NeuroToxicology 31 (2010) 509517

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NeuroToxicology

Cuprizone neurotoxicity, copper deciency and neurodegeneration

Federico Benetti a,1, Marcello Ventura a, Benedetta Salmini a, Stefano Ceola b,
Donatella Carbonera b, Stefano Mammi b, Andrea Zitolo c, Paola DAngelo c, Emanuela Urso d,
Michele Mafa d, Benedetto Salvato a,2, Enzo Spisni e,2,*
a

Department of Biology, University of Padova, via U. Bassi 58/B, 35121 Padova, Italy
Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy
Department of Chemistry, University of Rome La Sapienza, P. le A. Moro 5, 00185 Roma, Italy
d
Department of Biology and Environmental Science and Technology, University of Salento, Prov.le Lecce-Monteroni, 73100 Lecce, Italy
e
Department of Experimental Biology, University of Bologna, Bologna, Italy
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 24 March 2010
Received in revised form 17 May 2010
Accepted 18 May 2010
Available online 26 May 2010

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to
several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical
formula, its neurotoxic mechanism is still unknown. In this work, we examined both physicochemical
properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated
and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross
neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by
cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its
neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper
deciency.
2010 Elsevier Inc. All rights reserved.

Keywords:
Cuprizone
Copper deciency
Ceruloplasmin activity
Neurodegeneration

1. Introduction
Cuprizone (CZ), oxalic acid bis(cyclohexylidene hydrazide)
(Fig. 1), is a selective and sensitive copper chelating agent. Soon
after its discovery, CZ was used for serum copper detection
(Peterson and Bollier, 1955). Some years later, oral administration
of CZ was found to produce severe status spongiosus (Carlton,
1967; Suzuki and Kikkawa, 1969), central nervous system (CNS)
demyelination and hepatic lesions (Suzuki and Kikkawa, 1969).
Thus, CZ-treated animals have been used as models to investigate
demyelination-associated diseases as well as remyelination
processes. CZ-treated mice revealed diffuse status spongiosus in
the brain stem (largely in the tegmentum of the pons and
midbrain), in the diencephalon (particularly in the thalamic nuclei)
and in the cerebellar and cerebral white matter. CNS demyelination induced by CZ acts mainly on oligodendrocytes causing their

Abbreviations: CZ, cuprizone; CZ[Cu], cuprizonecopper; Cp, ceruloplasmin; Cu(II),

copper (II).
* Corresponding author. Tel.: +39 051 209 41338; fax: +39 051 209 4286.
E-mail address: enzo.spisni@unibo.it (E. Spisni).
1
Present Address: Neurobiology Sector, Scuola Internazionale Superiore di Studi
Avanzati - International School for Advanced Studies (SISSA-ISAS), I-34014 Trieste,
Italy.
2
These two authors contributed equally to this work.
0161-813X/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuro.2010.05.008

damage and death (Binder et al., 2008; Matsushima and Morell,

2001; Stidworthy et al., 2003) probably by activation of the
microglia (Raivich and Banati, 2004). Activated microglia secrete a
variety of proinammatory molecules, including radical oxygen
species and cytokines, which can ultimately damage oligodendrocytes (Bakker and Ludwig, 1987; Binder et al., 2008; Kondo
et al., 1987).
A relationship has been shown to exist between copper
deciency and microglial activation in the cerebral cortex and
thalamus (Hiremath et al., 1998; Pasquini et al., 2007; Zucconi
et al., 2007). Moreover, CZ-induced disease presents pathological
signs similar to inherited copper metabolism disorders such as
Menkes and Wilson diseases: growth retardation, hepatic failure,
demyelination with cerebellar and cerebral atrophy (Geller et al.,
1997; Kaler et al., 1995; Kodama et al., 1999; Leventer et al., 1997;
Madsen and Gitlin, 2007), degeneration of gray matter and
neuronal loss especially in hippocampus and cerebellum (Barnard
et al., 1978; Oder et al., 1991; Okeda et al., 1991).
Menkes and Wilson inherited disorders are due to an
impairment of copper homeostasis caused by loss of function
mutations of Menkes protein ATP7A and Wilson protein ATP7B,
respectively. ATP7A transports copper to copper-requiring proteins within the secretory pathway in enterocytes (Bertini and
Rosato, 2008; Chelly et al., 1993; Mercer et al., 1993; Vulpe et al.,
1993). The loss of its activity results in copper deciency. ATP7B,

[(Fig._1)TD$IG]

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F. Benetti et al. / NeuroToxicology 31 (2010) 509517

Fig. 1. Molecular structure of CZ (Muniz-Miranda et al., 2008). Color code: carbon

(gray), hydrogen (white), oxygen (red) and nitrogen (blue). (For interpretation of
the references to color in this gure legend, the reader is referred to the web version
of the article.)

localized in hepatocytes, assists the incorporation of copper into

ceruloplasmin (Cp) or copper transfer from the liver to bile caniculi
(Linder, 1991). Its loss of function in Wilson disease results in the
synthesis of apo-Cp that is rapidly degraded in the plasma
(Hellman and Gitlin, 2002). Cp is an essential ferroxidase that
contains more than 95% of the plasma copper (Holmberg, 1944;
Holmberg and Laurell, 1948; Vassiliev et al., 2005). Its impairment
affects cupro- and iron-enzymes.
The CZ neurotoxic mechanism remains still unclear, even if
available information suggest that it could be due to its copper
chelation capability. It has been proposed that CZ could pass
through biological membranes and acts directly on intraneuronal
copper, stabilizing it as copper (III) (Messori et al., 2007; Zatta et al.,
2005).
In this work, we examined both the physico-chemical and the
biological behaviours of CZ. In particular, we analyzed CZ-copper
binding, CZ transport through the mouse duodenal epithelium, CZ
accumulation in liver and brain of CZ-fed mice and, nally, its
interaction with both neuronal membranes and cell viability in
vitro. Our data demonstrate that the CZ neurotoxic mechanism
involves its ionic copper chelation properties only, producing the
same effects as a chronic copper deciency.
2. Materials and methods
2.1. Materials
Cuprizone [oxalic acid bis(cyclohexylidene hydrazide)] (CZ)
was purchased from SIGMA-Aldrich, Copper Acetate Monohydrate
and Copper Sulphate from J. T. Baker. Other chemicals were
purchased from SIGMA-Aldrich.
2.2. Dialysis of CZ
A solution of 2 mM CZ in ethanol 50%-phosphate buffer 20 mM,
pH 7.6 was dialyzed against the same buffer using benzoylated
cellulose dialysis bags with a cut off of 1000 Da (D7884-10FT,
SIGMA-Aldrich). The passage through the membrane of CZ was
monitored by adding Cu(II) to both solutions and recording the
CZ[Cu(II)] complex absorption at 600 nm.
2.3. Ussing chamber studies
Experiments were performed on the duodenal tract isolated
from untreated mice and immediately placed in cold isosmolar
buffer, as described by Ungell et al. (1998). Briey, fragments of
duodenal tissue were mounted in three chambers simultaneously.
Both the mucosal and the serosal sides of the tissue were perfused
with 8 mL Krebs solution (118 mM NaCl, 4.6 mM KCl, 2.5 mM
CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM D-glucose) gassed

with a mixture of 5% CO2/95% O2 resulting in a physiological pH

(7.4) and maintained at 37 8C. After a period of 30 min, when
electrophysiological parameters were stabilized, 0.001% CZ was
added to the apical side of the chambers to monitor its
permeability across the membrane in the mucosal to serosal
direction. Aliquots of 100 mL from both the apical and the serosal
compartments were taken at 15-min intervals for 90 min and their
CZ content was revealed by adding an appropriate amount of
copper sulphate and monitoring spectrophotometrically (600 nm)
the appearance of the CZ[Cu(II)] complex. To assess the ability of
CZ[Cu(II)] complex to cross the epithelial membrane, 0.001% CZ
and 18 mM CuSO4 were simultaneously added to the apical bathing
solution and 100 mL aliquots from both chamber compartments
were collected at 515 min intervals for 80 min. Sample absorbances at 600 nm were measured as an index of disappearance/
appearance of the CZ[Cu(II)] complex at the mucosal/serosal sides
of tissue. A preliminary assay employing a substrate that certainly
crosses the intestinal epithelium in the mucosal to serosal
direction Cu(II) was conducted to conrm the suitability of
the Ussing chamber technique to study the permeability properties
of CZ. Specically 60 mM CuSO4 was added apically and 0.001% CZ
was dissolved in the basolateral bathing solution. Delivery of
copper ions through the intestinal tissue was analyzed by optical
density recordings made as for the CZ[Cu(II)] complex. For each
experiment optical density readings at 0 time were arbitrarily set
to 1. The transepithelial electrical parameters of duodenal tissue
were regularly recorded to ascertain the integrity of the membrane
throughout the experiments.
2.4. Cell culture and CZ treatment
GN11 and SH-SY5Y neurons were grown in high glucose
complete DMEM, supplemented with 10% foetal calf serum (FCS),
2 mM L-glutamine, penicillin (100 U/mL) and streptomycin
(100 mg/mL). For atomic absorption analysis, subconuent neurons (1.0  104 cells/cm2) were cultured for 96 h in T75 asks, in
the presence or in the absence of CuSO4 (0200 mM) and CZ
(200 mM). Before atomic absorption measurements, neurons were
washed four times with HBSS. For viability, cell growth and cell
cycle analyses, GN11 and SH-SY5Y neurons were cultured for 3
weeks in the presence or in the absence of CZ 200 mM. CZ was
added at every medium change. After this time neurons were
harvested and seeded onto 24-well plates maintaining the
presence of 200 mM CZ in treated samples. After 12, 24, 48 and
72 h, the cell number was evaluated by using the acidic
phosphatase method (Spisni et al., 1995). For cell cycle analyses,
GN11 and SH-SY5Y neurons, were cultured for 3 weeks in the
presence or in the absence of CZ 200 mM. Then, neurons were
harvested and seeded in 6-well plates in the presence or in the
absence of CZ 200 mM and grown for 48 h in low conuence (less
then 50% coverage of the culture surface area) before the analysis.
Stock solutions containing 40 mM CZ in 50% ethanol were prepared
every week. An equal amount of ethanol 50% solution was added to
the untreated cultures.
2.5. Atomic absorption analysis
A Perkin-Elmer atomic absorption spectrophotometer (model
AAnalyst 100) equipped with a multiple-element hollow-cathode
lamp (30 mA) was used. The wavelength and slit settings were
324.8 nm and 0.7 nm, respectively. The pressure was 440 kPa for
acetylene and 130 kPa for compressed air. The measurement of
intracellular copper concentration by atomic absorption was
performed using 106 cells per point. The sample copper
concentrations were calculated using a 4 ppm copper solution
(Baker Analyzed, USA) as standard. For non-specic copper binding

F. Benetti et al. / NeuroToxicology 31 (2010) 509517

determination, GN11 neurons were xed in 4% paraformaldheide,

blocked in 5% FBS for 30 min and incubated with the same copper
dose range. The values obtained were subtracted from the total
cellular copper measurements to determine the specic uptake of
the metal, as previously described (Watt and Hooper, 2001).

511

2.9. Statistical analysis

Statistical signicance was determined by ANOVA one-way
variance test. Mean differences were considered statistically
signicant at p < 0.05 or at p < 0.01. All results are expressed as
mean  SE.

2.6. Cell cycle analysis

For the evaluation of the cell cycle, GN11 neurons were
incubated with 10 mM bromodeoxyuridine (BrdUrd, SigmaAldrich) for 60 min in a CO2 incubator at 37 8C. Harvested cells
were xed in 70% ethanol for 30 min. After xation, cells were
treated with 100 mg/ml RNase for 30 min at 37 8C. DNA denaturation was obtained by using 2N HCl for 30 min at room
temperature. After the incubation with a-BrdUrd (Sigma) diluted
1:10 as the primary monoclonal Ab, cells were stained with a
secondary antimouse FITC Ab (Sigma), diluted 1:40. Finally, cells
were stained with 20 mg/ml propidium iodide (Sigma) before FACS
analysis.
2.7. Ceruloplasmin activity
The oxidase activity of Cp-containing FCS, in the presence or in
the absence of CZ, was tested by using a modied Curzons protocol
(Curzon and Vallet, 1960). A volume of 100 mL of serum was added
to 4 mL of 100 mM acetate buffer, pH 6 and 500 mL of 2.3 mM pphenylenediamine (PPD) and incubated at 37 8C for 1 h. The
reaction was blocked by adding 1.84 mL of 0.5% (w/v) sodium azide
and the oxidized product formation was followed recording the
absorbance at 530 nm. Cp activity was determined both in the
presence and in the absence of 200 mM CZ. The CZ[Cu(II)] complex
oxidase activity, in the same experimental conditions, was also
tested.
2.8. In vivo experiments and mass spectrometry
CD1 mice were fed with 0.5% (w/w) CZ-containing fodder for
16 days. Brains and livers isolated from control and CZ-treated
mice (n = 10) were analyzed by using ESI QTOF mass spectrometry, and compared with tissues containing 200 mM CZ (positive
controls). Brain and Liver samples for mass spectrometry analysis
were obtained as previously described (Zatta et al., 2005). Briey,
tissues were homogenized in CHCl3/CH3CH2OH (Merck) (1:1 (v/
v), 1 ml for each mg of tissue) by using a Ultra-Turrax at
10,000 rpm. After centrifugation (10,000  g for 20 min), supernatants were collected, evaporated and resuspended in 50 ml of
ethanol for each mg of starting tissue. CZ-containing tissue
extracts (positive controls) were obtained by adding to the tissues,
before homogenization, an amount of 2 mM CZ solution corresponding to 2.78 mg of CZ added for each mg of starting tissue.
After homogenization, samples were centrifuged at 10,000  g for
20 min. Supernatants were collected, evaporated by using a
SpeedVac concentrator and resuspended in 50 ml of ethanol for
each mg of starting tissue. In this manner, CZ-containing tissue
extracts (positive controls), had maximum content of CZ
corresponding to 200 mM.
Electrospray mass spectra were collected by using the nano-ESI
QTOF Ultima (Waters, Manchester, UK). The QTOF hybrid mass
spectrometer was equipped with a nanolock Z-spray source, and
coupled online with a capillary chromatography system CapLC
(Waters). Samples were directly injected at a ow rate of 1 ml/min,
with a capillary voltage of 2000 V and a cone voltage of 100 V.
Spectra were collected in the m/z range from 100 to 1000,
calibrating the signals with GluFibrinopeptide B. The collision
energy during MS/MS experiments was set up between 10 and
30 V, using argon as a collision gas.

3. Results
3.1. Physico-chemical behaviour of cuprizone
Since CZ has a very complicated solution chemistry, before
starting in vitro and in vivo experiments, we decided to investigate
CZ physico-chemical properties. These chemical analyses are
partially reported as supplementary information. The CZ[Cu]
complex presents a maximum absorption at 600 nm because
nitrogens produce a stronger ligand eld than water molecules
(Fig. 2). The complex formation was analyzed as a function of time,
and copper concentration by using absorption spectroscopy
(Supplementary Fig. 1S). Two different trends were observed: in
the presence of low copper concentration the CZ[Cu] complex
show a maximum after 17 days (Fig. 1S, panel A), while at high
copper concentrations, the CZ[Cu] complex concentration
decreases as a function of time (Fig. 1S, panel B). The divalent
oxidation state of Cu in CZ[Cu] complex has been demonstrated by
using Electron Paramagnetic Resonance (EPR) and X-ray Absorption (XAS) spectroscopies (Benetti et al., unpublished results).
Therefore, it seems very unlikely that CZ toxicity depends on its
capability to stabilize Cu as copper (III), since CuK-edge XAS clearly
demonstrate that only Cu(II) is present in the CZ[Cu] complex.
Dialysis and NMR experiments revealed the formation of large
aggregates in aqueous solutions. These analyses demonstrate that
CZ in aqueous solutions, such as tissue culture media or gastrointestinal uids, form high molecular weight aggregates capable to
bind large amounts of Cu(II) but with the tendency to precipitate
from solution at high copper concentration.
3.2. Biological behaviour of cuprizone
It has been recently proposed that CZ could act directly on
neurons (Messori et al., 2007; Zatta et al., 2005). To clarify whether
the neurotoxic effect of the CZ diet is a result of its direct action on
CNS, we evaluated its absorption through the duodenal tissue in
the
[(Fig._2)TD$IG] apical-to-basolateral direction by using Ussing chambers. The

Fig. 2. Absorption spectra of the CZ[Cu(II)] complex (black line) and Cu(II) aq (red
line). (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of the article.)

[(Fig._3)TD$IG]

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F. Benetti et al. / NeuroToxicology 31 (2010) 509517

suitability of this procedure was preliminarily ascertained by

analyzing the transepithelial delivery of Cu(II) ions, applied to the
apical side of tissue as copper sulphate. After addition of 0.001% CZ
to the basolateral side, a constant increase in optical density
readings (600 nm) was observed (p < 0.01), indicating the
transepithelial ux of Cu(II) ions (Fig. 3A). The absorbance values
recorded for the apical side were not signicantly different from
zero and remained steady (p > 0.05; data not shown), indicating
that there was not any basolateral-to-apical translocation of CZ.
Subsequently, the permeability to CZ was examined by adding a
0.001% CZ solution to the apical side of tissue. The appearance of CZ
in the serosal compartment of the chambers was determined by
adding an excess of Cu(II) to samples collected from serosal
solution and measuring their absorbance at 600 nm. Optical
density readings referred to 15 min interval sampling, were
considered as a permeability index of CZ if compared to the
absorbance values registered at the beginning of the experiments
(time 0, Fig. 3B). The absorbance values recorded for the serosal
side were not signicantly different from zero and remained quite
steady. In addition, optical density measured in samples collected
from the mucosal side of the duodenal tissue was not found to be
signicantly decreased with respect to the starting value
(p > 0.05), as could be expected if CZ passed through the epithelial
membrane (Fig. 3B). To ensure the viability of the tissue
throughout the experiments, electrophysiological parameters
were recorded before and after the addition of CZ to the apical
compartment of the chambers (Fig. 3C). The transepithelial voltage
(Vm) and the short circuit current (Isc) of the duodenal tissue
resulted unaffected by the exposure to CZ. To investigate the ability
of CZ[Cu(II)] complex to cross the intestinal epithelium, both
0.001% CZ and 18 mM copper sulphate were added to the apical
compartment of the chambers. Optical density readings of the
mucosal side were monitored up to 80 min, and values were
identical to the starting value (p > 0.05; Fig. 3D). At the same time,
absorbance values of samples collected from the basolateral side
were not signicantly different from the time 0 value throughout
the whole experiment, indicating that also the CZ[Cu(II)] complex
is non epithelium-permeant.
To conrm our ex vivo results, we analyzed the accumulation of
CZ in brains and livers of CZ-fed mice, by performing ESI QTOF MS/
MS mass spectra. Livers and brains were collected from mice after
16 days of controlled diets (food supplemented or not with 0.5%
CZ), before CZ toxicity symptoms appear, in accordance with the
ECC Directive 86/609 and APA Ethical Principles for Animal
Experimentation. Tissue extracts were injected into a mass
spectrometry system and mass spectra were collected (see
Supplementary Figs. 2S, 3S and 4S). MS/MS spectra of the peak
279, corresponding to the molecular ion of CZ, were analysed in
brain and liver samples of control and CZ-fed mice. These spectra
were compared to the MS/MS fragmentation spectrum of CZ
molecular ion ([M+H]+ = 279 m/z). Results, reported in Figs. 4 and
5, clearly showed that no detectable CZ was found either in liver or
in brain of CZ-fed mice.

Fig. 3. Transepithelial delivery of Cu(II) ions and membrane permeability of CZ in

the duodenal tract of mouse. (A) 60 mM CuSO4 was added to the apical bathing
solution, while 0.001% CZ was applied to the basolateral chamber. Absorbance
values (SD) recorded at 600 nm are referred to the formation of CZ[Cu] complex at
the serosal side of tissue, indicating the apical-to-basolateral translocation of copper.

Data are representative of three independent experiments (n = 3). Statistical

signicance was determined by ANOVA followed by Bonferroni post-test
(**p < 0.01). (B) Optical density values (SD) recorded at 600 nm (CZ[Cu] complex)
show the presence of the chelating agent in the apical compartment of Ussing
chambers. Data demonstrate no evidence of a membrane transport mechanism for CZ
throughout the duodenal tract of mouse (n = 3; ANOVA followed by Bonferroni posttest, *p < 0.05). (C) Transepithelial electrophysiological parameters of duodenal tissue
before (black boxes) and after (white boxes) the addition of CZ to the apical side of
Ussing chambers. Vm (&, &) and Isc (~, ~) indicate the transepithelial voltage and the
short circuit current. (D) Membrane permeability of CZ[Cu(II)] complex in the
duodenal tract of mouse. 0.001% CZ and 18 mM CuSO4 were simultaneously added to
the mucosal bathing solution. Optical density (SD) values at 600 nm are reported for
the apical side of tissue (n = 3; ANOVA followed by Bonferroni post-test, *p < 0.05).

[(Fig._4)TD$IG]

F. Benetti et al. / NeuroToxicology 31 (2010) 509517

513

Fig. 4. MS/MS fragmentation spectra of the peaks with m/z = 279 performed on CZ (2 mM, A), on liver of CZ-fed mice (TL, B) or on control liver in which CZ (200 mM) was added
(C). Typical m/z signals of CZ fragmentation are 113, 139 and 184 (indicated with arrows). The fragmentation spectra of the peak m/z = 279 obtained in CZ-fed mice liver did
not correspond to CZ. On the contrary, the fragmentation spectra of the peak m/z = 279 of positive control livers containing 200 mM CZ showed typical CZ peaks. Other mass
spectrometry data are reported under supplementary materials.

Although duodenal absorption experiments indicate that CZ

does not cross the transepithelial barrier and mass spectrometry
measurements did not reveal its presence in tissues extracted from
CZ-fed mice, traces of CZ and/or CZ[Cu(II)] could pass into body.
Therefore, we directly studied the CZ and CZ[Cu(II)] complex
neurotoxicity on cultured neuronal cells. Specically, internalization of both CZ and CZ[Cu(II)] complex or their interaction with
neuronal plasma membranes as well as the proliferation rates and
cellular viability of CZ-treated neurons were evaluated in different
neuronal cell lines: GN11 and SH-SY5Y. The intracellular copper
content of cells exposed for 96 h to increased extracellular copper
concentrations, in the presence or in the absence of CZ, was
measured by using the atomic absorption spectroscopy. Exposure

to extracellular copper resulted in increased intracellular copper

levels (Fig. 6A). On the contrary, the addition of copper in the
presence of extracellular CZ did not increase the intracellular
copper accumulation, conrming that neither CZ nor the CZ[Cu(II)]
complex interact with or cross-neuronal plasma membrane. The
CZ effect on neurons was also studied evaluating their proliferation
rates, viability, and cell cycle distribution. Neurons were cultured
for 3 weeks in the presence or in the absence of CZ (200 mM). Both
the control and the CZ-treated neurons showed identical viability,
proliferation rates (Fig. 6B) and cell cycle distribution (Fig. 6C). We
also tested CZ toxicity on primary cultures of rat neonatal cortical
neurons, prepared from newborn Wistar rat as previously
described (Small et al., 2004), and we did not observe any

[(Fig._5)TD$IG]

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F. Benetti et al. / NeuroToxicology 31 (2010) 509517

Fig. 5. MS/MS fragmentation spectra of the peaks with m/z = 279 performed on brain of CZ-fed mice (TB, A) or on control brains in which CZ (200 mM) was added (B). The
fragmentation spectra of the peak m/z = 279 obtained in CZ-fed mice brains did not correspond to CZ. On the contrary, the fragmentation spectra of the peak m/z = 279 of
positive control brains containing 200 mM CZ showed typical CZ peaks (indicated with arrows). Other mass spectrometry data are reported under supplementary
materials.

modications in cellular viability after 48 h of incubation with CZ

(data not shown).
Since copper is a cofactor of Cytochrome c oxidase, its
deprivation should cause a strong impairment of energy production and a reduction of viability and proliferation rates in cultured
neurons. Instead, cultured neurons treated with CZ do not change
their viability and proliferation rates, indicating a CZ-independent
uptake of copper. Neurons were cultured in the presence of serum,
containing Ceruloplasmin (Cp). Cp is a physiologically appropriate
method to provide copper to cells in culture (Percival and Harris,
1990). Therefore, it seems that CZ is unable to chelate Cp-bound
copper. To conrm this hypothesis, the CZ chelation capability on
Cp-bound copper was evaluated by analyzing the Cp oxidase
activity. In the presence of CZ, the absorption at 530 nm of the
oxidized product was 0.112  0.012 while in the absence of CZ, it
was 0.113  0.011. Both in the presence and in the absence of CZ, the
Cp oxidase activity was the same (ANOVA one-way variance test,
p > 0.05). CZ[Cu(II)] complex did not show oxidase activity in the
experimental conditions used (Fig. 7). These results substantiate the
assumption that CZ is not a neurotoxic molecule capable to directly
act on neurons or CNS.

4. Discussion
CZ, a highly sensitive copper chelator, is considered a
neurotoxic compound because of its involvement in demyelination
and spongiotic processes. Currently, the CZ neurotoxic mechanism
remains unknown, and two alternative hypotheses have been
formulated. The rst-one (Venturini, 1973) stated that CZ acts in
the gut as a copper chelator, since CZ-treated mice showed a
decrease in copper content as well as in copper enzymes activity
(monoamine oxidase and Cytochrome c oxidase). The second-one
(Zatta et al., 2005) stated that CZ acts directly in the brain and
inside neurons as a toxic molecule capable to cross-neuronal
plasma membrane. Although CZ has a simple chemical formula, its
solution chemistry is very complicated and misunderstood. The CZ
toxicity molecular mechanism seems to involve copper chelation
with the subsequent decrease in copper bioavailability. Therefore,
in this work we rstly studied the physico-chemical behaviour of
CZ and CZ[Cu] complex. CZ is an amphipathic molecule because of
its hydrophobic cyclohexane rings and the hydrophilic oxalic and
hydrazonic groups. In polar environments, CZ tends to form high
molecular weight aggregates with the hydrophilic parts in contact

[(Fig._6)TD$IG]

[(Fig._7)TD$IG]

F. Benetti et al. / NeuroToxicology 31 (2010) 509517

515

Fig. 7. Oxidase activity of ceruloplasmin-containing FCS, measured in the presence

or in the absence of CZ. Oxidase activity of the CZ[Cu(II)] complex is also shown.

Fig. 6. Neuronal culture and CZ treatment. (A) CZ[Cu(II)] complex and CZ does not
interact with or cross-neuronal plasma membrane. Intracellular and extracellular
copper levels were evaluated using atomic absorption, after culturing GN11
neurons for 96 h with medium supplemented with different copper concentrations
(0200 mM), in the presence or in the absence of CZ (200 mM). Each point
represents the average (SEM) of two independent experiments, four experimental
points altogether, performed on GN11 neurons. Cellular copper stores increased when
extracellular copper concentration rose. Differences in cellular copper amounts,
compared to untreated cells, were analysed by the ANOVA test. The asterisk indicates
p < 0.05 while hashes indicates p < 0.01. (B) Effects of CZ on neuronal proliferation
rate. Extracellular CZ, 200 mM, was added to the culture media at each medium change.
After 3 weeks of culture into asks, in the presence or in the absence of CZ, cells were
trypsinised and reseeded onto 24-well plates maintaining the presence of 200 mM CZ
in treated samples. After 12, 24, 48 and 72 h, the cell number (SEM) was evaluated.
(C) Effects of CZ on GN11 cell cycle distribution. After 3 weeks of culture into asks, in
the presence or in the absence of CZ, GN11 neurons were seeded in multiwell plates,
maintaining the presence of 200 mM CZ in treated samples, and grown for 48 h in low
conuence before cell cycle analysis. Each point represents the media (SEM) of at
least two independent experiments and six experimental replicates. Similar results
were obtained by using SH-SY5Y cells.

with the surrounding solvent. Copper (II) ions within the CZ[Cu(II)]
complex are chelated by four nitrogen atoms that probably belong
to different CZ molecules. XAS analyses unequivocally indicate a
divalent oxidation state for the complex, in contrast of what
previously indicated (Messori et al., 2007).
In this work we show that CZ does not pass through the mouse
duodenal membrane. Since the absorption processes predominantly take place in the duodenal region of the intestine, we can
reasonably extend this concept to the entire intestinal tract. The
absence of mass spectrometry signals corresponding to CZ in both
liver and brain extracts of CZ-fed mice, further support the
impermeability of the gut to CZ. Certainly, CZ does not accumulate
in liver or brain of CZ-treated mice.
It is quite obvious to expect that a neurotoxic molecule is
capable to affect neuronal survival. So, we analyzed the effects of
CZ and CZ[Cu(II)] complex on neuronal viability and proliferation
rates as well as their capability to cross the neuronal plasma
membranes. It was a surprise to observe that both, CZ and
CZ[Cu(II)] complex, do not cross the neuronal plasma membranes
or alter the proliferation rate, the cell cycle or the viability of
cultured neurons. This suggest that CZ is not toxic for neurons and
that it is not capable to produce a condition of copper deciency in
cultured cells. Indeed, proliferation and viability processes require
metabolic energy produced by copper enzymes present in the
respiratory chain. In mammals, Cp contains about 95% of plasma
copper and it is involved both in conversion from iron(II) to iron(III)
and in copper transport from liver to peripheral cells and tissues
(Healy and Tipton, 2007; Lontie, 1984). An explanation for our
results could be that the addition of Cp to cell culture media, via
serum, represents an appropriate method for providing copper to
cells (Percival and Harris, 1990) also in the presence of CZ.
Therefore, we analyzed the CZ effect on Cp-bound copper by
testing the Cp oxidase activity. Foetal calf serum, in the presence or
in the absence of CZ, showed the same oxidase activity indicating
that CZ is not capable to chelate Cp-bound copper. These datum is
in agreement with the hypothesis that CZ can chelate copper ions
only and that it has no effect on protein-bound copper present in
body uids. Since in vivo experiments were performed by adding
CZ to commercially available mice food, containing copper in an
ionic form (copper sulphate, 11 ppm), we concluded that CZ
neurotoxic mechanism corresponds to that produced by a copper
decient diet.
The brain utilizes metal ions for several biochemical reactions,
and cortical neurons release ionically exchangeable copper and
zinc during depolarization and neurotransmission (Schlief et al.,
2005; Howell et al., 1984). The concentrations of copper, iron and
zinc in the gray matter are in the same order of magnitude as that

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F. Benetti et al. / NeuroToxicology 31 (2010) 509517

of magnesium: 0.10.5 mM (Bush, 2003). Since the brain has the

highest metabolic rate of all organs and depends predominantly on
oxidative metabolism for its energy (Strozyk and Bush, 2006), it is
not surprising that the deciency or dysmetabolism of these
metals dramatically affects CNS functions.
Transition metals are also involved in other metabolic processes
such as, for example, stabilization of collagen brils and protection
against free radicals. Therefore, alterations in their metabolism
cause damages in several physiological processes and may produce
various symptoms involving the entire organism, as in the case of
copper deciency (Harvey and McArdle, 2008). Recently, Werner
et al. (2010) investigated brain tissues following dietary CZ
administration by using a proteomic approach. The results of this
study clearly point out that the mitochondrial function, and in
particular oxidative phosphorylation and ubiquinone biosynthesis,
is the most altered cellular function following CZ treatment. Major
upregulated proteins, after 6 weeks of CZ treatment, include
several enzymes devoted to the electron transfer. Interestingly, CZtreated mice also show hepatic lesions which are characterized by
the formation of mega-mitochondria. In the light of our results,
these mitochondria impairment may be explained with the
reduction activity of the terminal cupro-enzyme of the electron
transport chain: Cytochrome c oxidase. The ndings that
mitochondrial impairment and mega-mitochondria have been
also observed in animal models of Menkes disease (Rossi et al.,
2001) further sustain our conclusions. If low bioavailability of
copper may result in demyelination and neurodegeneration, it is
important to keep in mind that also the excess of free extracellular
copper may induce oxidative stress, inammation and neurodegeneration (Spisni et al., 2009). The results of our study emphasizes
the role of copper deciency in demyelination processes as well as
its putative role in the pathogenesis of neurodegenerative diseases.
Moreover, they further underline the importance of copper
homeostasis in neurons and CNS.
Conict of interest
None.
Acknowledgements
Authors acknowledge the European Synchrotron Radiation
Facility and BM30B for provision of synchrotron radiation facilities
and Olivier Proux for the helpful support. Prof. S. Radovick
(University of Chicago, Chicago, IL) for providing GN11 cells. This
work was supported by grants from MIUR (FIRB 2003
RBNE03FMCJ).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.neuro.2010.05.008.
References
Bakker DA, Ludwig SK. Bloodbrain barrier permeability during cuprizone-induced
demyelination. Implications for the pathogenesis of immune-mediated demyelinating diseases. J Neurol Sci 1987;78:12537.
Barnard RO, Best PV, Erdohazi M. Neuropathology of Menkes disease. Dev Med Child
Neurol 1978;20:58697.
Bertini I, Rosato A. Menkes disease. Cell Mol Life Sci 2008;65:8991.
Binder MD, Cate HS, Prieto AL, Kemper D, Butzkueven H, Gresle MM, et al. Gas6
deciency increases oligodendrocyte loss and microglial activation in response to
cuprizone-induced demyelination. J Neurosci 2008;28:5195206.
Bush AI. The metallobiology of Alzheimers disease. Trends Neurosci 2003;26:20714.
Carlton WW. Studies on the induction of hydrocephalus and spongy degeneration by
cuprizone feeding and attempts to antidote the toxicity. Life Sci 1967;6:119.

Chelly J, Tumer Z, Tonnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, et al.

Isolation of a candidate gene for Menkes disease that encodes a potential heavy
metal binding protein. Nat Genet 1993;3:149.
Curzon G, Vallet L. The purication of human caeruloplasmin. Biochem J
1960;74:27987.
Geller TJ, Pan Y, Martin DS. Early neuroradiologic evidence of degeneration in Menkes
disease. Pediatr Neurol 1997;17:2558.
Harvey LJ, McArdle HJ. Biomarkers of copper status: a brief update. Br J Nutr
2008;99(Suppl. 3):S103.
Healy J, Tipton K. Ceruloplasmin and what it might do. J Neural Transm 2007;114:
77781.
Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function. Annu Rev Nutr
2002;22:43958.
Hiremath MM, Saito Y, Knapp GW, Ting JP, Suzuki K, Matsushima GK. Microglial/
macrophage accumulation during cuprizone-induced demyelination in C57BL/6
mice. J Neuroimmunol 1998;92:3849.
Holmberg CG. On the presence of a laccase-like enzyme in serum and its relation to the
copper in serum. Acta Physiol Scand 1944;8:2279.
Holmberg CG, Laurell CB. Investigations in serum copper II, isolation of the coppercontaining protein and a description of some of its properties. Acta Chem Scand
1948;2:5506.
Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc
in hippocampal slices. Nature 1984;308:7368.
Kaler SG, Buist NR, Holmes CS, Goldstein DS, Miller RC, Gahl WA. Early copper therapy
in classic Menkes disease patients with a novel splicing mutation. Ann Neurol
1995;38:9218.
Kodama H, Murata Y, Kobayashi M. Clinical manifestations and treatment of Menkes
disease and its variants. Pediatr Int 1999;41:4239.
Kondo A, Nakano T, Suzuki K. Blood-brain barrier permeability to horseradish peroxidase in twitcher and cuprizone-intoxicated mice. Brain Res 1987;425:18690.
Leventer RJ, Kornberg AJ, Phelan EM, Kean MJ. Early magnetic resonance imaging
ndings in Menkes disease. J Child Neurol 1997;12:2224.
Linder MC. Biochemistry of copper. New York: Plenum Press; 1991 pp. 113.
Lontie R. Copper proteins and copper enzymes. Boca Raton, FL: CRC Press; 1984.
Madsen E, Gitlin JD. Copper and iron disorders of the brain. Annu Rev Neurosci
2007;30:31737.
Matsushima GK, Morell P. The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathol
2001;11:10716.
Mercer JF, Livingston J, Hall B, Paynter JA, Begy C, Chandrasekharappa S, et al. Isolation
of a partial candidate gene for Menkes disease by positional cloning. Nat Genet
1993;3:205.
Messori L, Casini A, Gabbiani C, Sorace L, Muniz-Miranda M, Zatta P. Unravelling the
chemical nature of copper cuprizone. Dalton Trans 2007;21124.
Muniz-Miranda M, Pagliai M, Cardini G, Messori L, Bruni B, Casini A, et al. A multitechnique approach to predicting the molecular structure of cuprizone in the gas
phase and in the crystalline state. CrystEngComm 2008;10:41622.
Oder W, Grimm G, Kollegger H, Ferenci P, Schneider B, Deecke L. Neurological and
neuropsychiatric spectrum of Wilsons disease: a prospective study of 45 cases. J
Neurol 1991;238:2817.
Okeda R, Gei S, Chen I, Okaniwa M, Shinomiya M, Matsubara O. Menkes kinky hair
disease: morphological and immunohistochemical comparison of two autopsied
patients. Acta Neuropathol 1991;81:4507.
Pasquini LA, Calatayud CA, Bertone Una AL, Millet V, Pasquini JM, Soto EF. The neurotoxic
effect of cuprizone on oligodendrocytes depends on the presence of pro-inammatory cytokines secreted by microglia. Neurochem Res 2007;32:27992.
Percival SS, Harris ED. Copper transport from ceruloplasmin: characterization of the
cellular uptake mechanism. Am J Physiol 1990;258:C1406.
Peterson RE, Bollier ME. Spectrophotometric determination of serum copper with
Biscyclohexanoneoxalyldihydrazone. Anal Chem 1955;27:11957.
Raivich G, Banati R. Brain microglia and blood-derived macrophages: molecular
proles and functional roles in multiple sclerosis and animal models of autoimmune demyelinating disease. Brain Res Brain Res Rev 2004;46:26181.
Rossi L, De Martino A, Marchese E, Piccirilli S, Rotilio G, Ciriolo MR. Neurodegeneration
in the animal model of Menkes disease involves Bcl-2-linked apoptosis. Neuroscience 2001;103:1818.
Schlief ML, Craig AM, Gitlin JD. NMDA receptor activation mediates copper homeostasis in hippocampal neurons. J Neurosci 2005;25:23946.
Small CI, Lyles GA, Breen KC. Inducible form of nitric oxide synthase expression in rat
cortical neuronal cells in vitro. Neurobiol Dis 2004;17:706.
Spisni E, Bartolini G, Orlandi M, Belletti B, Santi S, Tomasi V. Prostacyclin (PGI2)
synthase is a constitutively expressed enzyme in human endothelial cells. Exp
Cell Res 1995;219:50713.
Spisni E, Valerii MC, Manerba M, Strillacci A, Polazzi E, Mattia T, et al. Effect of copper
on extracellular levels of key pro-inammatory molecules in hypothalamic GN11
and primary neurons. Neurotoxicology 2009;30(4):60512.
Stidworthy MF, Genoud S, Suter U, Mantei N, Franklin RJ. Quantifying the early stages of
remyelination following cuprizone-induced demyelination. Brain Pathol
2003;13:32939.
Strozyk D, Bush AI. The role of metal ions in neurology. An introduction. Metal Ions Life
Sci 2006;1:17.
Suzuki K, Kikkawa Y. Status spongiosus of CNS and hepatic changes induced by
cuprizone (biscyclohexanone oxalyldihydrazone). Am J Pathol 1969;54:30725.
Ungell AL, Nylander S, Bergstrand S, Sjoberg A, Lennernas H. Membrane transport of
drugs in different regions of the intestinal tract of the rat. J Pharm Sci 1998;87:
3606.

F. Benetti et al. / NeuroToxicology 31 (2010) 509517

Vassiliev V, Harris ZL, Zatta P. Ceruloplasmin in neurodegenerative diseases. Brain Res
Brain Res Rev 2005;49:63340.
Venturini G. Enzymic activities and sodium, potassium and copper concentrations in
mouse brain and liver after cuprizone treatment in vivo. J Neurochem
1973;21:114751.
Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for
Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat
Genet 1993;3:713.
Watt NT, Hooper NM. The response of neurones and glial cells to elevated copper. Brain
Res Bull 2001;55:21924.

517

Werner SR, Saha JK, Broderick CL, Zhen EY, Higgs RE, Dufn KL, et al. Proteomic
analysis of demyelinated and remyelinating brain tissue following
dietary cuprizone administration. J Mol Neurosci (April)2010 [Epub ahead of
print].
Zatta P, Raso M, Zambenedetti P, Wittkowski W, Messori L, Piccioli F, et al. Copper and
zinc dismetabolism in the mouse brain upon chronic cuprizone treatment. Cell Mol
Life Sci 2005;62:150213.
Zucconi GG, Cipriani S, Scattoni R, Balgkouranidou I, Hawkins DP, Ragnarsdottir KV.
Copper deciency elicits glial and neuronal response typical of neurodegenerative
disorders. Neuropathol Appl Neurobiol 2007;33:21225.