Wilson Disease at a Single Cell LevelINTRACELLULAR COPPER TRAFFICKING ACTIVATES COMPARTMENT-SPECIFIC RESPONSESIN HEPATOCYTES*□S

Received for publication, February 16, 2010, and in revised form, July 19, 2010 Published, JBC Papers in Press, July 20, 2010, DOI 10.1074/jbc.M110.114447

Martina Ralle‡1,2, Dominik Huster§¶1, Stefan Vogt�, Wiebke Schirrmeister¶, Jason L. Burkhead‡, Tony R. Capps‡,Lawrence Gray‡‡, Barry Lai�, Edward Maryon**, and Svetlana Lutsenko‡‡3

From the ‡Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239, the§Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig 04109, Sachsen, Germany, the ¶Department of Medicine,University of Magdeburg, 39104 Magdeburg, Germany, the �Argonne National Laboratory, Argonne, Illinois 60439, the**Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, and the‡‡Department of Physiology, The Johns Hopkins University, Baltimore, Maryland 21205

Wilsondisease (WD) is a severehepato-neurologicdisorder thataffectsprimarily childrenandyoungadults.WDiscausedbymuta-tions inATP7B and subsequent copper overload.However, copperlevels alone do not predict severity of the disease.Wedemonstratethat temporalandspatialdistributionofcopper inhepatocytesmayplay an important role inWDpathology.High resolution synchro-tron-basedx-ray fluorescence imaging in situ indicates that copperdoes not continuously accumulate in Atp7b�/� hepatocytes, butreaches a limit at 90–300 fmol. The lack of further accumulation isassociatedwith the lossofcopper transporterCtr1 fromtheplasmamembrane and the appearance of copper-loaded lymphocytes andextracellular copper deposits. The WD progression is character-ized by changes in subcellular copper localization and transcrip-tome remodeling. The synchrotron-based x-ray fluorescenceimaging andmRNAprofiling both point to the key role of nucleusin the initial response to copper overload and suggest time-depen-dent sequestration of copper in deposits as a protective mecha-nism.Themetabolicpathways,up-regulated inresponse tocopper,show compartmentalization that parallels changes in subcellularcopper concentration. In contrast, significant down-regulation oflipid metabolism is observed at all stages of WD irrespective ofcopperdistribution.Theseobservations suggest newstage-specificas well as general biomarkers forWD. Themodel for the dynamicrole of copper inWD is proposed.

Wilson disease (WD)4 is a severe disorder of copper metab-olism with varied manifestations and, if untreated, invariably

lethal outcome. In WD, copper excretion from the liver isblocked due to genetic inactivation of the copper transporterATP7B. Copper overload, which follows, induces markedchanges of liver morphology and function (1). AlthoughWD isunquestionably caused by copper accumulation, the preciserole of copper in inducing pathological changes remains poorlyunderstood. Increased oxidation of lipids, DNA damage, andenzyme inactivation have been reported for WD livers (2, 3).These effects are typically observed when pathology is fullydeveloped; therefore, it is uncertain whether the detectedchanges represent the cause of the disease or its consequences.Copper content in selected cell compartments of WD hepa-tocytes, such as lysosomes, has been measured (4–7); however,comparative quantitative analysis of copper distributionwithin liver tissue and individual cells is not available. Indiseased liver, large amounts of accumulating copper arefound in the cytosol, where copper is bound to metallothio-neins, high affinity metal-chelating proteins. Whether thissequestered copper is exchangeable and active is unclear. Fur-thermore, the severity and the onset of WD do not directlycorrelate with copper concentrations in the liver (6, 8, 9), sug-gesting that additional factors, such as spatial distribution ofcopper in the liver as well as environmental and genetic modi-fiers may play an important role in WD pathology. To beginaddressing these unresolved issues, we have utilized theAtp7b�/� mice, an animal model ofWD.We demonstrate thatthe disease progression is associated with specific changesin the intracellular distribution of accumulating copper ratherthan merely an increase in hepatic copper content. We showthat distinct cellular compartments become progressivelyinvolved in response to copper overload. The involvement ofcellular compartments in cellular response appears to correlatewith copper concentration in these compartments. We alsoidentified potential mechanisms that protect cells against cop-per overload. Our results begin to explain the lack of simplecorrelation between the total amount of copper in the liver andseverity of WD manifestations.

EXPERIMENTAL PROCEDURES

Tissue Preparation—Mice were housed according to theNational Institutes of Health guidelines; the procedures wereapproved by the Oregon Health & Science University Institu-

* This work was supported, in whole or in part, by National Institutes of HealthGrant P01 GM067166 (to S. L.). This work was also supported by GermanResearch Foundation Grant Hu932/3-2 (to D. H.). The use of the AdvancedPhoton Source was supported by the U.S. Department of Energy, Office ofScience Contract DE-AC-02-06CH11357.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 6 and Tables 1–3.

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: Dept. of Biochemistry and

Molecular Biology, Oregon Health & Science University, 3181 S.W. SamJackson Park Rd., Portland, OR 97239. Tel.: 503-494-3441; Fax: 503-494-8393; E-mail: rallem@ohsu.edu.

3 To whom correspondence may be addressed: Dept. of Physiology, JohnsHopkins University, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-4661; E-mail: lutsenko@jhmi.edu.

4 The abbreviations used are: WD, Wilson disease; SXRF, synchrotron-basedx-ray fluorescence.

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tional Animal Care and Use Committee. Mice were fed withRodent Diet 5001 (Lab Diet, St. Louis, MO), containing 13 ppmcopper, 70 ppm zinc, and 270 ppm iron. At given time points,the animals were euthanized and perfused with 10 ml of 0.9%saline injected in the left ventricle. For the SXRF analysis, liverpieceswere embeddedwithCryochrome (ThermoShandon) onan aluminum block, snap-frozen in dry ice-cooled isopentane,and cryosectioned (Leica CM1850). The 10-�m sections weretransferred onto a 2.5 mm-inch Mylar� window (Hyde, Chi-cago, IL) attached to a lucite sample holder (HuffstutterMachining, Hillsboro, OR), dried, and stored in a desicccator.Serum Cholesterol Analysis—Blood was collected by cardiac

puncture, and serumwas separated by centrifugation. The con-centration of cholesterol was determined after fractionation bysequential centrifugation as described by Teupser et al. (10).MicroarrayAnalysis—Animal ageswere selectedbasedonhis-

tology to represent stage I (6 weeks), stage II (46 weeks), and stageIII (60 weeks) of the disease. Immediately after removal of livers,total RNAwas isolated usingTRIzol reagent (Invitrogen) followedby anRNeasy cleanupprocedure (Qiagen). RNA integritywas ver-ifiedelectrophoreticallybyethidiumbromidestainingandabsorb-ance densities ratio (A260/280 nm �1.8). Each sample (n � 3/agegroup biological replicates) was hybridized to two MOE430AAffymetrix arrays (technical replicates). Image processing andanalysis were performed using AffymetrixMAS 5.0 software. Thedata were saved in a CEL format, imported into GeneSifter dataanalysis software (Geospiza), and subjected to robust multichipaverage normalization using published algorithms (11). Therobust multichip average-normalized dataset was then used toidentify changed genes and determine statistical significance andmagnitude of changes. Significance was established using Wilc-oxon t test with Benjamini andHochberg adjustment formultiplecomparisons. The hierarchical gene ontology analysis was per-formed by GeneSifter software using “biological function” and“cellular component” options. To confirm the mRNA profilingdata, changes in several transcripts were verified using real-timePCRwithGAPDHas internal standard (iCycler; Bio-Rad). The listof primers used for the experiments is given in supple-mental Table 1. The relative expression of target mRNA in theAtp7b�/� mouse liver compared with the wild-type mouse liverwas quantified using the 2��ct method (12).SXRF Imaging—SXRF imaging was performed on beamlines

2-ID-E and 2-ID-D at the Advanced Photon Source at theArgonne National Laboratory at incident x-ray energy of 10keV. X-rays weremonochromatized by a double-crystal Si(111)(Kohzu); a Fresnel zone plate focused beam to a spot of 0.5 (h)�0.5 (v) �m2 (13). Samples were raster-scanned, and the result-ing fluorescence of each element wasmeasured simultaneouslyat each pixel (dwell time, 1 s/pixel) using an energy-dispersivesilicon drift detector (SII NanoTechnology). For quantitation,the entire x-ray fluorescence spectrum was collected at everyposition during the scan. The spectra were then fitted individ-ually using modified gaussians for fluorescence peak descrip-tions and a version of the SNIP algorithm for backgrounddetermination (14). Conversion of fluorescence counts to two-dimensional densities of elements (�g/cm2) was done using acalibration curve based on the x-ray fluorescence of the thinfilm NBS standards 1832 (copper) and 1833 (zinc) (National

Institute of Standards and Technology, Gaithersburg, MD).The concentrations of copper (millimolar or �g/g wet weight)were calculated from two-dimensional densities (�g/cm2) bytaking into account the atomic mass of copper and using 1.035g/cm3 as sample density.

For the average elemental content or larger areas, spectrawere extracted from the original dataset, added up, fitted, andquantified as above. To estimate the amount of copper in indi-vidual cells, cellular volumewas calculated considering hepato-cytes as cylinders with identical height and diameter. Duringdisease progression, the diameter of hepatocytes increases dra-matically (from 18 up to 62 �m), but whether the cell heightincreases proportionally is unknown. Consequently, we calcu-lated a maximum cell volume (assuming the diameter and theheight of cells were equal) and a minimum cell volume (assum-ing the cell height remains the same as at 6 weeks). Nuclei in ahealthy hepatocyte were considered spheres. A nucleus issmaller than 10�m(the tissue thickness) in diameter, thereforeit comprises only a fraction of the total amount of copper in thecorresponding area. Consequently, we calculated the volume ofthe nucleus (�10% of the total cell volume) and subtracted itfrom the total cylindrical volume of the corresponding region.We then determined the amount of copper in the remaining(cytoplasmic) volume using cytoplasmic copper concentration,subtracted this contribution from the total amount of copper inthe cylinder to yield the amount and subsequently the concen-tration of copper in the nuclei. The calculations were per-formed for each scan (n � 6) individually. Data ranges (mini-mum and maximum) were used for samples with n � 10 andstandard deviation (S.D.) for all others. S.D. values were calcu-lated from themean averages; statistical significance was calcu-lated using Student’s t test with two-tailed distributions ineither paired or, where applicable, unpaired mode. Three-di-mensional copper concentrations were calculated from thearea concentrations and tissue thickness of 10�m. Shrinking ofthe samples in the x-y plane was determined to be negligible.Immunohistochemistry—Mounted 5-�m tissue sections

were hydrated in phosphate-buffered saline (PBS), blocked for30–60 min in blocking buffer (5% goat serum, 0.2% TritonX-100, 1% bovine serum albumin (BSA) in PBS), and then incu-bated with rabbit anti-hCtr1 (1:100 dilution in PBS containing0.25% BSA, 0.2% Triton X-100) for 2 h at room temperature.Following three 10-minwashes with PBS, slides were incubatedwith goat anti-rabbit Cy3-labeled antibody (at 1:10,000) for 2 hat room temperature. Sections were washed three for 10 mineach time with PBS, mounted with Vectashield-DAPI (VectorLaboratories, Burlingame, CA), and analyzed by confocalmicroscopy (LSM 5 Pascal, Carl Zeiss Microimaging, Thorn-wood, NY). The immunostaining of Na,K-ATPase was doneusing the same protocol except the sections were incubatedwith the rabbit anti-mouse Na,K-ATPase (1:1,000 dilution; giftfrom JackKaplan, University of Illinois at Chicago) at 4 °C over-night. The sections were washed four times for 15 min eachtime with 1 ml of PBS and incubated with Cy3-labeled goatanti-rabbit IgG (1:15,000 dilution) in the dark for 2 h. Afterincubation and two additional washes (10min each wash), withPBS the sections were mounted onto slides using Vectashieldwith DAPI and visualized.

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RESULTS

Quantitative Imaging of Copper in the Liver using SXRFMicroscopy—To understand the mechanisms of pathologydevelopment in WD, we have utilized the Atp7b�/� mice, ananimal model for hepatic course of WD (6). In Atp7b�/� liver,the disease progresses in three major stages: (i) minor/absentmorphological changes (up to 6–8 weeks); (ii) inflammation,necrosis, and swelling of hepatocytes (most pronounced at13–20 weeks); and (iii) liver regeneration withmarked prolifer-ation of bile ducts (after 30–46 weeks) (6). Although copper iselevated at all three stages of the disease, no correlation isobserved between the total copper content and the Atp7b�/�

liver morphology/function, similarly to human WD patients.Consequently, to understand better the role of copper in WDpathology we examined copper distribution within theAtp7b

�/�hepatocytes in physiologically relevant tissue context.

Until recently, such measurements have been very difficultbecause available methods lack sufficient sensitivity and/orspatial resolution. Histological staining with rhodanine is notquantitative (15) and does not detect all forms of copper in thelivers. An electron microprobe is highly sensitive and quantita-tive, but does not allow simultaneous comparison of individualcells and larger areas of tissue. SXRF has emerged as a promis-ing technology to overcome these problems (16–22). Third-generation synchrotron sources provide sufficient flux in afocused spot to measure the inherent fluorescence of metalswith exceptional sensitivity (down to the attomolar 10�18 M

range) and subcellular resolution. At the time of our experi-ments, SXRF had not yet been used for a single cell analysis insitu. Consequently, we have developed a necessary protocol(supplemental Fig. 1) and applied it to the Atp7b �/� liver.Because SXRF measurements and animal work depend oninstrumentation/animal availability, we initially focused onthree time points in the disease progression: 6 weeks (stage I),13 weeks (early stage II), and 20 weeks (mid-late stage II). Itshould be noted that the Atp7b�/� animals are born withgreatly reduced levels of copper in the liver compared with wildtype (8.1 � 1.3 compared with 249.5 � 54.3 �g/g dry weight at2–4 days) (23), and all copper accumulation occurs after birth.Changes of Copper Content in the Atp7b�/� Liver Differ from

the Kinetics of Copper Accumulation by Individual Hepatocytes—It is assumed that during the course ofWD, copper continues toaccumulate in the liver, facilitating pathology development.

However, our analysis of Atp7b�/�

livers revealed a more complex pic-ture. Integrated SXRF scans of tis-sue areas demonstrated that copperconcentration in the Atp7b�/� liv-ers is high at 6 weeks (228–530�g/g, or 6.4 � 2.3 mM, n � 5), butdecline subsequently to 60–159�g/g, or 2.3 � 1.1 mM, n � 5) at 20weeks (Fig. 1, left). Atomic absorp-tion spectroscopy analysis of liverpieces confirmed this observation(supplemental Fig. 2).This time-dependent decrease

could be due to the replacement ofhepatocytes with other cell types that accumulate less copper(fibroblasts, for example). Alternatively, unlimited copperaccumulation in the liver can be prevented through down-reg-ulation of copper uptake into hepatocytes or stimulation ofcopper efflux. To distinguish between these two scenarios (cellloss versus limited accumulation) we measured copper contentof individual cells using SXRF (Fig. 1, right). In contrast to tissuesections, the amount of copper in individual hepatocytes notonly did not decline, but increased 3–10-fold in the period from6 to 13 weeks (Fig. 1, right). Thus, during this time copper con-tinues to accumulate in hepatocytes, and the decrease in coppercontent in the liver tissue must be due to a decrease in thenumber of hepatocytes and the appearance of nonhepatic cells.(A lower number of hepatocytes at 13 weeks compared with 6weeks is apparent in the hematoxylin and eosin staining;supplemental Fig. 3, B and C.) However, after 13 weeks, nofurther increase of copper in individual hepatocytes wasobserved, indicating that copper accumulation had reached itsupper limit (90–300 fmol or 50–200 billion copper atoms/hep-atocyte). Thus, the pathology development from stage I (6weeks) to early stage II (13 weeks) is associated with the eleva-tion of cellular copper, whereas subsequent events in diseaseprogression cannot be due to copper increase and can be causedby intracellular copper redistribution.Decrease in Copper Accumulation Is Associated with the

Down-regulation of Copper Transporter Ctr1—To understandbetter the molecular mechanism behind limited copper accu-mulation, we examined the plasma membrane levels of themajor copper uptake transporter Ctr1 before and after hepaticcopper had reached its limit, i.e. at 6 and 20 weeks, respectively(Fig. 2A). At 6 weeks, Ctr1 was easily detected at the plasmamembrane of hepatocytes in either control orAtp7b �/� livers;and the patterns of Ctr1 staining were very similar (Fig. 2A, aand b). At 20 weeks, the staining of Ctr1 in the control liver wasthe same as at 6 weeks (supplemental Fig. 4), whereas theAtp7b�/� liver showed weaker and uneven staining (Fig. 2A, cand d). Closer examination of individual cells revealed thealmost complete lack of Ctr1 at the plasma membrane ofenlarged hepatocytes. Smaller hepatocytes that had retainedcell-cell contacts, and nonhepatic cells still showed some Ctr1staining. The loss of Ctr1 appeared specific because anotherplasma membrane protein, Na,K-ATPase, was present at theplasma membrane of all hepatocytes (Fig. 2B).

FIGURE 1. Time-dependent changes of copper levels in the Atp7b�/� liver differ from changes in individ-ual hepatocytes. SXRF measurements were performed for liver tissue sections (left) and individual hepato-cytes (right) at three different time points (n � 5 for each time point).

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Further analysis of mRNA levels demonstrated that the Ctr1transcript was decreased in the Atp7b�/� liver compared withcontrol at the stage I ofWD andwas significantly down-regulated

at the stage II (Fig. 2C), thusprovidingpossible explanation for the decreaseof theCtr1 protein. (TheCtr1mRNAdown-regulation was verified byreal-time PCR; supplemental Fig. 5.)The mRNA levels for another cop-per transporter, Ctr2, and forNa,K-ATPase were not changed,further suggesting specificity ofCtr1 down-regulation.Decreased Hepatic Accumulation

of Copper Is Accompanied by theAppearance of Copper-loaded In-flammatory Cells and ExtracellularCopper Deposits—The decrease ofCtr1 at the plasma membrane islikely to diminish copper uptakethus protecting liver against furthercopper overload. Coincidentally, in20-week Atp7b�/� livers (but notearlier), we observed yellowishextracellular deposits (Fig. 3A),which are visible in stained and evenunstained tissue (Fig. 3A). Theseextracellular deposits are highlyenriched in copper (maximum con-centration of 43 mM), sulfur, andiron. The co-clustering of three ele-ments is not observed within cells,where copper distribution does notparallel the distribution of iron andshows only partial correlation withsulfur. This result suggests that theextracellular copper deposits arenot merely a product of necrotichepatocytes, but are more likelyproduced due to the diminisheduptake by hepatocytes.It is not known whether in WD

liver cells other than hepatocytesaccumulate copper. Because thedecrease in copper uptake by hepa-tocytes coincides with the appear-ance of inflammatory cells, wemeasured copper concentration inthese cells (Fig. 3C). Lymphocytesand macrophages express homolo-gous copper-transporting ATPase,ATP7A, but not ATP7B, and a pri-ori are not expected to accumulatecopper. At 20 weeks, the copperconcentration in these cells wasindeed lower than in neighboringAtp7b�/� hepatocytes (1.4 mM

compared with 3.8 mM), but �12times higher compared with hepatocytes in healthy tissue.Thus, copper that is no longer taken by Atp7b�/� hepatocytesis redirected, at least partially, to other cells in the liver.

FIGURE 2. Ctr1 levels decrease in 20-week-old Atp7b�/� hepatocytes. A, confocal images of liver sectionsimmunostained with rabbit anti-Ctr1 antibody: a, 6-week-old wild type (WT); b, 6-week-old Atp7b�/�; and c andd, 20-week-old Atp7b�/� mice. Right panel, magnified views of areas in white rimmed boxes 1–3. B, confocalimages of 20-week-old control (left) and Atp7b�/� (right) liver sections immunostained with anti-Na,K-ATPaseantibody. The staining at plasma membrane is apparent in even enlarged hepatocytes. C, down-regulatedmRNA levels in Atp7b�/� samples compared with control (taken as 1). The average robust multichip averagenormalized fluorescence intensity of RNA hybridization signal from three biological replicates for Ctr1, anothercopper transporter Ctr2, and unrelated plasma membrane protein Na,K-ATPase (the time-dependent down-regulation of Ctr1 mRNA was additionally verified and confirmed by real-time PCR) is shown. Scale bar, 50 �m.

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Hepatic Nuclei Contain Copper,Which Became Highly Elevated atStage I of WD—So far, our resultsindicated that the development ofWD pathology (stage I and earlystage II) was associated with theincrease of hepatic copper content,but the data offered little informa-tion about the site of copper action.Also, the late stage II showed no fur-ther copper elevation, raising thequestion about the role of copper, ifany, at this stage of the disease.Consequently, to gain additionalinsight we compared the distribu-tion of copper within control andAtp7b�/� hepatocytes at all threetime points. At 6 weeks, in controlhepatocytes, copper was highest inthe cytosol (which contains abun-dant Cu,Zn-superoxide dismutaseand mitochondria). Unexpectedly,significant amount of copper wasalso detected in the nucleus (Fig.4A), even after the contribution ofcytosolic copper was subtracted.The nuclear copper concentrationwas in the range of 20–69 �M (n �6), and the average concentration

FIGURE 3. A and B, accumulation of copper in extracellular deposits and inflammatory cells in 20-week-oldAtp7b�/� liver. A, extracellular deposits (indicated by arrows) in the unstained SXRF section (left) and a hema-toxylin and eosin-stained tissue (right). Scale bar, 100 �m. B, hematoxylin and eosin (H&E) stain of the scan area(50 � 50 �m; bar, 10 �m) illustrating the absence of nuclei/cells in the area of deposit (circled). Elemental mapsof sulfur (S), iron (Fe), and copper (Cu) illustrate an increased concentration (lighter spots) of each element in thesame area. Concentrations are displayed using red temperature false coloring (on a logarithmic scale).C, inflammatory cells were identified by phosphorus map (left) and by hematoxylin and eosin staining (center),and the copper concentration in these areas was measured by SXRF (right).

FIGURE 4. Intracellular copper distribution in control and Atp7b�/� hepatocytes. A, two-dimensional fluorescence maps demonstrate the distribution oftwo elements (phosphorus and copper) within the same scanned area of control or Atp7b�/� livers at 6 weeks. Concentrations are displayed using redtemperature false coloring (on a logarithmic scale) with lighter color corresponding to higher concentrations. The phosphorus concentration in the nuclei(circled by a white dotted line) is higher than in the cytosol in either control or Atp7b�/� sample. Copper concentrations in the nucleus are lower compared withcytosol in control livers and equal to cytosolic copper in Atp7b�/� sample. B, SXRF scans of Atp7b�/� livers at three time points demonstrate a progressiveincrease in copper concentration in hot spots (marked by arrows) along with the decrease of average copper concentration in the remainder of cytosol (givenin numerical values above the images). C, quantitation of copper concentrations in hepatic nuclei, cytosol, and hot spots at different ages is shown. Data aregenerated using two livers for each age, two or three scans/sample.

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(40 �M) was only three times lower than copper concentra-tion in the cytosol (average: 120 �M; range: 60–170 �M, n �6). Considering that the volume of mouse nuclei is �340�m3, the total amount of copper in hepatic nucleus equals 10million atoms. This high amount of copper in healthy nucleisuggests an important but currently unknown role for cop-per in mammalian nuclei.In 6-week Atp7b�/� hepatocytes, a massive entry of copper

into the nucleus was observed (Fig. 4, A and B). The averagecopper concentration in this compartment was increased byabout 100-fold (to 7.2 mM; range 3.9–10.2 mM, n � 3) com-pared with control. The concentration of copper in the cytosoland nucleus became similar; the Cunuc/Cucyt ratio was close to1.02 (0.80–1.44, n � 8). This observation suggests that excesscopper is present in a form that easily shuttles between thecytosol and nucleus, leading to equilibrium. It further impliesthat this form of copper is not present in normal hepatocytes,where no such equilibrium is observed. (We examined byWestern blotting the levels of two potential copper carriers,metallothionein and Atox, in Atp7b�/� nuclei. Neither metal-lothionein nor Atox1, both detectable in a cytosol, were ele-vated in nuclei; data not shown.)Concentration of Copper in Cellular Compartments Changes

with Disease Progression—Further time course studies dem-onstrated that control liver had a fairly constant copper con-centration in either tissue sections (100–120 �M, n � 3 foreach age group) or individual hepatocytes (range: 90–133�M, n � 3). In contrast, we observed distinct changes in thecellular and subcellular copper concentrations when wecompared the 6- and 13-week-old Atp7b�/� hepatocytes(Fig. 4B). In older mice, the average nuclear and cytosoliccopper concentration decreased from initially 7.2 mM to 2.1mM (range 0.9–3.8 mM, n � 3). The decrease in concentra-tion was observed despite the elevation of total copper con-tent in a cell. This counterintuitive phenomenon was causedby two factors. One was a marked increase in a cellular vol-ume: hepatocytes expanded from 4,641 � 10 �m3 (n � 24) at6 weeks to 19,0590� 1,814 �m3 (n� 20) at 13 weeks. (Nucleiincreased from 340 � 2 �m3, n � 106, to 8,329 � 264 �m3,n � 50.) Another important factor was the appearance ofnumerous “hot spots” (localized deposits enriched in copper;Fig. 4B). At 6 weeks, only a few small (�0.5 �m) hot spotswere detected. The copper concentration in these spots wasslightly higher compared with surrounding cytosol (5.7–10.5mM; average 8.5 mM versus average 7.2 mM). At 13 weeks, hotspots became larger (1–6 �m, average 2 �m) andmore abun-

dant (Fig. 4B), although their copper concentration on aver-age did not change very significantly (5.0–15.8 mM, average9.7 mM, n � 3; Fig. 4C).

At 20weeks, copper concentration in the cytosol and nucleusdecreased further (Fig. 4B and supplemental Table 3), whereastheir volumes showed no further increase (in fact, somedecrease was detected). Thus, at 20 weeks copper was leavingthese compartments. In contrast, copper accumulationwithin the hot spots became truly pronounced, and the con-centration of copper in hot spots reached 10.0–35.7 mM

(average 22.4 mM, n � 3). (For the summary of all changes,see supplemental Table 2.)Physiological ProcessesUp-regulated inWDLiver ShowTime-

dependent Compartmentalization That Parallels CopperDistribution—Time-dependent changes in local copper con-centrations suggested that coppermay trigger distinct cellularresponses at different stages of the disease. Previously, wefound that the initial stage of WD is characterized by selec-tive up-regulation of machinery associated with the cell cycleand marked down-regulation of cholesterol metabolism (8).To determine whether these pathways remained perma-nently altered during pathology development (or changealong with the changes in copper distribution), we per-formed mRNA profiling using livers from Atp7b�/� mice atthe advanced stage II of the disease and from the regenerat-ing portion of Atp7b�/� liver at stage III. The profiles werecompared with the age-matched controls and our earlierdata collected at stage I. The accuracy of the gene arraymeasurements was verified by the real-time PCR analysis ofseveral altered transcripts (supplemental Fig. 6).The summary of gene ontology for the most significantly

up-regulated and down-regulated processes at all three stages isshown inTables 1 and 2, respectively.We found that the unreg-ulated processes differ considerably depending on the stage ofthe disease (Table 1). Cell cycle machinery is most affected atstage I; stage II is characterized by marked changes in cytoskel-eton and cell adhesion as well as up-regulation of glycerol andorganic ether metabolism; and stage III shows the hallmarks ofincreased immune response (Table 1). Themost striking obser-vation is an apparent time-dependent switch between cellularcompartments that house the up-regulatedmetabolic pathways(Fig. 5). Specifically, the nucleus is most noticeably involved atthe stage I but is not a significant player at the stage III of thedisease. In contrast, the processes in the lysosomes and endo-cytic vesicles are not activated at stage I but appear to play themajor role at the stage III (Fig. 5). The switch in the compart-

TABLE 1Most significantly up-regulated processes in Atp7b�/� livers

Stage I Stage II Stage IIIProcess Score Process Score Process Score

Reciprocal meiotic recombination 12.42 Acylglycerol metabolism 5.06 T cell activation 7.91Chromosome condensation 10.85 Complement activation, alternative pathway 5.01 Cell-substrate adhesion 7.56Meiosis I 10.82 B cell apoptosis 5.01 Processing, presentation of peptide antigen 7.12M phase 9.56 Neutral lipid metabolism 4.97 Lymphocyte activation during immune response 7.02Glycerol metabolic process 9.45 Cytoskeleton organization 4.90 Positive regulation of phagocytosis 6.50Alditol metabolic process 8.91 Glycerol ether metabolism 4.89 Cell activation during immune response 6.50Cell cycle phase 8.77 Nucleus localization 4.74 Leukocyte activation 6.50Mitosis 8.68 Positive regulation of cell adhesion 4.72 Cell-matrix adhesion 6.34Nuclear division 8.68 Organic ether metabolism 4.64 Processing, presentation of exogenous antigen 6.29Organelle fission 8.62 Response to chemical stimulus 4.57 Positive regulation of endocytosis 6.02

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mentalization of up-regulated pathways parallels changes incopper concentration in these compartments, suggesting thatintracellular copper localizationmayhave a significant effect oncellular response.LipidMetabolism Is theMajor Down-regulated Process in WD—

In contrast to up-regulated pathways that are time- and compart-ment-specific, lipid metabolism is most significantly down-regulated at all stages of WD (Table 2). Two other pathwaysdown-regulated at all stages of WD, although less significantly,were alcohol metabolism and reductive/oxidative processes. Thisobservation suggests that lipid metabolism is exquisitely sensitiveto copper misbalance and that monitoring lipid metabolism mayprovide useful information about liver function inWD.To verify that changes in the liver transcripts for enzymes

involved in cholesterol biosynthesis (supplemental Table 3) areaccompanied by metabolic changes, we compared choles-terol levels in the serum of control and Atp7b�/� animals.These measurements revealed significant cholesterol mis-balance (Fig. 6). Specific changes in different fractions ofserum cholesterol were similar at 6 weeks and 30 weeks. Asobserved previously for stage I (6), the VLDL cholesterol wasmarkedly decreased in Atp7b�/� serum at either 6 or 30weeks, representing the most sensitive measure of choles-terol misbalance. The HDL and LDL cholesterol levels werenot changed significantly at 30 weeks, although a downwardwas detected for the HDL fraction.

DISCUSSION

The goal of this study was to understand better the role ofcopper in pathology development inWD. The consequences

of complete Atp7b inactivation provided by a gene knockoutcan be more severe or manifest sooner compared with thoseof a single-site mutation. However, the role of copper intriggering the disease is likely to be the same. Specifically, wedemonstrate that the status of the Atp7b�/� liver is definedby the levels of copper in individual hepatocytes, the intra-cellular copper distribution, and the up-regulation of meta-bolic pathways in specific cell compartments. Our experi-ments identify lipid metabolism as the pathway mostsignificantly down-regulated in WD and therefore a likelycontributing factor to WD pathogenesis. The serum VLDLcholesterol could be a useful marker to monitor the func-tional state of the WD liver.Using high resolution imaging in situ, we have determined

the copper content of individual hepatocytes, showed thatdifferent cell compartments are preferentially involved inresponse to copper overload, and discovered the existence ofa limit for copper accumulation in individual hepatocytes.These results begin to explain the lack of simple correlationbetween the copper levels and the functional state of WDliver. We propose the following model for the role of copperin WD progression (Fig. 7). Early on, the inactivation ofAtp7b�/� is associated with copper accumulation by hepa-tocytes. At this stage copper enters the hepatic nuclei andtriggers the remodeling of liver transcriptome, whichinvolves up-regulation of cell cycle and down-regulation oflipid metabolism. Once copper levels are 250–800-foldabove the norm, copper uptake is ceased and/or additionalcopper-export mechanisms are activated to prevent furthercopper accumulation. Our data suggest that the lack of con-tinued copper accumulation could be due to down-regula-tion of Ctr1.Copper that is no longer absorbed by hepatocytes has

three potential routes of further distribution: it may beabsorbed by the inflammatory cells, deposited in extracellu-lar aggregates, or exported through filtering into urine (Fig.7). Together, these processes may lessen the copper load inhepatocytes and thus facilitate hepatic regeneration. Highcopper levels in lymphocytes compared with healthy hepa-tocytes support the proposed role for elevated copper ininflammation as a response to tissue injury (24–26). Giventhe observed up-regulation of transcripts associated withinflammatory processes observed at the later stages of thedisease, it is tempting to speculate that the redirection ofcopper uptake from hepatocytes to other cell types may facil-

FIGURE 5. Association of the significantly up-regulated pathways withcellular compartments. The compartmentalization of significantly up-regu-lated pathways was examined using the GeneSifter software. The Z-scoreabove 2 (green dotted line) indicates significant overrepresentation of pro-teins/pathways in a given compartment compared with the averagechange/compartment.

TABLE 2Most significantly down-regulated processes in Atp7b�/� livers

Stage I Stage II Stage IIIProcess Score Process Score Process Score

Isoprenoid biosynthesis 25.92 Sterol biosynthesis 11.22 Acyl-CoA metabolism 7.12Sterol biosynthesis 21.48 Sterol metabolism 10.53 Oxidation reduction 6.43Cholesterol biosynthesis 20.51 Cholesterol biosynthesis 8.86 Long chain fatty acid metabolism 4.62Isoprenoid metabolism 17.00 Cholesterol metabolism 8.71 Lipid metabolism 3.92Steroid biosynthesis 16.25 Steroid biosynthesis 8.37 Cholesterol metabolism 3.89Sterol metabolism 16.01 Steroid metabolism 7.91 Cytolysis 3.70Cholesterol metabolism 14.61 Isoprenoid biosynthesis 7.74 Sterol metabolism 3.63Alcohol metabolism 10.37 Isoprenoid metabolism 7.38 Amino sugar metabolism 3.61Pigment biosynthesis 6.50 Oxidation reduction 6.10 Cofactor metabolism 3.51Fatty acid metabolism 6.21 Alcohol metabolism 5.00 Organic anion transport 3.51Oxidation reduction 5.77 Regulation of fat cell differentiation 4.84 Fatty acid metabolism 3.19

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itate inflammation/contribute to activation of nonhepaticcells. Yet, the copper levels in lymphocytes are lower than inthe age-matched Atp7b�/� hepatocytes likely due to com-pensatory effects mediated by another copper-transportingATPase, ATP7A, expressed in these cells (27).At stage II, copper concentration in the nuclei and cytosol

decreases, and the intracellular deposits appear (Fig. 7). Thedriving force for copper accumulation in deposits is unknown.One possibility is a time-dependent polymerization of copper-bound metallothioneins (28), which are highly up-regulatedin Atp7b�/� liver (8), with a subsequent phagocytosis ofoligomers into lysosomes. This scenario is consistent with theprevious reports finding copper in electron dense lysosome-likecompartments at the late stage ofWD (29).We assume that the

hot spots represent copper within the lysosomes-like compart-ments because granular copper accumulation in lysosomes hasbeen reported in WD patients (5, 30), although this remains tobe formally established.It should be emphasized that the observed copper com-

partmentalization reflects preferential rather than exclusivedistribution. Elevated copper can be found (and presumablyact) in various cellular locations particularly at stage II whenthe disease phenotype is most severe. Nevertheless, thechange in local copper concentrations is very distinct and, atthe late stages of the disease, is likely to represent an impor-tant protective mechanism. As the result of copper redistri-bution, the “mobile” and likely active form of copper issequestered, minimizing the range of copper activity.The mechanism of further copper decrease in hepatocytes

and liver recovery remains to be characterized. The involve-ment of the ABC-type transporterMRP2 in copper excretionwas suggested based on the measurements of copper exportin the MRP2-deficient rats (31). It is interesting that severalABC transporters are up-regulated in Atp7b�/� livers (ourgene array, data not shown). Determining the molecular

FIGURE 6. Sterol metabolism is dysregulated at different stages of WD.Analysis of serum cholesterol fractions demonstrates a significant decrease ofVLDL cholesterol in Atp7b�/� animals at either 6 weeks or 30 weeks (n � 6 pergroup; *, p � 0.05).

FIGURE 7. Proposed role of copper in WD progression. The inactivation ofAtp7b�/� results in disrupted copper export from hepatocytes and copperaccumulation. Metallothionein (MT) is up-regulated as an early responseto copper overload and sequesters copper in the cytosol. Excess copperenters the nucleus and triggers the remodeling of liver transcriptome.Subsequently, copper leaves the nuclei, decreasing nuclear involvementand concentrating in the intracellular deposits. When copper reaches thelimit of accumulation, the uptake stops (or/and the efflux is activated), andmore intracellular copper is sequestered in the deposits (presumably lyso-somes). The copper that is not taken by hepatocytes is absorbed by theinflammatory cells and is deposited in extracellular aggregates. Metabolicpathways in compartments other than nucleus become activated.

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nature of the mechanism that facilitates loss of copper fromhepatocytes may open new avenues for treatment of WD.

Acknowledgments—We thank Cara Poage and Carolyn Gendron(Oregon Health & Science University Histology Core) for help withtissue sectioning and staining; Dr.Milton Finegold, Dr. AnnHubbard,and Dr. Jack H. Kaplan for critical reading of this manuscript andhelpful comments; and Wolfgang Wilfert for assistance with lipidmeasurements.

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Tony R. Capps, Lawrence Gray, Barry Lai, Edward Maryon and Svetlana LutsenkoMartina Ralle, Dominik Huster, Stefan Vogt, Wiebke Schirrmeister, Jason L. Burkhead,

HEPATOCYTESTRAFFICKING ACTIVATES COMPARTMENT-SPECIFIC RESPONSES IN

Wilson Disease at a Single Cell Level: INTRACELLULAR COPPER

doi: 10.1074/jbc.M110.114447 originally published online July 20, 20102010, 285:30875-30883.J. Biol. Chem. 

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