increased oxidation of certain glycolysis and energy metabolism enzymes in the frontal cortex in...

Increased Oxidation of Certain Glycolysisand Energy Metabolism Enzymes in theFrontal Cortex in Lewy Body Diseases

Anna Gomez and Isidre Ferrer*

Institut Neuropatologia, Servei Anatomia Patologica, Idibell-Hospital Universitari de Bellvitge,Hospitalet de Llobregat, Spain

Lipoxidative damage of aldolase A, enolase 1, andglyceraldehyde dehydrogenase (GAPDH) was found inthe frontal cortex in a percentage of aged controls bybidimensional gel electrophoresis, Western blot test, in-gel digestion, and mass spectrometry. Aldolase A andenolase 1 were altered in 12 of 19 cases, whereas oxi-dation of GAPDH was found in 6 of 19 controls. Thethree enzymes were oxidized in the frontal cortex in themajority of cases of incidental Parkinson’s disease(iPD), PD, and dementia with Lewy bodies (DLB). Differ-ences were statistically significant (v2 test) for GAPDHin PD and DLB. Densitometric studies have shown thatthe ratio of oxidized protein per spot is higher in iPD,PD, and DLB compared with controls. These findingsshow oxidation of three enzymes linked with glycolysisand energy metabolism in the adult human brain aswell as increased oxidation of aldolase A, enolase 1,and GAPDH in the frontal cortex in Lewy body dis-eases. Modifications of these enzymes may result indecreased activity and may partly account for impairedmetabolism and function of the frontal lobe in PD.VVC 2008 Wiley-Liss, Inc.

Key words: Parkinson disease; Lewy body disease;dementia with Lewy bodies; incidental Lewy bodydisease; aldolase A; enolase 1; glyceraldehydedehydrogenase; oxidative damage

Parkinson’s disease (PD) is a chronically progressiveage-related neurological disease that affects at least 1%of the population over 55 years of age (Olanow andTatton, 1999). Clinically, it is characterized by restingtremor, rigidity, bradikinesia, gait disturbance, and pos-tural instability. The neuropathological hallmarks of PDare degeneration and loss of dopamine neurons in thesubstantia nigra and other brain stem areas, together withaccumulation of proteinaceous intraneuronal inclusionsknown as Lewy bodies and aberrant neurites, mainlycomposed of a-synuclein (Forno, 1996; Gai et al., 2000;Goedert, 2001; Jellinger and Mizuno, 2003; Shults,2006). Diffuse Lewy body disease or dementia withLewy bodies (DLB) is clinically manifested as dementiaand parkinsonism. DLB is characterized by typical lesions

of PD together with Lewy bodies and neurites in thecerebral neocortex and diencephalic nuclei (Ince andMcKeith, 2003; McKeith et al., 2004). Parkinson-likepathology restricted to the medulla oblongata and pons,associated or not with mild midbrain involvement in theabsence of motor symptoms, is known as incidental orpreclinical PD (iPD) (Forno, 1996; Jellinger and Miz-uno, 2003). PD and DLB are considered as Lewy bodydiseases (LBD) within a common pathological spectrum.LBD brain stem type includes premotor (iPD) andmotor stages of PD (PD); LBD limbic covers PD withinvolvement of limbic structures, including amygdala;and LBD neocortical is categorized as DLB (McKeithet al., 2006).

Staging of brain pathology related to sporadic PDhas also been proposed (Braak et al., 2003). This instru-mental classification is useful because it delineates the to-pography of lesions in the different stages and correlatesand matches with clinical symptoms in most cases. Thus,stages 1 and 2 are coincidental with preclinical PD,stages 3 and 4 may manifest as PD, and stages 5 and 6are often manifested as PD with cognitive impairmentand DLB (Braak et al., 2004, 2005; Wolters and Braak,2006).

Several studies have shown that oxidative stressplays a crucial role in the pathogenesis of established PD(Markesberry et al., 2001; Jenner, 2003). Studies in thesubstantia nigra and midbrain have shown decreasedlevels of reduced glutathione (Perry et al., 1982; Sianet al., 1994), and increased Cu/Zn superoxide dismutaseI (SOD1) and Mn superoxide dismutase (SOD2) proteinand mRNA levels (Marttila et al., 1988; Saggu et al.,1989; Ceballos et al., 1990). Increased protein carbonyls(Alam et al., 1997; Floor and Wetzel, 1998), lipid

*Correspondence to: Isidre Ferrer, Institut Neuropatologia, Servei Anato-

mia Patologica, Idibell-Hospital Universitari de Bellvitge, carrer Feixa

Llarga sn 08907, Hospitalet de Llobregat, Spain.

E-mail: [email protected]

Received 4 June 2008; Revised 1 August 2008; Accepted 19 August

2008

Published online 14 October 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21904

Journal of Neuroscience Research 87:1002–1013 (2009)

' 2008 Wiley-Liss, Inc.

hydroperoxides (Dexter et al., 1986), 4-hydroxy-2-nonenal (Yoritaka et al., 1996; Shelley et al., 1998), andincreased generation of malondialdehyde and hydroper-oxides (Dexter et al., 1989; Jenner, 1998) have beenreported in the substantia nigra and related pathways inclinical PD. Advanced glycation end products have alsobeen found in the substantia nigra and locus ceruleus inPD (Castellani et al., 1996).

In spite of this important information, little isknown about the appearance of oxidative stress and sub-sequent oxidative damage in the frontal cortex at earlystages of PD. Yet this is crucial to understandingwhether oxidative damage is an early event occurring inregions lacking typical morphological markers (i.e.,Lewy bodies and neurites). Oxidative stress has beenreported in the cerebral cortex in incidental PD (Dexteret al., 1994). Moreover, recent studies have shownincreased lipoxidative damage, as revealed with malon-dialdehyde-lysine (MDA-Lys) and 4-hydroxynonenal-lysine markers, in the frontal cortex at early stages of PD(Dalfo et al., 2005). Proteomics have also revealed b-synuclein and SOD2 as targets of lipoxidative damage inthe frontal cortex in incidental PD (Dalfo et al., 2005).Further work has shown that a-synuclein is lipoxidizedin the frontal cortex in PD in the absence of Lewybodies and neurites, and that this modification is alsopresent in the frontal cortex in PD (Dalfo and Ferrer,2008).

In the present study, 4-hydroxynonenal (HNE), ana,b-unsaturated aldehyde produced during oxidation ofmembrane lipid polyunsaturated fatty acids, has beenused as a marker of lipoxidation in the frontal cortex ina large series of controls and in cases of Lewy body dis-ease, covering iPD, PD, and DLB. Results of the presentstudy further increase the number of proteins subject tooxidative damage in the frontal cortex PD. These resultsfurther help to increase understanding of possible bio-chemical substrates of cerebral cortex involvement in PDas revealed by different approaches including neuropsy-chological tests, neuroimaging, and positron emission to-mography scanning.

MATERIALS AND METHODS

Tissue Samples

Brain samples were obtained from the Institute ofNeuropathology Brain Bank following the guidelines of thelocal ethics committee. The causes of death in controls andpatients with disease were similar. Particular attention waspaid to exclude cases of sepsis, metabolic diseases (i.e., hepaticfailure, renal failure, acidotic diabetes), convulsions, fever, andprolonged agonal state. Because previous studies (Ferrer et al.,2008) have shown that the pattern of oxidized proteins ismodified with long postmortem delay, only samples withpostmortem delay between 2 and 12 hr were considered forthe present study. The pH of the brain at the time of studyranged between 6.8 and 7. Samples that exhibited no neuro-logical and neuropathological lesions were classified as con-trols. Cases of iPD were apparently nonneurologically affected,

and the neuropathological lesions were consistent with stages1 and 2 of Braak (Braak et al., 2003). Cases of clinically mani-fested PD and neuropathological lesions categorized as stages 3or 4 of Braak were classified as PD in the present study.Finally, cases of dementia and neocortical distribution of Lewybodies and neurites stages 5 and 6 of Braak were classified asDLB.

Alzheimer’s disease (AD) changes were categorized fol-lowing the nomenclature of Braak and Braak (1999). Amyloidplaques or tau deposits in the cerebral cortex, including basalregions (stage A of Braak and Braak for amyloid plaques) andentorhinal cortex (stages I and II of Braak and Braak for neu-rofibrillary pathology) were present in some cases. Sampleswith associated AD pathology stages IV–VI, B and C, werenot considered in the present study.

A summary of the main characteristics of iPD (cases 20–25; n 5 6), PD (cases 26–31; n 5 6), DLB (cases 32–37; n 56), and controls (cases 1–19; n 5 19) is provided in Table I.

Sample Preparation

Frozen samples (0.25 g) of the frontal cortex (area 8) iniPD, PD, DLB, and controls were homogenized separatelyin 7 volumes of ice-cold lysis buffer composed of 20 mMHEPES-KOH, 250 mM sucrose, 10 mM KCl, 1.5 mMMgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,50 mM sodium orthovanadate, and a tablet of protease inhibi-tors. Homogenates were first centrifuged at 770g for 30 minat 48C. Pellets were discarded, and the resulting supernatantswere centrifuged at 8,040g for 20 min at 48C. The pelletsobtained were resuspended in 300 lL of lysis buffer. Proteinconcentrations were determined by the Bradford method withbovine serum albumin as a standard. This protocol permittedan enrichment of mitochondria to permit a better visualizationof proteins linked with energy metabolism.

Gel Electrophoresis and Western Blot Test

Samples containing 30 lg of protein were loaded onto10% acrylamide gels. Proteins were separated in sodium do-decyl sulfate–polyacrylamide gel electrophoresis and electro-phoretically transferred to nitrocellulose membranes (200 mA/membrane, 90 min). Subsequently, the membranes werewashed with TBS (100 mM Tris-buffered saline, 140 mMNaCl, pH 7.4) and then incubated with a reducer solution of10 mM NaBH4 in TBS for 30 min. After washing in TBS,the membranes were blocked with a solution of 5% skim milkin 100 mM Tris-buffered saline, 140 mM NaCl, and 0.1%Tween-20, pH 7.4 (TBST buffer) for 1 hr at room tempera-ture. Then the membranes were incubated at 48C overnightwith the anti-HNE antibody (Calbiochem, Bionova, Madrid,Spain) used at a dilution of 1:1,000 in TBST containing 3%bovine serum albumin (Sigma, Barcelona, Spain). This wasfollowed by incubation with horseradish peroxidase–conju-gated secondary antibody (Dako) used at a dilution of 1:1,000,washing in TBST, and development with chemiluminescenceECL Hyperfilm (Amersham Biosciences).

Oxidation in Frontal Cortex in Lewy Body Diseases 1003

Journal of Neuroscience Research

2D Gel Electrophoresis and Western Blot Test

Samples (0.25 g) of the frontal cortex (area 8) of controland diseased cases were processed in parallel. A total of 200 lg ofprotein was mixed with 2D lysis buffer composed of 40 mM TrispH 7.5 containing 7 M urea, 2 M thiourea plus 0.2% Byolites(v/v), 4% CHAPS (Bio-Rad, Barcelona, Spain), 2 mM TBP,and 0.1% bromophenol blue in a final volume of 150 lL.

In the first dimension, sample solution was applied onimmobilized 7-cm IPG strips, pH 6–11, both linear gradient(Amersham Biosciences), at the basic and acidic ends of thestrip. After rehydration of the strips for 24 hr, proteins werefocused at 500 V for 1 hr, 1,000 V for 8 hr, 10,000 V for 4hr, and 50 V for 5 hr.

Strips were incubated in equilibration buffer composedof 50 mM Tris-HCl pH 6.8, 6 M urea, 1% sodium dodecylsulfate, 30% glycerol, and 2% dithiothreitol (Sigma, Madrid,Spain). A second equilibration step was performed incubating

strips for 10 min in equilibration buffer containing 2.5% iodo-acetamide (Bio-Rad). All strips were placed onto 10% poly-acrylamide gels and the second dimension gels were run at0.02 A per gel. For gel staining, a mass spectrometry–modifiedsilver staining method (Amersham Biosciences) was used asdescribed by the manufacturer. Control and diseased sampleswere run in parallel. In every case, one gel was stained silverwhile the other gel was transferred to a nitrocellulose mem-brane (200 mA/membrane for 90 min). The anti-HNE anti-body (Calbiochem) was used at a dilution of 1:1,000 in TBSTcontaining 3% bovine serum albumin. Antibodies to a-enolase(Abnova), aldolase A (Novus Biologicals), and GADPH (glyc-eraldehyde-3-phosphate dehydrogenase; Ambion) were usedat dilutions of 1:2,000, 1:1,000 and 1:1,000, respectively. Afterincubation with the primary antibody, the membranes werewashed for 5 min in TBST three times. The horseradish per-oxidase–linked secondary antibody (Dako) diluted 1:1,000 in5% skimmed milk in TBST was then added for 1 hr at roomtemperature. Membranes were washed and developed by ex-posure to ECL Hyperfilm (Amersham Biosciences).

Selection of Spots and In-gel Digestion

Parallel gel and membranes of every individual casewere scanned and pair images were superimposed and adapted

TABLE I. Summary of Cases*

Case Sex Age (y)

Postmortem

delay (hr)

Clinical

diagnosis Neuropathology

1 M 49 7 Control

2 F 49 6 Control

3 F 65 4 Control

4 M 54 3 Control

5 F 73 7 Control

6 F 66 8 Control

7 M 53 3 Control

8 M 55 5 Control

9 F 73 7 Control

10 M 52 4 Control

11 M 58 4 Control

12 M 79 7 Control

13 F 75 3 Control

14 M 67 5 Control

15 M 78 2 Control

16 M 55 6 Control

17 M 66 5 Control

18 M 61 3 Control

19 F 78 4 Control

20 F 54 10 iPD

21 M 72 8 iPD

22 M 66 8 iPD

23 F 77 3 iPD

24 F 70 10 iPD

25 M 78 10 iPD

26 M 88 2 PD PD

27 F 62 5 PD PD

28 M 78 5 PD PD

29 M 76 12 PD PD

30 M 81 5 PD PD

31 M 66 5 PD PD

32 M 60 8 Dementia DLB

33 M 85 7 Dementia DLB

34 M 67 3 AD DLB

35 M 80 3 AD DLB

36 F 77 5 DLB DLB

37 F 72 3 AD DLB

*iPD, incidental Parkinson’s disease; PD, Parkinson’s disease; DLB, de-

mentia with Lewy bodies; AD, Alzheimer’s disease.

Fig. 1. Gel electrophoresis and Western blot test of HNE of frontalcortex in control, iPD, PD, and DLB. Bands between 38 and 45kDa are detected in disease cases but not in these controls. b-Actin(46 kDa) is used as a marker of protein loading.

1004 Gomez and Ferrer


each other following predetermined marks in gels and mem-branes. Reference spots were checked in every case. Only gelsand membranes with net background and very visible silverspots were considered for study. Gels and membranes subopti-mally stained were discarded and corresponding samples proc-essed once again.

Proteins were in-gel digested with trypsin (sequencinggrade modified; Promega, Barcelona, Spain) in the automatic

Investigator ProGest robot of Genomic Solutions. Briefly,excised gels spots were washed sequentially with ammoniumbicarbonate buffer and acetonitrile. Proteins were reducedwith 10 mM dithiothreitol solution for 30 min and alkylatedwith 100 mM solution of iodine acetamide for 15 min. Aftersequential washings with buffer and acetonitrile, proteins weredigested overnight at 378C with trypsin 0.27 nM. Trypticpeptides were extracted from the gel matrix with 10% formic

Fig. 2. 2D gel electrophoresis and Western blot test of HNE of frontal cortex in control, iPD,PD, and DLB. A: Silver staining showing three parallel rows of stained spots, encircled in gels inone control. B: HNE antibody in membranes reveals three rows of spots: one of about 40 kDa(1–3), another of about 48 kDa (4 and 5), and the third of about 35 kDa (6 and 7) in Lewy bodydiseases.

TABLE II. HNE-modified Proteins in the Frontal Cortex

Site* Protein Molecular weight pI MOWSE score Peptides matched ID number

1 Aldolase A 39,706 8.34 139 2 gi 28614

2 Aldolase A 39,851 8.30 553 12 gi 4557305

3 Aldolase A 39,706 8.34 412 13 gi 28614

4 Enolase 1 47,481 7.01 157 4 gi 4503571

5 Enolase 1 47,481 7.01 98 3 gi 4503571

6 GADPH 36,201 8.57 116 7 gi 7669492

7 GADPH 36,201 8.57 134 6 gi 7669492

8 Aldolase A 39,851 8.30 439 15 gi 4557305

9 Aldolase A 39,851 8.30 146 5 gi 4557305

10 Aldolase A 39,851 8.30 193 7 gi 4557305

11 GADPH 36,202 8.26 502 10 gi 31645

12 GADPH 36,202 8.26 267 6 gi 31645

*Numbers indicate arbitrary assignation of spots in different gels.



acid and acetonitrile. The extracts were pooled and dried in avacuum centrifuge.

Acquisition of Mass Spectrometry and MS/MS Spectra

Proteins manually excised from the 2D gels weredigested and analyzed by CapLC-nano-ESI-MS-MS massspectrometry. The tryptic digested peptide samples were ana-lyzed by online liquid chromatography (CapLC, Micromass-Waters, Manchester, UK) coupled with tandem mass spec-trometry (Q-TOF Global, Micromass-Waters). Samples wereresuspended in 12 lL of 10% formic acid solution, and 4 lLwas injected for chromatographic separation into a reverse-phase capillary C18 column (75 lm internal diameter and 15cm in length, PepMap column, LC Packings, Amsterdam,The Netherlands). The eluted peptides were ionized viacoated nano-ES needles (PicoTip., New Objective, Woburn,MA). A capillary voltage of 1,800–2,200 V was applied to-gether with a cone voltage of 80 V. The collision in the colli-sion-induced dissociation was 25–35 eV and argon was usedas the collision gas. Data were generated in PKL file formatand submitted for database searching in the MASCOT server

(Matrix Science, Boston, MA). The NCBI database was usedwith the following parameters: trypsin enzyme, one missedcleavage, carbamidomethyl (C) as fixed modification and oxi-dized (M) as variable modification, and mass tolerance of 150–250 ppm.

A probability-based MOWSE score was used to deter-mine the level of confidence in the identification of specificisoforms from the mass spectra. This probability equals10(MOWSE score/10). MOWSE scores of >50 were consideredto indicate a high confidence of identification. All the experi-ments were performed in triplicate.

Statistical Analysis

Results were analyzed with v2 test. Values consideredsignificant were those reaching 95% of significance.

Densitometric Studies and Data Analysis

Membranes were scanned and saved in TIFF format byHPLaserJet 3052. The densitometric quantification of selectedWestern blot spots was carried out with the TotalLab v2.01software. Values were obtained for every spot resulting from

Fig. 3. 2D gels immunostained with anti–aldolase A antibodies. Rows of about 40 kDa, represent-ing aldolase A, are seen in controls and cases of disease (iPD, PD, and DLB).



the ratio between HNE densitometry and densitometry of thespecific protein (i.e., HNE/aldolase) in control and diseasedsamples. Samples were processed in parallel. Microsoft Excelsoftware was used for statistical analysis.

RESULTS

Monodimensional Gel Electrophoresis andWestern Blot Testing of HNE

Several HNE-immunoreactive bands were found inthe frontal cortex in control and diseased samples. Im-munoreactive bands between 38 and 45 kDa of molecu-lar mass were present in iPD, PD and DLB samples (Fig. 1).

2D Gel Electrophoresis, Western Blot Test,and Identification of Oxidized Proteins

IPG strips at pH ranging from 6 to 11 immuno-blotted with anti-HNE antibody showed oxidized spotsbetween 38 and 45 kDa in iPD, PD, and LBD (Fig. 2).Parallel silver-stained gels were used to identify the oxi-dized spots obtained by HNE Western blot test (Fig. 2).

Several spots were observed in control and diseasedcases. We selected three rows of spots because theyappeared most constantly and permitted quantificationand statistical processing. In-gel digestion and mass spec-trometry allowed identification of three oxidized pro-teins: aldolase A, a-enolase, and glyceraldehyde dehy-drogenase (GAPDH) (Table II).

2D gels run in IPG strips at pH ranging from 6 to11 immunoblotted with anti–aldolase A antibodies dem-onstrated the localization of aldolase A at the same posi-tion as the corresponding oxidized spots (Fig. 3). A simi-lar approach was carried out to show the localization ofenolase and GAPDH in IPG strips. Anti-a-enolase (Fig.4) and anti-GAPDH (Fig. 5) antibodies recognized thecorresponding spots in diseased cases.

Distribution of Oxidized Proteins in Controland Diseased Cases

Similar studies were carried out in a large seriesof control samples. The number of labeled spots was

Fig. 4. 2D gels immunostained with anti-enolase antibodies. Rows of about 45 kDa, representinga-enolase, are seen in controls and cases of disease (iPD, PD, and DLB).



variable from one case to another; Figure 6 is a represen-tative composite image of this variability. Identificationand validation of oxidized proteins was done as in path-ological cases (Table II).

A list of oxidized proteins in individual controlsand in iPD, PD, and DLB cases is shown in Table III.Oxidation of aldolase A and a-enolase was common incontrols, accounting for 12 of 19. GAPDH was oxidizedin 6 of 19 controls. These modifications were presentequally in diseased samples taken from patients aged 49–58 years (n 5 8) and in patients aged 61–78 years (n 510). The v2 test did not show significant differencesbetween younger vs. older controls. Aldolase A, a-eno-lase, and GAPDH were oxidized in practically all casesof PD and DLB, and in the majority of cases of iPD.Differences between control and diseased samples weresignificant only for GAPDH in the frontal cortex in PDand DLB at a level of significance of 95% (v2 test, val-ues: 6.02 and 9.56, respectively). Oxidation of GAPDHin the frontal cortex ion iPD reached a level of signifi-

cance of 90% when compared with controls. Finally, ox-idation of aldolase A in PD and DLB when comparedwith controls reached a significance of 90% (v2 values3.53 in both instances).

Densitometric Studies of Individual Spots

As seen in Figure 7, the ratio of oxidized proteinwas higher in diseased samples compared with controls,although with variable extent depending on the spots.This applies to aldolase, enolase, and GAPDH, thus indi-cating that the amount of oxidized protein of positivecases is higher in iPD, PD, and DLB compared withpositive spots in controls. Interestingly, values in DLBwere lower than in PD, thus suggesting higher ratio ofprotein oxidation in PD than in DLB.

DISCUSSION

Studies in the postmortem brain may be hamperedby artifacts related with several factors, including agonal

Fig. 5. 2D gels immunostained with anti-GAPDH antibodies. Rows of about 35 kDa, represent-ing GAPDH, are seen in controls and cases of disease (iPD, PD, and DLB).



state and postmortem delay and processing of the tissue.These points have been extensively analyzed in the con-text of the European Brain Net Consortium (Brain NetII), and several articles have been appeared focused onthe limitations, as well as the optimization, of postmor-tem brain tissue for protein study (Santpere et al., 2006;Ferrer et al., 2007). Recently, certain aspects have beenreviewed in detail that show, for relevance in the presentcontext, that a postmortem delay of <20 hr is accompa-nied by no increased or decreased oxidation and nitra-tion levels of proteins (Ferrer et al., 2008). The presentstudy was carried out within the strict limitations obligedby our previous studies in the context of Brain Net II.

The three proteins identified as targets of oxidationin the frontal cortex—a-enolase, aldolase A, andGAPDH—are enzymes related with glycolysis andenergy metabolism. a-Enolase (ENO1) is one of thesubunits composing the enzyme enolase, which inter-converts 2-phosphoglycerate and phosphoenolpyruvatein glycolysis (Keller et al., 1994). Enolase is present inmitochondria and contributes to mitochondrial function

(Poon et al., 2006). Aldolase A is a glycolytic enzymecatalyzing a reaction that converts fructose 1,6 biphos-phate to glyceraldehyde 3 phosphate and dihydroxyace-tone phosphate (Buono et al., 2001). GAPDH isinvolved in endocytosis and membrane fusion, vesicularsecretory transport and translation control, nucleartRNA transport, DNA replication, and DNA repair(Sirover et al., 2005). GAPDH is present as a tetramer inthe cytoplasm where the enzyme carries out its glyco-lytic activity. Yet the uracyl glycosylase activity ofGAPDH is associated with the GAPDH monomer inthe nucleus (Mazzola and Sirover, 2005). Interestingly,GADPH is involved in the formation of Lewy bodies(Tsuchiya et al., 2005; Olah et al., 2006).

Enolase oxidation has been found in the cerebralcortex and hippocampus in patients with mild cognitiveimpairment, and in advanced AD and related experi-mental models in association with b-amyloid deposition(Castegna et al., 2002; Boyd-Kimball et al., 2005b; But-terfield et al., 2006a, 2006b; Sultana et al., 2006). a-Enolase oxidation has recently been reported in a-synu-

Fig. 6. Representative membranes immunoblotted with anti-HNE antibodies in three controlsshowing variable presence of immunoreactive spots corresponding to enolase, aldolase, andGAPDH. Numbers represent the same spots as in Figure 2.



clein transgenic mice used as a model of PD (Poonet al., 2005). GAPDH oxidation has been observed in thecerebral cortex in AD (Boyd-Kimball et al., 2005a) and af-ter traumatic brain injury (Opii et al., 2007) Lipoxidationof aldolase A has been described in the frontal cortex inprogressive supranuclear palsy (Martınez et al., 2008).

A crucial point derived from the present study incontrol and diseased brains is that oxidation of aldolaseA, a-enolase, and GAPDH may occur in the frontalcortex in certain control individuals. Oxidation of aldol-ase A and enolase 1 is not uncommon, accounting for12 of 19 cases. Lipoxidation of GAPDH was found in 6of 19 cases. Although age is probably a risk factor, nosignificant differences (v2 test) have been observed

between individuals aged 49–58 years and those aged61–78 years. Therefore, age is not the unique factorassociated with aldolase A, a-enolase and GAPDH oxi-dation in control brains.

The present study has also shown that a-enolase,aldolase A, and GAPDH are oxidized in the frontal cor-tex in iPD, PD, and DLB, thus indicating oxidation ofthese proteins throughout the various stages of PD-likepathology. Significant differences of 95% (v2 test)between control and diseased samples were only seen forGAPDH in PD and DLB when compared with controls.However, significant differences at the level of 50%were also observed for GAPDH in iPD when compared

Fig. 7. Graphs representing the ratio of HNE and the specific pro-tein (aldolase A, enolase, or GAPDH) in the different spots in con-trol, iPD, PD, and DLB. The amount of oxidized protein is higherin diseased cases when compared with controls. Interestingly, theoxidation ratio is higher in PD than in DLB.

TABLE III. Oxidized Proteins in the Frontal Cortex in Controls

and Cases of Disease*

Case No. NP Aldolase A Enolase 1 GADPH Age (y)

1 C 2 2 2 49

2 C 1 1 2 49

3 C 2 2 2 65

4 C 1 2 2 54

5 C 1 1 1 73

6 C 1 2 2 63

7 C 1 1 1 53

8 C 1 1 1 55

9 C 1 1 1 73

10 C 1 1 2 52

11 C 2 1 2 58

12 C 1 1 1 79

13 C 2 2 2 75

14 C 2 2 2 67

15 C 1 1 2 78

16 C 1 1 1 55

17 C 1 1 2 66

18 C 2 1 2 61

19 C 2 2 2 78

20 iPD 2 2 2 54

21 iPD 1 1 2 72

22 iPD 1 1 1 66

23 iPD 1 1 1 77

24 iPD 1 1 1 70

25 iPD 1 1 1 78

26 PD 1 1 1 88

27 PD 1 1 1 62

28 PD 1 1 1 78

29 PD 1 1 1 76

30 PD 1 1 1 81

31 PD 1 1 2 66

32 DLB 1 1 1 60

33 DLB 1 1 1 85

34 DLB 1 1 1 67

35 DLB 1 1 1 80

36 DLB 1 1 1 77

37 DLB 1 1 1 62

*NP, neuropathological diagnosis; 1 and 2 indicate, respectively, the

presence or absence of oxidative damage of the corresponding protein.

Although the mean age of controls is lower than that of patients with dis-

ease, note that control patients 7, 8, and 16 (all of them aged about 50

years) bear oxidative damage in aldolase A, enolase 1, and GADPH.



with controls. Therefore, increased oxidation ofGAPDH in the frontal cortex in PD occurs in the ab-sence of Lewy pathology in this region, as well as inDLB with accompanying Lewy pathology. Oxidation ofaldolase A in PD and DLB compared with controlsreached a significance of 90% (v2 values 3.53 in bothinstances), thus indicating a trend following the limits ofour restricted conditions.

Densitometric studies of specific spots directed tolearn the ratio of oxidized protein in a given spot dis-closed higher values in iPD, PD, and DLB comparedwith positive spots in controls. That means that not onlyis there an increase in the number of positive (oxidized)spots in Lewy body diseases, but also that the amount ofoxidized protein is higher in cases of disease comparedwith controls.

Yet the present observations support the conceptthat several metabolic pathways are damaged by oxida-tion in the frontal cortex in normal aging, and that thisdamage is increased in Lewy body diseases, even inregions with no Lewy pathology. These results indicatesubtle differences between control and diseased brains,and they suggest that brain dysfunction and derived clin-ical manifestations, once a certain individual threshold isreached, are probably not only related to classic patho-logical events, but also to disturbed basal metabolic path-ways. Along these lines, it is worth stressing that PD isno longer considered as a neurodegenerative disease withmotor-only involvement. Cognitive changes and abnor-mal behavior are well documented even at early stagesof the disease (Levin and Katzen, 2005; Caballol et al.,2007; McNamara et al., 2007). Impaired frontal tasks inPD are independent of the presence or absence ofdepression (Silberman et al., 2007). Interestingly, meta-bolic changes, as seen by positron emission tomographyscanning, have been observed in the frontal and parietalcortex in PD, often accompanied by little changes inbehavior, and appearing at very early stages of the disease(Huang et al., 2007, 2008). Furthermore, neuroimaginghas revealed early morphological modifications in thesame cortical regions in PD cases (Tessa et al., 2008),and diffusion tensor imaging has shown decreased frac-tional anisotropy in subjects with PD bilaterally in thefrontal lobes without volume loss (Karagulle Kendiet al., 2008). These findings confirm that neurodegener-ative processes extend beyond the basal ganglia in PD.

Taken together, the present metabolic deficits areconsistent with previous clinical, neuroradiological, andfunctional observations demonstrating abnormal cerebralcortex in PD. More important, they offer informationabout a part of the biochemical substrates probably dam-aged in the frontal cortex at early stages of PD. Identifi-cation of impaired metabolic pathways will permit amore rational therapeutic approach of cognitive impair-ment and behavioral disturbances in PD.

ACKNOWLEDGMENTS

This work was funded by the PI05/1570 grantfrom the Spanish Ministry of Health, Instituto de Salud

Carlos III, and supported by the European Commissionunder the Sixth Framework Programme (BrainNetEurope II, LSHM-CT-2004-503039; and INDABIP).We thank E. Dalfo for help at the beginning of thework, S. Boluda for tissue sampling, and T. Yohannanfor editorial assistance. Brain samples were obtained fromthe Institute of Neuropathology following the guidelinesand approval of the local ethics committees.

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