Augmenter of liver regeneration, a protective factor against ROS-induced oxidative damage in muscle tissue of mitochondrial myopathy affected patients

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The International Journal of Biochemistry & Cell Biology 45 (2013) 2410– 2419

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

journa l h om epa ge: www.elsev ier .com/ locate /b ioce l

ugmenter of liver regeneration, a protective factor againstOS-induced oxidative damage in muscle tissue ofitochondrial myopathy affected patients

orenzo Polimenoa,b,c,∗, Roberta Rossid, Maria Mastrodonatoe, Monica Montagnani f,omenico Piscitelli d, Barbara Pesettib, Leonarda De Benedictis f, Bruna Girardid,eonardo Restad, Anna Napolid, Antonio Francavillab

Section of Gastroenterology, Dept. of Emergency and Transplant of Organs (DETO), Univ. of Bari, ItalyIRSSC “S. de Bellis” Castellana Grotte, Bari, ItalyCenter Interdept. of Res. on Gastroent. and Evolut. Hepatology (CIRGEE), ItalySection of Anatomy-Pathology, DETO, Univ. of Bari, ItalyDept. of Animal and Environmental Biology, Univ. of Bari, ItalyDept. of Biomedical Sciences and Human Oncology, University of Bari, Italy

r t i c l e i n f o

rticle history:eceived 9 March 2013eceived in revised form 24 June 2013ccepted 9 July 2013vailable online 31 July 2013

eywords:LRyopathies

OSpoptosis

a b s t r a c t

Mitochondria-related myopathies (MM) are a group of different diseases defined by a varying degreeof dysfunctions of the mitochondrial respiratory chain which leads to reactive oxygen species (ROS)generation followed by oxidative stress and cellular damage. In mitochondrial myopathy muscle tissuean overexpression of antioxidant enzymes has been documented probably as an attempt to counteractthe free radical generation.

We previously documented, in human non-pathological muscle fibres, the expression of the augmenterof liver regeneration (ALR), a sulfhydryl oxidase enzyme, whose presence is related to the mitochondria;indeed it has been demonstrated that ALR mainly localizes in the mitochondrial inter-membrane space.Furthermore we reported, in different experimental models, in vivo and in vitro, the anti-apoptotic andanti-oxidative capacities of ALR, achieved by up-regulating Bcl-2 anti-apoptotic family factors and theanti-apoptotic/anti-oxidative secretory isoform of clusterin (sClu).

With the present study we aimed to determine ALR, Bcl-2 protein, clusterin and ROS expression in

muscle tissue biopsies from MM-affected patients. Non-pathological muscle tissue was used as con-trol. Enzymatic, histochemical, immunohistochemical and immune electron microscopy techniques wereperformed.

The data obtained revealed in MM-derived muscle tissue, compared to non-pathological tissue, theover-expression of ROS, ALR and Bcl-2 and the induction of the nuclear, pro-apoptotic, isoform of clusterin(nCLU).

. Introduction

The mitochondria-related myopathies (MM) represent a hetero-eneous group of human diseases characterized by dysfunction inultiple organs systems and extensive variability in clinical pre-

entation (Pfeffer and Chinnery, 2013). MM are defined by a varying

egree of dysfunctions of the mitochondrial respiratory chain,evealed through rigorous mitochondrial morphological investi-ation, and biochemical and genetic evaluations (Bernier et al.,

∗ Corresponding author at: Section of Gastroenterology, DETO, University of Bari,oliclinico, Piazza G. Cesare, 11, 70124 Bari, Italy. Tel.: +39 805478641.

E-mail address: [email protected] (L. Polimeno).

357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.biocel.2013.07.010

© 2013 Elsevier Ltd. All rights reserved.

2002; Taylor et al., 2004; Medja et al., 2009; Pfeffer and Chinnery,2013). The histological analysis of the skeletal muscle fibres ofpatients affected by MM reveals the presence of ragged red fibres(RRF), due to the accumulation of proliferating mitochondria, and ofcytochrome c oxidase (COX)-negative fibres (Pfeffer and Chinnery,2013).

The prognosis for these disorders ranges in severity fromprogressive weakness to death. Most mitochondria-relatedmyopathies occur before the age of 20, and often begin with exer-cise intolerance or muscle weakness. In the infancy, the clinical

demonstrations of these illnesses can include deceleration orarrest of the growth, recurrent myoglobinuria, renal damages, lowstature, endocrine dysfunctions as diabetes mellitus, insipid dia-betes, optic atrophy and deafness or progressive encephalopathy.

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ome of the most common mitochondrial myopathies are theearns-Sayre syndrome, the myoclonus epilepsy with raggedbres and the MELAS syndrome, a mitochondrial encephalomy-pathy with lactic acidosis and stroke-like episodes (Mita et al.,995; Sarnat and Marín-García, 2005; Phadke et al., 2012;oodfellow et al., 2012).

Mitochondrial dysfunction is most often considered in terms oflteration in electron transport chain with increased oxidant pro-uction, decreased ATP production causing ROS generation withonsequent oxidative stress and abnormal activation of apopto-is, whose biochemical players are still not well defined (Pitkanennd Robinson, 1996; Brambilla et al., 1997; Lenaz, 1998; Wei et al.,998; Esposito et al., 1999; Filosto et al., 2002). It is known thathe “oxidative stress” can cause DNA, protein, and/or lipid dam-ge, leading to changes in chromosome stability, genetic mutation,nd/or modulation of cell growth, but its role in the pathogenesisf MM is still controversial (Tews, 2002; Mancuso et al., 2010). Inddition, even if a pivotal role in the disease process of MM haseen attributed to the ROS (Esposito et al., 1999; Tarnopolsky andaha, 2005; St-Pierre et al., 2006), so far, to our knowledge, noata definitively show a direct presence of ROS in human muscleissue from MM patients. ROS induction has mainly been indi-ectly demonstrated, in isolated mitochondria associated to theROS detoxification enzymes” over-expression, such as SOD andSHPxa (Esposito et al., 1999), or in COX-deficient myofibres fromM-affected patients in which an oxidative damage to skeletaluscle DNA and/or an intense Mn and Cu/Zn SOD immunostain-

ng have been reported (Ohkoshi et al., 1995; Mitsui et al., 1996;arrier et al., 2000). However it is important to underline that allhese data strongly indicate a linkage between COX defect, apopto-is, and over-expression of antioxidant mechanisms, as an attempto compensate free radical overproduction.

We previously documented, in human muscle tissue fromealthy patients (Polimeno et al., 2000), the presence of a sulfhydrylxidase enzyme, ALR (Lisowsky et al., 2001), which demonstrated,n different experimental models in vivo and in vitro, strong anti-poptotic and anti-oxidative activities (Thirunavukkarasu et al.,008; Polimeno et al., 2009b; Cao et al., 2009; Todd et al., 2010;iao et al., 2010; Polimeno et al., 2011). The anti-apoptotic activ-ty of ALR is induced by its capacity to up-regulate the expressionf the anti-apoptotic gene (Bcl-2), to inhibit the expression ofhe pro-apoptotic gene (Bax) and to preserve the integrity of theuter mitochondrial membrane, the anti-oxidative activity of ALRs achieved mainly by the induction of the secretory isoform oflusterin (sCLU) (Polimeno et al., 2012).

Clusterin (CLU) is a multifunctional factor that has secretorysCLU) and nuclear (nCLU) isoforms, which play opposite roles inhe cell survival/death. The sCLU isoform appears to be an anti-poptotic factor (Zhang et al., 2005; Moretti et al., 2007; Kim andhoi, 2011; Polimeno et al., 2011, 2012), function achieved by inter-cting with Ku70 and Bax (Djeu and Wei S, 2009; Trougakos et al.,009), whereas the nCLU isoform is thought to retain pro-apoptoticctivity (Leskov et al., 2003) and to favour cell death by the inhi-ition of nuclear factor-kB-dependent Bcl-XL expression (Scaltritit al., 2004; Takase et al., 2008).

It is known that the balance between pro- and anti-apoptoticactors is crucial for mitochondrial functions and for cell viabil-ty; to improve the knowledge and the role of the apoptosis onhe pathogenesis and on the progression of mitochondria-related

yopathies, we studied the behaviour of ALR, ROS and clusterin,hree factors implicated in the apoptotic process, in muscle tissueamples from MM-affected patients. Muscle tissue sample from

ubjects treated for non-traumatic orthopaedic diseases and allegative for the histochemical (COX negative fibres) and histologi-al (ragged red fibres) typical features of mitochondrial dysfunctionrequently recognized in MM muscle tissue, were used.

Fig. 1. RRF and COX-negative fibres evidenced in muscle fibres of MM human muscletissue.

2. Materials and methods

2.1. Chemicals

If not specifically reported, the chemical reagents were pur-chased by Sigma–Aldrich, Milan, Italy. The polyclonal antibodyagainst ALR (MultiBind GmbH, Koln, Germany; a gift from T.Lisowsky) had already been employed for specific identification ofALR (Klissenbauer et al., 2002; Thasler et al., 2005; Polimeno et al.,2009a, 2009b; Polimeno et al., 2011). For raising the ALR antibody apurified hexahistidinyl-tagged carboxyl-terminal fragment of ALR(residues 81-205) was used (Thasler et al., 2005; Polimeno et al.,2009a, 2012).

2.2. Muscle tissue samples

Tissue biopsies from 10 specimens of non-pathological mus-cle tissues and 10 specimens of pathological (mitochordria-relatedmyopathies) muscle tissues were collected. The non-pathologicalskeletal muscle tissues were from patients treated for non trau-matic orthopaedic disease who attended the Orthopaedic Unitof the Medical School of University of Bari and who gave theirinformed consent to take a muscle tissue sample for the study.Muscular tissues with various mitochondria-related myopathieswere retrieved from the Ultrastructure Laboratory archive of theDept of Anatomo-Pathology, University of Bari and/or from theNeurological Laboratory of the University of Bari. For the presentstudy we selected muscle tissue sample, mainly taken from the leftbrachial biceps, which were strongly characterized by the presenceof RRF and COX-negative fibres (Fig. 1). All the patients identi-

fied as eligible for the study referred, singularly or in association,one of the following clinical features, stated in literature to besuspected for MM diagnosis: progressive external opthalmoplegia,ptosis, retinitis pigmentosa, limb myopathy, cardiac abnormality,

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pilepsy, stroke-like episodes, neurosensory hearing loss, cere-ellar and/or extrapyramidal dysfunction, cognitive impairment,europathology, short stature, diabetes, gastrointestinal dysfunc-ion (Zeviani et al., 1991; Jackson et al., 1995; DiMauro et al., 1998;apadimitriou et al., 1998).

At the time of the enrolment to the study, as stated by thelinical protocol, the patients underwent to muscle biopsies foristological and histochemical diagnostic purposes and for elec-ron microscopy analysis to provide, by the recognition of ultratructural alterations of mitochondria, more criteria for the diag-osis of the mitochondria-related myopathy (Bernier et al., 2002;feffer et al., 2011; Pfeffer et al., 2013). For all these purposes part ofissue samples were suddenly sneep-frozen in isopentane-coolediquid nitrogen (for histological, histochemical and for oxidativeuorescence studies), part in formalin 10% for immunological stud-

es and part (for EM study) was fixed in 2.5% glutaraldehyde in.1 M saline phosphate buffer (PBS), at pH 7.4, for 4 h at 4 ◦C, afterhich were postfixed in 1% OsO4 in PBS for 20 min at 4 ◦C. Fixed

amples were washed in several changes of PBS and dehydratedn graded alcohols and propylene oxide and finally embedded inpon-Araldite (TAAB, Reading, UK). The semi-thin sections, hav-ng a thickness of 1 �m, were heat stained with toluidine blueorate. Ultrathin sections for EM were mounted on formvar-coatedickel grids and stained routinely with uranyl acetate and leaditrate.

.3. Histology/histochemistry

Serial cryostat sections (8/10 �m) of frozen in isopentane-ooled liquid nitrogen fresh skeletal muscle tissue biopsies wererocessed according to standard histological techniques, Modi-ed Gomori Trichrome (Dubowitz, 1985; Sciacco et al., 2001) andnzymatic procedures to detect ragged red fibres (RRF are specif-cally demonstrated by succinate dehydrogenase-SDH stain) andytochrome c oxidase (COX) positive or negative fibres (Dubowitz,985; Sciacco et al., 2001). Indeed in MM-affected muscle tis-ue samples, for the diagnosis of mitochondria-related myopathy,he most informative light microscopic change is the presence ofRF, which reflects massive mitochondrial proliferation, and thebsence or reduction of COX activity.

.4. ROS detection

Reactive Oxygen Species presence in human muscle tissue wasvaluated on frozen sample maintained in liquid nitrogen, as pre-iously described (Whitehead et al., 2008; Potenza et al., 2009;ozzoli et al., 2011). Dihydroethidium (DHE; Molecular Probes Lifeechnologies Inc., Monza, Italy) was used to evaluate superoxideO−

2 ) production in muscles in situ; DHE freely enters cells where, inhe presence of (O−

2 ), it is oxidized to ethidium bromide that interca-ates within nuclear DNA and emits red fluorescence in proportiono the amount of (O−

2 ) present (excitation at 488 nm, emissiont 610 nm). We already reported ROS determination in differentxperimental situations (Polimeno et al., 2009b, 2011, 2012) ande know that the fluorescent emission of DHE is not the easiestethod available but, considering the scarcity of muscle tissue fromM patient, to our knowledge, this is the most affordable method

o directly detect ROS level in fresh human tissue.Briefly reporting the experimental procedure, unfixed frozen

uscles were cut into 10-�m-thick cross-sections in cryostat andlaced on poly-L-lysine coated microscope slides (Polysciencesurope GmbH, D-69214 Eppelheim, Germany). Sections were

re-hydrated with PBS (10 min) and then incubated with DHE2 �M, 30 min, 37 ◦C) in a dark, humidified chamber. The sectionsere subsequently washed, stained with TO-PRO®-3 (Invitrogen

rl, Monza, Italy) diluted 1:7000 in PBS for 20 min for nuclear

mistry & Cell Biology 45 (2013) 2410– 2419

staining. The slides were then mounted with Fluoromount K024,an anti-fading agent (Diagnostic BioSystems, Pleasanton, CA, USA),and analyzed with the confocal microscope Leica TCS SP2 (LeicaMicrosystems Srl, Milan, Italy). We repeated the experiments threetimes. The red colour identifies ethidium bromide presence andthe blue colour the nuclei.

2.5. Bcl-2 determination

Bcl-2 expressions were evaluated using immunohistochemicalanalysis. Bcl-2 presence in tissue sections was detected using aspecific monoclonal mouse anti-Bcl-2 antibody (NCL-bcl-2, raisedagainst amino acids 41–55 of human Bcl-2), purchased from Novo-castra Laboratories (Newcastle, UK). Six-�m-thick human frozenmuscle slides were fixed in acetone for 10′. Endogenous perox-idase was blocked in H2O2/methanol 1:10, v/v for 7′ at roomtemperature. After three washes in PBS (pH 7.4) (Sigma–Aldrich,Milan, Italy), sections were incubated in a solution of 3% GoatSerum (Sigma–Aldrich) in PBS (blocking solution) for 30′ at roomtemperature. Sections were then incubated in blocking solutioncontaining the mouse monoclonal Bcl-2 antibody diluted 1:100 at4 ◦C overnight. Sections were then washed three times for 5′ in PBSand incubated 1 hr at room temperature with the EnVision SystemLabelled Polymer HRP anti-mouse (Dako, Carpinteria, CA, USA). Sec-tions were then washed three times for 5′ in PBS and incubated ina solution containing 3-amino,9-ethyl-carbazole chromogen (AEC)(Vector Labs, Burlingame, CA, USA) that provides the brown chro-mogen deposition at the reaction sites on tissue. After three washesof 5′ in PBS, slides were mounted. Slides were considered positivefor this reaction when a brown colour was developed. Appropriatecontrols to verify the specificity of Bcl-2 identification were doneincubating the tissue samples only in the presence of secondaryantibody.

2.6. ALR evaluations

2.6.1. Immunofluorescence microscopyFor the immunefluorescent analysis five-�m-thick tissue slides

were deparaffinised in xylene for 1hr and rehydrated in decreasingconcentrations of ethyl alcohol, in water and finally in PBS. Therehydrated tissue sections were microwave-pretreated in pH 6.0citrate buffer for three of five-minute cycles for antigen retrieval.The tissue sections were then treated with blocking solution (1%normal goat serum in PBS) for 30 min at room temperature, andsuccessively incubated, at 4 ◦C, overnight with a polyclonal rabbitanti-ALR antibody (MultiBind GmbH, Koln, Germany) diluted 1:200in the blocking solution. This antibody has been already demon-strated to be able to immunodetect ALR (Klissenbauer et al., 2002;Thasler et al., 2005; Polimeno et al., 2009a, 2009b, 2011). After sev-eral rinses in PBS, tissue sections were incubated with secondaryantibody anti-rabbit ALEXA Fluor 488 (Molecular Probes, Eugene,Ore, USA) diluted 1:500 for 5 h at room temperature and withTO-PRO®-3 diluted 1:7000 in PBS for 20 min for nuclear staining.Subsequently the tissue sections were mounted with an anti-fadingagent (Fluoromount K024, Diagnostic BioSystems, Pleasanton, CA,USA) and analyzed with the confocal microscope Leica TCS SP2(Leica Microsystems, Wetzlar, Germany). To verify the specificityof the immunoreaction, appropriate controls were performed incu-bating the cells with the blocking solution short of the ALR specificprimary antibody or with only the secondary antibody or using thepre-immune rabbit serum as primary antibody. We repeated theexperiments three times. The green colour identifies ALR immuno-detection and the blue colour the nuclei.

2.6.2. Immunoelectron microscopyImmunogold labelling of ultrathin sections was conducted using

a rabbit polyclonal anti-ALR, diluted 1:350 in PBS (MultiBind GmbH,

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ig. 2. Electron micrographs of non-pathological (28,000× magnifications) and myoissue samples considered.

öln, Germany), as primary antiserum. The secondary antiserumas 10 nm gold conjugate rabbit antiserum (Sigma–Aldrich, Milan,

taly) diluted 1:10 in PBS. Briefly, thin tissue sections were incu-ated in blocking solution, consisting of saline phosphate bufferH 7.4 added with 2% bovine serum albumin (BSA, Sigma–Aldrich,ilan, Italy) for 15 min at 37 ◦C, and then incubated with 2% normal

oat serum (NGS, Sigma–Aldrich, Milan, Italy) in PBS added with 2%SA for 15 min at 37 ◦C. The sections were then incubated with pri-ary antiserum for 2 hrs, washed several times with PBS and then

ncubated for 1 hr at 37 ◦C with 10 nm gold conjugated rabbit anti-erum. Grids were then washed with ddH2O, stained with uranylcetate and lead citrate and observed with a TEM Morgagni 268.ontrol samples were incubated with blocking solution plus 10 nmold conjugate rabbit antiserum.

.7. Clusterin immunofluorescence detection

Five-�m-thick tissue slides, from healthy and MM-patientsuscle tissue samples, were deparaffinised in xylene for 1hr and

ehydrated in decreasing concentrations of ethyl alcohol, in waternd finally in PBS. Antigen retrieval was performed by microwaves,ncubating the slides in buffer citrate (pH 6.0) 3 times for 3 minach at 750 W. After washing in PBS, endogenous peroxidase wasuenched with H2O2 for 10 min. Non-specific sites were blockedy incubating the slides with a blocking solution (FCS + BSA inBS) for 1 h. Slides were then incubated with the primary anti-lusterin antibody (sc-8354, Santa Cruz, Inc., Santa Cruz, CA, USA)iluted 1:100, over night at 4 ◦C. After 3 washes in PBS, slides were

ncubated for 2 h with the secondary anti-rabbit ALEXA Fluor 488Invitrogen, Molecular Probes, Eugene, OR, USA) antibody diluted:200, and after 3 washes in PBS, with TO-PRO®-3 (Invitrogen,olecular Probes, Eugene, OR, USA) diluted 1:7000 for 20 min.

inally slides were mounted with an anti-fading mounting andovered with a coverslip and observed at a confocal microscopeLeica TCS SP2, Leica Microsystems Srl, Milan, Italy). To verify thepecificity of the immunoreaction, appropriate controls were done

ncubating the cells with only the secondary Ab or using the pre-mmune rabbit serum as primary Ab. We repeated the experimentshree times. The green colour identifies the clusterin immunode-ection and the blue colour the nuclei.

es (18,000× magnifications) muscle tissues. The figures are representative of all the

2.8. Statistical analysis

The data reported are expressed as mean ± standard deviation(M ± S.D.). Statistical comparison among groups was determinedusing analysis of variance (ANOVA). Where indicated, individualcomparisons were performed using Student’s t test. Statistical sig-nificance was ascribed to the data when p < 0.05. If not specificallyreported, each datum is representative of at least three differentand separated experiments.

3. Results

Fig. 1 reports the identification of pathological fibres charac-teristic of muscle tissue from MM-affected patients. Enzymatic-histochemical staining of SDH activity revealed “ragged bluefibres” (upper part). In the lower part of the Figure the diagno-sis of mitochondrial diseases is confirmed by a modified Gomoritrichrome histochemical stain which allows the detection of abnor-mal deposits in mitochondria by light microscopy, revealing in thesubsarcolemmal region abnormal fibres that stain red, the so-called‘ragged red fibres’ (red arrows), which are considered the distin-guishing morphological features of mitochondrial myopathies. Inthe same slide the COX negative muscle fibres are identified (blackarrows).

Fig. 2 reports the electron microscopy observations foundin human muscle tissue from the two groups of subjects. Innon-pathological muscle fibres (28,000 × magnifications), eachmyofibril with its repetitive sequence of sarcomeres is sur-rounded by specialized cytoplasm, the sarcoplasm. It containsseveral organelles, including mitochondria (arrows), the sarcoplas-mic reticulum and the T-tubule membrane systems, the Golgiapparatus and a cytoskeleton of microtubules, intermediate fila-ments and actin microfilaments, as well as glycogen (15–30 nmin size), free ribosomes, lipid droplets and lipofuscin. RegardingMM-derived muscle tissues, it is known that MM are a complexand heterogeneous group of neuromuscular disorders, in whichabnormalities of mtDNA and mitochondrial metabolic function

may be associated to structural abnormalities in the mitochondria(arrows). Our observation revealed mitochondria to be particu-larly numerous, mainly located in the sub-sarcolemmal space, butalso present within fibres, different in size and shape, sometimes

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ig. 3. ROS immunodetection in MM- and non-pathological tissue samples. The fluormed by the amount of the (O−

2 ) present in the muscle fibres. The blue colour is re

ssuming bizarre and/or giant forms and containing characteris-ic paracrystalline inclusions. In the basal cytoplasm, numerouslectron-lucent lipid droplets were frequently found (18,000×agnifications).Fig. 3 reports the red fluorescence signal of ROS determined

y confocal microscopy in non-pathological and in MM-affecteduscle tissue. A very weak red signal was evidenced in the

on-pathological fibres, soften dispersed in the cytoplasmarrows), instead an important and stable red signal is evident

Fig. 4. Clusterin immuno-detection (green colour) in MM and non-pat

ent red signal is determined by the presence of ethidium bromide, proportionally to the nuclei.

in muscle fibres from MM-affected subjects, detected in thecytoplasm such as in the nucleus (arrows).

Fig. 4 reports the immuno-detection of clusterin, determinedby confocal microscopy, in non-pathological muscle tissue and inMM-patients muscle tissue. A very weak fluorescent green signal

was detected in non-pathological tissue, soften dispersed in thecytoplasm (arrows), that certainly does not co-localizes with thenuclear blue signal. On the contrary a strong green signal is evidentin the muscle tissue from MM-affected patients which is specifically

hological muscle tissues. The blue colour is related to the nuclei.

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ig. 5. Immunofluorescence micrographs of ALR-dependent reaction on pathologepresentative of all the tissue samples considered.

resent in the nucleus (arrows) supporting the idea of the presencef the nuclear isoform of clusterin (nClu) in MM-affected patientsuscle tissue.Fig. 5 reports the ALR immuno-detection in the muscle tis-

ue from the two groups of subjects (400× magnifications). Themmunoreaction is more evident in pathological than in nonathological muscle fibres. Immunoreactivity is present in the sub-arcolemmal space and cytoplasm in both fibres I and II. Labellingas not observed when the primary antibody was substituted

ith PBS, nor when antibodies used were pre-adsorbed with their

espective antigens, neither when control immunostain were done.Fig. 6 reports electron microscopy ALR-related immuno-

old markers in non-pathological (56,000× magnifications) and

ig. 6. Electron micrographs from non-pathological or mitochondrial-related myopathiLR-dependent immunoreactions (56,000× magnifications).

d non-pathological skeletal muscle tissue (400× magnifications). The figures are

pathological (56,000× magnifications) muscle tissue. In bothgroups the ALR presence was detected, much more evident in theMM-derived muscle tissue. The gold marker is localized in the IMSand on mitochondrial cristae, as well as in the cytoplasm.

Fig. 7 reports a statistical analysis of the gold ALR-related par-ticles observed, by EM, in the muscle tissue samples from thetwo groups of subjects. A mean value of 6.4 ± 3.1 of gold par-ticles/mitochondrion was detected in the inner mitochondrialmembrane of non-pathological muscular tissue. On the other

hand, a statistically significant (p < 0.01) increased level (24.5 ± 6.7gold particles/mitochondrion) of gold particles was found in themitochondrial inter-membrane space (IMS) of mitochondria ofMM-derived human muscle tissue. The data are from a random

es human muscle tissue. The black points (gold markers) are determined by the

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ig. 7. Statistical evaluation of the gold, ALR-dependent, markers observed in non-athological and mitochondria-related myopathies human muscle tissue.

valuation of 100 mitochondria of each experimental group carriedut blindly by two different pathologists.

Fig. 8 reports the Bcl-2 evaluation in MM- (Fig. 8A) and in non-athological-derived (Fig. 8B) muscle tissue. A clear over-presencef brown particles (arrows), determined by the Bcl-2 expression,

ig. 8. Immunohistochemical identification of Bcl-2 protein (dark brown colour,rrows) in MM- and non-pathological-derived human tissue (20× magnifications).he figures are representative of all the tissue samples considered.

mistry & Cell Biology 45 (2013) 2410– 2419

was detected in MM-derived muscle fibres compared to the non-pathological muscle tissue (20× magnifications).

4. Discussion

The relationship between the functions and biogenesis ofmitochondria and ALR, a factor phylogenetically preserved fromyeast to mammals (Polimeno et al., 1999), is experimentally provedand universally accepted (Lisowsky et al., 1994; Polimeno et al.,1999, 2000; Lange et al., 2001; Allen et al., 2005; Polimeno et al.,2009b, 2012; Kallergi et al., 2012). Indeed the molecular similar-ity between ALR and ERV1, a protein present in yeast, essential forcell viability and for mitochondrial biogenesis and functionality,allows to ALR to restore the biological functions and the mitochon-drial integrity of ERV1-depleted yeast (Lisowsky, 1994; Polimenoet al., 1999). Since the beginning, these data supported the ideathat, like ERV1, ALR is a factor necessary for cell survival, princi-pally preserving mitochondrial structure and functions (Lisowsky,1994; Polimeno et al., 2009b, 2012). Furthermore both proteins, incooperation with Mia40 (Allen et al., 2005), improve mitochondrialcrystal structure and support mitochondrial protein import into theintermembrane space as well as iron-sulphur cluster assembly inthe cytosol (Lange et al., 2001; Kallergi et al., 2012).

In 1994 our group isolated and sequenced ALR which, as thename suggests, was at first thought to be a factor that stimu-lates hepatocyte proliferation (Francavilla et al., 1994, Hagiya et al.,1994). Later we demonstrated other functions of ALR in the liveras in other tissues: e.g. in mitochondria isolated from intact liverof ALR-treated rats, ALR stimulates ATP production, oxygen con-sumption and induces mtTFA expression, a factor that regulatesmitochondrial DNA (Polimeno et al., 2000). Successive studies con-firmed our data adding new discoveries (Thirunavukkarasu et al.,2008; Polimeno et al., 2009b; Cao et al., 2009; Todd et al., 2010;Liao et al., 2010; Polimeno et al., 2011, 2012; Gandhi, 2012). In arecent paper we reported, in healthy human muscle tissues, thepresence of ALR in the mitochondrial intermembrane space (IMS)close to the mitochondrial cristae (Polimeno et al., 2009a). Theidentification of ALR in the IMS of eukaryotic cells strongly jus-tifies and supports the importance of such factor, as well as forthe yeast Erv1, on the mitochondrial biogenesis, to which ALR par-ticipates favouring the import and folding, in the IMS, of proteincontaining specific SH-spacer-SH sequence. It has recently evi-denced that protein oxidative folding, through cysteine oxidation,occurs also in the IMS and not only in the eukaryotic endoplas-mic reticulum or in the bacterial periplasmatic space (Deponteand Hell, 2009; Endo and Yamano, 2009; Riemer et al., 2011) andthat, in this process, a central role is played by the oxidoreductasiMia40 (Mitochondrial Intermembrane space Import and Assem-bly) protein catalyzing the formation of disulfide bonds into targetprotein. A new class of FAD-linked sulfhydryl oxidase (Evr1/ALR)restores the functional state of Mia40 leading to its participation toa new reaction cycle (Kallergi et al., 2012; Fraga and Ventura, 2013).Furthermore, in a recent report, the group of Chacinska demon-strated that human ALR is a factor that controls not only the abilityof Mia40 to bind and oxidize protein clients, but also the local-ization of human Mia40 in the mitochondria (Sztolsztener et al.,2013).

Two effects of ALR are currently shared by different authors andexperimentally supported: (1) the anti-apoptotic effect, with theinduction of anti-apoptotic factors, such as Bcl-2, and the inhibitionof pro-apoptotic factors, like Bax, and (2) the anti-oxidative effect,

due to the sulfhydryl-oxidase enzymatic activity of ALR (Lisowskyet al., 2001) and to its capacity to stimulate the expression ofsecreted clusterin isoform (sClu), a powerful anti-oxidant agent(Polimeno et al., 2009b, 2011, 2012).

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Fig. 9. Biological effects of ROS, ALR and n

The importance of ALR/ERV1 in the biogenensis and regula-ion of mitochondrial function has been recently demonstratedlso in humans by the identification of a muscle disease linkedo the alteration of GFER, the codifying gene for ALR (Di Fonzot al., 2009). These subjects show, in muscle tissue, a low con-ent of cysteine-rich proteins with appearance of mitochondrialisruption. Furthermore, Lamperti identified, in a heterogeneusroup of human muscle diseases, GFER as the gene involved in aitochondrial DNA depletion syndrome. These disorders are char-

cterized by a myopatic form and a fatal infantile hepato-cerebralyndrome which rapidly leads to progressive liver and brain fail-re (Lamperti and Zeviani, 2009). To our knowledge, no data arevailable on the physiological role of ALR in the muscle tissue inealthy and/or in pathological conditions. In this study, in order tovaluate the role of three factors (ALR, ROS and clusterin) involvedn the apoptotic process and in the oxidative stress, character-stic of mitochondria-related myopathies, we determined theirxpression in muscle tissue samples from MM-affected patients.ur study revealed: (i) an induction of oxidative factors (ROS,ig. 3) and an apoptosis-inducing factor (nClu, Fig. 4), and (ii) thever-expression of ALR, a new anti-apoptotic/anti-oxidative factorFig. 5) and of Bcl-2 (Fig. 8). Furthermore, we directly demon-trated, for the first time, the presence of ROS in human muscleissue (Fig. 3). So far, to our knowledge, ROS expression in theseathologies has been only indirectly demonstrated mainly asso-iated to the induction of detoxifying factors like SOD, GSHPx orn and Cu/Zn SOD (Esposito et al., 1999). Some authors reported,

n muscle tissue from mitochondrial myopathy affected patients:i) increased SOD immunostaining in COX-deficient myofibres, (ii)ntense Mn and Cu/Zn SOD immunostaining, (iii) mtDNA muta-ions and (iv) respiratory chain defects (Ohkoshi et al., 1995; Mitsuit al., 1996; Carrier et al., 2000). These data strongly indicate, evenf indirectly, a linkage between COX defect and over-expressionf oxidant agents, which could induce ALR over-production as a

rotective need against the oxidative stress and related apoptosisPolimeno et al., 2009b, 2012). Previous reports demonstrated anncreased susceptibility to apoptosis in muscle tissue from patients

ith mitochondrial DNA (mtDNA) disorders included the MM (Auré

n mitochondrial myopathy muscle tissue.

et al., 2006). Aurè and co-workers reported the analysis of apop-totic features in parallel to cytochrome c oxidase and succinatedehydrogenase activity showing that apoptosis occurred only inmuscle fibres with mitochondrial proliferation (ragged red fibres).This observation gives a sense to the different levels of ALR expres-sion in the muscle fibres from MM-affected patients (Fig. 5, lowerpart). It has been already demonstrated a similar mosaicism forALR in non-pathological human muscle tissue associated to a differ-ent number of mitochondria as occurs in type I and type II musclefibres (Polimeno et al., 2009a). To this purpose it could be con-venient to remind that ALR mainly localizes in the mitochondrialinter-membrane space (Yang et al., 1997).

We believe that the increased ALR expression in muscle fibresfrom MM-affected patients (Figs. 5, 6 and 7) could be, as for theanti-oxidative agents, a protective need, maybe associated also tothe presence of nClu, against the pro-apoptotic susceptibility ofmuscle tissue derived from MM-affected patients, process in whichthe nClu pro-apoptotic factor seems to be involved. All these datatogether support and in part clarify the metabolic pathway of theapoptotic process which takes place in the muscle tissue of MM-affected subjects, underlining the hypothesis that apoptosis mostprobably contributes to mitochondrial pathology.

A different interpretation of the present data finds its support inour personal data, submitted recently for publication, in which weshow, in human-derived colon cancer tissue, a different behaviourof anti-apoptotic (ALR and STAT3 – Signal Transducer and Activa-tor Factor) and pro-apoptotic factors (PIAS3 – Protein Inhibitor ofActivated STAT) in relation to the degree of tissue differentiationfrom benign (histological grade G1) to malignant tumour (histo-logical grade G3). Indeed in colorectal tissues samples we observedthat ALR and STAT3 are highly expressed and are accompanied bya low expression of PIAS3, when tissues are from patients with G1degree of disease, while in colorectal tissue samples from patientswith G3 degree of disease this relationship changes showing an

higher presence of PIAS3 and a significantly decreased presence ofALR and STAT3. We sustain the idea that, since apoptosis representsthe end of cell life, the balance between pro- and anti-apoptotic fac-tors is an important tool in health as in pathological conditions. We

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ypothesize that, as it happens in tumours, in which genetic alter-tions play a crucial role in determining the stage of the disease,imilar biochemical behaviour may realize in muscle tissue fromM-affected patients, certainly determined by the complexity of

enetic alterations to which these tissues are subjected.We are aware that our present data may lack of a complete eval-

ation of a pro- and anti-apoptotic agents in the MM muscle tissue,ut the small quantity of human tissue and ethical reasons make

t difficult to realize. However the detection of the nClu isoform, aro-apoptotic factor (Kim and Choi, 2011) in the nucleus of MM-ffected muscle tissue is a clear index of a pro-apoptotic impulsen these muscle tissues. On the other hand, in tissues in which biol-gy is completely subverted and cells are dying, it is easy to findro- and anti-apoptotic gene expression subverted, compared toealthy tissues (Ouyang et al., 2012).

In conclusion we suggest that the up-regulation of ALR and Bcl-2,videnced in muscle tissues of patients affected by MM, representsn attempt to compensate the pro-apoptotic stimulus of nClu andhe oxidative stress (Fig. 9), typical features of MM pathologiesOhkoshi et al., 1995; Mitsui et al., 1996; Carrier et al., 2000) andertainly determinants for the ethiopathogenesis of the disease.

onflict of interest

No actual or potential conflict of interest.

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