doi:10.

1093/brain/awt303 Brain 2014: 137; 137–152 | 137

BRAIN
A JOURNAL OF NEUROLOGY

Zinc deficiency dysregulates the synaptic

ProSAP/Shank scaffold and might contribute
to autism spectrum disorders
Stefanie Grabrucker,1,2,* Linda Jannetti,1,* Matti Eckert,1 Simone Gaub,2 Resham Chhabra,1
Stefanie Pfaender,3 Katharina Mangus,1 Parameshwar Pasham Reddy,4 Vladan Rankovic,4
Michael J. Schmeisser,3 Michael R. Kreutz,4 Günter Ehret,2 Tobias M. Boeckers3 and
Andreas M. Grabrucker1,3

1 WG Molecular Analysis of Synaptopathies, Neurology Department, Neurocentre of Ulm University, Ulm, Germany
2 Institute of Neurobiology, Ulm University, Ulm, Germany
3 Institute for Anatomy and Cell Biology, Ulm University, Ulm Germany
4 RG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany

*These authors contributed equally to this work.

Correspondence to: Prof. Dr. Andreas M. Grabrucker,

WG Molecular Analysis of Synaptopathies,
Neurology Dept. of Ulm University,
Albert Einstein Allee 11,
89081 Ulm Germany
E-mail: andreas.grabrucker@uni-ulm.de

Proteins of the ProSAP/Shank family act as major organizing scaffolding elements within the postsynaptic density of excitatory
synapses. Deletions, mutations or the downregulation of these molecules has been linked to autism spectrum disorders, the
related Phelan McDermid Syndrome or Alzheimer’s disease. ProSAP/Shank proteins are targeted to synapses depending on
binding to zinc, which is a prerequisite for the assembly of the ProSAP/Shank scaffold. To gain insight into whether the
previously reported assembly of ProSAP/Shank through zinc ions provides a crossing point between genetic forms of autism
spectrum disorder and zinc deficiency as an environmental risk factor for autism spectrum disorder, we examined the interplay
between zinc and ProSAP/Shank in vitro and in vivo using neurobiological approaches. Our data show that low postsynaptic
zinc availability affects the activity dependent increase in ProSAP1/Shank2 and ProSAP2/Shank3 levels at the synapse in vitro
and that a loss of synaptic ProSAP1/Shank2 and ProSAP2/Shank3 occurs in a mouse model for acute and prenatal zinc
deficiency. Zinc-deficient animals displayed abnormalities in behaviour such as over-responsivity and hyperactivity-like behav-
iour (acute zinc deficiency) and autism spectrum disorder-related behaviour such as impairments in vocalization and social
behaviour (prenatal zinc deficiency). Most importantly, a low zinc status seems to be associated with an increased incidence rate
of seizures, hypotonia, and attention and hyperactivity issues in patients with Phelan-McDermid syndrome, which is caused by
haploinsufficiency of ProSAP2/Shank3. We suggest that the molecular underpinning of prenatal zinc deficiency as a risk factor
for autism spectrum disorder may unfold through the deregulation of zinc-binding ProSAP/Shank family members.

Keywords: PSD; ASD; Shank3; synapse; Zn2 +

Abbreviation: MT = metallothionein

Received May 24, 2013. Revised September 2, 2013. Accepted September 8, 2013. Advance Access publication November 25, 2013
ß The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
138 | Brain 2014: 137; 137–152
Introduction
The three members of the ProSAP/Shank family of scaffolding
proteins are key molecules for the spatial organization of the post-
synaptic density of excitatory synapses (Boeckers et al., 2002).
Recent studies report mutations in ProSAP1/Shank2, ProSAP2/
Shank3 and Shank1 associated with autism (Durand et al.,
2007; Moessner et al., 2007; Gauthier et al., 2009; Berkel
et al., 2010; Pinto et al., 2010; Leblond et al., 2012; Sato
et al., 2012). Moreover, the loss of one copy of ProSAP2/
Shank3 in humans is correlated with Phelan-McDermid syndrome
(22q13 deletion syndrome) (Bonaglia et al., 2001; Phelan et al.,
2001; Wilson et al., 2003; Manning et al., 2004). This syndrome is
characterized by moderate to profound mental retardation, neo-
natal hypotonia, global developmental delay, absent or severely
impaired speech (Manning et al., 2004), occurrence of seizures
and ‘autistic-like’ behaviour. Besides genetic factors, it has been
suggested previously that a dysregulation of metal-ion homeosta-
sis might contribute to the pathogenesis of autism spectrum
disorders (Curtis and Patel, 2008). Intriguingly, the incidence
rate of Zn2 + -deficiency is as high as 50% for autistic patients
between 0 and 3 years of age compared with 51% in a healthy
control group (Yasuda et al., 2011).
Recent reports demonstrate a tight interplay between
Zn2 + -binding of ProSAP1/Shank2 and ProSAP2/Shank3, but not
Shank1 (Baron et al., 2006; Gundelfinger et al., 2006; Grabrucker
et al., 2011a) and their synaptic association and function
(Grabrucker et al., 2011a). Zn2 + is enriched in cortical, striatal
and hippocampal regions of mammals and selectively stored in,
and co-released with glutamate from presynaptic vesicles (Lin
et al., 2001; Wei et al., 2004). It has been argued that Zn2 +
released from presynaptic vesicles might influx into the postsynap-
tic compartment through NMDA and AMPA receptors, and
Ca2 + -channels (Frederickson et al., 2005) and thereby provide a
pool of free chelatable Zn2 + . The postsynaptic density has an
unusually high Zn2 + -content (Jan et al., 2002) and postsynaptic
Zn2 + is probably bound to metallothioneins (MTs) as well as to
structural proteins such as ProSAP1/Shank2 and ProSAP2/Shank3
(Lee et al., 2003; Grabrucker et al., 2011a). Although indirect
evidence for an activity-dependent increase of postsynaptic
Zn2 + -levels was reported (Bitanihirwe and Cunningham, 2009),
it is currently unknown whether the postsynaptic Zn2 + -pool is
dynamic and whether free Zn2 + can contribute to the folding
and synaptic association of ProSAP1/Shank2 and ProSAP2/
Shank3. We therefore studied whether the postsynaptic density
scaffold at the synapse is regulated by Zn2 + -levels, and whether
Zn2 + -deficiency is accompanied by a dysregulation of the ProSAP/
Shank postsynaptic density scaffold in vitro and in vivo.
Moreover, we addressed the possibility that this mechanism
might constitute a plausible interplay between an environmental
risk factor and a known molecular pathway associated with autism
spectrum disorder. To that end, we used acute and prenatal
Zn2 + -deficient animals and performed behavioural analyses testing
for autism-related phenotypes. Finally, we investigated, whether
Zn2 + -deficiency augments specific symptoms observed in patients
with Phelan-McDermid syndrome.
S. Grabrucker et al.
Materials and methods
Materials
ZnCl2, CaEDTA and TPEN [N,N,N0 ,N0 -tetrakis(2-pyridylmethyl) ethyl-
enediamine], Zinquin ethyl-ester and Zinpyr1 were purchased from
Sigma-Aldrich as were all other chemicals unless indicated otherwise.
Primary antibodies were purchased from Synaptic Systems (Gephyrin,
Homer1, Homer1b/c, GluA1, GluA2, GluA3), Stressgen (bassoon),
Abcam (PSD95) Novus Biological [GKAP/SAPAP, Shank1 (immuno-
fluorescence, immunohistochemistry), mGluR5], Sigma [b-actin,
GluN1, Shank1 (western blotting)], Santa Cruz (MT-3), Chemicon
(MAP2, mGluR5) and Millipore (GluN2A, GluN2B). ProSAP1/Shank2
and ProSAP2/Shank3 antibodies have been described previously
(Schmeisser et al., 2012). Secondary Alexa FluorÕ conjugated antibo-
dies were purchased from Invitrogen.
Expression constructs and transfection
The pEGFP (C1-3) vector system (Clontech) was used for MT-3 ex-
pression constructs and the pLVX vector system (Clontech) for MT-3
knockdown. Short hairpin RNA oligonucleotides were purchased from
MWG-Eurofins. Two sequences were used for MT-3 knockdown:
AAGGGCTGCAAATGCACGA and CCTGCCCCTGTCCTACTGG.
Hippocampal cells were transfected at Day 10 and fixed on Day 14
in vitro using OptiFectTM (Invitrogen).
Immunohistochemistry
Cryosections were thawed for 20 min and fixed in paraformaldehyde
for 20–30 min. After fixation, sections were washed 3  10 min with
PBS. Sections were permeabilized with 0.2% Triton in PBS for 2 h and
washed again for 3  10 min with PBS containing 0.05% Triton.
Sections were then placed in 10% foetal calf serum in PBS for 2 h.
After blocking, sections were incubated with the primary antibody at
37 C for 2 h before 3  10 min washing with PBS containing 0.05%
Triton. Sections were incubated with the secondary antibody at 37 C
for 1.5 h. After washing 3  15 min with PBS containing 0.05% Triton,
sections were washed for 2  5 min with PBS containing DAPI, rinsed
with distilled H2O and mounted with VectaMountTM (Vector
Laboratories).
Timm’s staining was performed as described by Jaarsma and Korf
(1990) (Supplementary material). Zinc staining was performed using
10 mM Zinpyr1 for 1 h at room temperature.
Hippocampal culture, stainings and
treatments
Hippocampal cultures were prepared from rat (embryonic Day 18) as
described previously (Grabrucker et al., 2009). For fluorescent
Zn2 + -staining, growth medium was discarded and cells were washed
3  with Hank’s Balanced Salt Solution. Coverslips were incubated
with a solution of 25 mM Zinquin ethyl ester or 5 mM Zinpyr1 in
Hank’s Balanced Salt Solution for 40 min at 37 C (27). Immunofluor-
escence was performed as described previously with minor modifi-
cations (Grabrucker et al., 2011a) (Supplementary material).
Growth media were supplemented with Zn2 + -chelators (TPEN or
CaEDTA) or ZnCl2 (10 mM). CNQX (10 mM) and AP5 (50 mM) were
used to block synaptic activity. For stimulations, 10 mM ZnCl2, 50 mM
KCl (HiK + ) or 30 mM glutamate was used. Generation of nitric oxide
Zn deficiency dysregulates ProSAP/Shank
(NO) was manipulated using the NO agonist spermine nonoate (1 mM)
and antagonist N-nitro-L-arginine (100 mM).
Quantitative real-time polymerase
chain reaction
Isolation of total RNA from three control and three Zn2 + -deficient
mice as well as 10 pups from control and Zn2 + -deficient mice each
was performed using the RNeasyÕ kit (Qiagen). First strand synthesis
and quantitative real-time-PCR amplification were carried out in a
one-step, single-tube format using the QuantiFastTM SYBRÕ Green
RT-PCR kit (Qiagen). Thermal cycling and fluorescent detection were
performed using the Rotor-GeneÕ Q real-time PCR machine (model 2-
Plex HRM) (Qiagen). The SYBRÕ Green I reporter dye signal was
measured against the internal passive reference dye (ROX) to normal-
ize non-PCR-related fluctuations (Supplementary material). Resulting
data were analysed using the hydroxymethylbilane synthase gene as
an internal standard to normalize transcript levels. Cycle threshold (ct)
values were calculated by the Rotor-GeneÕ Q Software (version
2.0.2). All quantitative real-time PCR reactions were run in technical
triplicates and mean ct-values for each reaction were taken into
account for calculations.
Protein biochemistry
Subcellular fractions from mouse brain were isolated as described
previously with minor modifications (Schmeisser et al., 2012)
(Supplementary material). Proteins were separated by SDS-PAGE and
blotted onto nitrocellulose membranes. Immunoreactivity was visua-
lized using horseradish peroxidase-conjugated secondary antibodies
and the SuperSignalÕ detection system (Pierce).
Intrinsic fluorescence spectroscopy
Tryptophan fluorescence emission spectra were recorded for the
ProSAP2/Shank3-SAM domain (rat ProSAP2/Shank3 aa1464–1815,
Genscript) on a Hitachi F7000 spectrofluorimeter. The quartz cuvette
was cleaned with chromic acid and double-distilled H2O. EDTA
(100 mM) solution was used to rinse the cuvette followed by double-
distilled H2O. One millilitre of protein sample (0.1–0.3 mg/ml) in
50 mM Tris-HCl pH 7.4, 100 mM KCl was used for measurements.
Excitation wavelength of 295 nm was used and spectra were recorded
in the range of 300–450 nm by subtracting time-dependent reduction
of fluorescence emission. All spectra were recorded at room tempera-
ture in corrected spectra mode using an excitation and emission band
pass of 10 nm and 10 nm, respectively. The response-time was set to
2 s with a scan speed of 240 nm/min. The effects of Mg and Zn
standard solutions (Fluka) were studied by titrating them into protein
solution after 1–2 min incubation periods.
Animals
Ten-week-old mice were purchased from Janvier and housed in plastic
cages under the standard laboratory conditions (average temperature
of 22 C, food and water available ad libitum). Lights were automat-
ically turned on/off in a 12 h rhythm (lights on at 7 am). After one
week of acclimation, mice were divided into two groups, one group
(12 females) was fed a Zn2 + -deficient diet (4 ppm zinc, TestDiet) with
distilled, demineralized drinking water, whereas the control group (12
females) was fed with standard laboratory food (35 ppm zinc) and tap
water. To prevent zinc contamination, feeding jars, water bottles and
Brain 2014: 137; 137–152 | 139
plastic cages were rinsed with HCl and deionized water. After 5 weeks,
females of the control and Zn2 + -deficient group were mated. For
behavioural tests, acute Zn2 + -deficient animals were tested as well
as their offspring (prenatal Zn2 + -deficient) at the same age and
compared with control animals. A detailed description of the behav-
ioural tests performed can be found in the Supplementary material.
All animal experiments were performed in compliance with the
guidelines for the welfare of experimental animals issued by the
Federal Government of Germany and the local ethics committee
(Ulm University) ID Number: 0.103.
Human subjects
Blood samples from 37 participants diagnosed with Phelan-McDermid
syndrome were obtained from Nelson Biological Laboratories at
Rutgers University and atomic absorption spectroscopy performed at
the ‘ZE klinische Chemie’ of Ulm University Hospital to quantify Cu2 +
and Zn2 + levels. Blood samples were mostly from fasting blood dona-
tions and participants were evaluated regarding their status on dietary
zinc or vitamin supplementations. All experiments were performed in
compliance with the local ethics committee (Ulm University, ID: 282/
12) and informed consent was obtained from all subjects. Clinical and
developmental data of the participants were obtained through the
Phelan-McDermid syndrome registry. A total of 26 parameters were
evaluated based on the categorization of participants into three groups
(no, mild and severe Zn2 + -deficiency), including age, gender, diagno-
sis, birth weight, number of ear infections, possible signs of primary
immunodeficiency, occurrence of: hypotonia, seizures, nose and throat
problems, scoliosis, dysplastic finger/toenails, eczema, gastrointestinal
problems, allergy-related problems, endocrine conditions, abnormal
involuntary movements, sensitivity problems, other neurological con-
ditions, abnormal MRI scans, abnormal CAT scans, feeding disorder,
anxiety disorder, diagnosed language disorder, and attention deficit
disorder/attention deficit hyperactivity disorder or other attention or
hyperactivity issues. The overall occurrence of a parameter was calcu-
lated and the fraction of each Zn2 + status-group showing the param-
eter identified. Zn2 + and Cu2 + levels were compared with (whole
blood) reference values for age matched healthy controls (age 0–1:
41–58 mmol/l; age 1–4: 40–50 mmol/l; age 4–6: 49–52 mmol/l; age
6–14: 51–60 mmol/l; age 14–18; 50–56 mmol/l; age 18 and up:
66–117 mmol/l). Adult Zn2 + concentrations show no significant correl-
ation with age, although some studies report a decrease in very old
age (Rükgauer et al., 1997).
Statistics
Cell culture experiments
For cell culture experiments, 10 cells of each condition were imaged.
For brain sections, three animals of each group (control,
Zn2 + -deficient) were used and each of the four brain regions (cere-
bellum, cortex, hippocampus, striatum) imaged by at least three optic
fields. Fluorescence images were obtained with an upright Axioscope
microscope equipped with a Zeiss CCD camera (16 bits; 1280  1024
ppi) using Axiovision software (Zeiss). The signal intensity of all signals
within the optic fields was quantified using ImageJ 1.46p. Statistical
analysis was performed using Microsoft Excel and tested for signifi-
cance using t-tests followed by ANOVA.
Western blot data
Evaluation of western blot bands was performed using ImageJ 1.46p.
Three independent experiments were performed and the integrated
140 | Brain 2014: 137; 137–152
density of bands was measured. All bands were normalized to b-actin
and the ratios averaged and tested for significance. The  level of
significance was set at 0.05 (*P 5 0.05; **P 5 0.01; ***P 5 0.001).
Behavioural data
Normal distribution of data was determined by Shapiro-Wilk test. Two
sets of data were compared with t-test, if normally distributed, or
Wilcoxon-Mann-Whitney U-test if not normally distributed. Several
data sets were compared with one-way ANOVA or ANOVA on
ranks and Chi-square test. Although not all data are normally distrib-
uted, data are sometimes shown in figures as means  SEM to facili-
tate comparisons. Statistical tests were performed using absolute
values, despite some diagrams displaying percentages for better
comparison. All tests were run by SPSS (version 19) and SigmaPlot
software (version 2.0). Statistical tests were two-tailed with a signifi-
cance level of  4 0.05 (*P 5 0.05; **P 5 0.01; ***P 5 0.001).
Results
The local zinc concentration regulates
ProSAP/Shank protein levels at the
postsynaptic density
We first investigated whether alterations of Zn2 + -concentrations
regulate ProSAP1/Shank2 and ProSAP2/Shank3 levels at the syn-
apse. Therefore, we supplemented hippocampal primary neurons for
1 h with 10 mM ZnCl2, which leads to an increase of postsynaptic
Zn2 + concentration as evidenced by elevated Zinquin ethyl-ester
fluorescence (Coyle et al., 1994) (Supplementary Fig. 1A).
Supplementation of neurons with Zn2 + leads to a significant
increase in synaptic ProSAP1/Shank2 and ProSAP2/Shank3
immunofluorescence levels (Supplementary Fig. 1B and Fig. 1A
and C), which was blocked in the presence of AMPA and NMDA
receptor antagonists (Supplementary Fig. 1C). Increasing neuronal
activity with 2 min bath application of HiK + (Fig. 1A–C) or 30 mM
glutamate (Supplementary Fig. 1D) induces similar changes in syn-
aptic ProSAP/Shank immunofluorescence, and protein levels in a
synapse-enriched P2 fraction after 30 min without increase in overall
protein levels (Supplementary Fig. 1F). Application of HiK + increased
synaptic Zn2 + measured by Zinquin (Supplementary Fig. 1E). HiK +
stimulation in presence of Zn2 + leads to a similar increase and does
not act in an additive manner hinting towards a mechanism triggered
by stimulation similar to those observed with increasing Zn2 + levels
(Fig. 1C). To exclude toxic effects of HiK + stimulation in the pres-
ence of Zn2 + , we quantified the number of cells per optic field. Our
data show no significant increase in cell death (Supplementary Fig.
1G), nor could we detect significantly more swelling or pinching off
of dendrites. Most important, bath application of HiK + in the pres-
ence of a Zn2 + -chelator (CaEDTA or TPEN) was unable to elicit
activity dependent increases in synaptic ProSAP1/Shank2 and
ProSAP2/Shank3 levels and the application of CaEDTA or TPEN
even led to a decrease in ProSAP1/Shank2 and ProSAP2/Shank3
levels (Fig. 1C). Although CaEDTA is cell impermeable, at the con-
centration used it will also deplete intracellular Zn2 + (Frederickson
et al., 2002). Lower CaEDTA concentrations in contrast inhibited the
S. Grabrucker et al.
activity-dependent upregulation of ProSAP1/Shank2 and ProSAP2/
Shank3 to a lesser extent (Supplementary Fig. 1H).
An activity-dependent modification of the postsynaptic density
by a Zn2 + -dependent assembly of ProSAP/Shank proteins requires
a modest Zn2 + binding affinity in a range that does not allow
multimerization with cytoplasmic free Zn2 + levels, which are in
the low picomolar range (Frederickson, 1989; Outten and
O’Halloran, 2001; Krezel and Maret, 2006). Thus, to determine
the dynamic range in which Zn2 + -dependent conformational
changes and oligomerization are apparent, we performed intrinsic
fluorescence spectroscopy and Dynamic light scattering experi-
ments with the ProSAP2/Shank3 SAM domain (Fig. 1E and F
and Supplementary Fig. 1I–L). Taken together, we found that
the Zn2 + -dependent multimerization of ProSAP2/Shank3
occurs at nanomolar Zn2 + -concentrations and it is therefore
plausible that a cytoplasmic pool of ProSAP2/Shank3 exists that
is not Zn2 + bound.
A regression correlation analysis revealed that
Zn2 + -supplementation or depletion had significantly stronger
effects on the synaptic localization of ProSAP2/Shank3 than
ProSAP1/Shank2 (Supplementary Fig. 2A). Given that dissociated
hippocampal cell cultures are low in presynaptic vesicular Zn2 +
and that application of Zn2 + -chelators during 1 min HiK + stimu-
lation and subsequent re-supplementation with conditioned
medium still results in an increase of synaptic ProSAP1/Shank2
and ProSAP2/Shank3 levels (Supplementary Fig. 2B), we also
investigated whether releasable postsynaptic Zn2 + is a potential
source for binding to ProSAP/Shank. Important intracellular Zn2 +
buffers are MTs, in particular MT-3, which is prominently
expressed in neurons (Lee et al., 2003). It has been reported
that Ca2 + transients can rapidly and persistently release Zn2 +
from MT through endogenously generated NO (Wang et al.,
2008). As such a mechanism can conceivably also occur in syn-
apses we therefore overexpressed MT-3 and applied a NO antag-
onist (N-nitro-L-arginine) as well as an agonist (spermine nonoate)
(Fig. 1G and H). Stimulation of neurons in the presence of the NO
antagonist inhibited an increase in synaptic ProSAP1/Shank2 and
ProSAP2/Shank3 levels, whereas application of the NO agonist
slightly increased ProSAP2/Shank3 platform formation (Fig. 1G).
Moreover, analysis of MT-3 overexpressing neurons compared
with control cells showed an increase in ProSAP2/Shank3 levels
in transfected cells (Fig. 1H), indicating that releasable postsynap-
tic Zn2 + can also contribute to the assembly of the postsynaptic
ProSAP/Shank scaffold. Stimulation with HiK + further increased
synaptic ProSAP1/Shank2 and ProSAP2/Shank3 levels in MT-3
overexpressing cells, whereas knockdown of MT-3
(Supplementary Fig. 2C) blocked activity-dependent increases
(Fig. 1H).
Finally, the synaptic localization of Shank1 (which does not
bind Zn2 + as well as Homer1b/c) PSD95, mGluR5 and gephyrin
(a scaffold protein of inhibitory synapses that does not contain
ProSAP/Shank proteins) were analysed. The results show that
there is no general increase in postsynaptic density protein levels
after Zn2 + -supplementation. Shank1 concentrations at the postsy-
naptic density are not affected by either Zn2 + supplementation
or HiK + stimulation (Fig. 1D), whereas Homer1b/c seems to be
negatively regulated by activity and Zn2 + supplementation, but
Zn deficiency dysregulates ProSAP/Shank Brain 2014: 137; 137–152 | 141

Figure 1 Synaptic levels of zinc-binding ProSAP/Shank family members change under Zn2 + -supplementation and depletion in a MT-3
and NO-dependent manner. Hippocampal neurons (14 days in vitro) were treated with HiK + and/or Zn2 + -chelators (TPEN, CaEDTA)
and/or were supplemented with Zn2 + . (A, C, D, G and H) A puncta by puncta analysis of the synaptic signal intensity was performed from
10 cells (14 days in vitro, n = 10) of each treatment group. Signal intensities were normalized against untreated cells (Control) or in case of
transfected cells (H) against an untransfected cell in the same optic field of view. (A) Example images are shown; merged images
additionally show DAPI staining of the nucleus (scale bar = 20 mm). (B) Protein biochemistry confirms an increase of ProSAP2/Shank3

(continued)
142 | Brain 2014: 137; 137–152 S. Grabrucker et al.

unaffected from Zn2 + depletion. In contrast, synaptic immuno-
fluorescence for PSD95 and mGluR5 receptors, was also elevated
following Zn2 + supplementation or HiK + stimulation (Fig. 1D),
although only the increase in mGluR5, but not PSD95 was blocked
by stimulation in the presence of CaEDTA.
Zinc deficiency leads to synaptic deficits
associated with the loss of synaptic
ProSAP2/Shank3 in vivo
Autism spectrum disorders are neurodevelopmental disorders
and we therefore asked whether acute or chronic
Zn2 + -supplementation or depletion influence synapse maturation
during development. Cultivation of hippocampal neurons under
Zn2 + -deficient conditions leads to a decrease in synapse density,
whereas the number of inhibitory synapses was not altered
(Supplementary Fig. 3). In contrast, we observed a significant
upregulation of ProSAP1/Shank2 signal density in cells supple-
mented with ZnCl2 (Supplementary Fig. 3). Thus, chronic
Zn2 + -deficiency might contribute to defects that are observed
in ProSAP/Shank associated brain disorders such as autism
spectrum disorder. We therefore analysed the impact of
Zn2 + -deficiency on ProSAP/Shank scaffold formation in vivo by
inducing Zn2 + -deficiency in mice.
To this end, mice were fed a Zn2 + -deficient diet for 8 weeks.
Timm’s staining to visualize free Zn2 + ions revealed a significant
reduction of brain Zn2 + levels in cortical, striatal and hippocampal
regions (Supplementary Fig. 4A and B). The significant reduction
in Zn2 + levels was further confirmed in brain sections using
Zinpyr1, a Zn2 + -staining fluorophore (Supplementary Fig. 4C
and D). Given that levels of vesicular Zn2 + are generally low in
cerebellum (Frederickson and Moncrieff, 1994), we failed to
detect a significant reduction (Supplementary Fig. 4A–D).
The general brain morphology and cell density assessed by 4’,6-
diamidino-2-phenylindole (DAPI) and Nissl staining was not
altered in Zn2 + -deficient animals (Supplementary Fig. 4E–H).
Labelling of the pre- and postsynaptic marker proteins bassoon,
homer1 and gephyrin in brain sections of Zn2 + -deficient animals
shows that compared with control animals, a general decrease in
the number of homer1- and bassoon-positive signals (excitatory
synapses), visible as a trend (P = 0.09) in the hippocampus but
statistically significant in the cortex and striatum (Fig. 2A), can
be detected. The number of gephyrin- and bassoon-positive
signals (inhibitory synapses) was unchanged. Next, we performed
immunohistochemistry to label ProSAP/Shank proteins. The
immunofluorescence intensity correlating to protein levels of
ProSAP2/Shank3 was significantly reduced in all four brain
regions: striatum, hippocampus, cortex and cerebellum (Fig. 2B
and C). Moreover, we found a significant reduction of ProSAP1/
Shank2 immunoreactivity in cortex (Fig. 2C). The density and in-
tensity of gephyrin immunofluorescence puncta used as a marker
of inhibitory synapses was not affected in mice fed with a Zn2 +
deficient diet (Fig. 2A and C).
Analysis of P2 fractions from striatum, hippocampus, cortex and
cerebellum also revealed a significant reduction of protein levels of
ProSAP1/Shank2 in cortex and striatum, ProSAP2/Shank3 in all
four brain regions, and of Shank1 in hippocampus (Fig. 2D) in
Zn2 + -deficient mice. Along with this reduction we observed a
shift from the insoluble P2 to the soluble S2 fraction for several
synaptic proteins including ProSAP/Shanks (Supplementary Fig. 5A
and B). Importantly, the loss of ProSAP1/Shank2 in the synapse
enriched P2 fraction translates into a decrease of excitatory

Figure 1 Continued
normalized against actin after HiK + stimulation and Zn2 + -supplementation in the P2 fraction of hippocampal neurons (14 days in vitro,
n = 3). (C and D) Quantification of the signal intensities obtained by immunofluorescence. A Zn2 + -concentration dependent regulation of
ProSAP1/Shank2 and ProSAP2/Shank3 can be observed. HiK + stimulation and Zn2 + -supplementation do not show additive effects. Zn2 +
chelation prevents changes induced by HiK + [no significant differences were detected between HiK + stimulation, Zn2 + -supplementation
and HiK + stimulation plus Zn2 + -supplementation, whereas treatment with CaEDTA or TPEN with or without HiK + stimulation leads to a
highly significant (P 5 0.001) decrease compared to the HiK + , Zn2 + , and HiK + + Zn2 + conditions]. (D) Along with the regulation of
ProSAP1/Shank2 and ProSAP2/Shank3, the level of PSD95 and mGluR5 receptors at the synapse changes. However, Shank1 and
Gephyrin signals do not increase with Zn2 + -supplementation. Homer1b/c levels seem to decrease with synaptic stimulation and
Zn2 + -supplementation, but are unaffected by Zn2 + -depletion. (E and F) Intrinsic fluorescence spectroscopy experiments show Zn2 + and
Mg2 + titration for the ProSAP2/Shank3 SAM domain. Mg2 + and Zn2 + titration of the ProSAP2/Shank3 SAM domain led to a decrease in
Trp fluorescence emission. The apo-SAM domain exhibited a decrease in Trp fluorescence in the range of 3–4 nM. Higher
Zn2 + -concentrations led to precipitation of the protein (data not shown). As Zn2 + -binding of the protein will always occur in the presence
of Mg2 + titration we also performed Zn2 + titration of the Mg2 + bound-SAM domain and found here a decrease in Trp fluorescence in the
lower nanomolar but not picomolar range. (E) Zn2 + titration of the apo-SAM domain with low nM Zn2 + decreases fluorescence intensity.
(F) Mg2 + titration of the apo-SAM domain decreases fluorescence intensity and Zn2 + titration in the low nanomolar range to Mg2 +
bound protein further decreases the fluorescence intensity. (G) HiK + stimulation of cells significantly increases ProSAP1/Shank2 and
ProSAP2/Shank3 levels compared with control cells treated with N-nitro-L-arginine, and cells treated with spermine. Stimulation of cells in
the presence of a NO agonist or antagonist shows that HiK + together with NO antagonist (N-nitro-L-arginine) blocks the increase in
ProSAP1/Shank2 and ProSAP2/Shank3. (H) Overexpression of MT-3 (10–14 days in vitro) increases synaptic ProSAP1/Shank2 and
ProSAP2/Shank3 levels. Stimulation of neurons increases ProSAP1/Shank2 and ProSAP2/Shank3 levels in control (GFP transfected) cells
and even further increases the levels in MT-3 overexpressing cells. In contrast, stimulation-induced changes are prevented by knockdown
of MT-3 (10–14 days in vitro). No significant differences were detected between HiK + stimulated and unstimulated GFP-MT-3 shRNA
transfected cells. The ratio between transfected and untransfected neurons within the same optic field are shown. All values were normally
distributed and tested for significance using t-test followed by ANOVA. *P 5 0.05; **P 5 0.01; ***P 5 0.001.
Zn deficiency dysregulates ProSAP/Shank Brain 2014: 137; 137–152 | 143

Figure 2 Dietary Zn2 + deficiency reduces the amount of ProSAP/Shank and postsynaptic density (PSD) proteins. (A) Sections of
three control and three Zn2 + -deficient mice (18 weeks of age) were stained for bassoon, homer1 and gephyrin and the number of
immune-reactive signals counted in at least three optic fields of view per region. Bassoon- and gephyrin-positive signals correspond to
inhibitory synapses, whereas bassoon- and homer1-positive signals were considered as excitatory synapses. A significant decrease in
excitatory synapse density in cortex and striatum can be seen in Zn2 + -deficient animals. A trend towards an increase in inhibitory synapse
numbers can be seen, although not significant in Zn2 + -deficient animals. (B and C) Sections of three control and three Zn2 + -deficient mice

(continued)
144 | Brain 2014: 137; 137–152 S. Grabrucker et al.

receptors in this fraction, including GluA1 (striatum and hippocam-
pus), GluA2 (cerebellum and striatum), GluA3 (striatum), GluN1
(striatum and hippocampus) and GluN2B (cortex) (Fig. 2D).
Interestingly, mGluR5 levels were significantly decreased in hippo-
campus but significantly increased in striatum (Fig. 2D).
We also prepared synaptic junction fractions of whole brain
(without cerebellum) of Zn2 + -deficient animals and compared pro-
tein levels to those of control mice (Fig. 2E). We detected a sig-
nificant loss of ProSAP2/Shank3 proteins and GluA1 receptors in
Zn2 + -deficient animals. However, we could not see the decrease
in ProSAP1/Shank2 and Shank1 (Fig. 2E). Quantitative real-time
PCR confirmed that the observed changes occurred on protein
rather than on messenger RNA level, since the transcriptional
levels of ProSAP1/Shank2 and ProSAP2/Shank3 were not altered
between control and Zn2 + -deficient mice (Fig. 2F).
Analysing the pups of control and Zn2 + -deficient animals on
post-natal Day 3, we also detected a significant reduction in
brain Zn2 + -levels in pups from Zn2 + -deficient mothers (Fig. 3A
and B). However, all pups from and nursed by Zn2 + -deficient mice
died on post-natal Day 20, whereas pups from Zn2 + -deficient
mice but nursed by control mice survived. The brain Zn2 + levels
of Zn2 + -deficient pups nursed by control mice were no longer
significantly different from those of control pups (Fig. 3C).
Zn2 + -deficient pups did not show significantly fewer synapses
(Fig. 3D), but displayed a trend towards a decrease, and the tran-
scriptional levels of ProSAP1/Shank2 and ProSAP2/Shank3 were
not altered in Zn2 + -deficient pups (Fig. 3E). However, protein
biochemistry using P2 fractions from post-natal Day 3 pups
shows that a significant loss of all ProSAP/Shank family members
occurred in pups from Zn2 + -deficient mothers (Fig. 3F). Along
with this, we again detected a significant decrease in GluA1,
GluN1 and GluN2B levels. In contrast, pups from Zn2 + -deficient
mothers that have been nursed by control animals (fed a normal
diet) after birth show a significant rescue of ProSAP/Shank and
GluA1, GluN1 and GluN2B protein levels. However, Shank1,
GluA1 and GluN1 still were significantly reduced compared with
control animals (Fig. 3F). Although the amount of mGluR5 was
significantly decreased in Zn2 + -deficient pups, a significant
increase was detected in pups from Zn2 + -deficient mothers that
have been nursed by control animals (Fig. 3F). Using immunohis-
tochemistry, similar results were obtained for ProSAP/Shank pro-
teins (Fig. 3G). Although all synaptic concentrations of ProSAP/
Shank family members are decreased in Zn2 + -deficient pups
(nursed by Zn2 + -deficient mothers), the signal intensity of labelled
ProSAP/Shank puncta in different brain regions of Zn2 + -deficient
pups nursed by control mothers was not significantly different for
ProSAP1/Shank2 and ProSAP2/Shank3. In contrast, Shank1 levels
were significantly decreased averaging all brain regions. Total
gephyrin levels were again not altered. The number of signals
per optic field was not significantly different for all ProSAP/
Shank family members (Fig. 3H), whereas a slight decrease of
gephyrin positive puncta in the cerebellum was measured
(Fig. 3H).
Mouse models for acute and prenatal
zinc deficiency are hyper-responsive,
hyperactive and show impairments in
social behaviour and ultrasonic
vocalization
To investigate the influence of Zn2 + -deficiency on behaviour, we
generated acute and prenatal Zn2 + -deficient mice. In a first set of
experiments, we analysed the influence of acute Zn2 + -deficiency
in a test for maternal behaviour (response to wriggling calls)
(Fig. 4A–E and Supplementary Fig. 6). Zn2 + -deficient mice
demonstrated increased activity and reactivity in the situation of
maternal care compared to normal mothers (Fig. 4A–E and
Supplementary Fig. 6D–I).
In a second set of experiments, we performed the same test for
maternal behaviour, this time using mice that were exposed to a
prenatal Zn2 + -deficiency (Supplementary Fig. 7A–D). In contrast
to acute Zn2 + -deficient mothers, mothers that had experienced a
prenatal Zn2 + -deficiency during development showed no hyper-
responsivity (Supplementary Fig. 7E–H). However, prenatal
Zn2 + -deficient mothers showed less maternal behaviour in
response to calls (Fig. 4F).
This decrease of maternal behaviour prompted us to analyse
prenatal Zn2 + -deficient mice, performing behavioural tests (pup
retrieval, analysis of ultrasonic vocalization and maternal resident
intruder test) indicative for autism spectrum disorder-related be-
haviour. The results show that although we could not detect sig-
nificant changes in the pup retrieval test between prenatal
Zn2 + -deficient and control mice regarding the latency to respond
to the stimulus, the number of control walks performed and the
number of pup retrievals (Supplementary Fig. 7I–K), prenatal
Zn2 + -deficient mice showed an impairment in auditory

Figure 2 Continued
were stained for ProSAP1/Shank2, ProSAP2/Shank3, Shank1 and gephyrin and the signal intensity of synaptic puncta evaluated [scale
bar = 500 mm (overview and 5); scale bar = 20 mm ( 40 and enlargements)]. A significant decrease of ProSAP2/Shank3 (all brain
regions) and ProSAP1/Shank2 (cortex) could be detected. The levels of Shank1 and gephyrin were not altered. (D) Protein biochemistry
using P2 fractions of four brain regions of Zn2 + -deficient and control mice (n = 3). Protein concentrations were normalized to actin and are
shown relative to the expression in control mice. A decrease of ProSAP2/Shank3 in all brain regions can be confirmed as well as a loss of
GluA1 and GluN1 receptors most prominent in hippocampus and striatum. (E) Protein biochemistry using synaptic junction fractions of
three Zn2 + -deficient and three control mice normalized against actin and relative to the expression levels of control mice. A significant
decrease in ProSAP2/Shank3 and GluA1 levels can be seen. (F) Quantitative real-time PCR shows no differences in ProSAP1/Shank2 and
ProSAP2/Shank3 messenger RNA expression levels between Zn2 + -deficient and control mice (n = 3). All values were normally distributed
and tested for significance using t-test followed by ANOVA. *P 5 0.05; **P 5 0.01; ***P 5 0.001.
Zn deficiency dysregulates ProSAP/Shank Brain 2014: 137; 137–152 | 145

Figure 3 Maternal Zn2 + deficiency reduces the amount of ProSAP/Shank and postsynaptic density proteins in pups. (A) Using the Zn2 +
fluorophore Zinpyr1 to evaluate Zn2 + -levels reveals a significant reduction in signal intensity in brains from pups (post-natal Day 3, n = 3)
from Zn2 + -deficient mothers and nursed by Zn2 + -deficient mothers. Sections of control and Zn2 + -deficient mice were imaged and
quantified by measuring at least three optic fields of view (scale bar = 300 mm) and controls were set to 100%. (B) Timm’s staining
similarly shows a significant reduction in signal intensity in pups from Zn2 + -deficient mice (post-natal Day 3, n = 3, controls set to 100%)
(scale bar = 150 mm). (C) Pups (post-natal Day 3) nursed by control mothers show a rescue in Zn2 + -levels almost up to brain

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146 | Brain 2014: 137; 137–152 S. Grabrucker et al.

discrimination between a meaningful (50 kHz) and neutral
(20 kHz) sound stimulus (Fig. 4G).
The most prominent impairments were observed evaluating
parameters of ultrasonic vocalizations of adult males to female
urine (Fig. 4H–L). Besides several parameters that were not
altered in prenatal Zn2 + -deficient mice compared with control
mice (Supplementary Fig. 8A–H), we found that prenatal
Zn2 + -deficient mice vocalized significantly less calls per minute
observation time (Fig. 4H), had a significantly reduced loudness
of calls (Fig. 4I), a significantly increased latency to start calling
(Fig. 4J) and a trend to a reduced average call duration compared
to control animals (Fig. 4K). Moreover, the number of overtones
and harmonics was significantly reduced in the calls (Fig. 4L).
Finally, we conducted a maternal resident intruder test. Besides
parameters that were not altered (Fig. 4M and Supplementary Fig.
8I–L), the number of attacks performed by the mother was
significantly increased in prenatal Zn2 + -deficient mice (Fig. 4N).
Zinc deficiency in Phelan-McDermid
syndrome is associated with a higher
incidence rate of seizures, hypotonia
and the occurrence of attention
deficits and hyperactivity
Given that patients with Phelan-McDermid syndrome suffer from
a haplo-insufficiency of ProSAP2/Shank3 we investigated whether
Zn2 + -deficiency in patients with Phelan-McDermid syndrome
leads to a more pronounced phenotype. Using atomic absorption
spectroscopy, we evaluated Cu2 + and Zn2 + -levels in blood
samples of 38 participants diagnosed with Phelan-McDermid syn-
drome. Similar to former studies with autistic patients (Faber et al.,
2009; Lakshmi Priya and Geetha, 2011; Yasuda et al., 2011), we
detected an increased incidence rate for Zn2 + -deficiency in
patients with Phelan-McDermid syndrome primarily in the age
group 512 years (Supplementary Fig. 9A) as previously reported
for autistic children (Yasuda et al., 2011), with the exception of
two cases at 19 and 46 years of age. From 37 participants,
30 were found to have normal blood Zn2 + -levels, four showed
a mild Zn2 + -deficiency (1–20% less than the lowest concentration
of the age matched reference values for healthy controls)
and three a severe Zn2 + -deficiency (420% less than the lowest
concentration of the age matched reference values for healthy
controls) (Fig. 5A and B). Zn2 + -deficiency was not correlated
with gender (Supplementary Fig. 9B) and the participants
had no dietary problems (no underweight, no physical disability
hampering food consumption, normal dietary habits), that might
explain the deficiency.
Participants were evaluated based on 26 categories (see
‘Materials and methods’ section.). We found that 61% of partici-
pants suffering from seizures showed a Zn2 + -deficiency (mild
and severe combined) compared with only 12% of participants
without seizures (Fig. 5C). Additionally, 60% of participants
with attention deficit disorder/attention deficit hyperactivity
disorder or other attention or hyperactivity issues were
severely Zn2 + -deficient and 27% of participants with hypotonia
compared with 15% and 0%, respectively, of participants with-
out the symptom (Fig. 5C). Additionally, the occurrence
of Zn2 + -deficiency was positively correlated with signs of
immunodeficiency (Fig. 5C).

Figure 3 Continued
Zn2 + -concentration of control pups. Again Timm’s staining (left) and Zinpyr1 labelling (right) was performed as shown in A and B.
(D) Sections of three control and three Zn2 + -deficient mice were stained for bassoon and homer1 and the number of positive signals
counted in multiple fields of view. Bassoon- and homer1-positive signals were considered as excitatory synapses. A trend towards a
decrease in excitatory synapse density, although not significant, can be seen in Zn2 + -deficient animals. (E) Quantitative real-time PCR
shows no differences in ProSAP1/Shank2 and ProSAP2/Shank3 messenger RNA expression levels normalized to HMBS expression
between pups (post-natal Day 3) from Zn2 + -deficient and control mice (n = 3). (F) P2 fractions from post-natal Day 3 pups show a
significant reduction of all ProSAP/Shank family members in pups from Zn2 + -deficient mothers. Along with this, a significant decrease in
GluA1, GluN1 and GluN2B can be observed. Pups from Zn2 + -deficient mothers that have been nursed by control animals show a
significant rescue of ProSAP/Shank and GluA1, GluN1 and GluN2B levels, although Shank1, GluA1 and GluN1 stay significantly reduced
compared with control animals. Expression levels were normalized against actin and are shown relative to the expression of control
mice (n = 3). (G) Sections from three control, Zn2 + -deficient pups (post-natal Day 3) raised by control mothers, and Zn2 + -deficient pups
(post-natal Day 3) raised by Zn2 + -deficient mothers were stained for ProSAP1/Shank2, ProSAP2/Shank3, Shank1 and gephyrin and the
signal intensity of synaptic puncta evaluated. Similar to the western blot results shown in (F), a significant decrease of ProSAP1/Shank2,
ProSAP2/Shank3 and Shank1 (total of all brain regions) could be detected in Zn2 + -deficient pups. The levels of ProSAP1/Shank2 and
ProSAP2/Shank3 in rescued Zn2 + -deficient pups (raised by control mothers) were not different from control animals. The levels of Shank1
however, were still significantly reduced considering the average values of all brain regions. The synaptic concentration of gephyrin for all
brain regions is not altered, although a decrease in cerebellum and an increase in hippocampus is seen in rescued Zn2 + -deficient pups. The
data are shown normalized to signal intensity values of controls. (H) Sections of three control and three Zn2 + -deficient pups (post-natal
Day 3) raised by control mothers were stained for ProSAP1/Shank2, ProSAP2/Shank3, Shank1 and gephyrin and the mean number of
positive signals counted per optic field from five images. The data are shown normalized to signal density values of controls. A significant
decrease in gephyrin-positive signals can be seen in Zn2 + -deficient pups raised by control mothers. The signal density of ProSAP/Shank
immunoreactive puncta was not significantly different from control pups [images show exemplary stainings of ProSAP1/Shank2 (green),
ProSAP2/Shank3 (red) and DAPI (blue) of the analysed brain regions] (scale bar = 10 mm). Although ratios are shown, statistical tests were
performed using the actual signal density of the Zn2 + -deficient (rescued) animals compared to the actual signal density of control animals.
(A–H) All values were normally distributed and tested for significance using t-test followed by ANOVA. *P 5 0.05; **P 5 0.01;
***P 5 0.001.
Zn deficiency dysregulates ProSAP/Shank Brain 2014: 137; 137–152 | 147

Figure 4 Behavioural deficits in a mouse model for acute and prenatal Zn2 + -deficiency. (A–E) Acute Zn2 + -deficient mice demonstrated
increased activity and reactivity in the situation of maternal care compared to normal mothers. (A) In a test for maternal behaviour, adult
Zn2 + -deficient mice (n = 20, A–E) demonstrate a significantly higher number of interruptions of nursing/warming position compared to
controls (n = 18, A–E, t-test). Additionally, Zn2 + -deficient mice showed significantly more spontaneous attention behaviour (lifting or
turning the head, U-test) (B) and spontaneous maternal behaviour (C) in the absence of calls independent of the Zn2 + status of pups
(U-test) (Supplementary Fig. 6D–F). (D) The attention response to natural wriggling calls and the synthesized call model was significantly

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148 | Brain 2014: 137; 137–152 S. Grabrucker et al.

Zn2 + -deficiency in adult mice only significantly changes Shank1

Discussion
Recent first in vitro evidence indicated Zn2 + incorporation in large
macromolecular platforms that are built by the SAM domains of
the autism-associated postsynaptic density scaffolding molecules
ProSAP2/Shank3 and ProSAP1/Shank2. Here, we propose a
model where the synaptic assembly of ProSAP1/Shank2 and
ProSAP2/Shank3 proteins is influenced by Zn2 + -deficiency.
Zn2 + -depletion from neuronal cultures or Zn2 + -sequestration by
amyloid-b decreased the synaptic levels of Zn2 + -binding ProSAP/
Shank family members as shown here and in former studies
(Grabrucker et al., 2011a, b) and results in a reduced synapse
density. Analysing Zn2 + -deficient mice, here, we also detected a
reduction in synapse number in the cortex and striatum. Along
with this, we could show a significant decrease of ProSAP1/
Shank2 (cortex) and ProSAP2/Shank3 (cortex, hippocampus, stri-
atum, cerebellum) through immunofluorescence and a significant
decrease of ProSAP1/Shank2 (cortex, striatum), ProSAP2/Shank3
(cortex, hippocampus, striatum, cerebellum) and Shank1 (hippo-
campus) through protein biochemistry in Zn2 + -deficient mice. This
loss of postsynaptic density scaffold proteins was accompanied by
a downregulation of receptors, especially GluA1 and GluN1
(hippocampus and striatum) reminiscent of a similar downregula-
tion of AMPA receptors reported in heterozygous and homozy-
gous ProSAP2/Shank3b knockout mice (Bozdagi et al., 2010;
Wang et al., 2011). ProSAP2/Shank3b knockout and ProSAP2/
Shank3
C mice were also reported to have decreased GluN1 and
GKAP levels (Bangash et al., 2011; Wang et al., 2011).
Furthermore, ProSAP1/Shank2 knockout mice were reported to
display a decrease in NMDA receptors (Won et al., 2012).
Previous studies have shown that especially nascent synapses
are sensitive to Zn2 + -depletion (Grabrucker et al., 2011a) and a
model was proposed where ProSAP1/Shank2 is the first ProSAP/
Shank family member found at forming synapses followed
by ProSAP2/Shank3. Only if a sufficient stabilized synapse
with preformed scaffold is established, Shank1 is targeted to post-
synaptic densities, which leads to less Zn2 + -sensitive mature
synapses. The results of the present study show that although
levels in hippocampus, a much more pronounced reduction
of Shank1 is seen in pups from Zn2 + -deficient mothers.
One might speculate that in adult mice, synapses have already
been stabilized by Shank1 and that loss of Shank1 because
of impaired synapse maturation needs longer periods of
Zn2 + -deficiency in less plastic brain regions than the hippocampus.
In contrast, in Zn2 + -deficient pups upon initial synapse formation,
postsynaptic densities might not be targeted by Shank1 from
the beginning because of their weakened ProSAP1/Shank2 and
ProSAP2/Shank3 scaffold.
Intriguingly, we observed most alterations in hippocampus and
especially striatum, being the brain regions with the highest
expression levels of ProSAP2/Shank3 compared with other
ProSAP/Shank family members (Peça et al., 2011). Similar to a
ProSAP2/Shank3 knockout mouse model (Peça et al., 2011), we
detected a reduction in spine density in the striatum. Given that
the striatum contains a low amount of presynaptic Zn2 + but has a
high expression of ProSAP2/Shank3 scaffolds that can only be
built in presence of Zn2 + , indeed Zn2 + may be bound within
the postsynaptic density in all brain regions regulated by postsy-
naptic Zn2 + -stores. Thus, if postsynaptic stores are affected and
ProSAP2/Shank3 has a predominant role in the striatum, it is likely
that synapse density in this brain region is most affected by
Zn2 + -deficiency, independent from the presynaptic pool of Zn2 +
ions. This might also explain the reduction of ProSAP2/Shank3
levels in cerebellum.
As it was also shown before that there is a rapid turnover of
ProSAP/Shank proteins at the synapse (Tsuriel et al., 2006) and
that ProSAP1/Shank2 and ProSAP2/Shank3 can be detected in
the soluble (S2) as well as postsynaptic density-bound (P2) frac-
tion of brain lysates, it is plausible that both proteins undergo a
shift from P2 to S2 upon Zn2 + -depletion (Grabrucker et al.,
2011a). One can therefore assume that the soluble and postsy-
naptic density-bound pool exist in equilibrium. Synaptic activity
and Zn2 + -release may rapidly shift this equilibrium to a more
postsynaptic density-bound pool of ProSAP/Shank (Grabrucker,
2013).

Figure 4 Continued
increased in Zn2 + -deficient mothers independent of the Zn2 + status of pups (Chi square test) (Supplementary Fig. 6G). (E) Maternal
behaviour (licking, change in nursing position, nest building) of both groups of mothers was increased in response to calls of
Zn2 + -deficient pups and to the call model (Chi square test) (Supplementary Fig. 6H and I). (F) In the same test for maternal behaviour
(A–E) conducted with mice that suffered from prenatal Zn2 + -deficiency, over-responsiveness and hyperactivity were not detected.
However, prenatal Zn2 + -deficient mice (n = 6, 15 weeks of age) showed significantly less maternal behaviour in response to the natural
calls of pups compared to control mice (n = 7, Chi square test) (F). (G) A pup retrieval test revealed that prenatal Zn2 + -deficient mice
(n = 24, 14 weeks of age with pups post-natal Day 4–5) responded significantly more to the neutral 20 kHz sound stimulus and thus
significantly less to the biologically meaningful 50 kHz sound compared with control mice (n = 42). Data of both groups were compared
with the sign test according to Dixon and Mood. The comparison of the number of choices between the two tones was done using
Chi-square test. Moreover, prenatal Zn2 + -deficient male mice (n = 7, 15 weeks of age) showed impaired ultrasonic vocalization compared
with control mice (n = 13) (H–L). The number of calls per minute (H) was significantly decreased (t-test), the loudness was significantly
reduced (U-test) (I) and the latency to start calling significantly increased (U-test) (J) compared with control animals. Prenatal Zn2 + -
deficient males additionally showed a trend towards a reduced call duration (#P = 0.073, t-test) (K) and significantly less vocalizations with
overtones and harmonics (L) compared with control animals (Chi-square test). (M) A maternal resident intruder test revealed that,
although the time a mother spent in contact with an intruding male was not significantly different between prenatal Zn2 + -deficient and
control mothers (U-test) (M), the number of attacks was significantly increased in prenatal Zn2 + -deficient mothers (n = 5, t-test) (N).
*P 5 0.05; **P 5 0.01.
Zn deficiency dysregulates ProSAP/Shank Brain 2014: 137; 137–152 | 149

Figure 5 Zn2 + deficiency is associated with an increased rate of seizures, attention or hyperactivity issues, hypotonia and possible signs of
primary immunodeficiency in patients with Phelan-McDermid syndrome. (A and B) Using atomic absorption spectroscopy, Cu2 + and
Zn2 + -levels in blood samples of 38 participants diagnosed with Phelan-McDermid syndrome were evaluated. (A) From 37 participants,
30 were found to have normal blood Zn2 + -levels, four showed a mild Zn2 + -deficiency and three a severe Zn2 + -deficiency. (B) A copper
overload was detected in four participants. (A and B) The measured values are shown in per cent compared with age matched control
reference values. (C) A total of 26 categories were analysed based on the participant’s data provided by the Phelan-McDermid Syndrome
Registry. The results show that 61% of participants suffering from seizures have Zn2 + -deficiency compared with only 12% of participants
without seizures (participants with mild and severe Zn2 + -deficiency combined) (n = 32 total). Sixty per cent of participants with attention
deficit disorder/attention deficit hyperactivity disorder (ADD/ADHD) or other attention of hyperactivity issues were Zn2 + -deficient and
27% of participants with hypotonia compared with 15% and 0%, respectively, of participants without the symptom (participants with
mild and severe Zn2 + -deficiency combined; n = 17 and 24 total, respectively). In the group of participants showing possible signs of
primary immunodeficiency (four or more ear infections within 1 year, two or more serious sinus infections within 1 year, two or more

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150 | Brain 2014: 137; 137–152
The present results raise a number of questions concerning the
source of postsynaptic free Zn2 + , the dynamics of the ProSAP/
Shank scaffold and the pathomechanism of autism spectrum dis-
order and related disorders. Knockout mice for ZnT3, the major
and possibly sole synaptic vesicular Zn2 + -transporter, were not
reported yet to display autism-like features, although they show
an absence of free Zn2 + in synaptic vesicles (Cole et al., 1999).
Therefore the question arises whether presynaptically released
Zn2 + is the sole source of Zn2 + ions involved in the synaptic
targeting of ProSAP/Shank. Alternatively, local elevation of
Zn2 + -concentrations could arise by activation of intracellular
Zn2 + -stores through NO. In the brain, NO has been shown to
displace Zn2 + from protein-binding sites (Cuajungco and Lee,
1998; Aravindakumar et al., 1999), causing free or weakly
bound Zn2 + to be present in the cytoplasm that could play a
role in intracellular signalling (Bossy-Wetzel et al., 2004; Lin
et al., 2007). Our in vitro experiments indicate that MT-3 might
be a candidate for postsynaptic activity-dependent Zn2 + -release
due to activity-dependent nitrosylation or oxidation of the thiol
ligands by NO (Maret, 2000). In line with this, NO antagonists
prevented the stimulation-induced upregulation of Zn2 + -binding
ProSAP/Shank proteins at the synapse. Overexpression of MT-3,
because of spontaneous synaptic activity or more so after HiK +
stimulation, increased synaptic ProSAP2/Shank3 levels, whereas
MT-3 knockdown prevented an activity-dependent increase. The
slight increase in ProSAP2/Shank3 by MT-3 knockdown in
absence of stimulation might be because of a higher baseline
availability of free Zn2 + that would have been bound to MT-3
otherwise. Intriguingly, mice lacking MT-3 show a reduction in
brain Zn2 + -concentrations as a result of the absence of Zn2 +
bound to MT-3. However, the histochemically-reactive
Zn2 + -content is unaffected, providing evidence that MT-3 does
not influence presynaptic vesicular Zn2 + (Erickson et al., 1995,
1997). MT-3 knockout mice show abnormalities in behaviour
(Koumura et al., 2009) and have been suggested as suitable sub-
jects to investigate psychological disorders (Koumura et al., 2009).
Interestingly, previous studies have reported a high rate of
Zn2 + -deficiency in autistic children (Walsh et al., 1997; Jen and
Yan, 2010; Yasuda et al., 2011), suggesting that infantile
Zn2 + -deficiency might contribute to the pathogenesis of autism
(Yasuda et al., 2011). Along with a reduction in synaptic
ProSAP1/Shank2 and ProSAP2/Shank3 levels, Zn2 + -deficient
animals developed behavioural abnormalities.
Acute Zn2 + -deficiency led to increased hyperactivity-like spon-
taneous behaviour and over-responsiveness to acoustic stimuli.
This suggests lability of behavioural coordination in the highly af-
fected striatum (see above) as changes both in sound perception,
which could be related to cortical processing, and in goal-directed
Figure 5 Continued
pneumonias within 1 year, persistent thrush in mouth or fungal infection of skin, or the need of intravenous antibiotics to clear infections),
38% of participants were found with mild or severe Zn2 + -deficiency compared to 7% of participants without any of these symptoms
(n = 31 total). Differences between patients with normal Zn2 + -levels/mild Zn2 + -deficiency and patients with severe Zn2 + -deficiency are
significant for the occurrence of seizures (P 5 0.001), attention deficit disorder/attention deficit hyperactivity disorder or other attention or
hyperactivity issues (P 5 0.01), possible signs of immunodeficiency (P 5 0.01), while only a trend can be observed for hypotonia
(P = 0.058) (chi-square Goodness of fit).
S. Grabrucker et al.
instinctive behaviour, which might reflect cerebellar action, were
not observed. Because autism spectrum disorders are developmen-
tal disorders, induction of Zn2 + -deficiency in adulthood is not
expected to generate autism in mice. Nevertheless, the results
show that Zn2 + -deficiency might be responsible for co-morbidities
seen in autism spectrum disorders such as increased anxiety and
sensory over-responsiveness, which is underlined by the increased
rate of hyperactivity issues in patients with Phelan-McDermid syn-
drome and Zn2 + -deficiency. For instance, in addition to the core
diagnostic features for autism spectrum disorder, atypical stimuli-
evoked responses have been reported in up to 95% of children
with autism spectrum disorder (Lane et al., 2012).
Intriguingly, our data from pups from Zn2 + -deficient mice indi-
cate that maternal Zn2 + -deficiency is also able to induce changes
in ProSAP/Shank levels in the offspring, similar to those reported
in adult Zn2 + -deficient mice here. Although Zn2 + -deficiency led to
the death of pups on post-natal Day 20 hinting towards a devel-
opmental milestone that Zn2 + -deficient pups were unable to
achieve, provision of adequate Zn2 + supply by control mothers
interestingly led to survival and a rescue of the observed alter-
ations in most, but not all postsynaptic density proteins. At the
age of 17–22 days, pups usually start eating solid food and during
this weaning period, the mothers begin to withdraw from their
litter to reduce suckling attempts by pups (Koenig and Markl,
1987). Zn2 + -deficiency is known to have an influence on motor
development (Black, 1998) and a possible reason for the death of
pups on post-natal Day 20 might be an inability to reach sufficient
amounts of food and water at the top of the cage.
Prenatal Zn2 + -deficient mice, in contrast to acute
Zn2 + -deficient mice, did not show signs of increased hyperactiv-
ity-like and over-responsive behaviour. We observed significantly
decreased maternal behaviour along with impairment in auditory
discrimination of sound stimuli, whereas sound perception per se
was not altered. Most prominent, we could detect significant dif-
ferences in the vocalization of prenatal Zn2 + -deficient males.
Intriguingly, a reduction in ultrasonic vocalizations has been re-
ported previously for heterozygous ProSAP2/Shank3b (Bozdagi
et al., 2010) and homozygous ProSAP1/Shank2 knockout mice
(Schmeisser et al., 2012; Won et al., 2012). Additionally, prenatal
Zn2 + -deficient mice showed alterations in the social context of a
maternal resident intruder test. We observed a significant increase
in attacks towards the intruder. Increased aggressiveness has been
reported in prenatal Zn2 + -deficient animals (Sandstead et al.,
1977). Taken together, prenatal Zn2 + -deficient animals indeed
display some behavioural features observed in mouse models for
autism.
Patients with Phelan-McDermid syndrome already lack one
functional copy of ProSAP2/Shank3, so that they should be
Zn deficiency dysregulates ProSAP/Shank
more susceptible to changes in body Zn2 + -levels. Although,
given the low incidence rate of Phelan-McDermid syndrome
with 2.5–10 per million births, the number of participants in our
study was rather low and serum Zn2 + -levels are only indicative of
brain Zn2 + -levels, the results complement those from our
Zn2 + -deficient mouse model with regard to increased rate of
attention and hyperactivity issues compared with patients without
these symptoms. Similarly, we found a higher proportion of
Zn2 + -deficient participants in the group of patients suffering
from hypotonia and/or seizures and signs of primary immunodefi-
ciency. Immune system abnormalities have been linked to
Zn2 + -deficiency before and both have been reported as risk
factors for the development of autism (Grabrucker, 2012).
Although an alternative explanation would be that patients with
more severe clinical presentations are more susceptible to
Zn2 + -deficiency, it is likely that Zn2 + -deficiency is influencing
the severity of these symptoms.
In conclusion, our data predict that autistic children on
Zn2 + -therapy should improve regarding attention, hyperactivity,
seizures, sound and tactile sensitivity, among others. Recent data
confirm this therapeutic outlook (Russo and Devito, 2011).
Acknowledgements
The authors gratefully acknowledge the professional technical
assistance of Elisabeth Pica and thank Geraldine Bliss, Megan
O’Boyle and the Phelan McDermid Foundation for their help as
well as the Phelan-McDermid syndrome families for participating
in blood donations.
Funding
This work was supported by Baustein 3.2 (L.SBN.0083) and the
DAAD (to A.M.G). T.B. was funded by the ANR (ANR-08-MNPS-
037-01 – SynGen), Neuron-ERANET (EUHF-AUTISM), Fondation
Orange and the Fondation FondaMentale. M.R.K by the DFG (SFB
779/TPB8; Kr1879/3-1). S.G. is a member of the International
Graduate School in Molecular Medicine at Ulm University.
Supplementary material
Supplementary material is available at Brain online.
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