Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer's disease and in other neurodegenerative diseases

Neurobiology of Aging 26 (2005) 587–595

Authors’ response to commentaries

Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer’s disease and in other neurodegenerative diseases L. Migliorea,∗ , I. Fontanaa , R. Colognatoa , F. Coppedea , G. Sicilianob , L. Murrib a

Department of Human and Environmental Sciences, University of Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy b Department of Neurosciences, University of Pisa, Italy Received 14 October 2004; accepted 29 October 2004

Abstract The contribution of oxidative stress to neurodegeneration is not peculiar of a specific neurodegenerative disease, oxidative stress has been found implicated in a number of neurodegenerative disorders among which Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS). Even increasing are studies dealing with the search for peripheral biomarkers of oxidative stress in biological fluids or even in peripheral tissues themselves such as fibroblasts or blood cells. The application of the modified version of the comet assay for the detection of oxidised purines and pyrimidines in peripheral blood leukocytes results particularly useful if the study requires repeated blood drawn from the same individual, for instance if a clinical trial is performed with a preventive therapy. Likely damage occurs to every category of biological macromolecules and we consider, in the context of neurodegenerative diseases, particularly critical the proteic level. The identification of subjects at risk to develop AD or with pre-pathogenic conditions, the possibility to use “a battery of assays” for the detection of oxidative damage at peripheral level, together with recent advances in brain imaging, will allow to better address studies aimed not only to therapeutic purposes but also mainly to primary prevention. © 2004 Elsevier Inc. All rights reserved. Keywords: Alzheimer; Oxidative stress; Peripheral biomarkers; Neurodegenerative diseases

1. Oxidative stress in Alzheimer’s disease and in other neurodegenerative diseases Starting from the last but one sentence of our article [60], where we stated that the peripheral marker of oxidative stress we assessed cannot be considered peculiar only for Alzheimer’s disease (AD) and Mild Cognitive Impairment (MCI) individuals, as Pratic`o in his commentary observed [75], we would like to enlarge our view to the broader field of neurodegenerative diseases. Recently, we published a review summarising the last findings in the field of carcinogenesis and neurodegenerative diseases [57] indicating in oxidative stress a common factor predisposing to both these age-related complex pathologies in whose etiology either genetic and environmental factors have been found involved. In ∗

Corresponding author. Tel.: +39 050 836 223; fax: +39 050 551 290. E-mail address: [email protected] (L. Migliore).

0197-4580/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2004.10.002

brief the parallel between carcinogenesis and neurodegeneration, is based on the fact that if it is well established that carcinogenesis is a multi-step process caused by a series of mutations occurring into a cell and conferring to this cell a growth advantage, similarly pathophysiology of neurodegenerative diseases involves multiple pathways of neuronal damage, characterised by the generation of abnormal proteins, in some instance due to mutations in corresponding gene(s), and by their subsequent accumulation into (or outside) specific areas of the brain, often with selected neuronal cell death as final endpoint. Dealing with this hypothesis, the contribution of oxidative stress to neurodegeneration is necessarily to be considered not peculiar of a specific neurodegenerative disease. This “multistep” or “more than one hit” hypothesis quite deals with Smith’s et al. comments [87] who, even if their discussion is limited to AD, state that “oxidative stress could be a necessary, but insufficient factor such that the development of disease is dependent upon

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an additional factor(s) for the onset of occult pathogenesis”. It should be matter of reflection at this regard that oxidative stress has been found increasingly implicated in a number of neurodegenerative disorders including AD, Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and others [71]; however, for all these disorders the main question as to whether oxidative stress is involved in the onset and progression of the pathology or is merely a consequence of neurodegeneration is still debated. At present literature evidences indicate that loss of neurons in such disorders results from a complex interplay among oxidative injury, excitotoxic stimulation, dysfunction of critical proteins, and genetic factors. Because of its high-metabolic rate, the brain is believed to be particularly susceptible to reactive oxygen species (ROS), and the effects of oxidative stress on “post-mitotic cells” such as neurons might be cumulative. Peroxidation of cellular membrane lipids generates highly reactive aldehydes, such as 4-hydroxynonenal (HNE). Increased levels of HNE have been observed in AD but also in PD, ALS and other neurodegenerative pathologies, confirming a pathophysiological role of oxidative stress in these diseases [103]. It has been demonstrated that PD is characterized by a reduction of glutathione (GSH), an important intracellular antioxidant, in the substantia nigra [69,70], and such depletion is the earliest known biochemical indicator of nigral degeneration; moreover, protein oxidation has been demonstrated to be elevated in Lewy bodies in cases of PD [35]. Another consistent finding in PD is a defect in oxidative phosphorylation due to a decrease in complex I activity of the electron transport chain, and an impaired complex I activity leads to free radical stress, making neurons vulnerable to glutamate excitotoxicity [83]. Several pesticides and other environmental toxins inhibiting complex I are thought to be involved in the pathogenesis of PD, suggesting that impairments in complex I may be central to the neuronal death in sporadic PD [22]. Markers of oxidative stress associated with ALS are intracellular levels of ROS, lipid peroxidation adducts and mitochondrial DNA oxidation adducts, protein nitration and mitochondrial DNA mutations (for a review, see Carr`ı et al. [17]). Further support to the involvement of oxidative stress in ALS is provided by the observation that mutations in the gene coding for the antioxidant enzyme Cu, Zn, superoxide dismutase (SOD1) are responsible for 10–15% of familial ALS cases [36].

2. Peripheral biomarkers for the detection of oxidative stress It is to our opinion of extreme importance to have demonstrated that significant biological changes related to a condition of oxidative stress have been found not only in brain tissue but also in peripheral tissues of AD individuals. Even increasing are studies dealing with the search for soluble peripheral biomarkers of oxidative stress in biological fluids,

mainly cerebrospinal fluid (CSF) but also peripheral blood (PB) (serum/plasma) or urines or even in peripheral tissues themselves such as fibroblasts or blood cells. HNE was found increased in the CSF [51] and plasma of AD patients [54,81]. Elevated levels of F2-isoprostane were observed in the CSF [62], plasma [97] and urines [95] of AD individuals and higher levels of a specific isoprostane, 12iso-iPF2alpha-VI in plasma, urines and CSF of subjects with probable AD, compared with controls, have been found by Pratic`o et al. [74]. Cecchi et al. [18] showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4HNE in fibroblasts and lymphoblasts of familial Alzheimer’s disease (FAD) patients, compared to controls. Other studies reported increased levels of total oxidized proteins in AD plasma compared to controls [19,21]. For an exhaustive review of biochemical markers of oxidative damage for serum and CFS of AD see Taunissen et al. [91]. Using HPLC analysis Mecocci et al. [55,56] found a significantly higher lymphocyte concentration of the oxidized purine 8-hydroxy-2-deoxyguanosine (8-OHdG) at DNA level, besides a significantly lower plasma levels of antioxidants in AD compared to controls. These findings were recently confirmed by applying the modified version of the comet assay for the detection of oxidised purines and pyrimidines in leukocytes of AD patients [43,64] and both in AD and MCI individuals [60]. Also, studies performed in living patients with a clinical diagnosis for other different types of neurodegenerative pathologies such as PD, ALS, and Friedreich’s Ataxia have evaluated the levels of biomarkers of oxidative damage in peripheral tissues. An increased concentration of MDA in serum, plasma and CSF of patients with PD has been reported [41,44]. Another study showed elevated plasma thiobarbituric acid reactive substances (TBARS) in PD as sign for increased lipid peroxidation [82]. More recently, Buhmann et al. [12] found higher levels of lipoprotein oxidation in plasma and CSF of PD patients compared to controls. Moreover, increased levels of dihydroxybenzoate, an index of hydroxyl radical generation, in isolated platelets of patients with PD have been documented [5]. Significantly elevated levels of 8-OHdG/8-OHG in serum and CSF of PD patients compared to controls have been found [47]; these data are consistent with our observation of increased oxidized purines in leukocytes of PD individuals compared to controls [58]. In studies performed in living patients with a diagnosis of amyotrophic lateral sclerosis, levels of 3-nitrotyrosine [93] and HNE [88] but not of F2-isoprostane [62] were found elevated in CSF compared to controls. More recently, Simpson et al. [85] found significantly higher levels of HNE in the sera and spinal fluid of ALS patients compared to controls. Other investigators have reported increased levels of free 8OHdG in urine, plasma, and CSF of ALS patients [8]. Other studies reported a high level of free radicals identified by electron spin resonance [40], elevated concentrations of TBARS [2,10,68], and increase in protein-associated carbonyls [68] in the blood of ALS patients.

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3. Use of different peripheral cell types for the detection of oxidative damage We agree with the comments of Gibson and Haung [33] on the fact that fibroblasts are an excellent substrate to perform mechanistic studies, since circulating leukocytes are more prone to be susceptible to confounding factors linked to diet or drugs. Also, primary cultures of human olfactory neurons from AD patients proved to be an interesting model for studying oxidative damage [29]. However, the comet assay in our hand (and in most laboratories who perform this assay) was primarily set up in leukocytes, to this respect, we trust that it results easier to take repeated blood drawn from the same individual as frequently as the study requires, for instance, if a clinical trial is performed with a preventive therapy (see a further paragraph). In fact, since among desirable characteristics of a good marker, there is the need to be non-invasive enough to allow repeated measures at yearly intervals (or less) [48], we consider the use of peripheral blood more close to those practical requests. On the other hand, results obtained with fibroblasts are often comparable with those obtained with white blood cells, cytogenetic features we studied in fibroblasts of FAD individuals resulted similar to those observed in lymphocytes of sporadic AD patients [94]. The possibility to look at the oxidative damage in different lymphocyte subtypes, addressed by Gibson and Haung [33], is of concern. Morillas et al. [63] reported indeed that CD4+ lymphocytes are the fraction with the highest induced level of spontaneous genetic damage, evaluated by the comet assay (classical method, without the use of enzymes for the detection of oxidative damage). However, the measurement of DNA fragmentation can be increased during cell subtype isolation and the procedure is time consuming and results prone to artifacts. Attempts to avoid artifacts and improve time for isolation procedure are in progress. For instance, a methodology to measure both DNA strand breaks and oxidized purines (fpg-sensitive sites) in other white blood cells, i.e. polymorphonucleates and in mononucleates, without previous isolation of these subtypes, has been proposed by Giovannelli et al. [34]. We consider however that, for screening purposes, the use of whole blood in the comet assay to measure DNA damage in peripheral leukocytes is simpler and avoids cell manipulations that can interfere with the sensitivity of the assay. Gibson and Haung [33] observed that in experiments carried out by Mecocci et al. [55], a significantly higher concentration of 8-OHdG in AD patients with respect to controls was found; moreover, in controls 8-OHdG, levels increased progressively with age. However, after acute oxidative stress induced by H2 O2 , the levels of formed 8-OHdG did not differ between patients and controls. Morocz et al. [64] demonstrated, with another technique (comet assay, in the modified version for the detection of oxidized bases, the same we used), that AD lymphocytes are more prone to H2 O2 induced oxidative damage than healthy controls, and AD patients

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also showed a diminished repair of H2 O2 -induced oxidized purines. Those results seem to be conflicting but they have been obtained in different experimental conditions. Mecocci et al. [55] used higher concentrations of H2 O2 (mM) and blood cells were frozen immediately after the treatment, without allowing cells time to repair damaged bases; on the contrary M`orocz et al. [64] used lower concentrations of H2 O2 (␮M) and incubated the cells at 37 ◦ C for 1 h after the treatment to allow the progression of repair. These last authors indeed observed different levels of damage in AD patients compared to controls, and supposed that lymphocytes of AD patients could have a diminished repair activity. Mecocci et al. think to a sort of already presence of oxidative stress in AD individuals. This is in agreement with another interesting hypothesis on the existence of an adaptative response toward oxidative stress in neurons of AD individuals, chronically exposed to low levels of oxidative stress [105].

4. Biomarkers of DNA damage The comet assay or single cell gel electrophoresis (SCGE) is a rapid, simple and sensitive device able, in its classical alkaline version, to quantify genomic damage at the individual cell level and specifically to detect DNA strand breaks. In the last years, its use has become a tool even more employed in human biomonitoring studies having also increasing applications in the assessment of DNA damage in subjects affected by various pathological conditions [1,38] and in the investigation of the protective effects of antioxidant supplementation [45,78,84]. In their recent paper, Faust et al. [26] reviewed 45 alkaline comet assay studies with lymphocytes published in the last 3 years and concluded that a remarkable concordance between comet assay and other classical cytogenetic assay data was proved. In order to obtain specific information about oxidative DNA damage, the alkaline SCGE procedure was modified with the digestion of DNA by two bacterial repair endonucleases [20]. These lesion-specific enzymes are endonuclease III (endo III), which recognises oxidised pyrimidines, and formamidopyrimidine-DNA glycosylase (fpg), specific for altered purines, particularly the 8-OHdG, the most common biological marker of oxidative stress in DNA. The modified version of this assay has been applied until now to a less extent, but an increasing number of articles dealing with it can be found in the literature [6,7,11,24]. The identification of valuable reliable peripheral markers that may have utility in the diagnosis of AD is of fundamental importance. However, we do not believe that the detection of oxidized purines and pyrimidines in the DNA can at present be used as a unique diagnostic biomarker for several reasons. First of all, because if we consider a single individual, the entity of the increase of these endpoint is not always high enough to clearly differentiate between healthy controls and affected individuals (e.g. AD or MCI). In general, endpoints

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of genotoxicity are used for biomonitoring individuals, environmentally exposed to potential mutagens/carcinogens. Recent prospective studies on human populations have indicated a positive correlation between the frequencies of spontaneous chromosome aberrations in peripheral lymphocytes and subsequent onset of cancer, but the fact that individuals with higher spontaneous frequency of chromosome aberrations have an increased risk to develop a tumor (certain types of cancer) does not confer to the chromosome aberration assay a value as a diagnostic tool [9]. Sometimes cytogenetic endpoints can be indeed used as diagnostic biomarker of diseases such as Bloom’s syndrome (BS), where an abnormally great amount of exchange between sisters’ chromatids is a highly characteristic feature of cultured blood lymphocytes. In this case, sister chromatid exchanges represent a cytological surrogate marker useful for diagnosis including prenatal diagnosis. Similarly, in Fanconi’s anemia (FA), a high either spontaneous or diepoxybutane and mitomycin C induced level of chromosomal aberrations (in particular exchanges between non-homologous chromosomes) is normally found and usually it is considered a reliable technique to identify FA homozygotes. However, in those two monogenic diseases, the cytogenetic features are mainly due to the specific loss-of-function mutations (in omozygosis in affected individuals) in genes that are involved in chromosome stability. In fact, in BS patients, mutations are in a gene which encodes for RecQ helicase homolog protein, a member of the RECQL gene family and FA is an heterogenous disorder, with at least nine complementation groups having been identified, but FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA. The interaction between ROS and DNA can lead to many premutational lesions, such as sugar lesions, abasic sites, single- and double-strand breaks or oxidised bases that can be fixed into mutations. But it is not possible, as proposed by Gibson and Haung [33], to exactly characterize from a molecular point of view the AD related changes neither compare them by adding selected oxidants. For instance, we know that in the carcinogenic process, environmental mutagens/carcinogens can cause the mutational changes in genes. Some of them have deeply been studied; this is the case of a key gene for the onset of many cancers, TP53 gene, and the mutation spectra of this gene vary with the different carcinogens and cancer type. It is well known that the presence of specific chromosome aberrations (e.g. dicentrics) in peripheral lymphocytes are related to ionizing radiation exposure; this technique is being routinely used all over the world for dosimetric purposes, especially when radiation incidents occur. However, the relationship is not univocal; dicentrics can be induced also by other mutagenic chemicals, like alkylating agents or typically the so-called radiometric agents such as bleomycin. Thus, it appears quite unlikely to relate a specific cytogenetic endpoint to a definite exposure. An exception is the assessment of another endpoint detectable at molecular level, the

so-called DNA adducts. Specific DNA adducts are induced by exposure to certain chemicals but this is true only for selected well-studied chemicals (e.g. Benzo(a)pyrene). Thus, biomarkers of genotoxicity can be usefully employed for biomonitoring individuals in certain exposure conditions to establish if those exposures can render them more susceptible at group level. Analogously, we think that these endpoints can be used for identify groups of individuals at risk to develop degenerative diseases and for monitoring them for instance during a therapy.

5. Oxidative stress and the functionality of the proteasome If, as it results from many literature evidences, oxidative stress is a common feature of neurodegenerative diseases, what can be the consequences at molecular level? We found that either in AD, MCI or PD, there is an increase in oxidatively-damaged DNA at peripheral level, but likely damage occurs to every category of biological macromolecule prior to lesion formation and we consider in the context of neurodegenerative diseases, particularly critical the proteic level. As reviewed by Butterfield [14], the use of proteomics to specifically identify oxidatively-modified proteins in AD brain allows to determine which proteins are more affected by oxidation in AD, providing insights into potential mechanisms of neurodegeneration. Proteomic studies indicated several such proteins that can be classified as those dealing with energy metabolism, glutamate reuptake, recycling of damaged or aggregated proteins through the proteasome such as Ubiquitin Carboxyl terminal Hydrolase L1 (UCH L-1). This latter enzyme is specifically oxidatively modified in AD leading to loss of activity of the proteasome, and accumulation of damaged or aggregated proteins. Other such proteins are involved in the maintenance of membrane structure and function and in directing dendrites to adjacent neurons. All these functions are compromised in AD and the use of proteomics identified proteins whose decreased functions are consistent with the pathophysiology of AD [14]. The proteasome is a large intracellular protease, present in all cells of the central nervous system (CNS), that is responsible for the degradation of oxidized, aggregated, and misfolded proteins, playing an important role in maintaining neuronal homeostasis; moreover, alterations in proteasome activity may play an important role in neurodegeneration [46]. Lipid peroxidation products including HNE have been demonstrated to be potent inhibitors of proteasome activity [28]. Since HNE is capable to covalently cross-link proteins, it is believed that it inhibits proteasome activity in AD through the formation of crosslinks between protein-bound HNE and the proteasomal proteins, providing steric interference that blocks the entry of the pore of the proteasome [13]. Moreover, the proteasomal system might be compromised in AD due to oxidation of proteins such as UCH L-1 [14]. Recent studies have demonstrated that many subunits of

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the proteasome complexes contain heat shock proteins (HSP) binding sequences, implicating a possible role for HSPs in proteasome function [52], and the protein heat shock cognate 71 was found to be specifically oxidatively modified in AD brain [14]. Inibition of the proteasome activity seems to be a necessary step leading to A␤ neurotoxicity, and A␤1–42 induced suppression of proteasome activity was showed to be mediated by the ubiquitin-conjugating enzyme E2-25K/Hip2 protein [89]. Molecular misreading is a recently discovered process leading to the formation of aberrant transcripts formed as a result of dinucleotide deletion during or after transcription, and the resulting proteins are called +1 proteins [96]; among them the aberrant ubiquitin UBB + 1 is a potent inhibitor of the proteasome in AD brain and was found to colocalyze and interact with E2-25K/Hip-2 in mediating A␤ neurotoxicity [89]. It has been hypothesized that quality control mechanisms work less efficiently during aging, leading to +1 proteins formations, however, being UBB + 1 protein degraded by the proteasome under non-pathological circumstances, its presence can be considered a marker of proteasomal dysfunction [96]. A decrease of the proteasome function induced by oxidative stress could results in an increased level of UBB + 1 protein, thus mediating the A␤1–42 induced neurotoxicity. As observed by Pratic`o [75] the “oxidative hypothesis of AD” formulated by Markersbery [53] leads to the question as to whether oxidative stress is an early event triggering the formation of senile plaques (SPs) and neurofibrillary tangles (NFTs), or a response to SPs and NFTs. Recent evidence seems to highlight that even if not the primary initiating event, oxidative stress is an earlier event associated with neurodegeneration, required for the propagation of an integrated series of cellular phenomena, all contributing to the neuronal death. Extensive evidence supports an A␤1–42 induction of lipid peroxidation and protein oxidation [15,101]; moreover, there is evidence that small aggregates of A␤1–42 , rather than fibrils, are the toxic species of this peptide [98]; small aggregates of A␤1–42 are believed to penetrate the lipid bilayer inducing lipid peroxidation, and peptide induced ROS formation [49]. Oxidative stress so generated might lead to proteasome inhibition and/or to the alteration of several other cellular pathways, resulting in neuronal death. As reported by Beal [4], A␤ interacts with proteins localized in the mitochondrial matrix contributing to oxidative damage, and A␤ might directly contribute to oxidative stress in mitochondria. Moreover, data obtained by the group of Beal [50] in Tg19959 mice overexpressing a doubly mutated human beta-amyloid precursor protein, indicate that a partial deficiency of manganese superoxide dismutase (MnSOD), which causes elevated oxidative stress, significantly increased brain A␤ levels and A␤ plaque burden. These results, approaching the answer to the question of whether oxidative stress also contributes to A␤ deposition, appear to be really interesting and reinforce the role of oxidative stress in the propagation of several cellular phenomena leading to neuronal death.

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Disturbance of protein degradation by the ubiquitinproteasome system might have a critical role also in PD, and in neurodegeneration in general, suggesting that uncovering the mechanisms of protein degradation should add importantly to understanding the neurodegenerative process [39]. Due to increasing evidence suggesting that mitochondrial dysfunction, increased oxidative stress, and dysfunction of the ubiquitin-proteasome system are involved in alphasynuclein aggregation and Lewy body formation in PD, a proteomic approach to investigate these pathways has been recently proposed [104].

6. Individuals at risk to develop AD as human model To better address basic, therapeutic and mainly preventive approaches, studying pre-symptomatic subjects or individuals more prone to develop AD represents the best tool. For this purposes, as Gibson and Haung [33] suggest, patients with predisposition to develop AD can be a good model to verify the onset of biological changes due to oxidative stress. Examples for that are MCI subjects but also pre-synthomatic individuals belonging to families with FAD and carrying the causative mutation in APP, PS1 or PS2 genes, even if for those subjects, the proper ethical issues cannot be disregarded. Also, Down syndrome (DS) patients represent an excellent model for studying the temporal sequence of events that triggers the onset and progression of AD since the majority of DS individuals develops neuropathological features of AD by the age of 40, presumably due to the presence and the expression of three copies of the APP gene located on chromosome 21 [73]. Insights into the similarity or divergence of mechanisms leading to dementia in DS and AD support the involvement of oxidative stress in progression of DS. The levels of 8-OHdG and MDA are significantly elevated in urine samples of DS patients compared to controls [42]. Odetti et al. [67] studied Down’s fetal brain cortex to evaluate the presence and amount of lipid and protein oxidation markers; all parameters were significantly increased in Down’s fetal brains in comparison to controls, providing the evidence that accelerated brain glycoxidation occurs very early in the life of Down’s syndrome subjects. Our findings of an increased oxidative damage in leukocytes of MCI patients, a good model of subjects at risk to develop AD, are consistent with the observed increased level of oxidative DNA damage in DS individuals compared to controls [27]. Another group of individuals prone to undergo AD is also that of mothers who had a DS child in young age. An increased frequency of AD (about five-fold) among young mothers of individuals with DS is reported [80]. We recently performed cytogenetic studies on peripheral blood lymphocytes of a group of those mothers showing that they are particularly prone to chromosome malsegregation, phenomenon likely related with aging and with aneuploid meiotic products. They could be biologically more elderly than their real age, showing a specific vulnerability to AD [61].

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7. Antioxidant treatments for neurodegenerative diseases The intense investigations on the mechanisms by which neurons die, and the recent findings on the involvement of oxidative stress in many neurodegenerative diseases, have led to the idea that the therapeutic use of antioxidants could be of help in aging and neurodegenerative diseases. Novel therapeutic neuroprotective strategies support the application of ROS scavengers, transition metal, like iron and copper, non-steroid anti-inflammatory drugs (NSAID), antiapoptotic drugs, and bio-energetic drugs in monotherapy or as part of an antioxidant cocktail formulation for the treatment of neurodegenerative diseases [86]. The main tendency in the scientific community is to divide the potential therapeutical treatments in two different categories: vitamin antioxidants and non-vitamins “cocktail”. Among the antioxidant treatments using vitamins, Vitamin E, Vitamin E analogs, and Vitamin C have been well characterized by literature since several years. Many vitamins directly scavenge ROS and in parallel can upregulate the antioxidant capacity of oxidative defence system of the body. Among them, Vitamin E or trolox (a water-soluble Vitamin E analog) has been recognized as one of the most important antioxidants also in “in vitro” models such as AD fibroblasts [30]. Vitamin E has been also recognized to inhibit ROS-induced generation of lipid peroxyl radicals, thereby protecting cells from peroxidation. Moreover, a dietary deficiency reduces the activity of many scavengers’ enzymes like hepatic catalase, GSH peroxidase, and glutathione reductase, induces liver lipid peroxidation and causes neurological and cardiovascular disorders [16,32,65]. Yokota et al. [102] have shown an increase in ROS accumulation in neurodegeneration in mice with a deficiency in the ␣-tocopherol transfer protein. Also, the role of physiological concentration of Vitamin C has been well established in the literature [3]. In the antioxidant treatments for neurodegenerative diseases, a hot topic is also the utilization of antioxidant compounds mainly belonging to the phytochemicals in the non-vitamins category. Among them, phenolic and polyphenolic compounds, such as flavonoids and catechin in edible plants, exhibit potent antioxidant activities [23,25]. Demonstrating this tendency, Mecocci et al. [56] have shown that in lymphocyte DNA 8-OHdG content was significantly higher and plasma levels of antioxidants were significantly lower in patients with AD compared with controls. Gibson et al. [30,31] have also shown that a variety of antioxidants like Ginko biloba, DMSO, N-acetylcysteine, trolox, and alpha-keto-beta-methylvalerato are able to diminish the effects of oxidants in bombesin releasable calcium stores (BCRS). Many of the biological actions of flavonoids have been attributed either through their reducing capacities per se or through their possible influences on intracellular redox status. The precise mechanisms by which flavonoids exert their beneficial or toxic actions remain unclear. However, recent studies have speculated that their classical hydrogendonating antioxidant activity is unlikely to be the sole ex-

planation for cellular effects [76,79,90]. Usual dietary antioxidants, in particular tea and tea flavonoids, have been reported to possess potent radical scavenging [66,77] and anti-inflammatory activities [37]. Among the various bioactive compounds which got a wide range of literature data, the tea extract is particularly rich in flavonoids, a family of polyphenols found in fruits and vegetables, as well as in plant beverages, including tea and pomegranate juice. Fresh tea (Camellia sinensis) leaves contain a high amount of catechins, a group of flavonoids or flavanols known to constitute 30–45% of the solid green tea extract [99,100]. Among other substances ␤-carotene and other carotenoids, such as ␣-carotene, ␥-carotene and ␤-cryptoxanthin, are potent antioxidants of plant origin [3]. A recent work performed by Theriault et al. [92] also shows that tocotrienol is able to reduce at plasma level the atherogenic apolipoprotein A, exerting hypocholesterolemic activity. As already mentioned above the experimental approaches to investigate the decrease of DNA damage triggered by antioxidant treatments for some diseases has shown their efficacy. Also, cytogenetic endpoints have been shown to be a useful marker to detect a decrease of chromosomes damage due to antioxidant therapy. A group of mitochondrial disease patients, affected by disorders clearly characterized by an impairment of oxidative metabolism, showed an increased level of chromosome damage, expressed as frequency of micronucleated lymphocytes, in comparison with healthy individuals. Patients receiving a two week therapy with ubidecarenone (coenzyme Q10) showed a statistically reduction in the frequency of micronucleated cells after therapy [59]. Although the recent advance in therapeutic agents are in various stages of development for several neurodegenerative diseases, the design of clinical trials has been “slowed-up” by the difficulty to identifying patients in the early onset of the pathology enough on the disease continuum to analyze and test new drugs for effectiveness in slowing the progression. Combining trophic factors with antioxidants or immuno-suppressants has shown enhanced survival and improved graft-induced functional effects and combining metabolic supplements with antioxidants or antiexcitotoxins shows greater protection than does either strategy independently. These treatments in a ‘cocktail’ approach, may provide an improved beneficial therapeutic intervention for the treatment of neurodegenerative diseases.

8. Conclusions We considered our study a contribution to the understanding of the molecular basis of AD because only if causative mechanisms of the disease are deeply understood, therapeutic but mainly preventive studies can be better addressed. We are aware that for the scientific community and mainly for clinicians the search for valuable reliable peripheral markers of a disease can be of fundamental value. At least a set of biomarkers for the detection of oxidative stress (such as

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oxidative DNA damage but also lipid peroxidation) can be identified and proposed as a battery of assays until the “ideal biomarker”, augured by Pratic`o [75] could be found. Increasing studies in parallel with multiple endpoints should be encouraged. Formal characterization of Mild Cognitive Impairment [72], recent advances in brain imaging and the identification of such a pre-pathogenic condition either through the analysis of the oxidative stress peripheral biomarkers are paving the way for better-addressed studies aimed not only to therapeutic purposes but mainly to primary prevention.

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