even without A?. Here, we tested the hypothesis that calcineurin mediates neurodegenerative effects via activation of the nuclear
transcription factor of activated T-cells (NFAT). We found that both spine loss and dendritic branching simplification induced by A?
of NFAT transcriptional activity in A?-related neurotoxicity. In vivo, when VIVIT or its nuclear counterpart were overexpressed in a
Amyloid ? (A?) soluble oligomers are thought to be an impor-
tant source of neurotoxicity in Alzheimer’s disease (AD) (Walsh
et al., 2002; Lesne ´ et al., 2006; Shankar et al., 2007; Koffie et al.,
2009). While synaptic loss correlates best with cognitive decline
(Terry et al., 1991; DeKosky et al., 1996), the molecular mecha-
nisms underlying A? synaptotoxicity and memory impairment
ing to protect neurons toward A?-related injuries need to be
cineurin (CaN, or protein phosphatase 2B) may trigger A?-
related pathological effects (Liu et al., 2005; Shankar et al., 2007;
Kuchibhotla et al., 2008; Wu et al., 2010). An increased CaN
activity has been described in AD brains (Liu et al., 2005; Wu et
al., 2010), and CaN activation was reported in astrocytes sur-
rounding amyloid deposits, thus exacerbating neuroinflamma-
tion (Norris et al., 2005). In neurons, CaN activation leads to
pathological morphological changes, which could be blocked by
Rozkalne et al., 2011). FK506 also improves cognitive deficits in
ing calcineurin a potential therapeutic target. However, because
roprotection are unclear.
Calcineurin is a unique neuronal Ca2?/Calmodulin-dependent
serine/threonine phosphatase that plays fundamental physiolog-
ical roles during development and regulates processes such as
neurotransmitter release, synaptic plasticity, and learning (Klee
et al., 1979; Groth et al., 2003). Upon activation, CaN leads to
post-translational modification of postsynaptic proteins such as
cofilin (Zhou et al., 2004) and AKAP79, which is associated with
long-term depression (Bhattacharyya et al., 2009; Jurado et al.,
2010). In addition, CaN dephosphorylates the nuclear factor of
and the expression of target genes implicated in neuronal sur-
vival, axonal outgrowth, and dendritic complexity (Benedito et
al., 2005; Nguyen and Di Giovanni, 2008; Schwartz et al., 2009).
synapse as well as long-term modifications of synaptic plasticity
by modulating gene expression through NFAT.
To decipher more precisely the role of CaN activation in Alz-
heimer disease, we tested the hypothesis that neuronal morpho-
logical changes caused by A? may be improved by inhibiting the
CaN-induced NFAT pathway. We used a genetically encoded
Author contributions: E.H., H.-Y.W., T.L.S.-J., and B.T.H. designed research; E.H. performed research; H.-Y.W.,
Norris (Sanders-Brown Center on Aging, Lexington, KY) for providing the VIVIT-GFP constructs and Dr. Ruthazer
We thank Dr. Alberto Serrano-Pozo for helpful comments during manuscript preparation and Daniel Joyner for
3176 • TheJournalofNeuroscience,February29,2012 • 32(9):3176–3192
CaN phosphatase activity (Aramburu et al., 1999). We estab-
lished that (1) VIVIT efficiently inhibits the neurotoxic effects
associated with a constitutively activated CaN (CACaN); (2) ex-
nocopy of the effects induced by CACaN or by A? exposure; (3)
inhibited by VIVIT; (4) restricted expression of VIVIT in the
nucleus retains the beneficial effects of VIVIT, whereas a
membrane-bound version of this peptide has no effect; (5)
changes associated with amyloid plaques are alleviated when VI-
VIT or NLS-VIVIT were introduced by AAV-mediated gene de-
livery in the cortex of an AD mouse model in vivo.
targeting A? downstream events through inhibition of NFAT
Culture of primary cortical neurons and collection of neuronal conditioned
media. Primary neurons were cultured from cerebral cortices of embry-
onic day 16 mice (Charles River Laboratories). Embryos resulted from
the mating between a Tg2576 male that heterozygously overexpresses a
transgenic (Tg) and littermate (Wt) cultures. The genotype of each em-
bryo was determined by PCR. Neurons were prepared as previously de-
scribed (Wu et al., 2010) and plated to a density of 6.7 ? 105viable
Aldrich). The medium of the cells was not renewed to allow A? peptides
to accumulate and conditioned media were collected from 14 DIV cul-
by a mouse/human ELISA kit (Wako) and reached ?7000 pMol.
Transfection experiments were done at 5 DIV using Lipofectamine
Spine density and Sholl analyses. At 5 DIV, primary neurons were trans-
fected with either pEGFP-N1 or pDsRed-Express-N1 plasmids (Clontech
structs generously provided by Dr. Norris (Sanders-Brown Center On Ag-
ing, University of Kentucky, Lexington, KY) and Dr. Ruthazer (McGill
University, Montreal, QC, Canada), respectively. For a subset of experi-
ments, cells were also transfected with a wild-type or a constitutively active
form of calcineurin described previously (Wu et al., 2010). Live imaging of
GFP- or DsRed-expressing primary neurons (18 DIV) was used to analyze
the morphological parameters of the cells. Images of the whole cell and of
dendritic segments were captured using a LSM 510 Zeiss microscope to
determine the neuritic complexity and the spine density. GFP and DsRed
between 500–550 nm and 565–615 nm. Dendritic spines were analyzed
using the NeuronStudio software (CNIC tools) that automatically detects
each spine) (Rodriguez et al., 2008). The complexity of the neuronal den-
reporting the number of branch points with respect to the distance
cells that did not show any obvious morphological alterations such as
Immunocytochemistry. Immunostaining was performed using a stan-
4% paraformaldehyde in PBS, pH 7.4, for 15 min, permeabilized with
albumin at room temperature for 1 h. Primary antibodies to detect
NFATc4 (polyclonal s.c.-13036, 1:200 dilution, Santa Cruz Biotechnol-
ogy), laminin-B1 (polyclonal ab16048, 1:1000, Abcam), HA tag (Clone
16B12, 1:200 dilution, Covance), and GFP (polyclonal ab6556, 1:1000,
Abcam or chicken anti-GFP, Aves) were incubated overnight at 4°C.
Secondary antibodies conjugated to either cyanine 3 (Cy3, 1:1000; Jack-
son ImmunoResearch), cyanine 5 (Cy3, 1:500; Jackson ImmunoRe-
search), or Alexa 488 (1:1000; Invitrogen, Eugene, OR) were then
endogenous protein in primary neurons or in mouse brain slices. GFP-
positive neurons that were previously transfected or transduced with
GFP, VIVIT-GFP, or NLS-VIVIT-GFP were specifically analyzed. Using
ImageJ (National Institutes of Health open software), the fluorescence
intensity of NFATc4 in the nucleus was determined by overlap with the
nuclear staining DAPI (Vector Laboratories), whereas the fluorescence
intensity in the cytoplasm was quantified in the rest of the cell body,
excluding the nuclear compartment (Fig. 1C). The intensity of nuclear
NFATc4 was then divided by the intensity of cytoplasmic NFATc4.
Evaluation of NFAT transcriptional activity using a Luciferase reporter
NFAT–TA–Luc, altogether with AAV-wtCaN or AAV-CACaN and
AAV-GFP, AAV-VIVIT-GFP, or AAV-NLS-VIVIT-GFP. Three days af-
sured with a luminometer using a reagent kit (Luciferase Assay System
with Reporter Lysis Buffer; Promega). The background luciferase activ-
ity, calculated when pNFAT-Luc alone was added, was subtracted from
Animals. In vivo experiments were performed using APPswe/PS1d9
double transgenic mice (APP/PS1, obtained from Jackson Laboratory)
that overexpress a human mutant amyloid precursor protein gene con-
taining the Swedish mutation K594N/M595L and a variant of the Prese-
nilin 1 gene deleted for the exon 9, both under the control of PrP
promoter (Jankowsky et al., 2004). Substantial amyloid deposition is
visible by 6 months of age and we used 7-month-old animals. Wild-type
dance with NIH and institutional guidelines.
Viral vectors construction and production. Plasmids containing VIVIT-
enzymes and subcloned into an AAV backbone containing the
chicken ?-actin promoter and a Woodchuck Hepatitis Virus Post-
Transcriptional Regulatory Element (WPRE). The Myr-tagged VIVIT-
backbone using Xho1 and BsrG1 restriction sites. The AAV-NFAT–TA–
Luc plasmid was purchased from Clontech Laboratories and the con-
structions of AAV-CBA-HA-wtCaN and pAAV-CBA-HA-CACaN
backbones were described previously (Wu et al., 2010). As a control, we
High titer of AAV serotype 2 vectors (AAV2-GFP, AAV2-VIVIT-GFP
obtained after amplification of HA-mNFATc4 (WtNFAT) and HA-CA-
mNFATc4 (CANFAT) by PCR. Both PCR products were then digested
lentiviral backbone. Lentiviral vectors were produced by the Vector De-
velopment and Production Core at Massachusetts General Hospital. All
the constructs were verified by sequencing.
Stereotactic intracortical injections. Stereotactic intracortical injections of
2005). Animals were anesthetized by intraperitoneal injection of ketamine/
xylazine (100 and 50 mg/kg body weight, respectively) and positioned on a
a 10 ?l Hamilton syringe (Hamilton Medical) at a rate of 0.12 ?l/min. Ste-
Cranial window implantation and multiphoton imaging. Four weeks
(1.5%) and a cranial window was implanted by replacing a piece of skull
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3177
by a glass coverslip of 8 mm diameter [as described previously (Spires et
al., 2005)]. For imaging, a wax ring was built on the edge of the window
to create a well of water for the objective (Olympus XLPlan N 25?
objective with a numerical aperture of 1.05). To visualize the amyloid
deposits, transgenic animals received an intraperitoneal injection of
methoxy-XO4(5 mg/kg) 24 h before surgery, a fluorescent compound
skai et al., 2002; Klunk et al., 2002). When needed, Texas Red dextran
(70,000 Da molecular weight; 12.5 mg/ml in sterile PBS; Invitrogen) was
injected into a lateral tail vein to provide a fluorescent angiogram.
GFP-filled neuronal processes, amyloid deposits (in the case of
APP/PS1 animals), and blood vessels were imaged using the Olympus
FluoView FV1000MPE multiphoton laser-scanning system mounted
on an Olympus BX61WI microscope (Olympus). A DeepSee Mai Tai
Ti:sapphire mode-locked laser (Mai Tai; Spectra-Physics) generated
two-photon fluorescence with 860 nm excitation. Emitted light was
detected through three filters in the range of 420–460, 495–540, and
575–630 nm (Hamamatsu). Neurites were imaged at depth of 50–200
?m from the surface of the brain. High-resolution images were cap-
tured using the optical zoom feature in the Fluoview software (63?
63? 1 ?m sections; 40–100 sections per stack).
Image processing and analysis. Two-dimensional projections of GFP-
filled neurites were obtained using ImageJ. Only dendrites that were at
least 20 ?m long and had prominent dendritic spine protrusions were
considered for analysis. Spine density and morphology were evaluated
using the NeuronStudio software, as described previously (Rodriguez et
3178 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
al., 2008). Distances to plaques were evaluated by the average of three
and one measure at the midpoint) and the edge of the nearest amyloid
nial window, mice were killed by CO2inhalation. One cerebral hemi-
sphere was fixed in 4% paraformaldehyde in PBS whereas the other
hemisphere was snap frozen in liquid nitrogen. To detect GFP and
NFATc4, paraffin-embedded sections (10 ?m) were sequentially depar-
Sodium Citrate, 0.05% Tween 20, pH 6.0), permeabilized in PBS with
for 2 h at room temperature the next day.
Statistical analysis. Except for the Sholl’s plot analyses, statistical anal-
yses were done using Statview and JMP softwares. In vitro experiments
per condition. Equality of variances (F test) was verified between each
date of experiment and between animals of the same group. We made
comparisons between groups by one- or two-way ANOVA followed by
post hoc Bonferroni correction for multiple comparisons among means.
Data are presented as mean ?SD.
Sholl’s plot analyses were represented as the mean of the number of
intersections according to the distance from the cell body, and statistics
(Spires-Jones et al., 2011)]. To assess the difference in length and com-
plexity of dendritic arborization between each treatment, the area under
the curve (AUC) was estimated through a trapezoidal method, which
reflects the overall complexity of the dendritic arborization. A Wilcoxon
applied. For all the analyses, differences with a p value of ?0.05 were
considered statistically significant.
able to interact with the CaN docking motif of NFAT with a high
In vitro, we first tested whether the overexpression of VIVIT-
GFP in cortical primary neurons was able to counteract the nu-
clear translocation and the transcriptional activity of NFATc4
[the most prominent NFAT isoform in neurons (Ho et al.,
1994)], as well as the neuronal morphological changes induced
stitutively active CaN was initially identified as a calpain-
dependent truncated product of calcineurin (45 kDa), in which
the regulatory autoinhibitory domain was removed, so that CA-
CaN has an enhanced phosphatase activity (Wu et al., 2004). By
contrast, overexpression of wild-type calcineurin (WtCaN) is
tightly regulated and does not lead to an increase of calcineurin
activity. At 5 DIV, cortical primary neurons were cotransfected
with either CACaN or WtCaN and with either GFP or VIVIT-
GFP. Cells were cultured for 16–18 DIV before analysis. Because
of the presence of an HA-tag in the WtCaN and CACaN con-
structs, we verified that ?90% of GFP-filled neurons also coex-
pressed each form of CaN (data not shown). As expected, the
nuclear/cytoplasmic ratio of NFATc4 immunofluorescence in-
tensity was significantly higher when CACaN was overexpressed
(0.94 ? 0.025) compared with WtCaN (0.62 ? 0.028), whereas
WtCaN by itself did not affect NFAT subcellular localization
compared with GFP transfected cells (0.67 ? 0.08) (Fig. 1A,B).
This suggests that increased CaN activity leads to an accumula-
tion of NFATc4 in the nucleus. By contrast, co-transfection of
CACaN with VIVIT-GFP prevents the nuclear accumulation of
NFATc4, as indicated by lower nuclear/cytoplasmic ratios
change could be detected in the subcellular localization of
to GFP-transfected cells (data not shown). To further evaluate
the effect of VIVIT on NFAT transcriptional activity, a reporter
system containing several NFAT transcriptional response ele-
rons were transduced with AAV-GFP, AAV-WtCaN?GFP,
AAV-CACaN?AAV-GFP, AAV-CACaN?AAV-VIVIT-GFP at
NFAT transcriptional activity compared with WtCaN. When
VIVIT-GFP was co-transduced with CACaN, a significant decrease
of the Luciferase activity was observed, suggesting that VIVIT effi-
To determine whether VIVIT-GFP was able to prevent the
pathological morphological changes induced by CACaN, we an-
alyzed the spine density and dendritic complexity of GFP or
VIVIT-GFP-expressing neurons that were also transfected with
WtCaN or CACaN plasmids. The density of spines in CACaN/
GFP overexpressing neurons (0.18 ? 0.046 spines/?m) was sig-
(0.42 ? 0.09 spines/?m; Fig. 1D,E). Sholl’s plot analyses also
indicated that uncontrolled activation of calcineurin in neurons
induced a marked dendritic simplification compared with wild-
type calcineurin (Fig. 1D,G). However, when VIVIT-GFP was
introduced together with CACaN, a significantly higher spine
density (0.4 ? 0.081 spines/?m) and a more complex neuritic
arborization were observed, so that these morphological param-
eters reached those of WtCaN transfected neurons (Fig. 1D,E,
G). Importantly, no difference was observed between cells that
were transfected with GFP alone (0.43 ? 0.13 spines/?m) or
WtCaN?GFP (0.42 ? 0.09 spines/?m), suggesting that WtCaN
did not change the spine density and dendritic complexity of
cortical neurons in culture (Fig. 1D,E,G). Similarly, overexpres-
sion of VIVIT-GFP alone did not affect the morphological pa-
rameters of the cells (data not shown). The beneficial effect of
VIVIT-GFP was therefore specifically related to the inhibition of
CACaN-dependent NFAT activation.
with CACaN?GFP (38% ? 1.8%) compared with GFP (47% ?
2.8%), WTCaN?GFP (44% ? 2.3%), or CACaN?VIVIT-GFP
collapse of mature, mushroom-shaped spines that are known to
be more stable than thin spines (Holtmaat et al., 2005).
As both spine loss and dendritic simplification were rescued
when a genetically encoded VIVIT-GFP was overexpressed in
neurons, we postulated that the morphological effects of
CACaN were related to NFAT activation. We therefore inves-
tigated the specific effects of NFATc4 on these morphological
parameters. We transduced 14 DIV cortical neurons with ei-
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3179
ther a lentiviral vector encoding a HA-tagged wild-type
(WtNFAT) or constitutively activated (CANFAT) form of
NFATc4 that lacks its N terminus regulatory CaN binding
domain. This truncated NFATc4 is therefore activated with-
out calcineurin (Molkentin et al., 1998). To detect morpho-
logical variables, neurons were previously transfected with
GFP at 5 DIV and we verified that a high percentage (80%) of
the GFP-filled cells were also positive for HA. As observed in
was excluded from the nucleus (delimited by the marker of
the nuclear membrane laminin-B1), whereas, as expected, the
constitutively active form of NFATc4 was concentrated in the
nucleus. Importantly, CaN activity was not previously in-
duced in these cells (either by using a calcium ionophore or
CACaN overexpression), demonstrating that CANFAT did
contrast to CACaN-induced NFATc4 nuclear recruitment, ac-
cumulation of CANFAT into the nucleus could not be pre-
vented by VIVIT-GFP (Fig. 2A). Overexpression of CANFAT
led to a significant decrease of spine density (0.22 ? 0.045
spines/?m) and dendritic simplification compared with Wt-
NFAT (0.39 ? 0.07 spines/?m). None of these changes could
be improved by co-transfection with VIVIT-GFP (0.24 ?
0.023 spines/?m) (Fig. 2B–D), thus demonstrating that
VIVIT acts upstream of NFAT activation and does not have an
effect nonrelated to calcineurin. We therefore concluded that
sufficient to induce spine loss and dendritic simplification in
primary neurons, suggesting that NFATc4-related transcrip-
3180 • J.Neurosci.,February29,2012 • 32(9):3176–3192 Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
We previously observed that both transgenic neurons cultured
from Tg2576 embryos and wild-type neurons treated with A?-
containing conditioned media develop AD-like pathological
ritic simplification. The involvement of CaN in these processes
was suggested by the fact that both phenotypes could be rescued
(Wu et al., 2010; Rozkalne et al., 2011). Here we showed that the
overexpression of constitutively activated CaN or NFAT pheno-
copy the morphological changes induced by A?. Based on these
observations, we asked whether the inhibition of NFAT activa-
tion by calcineurin can also alleviate A? synaptotoxicity.
We first tested the efficiency of VIVIT-GFP in transgenic
mutated APP transgene was present as a heterozygous state,
transgenic (Tg) or littermate (Wt) cultures could be analyzed
in parallel. Cells were transfected with either GFP or VIVIT-
GFP at 5 DIV and analyzed at 18 DIV. A decreased density of
dendritic spines was observed in Tg neurons (0.21 ? 0.11
spines/?m) compared with their wild-type counterpart
(0.45 ? 0.1 spines/?m) (Fig. 3A,B). The spine loss detected in
Tg neurons was also associated with a simplification of the
dendritic tree (Fig. 3C). Importantly, when VIVIT-GFP was
overexpressed in Tg neurons, both parameters were rescued to
normal wild-type levels (spine density: 0.39 ? 0.12 spines/
?m) and significantly improved compared with Tg neurons
transfected with GFP alone.
To study the effect of exogenous A? peptides on neuronal
morphological parameters, A?-containing conditioned medium
(TgCM) was obtained by collecting the medium of Tg neurons
rable to the levels observed in AD brains (Ingelsson et al., 2004).
Western blot analysis showed that TgCM mainly contained sol-
uble oligomeric species (data not shown) that have been de-
scribed to have important neurotoxic effects (Walsh et al., 2002;
Lesne ´ et al., 2006; Shankar et al., 2007, 2008). Wild-type primary
DIV were exposed for 24 h to either wild-type (WtCM) or trans-
genic (TgCM) conditioned media diluted 1:1 in the initial me-
dium. As observed in Figure 3, D and E, 24 h exposure to A? was
sufficient to induce a significant loss of spines in wild-type pri-
mary neurons (0.28 ? 0.075 spines/?m) compared with WtCM
treatment (0.48 ? 0.12 spines/?m). However, no change of the
dendritic complexity was observed, suggesting that such a short
arbor of the cells. A? immunodepletion (using the anti-
amyloid-? monoclonal antibody 6E10) significantly inhibited
the effect of TgCM on spines (0.39 ? 0.09 spines/?m), demon-
strating that the spine loss was directly related to the presence of
amyloid ? species (immunodepletion led to a 70% decrease in
effect on spine density was greatly ameliorated (0.41 ? 0.108
This suggests that blocking CaN-mediated activation of NFAT
protects against neurotoxic effects of soluble exogenous A?
VIVIT was initially identified as a selective blocker of the CaN/
is required to bind calcineurin. Although Aramburu et al. (1999)
showed that calcineurin activity on critical substrates (such as
CREB) was not affected by VIVIT, several recent studies sug-
gested that it can also disrupt the interaction between CaN and
other important cytosolic targets implicated in the regulation of
synaptic activity such as AKAP or cabin (Dell’Acqua et al., 2002;
Liu, 2003). In an attempt to precisely decipher the role of the
transcription factor NFAT in A?-related neurotoxic events, we
used a nuclear-targeted version of VIVIT that was shown to have
a restricted localization within the nucleus and that was previ-
ously used to specifically counteract NFATc4-related transcrip-
tional activity (Schwartz et al., 2009). As a negative control, we
fused a myristoylation (Myr) tag to the VIVIT-GFP plasmid to
direct the localization of this peptide toward the cell membrane.
Thus, these constructs would disambiguate the transcriptional
effects of VIVIT from other non-transcriptional functions at the
We first tested whether these two constructs, NLS-VIVIT-
GFP and Myr-VIVIT-GFP, showed the expected subcellular lo-
calization. At 5 DIV, primary cortical neurons were transfected
Figure 4A, NLS-VIVIT-GFP was strictly restricted to the nuclear
compartment and limited by the nuclear membrane marker
laminin B1. By contrast, the fluorescent signal of Myr-VIVIT-
mid was transfected altogether with DsRed (as a common
denominator to obtain the full morphology of each cell), both
GFP and Myr-VIVIT-GFP could be detected in the processes of
neurons and in the dendritic spines, which was not the case for
NLS-VIVIT-GFP (Fig. 4B).
transcriptional activity, primary neurons were transduced with
an AAV-pNFAT-Luc reporter vector, altogether with AAV-
WtCaN?GFP, AAV-CACaN?AAV-GFP, AAV-CACaN?AAV-
NLS-VIVIT-GFP, or AAV-CACaN?AAV-Myr-VIVIT-GFP. After
overexpression of a constitutively active form of calcineurin led
to an increase of NFAT transcriptional activity compared with
WtCaN. This increase was significantly diminished when NLS-
VIVIT-GFP was overexpressed, but no change could be detected
with Myr-VIVIT-GFP. This suggested that only a nuclear-
ity in vitro (Fig. 5A). We next determined whether CaN-induced
morphological changes could be rescued by the different VIVIT-
GFP constructs by co-transfecting neuronal cultures with either
WtCaN or CACaN, altogether with GFP, NLS-VIVIT-GFP, or
Myr-VIVIT-GFP. Because dendrites and spines were not detect-
able from the GFP signal when NLS-VIVIT-GFP was overex-
pressed, we used DsRed as a common denominator to evaluate
spine density and dendritic complexity. At 18 DIV, both spine
loss and dendritic simplification associated with CACaN overex-
transfected (Fig. 5B–D). By contrast, no effect could be detected
when Myr-VIVIT-GFP was used, consistent with the hypothesis
that the neuroprotection of VIVIT against CACaN-mediated
neurodegeneration is related to transcriptional effects in the nu-
cleus rather than non-transcriptional effects in the cytoplasm or
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity J.Neurosci.,February29,2012 • 32(9):3176–3192 • 3181
3182 • J.Neurosci.,February29,2012 • 32(9):3176–3192 Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
pathological changes induced by CACaN. Considering the fact that
downstream events to A? neurotoxicity (Busche et al., 2008;
mary neurons that were co-transfected with DsRed (Fig. 6A). Both
when NLS-VIVIT-GFP was overexpressed in Tg neurons, but no
Similarly, when Wt primary neurons were transfected with
GFP?DsRed, NLS-VIVIT-GFP?DsRed, or Myr-VIVIT-GFP?
DsRed and then treated for 24 h with A?-containing TgCM (Fig.
6D), the TgCM-induced spine loss was attenuated by NLS-VIVIT-
GFP (Fig. 6E), but no change could be observed in TgCM-treated
neurons transfected with GFP or Myr-VIVIT-GFP. Again, this
short-term exposure to A? oligomers did not affect the dendritic
transcriptional events play a role in A?-related neurotoxicity, thus
neurons transfected with GFP). D, Representative images of wild-type cortical neurons (left) and
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3183
In vitro, we observed that a selective disruption of the interac-
tion between CaN and NFAT may alleviate A?-related neuro-
be reproduced in vivo. Neurites surrounding amyloid deposits
have an increased calcium overload, decreased spine density,
and an increased probability to develop neuritic dystrophies
(Spires et al., 2005; Kuchibhotla et al., 2008). Interestingly,
overexpression of a constitutively activated CaN in vivo leads
to similar abnormal neuronal changes (Wu et al., 2010). We
therefore hypothesized that amyloid aggregates may be re-
sponsible for an abnormal increased activation of CaN/NFAT
pathway in vivo, which would compromise neuronal integrity.
We revisited this hypothesis by delivering VIVIT in the brains
using the pNFAT-luciferase reporter system shows that overexpression of NLS-VIVIT-GFP, altogether with CACaN, is associated with a decreased luciferase activity. No effect is observed when
were co-transfected with a DsRed fluorescent reporter. Scale bar, 100 ?m. C, The decreased spine density associated with the overexpression of a constitutively activated CaN (CACaN) can be
3184 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3185
of APP/PS1 transgenic mice and exam-
To investigate the effect of VIVIT in
vivo, AAV vectors encoding for VIVIT-
GFP, NLS-VIVIT-GFP (both inhibitors
having proven beneficial effects in vitro),
and GFP (as a control) were stereotacti-
littermates or APP/PS1 mice, when amy-
loid deposits are already present. In the
case of AAV-NLS-VIVIT-GFP injected
mice, we first verified that the recombi-
nant protein was properly addressed to
the nuclear compartment and colocalized
with Hoechst-stained nuclei (Hoechst so-
lution was applied topically; Fig. 7A).
AAV-NLS-VIVIT-GFP and AAV-GFP
that a high proportion of GFP filled neu-
rons were also transduced by the AAV-
NLS-VIVIT-GFP vector. One month
later, a cranial window was implanted.
Neurites and spines were detected and
magnification images were taken to visu-
alize dendritic spines. Spine density was
the NeuronStudio software (see Materials
and Methods). Importantly, no signifi-
cant difference was observed when com-
paring the spine density in wild-type
littermate mice injected with either
AAV-GFP (0.47 ? 0.11 spines/?m),
AAV-VIVIT-GFP (0.49 ? 0.107 spines/
?m), or AAV-NLS-VIVIT-GFP (0.53 ?
ing that VIVIT-GFP does not have an effect on spine density by
itself, i.e., independently of A?.
mates (0.47 ? 0.11 spines/?m), dendrites in the vicinity of amy-
loid deposits (?100 ?m away from plaque) in APP/PS1 mice
exhibited a decreased spine density (0.31 ? 0.12 spines/?m).
This amyloid-associated spine loss was restored to essentially
?m) or AAV-NLS-VIVIT-GFP?AAV-GFP (0.44 ? 0.1 spines/
?m) were injected in APP/PS1 mice (Fig. 8B). In both APP/PS1
animals and human AD brains the spine densities are correlated
with the distance from the edge of the amyloid deposits (correla-
tion coefficient: 0.41) (Koffie et al., 2009) (Spires et al., 2005).
However, this local effect of amyloid plaques was nearly abol-
ished when AAV-NLS-VIVIT-GFP (correlation coefficient:
0.22) or AAV-VIVIT-GFP was expressed (correlation coeffi-
cient: 0.09; Fig. 8C).
a marked recovery of spine density around amyloid deposits in
vivo. A significant beneficial effect was also observed in AAV-
NLS-VIVIT injected mice, even though this improvement did
not reach the levels of VIVIT-GFP. This difference might be due
to the fact that some of the GFP-filled neurites observed in ani-
mals coinjected with AAV-NLS-VIVIT-GFP and AAV-GFP had
only been transduced by the later vector. To examine this possi-
bility, we coinjected the AAV-NLS-VIVIT-GFP and an AAV-
TdTomato with the same 3:1 ratio as previously used, and we
observed that 86% of the red fluorescent cells also contained a
nuclear GFP signal (Fig. 7B), suggesting that the vast majority of
transduced neurons expressed both recombinant proteins.
The presence of amyloid deposits not only affects the density of
neuritic shaft itself (Knowles et al., 1999). We therefore asked
development of neuritic dystrophies, which can be visualized by
(Figure legend continued.)
neurons (left) or dendritic segments (right) after co-transfection by GFP, NLS-VIVIT-GFP, or
spine density was induced by TgCM, which could be inhibited by NLS-VIVIT-GFP but not by
complexity compared with GFP-transfected Tg neurons and
efficient approach to be able to detect spines in vivo and to ascertain the fact that the observed Tomato-transduced cells also
3186 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
amyloid plaques that were analyzed was comparable between all
the injected animals, plaques from AAV-VIVIT-treated mice
were associated with fewer dystrophies (5 ? 4 dystrophies/
plaque) compared with those from AAV-GFP (14 ? 9 dystro-
phies/plaque) and AAV-NLS-VIVIT (18 ? 10 dystrophies/
plaque; Fig. 9A,B) injected mice. Because the density of GFP-
filled neurons around plaques was similar among all the groups
(data not shown), we conclude that the difference in the number
of dystrophies was not due to a decreased amount of GFP-filled
neurites between AAV-GFP and AAV-VIVIT-GFP treated ani-
mals. This finding suggests that dystrophies, unlike dendritic
spine loss, might not be recovered by a restricted inhibition of
NFAT transcriptional activity in the nucleus. Interestingly, like
in neuritic dystrophies (Rozkalne et al., 2011). The remaining
dystrophies in AAV-VIVIT-treated mice were similar in size to
those from AAV-GFP and AAV-NLS-VIVIT-injected mice.
A third morphological characteristic of neurites around amy-
loid deposits is a subtle change in their trajectories (Knowles et
al., 1999). This abnormal neuritic curvature was assessed on
paraffin-embedded sections after immunostaining for GFP and
the length of a neurite divided by the end-to-end length of the
both AAV-VIVIT-GFP (1.054 ? 0.038) and AAV-NLS-VIVIT-
GFP (1.059 ? 0.048) injected mice were improved around amy-
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3187
0.07) (Fig. 9C,D).
trophies and curvature) that are abnormal in the vicinity of
amyloid deposits were significantly improved when either AAV-
VIVIT-GFP or AAV-NLS-VIVIT-GFP was injected in the cortex
to the efficient inhibition of NFATc4 recruitment into the nucleus,
Brain sections of injected mice were stained to detect both
in the nucleus versus cytoplasm in transduced neurons. We
found that the distribution of NFATc4 nuclear/cytoplasmic ra-
tios was shifted toward lower values in APP/PS1 mice injected
with AAV-VIVIT-GFP treated animals (0.8 ? 0.08) compared
with AAV-GFP (0.89 ? 0.06). AAV-NLS-VIVIT-GFP-injected
mice exhibited an intermediate NFATc4 nuclear/cytoplasmic ra-
tio between AAV-GFP and AAV-VIVIT-GFP-injected animals
(0.83 ? 0.09) (Fig. 10A,B).
The biological mechanisms that sculpt the fine structure of the
adult brain and their alterations in neurodegenerative diseases
neurotoxic damage can be prevented or even reversed by inhib-
iting calcineurin-mediated activation of NFAT. Importantly, the
same profound morphological changes occur when a constitu-
3188 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
effects of A? are mediated, at least in part, by the transcriptional
activation of NFAT downstream target genes. Although the con-
tribution of NFAT to neurological function or dysfunction has re-
ceived little attention, recent findings have demonstrated a role of
NFAT in synaptic plasticity during development (Nguyen and Di
Giovanni, 2008; Schwartz et al., 2009). Moreover, NFAT shows an
and the severity of cognitive impairment (Abdul et al., 2009).
Our study provides evidence supporting the idea that NFAT is
a specific molecular mediator of transcription-dependent
modifications of the CNS structure in the context of Alzhei-
In vitro, we observed that the morphological changes induced
by constitutively active calcineurin or by constitutively active
sity and dendritic simplification induced
by the neurotoxic A? peptides. Along
with recent data indicating that calcineu-
rin is activated both in human AD brain
we tested the hypothesis that inhibition
of the excessive NFAT activity mightim-
in AD models. We demonstrate that VI-
the complexity of dendritic arbors in
APPswe-transgenic neurons or in TgCM-
treated wild-type neurons. In vivo, a gene
transfer approach using adeno-associated
vectors enabled the delivery of VIVIT to
neurons and significantly increased the
spine density in the vicinity of senile
plaques. Transduction with an inhibitor
that, due to a nuclear localization signal
(NLS-VIVIT), specifically blocked the nu-
clear activation of NFAT, was also able to
restore to nearly normal the morphological
ity of NLS-VIVIT to reproduce the same
beneficial effects as VIVIT is intriguing, as
this peptide is thought to interact primarily
with the docking site of NFAT upon CaN.
activated CaN is able to translocate to the
nucleusinneurons(Pujoletal.,1993;Sola ` et
al., 1999; Yang et al., 2005; Schwartz et al.,
2009), a phenomenon we also previously
described in AD patients (Wu et al., 2010).
We therefore propose that both CaN and
NFAT translocate to the nuclear compart-
ment, where VIVIT would compete with
NFAT for CaN binding. Considering the
compared with NFAT (Aramburu et al.,
1999)], the CaN/NFAT interaction would
thus be interrupted and NFAT would rap-
the cytoplasm. This hypothesis is in agreement with the previous
observation that NLS-VIVIT efficiently inhibited the expression of
of a significant decrease in the NFAT nuclear/cytoplasmic ratio not
only in AAV-VIVIT-GFP-injected animals but also in AAV-NLS-
Our results indicate that (1) NFAT activation is a likely con-
sequence of A? accumulation in Alzheimer’s disease, as sug-
gested by its prominent nuclear localization in the brain of AD
VIVIT to restore A?-associated morphological neurodegenera-
tive changes; (2) once activated and translocated to the nucleus,
NFAT presumably induces the transcription of target genes in
mature neurons that lead to a pathological remodeling of den-
drites and dendritic spines. The beneficial effect of a nucleus-
directed NFAT inhibitor supports the role of transcriptional
events in the regulation of dendritic spine stability in vitro and in
in all three groups tested. However, there is a shift toward higher values in AAV-GFP-injected APP/PS1 mice compared with
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity J.Neurosci.,February29,2012 • 32(9):3176–3192 • 3189
vivo. To some extent, these findings are unexpected, as the mod-
ulation of spine morphology has been largely focused on endo-
cytosis and stability of AMPAR and NMDAR receptors, rather
than transcriptionally regulated phenomena (Lau and Zukin,
2007; Zhang et al., 2008; Bhattacharyya et al., 2009; Goebel-
Goody et al., 2009). Although local spine-specific phenomena
will ultimately determine the stability of individual spines, the
current data emphasize that dendritic trees and spine density are
also impacted at a transcriptional level. This conclusion is in
modulation can affect memory function (Saura and Valero, 2011),
and with recent data demonstrating that alterations in mRNA traf-
eration in disorders as disparate as autism and amyotrophic lateral
The literature addressing the question of NFAT target genes
specifically regulated in neurons is quite limited, but few inter-
esting findings might be relevant in the context of Alzheimer’s
disease. For example, NFAT was shown to contribute to the in-
duction of apoptosis in neuroblastoma cells through upregula-
2011), a gene that was also reported to increase in AD brains,
especially in the hippocampal formation and entorhinal cortex
peptides induce a pathological increase in Fas-L in cortical pri-
dystrophies surrounding the amyloid deposits (Su et al., 2003).
phology has been reported so far, one of the down-stream target
of Fas-L, capsase-3, was recently shown to trigger early synaptic
dysfunction and spine loss in AD (D’Amelio et al., 2011). This
would suggest that activation of caspase-3 may not only be in-
volved in cell death but is also closely associated with the regula-
tion of synaptic plasticity. It is therefore possible that the
activation of Fas-L expression by NFAT indirectly causes a syn-
way. The expression of the potassium channel Kv2.1 can also be
driven by NFAT and was shown to be upregulated by 72% in the
hippocampus of rat injected with A?25–35.(Pan et al., 2004). In-
terestingly, the use of several potassium channel openers (mi-
noxidil, pinacidil, cromakalim) can induce amnesia in mice
activity of the potassium channels might impair memory encod-
ing, one of the best hallmark of AD. As learning and memory
deficits mainly reflect a default in spine-mediated plasticity, we
can hypothesize that dysregulation of the potassium channel
Last, the upregulation of the inositol 1,4,5-trisphosphate type 1
receptor (InsP3R) gene by NFAT is of particular interest in the
al., 2004). Indeed, InsP3R was shown to directly interact with
presenilins 1 and 2, increasing its activity in response to Inositol-
et al., 2011). The upregulation of InsP3R might therefore partic-
ipate to exacerbate the increase of the resting calcium levels. The
of AD, but other NFAT target genes previously identified does
not seem relevant in a pathological context. Indeed, NFAT tran-
the neurotrophic factor BDNF in Purkinje cells, an important
mediator of axonal outgrowth during development (Graef et al.,
2003). These varied results could be explained by the ability of
which the pattern of NFAT-induced target genes can be modu-
on the physiological or pathological context. It is therefore con-
ceivable that a particular set of NFAT coactivators are present in
cortical neurons exposed to A?, so that its activation leads to the
upregulation of genes implicated in dendritic spine shrinkage.
Although the transcriptional effects of NFAT (and its inhibi-
tion) await further investigation, the present study provides a
neurotoxic effects of amyloid-? and confirms the feasibility to
pharmacologically arrest the “amyloid cascade” of neurodegen-
eration at a step after amyloid deposition has already occurred.
The latter has critical therapeutic implications, since most amy-
loid deposition is thought to occur years or even decades before
the onset of cognitive decline, and therefore, before AD diagno-
sis, whereas synaptic and neuron loss largely occurs in a second
stage and correlates with the severity of cognitive impairment
(Sperling et al., 2009). In this scenario, amyloid-directed thera-
pies would not be sufficiently effective once cognitive symptoms
(and the underlying neurodegenerative processes) have begun.
damage would be needed.
Abdul HM, Sama MA, Furman JL, Mathis DM, Beckett TL, Weidner AM,
Patel ES, Baig I, Murphy MP, LeVine H 3rd, Kraner SD, Norris CM
(2009) Cognitive decline in Alzheimer’s disease is associated with selec-
tive changes in calcineurin/NFAT signaling. J Neurosci 29:12957–12969.
Alvarez S, Blanco A, Fresno M, Mun ˜oz-Ferna ´ndez MA´ (2011) TNF-alpha
contributes to caspase-3 independent apoptosis in neuroblastoma cells:
role of NFAT. PLoS One 6:e16100.
AmbergGC,RossowCF,NavedoMF,SantanaLF (2004) NFATc3regulates
Kv2.1 expressionin arterial
Aramburu J, Yaffe MB, Lo ´pez-Rodríguez C, Cantley LC, Hogan PG, Rao A
(1999) Affinity-driven peptide selection of an NFAT inhibitor more se-
lective than cyclosporin A. Science 285:2129–2133.
Bacskai BJ, Klunk WE, Mathis CA, Hyman BT (2002) Imaging amyloid-
beta deposits in vivo. J Cereb Blood Flow Metab 22:1035–1041.
A (2005) The transcription factor NFAT3 mediates neuronal survival.
J Biol Chem 280:2818–2825.
Bhattacharyya S, Biou V, Xu W, Schlu ¨ter O, Malenka RC (2009) A critical
role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA
receptors. Nat Neurosci 12:172–181.
Busche MA, Eichhoff G, Adelsberger H, Abramowski D, Wiederhold KH,
Haass C, Staufenbiel M, Konnerth A, Garaschuk O (2008) Clusters of
hyperactive neurons near amyloid plaques in a mouse model of Alzhei-
mer’s disease. Science 321:1686–1689.
Crabtree GR, Olson EN (2002) NFAT signaling: choreographing the social
lives of cells. Cell 109 [Suppl]:S67–S79.
D’Amelio M, Cavallucci V, Middei S, Marchetti C, Pacioni S, Ferri A, Dia-
mantini A, De Zio D, Carrara P, Battistini L, Moreno S, Bacci A,
Ammassari-Teule M, Marie H, Cecconi F (2011) Caspase-3 triggers
early synaptic dysfunction in a mouse model of Alzheimer’s disease. Nat
DeKoskyST,ScheffSW,StyrenSD (1996) Structuralcorrelatesofcognition
in dementia: quantification and assessment of synapse change. Neurode-
Dell’AcquaML,DodgeKL,TavalinSJ,ScottJD (2002) Mappingtheprotein
phosphatase-2B anchoring site on AKAP79. Binding and inhibition of
phosphatase activity are mediated by residues 315–360. J Biol Chem
DineleyKT,HoganD,ZhangWR,TaglialatelaG (2007) Acuteinhibitionof
calcineurin restores associative learning and memory in Tg2576 APP
transgenic mice. Neurobiol Learn Mem 88:217–224.
smoothmuscle.J Biol Chem
3190 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity
Gatto CL, Broadie K (2010) Genetic controls balancing excitatory and in-
hibitory synaptogenesis in neurodevelopmental disorder models. Front
Synaptic Neurosci 2:4.
Genazzani AA, Carafoli E, Guerini D (1999) Calcineurin controls inositol
Sci U S A 96:5797–5801.
Ghelardini C, Galeotti N, Bartolini A (1998) Influence of potassium
channel modulators on cognitive processes in mice. Br J Pharmacol
Goebel-Goody SM, Davies KD, Alvestad Linger RM, Freund RK, Brown-
ing MD (2009) Phospho-regulation of synaptic and extrasynaptic
N-methyl-d-aspartate receptors in adult hippocampal slices. Neuro-
GR (2003) Neurotrophins and netrins require calcineurin/NFAT sig-
naling to stimulate outgrowth of embryonic axons. Cell 113:657–670.
Groth RD, Dunbar RL, Mermelstein PG (2003) Calcineurin regulation of
neuronal plasticity. Biochem Biophys Res Commun 311:1159–1171.
Ho AM, Jain J, Rao A, Hogan PG (1994) Expression of the transcription
factor NFATp in a neuronal cell line and in the murine nervous system.
J Biol Chem 269:28181–28186.
Holtmaat AJ, Trachtenberg JT, Wilbrecht L, Shepherd GM, Zhang X, Knott
GW, Svoboda K (2005) Transient and persistent dendritic spines in the
neocortex in vivo. Neuron 45:279–291.
Ingelsson M, Fukumoto H, Newell KL, Growdon JH, Hedley-Whyte ET,
Frosch MP, Albert MS, Hyman BT, Irizarry MC (2004) Early Abeta ac-
AD brain. Neurology 62:925–931.
land NG, Lee MK, Younkin LH, Wagner SL, Younkin SG, Borchelt DR
(2004) Mutant presenilins specifically elevate the levels of the 42 residue
beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific
gamma secretase. Hum Mol Genet 13:159–170.
Jurado S, Biou V, Malenka RC (2010) A calcineurin/AKAP complex is re-
quired for NMDA receptor-dependent long-term depression. Nat Neu-
Kao SC, Wu H, Xie J, Chang CP, Ranish JA, Graef IA, Crabtree GR (2009)
Calcineurin/NFAT signaling is required for neuregulin-regulated Schwann
Klee CB, Crouch TH, Krinks MH (1979) Calcineurin: a calcium- and
calmodulin-binding protein of the nervous system. Proc Natl Acad Sci
U S A 76:6270–6273.
Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP,
Debnath ML, Holt DP, Wang Y, Hyman BT (2002) Imaging Abeta
plaques in living transgenic mice with multiphoton microscopy and
methoxy-X04, a systemically administered Congo red derivative. J Neu-
ropathol Exp Neurol 61:797–805.
HE, Hyman BT (1999) Plaque-induced neurite abnormalities: implica-
tions for disruption of neural networks in Alzheimer’s disease. Proc Natl
Acad Sci U S A 96:5274–5279.
Koffie RM, Meyer-Luehmann M, Hashimoto T, Adams KW, Mielke ML,
Garcia-Alloza M, Micheva KD, Smith SJ, Kim ML, Lee VM, Hyman BT,
Spires-Jones TL (2009) Oligomeric amyloid beta associates with post-
synaptic densities and correlates with excitatory synapse loss near senile
plaques. Proc Natl Acad Sci U S A 106:4012–4017.
in vivo resulting in structural and functional disruption of neuronal net-
works. Neuron 59:214–225.
Lau CG, Zukin RS (2007) NMDA receptor trafficking in synaptic plasticity
and neuropsychiatric disorders. Nat Rev Neurosci 8:413–426.
Lee SH, Kim BC, Yang DH, Park MS, Choi SM, Kim MK, Cho KH (2008)
Calcineurin inhibitor-mediated bilateral hippocampal injury after bone
marrow transplantation. J Neurol 255:929–931.
Lesne ´ S,KohMT,KotilinekL,KayedR,GlabeCG,YangA,GallagherM,Ashe
KH (2006) A specific amyloid-beta protein assembly in the brain im-
pairs memory. Nature 440:352–357.
Liu F, Grundke-Iqbal I, Iqbal K, Oda Y, Tomizawa K, Gong CX (2005)
Truncation and activation of calcineurin A by calpain I in Alzheimer
disease brain. J Biol Chem 280:37755–37762.
Liu JO (2003) Endogenous protein inhibitors of calcineurin. Biochem Bio-
phys Res Commun 311:1103–1109.
Luoma JI, Zirpel L (2008) Deafferentation-induced activation of NFAT
(nuclear factor of activated T-cells) in cochlear nucleus neurons during a
the CNS. J Neurosci 28:3159–3169.
Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant
SR, Olson EN (1998) A calcineurin-dependent transcriptional pathway
for cardiac hypertrophy. Cell 93:215–228.
Mu ¨ller M, Cheung KH, Foskett JK (2011) Enhanced ROS generation medi-
ated by Alzheimer’s disease presenilin regulation of InsP3R Ca2?signal-
ing. Antioxid Redox Signal 14:1225–1235.
Nguyen T, Di Giovanni S (2008) NFAT signaling in neural development
and axon growth. Int J Dev Neurosci 26:141–145.
Norris CM, Kadish I, Blalock EM, Chen KC, Thibault V, Porter NM, Land-
field PW, Kraner SD (2005) Calcineurin triggers reactive/inflammatory
processes in astrocytes and is upregulated in aging and Alzheimer’s mod-
els. J Neurosci 25:4649–4658.
Ogata K, Sato K, Tahirov TH (2003) Eukaryotic transcriptional regulatory
complexes: cooperativity from near and afar. Curr Opin Struct Biol
Pan Y, Xu X, Tong X, Wang X (2004) Messenger RNA and protein expres-
sion analysis of voltage-gated potassium channels in the brain of
Abeta(25–35)-treated rats. J Neurosci Res 77:94–99.
Ponticelli C, Campise MR (2005) Neurological complications in kidney
transplant recipients. J Nephrol 18:521–528.
Pujol MJ, Bosser R, Vendrell M, Serratosa J, Bachs O (1993) Nuclear
Rodriguez A, Ehlenberger DB, Dickstein DL, Hof PR, Wearne SL (2008)
Automated three-dimensional detection and shape classification of den-
dritic spines from fluorescence microscopy images. PLoS One 3:e1997.
RozkalneA,HymanBT,Spires-JonesTL (2011) Calcineurininhibitionwith
FK506 ameliorates dendritic spine density deficits in plaque-bearing Alz-
heimer model mice. Neurobiol Dis 41:650–654.
SauraCA,ValeroJ (2011) TheroleofCREBsignalinginAlzheimer’sdisease
and other cognitive disorders. Rev Neurosci 22:153–169.
SchwartzN,SchohlA,RuthazerES (2009) Neuralactivityregulatessynaptic
naling. Neuron 62:655–669.
Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini
BL (2007) NaturaloligomersoftheAlzheimeramyloid-betaproteinin-
duce reversible synapse loss by modulating an NMDA-type glutamate
receptor-dependent signaling pathway. J Neurosci 27:2866–2875.
Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I,
Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM,
Sabatini BL, Selkoe DJ (2008) Amyloid-beta protein dimers isolated di-
rectly from Alzheimer’s brains impair synaptic plasticity and memory.
Nat Med 14:837–842.
Sola ` C,TusellJM,SerratosaJ (1999) Comparativestudyofthedistribution
Sperling RA, Laviolette PS, O’Keefe K, O’Brien J, Rentz DM, Pihlajamaki M,
Marshall G, Hyman BT, Selkoe DJ, Hedden T, Buckner RL, Becker JA,
Johnson KA (2009) Amyloid deposition is associated with impaired de-
fault network function in older persons without dementia. Neuron
Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J, Nguyen PT,
BacskaiBJ,HymanBT (2005) Dendriticspineabnormalitiesinamyloid
precursor protein transgenic mice demonstrated by gene transfer and
intravital multiphoton microscopy. J Neurosci 25:7278–7287.
Spires-Jones TL, Kay K, Matsouka R, Rozkalne A, Betensky RA, Hyman BT
(2011) Calcineurin inhibition with systemic FK506 treatment increases
dendritic branching and dendritic spine density in healthy adult mouse
brain. Neurosci Lett 487:260–263.
Su JH, Anderson AJ, Cribbs DH, Tu C, Tong L, Kesslack P, Cotman CW
AD brain and participate in beta-amyloid-induced neuronal death. Neu-
robiol Dis 12:182–193.
Taglialatela G, Hogan D, Zhang WR, Dineley KT (2009) Intermediate- and
long-term recognition memory deficits in Tg2576 mice are reversed with
acute calcineurin inhibition. Behav Brain Res 200:95–99.
Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicityJ.Neurosci.,February29,2012 • 32(9):3176–3192 • 3191
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Download full-text
Katzman R (1991) Physical basis of cognitive alterations in Alzheimer’s
disease: synapse loss is the major correlate of cognitive impairment. Ann
SelkoeDJ (2002) Naturallysecretedoligomersofamyloidbetaproteinpo-
tently inhibit hippocampal long-term potentiation in vivo. Nature
A, Matsui H (2004) Critical role of calpain-mediated cleavage of cal-
cineurin in excitotoxic neurodegeneration. J Biol Chem 279:4929–4940.
Wu HY, Hudry E, Hashimoto T, Kuchibhotla K, Rozkalne A, Fan Z, Spires-
Jones T, Xie H, Arbel-Ornath M, Grosskreutz CL, Bacskai BJ, Hyman BT
of spine loss, dendritic simplification, and neuritic dystrophies through
calcineurin activation. J Neurosci 30:2636–2649.
Yang Y, Fischer QS, Zhang Y, Baumga ¨rtel K, Mansuy IM, Daw NW (2005)
Reversible blockade of experience-dependent plasticity by calcineurin in
mouse visual cortex. Nat Neurosci 8:791–796.
GL, Lombroso PJ (2008) The tyrosine phosphatase STEP mediates
AMPA receptor endocytosis after metabotropic glutamate receptor stim-
ulation. J Neurosci 28:10561–10566.
Zhou Q, Homma KJ, Poo MM (2004) Shrinkage of dendritic spines associ-
ated with long-term depression of hippocampal synapses. Neuron 44:
3192 • J.Neurosci.,February29,2012 • 32(9):3176–3192Hudryetal.•NFATInhibitionAlleviatesAmyloidBetaToxicity