,1 ,1

*DIFAR, University of Genoa, Italy

†DINOGMI, University of Genoa, Italy

AbstractMyelin sheath is the proteolipid membrane wrapping the axonsof CNS and PNS. We have shown data suggesting that CNSmyelin conducts oxidative phosphorylation (OXPHOS), chal-lenging its role in limiting the axonal energy expenditure. Here,we focused on PNS myelin. Samples were: (i) isolated myelinvesicles (IMV) from sciatic nerves, (ii) mitochondria fromprimary Schwann cell cultures, and (iii) sciatic nerve sections,from wild type or Charcot-Marie-Tooth type 1A (CMT1A) rats.The latter used as a model of dys-demyelination. O2 con-sumption and activity of OXPHOS proteins from wild type (Wt)or CMT1A sciatic nerves showed some differences. In partic-ular, O2 consumption by IMV from Wt and CMT1A 1-month-oldrats was comparable, while it was severely impaired in IMV

from adult affected animals. Mitochondria extracted fromCMT1A Schwann cell did not show any dysfunction. Trans-mission electron microscopy studies demonstrated anincreased mitochondrial density in dys-demyelinated axons,as to compensate for the loss of respiration by myelin.Confocal immunohistochemistry showed the expression ofOXPHOS proteins in the myelin sheath, both in Wt and dys-demyelinated nerves. These revealed an abnormal morphol-ogy. Taken together these results support the idea that alsoPNS myelin conducts OXPHOS to sustain axonal function.Keywords: bioenergetics, FoF1-ATP synthase, myelin,OXPHOS.J. Neurochem. (2013) 10.1111/jnc.12253

The Nervous System (NS) displays a great energy demand,and critically depends on oxygen (O2) supply (Ames 2000).Faster transmitting axons are surrounded by myelin, amultilamellar membrane produced by highly specializedglial cells, i.e., Schwann cells (SC) in PNS and oligoden-drocytes in CNS. Besides being an insulating wrap, myelinhas been proposed to play a trophic role, as its loss causesaxonal degeneration (Ferguson et al. 1997; Trapp and Nave2008). We have recently proposed the existence of anextramitochondrial aerobic metabolism in myelin frombovine forebrain (Ravera et al. 2011, 2009; Morelli et al.2011; and in retinal rod outer segment disks (Panfoli et al.2008, 2009), both devoid of mitochondria. The expression ofmitochondria-derived proteins of the oxidative phosphoryla-tion (OXPHOS) was reported in many proteomic analyses ofCNS (Taylor et al. 2004),(Vanrobaeys et al. 2005; Werneret al. 2007; Yamaguchi et al. 2008; Ishii et al. 2009; Jahnet al. 2009) and in the recent analysis of PNS myelinproteome (Patzig et al. 2011). The presence of proteins

involved in aerobic respiration in the myelin fraction wasalso reported by large-scale proteomics screening of cere-brospinal fluid children during initial presentation of CNSinflammation (Dhaunchak et al. 2012). Consistently, it hasbeen suggested that CNS myelin would facilitate nerveimpulse transmission supporting the axonal mitochondria in

Received November 28, 2012; revised manuscript received March 13,2013; accepted March 14, 2013.Address correspondence and reprint requests to Prof Isabella Panfoli,

University of Genova, Faculty of Sciences, Department of Pharmacy-Biochemistry Lab, Viale Benedetto XV, 3, 16132 Genova.E-mail: Isabella.Panfoli@unige.it1These authors equally contributed to this work.Abbreviations used: ANT, adenosine nucleotide translocase; CMT1A,

Charcot-Marie-Tooth type 1A; COX II, cytochrome c oxydase, subunitII; EM, electron microscopy; ETC, electron transfer chain; IMV, isolatedmyelin vesicles; MPZ, myelin protein zero; OXPHOS, oxidativephosphorylation; PBS, phosphate-buffered saline; SC, Schwann cells;TIM, inner membrane translocase, subunit 8A; TOM, outer membranetranslocase, subunit 20; Wt, wild type.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12253 1

JOURNAL OF NEUROCHEMISTRY | 2013 doi: 10.1111/jnc.12253

supplying ATP rather than by limiting energy consumption(Ravera et al. 2009, 2011; Morelli et al. 2011). In thisarticle, we focused on PNS myelin to investigate whether itplays the same energetic role as the CNS one. A transgenicrat, reproducing Charcot-Marie-Tooth type 1A (CMT1A)disease, was utilized as a model of dys-demyelination(Sereda et al. 1996). Isolated myelin vesicles (IMV) fromrat sciatic nerves were assayed for their respiratory propertiesby biochemical and confocal scanning or transmissionelectron microscopy techniques. IMV from the CMT1Asciatic nerves lose the capacity to synthesize ATP andconsume oxygen, with time. Moreover, an increment inmitochondrial density was observed in dys-demyelinatedaxons. Overall data are consistent with the idea of a pivotalrole of myelin, besides axonal mitochondria, in conductingoxidative phosphorylation in PNS, and its impairment in amodel of dys-demyelination.

Materials and methods

Antibodies

Primary antibodies (Ab) were: monoclonal antibody against myelinprotein zero (MPZ) (P07 extracellular domain, Astexx Ltd. & Co.KEG, Graz, Austria); polyclonal antibodies against: adenosinenucleotide translocase (ANT) (Q-18, sc-9300 Santa Cruz Biotech-nology, Santa Cruz, CA, USA); mitochondrial import inner mem-brane translocase, subunit 8A (TIM) (N-20, sc-17052, Santa CruzBiotechnology); mitochondrial import outer membrane translocase,subunit 20 (TOM) (C-20, sc-11021, Santa Cruz Biotechnology);FoF1-ATP synthase a-subunit (A9585, Sigma-Aldrich, St. Louis,MO, USA); NADH-ubiquinone oxidoreductase subunit (ND4L)(H-94, sc-20665, Santa Cruz Biotechnology), and cytochrome coxydase, subunit II (COX II) (Q-18, sc-23983, Santa Cruz Biotech-nology). Secondary Ab for immunofluorescence were: Alexa-488-conjugated anti-rabbit or anti-goat (Molecular Probes, Grand Island,NY, USA) or Cy3-conjugated anti mouse (Sigma-Aldrich). Horse-radish peroxidase-conjugated mouse or rabbit IgG Ab (Santa CruzBiotechnology) were used for western blot experiments;

Animal model

Transgenic Sprague–Dawley rats over-expressing PMP22 (CMT1Arats) were genotyped by PCR (Sereda et al. 1996). Heterozygousanimals and normal age-matched littermates were used for exper-iments. Rearing conditions were consistent with the guidelines ofthe Italian Health Ministry relating to the use and storage oftransgenic organisms. Experiments were carried out in compliancewith animal care requirements requested by Italian law (law D.L.27.1.1992 n. 116, in agreement with the European Union directive86/609/CEE).

Primary Schwann cell cultures

SC for primary cultures were isolated from 3-day-old rat sciaticnerves, as previously described (Brockes et al. 1979; Nobbio et al.2004). SC were grown in Dulbecco’s modified Eagle’s medium/F12medium (Invitrogen, Grand Island, NY, USA) supplemented with10% fetal calf serum (FCS), penicillin and streptomycin for 48 h.

Cytosine arabinoside (Ara-C) 10�5 M was added after 48 h and thetreatment prolonged for further 48 h resulting in SC cultures thatwere 99% pure and were used for biochemical experiments.

Isolation of Schwann cell mitochondria-enriched fractions

Mitochondria-enriched fractions from SC cultures were isolated bystandard differential centrifugation techniques, as described (Raveraet al. 2007). SC were homogenated in 0.25 M sucrose, 5 mM N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid (HEPES) pH 7.2,protease inhibitor cocktail, 30 nM Cyclosporin A, 30 nM 2-Aminoethoxydiphenyl borate and 0.060 mg/mL Ampicillin, andcentrifuged at 700 g for 10 min, in Heraeus centrifuge. Pellet wasdiscarded, and supernatant centrifuged at 10 900 g for 10 min.Pellet was retained as a mitochondria-enriched preparation, storedon ice and used within 4 h of isolation. All steps were performed at4°C.

Myelin isolation

IMV were purified from sciatic nerves of 1- or 12-month-old wildtype (Wt) or CMT1A rats by a modification of the method of Nortonand Poduslo (Haley et al. 1981; Larocca and Norton 2007; Nortonand Poduslo 1973). Briefly, sciatic nerves were homogenized by aPotter-Elvehjem procedure in 20 vol. (w/v) of 0.32 M sucrose in2 mM EGTA and 100 mM Tris-HCl pH7.5. Myelin enrichedfractions were obtained by differential centrifugation after hypotonicshock of the nerve homogenate. The sciatic nerve homogenate waslayered onto 0.85 M sucrose in 2 mM EGTA and centrifuged at75 000 g for 30 min in a TL 100 ultracentrifuge (Beckman Coulter,Brea, CA, USA). Protease inhibitor cocktail (Sigma-Aldrich),30 lg/mL 5-fluorouracil, and 20 lg/mL ampicillin were presentthroughout. Oxygen consumption and ATP synthesis were observedin the absence of mitochondrial permeability transition poreinhibitors (Berman et al. 2000), as cyclosporin A or 2-amin-oethoxydiphenyl borate (Chinopoulos et al. 2003; Crompton et al.1988). The method utilized to obtain IMV minimizes contamination.In fact, during the first step the membrane fraction is separated fromthe cellular organelles that are found in the precipitate. Moreover,shocks in distilled water eliminate the contaminating mitochondria,if any, both physically and functionally. In fact, we have shown thatmitochondria subjected to hypotonic treatment do not respire norsynthesize ATP (Panfoli et al. 2009).

Western blot

Equal protein amounts (20 lg) from both Wt and transgenic animalsamples were loaded onto gradient 8–12% polyacrylamide gels andseparated by sodium dodecyl sulfate–polyacrylamide gel electropho-resis. Proteins were transferred to nitrocellulose membranes (Amer-sham Biosciences, Glattbrugg, Switzerland) and probed overnightwith primary antibodies against MBP (1 : 10 000); PMP22(1 : 200); MPZ (1 : 400); ANT (1 : 400); TIM (1 : 200); TOM(1 : 200); Adenylate kinase, isoform 3 (AK3) (1 : 200). SecondaryHorseradish peroxidase-conjugated Abs were diluted in phosphate-buffered saline (PBS) (1 : 5000 for anti-mouse IgG; 1 : 7500 foranti-rabbit and anti-goat IgG). Bands were visualized and quantifiedby ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA,USA). Total protein content of mitochondria-enriched fractions IMVor sciatic nerves homogenates was determined by Bradford method,using bovine serum albumin as a standard protein.

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2 S. Ravera et al.

Adenylate kinase, isoform 3 assay

Spectrophotometric assay of AK3, a protein typical of mitochondrialmatrix, was performed according to Tomasselli et al. (1979).

Mitochondrial DNA quantification in IMV

To assess the presence/absence of mitochondrial DNA, a PCRanalysis against a typical sequence of bovine mitochondrial DNAwas carried out. The primer sequences were designed by the on-linePrimer3 software program (Rozen and Skaletsky, 2000) and were:forward 5′-GCTAAGACCCAAACTGGGATT-3′, reverse 5′-AG-CCCATTTCTTCCCATTTC-3′. Amplification reactions werecarried out in a total volume of 20 lL, in a reaction mixturecontaining 5 9 PCR-Buffer, 2 mM MgCl2, 1 pmol of each primer,0.2 mM deoxynucleoside triphosphates, and 1 U of AmpliTaqDNA polymerase (PerkinElmer, Emeryville, CA, USA). Thesamples were subjected to a PCR program consisting of 1 cycle at95°C for 2 min, then 12–27 cycles at 95°C for 30 s for denaturing,56°C for 45 s for annealing, and 72°C for 30 s for extension, finally1 cycle at 72°C for 7 min. The PCR products were separated onto1% agarose gels in Tris-acetate-EDTA buffer and stained withethidium bromide (0.4 mg/mL).

Oxygraphic measurements

O2 consumption by IMVwas assayed in a thermostatically controlledoxygraph equipped with an amperometric electrode (Unisense –Microrespiration, Unisense A/S, Copenhagen, Denmark), accordingto manufacturer’s instructions, as previously described (Ravera et al.2009). To observe uncoupled respiration rates, 30 lM nigericin wasadded before the addition of 0.7 mM NADH (to stimulate the ETC. I+ III + IV) and 20 mM succinate (to stimulate the ETC. II +III +IV).To verify that the oxygen consumption was because of the electrontransfer chain (ETC.) activity, 40 lM rotenone (ETC. I inhibitor) and50 lM Antimycin A (ETC. III inhibitor) were added. Respiratoryrates are expressed as lM O2/min/mg.

Analysis of respiratory complexes

The ETC. complexes were tested on IMV by spectrophotometricassays: ETC. I (NADH-ubiquinone oxidoreductase) following thereduction of ferricyanide at 420 nm (Sottocasa et al. 1967); ETC. II(Succinic dehydrogenase) following the reduction of dichloroind-ophenol at 600 nm, (Janssen et al. 2007); ETC. III (cytochrome creductase) following the reduction of oxidized cytochrome c at550 nm (Sottocasa et al. 1967); ETC. IV (cytochrome c oxydase)following the oxidation of Ascorbate-reduced cytochrome c at550 nm (Baracca et al. 2003).

ATP synthesis assay

ATP synthesis by IMV was assayed by the luciferin/luciferasechemiluminescent method (Roche Applied Science, Indianapolis,IN, USA), according to Sgarbi et al. (2006). Samples (0.01 mgprotein/mL) were incubated for 5 min at 37°C in 10 mM Tris/HCl(pH 7.4), 100 mM KCl, 1 mM EGTA, 2.5 mM EDTA, 1 mMNaH2PO4, 2 mM MgCl2, 0.7 mM NADH, 20 mM succinate and25 lg/mL ampicillin. To inhibit adenylate kinase and Na+/K+

ATPase activity, 0.2 mM di(adenosine-5′) penta-phosphate and0.6 mM ouabain were added, respectively. ATP synthesis wasinduced by adding 0.5 mM ADP to the sample, at the same pH ofthe mixture. 10 lM Oligomycin, 0.5 mM potassium cyanide or

50 lM antimycin A (Ravera et al. 2009) were added to theincubation mixture as inhibitors. ATP standard solutions between10�9 and 10�7 M were used for calibration (Roche DiagnosticsCorp., Indianapolis, IN, USA).

Immunohistochemistry

Sciatic nerves were fixed in 4% paraformaldehyde in PBS (pH 7.4)for 12 h, then dehydrated and embedded in Paraplast. Transversalsections (5 lm thick) were cut, rehydrated, dewaxed, and incubatedwith rabbit antibodies against FoF1-ATP synthase (1 : 100), ND4L(1 : 100) or COX II (1 : 100) together with a mouse antibodyagainst MPZ, in a moist chamber at 4°C, overnight. After washing,sections were incubated with the secondary antibodies: Alexa-488-conjugated anti-rabbit and Cy3-conjugated anti-mouse. Sectionswere then washed in PBS, mounted in a glycerol/PBS (1 : 1)solution, and examined under an inverted Leica TCS SP5 AOBSconfocal laser scanning microscope, equipped with a Leica 63XPLAPO NA 1.40 oil immersion objective (Leica Microsystems CMS,Mannheim, Germany).

Electron microscopy

For transmission electron microscopy (EM) examination, 6-month-old CMT1A rats were killed and sciatic nerves were quicklyremoved. Specimens were fixed in 2.5% glutaraldehyde in cacody-late buffer, pH 7.4, for 2 h, post-fixed with 1% osmium tetroxide incacodylate buffer, pH 7.4, for 1 h, dehydrated in alcohol andembedded in epoxy resin. Ultrathin sections, stained with 5% uranylacetate and lead citrate, were then prepared from these specimens,and examined under a Zeiss EM 109 (Oberkochen, Germany).

Statistical analysis

Data are expressed as Mean � SD Differences between groupswere determined using unpaired Student’s t-test. Multiple-parametercomparisons were performed by one-way ANOVA followed byBonferroni’s post hoc test. A value of p < 0.05 was consideredstatistically significant. GraphPad Prism 5.0 Software (GraphPadSoftware Inc., La Jolla, CA, USA) was used for statistical analysis.

Results

Characterization of IMV from sciatic nerve

The method utilized to purify myelin from sciatic nerves(Larocca and Norton 2007) IMV by hypotonic shock of thehomogenate, minimizing contamination by subcellular orga-nelle. In fact the membrane fraction and the organelle lay indifferent layers of the sucrose step gradient (see Material andMethods). Fig. 1 shows a characterization of the IMVpurified from Wt sciatic nerves from 12-month-old ratsconducted by semiquantitative WB analysis. Panel (a) showsthe protein pattern of mitochondria-enriched fractions (lane1) sciatic nerve homogenate (lane 2) and IMV (lane 3), asstained with Colloidal Blue Coomassie. SemiquantitativeWB analyses were conducted with antibodies against MBP(Panel b) a marker of myelin; PMP22 (Panel c) and MPZ(Panel d), markers of peripheral myelin; ANT (Panel e) andTIM (Panel f), two proteins typical of the inner mitochondrialmembrane; TOM (Panel g), a marker of the outer

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Oxidative phosphorylation in PNS myelin 3

mitochondrial membrane; AK3 (Panel h), a protein typical ofmitochondrial matrix. The intensity of the chemiluminescentWB signal of myelin markers increased in parallel to theisolation of myelin. In contrast, in IMV the signal of typicalmitochondrial proteins is absent. Data are confirmed by thedensitometric analysis of chemiluminescent signal, reportedin Panel (i). To confirm the absence of mitochondrial matrixproteins, AK3 was also assayed. Panel (j) shows that AK3activity was present in mitochondria-enriched fractions andin the pellet obtained after the first step of WT or CMT1Amyelin isolation (composed by nuclei, mitochondria, andcellular debris), decreased in sciatic nerve homogenate andwas absent in isolated myelin fractions.

To demonstrate the absence of mitochondrial DNA inisolated myelin vesicles, a PCR analysis was performed,using primers specific for a mitochondrial DNA sequence(see Material and Methods). Panel (k) shows that it wasalready possible to observe the signal after 10 cycles ofamplification exclusively in the mitochondrial sample. Incontrast, in IMV the signal was still absent after 30 cycles.

OXPHOS proteins are functional in IMV from Wt rats, but

impaired in a model of peripheral dys-demyelination

Having reported that CNS myelin expresses the fivecomplexes of respiration and is able to consume O2

(Ravera et al. 2009), we tested IMV from sciatic nerve of

(a)

(i)

(j)

(k)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Fig. 1 Characterization of rat sciatic nerve-

derived fractions. Figure shows thesemiquantitative WB analysis of typicalmyelin proteins, as MBP, PMP22, myelin

protein zero (MPZ) and typical mitochon-drial proteins, as adenosine nucleotidetranslocase (ANT), inner membrane tran-slocase, subunit 8A (TIM), outer mem-

brane translocase, subunit 20 (TOM) andAdenylate kinase, isoform 3 (AK3) inmitochondria-enriched fractions (lane 1),

sciatic nerve homogenate (lane 2) andisolated myelin vesicles (IMV, lane 3) from12-month-old wild type (Wt) rats. Panel (a)

reports the protein pattern stained withColloidal Blue Coomassie. In panels (b–h)WB analysis against MBP, PMP22, MPZ,

ANT,TIM, TOM and AK3 is shown,respectively. The densitometric analysis ofWB signals is reported in Panel (i). Tofurther assess the absence of contami-

nation from mitochondrial matrix wechecked for AK3 activity in PNS IMV. AK3activity was strongly impaired in SN, and

undetectable in IMV compared to MF(Panel j). Panel (k) shows the amplifi-cation of a specific mitochondrial DNA

sequence. The signal is present only inthe mitochondrial sample, used as positivecontrol, and absent in IMV. Each Panel isrepresentative of at least 10 different

experiments.

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Wt 12-month-old rats for their ability to consume O2, as SCand oligodendrocytes do not share the same embryonicorigin. O2 consumption of nigericin-uncoupled on IMV fromsciatic nerves from Wt and CMT1A rats aged 1 and12 months, respectively, was measured at 23◦C in anamperometric electrode (Unisense A/S, Denmark). Resultsare in Fig. 2. The maximal respiratory fluxes elicited byNADH and succinate were measured as rates sensitive toinhibition by rotenone and antimycin A, respectively, todemonstrate that the oxygen consumption is because of theOXPHOS proteins. Panels (a, and c) show the respiratoryrates IMV from 12-month-old or 1-month-old Wt rats. O2

consumption in IMV from CMT1A 12-month-old rats wasseverely impaired (Panel b). In contrast, respiratory rates ofIMV from Wt and CMT1A 1-month-old rats, and from Wt12-month-old rats appear comparable (Panels a, c, and d).Data suggest that peripheral myelin sheath is metabolicallyfunctional in the first stages of the dys-demyelinating

process, but with the progression of the pathology, itbecomes unable to conduct oxidative phosphorylation.Mitochondria-enriched fractions from primary cultures ofboth wild type and CMT1A SC were used as controls toexclude mitochondrial contribution in the impairment ofrespiration in affected IMV. Respiratory rates of mitochon-dria from primary cultures of SC cells from both Wt andCMT1A 12-month-old rats were also assayed. Panels (e andf) show that these are fully functional.

IMV ETC. I and IV are functional in Wt but are

impaired in dys-demyelinated animals

The above data suggest that the impairment in O2 consump-tion observed in IMV from 12-month-old CMT1A rats is notbecause of damage of SC mitochondria, but to structuralmyelin defects. To assess whether the ETC. is active inmyelin isolated from Wt or affected 12-month-old animals,activity of ETC. I (NADH-Ubiquinone oxidoreductase, Panel

(a) (b)

(c) (d)

(e) (f)

Fig. 2 Oxygen consumption. (a–f) Amperometric tracings of nigericin-uncoupled respiratory rates in isolated myelin vesicles (IMV) from Wtand CMT1A rat sciatic nerves, and mitochondria-enriched fractions

extracted from Schwann cell (SC). (a) O2 consumption by IMV comingfromWt 12-months-old rats. (b) O2 consumption was severely impaired

in IMV from CMT1A 12-months animals. No difference was found in O2

consumption by Wt IMV and CMT1A IMV in the early stages ofmyelination (1-month-old rats) (c, d) and by mitochondria extracted

from primary SC cultures from both 12-months-old normal and CMT1Arats (e, f).

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Oxidative phosphorylation in PNS myelin 5

a) and IV (cytochrome c oxydase, Panel b), was assayed inIMV, in the presence or absence of specific inhibitors(rotenone for I and cyanide for IV). Activity of I and IV wasreduced in 12-month-old CMT1A (Fig. 3, Panels a and b)but not in IMV from 1-month-aged CMT1A rats (data notshown). In particular, NADH oxidoreductase lost about 86%of its activity (Panel a, third column), while cytochrome coxydase, about 65% (Panels d, third column). These data areconsistent with the impairment in O2 consumption by IMVfrom CMT1A 12-month-old rats (see Fig. 2, Panel b).Inhibition by rotenone or cyanide ranged around 80% and98%, respectively, suggesting that the activities are specific.

IMV synthesize ATPIn coupled conditions, the ETC. activity is linked to ATPsynthase functioning, i.e. O2 consumption elicits an ATPproduction, similarly to mitochondria. Fig. 4, Panel (a),shows the extra vesicular ATP synthesis by IMV from Wt12-month-old rats. Activity accounted for 37 nmol ATP/min/mg of total proteins and was sensitive to the FoF1-ATPase/H+-pump inhibitor oligomycin (90%), suggesting specificityof ATP production. Moreover, ATP synthesis was inhibitedby KCN (99%) and Antimycin A (64%), inhibitors of ETC.IV and III, respectively, indicating that ATP synthesis iscoupled with the electron transport chain. Panel (b) showsthat, ATP synthase activity in IMV from affected 12-month-old rats is significantly impaired, as compared to the Wt ateach time point.

The OXPHOS proteins colocalize with MPZ in 12-month-old

Wt rat sciatic nerves and in the model of dys-demyelinationThe possibility to assay the ETC. in IMV implies theexpression in the PNS myelin sheath of the mitochondrial

respiratory complexes, as reported in its CNS counterpart(Ravera et al. 2009). To verify this hypothesis an immu-nohistochemical analysis with antibodies against MPZ,ND4L (a subunit of ETC. I) (Panel a), COX-II (a subunitof ETC. IV) (Panel b) or b-subunit of FoF1-ATP synthase(Panel c) was conducted on transversal sections of sciaticnerves from 12-month-old Wt or CMT1A rats. The greenfluorescent signal of each of the OXPHOS proteins assayedcolocalizes, in normal and CMT1A nerves, with the redfluorescent signal of MPZ suggesting that these mitochon-drial subunits are present in the myelin sheath of bothconditions. As expected, CMT1A tissue shows fewermyelinated fibers and some derangement of the myelinsheath in the surviving fibers. Localization studies thensuggest that the impairment of Oxygen consumption in IMVof 12-months-old-CMT1A rats (see Figs 2–4) is not becauseof the absence of the OXPHOS proteins, rather to theiraltered function.

Mitochondrial density is increased in demyelinated fibers of

affected sciatic nerves

The above data prompted us to verify whether mitochondriaare recruited into the affected axons. The histogram(Figure S1) shows that the number of axonal mitochondriain demyelinated fibers from CMT1A rat was significantlyincreased, as compared to the number of mitochondria in thenormally myelinated fibers of the Wt counterparts. It hasbeen reported both in acquired and hereditary demyelinatingdisorders that mitochondria are recruited to the demyelinat-ed regions to meet the increased energy requirementsnecessary to maintain conduction as an adaptive change(Trapp and Nave 2008; Hogan et al. 2009; Saporta et al.2009).

(a) (b)

Fig. 3 Activity of electron transfer chain (ETC.) I and IV. (a, b) Sciatic-nerve-derived isolated myelin vesicles (IMV) from Wt rats show thetypical mitochondrial activity ETC. I and IV. In fact, the specific inhibition

of each of the two complexes by rotenone and cyanide, respectively,dramatically reduced the respiratory properties of theWt IMV comparedto the untreated normal controls (ETC. I: mean � SD: 0.37 � 0.05 vs.

1.82 � 0.11 U/mg, respectively; n = 5; ***p < 0.0001), (ETC. IV:Mean � SD: 0.0024 � 0.0005 vs. 0.09 � 0.008 U/mg, respectively;

n = 5; ***p < 0.0001). Interestingly, IMV from CMT1A rats showed asignificantly reduced activity of ETC. I and IV compared to the Wt ones(ETC. I: mean � SD: 0.26 � 0.04 vs. 1.82 � 0.11 U/mg, respectively;

n = 5; ***p < 0.0001), (ETC. IV: mean � SD: 0.03 � 0.004 vs.0.09 � 0.008 U/mg, respectively; n = 5; ***p < 0.0001), resemblingthe respiratory rates observed in IMV from Wt rats following

specific inhibition of complexes I and IV by rotenone and cyanide,respectively.

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Discussion

Data show that IMV purified from rat sciatic nerve consumeO2 (Fig. 2), and display functional ETC. proteins and ATPsynthase, sensitive to classical mitochondrial inhibitors(Figs 3 and 4), supporting the hypothesis that PNS myelinmay metabolically supply the axons with aerobicallysynthesized ATP, similarly to what reported for CNS (Raveraet al. 2009; Morelli et al. 2011). Therefore, myelin proteincomplement would be determined by its functional require-ment of ATP supplier, regardless of the embryonic origin ofthe producing cell. A mitochondrial contamination of IMVhas to be considered, however, absence of ANT, TIM, TOM,and AK3, as well as the absence of mitochondrial DNA(Fig. 1) suggests that our IMV preparations are negligiblycontaminated. Moreover, to be fully functional, a metabolicprocess such as ATP synthesis associated to O2 consumption,needs a perfectly coupled system, which would not berepresented by mere contaminating mitochondrial vesicles.Oligodendrocytes-synthesized CNS myelin has been pro-posed to act as an energy supplier for the axons (Raveraet al. 2009; Morelli et al. 2011), with a pivotal role ofconnexins in radial transport of the aerobically synthesizedATP to the axoplasm (Adriano et al. 2011). Data are alsoconsistent with several proteomic studies, (Taylor et al.2004; Vanrobaeys et al. 2005; Werner et al. 2007; Yamag-uchi et al. 2008; Ishii et al. 2009; Jahn et al. 2009; Patziget al. 2011; Dhaunchak et al. 2012), reporting the presenceof proteins involved in aerobic respiration in purified myelin.The proteomic analysis of IMV from PNS, isolated by thesame technique used here, suggested that some of the

identified mitochondrial proteins may be additional myelincomponents (Werner et al. 2007). Also, glucose metabolismwas considered over-represented in the myelin proteome(Patzig et al. 2011).The idea of myelin sheath conducting an oxidative

phosphorylation agrees with the observation that IMV froman animal model of dys/demyelination, the CMT1A rat,display an impaired O2 consumption, and OXPHOS proteinactivity at later stages (12-months-old animals), but similar toWt in the early stages of myelination (1-month-old ani-mals) (Figs 2–4). O2 consumption is impaired in IMV from12-month-old CMT1A rats, with respect to Wt animals, butnot in 1-month-old CMT1A rats. However, mitochondriafrom cultured Schwann cells from Wt and CMT1A rats (both12-months old) showed normal O2 consumption rates(Fig. 2). Activity of the ETC. I and IV in IMV from sciaticnerve (Fig. 3) is consistent with the reports that myelincontains cardiolipin, (Heape et al. 1986), a lipid necessaryfor COX function (Zhang and Sejnowski 2000).The sensi-tivity of ATP synthesis to KCN, that was also able to inhibitETC. IV (Fig. 3) and to antimycin A, an inhibitor of ETC. III(Fig. 4), suggests that ATP synthesis by IMV is coupled tothe formation of a proton gradient, similarly to mitochondria(Boyer 1997). Confocal immunofluorescence imaging ofsciatic nerve sections (Fig. 5) is suggestive of a colocaliza-tion of OXPHOS proteins with MPZ, in myelin from eitherWt or CMT1A rats. Even though confocal microscopy doesnot possess the necessary resolution, such data are similar tothose reported by the same technique in CNS (Ravera et al.2009). A specific interaction between Schwann cells andaxons was reported at the nodes of Ranvier (Gatzinsky et al.

(a) (b)

Fig. 4 ATP synthesis in isolated myelin vesicles (IMV) from sciaticnerves. IMV from Wt rats synthesize ATP through FoF1-ATP synthase

activity (a). In fact, in the presence of classical inhibitors of mitochon-drial ATP synthase, they lose this ability (oligomycin: Mean � SD:38 � 4 vs. 4.3 � 0.4 nmol ATP/min/mg; n = 5; ***p < 0.0001), (KCN:

mean � SD: 38 � 4 vs. 0.4 � 0.05 nmol ATP/min/mg; n = 5;***p < 0.0001), antimycin A: mean � SD: 38 � 4 vs. 13.6 � 0.8 nmol

ATP/min/mg; n = 5; ***p < 0.0001). (b) Time course of ATP synthesiswas significantly impaired in IMV from CMT1A adult rats, as compared

to the Wt ones at each time point (30 s: Mean � SD: 18.13 � 1.9 vs.3.3 � 0.3 nmol ATP/min/mg; n = 4; ***p < 0.0001), (60 s: mean �SD: 37.7 � 2.1 vs. 6.1 � 0.7 nmol ATP/min/mg; n = 4;

***p < 0.0001), (90 s: mean � SD: 55.3 � 5.1 vs. 8.6 � 0.8 nmolATP/min/mg; n = 4; ***p < 0.0001).

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12253

Oxidative phosphorylation in PNS myelin 7

2003), which may function in disposal of organelles. Furtherexperiments may tell whether the OXPHOS proteins are truecomponents of myelin sheath. Nevertheless, Fig. 5 wouldsuggest that the impairment in O2 consumption, ATPsynthesis and ETC. I and IV activity in CMT1A 12-month-old rats may not depend on the absence of the OXPHOSproteins, but to the dys/demyelinated phenotype, displaying adefective sheath. Indeed OXPHOS proteins were foundectopically expressed in many different cell lines membranes

and in some cases they were active (Champagne et al. 2006;Panfoli et al. 2011a,b). Several reports suggest that mito-chondrial dynamics involve a close contact between organ-elles like mitochondria and endoplasmic reticulum (Crottyand Ledbetter 1973; Hales 2004; Soltys and Gupta 2000;Giorgi et al. 2010). In this view, it was speculated thatmitochondrial proteins may be transferred to the sheathduring its development (Morelli et al. 2011). The phenotypeof mitochondrial disorders (Nicholls and Budd 2000), a

(a)

(b)

(c)

Fig. 5 Immunofluorescence microscopy

reveals electron transfer chain expressionin rat sciatic nerves. A normal distribution ofmyelin protein zero (MPZ) was detected in12-month-old rat sciatic nerves from both

Wt and CMT1A animals, Panels (a–c, redsignal). ND4L (a subunit of mitochondrialNADH oxidoreductase, Panel a), cyto-

chrome c oxydase, subunit II (a subunit ofmitochondrial Cytochrome oxydase, Panelb) and FoF1-ATP synthase b subunit (Panel

c) are expressed in myelin (green signals),colocalizing with MPZ (Panels a–c, yellowsignal). This suggests that the oxidativephosphorylation proteins decrement is not

because of absence, but a lack of function-ality. Images were recorded onto aninverted Leica TCS SP5 AOBS confocal

laser scanning microscope.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12253

8 S. Ravera et al.

group of human diseases characterized by defects of theOXPHOS, primarily affect visual system, CNS, and PNS(Zeviani and Di Donato 2004), also supports the ideahypothesis that myelin exerts its trophic function byexpressing OXPHOS proteins. Interestingly, mutations orknockout of ANT, a mitochondrial inner membrane proteinnot directly involved in oxidative phosphorylation, but inATP delivery from the mitochondrion to the cytosol (Yanget al. 2007), do not affect the nervous tissue, but result inviable phenotypes involving cardiac and muscle dysfunction(Sharer 2005; Jang and Lee 2006). Along this line, it wasshown that the energy budget for the myelinated axon isconsistent with its reduced cost of action potential (however,assuming that myelinated axons use 23% of the ATPconsumed by unmyelinated axons (Morelli and Panfoli2012), but it was not excluded that some ATP productionis driven by metabolic cooperation with glia (Harris andAttwell 2012a).Our observation that myelin from a model of dys/

demyelination is not able to aerobically produce ATP inspite of a normal functioning of Schwann mitochondria, evenin the presence of an increased mitochondrial number,supports the idea of an energetic role played by peripheralmyelin. Supposing that in dys-demyelinating disorders, likeCMT1A, a considerable impairment in myelin function isbecause of structural and functional alterations (Nobbio et al.2004; Avila et al. 2005), these abnormalities might compro-mise the activity of the OXPHOS proteins, which wouldbecome unable to energetically support the axon. Recently,the hypothesis of myelin sheath able to conduct aerobicmetabolism has been challenged (Harris and Attwell 2012b),however, other Authors reported an extramitochondrial ATPsynthesis and expression of the ETC. and ATP synthase onthe plasma membrane of cancer cells, a site characterized byan inverted membrane potential with respect to that presentin the inner mitochondrial membrane (Chang et al. 2012).Also, a less compact myelin sheath, might negativelyinfluence the radial diffusion of small molecules as ATPbetween the axonal and perinuclear SC cytoplasm (Balice-Gordon et al. 1998; Adriano et al. 2011). As recentlysuggested, an incorrectly assembled myelin can indeed be‘toxic’ for the axon (Nave 2010). Consistently, in sciaticnerves from affected 12-month-old rats transmission EMshowed a significantly increased number of mitochondria(Figure S1) with respect to matching unaffected controls.Therefore, based on our hypothesis that myelin is responsiblefor an aerobic metabolism in peripheral myelinated fibers, itis tempting to speculate that recruitment of mitochondria atthe site of demyelination, represents an attempt to compen-sate for the aliquot of energy supply because of myelin.In conclusion, the idea of a new energetic function of

myelin is emerging (Ames 2000; Morelli et al. 2011). Ourobservation that IMV from peripheral nerves express func-tional ETC. proteins and FoF1-ATP synthase, as well as O2

consumption, ATP synthesis and OXPHOS functionalitystrongly support this hypothesis.

Acknowledgments

This study was supported by a Grant from the ‘Compagnia di SanPaolo’- Neuroscience Program, for the research project entitled:‘Energetic metabolism in myelinated axon: a new trophic role ofmyelin sheath’. The Authors have no conflict of interest to declare.

Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web-site.

Figure S1. Evaluation of mitochondrial recruitment in CMT1Asciatic nerves.

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