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Oxidative Stress and Movement Disorders 339

Oxidative Stress and Movement DisordersI G Gazaryan and R R Ratan, Burke Medical Research Institute, Weill Medical College of Cornell University,NY, USA

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

Complex I deficiency – The mitochondria generate

ATP via chemical coupling of a gradient of protons to

ATP synthesis. A gradient of protons (between the

outer mitochondrial membrane and the inner

mitochondrial membrane) is generated by the

transport of electrons from Complex I to IV.

Abnormalities of complex I in PD lead to a buildup of

electrons (which are passed on to oxygen to

generate superoxide) and a decreased proton

gradient leading to decreased ATP.

Free radical – Stable molecules contain two paired

electrons of opposite spin in one or more of their

outer most orbitals. Radicals contain an unpaired

electron in their outer most orbitals. They are called

‘free,’ because they are stable enough for

independent existence. Radicals move around the

cell, looking for electrons to steal from cellular

constituents such as lipid, protein, or DNA and

thereby leave the target irreversibly altered.

Glutathione – An endogenous tripeptide,

g-glutamyl-cysteinyl-glycine, that is present in brain

and peripheral tissues in concentrations approaching

millimolar. It is a versatile antioxidant that along with

enzymes such as glutathione peroxidase and

glutathione reductase protects neurons. Of note,

glutathione is one of the earliest known changes in

Parkinson’s disease – its levels are decreased.

Oxidative stress – Operationally defined as an

imbalance between oxidants (free radicals) and

antioxidants in the cell in favor of oxidants and above

a threshold that leads to damage or death of a cell.

Superoxide dismutase – Superoxide is produced

as a result of an addition of one electron to oxygen.

Superoxide is produced as a consequence of many

reactions, particularly as a byproduct of oxygen

utilization in the mitochondria to generate ATP.

Superoxide is thus a free radical that is produced as

a consequence of normal metabolism. Mitochondrial

dysfunction in PD leads to increase superoxide

production.

Prototypic neurodegenerative movement disordersinclude Parkinson’s disease (PD) and Huntington’s disease(HD). Both the disorders are linked to inherited mutationsresulting in accumulation of the damaged proteins or theirwrongly processed variants. However, sporadic neurode-generative conditions are also associated with accumula-tions of misfolded proteins and their aggregates resultingin the endoplasmic reticulum (ER) stress. The ER has aquality-control function with these proteins; only cor-rectly folded proteins are excreted from the ER, whileunfolded or misfolded proteins are degraded via ER-associated protein degradation, which is mediated by theubiquitin–proteasome system. The accumulation ofunfolded or misfolded proteins in the ER is one of themajor causes of ER dysfunction. Downstream of theseevents, metal-catalyzed oxidation leading to oxidative stresshas been implicated as a common final pathway of injury(Scheme 1). Despite the rarity of the familial forms of PDandHD, the identification of the genes and their defects hasfueled our understanding of the pathogenic mechanisms,

ROS

Aggregated protein Gain-of-function: ROS generation

ROS

Fe2+, Fe3+ Fe2+/+3, Cu2+, Zn2+ O2

Cross-linked aggregate

Scheme 1 Widely accepted hypothetic gain-of-function transformation of cross-linked aggregates of misfolded proteins.

Confirmed for AD, PD, ALS, and supposed for HD.

340 Oxidative Stress and Movement Disorders

which include ubiquitin–proteasome system malfunction,oxidative stress, and mitochondrial dysfunction.

Oxidative stress can be operationally defined as animbalance of cellular oxidants and antioxidants in favor ofoxidants. However, cellular oxidants and the injuries theyperpetrate are not an undifferentiated whole. Rather dis-tinct oxidants act in distinct cell types and subcellular loca-tions to trigger a continuum of responses from adaptation toapoptosis to necrosis. Here, we provide a 30 000 foot viewofoxidative injury in the nervous system, as it relates tomovement disorders and from this hope to build a concep-tual framework for understanding how changes in redoxbalance can mediate dysfunction in the CNS.

Oxidative Stress: Inescapable Componentof Mitochondrial Respiration

Mitochondria as a Major Source of Superoxideunder Normal Conditions

Neurodegeneration is directly modified by cell aging.Some theories invoke the mitochondria as the major siteof generation of deleterious free radicals that promoteaging. The chemistry of reactive species that could resultin oxidative damage under normal physiological condi-tions is the same for any cell. The major species in cells issuperoxide radical produced as a byproduct of the mito-chondrial respiration, in particular by Complex I and IIIof the respiratory chain. Complex III breaks 2e reduc-ing equivalent into two single-electron reducing equiva-lents inside the membrane (Q-cycle), and the resultingquinone radical reacts with oxygen, giving rise to super-oxide radical. This side reaction is known as ‘mitochon-drial leakage’ and comprises up to 3–4% of the totaloxygen consumption. If in a lifetime, we consume nearly60 000 L of oxygen per kg weight, it means that we pro-duce�2000 L superoxide per kg weight. What happens tothe released superoxide and how does cell handle theconsequent ‘oxidative’ load?

Antioxidant/Antiaging Mechanisms: SuperoxideScavenging

In both cytosol and mitochondria, superoxide is scav-enged and converted into hydrogen peroxide and oxygen

by superoxide dismutase (SOD), although the nature ofSOD in mitochondria and cytosol is different (MnSODand CuZnSOD). The formed hydrogen peroxide is fur-ther decomposed into water with the help of catalase orreduced by the glutathione peroxidase/glutathione sys-tem. Very recent studies show that thioredoxin reductase/thioredoxin system is also capable of reducing hydrogenperoxide to water.

There are a number of theoretical reasons why thenervous system in general and neurons in particular areunder the biggest load of oxidative stress under basalconditions. Neurons have a very high metabolic rate inorder to meet the demands of electrical signaling. Specif-ically, our brain uses 20% of the total oxygen consumed,although brain is only 2% of the body weight. Anexpected corollary of high oxygen utilization is 10-foldhigher level of radical production. Paradoxically, despitehigher radical production, catalase is absent in brainmitochondria. This is not the case for heart mitochondria.

Given the higher level of radical production and theabsence of catalase in brain mitochondria, what are themechanisms at play inside neuronal mitochondria to neu-tralize the formed hydrogen peroxide and to keep oxida-tive damage to tolerable levels? In addition to glutathioneperoxidase, the only other known system that neutralizeshydrogen peroxide is that of thioredoxin and pero-xiredoxin. Reduced thioredoxin provides peroxiredoxinwith reducing equivalents to reduce hydrogen peroxide towater. Oxidized thioredoxin is reduced back by mitochon-drial thioredoxin reductase, selenocysteine flavo-dithioloxidoreductase. Why mitochondrial catalase is absent inneurons is not well understood, but could be related tosignaling functions of peroxide in neurons that are notnecessary in other tissues. The absence of catalase fromneuronal mitochondria places the burden of neutralizinghydrogen peroxide generated in mitochondria squarelyon the shoulders of the thiol-based detoxification systems,particularly glutathione, thioredoxin, and periredoxin.A causal role for mitochondrial peroxide in aging wassupported by the generation of transgenic mice that over-express catalase in the nucleus, the peroxisome, and themitochondria. Significant extension of lifespan was seenonly in animals in which catalase was overexpressed inthe mitochondria. The extension of lifespan was associated


with improvement in cardiac function, decreased develop-ment of cataracts, and diminished mitochondrial DNAmutations. It is unclear to what extent the increased life-span reflected improvements in brain function.

Antioxidant/Antiaging Mechanisms: Repair ofOxidized Residues

Unscavenged radicals are thermodynamically driven tooxidize cellular constituents including protein cysteinesand methionines. These critical amino acid residues canbe reduced back only with the help of specialized enzymesystems (Scheme 2). Cysteines are reduced back withglutaredoxin-glutathione system, and methionines arereduced back with methionine sulfoxide reductase(MSR) system. The MSR system includes two enzymes,only one of which belongs to the same enzyme class asglutathione reductase and thioredoxin reductase.

The glutaredoxin-glutathione enzyme system has ben-eficial effects on the functional activity of a number ofproteins, including the thiol-containing mitochondrialComplex I, the inhibition of which was caused by admin-istration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), a neurotoxin that produces PD-like symptomsin primates, including humans. Interestingly, glutathi-one depletion is an early feature of PD and appears to

Scheme 2 ROS/RNS generation and neutralization reactions & syst

superoxide dismutase (SOD), catalase, thioredoxin reductase (TR), pe

by thioredoxin (Trx) and glutathione (GSH). Center: Oxidation statesmost of the oxidative modifications by glutathionylation followed by i

repaired with methionine sulfoxide reductase (Msr) /Trx system; sulfo

repaired with the use of a recently characterized ATP- and Mg2+-depreducing the sulfinic form of peroxiredoxin. Lower level: peroxynitrite

proteins, DNA and lipids.

be disease specific. The depletion of glutathione mayexplain defects in Complex I function found in sporadicforms of PD.

MSR, another important enzyme in repair of proteins,is proposed to play a central role in neurodegenerationand aging. Genetically engineered organisms overexpres-sing the enzyme live longer. On the contrary, the reducedor suppressed activity of the enzyme results in shortenedlife span, hippocampal degeneration, and increased sensi-tivity to oxidative stress. The enzyme can repair oxidizedmethionines and restore the function of many importantproteins, for example, calmodulin. MSRs act on oxidizedcalmodulin and repair all the eight methionine sulfoxideresidues initially present in the inactive protein.

As mentioned earlier, neuronal mitochondria protectthemselves against hydrogen peroxide formed inside thematrix solely with the thiol-dependent enzyme systems.Moreover, repair systems also largely depend on reducedthiols (see above). In many cases, the catalytically impor-tant enzyme thiols were shown to be protected againstoxidative damage by glutathionylation. Glutathionylationis a posttranslational, reversible redox modification ofproteins by thiol/disulfide exchange. Indeed, glutathioneused to be a dominant theme in brain neurodegeneration,and it was only recently that the significance of thiore-doxin-based systems has been fully appreciated. Measure-ments of NAD(P)H-dependent thiol reducing activity in

ems: Upper level: Superoxide scavenging reactions catalyzed by

roxiredoxin (Prx), and glutathione peroxidase (GPx) andmediated

of cysteine and methionine and their repair; glutathione repairsts removal with glutaredoxin (Grx) system; methionine is

nic acid modification is irreversible, while sulfinic acid can be

endent enzyme, sulfiredoxin (Srx), which is capable of-generated radicals cause largely non-repairable damage to


brain mitochondria show that glutathione reductase andthioredoxin reductase are equally important. Thiol-depen-dent systems of hydrogen peroxide neutralization andsubsequent repair by themselves are targets for oxidativemodification, and this obviously creates a threshold of oxi-dative damage that can be handled by neuronal mitochon-dria. Once reached, it results in mitochondria failure andthe subsequent cell death by either apoptotic or necroticpathways.

Oxidative Stress and Aging

Chronic Oxidative Stress and Ischemia

The delicate balance existing under normal conditionsbetween oxidant production and antioxidant defensivemechanisms (Scheme 3) can be either slowly shiftedwith aging or disturbed at once upon acute injury. The‘slow acting’ factor is a progressive mitochondrial mal-function, which originates mainly from the damage of themitochondrial DNA: the superoxide released by mito-chondria is directed both ‘in’ and ‘out’ of the mitochon-drial inner membrane, and therefore, is capable ofdamaging mitochondrial DNA, matrix proteins, innermembrane proteins, and lipids. The progressive ‘oxida-tion’ eventually results in mitochondrial inability to uti-lize oxygen efficiently and makes them less competitivewith respect to other intracellular processes consumingoxygen, particularly those controlling the ratio betweenaerobic and anaerobic respiration. Aging cells are moreand more dependent on mitochondrial respiration, andconsequently are less prepared for the increased risk ofischemia. Paradoxically, ischemia results in the increased

SODCatalase, GPx

GR, TR/TrxPrx/Grx/Srx

MsrVitamin ELipoic acid

Ascorbic acidThionein

GSH

Pro-survival

Proliferation Differen

−260 −210

Scheme 3 Delicate balance between pro-death and pro-survival fa

cells, the depletion of intracellular redox potential (shown as that forthe cells will differentiate or die.

production of ROS and excessive damage upon reperfu-sion. The master regulator of the hypoxic adaptation,hypoxia-inducible factor (HIF), has been recently shownto activate one of the key antioxidant proteins in mito-chondria, metallothionein-3. HIF is a widespread tran-scription factor activating a battery of genes includingthose involved in glucose uptake and metabolism, extra-cellular pH control, angiogenesis, erythropoiesis, andmitogenesis, acting to enhance the cell survival ability.More and more new genes are found to be regulated byHIF. Tyrosine kinase receptor B (TrkB) for Brain-derivedneurotrophic factor (BDNF) is also a HIF target implicat-ing HIF in the regulation of neurotrophin signaling. Themost exciting finding was the upregulation of tyrosinehydroxylase, the rate-limiting enzyme in the synthesis ofdopamine, by HIF. However, the link between hypoxiaand mitochondrial biogenesis activator PGC-1a as wellas the mechanism of hypoxic upregulation of mitochon-drial uncoupling protein-3 is still controversial.

The predominant O2-dependent regulation of HIF-1ais mediated by posttranslational mechanisms, amongwhich hydroxylation of Pro564 (HIF-1a is the major reg-ulator and is catalyzed by nonheme iron aKG-dependentdioxygenases known as the HIF prolyl hydroxylases (HIFprolyl hydrodxylases isozymes 1–3). Deficiency or inhibi-tion of PHD1 induces hypoxia tolerance in skeletal muscleby reprogramming basal metabolism through activation ofHIF-2a – > Ppara – > Pdk4 (pyruvate dehydrogenasekinase isozyme-4, restricts entry of glycolytic intermedi-ates into TCA cycle), although no direct link betweenHIF-2a and Ppara has been established.

PHD3 only recently gained full attention because oftwo major findings. First, PHD3 was shown to accumulate

ROS and RNS: Superoxide

Hydrogen peroxideHydroxy-radical

PeroxynitritePeroxynitriteNitric oxide

Nitrosoglutathione4-hydroxynonenal

GSSG

tiation Cell death

Pro-death

mv−150

ctors determines the cell fate in vivo. For the cultured nerve

GSSG/GSH couple) below a certain level determines whether


with age in different tissues. Age-associated changes inPHD3 expression inversely correlate with the expressionof HIF-target gene macrophage migration inhibitoryfactor (MIF), which was described to be involved incellular HIF-mediated antiaging effects. In a recentstudy, evidence was provided that HIF-1 plays a criticalrole in delaying the onset of senescence in rodent cellsvia transcriptional activation of MIF and thereby inhibitionof the p53-mediated pathway. It is worth mentioning hereagain that PHD3 in mice has a mitochondria-targetingleader sequence and that PHD3 seems to be most flexi-ble PHD isoform regarding stimuli-induced change inexpression.

Second, PHD3was found to form subcellular aggregates.The most intriguing finding was that PHD3 inhibitionprevented it from forming aggregates. The PHD3 aggre-gates were dependent on microtubular integrity andcontained components of the 26S proteasome, chaperones,and ubiquitin, thus demonstrating features that are charac-teristic of aggresome-like structures. Forced expression ofthe active PHD3 induced the aggregation of proteasomalcomponents and activated apoptosis under normoxia. Theapoptosis was seen in cells prone to PHD3 aggregation andthe PHD3 aggregation preceded apoptosis. The data dem-onstrate the cellular oxygen sensor PHD3 as a regulator ofprotein aggregation in response to varying oxygen availabil-ity. Given the fact that PHD3 expression is upregulatedwith aging, it may actually contribute to the reduced celltolerance to hypoxia-reoxygenation and other pathologicalscenarios of oxidative stress.

Acute Oxidative Stress

The neurotoxins (rotenone, MPTP) and neurotoxic animalmodels of PD renewed interest in possible environmentalcauses of PD. The most common form of neurodegenera-tion occurs after an acute injury. The causes of neuronalinjury are many and include trauma, DNA damage fromradiation, chemotherapeutic agents, exposure to environ-mental neurotoxins, and others. Acute neuronal damageinvolves a complex combination of processes includingexcitotoxicity, inflammation, necrosis, and apoptosis. The

NO signaling: induction of “v

Heme oxygenase−1

Heme Fe2+, CO Chelates Zn2+ and C

mediates Msr activiBiliverdin

Bilirubin

Thionein

Scheme 4 Pro-survival role of NO: induction of synthesis of (a) hem

bilirubin/biliverdin/biliverdin reductase system; (b) thioneins, which ch

methionine sulfoxide reduction by Msr and Trx; and (c) heat shock pr

adaptive response to tissue damage includes the activa-tion of transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) andothers to switch on the inflammation response to limitdamage and promote repair.

Excitotoxicity

Excitotoxicity is a particularly important event that initi-ates acute neurodegeneration. Accumulation of glutamatewithin the synaptic cleft leads to Ca2+ influx via hyper-activation of N-methyl-D-aspartate receptors, voltage-gated Ca2+ channels, and nonspecific cation conductances.The latter non-specific cation channels are represented bythe recently discovered transient receptor potential (mel-astatin) (TRPM) ion channels, among which TRPM7 andTRPM2, shown to be permeable for Ca2+ and inhibitedby gadolinium, appear to play a critical role in anoxiccell death.

Excess Ca2+ via ionotropic glutamate receptors resultsin the initiation of neurotoxic signaling cascade by acti-vating calmodulin and neuronal NO synthase (nNOS).Current opinion holds that the intracellular redox state isthe critical factor determining whether in brain cells NOis toxic or protective (Scheme 4). NO is known to signalthe induction of heme-oxygenase-1, which is consideredas a prosurvival enzyme. It degrades heme yielding fer-rous iron, CO, and biliverdin, which cycles between theoxidized and reduced form, bilirubin. The latter exhibitsstrong antioxidant properties. Biliverdin reductase is pres-ent in brain in large functional excess, suggesting thatsuch redox cycling amplifies antioxidant effects of heme-oxygenase expression. On the other hand, NO also reactswith glutathione, generating nitrosoglutathione, which isable to modify and thus inactivate protein thiols. However,the most dangerous species, peroxynitrite, is generatedvia the direct interaction of superoxide radical and NO,a comparatively stable and harmless molecule. Peroxyni-trite is extremely reactive and capable of nitrating tyrosineresidues in proteins (Scheme 2). A downstream effect ofperoxynitrite production is the activation of TRPM chan-nels. Molecular deletion of TRPM2 or TRPM7 channelsrenders neurons resistant to hypoxia/aglycemia. Whether

itagenes”

Heat shock proteins (HSPs)

u2+

ty

Hsp 70 – general neuroprotectionHsp 47 – microglia

Hsp 27 – astrocytes Hsp 60, Hsp 10 – mitochondria

e oxygenase, which provides cells an antioxidant

elate metal ions and in addition can mediate/enhance

oteins.


peroxynitrite induces activation of TRPM channels vianitration of its critical components or some upstreammodulatory protein is unclear. Protein nitration has beenshown to take place in brain injuries and some but not allneurodegenerative diseases.

Superoxide and especially peroxynitrite-inducedmodification of the metallothionein thiols results in therelease of zinc. Neurodegenerative diseases are charac-terized by a mobilization of intracellular zinc. The lat-ter was shown to mediate NO-induced neuronal deathby directly affecting mitochondrial respiration throughinhibiting Complex III. In addition, it has recently beenshown that zinc is capable of entering mitochondria anddirectly inactivating lipoamide dehydrogenase, the termi-nal enzyme of major multienzyme energy-producing com-plexes, as well as glutathione reductase and thioredoxinreductase. The inactivation is irreversible, and only thenewly synthesized proteins transported into mitochondriamay compensate for the damage (Scheme 5). Thus, acuteoxidative damage resulting in massive release of intra-cellular zinc will cause the mitochondrial failure andcell death.

InflammationOxidative stress can directly or indirectly initiate aninflammatory cascade. Collective evidence from manyrecent studies suggests that increased phospholipase A2activity plays a central role in acute inflammatoryresponses in the brain as well as in oxidative damageassociated with AD, PD, and multiple sclerosis. PLA2contributes to the pathogenesis of neuroinflammation byattacking neural membrane phospholipids to yield arachi-donic acid and lysophospholipids. These are subsequently

Activation oftranscription factorsROS, RNS

JNK/SAPKLipid

peroxidation

NFkB

ILE-6PLA2

MalondialdehydePeroxidized

arachidonate

4-hydroxy-noneal (4-HNE)

Etheno-modified DNA(epsilon adducts)

Protein modification

(Lys, Cys, His)

Mitochondrialuncoupling

Scheme 5 4-hydroxy-nonenal: the most damaging product of

lipid peroxidation. Phospholipase A2 (PLA2) inhibitors wereshown to be neuroprotective.

metabolized to a variety of proinflammatory lipid media-tors such as prostaglandins, leukotrienes, thromboxanes,and platelet activating factor. Arachidonic acid metabo-lism is also one of the major sources of oxidative damage(Scheme 6). Arachidonic acid undergoes catalytic oxida-tion by cyclooxygenases 1 and 2, and in addition, can reactwith superoxide or peroxynitrite to generate one ofthe most potent modifying agents, 4-hydroxy-2-nonenal(HNE). The latter can modify lysine, cysteine, histidineresidues in proteins and can bind to free amino acids anddeoxyguanosine. Immunostaining for HNE-modifiedproteins shows that such modification is characteristic ofacute oxidative stress, occurring during brain and spinalcord injury.

Inflammation is characterized by the increased deposi-tion of iron. Iron, especially in the ferrous or unboundform, is able to catalyze the formation of free radicals andcould be a cause of neuronal injury. Depletion of antiox-idants in the brain and rise in iron-dependent oxidativestress and monoamine oxidase B (MAO B) activity are keycharacteristics of aging that contribute to the onset ofneurodegenerative disorders. MAO B is a mitochondrialflavin-dependent enzyme that catalyzes oxidative deami-nation of neurotransmitters and exogenous arylalkyla-mines. MPTP, an impurity in synthetic heroin, beingactivated by MAO B, gives a widely used chemicallyinduced model of PD.

1-methyl-4-phenylpyridinium (MPP+), the toxicproduct of MPTP conversion, increases superoxide for-mation by suppressing activity of NADH dehyrogenase(Complex I) and increasing leak of electrons to oxygen.Superoxide is then available to attack Fe/S cluster pro-teins such as aconitase. Destabilization of cytosolic as wellas mitochondrial aconitase results in an increase in ironregulatory protein-1 (IRP-1), an RNA-binding proteinthat signals cellular iron deficiency. This leads to theparadoxical and maladaptive increase in iron in the cell.The mechanisms underlying iron cellular toxicity areonly beginning to emerge. A representative trend in neu-roprotective drug development is to combine an ironchelator and a MAO inhibitor in the same compound.Iron chelators have recently been shown to have the abilityto induce adaptive gene expression via the stabilization offactors such as HIF-1. HIF is a heterodimeric transcrip-tional complex that mediates the induction of more than 70genes involved in hypoxic compensation including vascularendothelial growth factor (VEGF) and Epo.

Activated neutrophils and macrophages generate wide-spread secondary damage at the traumatic site by releasingcytokines and free radicals. They produce inducible NOsynthase and NAD(P)H oxidase (Phox) generating non-mitochondrial superoxide, both the enzymes cooperate togenerate peroxynitrite and thus, expose cells to furtheroxidative damage. In addition to peroxynitrite, leukocytespossess myeloperoxidase, which generates hypochlorite

ROS/RNS

Reversibleinhibition

Zn2+

MetallothioneinCa-uniporter Complex III

TRGR LADH

Irreversibleinhibition:

inactivationThionein

Scheme 6 Zn-induced damage to mitochondrial enzymes of energy production and antioxidant defense.


from hydrogen peroxide and chloride anion. Hypochloriteis a strong oxidizing agent capable of chlorinating proteinresidues and oxidizing membrane lipids. In all cases ofrodent neurodegeneration, medications reducing inflam-matory responses were shown to exhibit beneficial effectson the disease progression.

Apoptosis

The discovery that oxidative stress can trigger a programof cell death in neurons with features of apoptosis wassignificant in several aspects. First, it showed that oxida-tive stress does not always result in random and disor-dered cell damage. Second, it demonstrated the possibilityof free radical triggering an endogenous program of cellsuicide.

Oxidative stress has been shown to activate a host ofdownstream signaling pathways leading to apoptosis. Insome schemes, c-Jun N-terminal kinases (JNKs) signal-ing pathway resulting in Bax translocation, cytochrome crelease, and apoptosis. In other schemes, Erk activation orPARP activation leads to translocation of apoptosis induc-ing factor and caspase-independent cell death. Oxidativestress has been implicated in the activation of cell cycleresulting in cell death, although two studies in whichoxidative stress has been induced by downregulatingantioxidant defenses failed to demonstrate a protectiveeffect of cell cycle inhibitors.

The superoxide released by mitochondria is directedboth ‘in’ and ‘out’ of the mitochondrial inner membraneand, therefore, is capable of damaging mitochondrialDNA, matrix proteins, inner membrane proteins, andlipids, and cytosolic proteins and nuclear DNA. Withrespect to DNA damage, more than 1000 DNA damagingevents occur in each mammalian cell every day fromreplication errors and cellular metabolism. To cope withthe deleterious consequences of DNA lesions, cells areequipped with efficient defense mechanisms to removeDNA damage by DNA repair pathways, control cell cycleprogression, and eliminate damaged cells via apoptosis.

The complicated network of DNA repair mechanismsincludes base excision repair, transcription-coupled repair,global genome repair, mismatch repair, homologous re-combination, and nonhomologous end-joining damage.Evolution has overlaid the core cell cycle machinerywith a series of surveillance pathways termed cell cyclecheckpoints. Checkpoints in proliferating cells tightlycontrol progress through the cell cycle; cells may bearrested at any of the checkpoints and either DNA willbe repaired or cells will die by apoptosis. It appears thatapoptosis induced by oxidative damage may be promotedby multiple pathways, both cell cycle dependent and inde-pendent. The determining factor for the pathway(s)induced is an area of active exploration (Scheme 7).

Nature of Aggregated Deposits inNeurodegenerative Diseases

PD is a common neurodegenerative disorder affecting 1%of the population over the age of 65. Clinically, PD gen-erally presents with bradykinesia, resting tremor, muscu-lar rigidity, and postural instability. PD is a heterogeneousdisease, and the majority of the cases appear to havesporadic origins. The disease is characterized by the lossof dopaminergic neurons in the substantia nigra parscompacta, as well as the presence of Lewy body inclusionin these cells. At least 20% of Parkinson’s cases are famil-ial. Ten different genetic loci have been linked withfamilial PD, and the genes responsible for PD at theseloci include a-synuclein and dardarin/LRRK2 associatedwith dominantly inherited PD, and parkin, DJ-1, andPINK1 causing recessively inherited PD. a-synucleinreadily aggregates and is a major fibrillar component ofLewy bodies. Aggregation of a-synuclein is enhancedwith tissue transglutaminase: Lewy bodies in PD patientsare positively immunostained with antibodies recog-nizing isodipeptide bonds, a marker of tissue transgluta-minase cross-linking. Phosphorylation also promoted

Scheme 7 Cell fate depends on the level of oxidative stress.


a-synuclein aggregation. Culture cells overexpress-ing a-synuclein generate reactive oxygen species. Onemechanism by which aggregated synuclein leads to ROSgeneration in PD is via trapping trace metals into theaggregates. These metals can then easily accept electronsfrom reducing substances such as superoxide or glutathioneand transfer them to oxygen to form superoxide radical.

Two types of disruptions of the DJ-1 gene have beenidentified in PD patients. One is a deletion of several of itsexons, which abolishes the production of the DJ-1 protein.The other disruption is a single point mutation giving riseto the L166P mutant at the protein level, which destroysDJ-1 dimeric structure. The monomer apparently loses itsantioxidant properties and redistributes from cytosol tothe mitochondria and nucleus.

DJ-1 inactivation promotes a-synuclein aggregationstate in a cellular model of oxidative stress.

Polyglutamine diseases, the CAG trinucleotide repeat/polyglutamine diseases, are characterized by the occurrenceof protein aggregates within neurons. The most well-characterized among 10-known diseases of this origin areHD, dentatorubral and pallidoluysian atrophy (DRPLA),spinal and bulbar muscular atrophy, and multiple forms ofspinocerebellar ataxia. Each disease is caused by a distinctgene product with expanded polyglutamine repeats.

HD : The hallmarks of this genetic disorder are a pro-gressive chorea combined with dementia. Historically, thelurching madness was mistaken for possession by witch-craft (some of the Salem witches burnt in 1693 may haveactually had HD). It is caused by a single, dominant gene,that is, only one copy is sufficient to cause the diseaseunlike most genetic disorders which are recessive, that is,two ‘bad’ copies are needed to cause the disease. Theaverage onset is from 35 to 40 years. Huntingtin is alarge (350 kDa) protein of unknown function; once the

polyglutamine tail crosses the threshold of 38 residues,the mutant begins to form aggregates. The latter precipi-tate in cytosol and also form nuclear inclusions. Themechanisms by which huntingtin aggregates launch thedisease are still disputable. It is supposed to be a gain offunction event. It is documented that mutant huntingtincan interfere with gene expression that is associated withadaptation to oxidative stress or mitochondrial dysfunc-tion. Thus mutant huntingtin may be directly toxicto mitochondria and this toxicity may be sustained bymutant huntingtin’s suppression of compensatory geneexpression. While defects in energy metabolism arewidely documented in human HD and associated animalmodels, the only evidence for oxidative stress is oxidativeDNA damage.

DJ-1 in Focus: Linking AntioxidantDefense, HIF Prolyl Hydroxylase, andER Stress

DJ-1 antioxidant activity is still a mystery. DJ-1 containsan active cysteine 106 (see Fig. 1) which redox cycling isindispensable of DJ-1 functioning. The adjacent His 126and Glu 18 residues may form a putative active site(see Fig. 1).

Cytoprotective binding of DJ-1 to apoptosis signal-regulating kinase-1 (ASK1) depends on the centralredox-sensitive Cys-106 and may be modulated byperipheral cysteine residues. ASK1 is a member of themitogen-activated protein kinase family, which activatesc-Jun N-terminal kinase and p38 in response to a diversearray of stresses such as oxidative stress, ER stress, andcalcium influx. In the past decade, various regulatorymechanisms of ASK1 have been elucidated, including its

Figure 1 Redox active cyseine 106 and neighborous His126 and Glu18 residues may form a putative active site in DJ-1.


oxidative stress-dependent activation. Recently, it hasemerged that ASK family proteins play key roles in cancer,cardiovascular diseases, and neurodegenerative diseases.

DJ-1 is required for the activity of Nrf2 (nuclear factorerythroid 2-related factor), a master regulator of responseto oxidative stress. Nrf2 is a member of the cap’n’ collarfamily of basic leucine zipper transcription factors thatregulate the expression of many antioxidant pathwaygenes. Nrf2 is maintained at basal levels in cells by bind-ing to its inhibitor protein, Keap1. Keap1 is a BTB (Broadcomplex, Tramtrack, Bric-a-Brac) domain-containingprotein that targets Nrf2 for ubiquitination by Cul3, lead-ing to its constitutive degradation. Upon exposure tooxidative stress, xenobiotics, or electrophilic compounds,Nrf2 protein is stabilized and translocates to the nucleus.There, it forms heterodimers with other transcriptionregulators, such as small Maf proteins, and induces theexpression of antioxidant genes. Nrf2 drives the expres-sion of detoxification enzymes, such as NAD(P)H qui-none oxidoreductase-1, heme oxygenase-1, thioredoxin

reductase, and other enzymes that generate antioxidantmolecules, such as glutathione. DJ-1 is indispensable forNrf2 stabilization by affecting Nrf2 association withKeap1, an inhibitor protein that promotes the ubiquitina-tion and degradation of Nrf2.

Finally, DJ-1 was identified as the regulatory subunitof a 400-kDa RNA-binding protein complex and its pres-ence inhibits the binding of RNA by the complex. It isworth emphasizing that the large subunit of RNA poly-merase II, Rpb1, has been very recently shown to behaveas a substrate for hydroxylation (P1465) by HIF prolylhydrodxylase-1 (PHD1), in response to low-grade oxida-tive stress.

Neuroprotection Strategies: Problemsand Perspectives

By the time the patient is diagnosed as having a neuro-logical illness, extensive neuronal damage has usually


already occurred. Consequently, there is a great needfor the discovery of biomarkers that would allow ear-lier diagnosis and intervention. Common features amongneurodegenerative diseases, that is, genetic mutations,protein misfolding and aggregation, mitochondrial dys-function, and apoptosis have implications for disease pre-vention and development of effective therapies. Despitecommon final pathways, it is unlikely that a single drug ortargeting a single mechanism will be sufficient to haltneurodegenerative processes.

Chronic neurodegeneration is age-related, and thus,delay in biological aging will decrease the occurrence ofage-related diseaseswith resulting prolongation of a healthylife span. Available evidence suggests that to delay aging onehas to maintain healthy mitochondria and reduce oxidativestress. One of the proposed approaches is to maintain orrecover the activity of the so-called vitagenes. The positiveeffect of heme-oxygenase is linked to the production ofbilirubin and biliverdin (already mentioned), which admin-istration after the first few weeks of life in the doses slightlyabove normal levels resulted in cytoprotective effects.

Another approach to activating vitagenes is adminis-tration of nutritional antioxidants. Curcumin, the mostprevalent nutritional and medicinal compounds used byIndian populations, has the potential to inhibit lipid per-oxidation, and efficiently neutralize reactive oxygen andnitrogen species. It has been recently shown that curcumininhibits NF-kB activation and induces heme-oxygenase-1.Caffeic acid phenethyl ester, an active component of prop-olis, has been shown to induce hemeoxygenase-1 in astro-glial cells. Gene induction in both the cases occurs throughthe antioxidant response element (ARE), and this ledto the conclusion that the increased expression of genesregulated by the ARE may provide CNS with protectionagainst oxidative stress. Indeed numerous studies havesupported a role for ARE activators in the prophylaxisagainst acute and chronic neurodegenerative conditions.

Antiinflammatory and antiapoptotic treatments willhave also shown benefit for many forms of neurodegen-eration, although it is also becoming clear that the parts ofthe inflammatory response must be maintained to facili-tate repair.

A recent development in chelators involves the designand synthesis of multifunctional drugs that have the abil-ity to bind iron, inhibit a particular enzyme, and exhibitantioxidant properties (free radical scavengers). Metal,and in particular, iron chelation therapy has been pro-posed as a way of reducing the level of redox active metalsin neurodegenerative diseases. The green tea catechin,EGCG, which is known for its iron-chelating and antiox-idant properties, the antibiotic iron chelator clioquinol,and intracerebroventricularly injected desferal (DFO) arepotent neuroprotective agents. Obviously, iron chelationtargets not only unbound iron and that in the aggregateddeposits, but also, more significantly, iron-dioxygenases

such as HIF prolyl hydroxylase. The latter is emergingtarget for neuroprotection although HIF may be notthe only substrate of this enzyme. More than 70 genesof putative nonheme iron oxygenases have been identifiedin the human genome, but only a number of them havethe physiological functions ascribed. An assumptionon the uniqueness of an inhibitory action of a particulardrug selected among others may be wrong, if only oneenzyme candidate has been tested. The best exampleis probably EGCG, which targets HIF prolyl hydroxy-lase, MAO B, and MICAL, a flavin monooxygenaseimplicated into axonal guidance and highly homologousto MAO B. The recently discovered M30 as an antiox-idant/chelator/MAO inhibitor was also shown to stim-ulate neurite outgrowth and thus may actually targetMICAL as well.

Conclusion

Therapies targeted at reducing oxidative damage in thenervous system must achieve several goals in order to beeffective. First, they must interdict pathological oxidantinteractions without affecting physiological signaling byradicals. Over the past two decades, peroxide and nitricoxide were shown to play the role of messengers in thenervous system. Antioxidants that inadvertently abrogatethese signaling functions would not be desirable. Second,antioxidants or alternatively effective repair strategiesmust be augmented in distinct cell types and subcellularcompartments. Oxidative and nitrosative stress are not anundifferentiated whole and are mediated by distinct spe-cies produced in distinct cellular compartments and dis-tinct cell types. The great challenge has been to divine amultimodal strategy that inhibits a cassette of targetswithout the expected toxicity that arises as the specificityof the therapy decreases. We propose that understandingendogenous homeostatic pathways for protecting againstoxidative and nitrosative stress is the way forward. Thesehomeostatic pathways involve the activation of preexist-ing proteins as well as de novo gene expression. Smallmolecules that activate homeostatic responses to oxidantstress are expected to reap large therapeutic benefits.Evidence that such an approach is effective and safecontinues to emerge from preclinical studies. The ulti-mate proof will be the demonstration of neuroprotectionin a human clinical trial. Such success will also providelong overdue evidence supporting a role for oxidativedamage in human neurological disease.

See also: Complex I Deficiency; Dopamine; Mitochondri-

al Dysfunction; Monoamine Oxidase Type B Inhibitors;

Nitric Oxide; Parkinson’s Disease: Genetics; Proteasome

Function in Movement Disorders.


Further Reading

Aarts M, Iihara K, Wei WL, et al. (2003) A key role for TRPM7 channels inanoxic neuronal death. Cell 115: 863–877.

Becker EB and Bonni A (2004) Cell cycle regulation of neuronal apoptosisin development and disease. Progress in Neurobiology 72: 1–25.

Binda C, Hubalek F, Li M, Castagnoli N, Edmondson DE, and Mattevi A(2006) Structure of the human mitochondrial monoamine oxidase B:New chemical implications for neuroprotectant drug design.Neurology 67: S5–S7.

Calabrese V, Butterfield DA, Scapagnini G, Stella AM, and Maines MD(2006) Redox regulation of heat shock protein expression bysignaling involving nitric oxide and carbon monoxide: Relevance tobrain aging, neurodegenerative disorders, and longevity.Antioxidants & Redox Signaling 8: 444–477.

Farooqui AA, Ong WY, and Horrocks LA (2006) Inhibitors of brainphospholipase A2 activity: Their neuropharmacological effects andtherapeutic importance for the treatment of neurologic disorders.Pharmacological Reviews 58: 591–620.

FernandoMR, Lechner JM, Lofgren S, Gladyshev VN, and LouMF (2006)Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependentand thioredoxin reductase-dependent peroxidase activities in vitro andin lens epithelial cells. FASEB Journal 20: 2645–2647.

Fratelli M, Demol H, Puype M, et al. (2002) Identification by redoxproteomics of glutathionylated proteins in oxidatively stressedhuman T lymphocytes. Proceedings of the National Academy ofSciences of the United States of America 99: 3505–3510.

Ghezzi P and Bonetto V (2003) Redox proteomics: Identification ofoxidatively modified proteins. Proteomics 3: 1145–1153.

Jenner P, Dexter DT, Sian J, Schapira AHV, and Marsden CD (1992)Oxidative stress as a cause of nigral cell death in Parkinson’s diseaseand incidental Lewy body disease. Annals of Neurology 32:S82–S87.

Johansson C, Kavanagh KL, Gileadi O, and Oppermann U (2007)Reversible sequestration of active site cysteines in a 2Fe–2S-bridgeddimer provides a mechanism for glutaredoxin 2 regulation in humanmitochondria. The Journal of Biological Chemistry 282: 3077–3082.

Johnson MD, Yu LR, Conrads TP, et al. (2005) The proteomics ofneurodegeneration. American Journal of Pharmacogenomics 5:259–270.

Kruman II (2004) Why do neurons enter the cell cycle? Cell Cycle3: 769–773.

Langley B and Ratan RR (2004) Oxidative stress-induced death in thenervous system: Cell cycle dependent or independent? Journal ofNeuroscience Research 77: 621–629.

Lehtinen MK, Yuan Z, Boag PR, et al. (2006) A conserved MST-FOXOsignaling pathway mediates oxidative-stress responses and extendslife span. Cell 125: 987–1001.

Maher P (2006) Redox control of neural function: background,mechanisms, and significance. Antioxidants & Redox Signaling8: 1941–1970.

Mikhaylova O, Ignacak ML, Barankiewicz TJ, et al. (2008) The vonHippel-Lindau tumor suppressor protein and Egl-9-Type prolinehydroxylases regulate the large subunit of RNA polymerase II inresponse to oxidative stress. Molecular and Cellular Biology 28(8):2701–2717.

Poole LB, Karplus PA, and Claiborne A (2004) Protein sulfenic acids inredox signaling. Annual Review of Pharmacology and Toxicology44: 325–347.

Sagher D, Brunell D, Hejtmancik JF, Kantorow M, Brot N, andWeissbach H (2006) Thionein can serve as a reducing agent for themethionine sulfoxide reductases. Proceedings of the NationalAcademy of Sciences of the United States of America 103:8656–8661.

Schwartz EI, Smilenov LB, Price MA, et al. (2007) Cell cycle activation inpostmitotic neurons is essential for DNA repair. Cell Cycle6: 318–329.

Siddiq A, Aminova LR, and Ratan RR (2007) Hypoxia inducible factorprolyl 4-hydroxylase enzymes: Center stage in the battle againsthypoxia, metabolic compromise and oxidative stress.Neurochemical Research 32: 931–946.

Wang JY, Wen LL, Huang YN, Chen YT, and Ku MC (2006) Dualeffects of antioxidants in neurodegeneration: Directneuroprotection against oxidative stress and indirect protectionvia suppression of glia-mediated inflammation. CurrentPharmaceutical Design 12: 3521–3533.

Zheng H, Gal S, Weiner LM, et al. (2005) Novel multifunctionalneuroprotective iron chelator-monoamine oxidase inhibitor drugs forneurodegenerative diseases: In vitro studies on antioxidant activity,prevention of lipid peroxide formation and monoamine oxidaseinhibition. Journal of Neurochemistry 95: 68–78.

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