transcription

Inhibition ofcell death

Anti-inflammatoryactivity

Neurotrophiceffects

Estrogen receptors(intracellular)

-ERαααα-ERββββ

Interaction withneurotrophin

signal transductionpathways

Rapid non-genomicintracellularresponses

Modulation ofneurotransmitter

systems

Neurotrophinreceptors

Membranebinding sites

Neurotransmitterreceptors

Antioxidanteffects

Main results-No significant effect on theprimary outcome measure wasobserved in a meta-analysis ofantioxidants in general whencombining the results.-No significant differenceswere demonstrated in secondaryoutcome measures

Author’s conclusion-While there is no substantialclinical trial evidence to supporttheir clinical use, there is no clearcontraindication.

The Cochrane Library2005, Issue 3

Antioxidant treatment for ALS

Antioxidant treatment for HD

Antioxidant efficacy was not observed

in human clinical trial. Studies have

been planned for other free-radical

scavengers. (Gardian & Veesei, J Neural Trans 2004)

Antioxidants and neurology

Clinical evidence that antioxidants agentsmay prevent or slow the course of thesediseases is still relatively unsatisfactory,

and unsufficient to strongly modifythe clinical practice.

(Casetta et al., Curr Pharm Des. 2005)

(Wong et al., Retina, 2007)

AMD = age-related macular degeneration

Normal

Non-exudated AMD

Exudated AMD

Fe2+ Fe3+

H2O2 OH-+OH●

Free RadicalDamage

Fenton reaction

Iron positive

Estrogen & Brain PlasticityEstrogen supplement

increase dendritic knobEstrogensupplement Control

Rat’s brain: cognitive area

Estrogen Replacement Therapy (ERT):risk (uterine & breast cancer) VS benefit?

A brain selective estrogenreceptor modulator

(NeuroSERM) (Brinton, 2004)

A non-feminizing estrogen,2-(1-adamantyl)-4-

methylestrone (ZYC-26)(Perez et al., 2005)

ERT

Phytoestrogens=natural SERMs

Therapeutic trials with melatonin:

slowing the progression of AD but not of PD.(Srinivasan et al., Neurotox Res 2005)

(Ozdemir et al.,Neurosci Lett 2005)

CA1

CA3

DG

Melatonin protect hippocampus from theeffect of traumatic Brain Injury

Model for MS: oral flavonoids fail tobeneficially influence the course of EAEin mice but, instead, suppress recovery

from acute inflammatory damage.(flavonoids tested-apigenin, luteolin,

quercetin, hesperitin, morin,fisetin & curcumin)

Biochemical Pharmacology 70 (2005) 220-228

(Mayo et al., 2005)

Pueraria mirifica (����N�R�) :isoflavonoids

Soy isoflavones:Genistein, Daiazein,Glycitein etc.

Caenorhabditis elegans (C. elegans)

Soy isoflavone glycitein protects against betaamyloid-induced toxicity and oxidative stress in transgenic

Caenorhabditis elegans. Gutierrez-Zepeda et al., BMC Neurosci 2005

Glycitein

May have therapeutic potentialfor prevention of Aβ associatedneurodegenerative disorders

http://www.hsrmagazine.com/articles/2c1specialty2.html

ORAC=Oxygen Radical Absorbance Capacity

Blackcurrant

Strawberry

Raspberry

Normal neurons Damaged neurons

Neuronal Cell Death

Oxidative stress & Neuronal damageOxidative stress & Neuronal damage