much of the research performed on alzheimer disease ( ad ) over the past decade has focused on oxidative stress mechanisms and its importance in 
 disease pathogenesis . multiple lines of evidence have implicated oxidative stress and free radical damage to the pathogenesis and possible etiology of ad . such damage found in 
 ad includes advanced glycation end products , nitration , lipid peroxidation adduction products [ 3 , 4 ] as well as 
 carbonyl - modified neurofilament protein and free carbonyls 
 [ 5 , 6 ] . importantly , this damage involves all neurons vulnerable to death in ad , not just those containing neurofibrillary tangles . in fact , the spatio - temporal distribution 
 of specific types of damage elegantly reflects the biology and 
 chemistry of each modification . the cytopathological significance of oxidative damage is seen by 
 the up - regulation of antioxidant enzymes . heme oxygenase-1 ( ho-1 ) is among the most sensitive and selective indicators of the cellular oxidative stress response and , in ad , we demonstrated that both ho-1 protein and its mrna are increased in brains of ad patients [ 7 , 8 ] . in later studies , we demonstrated that increased ho-1 expression co - localizes with the altered form of tau (  ) . there is much substantive evidence for progressive oxidative modification of proteins as a consequence of aging . however , the most crucial aspects of the cellular oxidative damage in ad pathogenesis appear to be the cytoskeletal modifications in neurons susceptible to ad , which in turn play a key role in the irreversible cellular dysfunction that ultimately leads to 
 neuronal death . the most obvious of these cytoskeletal abnormalities are the nft , which are arguably the most important aspect of the degenerating phenotype in ad - affected neurons . these 
 inclusions largely consist of modified medium and high molecular 
 weight neurofilaments ( nfm and nfh , respectively ) as well as the 
 microtubule - stabilizing protein . in nft , these two families 
 of protein have an abnormally high level of phosphorylation , which 
 may be explained by their conserved kinase consensus sequences 
 . in addition to hyperphosphorylation , proteins in both nft and senile plaques have been shown to be modified by the addition 
 of advanced glycation endproducts ( age )   and 
 hydroxynonenal . some of the antibodies used to detect nft specifically recognize 
 oxidation - dependent epitopes . we have demonstrated that two 
 antibodies to nft recognize carbonyl - modified epitopes of high 
 molecular weight neurofilaments ( nfh ) . although both of these antibodies recognized neurofilaments of the axon in 
 aldehyde - fixed sections , they demonstrated no reactivity in 
 immunoblots with normal axonal protein prepared from tissue 
 unexposed to fixatives . in contrast , if these immunoblots were 
 carbonyl - modified , both antibodies recognized nfh . significantly , 
 in samples from ad brains , the antibodies recognized nft epitopes 
 in both tissue sections and nft - derived nfh in immunoblots 
 without need of carbonyl modification . furthermore , 
 carbonyl - modified nfts could be isolated from homogenates of ad 
 brain tissue but not from control tissue . taken together , these 
 results suggest that oxidized nfh is a prominent antigen in nft 
 . the oxidative conversion of protein side - chains to reactive 
 carbonyl or acylating moieties , or adduction of bifunctional 
 sugar- or lipid - derived products can result in abnormal protein 
 cross - linking . both neurofilaments and however , they have a 
 limited lifetime , and the protein - derived reactive moieties will 
 preferentially react with solvent or other molecules if no 
 suitably reactive and/or positioned group is nearby . we 
 hypothesize that the pathogenic role of oxidative stress , 
 involving direct oxidation and/or modification by oxidation , may 
 be primarily in the covalent  cementing  of reversible 
 associations normally reflecting simple noncovalent affinities . it has been found that many proteins associated with nft are 
 apparently covalently cross - linked to the filaments since they 
 can not be removed by denaturants . this is consistent with other 
 studies , which show that oxidative covalent cross - linking of 
 proteins occur predominantly when the proteins are preassociated . the in vitro enzymatic cross - linking of tau could be produced only 
 when the protein substrates were properly associated . since 
 oxidation - based cross - linking reduces susceptibility to 
 proteolytic degradation by providing nonpeptide bonds , covalent cross - linking may in fact represent a neurotoxic event by 
 inhibiting turnover . simultaneously , several oxidative 
 modifications may generate chemical species , which themselves 
 support further oxidative stress through  redox cycling  or 
 transition metal sequestration . closer examination of the spatio - temporal relationship between the 
 presence of oxidative modification and the hallmark ad lesions reveals an apparent paradox . it has been found that while stable glycation products are predominantly associated with nft and amyloid- ( a ) deposits , reversible or rapidly degraded adduction products are primarily in the cytoplasm of vulnerable neurons . in fact , when damage to short - lived molecules is examined , the damage is restricted to cytosolic compartments with nft and a being inversely correlated . to address where reactive oxygen species are produced , we focused our efforts to finding a marker that results from primary attack , 
 rather than more complex secondary reactions , and which involves damage to a cell constituent with a short half - life . proteins fail in the latter aspect of these criteria because cross - link modifications slow their turnover . therefore , modifications associated with cross - linking of proteins , while useful to assess 
 history , may reveal less of the current state . however , 8-hydroxyguanosine ( 8ohg ) , a nucleic acid modification predominantly derived from 
 hydroxide ( oh ) attack of guanidine , is amplified in cytoplasmic rna in vulnerable neuronal populations . notably , cases of ad with the most extensive a deposits show the lowest 8ohg levels . these findings seem markedly contradictory if we consider a to be the toxic agent in ad , which is likewise proposed to be a major source of 
 oxidative free radicals . however , neurons containing nft also have extremely low levels of 8ohg ( ie , current oxidative 
 stress status ) despite an obvious history of oxidative damage ( ie , advanced glycation endproducts or lipid peroxidation ) . this suggests that both a and nft may serve an antioxidant function , and thus be cellular compensations for increased oxidative stress . the hypothesis that a plays an antioxidant function is supported by a study of down syndrome patients , a disease where a deposits begin in the late teens and in which oxidative stress has been implicated . mitochondria are a potential major source of oxidative radicals and oxidative precursors , in the form of o2 and 
 h2o2 , respectively , since their production is linked to metabolism . in early studies , we found increases in mitochondrial 
 dna in the cell soma of ad susceptible neurons , which in itself 
 might cause increased oxidative potential . perhaps more importantly , in situ hybridization studies with a chimeric cdna probe to a common mitochondrial mutation ( 5 kilobase ( kb ) common deletion ) have shown at least a 3-fold increase in ad cases compared to controls . ultrastructural localization of mtdna with colloidal gold shows that deleted mtdna is mainly found in 
 abnormal mitochondria ( ie , those lacking cristae , swollen and in 
 many cases fused with lipofuscin ) . these findings suggest that the 
 mtdna in situ hybridization detected mtdna proliferation , 
 deletion , and duplication in abnormal mitochondria , many of which 
 have been fused with lysosomes , indicating that they are being 
 turned over . quantitative analysis of the colocalization of the mtdna deletion 
 and 8ohg in ad cases demonstrate a strong positive correlation 
 ( r = 0.934 ) . however , mitochondrial dna , even that containing 
 the 5  kb deletion , is relatively spared from oxidative 
 damage , like the formation of 8ohg , in comparison to cytoplasmic 
 nucleic acid ( ie , rna ) . we therefore suspect that mitochondrial 
 abnormalities correlate with , but do not directly cause , reactive 
 oxygen species ( ros ) . this may be due to the fact that 
 hydroxide radicals , which are responsible for the formation of 
 8ohg , have a sphere of diffusion of only 2  nm and are 
 fairly short lived . therefore , since damage is topographically 
 distinct , it is likely that oh radical formation occurs in the 
 cytoplasm rather than the mitochondria and that they are unable 
 to diffuse through the mitochondrial membrane to affect mtdna . however , abnormal mitochondria may produce excess h2o2 through the conversion of o2 by mitochondrial superoxide dismutase ( sod ) . such h2o2 is readily diffusible and relatively stable , that is , until 
 interacting with redox - active transition metals where the fenton 
 reaction produces hydroxyl radicals . several studies have implicated imbalances of trace elements , including al , si , pb , hg , zn , cu , and fe in ad . a disruption in the homeostasis 
 of the latter two redox - active metals is particularly significant in light of the increases in oxidative stress parameters , such as lipid peroxidation , and the oxidative damage to nfts , senile plaques [ 1 , 2 , 4 ] , and nucleic acids . micro particle - induced x - ray emission , recently found that zn(ii ) , fe(iii ) , and cu(ii ) are significantly increased in ad neuropil and that these metals are more concentrated within the core and periphery of senile plaques 
 . these results support earlier studies reporting 
 increased levels of iron , transferrin , and ferritin in ad . an in situ iron detection method revealed a significant association of redox - active iron with both 
 nft and senile plaques ( sp ) in ad . the association of iron with nft may be partly related to the binding of iron to  , their primary protein constituent . recent histochemical studies have demonstrated that the direct 
 detection of redox activity in ad lesions is inhibited by prior exposure of tissue sections to copper and iron selective chelators . re - exposure of the chelator - treated sections to either copper or iron salts can reinstate activity , suggesting that redox imbalance in ad is dependent on these metals . it is therefore probable that accumulation of iron and copper is a major source of the production of ros , which are in turn responsible for the more 
 global oxidative stress parameters observed in ad . free iron has been implicated in undergoing redox transitions in 
 vivo with the consequent generation of oxygen free radicals more than any other transition metal . abnormally high levels of iron and oxidative stress have been shown in a number of neurodegenerative disorders including ad , parkinson disease ( pd ) , multiple system atrophy , and progressive supranuclear palsy . therefore , experimental findings of increased total iron do not necessarily implicate increased oxidative stress if there are concomitant increases in proteins that store iron in redox inert forms . for example , ferritin ( which can be over - expressed in pd and ad ) complexes iron in its core to produce the insoluble , unreactive compound ferrihydrate . the fact that oxidative stress plays an important role in ad 
 pathogenesis is apparent , given all the evidence the research has recently provided . markers of oxidative damage including heme oxygenase-1 ( ho-1 ) and 8ohg are increased in the ad brain as compared with controls . in addition , nfts and senile plaques are altered in ways characteristic of oxidative damage including age - modification , protein cross - linking and carbonyl- and acyl - modification . although the source of the shift in oxidative homeostasis is still unclear , current evidence points to changes in the balance of redox transition metals , especially iron and copper . both fe and cu are present at significantly 
 elevated levels in ad neuropil , and detection of redox activity in the ad brain can be attenuated by chelators of these key metals . it has also been demonstrated that many of the proteins that are important in their regulation , including ferritin and ceruloplasmin , show altered expression in ad and other 
 neurodegenerative disorders . these changes could be responsible for the oxidative imbalance or may represent an attempted antioxidant response by affected cells . however , the key aspect of the degree of cognitive decline in 
 neurodegenerative disease is selective neuronal death . our findings that neurons displaying protein damage indicate an oxidative stress response is still in need of a mechanism that links damage and death . determining the relative contribution of sugar- or lipid - adduction reactions as well as direct side - chain 
 oxidation to the properties of abnormal inclusions will provide insight into the mechanistic link , but also into rational therapeutic protocols for each disease , for example , using water- versus lipid - soluble antioxidants or free - radical versus carbonyl scavengers . we are encouraged by the preliminary epidemiological and clinical studies suggesting that inhibiting oxidative stress 
 and glycation are effective in reducing the clinical manifestation of neurodegenerative diseases . the stage is now set to critically examine the importance of oxidative imbalance in the 
 pathogenesis of ad as antioxidants , and chelating agents take a primary role in clinical intervention of neurodegenerative diseases . this work was supported by grants from the national institute of 
 aging , the alzheimer association , and the american health assistance foundation .
research in alzheimer disease has recently demonstrated compelling evidence on the importance of oxidative processes in its pathogenesis . 
 cellular changes show that oxidative stress is an event that precedes the appearance of the hallmark pathologies of the disease , neurofibrillary tangles , and senile plaques . 
 while it is still unclear what the initial source of the oxidative stress is in alzheimer disease , it is likely that the process is highly dependent on redox - active transition metals such as iron and 
 copper . 
 further investigation into the role that oxidative stress mechanisms seem to play in the pathogenesis of alzheimer disease may lead to novel clinical interventions .