Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

Research ArticleRedox Imbalance and Morphological Changes inSkin Fibroblasts in Typical Rett Syndrome

Cinzia Signorini1 Silvia Leoncini12 Claudio De Felice3 Alessandra Pecorelli12

Ilaria Meloni4 Francesca Ariani4 Francesca Mari4 Sonia Amabile4

Eugenio Paccagnini5 Mariangela Gentile5 Giuseppe Belmonte6 Gloria Zollo12

Giuseppe Valacchi7 Thierry Durand8 Jean-Marie Galano8 Lucia Ciccoli1

Alessandra Renieri49 and Joussef Hayek2

1 Department of Molecular and Developmental Medicine University of Siena 53100 Siena Italy2 Child Neuropsychiatry Unit University Hospital Azienda Ospedaliera Universitaria Senese (AOUS) 53100 Siena Italy3 Neonatal Intensive Care Unit University Hospital AOUS Policlinico ldquoS M alle Scotterdquo 53100 Siena Italy4Medical Genetics University of Siena 53100 Siena Italy5 Department of Life Sciences University of Siena 53100 Siena Italy6Department of Medicine Surgery and Neuroscience University of Siena 53100 Siena Italy7 Department of Life Science and Biotechnologies University of Ferrara 44121 Ferrara Italy8 Institut des Biomolecules Max Mousseron (IBMM) UMR 5247-CNRS-UM I-UM II-ENSCM BP 14491 34093Montpellier Cedex 5 France

9Genetica Medica Azienda Ospedaliera Universitaria Senese 53100 Siena Italy

Correspondence should be addressed to Cinzia Signorini cinziasignoriniunisiit and Claudio De Felice genientegmailcom

Received 10 March 2014 Revised 9 May 2014 Accepted 12 May 2014 Published 29 May 2014

Academic Editor Ozcan Erel

Copyright copy 2014 Cinzia Signorini et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT) a neurodevelopmental disordermainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2 Little is known regarding the redox statusin RTT cellular systems and its relationship with the morphological phenotype In RTT patients (n = 16) we investigated fourdifferent oxidative stress markers F

2-Isoprostanes (F

2-IsoPs) F

4-Neuroprostanes (F

4-NeuroPs) nonprotein bound iron (NPBI)

and (4-HNE PAs) and glutathione in one of the most accessible cells that is skin fibroblasts and searched for possible changes incellularintracellular structure and qualitative modifications of synthesized collagen Significantly increased F

4-NeuroPs (12-folds)

F2-IsoPs (75-folds) NPBI (23-folds) 4-HNE PAs (148-folds) and GSSG (144-folds) were detected with significantly decreased

GSH (minus436) andGSHGSSG ratio (minus305 folds) Amarked dilation of the rough endoplasmic reticulum cisternae associatedwithseveral cytoplasmic multilamellar bodies was detectable in RTT fibroblasts Colocalization of collagen I and collagen III as well asthe percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses appears to be significantlyreduced in RTT cells Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skinfibroblast abnormalities in RTT patients

1 Introduction

Rett syndrome (RTT) predominantly affects females with anincidence of 1 in 10000ndash15000 female births [1 2] In itstypical form affected patients exhibit various neuropsychi-atric features after 6ndash18months [3] of apparently normal neu-rodevelopment Although phenotypical heterogeneity which

determines the clinical severity of the disease is a typicalfeature of RTT [4] key clinical aspects include autistictraits epileptic seizures breathing abnormalities gait ataxiastereotypies and loss of finalistic hands use [3] Mutationsin the gene encoding the Methyl-CpG-binding protein 2(MECP2) account for approximately 90of caseswith typicalRTT and are almost exclusively de novo [4] Nine most

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 195935 10 pageshttpdxdoiorg1011552014195935

2 Oxidative Medicine and Cellular Longevity

frequent mutations comprise more than 34 of all thereported pathogenic ones [5] in additionMECP2 genemuta-tions are usually categorized as missense or truncatingincluding nonsense frameshift and large deletions accord-ing to the type of sequence change

Oxidative stress (OS) indicates a combination of eventsresulting in a damage to biological molecules due to animbalance between cellular antioxidant defences and freeradicals production OS appears to be involved in a largenumber of human diseases including cancer [6ndash8] neurode-generative diseases [9 10] and inflammatory bowel disease[11] Furthermore cumulating evidence suggests the presenceof a redox imbalance in autism spectrum disorders (ASDs)a condition with a high social impact due to the explosiveincrease in its prevalence over the last four decades [12ndash18]Our group and other laboratories have reported enhancedOSmarkers levels in plasma and erythrocytes from patients withRTT thus suggesting the presence of a systemic OS in thedisease However to date it is unclear not only why but alsowhen and where this OS derangement may occur [19ndash26]

In particular to date little is known regarding the oxidant-antioxidant status in RTT cellular systems and its possiblerelationship with the morphological phenotype Here weinvestigated the levels of different OS markers (F

2-IsoPs F

4-

NeuroPs NPBI and 4-HNE PAs) and glutathione in primaryskin fibroblasts from patients with typical RTT as well aspossible changes in the cellularintracellular structure andorqualitative changes in the synthesized collagen

2 Materials and Methods

21 Subjects A total of 16 female patients with typical RTT(mean age 1306 plusmn 65) as well as 8 healthy female controlsof comparable age (mean age 132 plusmn 68) participated in thestudy Skin biopsies from the control group were obtainedfrom a skin biobank by selecting subjects without diagnosedskin or collagen diseases (responsibility Joussef Hayek)

All patients were consecutively admitted to the ChildNeuropsychiatry Unit of the University Hospital of Siena(Head Joussef Hayek) For all patients theMECP2mutationwas demonstrated (mutation categories missense mutation119899 = 8 early truncating mutation 119899 = 6 late truncatingmutation 119899 = 2) and a clinical severity score was calculatedusing the previously reported scaling system [27] (meanseverity score 159 plusmn 647 range 50ndash280) All the examinedsubjects were on a typical Mediterranean diet

A 3mm skin punch biopsy was performed after obtainedwritten informed consent of either the parents or the legaltutors of the patients (responsibility Joussef Hayek) Biopsiesfrom control subject were performed in the DermatologyUnit of the University Hospital of Siena The study was con-ducted with the approval of the Institutional Review Boardof the University Hospital Azienda Ospedaliera UniversitariaSenese

22 Fibroblasts Isolation and Culture Following informedconsent signature skin biopsies (about 3-4mm3) were per-formed using the Punch Biopsy procedure Fibroblasts were

isolated and cultured with standard protocols [28] Cells weregrown in DMEM (Biochrom) supplemented with 10 fetalbovine serum 100UmL penicillin 100 120583gmL streptomycinand 2mM L-glutamine Cells were incubated at 37∘C with5 CO

2until 80ndash90 confluence and routinely passed by

trypsin-EDTA (Irvine Scientific)Cells for analysis were seeded onto 100mm tissue culture

plates (for NPBI and IsoP evaluations) and 12-well plates(for 4-HNE PAs and GSHGSSG ratio) containing the indi-cated medium formulations Fibroblasts at low passage wereemployed for the analysis

At confluence the cells were scraped transferred in atube and washed twice with ice-cold PBS pH 74 Cellswere then centrifuged at 600 g for 10min and the pelletresuspended in a final volume of 2mL PBS

23 Nonprotein Bound Iron (NPBI) Nonprotein bound ironwas determined as desferoxamine- (DFO-) chelatable freeiron (DFO-iron complex ferrioxamine) DFO 25120583M wasadded to 1mL of cell suspension to obtain a final concen-tration of 25120583M DFO and the cells were ruptured by theaddition of 1mL water freeze-thawing and sonication Thesamples were then ultrafiltered in centrifuge filter devices(VIVASPIN 4 Sartorius Stedim Biotech GmbH GoettingenGermany) with a 30 kDa molecular weight cutoff and theultrafiltrate was stored at minus20∘C until analysis The DFOexcess was removed by silica (Silicagel 25ndash40120583m MerckDarmstadt Germany) column chromatography (Varian IncCA USA)TheDFO-iron complex was determined by HPLCand the detection wavelength was 229 nm [29]

24 Total (Sum of Free Plus Esterified) F2-Isoprostanes and F

4-

Neuroprostanes Determinations Butylated hydroxytoluenewas added to 900120583L of cell suspension as an antioxidantto obtain a final concentration of 90 120583M and the sampleswere frozen at minus70∘C until analysis All isoprostane andneuroprostane determinations were carried out by gas chro-matographynegative ion chemical ionization tandem massspectrometry (GCNICI-MSMS) analysis after solid phaseextraction and derivatization steps F

2-Isoprostanes and F

4-

neuroprostanes levels were normalized for the cell proteincontent

25 Solid Phase Extraction and Derivatization Procedures Atthe time of the determination the samples were sonicated byultrasound treatment for 30 seconds and then purified as pre-viously reported [29] Briefly aqueous KOH (1mM 450120583L)was added to the cellular suspension After incubation at45∘C for 45min the pH was adjusted to 3 by adding HCl(1mM 450 120583L) Each sample was spiked with tetradeuteratedprostaglandin F

2120572(PGF2120572-d4) (500 pg in 50120583L of ethanol)

as an internal standard and ethyl acetate (10mL) was addedto extract total lipids by vortex-mixing and centrifugation at1000 g for 5min at room temperature The total lipid extractwas applied onto an NH

2cartridge and the isoprostanes

in the final eluates were derivatized The carboxylic groupwas derivatized as the pentafluorobenzil ester whereas thehydroxyl groups were converted to trimethylsilyl ethers [30]

Oxidative Medicine and Cellular Longevity 3

26 F2-Isoprostane GCNICI-MSMS Measured ions were

the product ions at mz 299 and mz 303 derived fromthe [Mminus181]minus precursor ions (mz 569 and mz 573) pro-duced from 15-F

2t-IsoPs and the tetradeuterated derivative ofprostaglandin F

2120572(PGF2120572-d4) respectively [29 30]

27 F4-NeuroPs GCNICI-MSMS Measured ions were the

product ions at mz 323 and mz 303 derived from the[Mminus181]minus precursor ions (mz 593 and mz 573) producedfrom oxidized DHA and the tetradeuterated derivative ofPGF2120572 respectively [29 31]

28 Intracellular Redox Status Usually cellular glutathione(GSH) exists mainly in the reduced form whereas the oxi-dized disulfide form (GSSG) is present in small amountsThe GSHGSSG ratio is often taken as an indicator ofcellular redox status Intracellular GSH and GSSG levels weredetermined by an enzymatic recycling procedure accordingto Tietze [32] and Baker et al [33] The SH group ofthe molecule reacts with 551015840-dithiobis(2-nitrobenzoic acid)(DTNB) producing a yellow-coloured 5-thio-2-nitrobenzoicacid (TNB) and the disulfide is reduced by NADPH inthe presence of GSH reductase GSSG was determined afterderivatization step of GSH by reaction with 2-vinylpyridineThe rate of TNB formation was monitored at 420 nm GSHand GSSG levels were normalized for protein content

At confluence aftermedium removal cultured fibroblastswere washed twice with PBS pH 74 and treated with 5 5-sulfosalicylic acid (wv) solution for 30min at 4∘CThe acidicextracts were stored at minus70∘C until the assay The proteinextracts were obtained by cellular lysis with NaOH 01M

29 4-HNE Protein Adducts 4-HNE PAs are markers ofprotein oxidation due to aldehyde binding from lipid perox-idation sources [34] determined by Western blot techniqueCell proteins (30 120583g protein as determined by using Bio-Radprotein assay BioRad Hercules CA USA) were resolved on4ndash20 SDS-PAGE gels (Lonza Group Ltd Switzerland) andtransferred onto a hybond ECL nitrocellulosemembrane (GEHealthcare EuropeGmbHMilan Italy) After blocking in 3nonfat milk (BioRad Hercules CA USA) the membraneswere incubated overnight at 4∘C with goat polyclonal anti 4-HNE adduct antibody (cod AB5605 Millipore CorporationBillerica MA USA) Following washes in TBS Tween andincubation with specific secondary antibody (mouse anti-goat horseradish peroxidase-conjugated Santa Cruz Biotech-nology Inc CA USA) for 1 h at room temperature themembranes were incubated with ECL reagents (BioRadHercules CA USA) for 1min The bands were visualized byautoradiography Quantification of the relevant bands wasperformed by digitally scanning the Amersham HyperfilmECL (GE Healthcare Europe GmbH Milan Italy) and mea-suring immunoblotting image densitieswith ImageJ software

210 Transmission Electron Microscopy (TEM) Culturedfibroblasts were fixed in 25 glutaraldehyde in 01M cacody-late buffer pH 72 (CB) for 3 hours at 4∘C After a rinse inCB the material was postfixed in 1 osmium tetroxide in CB

for 2 hours at 4∘C dehydrated in a graded series of alcoholembedded in Araldite resins and polymerized in oven for48 hours at 60∘C Sixty nm thin sections obtained with aReichert ultramicrotome were routinely stained with uranylacetate and lead citrate and observed with a TEM Fei TecnaiG2 spirit at 100Kv

211 Immunofluorescence Double Staining The localizationof two main type of collagen synthetized by skin fibroblastswas evaluated by an immunofluorescence assay Primaryhuman fibroblasts grown on glass coverslips at a densityof 2 times 104 cellscm2 were fixed in 4 paraformaldehydefor 10min at 4∘C After fixation and permeabilization for10min at room temperature with 01 Triton X-100 cellswere blocked for 30min at room temperature with PBScontaining 5 BSA Fibroblasts were then incubated withprimary antibodies for collagen I and collagen III (ThermoFisher Scientific Inc Rockford IL USA) in PBS with 1BSA at 4∘C overnight After washing cells were incubatedwith secondary antibodies Alexa Fluor 568 and Alexa Fluor488 (Life Technologies Corporation Monza Italy) for 1 h atroom temperature Nuclei were stained with 1 120583gmL DAPI(Sigma-Aldrich Srl Milan Italy) for 1min Coverslips weremounted onto glass slides using antifade mounting medium14-diazabicyclooctane in glycerine (DABCO) Observationsand photographs were made with a Leitz Aristoplan lightmicroscope (Leica Milan Italy) equipped with fluorescenceapparatus Incubation in primary antibodies was omitted innegative controls

The relative intensity of fluorescence was measured inregions of interest (ROI) by using the Software LEICAAF6000 (Leica Microsystems-Germany)

212 Statistical Analysis Results were expressed as medianswith interquartile ranges or means plusmn standard deviationDifferences between groups were evaluated by the non-parametric Mann-Whitney rank sum test Wilcoxon ranktest or Kruskal-Wallis test analysis of variance (ANOVA)as appropriate The MedCalc ver 1214 statistical softwarepackage (MedCalc Software Mariakerke Belgium) was usedfor data analysis Two-sided 119875 values lt005 were consideredas significant

3 Results

31 Cell Oxidant and Antioxidant Status Biochemical signsof lipid and protein oxidative damage together with theantioxidant cellular defense were evaluated in skin fibroblastsfrom RTT and healthy control subjects Increased lipidoxidative damage was evidenced as indicated by increasedlevels of total (ie sum of free and esterified form) F

2-IsoPs

(75-folds) and F4-NeuroPs (12-folds) both deriving from

membrane polyunsaturated fatty acids that is arachidonic(AA) and docosahexaenoic (DHA) acid respectively (Figures1(a) and 1(b)) Biochemical signs of protein oxidative damageconsequent to a lipid peroxidation event were indicatedby increased (148-folds) 4-HNE PAs levels for which asignificant increase was also detected (Figure 1(c)) Oxidative


0

200

400

600

800

1000(p

gm

g pr

otei

n)

Healthy controls Typical RTT

Tota

lF2-I

soPs

lowast

(a)

0

200

400

600

800

1000

(pg

mg

prot

ein)

Tota

lF4-N

euro

Ps

lowast


(b)

0

5

10

15

20

25

4-H

NE

PAs (times106

au)

lowastlowast


(c)

0

02

04

06

08

1

NPB

I (nm

olm

g pr

otei

n)

lowast


(d)

Figure 1 Increased levels of total (ie sum of free and esterified form) F2-IsoPs total F

4-NeuroPs 4-HNE PAs and NPBI in RTT skin fibrob-

last as compared to the control cells lowast119875

lt 00001 lowastlowast119875

= 00013 Data are expressed as means plusmn standard deviation Legend F2-IsoPs

F2-isoprostanes F

4-NeuroPs F

4-neuroprostanes 4-HNE PAs 4-hydroxy-2-nonenal protein adducts NPBI nonprotein bound iron

0

04

08

12

16


Redu

ced

GSH

(n

mol

mg

prot

ein)

lowast

(a)

0

04

08

12

16

GSS

G

lowastlowast

(nm

olm

g pr

otei

n)


(b)

Figure 2 Significant reduction in cellular GSH and significant increase of GSSG in RTT skin fibroblast as compared to control cells lowast119875

lt

00001 lowastlowast119875

= 00033 Data are expressed as means plusmn standard deviation Legend GSH reduced glutathione GSSG oxidized glutathione

damage was concomitant to an imbalance in the principalantioxidant cytoplasmic agent in so far as a significantreduction (minus436) in cellular GSH a significant increase(144-folds) of GSSG (Figures 2(a) and 2(b)) and a significantreduction (minus305 folds) of GSHGSSG ratio were reportedLipid peroxidation events in RTT skin fibroblasts were foundto be related to the levels of NPBI a prooxidant agentNPBI was significantly increased (23-folds) (Figure 1(d))with significant positive correlations observed between NPBI

and total cellular F4-NeuroPs (Rho = 084 119875 = 0001) and

NPBI versus total cellular F2-IsoPs (Rho = 069 119875 = 0019)

No significant relationships between each of the investigatedmolecules (ie F

2-IsoPs F

4-NeuroPs 4-HNE Pas GSH and

GSSG) and theMECP2mutation categories were observed (119875value range 0461ndash0981)

32 Cell Morphology Study To evaluate the effect if presentof the oxidantantioxidant imbalance evidenced in RTT


M

RER

G

V

1120583m

(a)

RER

V

MLB

1120583m

(b)

Figure 3 Transmission electron microscopy of control (a) and RTT (b) fibroblasts cultures Skin fibroblasts either from control subjectsor RTT patients show a flattened morphology with extensive tapering cytoplasmic processes An euchromatic and oval-shaped nucleuswas present in central position of the cells with clumps of heterochromatin next to the nuclear envelope The cytoplasm contains manyvesicles with variable electron density a prominent Golgi complex and mitochondria Rough endoplasmic reticulum (RER) cisternae inRTT fibroblasts appear more dilated than in control Some large multilamellar bodies (MLB) are frequently detectable in the cytoplasm ofthe RTT fibroblast cells (G) Golgi complex (M) mitochondrion and (V) vesicle Bar = 1120583m

Hea

lthy

cont

rols

Typi

cal R

TT

Col III

Col III

Col I

Col I

Figure 4 Double immunofluorescence staining shows the localization of type I collagen (central column red color) and type III collagen(left column green color) Images are merged in the right panel and the yellow color indicates overlap of the staining The colocalization oftypes I and III collagen is reduced in RTT skin fibroblasts Legend Col I type I collagen Col III type III collagen

fibroblasts the physiological cellular condition was evaluatedby observation of two key typical features the morphologyand the collagen distribution

At transmission electron microscope significant ultra-structural differences between control and RTT fibroblastswere observed In particular a marked dilation of the roughendoplasmic reticulum cisternae was detectable in the skinRTT fibroblasts along with evidence of cytoplasmic multil-amellar bodies (Figures 3(a) and 3(b)) Immunofluorescencedouble staining showed a reduced degree of colocalizationof type III and type I collagen in RTT skin fibroblasts whencompared to control cells Staining for type I collagen wasfound to be more evident in RTT cells where perinuclear

granules were also detectable (Figure 4) Likewise in thesesame cells fluorescence relative intensity for type I collagenwas significantly reduced (119875 = 000997) (Figure 5)

4 Discussion

Our findings demonstrate for the first time the presenceof an extensive redox imbalance in primary skin fibroblastscultures obtained from patients with RTT by adding newevidence to the concept of an OS imbalance as key pheno-typical features of MeCP2 deficiency in RTT Earlier cluesfor an abnormal OS balance in RTT patients harbouringMECP2 gene mutations are to date limited to plasma and


0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast


Figure 5 Relative intensity of fluorescence for types I and typeIII collagen in RTT and control skin fibroblasts Software LEICAAF6000 (Leica Microsystems-Germany) Data are expressed asmedian plusmn semiinterquartile range lowast

119875

= 00062 NS no significantdifference (119875 = 04361) Legend Col I type I collagen Col III typeIII collagen

erythrocytes samples [26 35] Although enhanced plasmaticOS markers levels suggest a systemic OS status to date noindications are present in order to infer what tissuescellularsystems other than blood could be potentially damaged bythe OS imbalance in RTT A preliminary suggestion that anoxidative process should occur at the cellular level was con-tained in our recent study where oxidative posttranslationalmodifications on SRB1 receptor in primary fibroblast cultureswere observed [36] Fibroblasts have been used in the RTTscientific research to evaluate gene mutation and epigeneticprocess [37] differentiation to induced pluripotent stem (iPS)[38] cellular response subsequent to MECP2 mutation [39]reduction of stathmin-like 2 [40] and the effects of NB54and other rationally designed aminoglycoside derivatives aspotential therapeutic agents for nonsenseMECP2 mutationsin RTT [41] Fibroblasts have been extensively employedto investigate OS in different pathophysiological processessuch as genetic neurodegenerative diseases [42] Parkinsonrsquosdisease [43] aging [44] and response to etiological agents[45 46] Moreover lipid peroxidation events are known tobe relevant to the fibroblast function as relationship betweenmechanotransduction and lipid metabolites has been previ-ously investigated in keloids [47]

The biochemical markers so far employed for measuringOS are not to be considered as equal in terms of the con-veyed information In particular F

2-IsoPs the gold standard

molecules for the OS in vivo evaluation [35 48] are theend-products of arachidonic acid (AA) oxidation a polyun-saturated fatty acid abundant in both brain grey and whitematter On the other hand F

4-NeuroPs are the oxidative

end-products of docosahexanoic acid (DHA) abundant inneuronal membranes [49] NPBI is a prooxidant factor asso-ciated with hypoxia hemoglobin oxidation and subsequentheme iron release [50]

Our data demonstrate that both AA and DHA undergosignificant oxidation in RTT fibroblasts Although DHA isknown to be particularly abundant in neuronal membranes

this fatty acid represents a normal constituent of all cellmembranes In particular human fibroblasts are able tosynthetize DHA [51] and incorporate it in their membranephospholipids [52] Furthermore variations of the DHAcontent in the membrane of fibroblasts have been implicatedin neuropsychiatric disorders with lowered levels evidencedin patients with schizophrenia and bipolar disorder [53] Ourresults indicate an increased oxidation ofDHA inRTTfibrob-lasts thus underlying an increased susceptibility of these cellsto oxidative damage This process in turn could contributeto changes in the fatty acid composition of membranes withconsequent cellular damage

4HNE which is formed from arachidonic acid or otherunsaturated fatty acids following free radical attack can bindbyMichael addition to proteins particularly to cysteine his-tidine or lysine residuesThanks to its ability to form adductsto the proteins 4HNE is not only considered as a reliablemarker of OS but also has a biological impact by changingprotein function [34] Our findings of increased 4HNE-PAscan be considered as a long-term consequence of enhancedlipid peroxidation further contributing to cellular damage

Glutathione the main cellular antioxidant defence pre-venting the damage to key cellular components induced byreactive oxygen species exists in both reduced (GSH) andoxidized (GSSG) states In its reduced state the thiol group ofcysteine is able to donate a reducing equivalent (H+ + eminus) toother unstable molecules such as ROS whereas in donatingan electron glutathione itself becomes reactive but is ready toreact with another reactive glutathione to form GSSG

Our reported results of decreased GSH levels andGSHGSSG ratio indicate a reduced antioxidant defence inRTT cells and suggest that oxidative events are likely to bechronic thus determining a consumption of glutathione inits reduced form An alternative explanation could be that theMECP2mutation-harboring cellsmayhave coexisting defectsin the synthesis andor recycling of glutathione thus leadingto an uncontrolled free radicals action Of course furtherstudy is needed in order to clarify this point

Overall our data indicate that a significant oxidation ofDHA the precursor acid for F

4-NeuroPs and AA precursor

of F2-IsoPs likely triggered by NPBI occurs in skin fibrob-

lasts of patients withMECP2mutation and clinical RTT andconfirm the biological relevance of such keymediators of lipidperoxidation

Skin dermis is known to be consisting of 80 collagentype I with its remaining fraction being made mostly ofcollagen type III [54] In this context the fibroblast shows apivotal role for collagen production [55]

Besides being a major component of the extracellularmatrix in a variety of internal organs and skin in adults typeIII collagen is critical for fibrillogenesis in the developmentof apparatus such as skin and cardiovascular system [56 57]Overall in such tissues type III collagen is colocalized withinthe same fibril with themost abundantmember of the familytype I collagen and regulates the diameter of type I collagenfibrils which have to meet the physiological requirements ofdifferent tissues at different developmental stages [54 58ndash62]

The reported ultrastructural changes are to be consideredas a new finding to be added to the mitochondrial changes


previously reported in RTT skin fibroblasts [63] Since noabnormalities in wound repair andor healing have beenreported in RTT patients these ultrastructural features areto be considered as subclinical characteristics of the diseaseMarked dilation of the rough endoplasmic reticulum cister-nae and cytoplasmic multilamellar bodies were observed forthe first time in this study Dilated rough endoplasmic retic-ulum cisternae can be considered as a nonspecific adaptiveresponse due to either increased secretory activity or as theresult of the cellular response to noxae of different nature [6465] On the other hand the presence of multilamellar bodiescould be interpreted as an expression of cellular damageIn particular those structures suggest the occurrence ofautophagy phenomena of unclear pathogenesis [66]

A condition of oxidative stress has been previouslyreported in aging skin [67] Skin aging is known to be mainlydue to fragmentationloss of type I collagen fibrils conferringstrength and resiliency in association withmetalloproteinaseactivation involved in type I collagen degradation [68]Although no evidence of wound healing alterations or dermalaxity has not been reported in RTT an accelerated ageingprocess is known to occur in the affected patients Our resultssuggest the occurrence of a possible reduction in type Icollagen a feature of aging skin while they show a clear redoximbalance in RTT skin fibroblastsTherefore it is conceivablethat a premature skin aging may occur in RTT patients ahypothesis which is in line with prior evidence of prematuresenescence phenomena in the disease [69ndash72]

Given that OS is known to be a major determinant ofaging [73 74] it is conceivable that aging phenomena inRTT including skin fibroblasts morphological changes maybe caused through OS mechanisms

5 Conclusion

Our study demonstrates for the first time the occurrenceof lipid peroxidation in RTT fibroblasts together with areduced antioxidant cellular defense with a major impact oncell morphology We speculate about a possible functionalinvolvement of these changes in the skin fibroblasts of RTTpatients which must be taken into account when evaluatingthis cellular model of the disease Our findings suggest thatOS is a generalized phenomenon in RTT thus affectingcellular systems and tissues apparently unrelated to thecentral nervous system

Abbreviations

4-HNE 4-Hydroxy-2-nonenal4-HNE PAs 4-Hydroxy-2-nonenal protein adductsAA Arachidonic acidDHA Docosahexaenoic acidF2-IsoPs F

2-isoprostanes

F4-NeuroPs F

4-neuroprostanes

GSH Reduced glutathioneGSSG Oxidized glutathioneIsoPs IsoprostanesMECP2 Methyl-CpG-binding protein 2mdashhuman

gene

MeCP2 Methyl-CpG-binding protein 2mdashhumanprotein

NPBI Nonprotein bound ironOS Oxidative stressPUFAs Polyunsaturated fatty acidsROS Reactive Oxygen SpeciesRTT Rett syndromeTEM Transmission electron microscopy

Disclosure

Cofirsts Cinzia Signorini Silvia Leoncini Claudio De Feliceand Alessandra Pecorelli Colasts Lucia Ciccoli AlessandraRenieri and Joussef Hayek

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

The conception of the paper was performed by Cinzia Sig-norini Claudio De Felice Silvia Leoncini Lucia CiccoliAlessandra Renieri and Joussef Hayek The experimentaldesign was performed by Cinzia Signorini and Claudio DeFelice MECP2 mutation determinations were performedby Ilaria Meloni Francesca Ariani Francesca Mari SoniaAmabile and Alessandra Renieri Clinical assessement wasperformed by Joussef Hayek Claudio De Felice and Alessan-dra Renieri Skin biopsies were performed by Joussef HayekSamples were prepared by Cinzia Signorini Silvia LeonciniAlessandra Pecorelli Gloria Zollo Giuseppe Belmonte Mar-iangela Gentile Eugenio Paccagnini Sonia Amabile andIlariaMeloniNPBI assayswere performed by Silvia LeonciniIsoprostanes and neuroprostanes assays were performed byCinzia Signorini Thierry Durand and Jean-Marie Galano4-HNE-PAs assays were carried out by Alessandra Pecorelliand Giuseppe Valacchi Gluthatione assays were carried outby Silvia Leoncini and Gloria Zollo Immunocytochem-istry was performed by Giuseppe Belmonte and AlessandraPecorelli TEM was done by Eugenio Paccagnini and Mari-angela Gentile Data analysis was done by Claudio De FeliceCinzia Signorini and Silvia Leoncini All the authors equallycontributed in data interpretation paper drafting and paperapproval

Acknowledgments

This work was supported by the Grant no RF-TOS-2008-1225570mdashBando Malattie Rare to AR The present researchproject has beenmainly funded by the ItalianHealthMinistryand Tuscan Region This research is dedicated to all theRett girls and their families The authors thank Dr PierluigiTosi Dr Silvia Briani and Dr Roberta Croci from theAdministrative Direction of the Azienda Ospedaliera Senesefor continued support to our studies and for prior purchasingof the gas spectrometry instrumentation They also thank


professional singer Matteo Setti for his artistic contributionsto the exploration of Rett syndrome and his many charityconcerts dedicated to the Rett girls and their families

References

[1] B Hagberg J Aicardi K Dias and O Ramos ldquoA progressivesyndrome of autism dementia ataxia and loss of purposefulhand use in girls rettrsquos syndrome report of 35 casesrdquo Annals ofNeurology vol 14 no 4 pp 471ndash479 1983

[2] T Bienvenu C Philippe N De Roux et al ldquoThe incidence ofrett syndrome in Francerdquo Pediatric Neurology vol 34 no 5 pp372ndash375 2006

[3] B Hagberg ldquoClinical manifestations and stages of Rett syn-dromerdquo Mental Retardation and Developmental DisabilitiesResearch Reviews vol 8 no 2 pp 61ndash65 2002

[4] R EAmir I B VandenVeyverMWanCQ TranU Franckeand H Y Zoghbi ldquoRett syndrome is caused by mutations inX-linkedMECP2 encodingmethylmdashCpGmdashbinding protein 2rdquoNature Genetics vol 23 no 2 pp 185ndash188 1999

[5] J Christodoulou A Grimm T Maher and B Bennetts ldquoRet-tBASE the IRSA MECP2 variation database-a new mutationdatabase in evolutionrdquoHumanMutation vol 21 no 5 pp 466ndash472 2003

[6] B Halliwell ldquoOxidative stress and cancer have we movedforwardrdquoThe Biochemical Journal vol 401 no 1 pp 1ndash11 2007

[7] B Halliwell ldquoFree radicals and antioxidantsmdashquo vadisrdquoTrends in Pharmacological Sciences vol 32 no 3 pp 125ndash1302011

[8] B Halliwell Free Radicals in Biology and Medicine OxfordUniversity Press Oxford UK 4th edition 2007

[9] C D Aluise R A S Robinson J Cai W M Pierce W RMarkesbery and D A Butterfield ldquoRedox proteomics analysisof brains from subjects with amnestic mild cognitive impair-ment compared to brains from subjects with preclinicalAlzheimerrsquos disease insights into memory loss inMCIrdquo Journalof Alzheimerrsquos Disease vol 23 no 2 pp 257ndash269 2011

[10] A-M Enciu M Gherghiceanu and B O Popescu ldquoTriggersand effectors of oxidative stress at blood-brain barrier levelrelevance for brain ageing and neurodegenerationrdquo OxidativeMedicine and Cellular Longevity vol 2013 Article ID 297512 12pages 2013

[11] J C Brazil N A Louis and C A Parkos ldquoThe role ofpolymorphonuclear leukocyte trafficking in the perpetuation ofinflammation during inflammatory bowel diseaserdquo Inflamma-tory Bowel Diseases vol 19 no 7 pp 1556ndash1565 2013

[12] A Ghezzo P Visconti P M Abruzzo et al ldquoOxidative stressand erythrocyte membrane alterations in children with autismcorrelation with clinical featuresrdquo PLoS ONE vol 8 no 6Article ID e66418 2013

[13] L Ciccoli C De Felice E Paccagnini et al ldquoErythrocyte shapeabnormalities membrane oxidative damage and 120573-actin alter-ations an unrecognized triad in classical autismrdquo Mediators ofInflammation vol 2013 Article ID 432616 11 pages 2013

[14] A Pecorelli S Leoncini C De Felice et al ldquoNon-protein-bound iron and 4-hydroxynonenal protein adducts in classicautismrdquoBrain andDevelopment vol 35 no 2 pp 146ndash154 2013

[15] A Chauhan T Audhya and V Chauhan ldquoBrain region-specific glutathione redox imbalance in autismrdquoNeurochemicalResearch vol 37 no 8 pp 1681ndash1689 2012

[16] S J James S Rose SMelnyk et al ldquoCellular andmitochondrialglutathione redox imbalance in lymphoblastoid cells derivedfrom children with autismrdquo FASEB Journal vol 23 no 8 pp2374ndash2383 2009

[17] J Baio ldquoPrevalence of autism spectrum disordersmdashautism anddevelopmental disabilities monitoring network 14 sites UnitedStates 2008rdquoMorbidity andMortalityWeekly Report vol 61 no3 pp 1ndash19 2012

[18] S J James P Cutler S Melnyk et al ldquoMetabolic biomarkersof increased oxidative stress and impaired methylation capacityin children with autismrdquo The American Journal of ClinicalNutrition vol 80 no 6 pp 1611ndash1617 2004

[19] E Grillo C Lo Rizzo L Bianciardi et al ldquoRevealing thecomplexity of a monogenic disease Rett syndrome exomesequencingrdquo PLoS ONE vol 8 no 2 Article ID e56599 2013

[20] E Groszliger UHirt O A Janc et al ldquoOxidative burden andmito-chondrial dysfunction in a mouse model of Rett syndromerdquoNeurobiology of Disease vol 48 no 1 pp 102ndash114 2012

[21] C De Felice C Signorini T Durand et al ldquoPartial rescue ofRett syndrome by 120596-3 polyunsaturated fatty acids (PUFAs) oilrdquoGenes and Nutrition vol 7 no 3 pp 447ndash458 2012

[22] L Ciccoli C De Felice E Paccagnini et al ldquoMorphologicalchanges and oxidative damage in Rett Syndrome erythrocytesrdquoBiochimica et Biophysica Acta General Subjects vol 1820 no 4pp 511ndash520 2012

[23] S Leoncini C De Felice C Signorini et al ldquoOxidative stress inRett syndrome natural history genotype and variantsrdquo RedoxReport vol 16 no 4 pp 145ndash153 2011

[24] C De Felice C Signorini T Durand et al ldquoF2-dihomo-isoprostanes as potential early biomarkers of lipid oxidativedamage in Rett syndromerdquo Journal of Lipid Research vol 52 no12 pp 2287ndash2297 2011

[25] C De Felice L Ciccoli S Leoncini et al ldquoSystemic oxidativestress in classic Rett syndromerdquo Free Radical Biology andMedicine vol 47 no 4 pp 440ndash448 2009

[26] C De Felice C Signorini S Leoncini et al ldquoThe role ofoxidative stress in Rett syndrome an overviewrdquo Annals of theNew York Academy of Sciences vol 1259 no 1 pp 121ndash135 2012

[27] A Renieri F Mari M A Mencarelli et al ldquoDiagnostic criteriafor the Zappella variant of Rett syndrome (the preserved speechvariant)rdquo Brain and Development vol 31 no 3 pp 208ndash2162009

[28] N Dracopoli J Haines B Korf et al Current Protocols inHumanGenetics JohnWileyamp Sons NewYorkNYUSA 2000

[29] C Signorini L Ciccoli S Leoncini et al ldquoFree iron totalF2-isoprostanes and total F4-neuroprostanes in a model ofneonatal hypoxic-ischemic encephalopathy neuroprotectiveeffect of melatoninrdquo Journal of Pineal Research vol 46 no 2pp 148ndash154 2009

[30] C Signorini M Comporti and G Giorgi ldquoIon trap tandemmass spectrometric determination of F2-isoprostanesrdquo Journalof Mass Spectrometry vol 38 no 10 pp 1067ndash1074 2003

[31] C Signorini C De Felice S Leoncini et al ldquoF4-neuroprostanesmediate neurological severity in Rett syndromerdquo Clinica Chim-ica Acta vol 412 no 15-16 pp 1399ndash1406 2011

[32] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969

[33] M A Baker G J Cerniglia and A Zaman ldquoMicrotiter plateassay for the measurement of glutathione and glutathione


disulfide in large numbers of biological samplesrdquo AnalyticalBiochemistry vol 190 no 2 pp 360ndash365 1990

[34] G Valacchi A Pecorelli C Signorini et al ldquo4HNE proteinadducts in autistic spectrum disorders rett syndrome andautismrdquo in Comprehensive Guide to Autism V B Patel V RPreedy and C R Martin Eds pp 2667ndash2687 Springer NewYork NY USA 2014

[35] C Signorini C De Felice T Durand et al ldquoIsoprostanes and4-hydroxy-2-nonenal markers or mediators of disease focuson Rett syndrome as a model of autism spectrum disorderrdquoOxidative Medicine and Cellular Longevity vol 2013 Article ID343824 10 pages 2013

[36] C Sticozzi G Belmonte A Pecorelli et al ldquoScavenger receptorB1 post-translational modifications in Rett syndromerdquo FEBSLetters vol 587 no 14 pp 2199ndash2204 2013

[37] K Miyake C Yang Y Minakuchi et al ldquoComparison ofgenomic and epigenomic expression in monozygotic twinsdiscordant for rett syndromerdquo PLoS ONE vol 8 no 6 ArticleID e66729 2013

[38] M Amenduni R De Filippis A Y L Cheung et al ldquoIPS cells tomodel CDKL5-related disordersrdquo European Journal of HumanGenetics vol 19 no 12 pp 1246ndash1255 2011

[39] C Delepine J Nectoux N Bahi-Buisson J Chelly and TBienvenu ldquoMeCP2 deficiency is associated with impairedmicrotubule stabilityrdquo FEBS Letters vol 587 no 2 pp 245ndash2532013

[40] J Nectoux C Florian C Delepine et al ldquoAltered microtubuledynamics in Mecp2-deficient astrocytesrdquo Journal of Neuro-science Research vol 90 no 5 pp 990ndash998 2012

[41] M Vecsler B Ben Zeev I Nudelman et al ldquoEx vivo treatmentwith a novel synthetic aminoglycoside NB54 in primary fibrob-lasts from Rett syndrome patients suppresses MECP2 nonsensemutationsrdquo PLoS ONE vol 6 no 6 Article ID e20733 2011

[42] C Sandi M Sandi H Jassal et al ldquoGeneration and character-isation of friedreich ataxia YG8R mouse fibroblast and neuralstem cell modelsrdquo PLoS ONE vol 9 no 2 Article ID e894882014

[43] A Ferretta A Gaballo P Tanzarella et al ldquoEffect of resveratrolon mitochondrial function implications in parkin-associatedfamiliar Parkinsonrsquos diseaserdquo Biochimica et Biophysica Acta vol1842 no 7 pp 902ndash915 2014

[44] D M Boesten J M de Vos-Houben L Timmermans G J denHartog A Bast and G J Hageman ldquoAccelerated aging duringchronic oxidative stress a role for PARP-1rdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 680414 10 pages2013

[45] G-Y Yang C-L Zhang X-C Liu G Qian and D-Q DengldquoEffects of cigarette smoke extracts on the growth and senes-cence of skin fibroblasts in vitrordquo International Journal ofBiological Sciences vol 9 no 6 pp 613ndash623 2013

[46] H Yang C Liu D Yang H Zhang and Z Xi ldquoComparativestudy of cytotoxicity oxidative stress and genotoxicity inducedby four typical nanomaterials the role of particle size shape andcompositionrdquo Journal of Applied Toxicology vol 29 no 1 pp69ndash78 2009

[47] C Huang and R Ogawa ldquoRoles of lipid metabolism in keloiddevelopmentrdquo Lipids in Health and Disease vol 12 no 1 article60 2013

[48] E S Musiek H Yin G L Milne and J D Morrow ldquoRecentadvances in the biochemistry and clinical relevance of theisoprostane pathwayrdquo Lipids vol 40 no 10 pp 987ndash994 2005

[49] L J Roberts II J P Fessel and S S Davies ldquoThe biochemistry ofthe isoprostane neuroprostane and isofuran pathways of lipidperoxidationrdquo Brain Pathology vol 15 no 2 pp 143ndash148 2005

[50] LCiccoli S Leoncini C Signorini andMComporti ldquoIron anderythrocytes physiological and pathophysiological aspectsrdquo inOxidants in Biology G Valacchi and P Davis Eds pp 167ndash181Springer Heidelberg Germany 2008

[51] S A Moore E Hurt E Yoder H Sprecher and A A SpectorldquoDocosahexaenoic acid synthesis in human skin fibroblastsinvolves peroxisomal retroconversion of tetracosahexaenoicacidrdquo Journal of Lipid Research vol 36 no 11 pp 2433ndash24431995

[52] E R Brown and P V Subbaiah ldquoDifferential effects of eicos-apentaenoic acid and docosahexaenoic acid on human skinfibroblastsrdquo Lipids vol 29 no 12 pp 825ndash829 1994

[53] S P Mahadik S Mukherjee D F Horrobin K Jenkins EE Correnti and R E Scheffer ldquoPlasma membrane phospho-lipid fatty acid composition of cultured skin fibroblasts fromschizophrenic patients comparison with bipolar patients andnormal subjectsrdquo Psychiatry Research vol 63 no 2-3 pp 133ndash142 1996

[54] E H Epstein Jr and N H Munderloh ldquoIsolation and char-acterization of CNBr peptides of human [1205721(III)]3 collagenand tissue distribution of [1205721(I)]21205722 and [1205721(III)]3 collagensrdquoJournal of Biological Chemistry vol 250 no 24 pp 9304ndash93121975

[55] L-J Min T-X Cui Y Yahata et al ldquoRegulation of collagensynthesis in mouse skin fibroblasts by distinct angiotensin IIreceptor subtypesrdquo Endocrinology vol 145 no 1 pp 253ndash2602004

[56] B R Olsen ldquoThe roles of collagen genes in skeletal developmentand morphogenesisrdquo Experientia vol 51 no 3 pp 194ndash1951995

[57] R S Wong F M Follis B K Shively and J A Wernly ldquoOsteo-genesis imperfecta and cardiovascular diseasesrdquo The Annals ofThoracic Surgery vol 60 no 5 pp 1439ndash1443 1995

[58] E Vuorio and B de Crombrugghe ldquoThe family of collagengenesrdquo Annual Review of Biochemistry vol 59 pp 837ndash8721990

[59] K Piez and A Reddi Eds Extracellular Matrix BiochemistryElsevier New York NY USA 1984

[60] R Mayne and R Burgeson Eds Structure and Function ofCollagen Types Academic Press New York NY USA 1987

[61] P Bornstein and H Sage ldquoStructurally distinct collagen typesrdquoAnnual Review of Biochemistry vol 49 pp 957ndash1003 1980

[62] R Fleischmajer B R Olsen R Timpl J S Perlish and OLovelace ldquoCollagen fibril formation during embryogenesisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 80 no 11 pp 3354ndash3358 1983

[63] E Cardaioli M T Dotti G Hayek M Zappella and AFederico ldquoStudies on mitochondrial pathogenesis of Rett syn-drome ultrastructural data from skin and muscle biopsies andmutational analysis at mtDNA nucleotides 10463 and 2835rdquoJournal of Submicroscopic Cytology and Pathology vol 31 no2 pp 301ndash304 1999

[64] G Stabellini G Berti C Calastrini et al ldquoExperimental modelfor studying the effects of 2-ethylhexyl-phthalate and dialysateon connective tissuerdquo The International Journal of ArtificialOrgans vol 23 no 5 pp 305ndash311 2000

[65] G Ellender and K N Ham ldquoConnective tissue responsesto some heavy metals III silver and dietary supplements of


ascorbic acid Histology and ultrastructurerdquo British Journal ofExperimental Pathology vol 70 no 1 pp 21ndash39 1989

[66] V M Korkhov ldquoGFP-LC3 labels organised smooth endo-plasmic reticulum membranes independently of autophagyrdquoJournal of Cellular Biochemistry vol 107 no 1 pp 86ndash95 2009

[67] G J Fisher T Quan T Purohit et al ldquoCollagen frag-mentation promotes oxidative stress and elevates matrixmetalloproteinase-1 in fibroblasts in aged human skinrdquo Amer-ican Journal of Pathology vol 174 no 1 pp 101ndash114 2009

[68] W Xia C Hammerberg Y Li et al ldquoExpression of catalyticallyactive matrix metalloproteinase-1 in dermal fibroblasts inducescollagen fragmentation and functional alterations that resembleaged human skinrdquo Aging Cell vol 12 no 4 pp 661ndash671 2013

[69] B Hagberg ldquoRett syndrome long-term clinical follow-up expe-riences over four decadesrdquo Journal of Child Neurology vol 20no 9 pp 722ndash727 2005

[70] N Halbach E Smeets C Steinbusch M Maaskant D vanWaardenburg and L Curfs ldquoAging in Rett syndrome a longitu-dinal studyrdquo Clinical Genetics vol 84 no 3 pp 223ndash229 2013

[71] C S Ward E M Arvide T-W Huang J Yoo J L Noebels andJ L Neul ldquoMeCP2 is critical within HoxB1-derived tissues ofmice for normal lifespanrdquo Journal of Neuroscience vol 31 no28 pp 10359ndash10370 2011

[72] T Squillaro N AlessioM Cipollaro et al ldquoReduced expressionofMECP2 affects cell commitment andmaintenance in neuronsby triggering senescence new perspective for Rett syndromerdquoMolecular Biology of the Cell vol 23 no 8 pp 1435ndash1445 2012

[73] S Dato P Crocco P D rsquoAquila et al ldquoExploring the role ofgenetic variability and lifestyle in oxidative stress response forhealthy aging and longevityrdquo International Journal of MolecularSciences vol 14 no 8 pp 16443ndash16472 2013

[74] S I Liochev ldquoReactive oxygen species and the free radicaltheory of agingrdquo Free Radical Biology and Medicine vol 60 pp1ndash4 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


frequent mutations comprise more than 34 of all thereported pathogenic ones [5] in additionMECP2 genemuta-tions are usually categorized as missense or truncatingincluding nonsense frameshift and large deletions accord-ing to the type of sequence change

Oxidative stress (OS) indicates a combination of eventsresulting in a damage to biological molecules due to animbalance between cellular antioxidant defences and freeradicals production OS appears to be involved in a largenumber of human diseases including cancer [6ndash8] neurode-generative diseases [9 10] and inflammatory bowel disease[11] Furthermore cumulating evidence suggests the presenceof a redox imbalance in autism spectrum disorders (ASDs)a condition with a high social impact due to the explosiveincrease in its prevalence over the last four decades [12ndash18]Our group and other laboratories have reported enhancedOSmarkers levels in plasma and erythrocytes from patients withRTT thus suggesting the presence of a systemic OS in thedisease However to date it is unclear not only why but alsowhen and where this OS derangement may occur [19ndash26]

In particular to date little is known regarding the oxidant-antioxidant status in RTT cellular systems and its possiblerelationship with the morphological phenotype Here weinvestigated the levels of different OS markers (F

2-IsoPs F

4-

NeuroPs NPBI and 4-HNE PAs) and glutathione in primaryskin fibroblasts from patients with typical RTT as well aspossible changes in the cellularintracellular structure andorqualitative changes in the synthesized collagen

2 Materials and Methods

21 Subjects A total of 16 female patients with typical RTT(mean age 1306 plusmn 65) as well as 8 healthy female controlsof comparable age (mean age 132 plusmn 68) participated in thestudy Skin biopsies from the control group were obtainedfrom a skin biobank by selecting subjects without diagnosedskin or collagen diseases (responsibility Joussef Hayek)

All patients were consecutively admitted to the ChildNeuropsychiatry Unit of the University Hospital of Siena(Head Joussef Hayek) For all patients theMECP2mutationwas demonstrated (mutation categories missense mutation119899 = 8 early truncating mutation 119899 = 6 late truncatingmutation 119899 = 2) and a clinical severity score was calculatedusing the previously reported scaling system [27] (meanseverity score 159 plusmn 647 range 50ndash280) All the examinedsubjects were on a typical Mediterranean diet

A 3mm skin punch biopsy was performed after obtainedwritten informed consent of either the parents or the legaltutors of the patients (responsibility Joussef Hayek) Biopsiesfrom control subject were performed in the DermatologyUnit of the University Hospital of Siena The study was con-ducted with the approval of the Institutional Review Boardof the University Hospital Azienda Ospedaliera UniversitariaSenese

22 Fibroblasts Isolation and Culture Following informedconsent signature skin biopsies (about 3-4mm3) were per-formed using the Punch Biopsy procedure Fibroblasts were

isolated and cultured with standard protocols [28] Cells weregrown in DMEM (Biochrom) supplemented with 10 fetalbovine serum 100UmL penicillin 100 120583gmL streptomycinand 2mM L-glutamine Cells were incubated at 37∘C with5 CO

2until 80ndash90 confluence and routinely passed by

trypsin-EDTA (Irvine Scientific)Cells for analysis were seeded onto 100mm tissue culture

plates (for NPBI and IsoP evaluations) and 12-well plates(for 4-HNE PAs and GSHGSSG ratio) containing the indi-cated medium formulations Fibroblasts at low passage wereemployed for the analysis

At confluence the cells were scraped transferred in atube and washed twice with ice-cold PBS pH 74 Cellswere then centrifuged at 600 g for 10min and the pelletresuspended in a final volume of 2mL PBS

23 Nonprotein Bound Iron (NPBI) Nonprotein bound ironwas determined as desferoxamine- (DFO-) chelatable freeiron (DFO-iron complex ferrioxamine) DFO 25120583M wasadded to 1mL of cell suspension to obtain a final concen-tration of 25120583M DFO and the cells were ruptured by theaddition of 1mL water freeze-thawing and sonication Thesamples were then ultrafiltered in centrifuge filter devices(VIVASPIN 4 Sartorius Stedim Biotech GmbH GoettingenGermany) with a 30 kDa molecular weight cutoff and theultrafiltrate was stored at minus20∘C until analysis The DFOexcess was removed by silica (Silicagel 25ndash40120583m MerckDarmstadt Germany) column chromatography (Varian IncCA USA)TheDFO-iron complex was determined by HPLCand the detection wavelength was 229 nm [29]

24 Total (Sum of Free Plus Esterified) F2-Isoprostanes and F

4-

Neuroprostanes Determinations Butylated hydroxytoluenewas added to 900120583L of cell suspension as an antioxidantto obtain a final concentration of 90 120583M and the sampleswere frozen at minus70∘C until analysis All isoprostane andneuroprostane determinations were carried out by gas chro-matographynegative ion chemical ionization tandem massspectrometry (GCNICI-MSMS) analysis after solid phaseextraction and derivatization steps F

2-Isoprostanes and F

4-

neuroprostanes levels were normalized for the cell proteincontent

25 Solid Phase Extraction and Derivatization Procedures Atthe time of the determination the samples were sonicated byultrasound treatment for 30 seconds and then purified as pre-viously reported [29] Briefly aqueous KOH (1mM 450120583L)was added to the cellular suspension After incubation at45∘C for 45min the pH was adjusted to 3 by adding HCl(1mM 450 120583L) Each sample was spiked with tetradeuteratedprostaglandin F

2120572(PGF2120572-d4) (500 pg in 50120583L of ethanol)

as an internal standard and ethyl acetate (10mL) was addedto extract total lipids by vortex-mixing and centrifugation at1000 g for 5min at room temperature The total lipid extractwas applied onto an NH

2cartridge and the isoprostanes

in the final eluates were derivatized The carboxylic groupwas derivatized as the pentafluorobenzil ester whereas thehydroxyl groups were converted to trimethylsilyl ethers [30]
















3 Results


2-IsoPs




0

200

400

600

800

1000(p

gm

g pr

otei

n)


Tota

lF2-I

soPs

lowast

(a)

0

200

400

600

800

1000

(pg

mg

prot

ein)

Tota

lF4-N

euro

Ps

lowast


(b)

0

5

10

15

20

25

4-H

NE

PAs (times106

au)

lowastlowast


(c)

0

02

04

06

08

1

NPB

I (nm

olm

g pr

otei

n)

lowast


(d)






F2-isoprostanes F

4-NeuroPs F


0

04

08

12

16


Redu

ced

GSH

(n

mol

mg

prot

ein)

lowast

(a)

0

04

08

12

16

GSS

G

lowastlowast

(nm

olm

g pr

otei

n)


(b)


lt







2-IsoPs F





M

RER

G

V

1120583m

(a)

RER

V

MLB

1120583m

(b)


Hea

lthy

cont

rols

Typi

cal R

TT

Col III

Col III

Col I

Col I





4 Discussion



0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast



119875
























5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results


2-IsoPs




0

200

400

600

800

1000(p

gm

g pr

otei

n)


Tota

lF2-I

soPs

lowast

(a)

0

200

400

600

800

1000

(pg

mg

prot

ein)

Tota

lF4-N

euro

Ps

lowast


(b)

0

5

10

15

20

25

4-H

NE

PAs (times106

au)

lowastlowast


(c)

0

02

04

06

08

1

NPB

I (nm

olm

g pr

otei

n)

lowast


(d)






F2-isoprostanes F

4-NeuroPs F


0

04

08

12

16


Redu

ced

GSH

(n

mol

mg

prot

ein)

lowast

(a)

0

04

08

12

16

GSS

G

lowastlowast

(nm

olm

g pr

otei

n)


(b)


lt







2-IsoPs F





M

RER

G

V

1120583m

(a)

RER

V

MLB

1120583m

(b)


Hea

lthy

cont

rols

Typi

cal R

TT

Col III

Col III

Col I

Col I





4 Discussion



0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast



119875
























5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

200

400

600

800

1000(p

gm

g pr

otei

n)


Tota

lF2-I

soPs

lowast

(a)

0

200

400

600

800

1000

(pg

mg

prot

ein)

Tota

lF4-N

euro

Ps

lowast


(b)

0

5

10

15

20

25

4-H

NE

PAs (times106

au)

lowastlowast


(c)

0

02

04

06

08

1

NPB

I (nm

olm

g pr

otei

n)

lowast


(d)






F2-isoprostanes F

4-NeuroPs F


0

04

08

12

16


Redu

ced

GSH

(n

mol

mg

prot

ein)

lowast

(a)

0

04

08

12

16

GSS

G

lowastlowast

(nm

olm

g pr

otei

n)


(b)


lt







2-IsoPs F





M

RER

G

V

1120583m

(a)

RER

V

MLB

1120583m

(b)


Hea

lthy

cont

rols

Typi

cal R

TT

Col III

Col III

Col I

Col I





4 Discussion



0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast



119875
























5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















M

RER

G

V

1120583m

(a)

RER

V

MLB

1120583m

(b)


Hea

lthy

cont

rols

Typi

cal R

TT

Col III

Col III

Col I

Col I





4 Discussion



0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast



119875
























5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0100200300400500600700

Rela

tive i

nten

sity

NS

Col III Col I

lowast



119875
























5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion


Abbreviations


2-isoprostanes

F4-NeuroPs F

4-neuroprostanes


gene



Disclosure






Acknowledgments




References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References




















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

Documents

Transcript of Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome