Biochemical and Biophysical Research Communications xxx (2015) 1e6

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

The soluble extracellular fragment of neuroligin-1 targets Ab

oligomers to the postsynaptic region of excitatory synapses
Margarita C. Dinamarca a, 1, Monica Di Luca b, Juan A. Godoy a, Nibaldo C. Inestrosa a, c, d, e, *
a
Center for Aging and Regeneration (CARE), Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontical Catholic University of Chile,
Santiago, Chile
b
Department of Pharmacological Sciences, University of Milan, Milan, Italy
c
Centre for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia
d

lica de Chile, Santiago, Chile
Centro UC Sndrome de Down, Ponticia Universidad Cato
e
Centro de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 August 2015
Accepted 24 August 2015
Available online xxx

Amyloid-b oligomers (Abo) play a major role in the synaptic dysfunction of Alzheimer's disease (AD).
Neuroligins are postsynaptic cell-adhesion molecules, that share an extracellular domain with high
degree of similarity to acetylcholinesterase (AChE), one of the rst putative Abo receptors. We recently
found that Abo interact with the soluble N-terminal fragment of neuroligin-1 (NL-1). We report here that
Abo associate with NL-1 at excitatory hippocampal synapses, whereas almost no association was
observed with neuroligin-2, an isoform present at inhibitory synapses. Studies using puried hippocampal postsynaptic densities indicate that NL-1 interacts with Abo in a complex with GluN2Bcontaining NMDA receptors. Additionally, the soluble fragment of NL-1 was used as a scavenger for
Abo. Field excitatory postsynaptic potentials indicate that fragments of NL-1 protect hippocampal neurons from the impairment induced by Abo. To our knowledge, this is the rst report of the interaction
between this extracellular fragment of NL-1 and Abo, strongly suggest that NL-1 facilitates the targeting
of Abo to the postsynaptic regions of excitatory synapses.
2015 Published by Elsevier Inc.

Keywords:
Neuroligin-1
Ab oligomer
Excitatory synapses
Hippocampal neurons

1. Introduction
The degree of dementia in Alzheimer's disease (AD) patients
correlates closely with the level of soluble oligomers of the Ab
species in postmortem brains, especially in hippocampal and
cortical regions associated with learning and memory function

Abbreviations: Abo, Amyloid-b oligomers; AD, Alzheimer's disease; APP/PS1,

double-transgenic mice; LTP, long-term potentiation; PSD-95, postsynaptic density
protein-95; NMDA receptors, N-methyl-D-aspartate receptors; GluNR1, subunit of
NMDA receptors; NL-1, neuroligin-1 protein; NL-2, neuroligin-2 protein; AChE,
Acetylcholinesterase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; ACSF, articial cerebrospinal uid; GABAA, g-aminobutyric acid type A
receptor; CA1, Cornu Ammonis area 1; fEPSPs, eld excitatory postsynaptic potentials; PPF, paired pulse facilitation; S.E, standard error; DIV, day in vitro; CaMKII,
Ca2/calmodulin-dependent protein kinase II; IPP, co-immunoprecipitation assay.
* Corresponding author. CARE Biomedical Center, Faculty of Biological Sciences,
Ponticial Catholic University of Chile, Alameda 340, P.O. Box 8331150, Santiago,
Chile.
E-mail address: ninestrosa@bio.puc.cl (N.C. Inestrosa).
1
Present Address: Department of Biomedicine, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.

[1,2]. Assays of synaptic function and synaptic markers both in the

brains of AD patients [1,2] and transgenic mouse models, as reported by Buttini et al. [3], support the hypothesis that synaptic
degeneration and damage take place early during the development
of the disease. Light and electron microscopy studies have indicated
that the density and number of synapses per unit volume as well as
their morphology are affected in plaque-free regions of the dentate
gyrus of double-transgenic APP/PS1 mice [4]. Accumulating evidence suggests that soluble Ab oligomers (Abo) are responsible for
the cytotoxicity of Ab [5,6], selectively blocking long-term potentiation (LTP) and acutely disrupting cognitive function. Abos also
bind in a punctuate pattern to postsynaptic excitatory pyramidal
neurons [5,6] but not to the GABAergic components of neurons [7].
Several receptor proteins have been proposed to be capable of
binding various forms of Ab [8], including a7-nicotinic receptors
(a7-nAChR), N-methyl-D-aspartate receptors (NMDAR), metabotropic glutamate receptors (mGluR5), insulin receptors, cellular
prion protein (PrPC) and ephrin receptors [8]. In the case of NMDAR,
Abo induce receptor internalization and deregulation of these
signaling pathway [9,10]. Additionally, antibodies against the

http://dx.doi.org/10.1016/j.bbrc.2015.08.107
0006-291X/ 2015 Published by Elsevier Inc.

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107

M.C. Dinamarca et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6

extracellular domain of the GluNR1 subunit of NMDAR markedly

reduce the binding of Abo to neurons [10], suggesting that Abo bind
to synapses in close proximity to NMDAR. Given the major role of
Abo in the synaptic pathology of AD [3,5,8], it is critical to understand the mechanisms by which Abo are targeted to synaptic sites,
as well as their interaction with synaptic components. Regarding
synaptic proteins that might interact with Ab, important proteins
involved in cell-to-cell signaling between the pre- and postsynaptic
sites of chemical synapses have been recently implicated. NLs are a
family of neural cell-adhesion molecules that are involved in
excitatory/inhibitory synapses. NL-1 is preferentially enriched at
excitatory synapses, whereas NL-2 is present at inhibitory synapses
[12,23,24]. NLs belong to the a/b-hydrolase-fold superfamily of
proteins and are structurally related to AChE, however, this
cholinesterase-like domain of NL lacks important residues,
including a serine that is essential for the enzymatic function of
cholinesterases [13,25]. Previous work from our laboratory has
demonstrated that AChE interacts with Ab aggregates [14,26] and
senile plaques of AD brains [15]. NL has been proposed as a synaptic
protein candidate that may affect Ab accumulation in AD [8,11],
biochemical and physicochemical studies in vitro suggest that NL-1
could be a putative target for Abo at excitatory synapses [11]. NL-1,
but not NL-2, behaves as a nucleating factor for Ab by inducing the
accumulation of Abo [11]. These data show that NL-1 stabilizes
oligomeric assemblies of Ab, and endogenous NL-1 is enriched in
the postsynaptic membranes of glutamatergic synapses, it is
possible that Abo bind to NL-1 at these specic sites. Here, we
report that Abo associate with NL-1 in vitro, in primary cultures and
slices of rat hippocampus, suggesting that NL-1 is a relevant target
of Abo at the postsynaptic sites of excitatory synapses.
2. Materials and methods
2.1. Materials
Ab (1e40) and (1e42) peptides were purchased from Genemed
Biotechnology (San Francisco, CA). The monoclonal antibody 6E10,
antibody A11 against Ab total, Abo and the antibody against PSD-95
were purchased from Chemicon International (Temecula, CA). AntiGluN2A and Anti-GluN2B antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The soluble extracellular fragment of
NL-1 (sNL-1: fragment 1-691aa) and the full-length NL-1 and 2
plasmids were prepared as previously described [16]. Secondary
antibodies were labeled with Alexa-488, 543 or 633 (Afnity Bio
Reagents Inc., Golden, CO). Phalloidin-Alexa-633 from Molecular
Probes, (Eugene, OR), Abo were prepared as previously described
[11].
2.2. Postsynaptic density (PSD) preparation and
immunoprecipitation
To isolate PSDs from SpragueeDawley rat cortices, a modication of the method from Carlin et al. [17,18] was used. Briey,
cortices from adult rats were homogenized with 1 mM HEPES,
0.32 M sucrose, 1 mM MgCl2, 1 mM NaHCO3, 0.1 mM PMSF and a
protease inhibitor cocktail and centrifuged at 3000 rpm for 10 min.
The pellet (P) P1 was discarded. The supernatant was centrifuged at
10,000 rpm for 15 min. The P2 was re-suspended in 0.32 M sucrose
and then loaded onto a 0.85 M/1.0 M/1.2 M sucrose gradient and the
interface between the 1.0 M and 1.2 M layers was recovered (synaptosomal fraction). This fraction was diluted in 1 mM HEPES, 1%
Triton X-100 and 0.32 M sucrose and centrifuged at 24,800 rpm for
1 h. The pellet fraction (Triton X-100 insoluble fraction, TIF) was resuspended in 0.32 M sucrose, loaded onto a 1.0 M/1.5 M/2.1 M
sucrose gradient and centrifuged. The fraction at the 1.5 M/2.1 M

interface was recovered and re-suspended in 0.5% Triton X-100 and

75 mM KCl, and centrifuged at 40,000. The nal pellet (PSD) was resuspended in 20 mM HEPES. Immunoprecipitation (IPP) assays
were carried out using lysis buffer containing 0.32 M sucrose, 1 mM
NaHCO3, 0.1 mM PMSF, 0.1 mM MgCl2, 1 mM Hepes buffer, 1 mM
NaF, 1% SDS and a complete protease inhibitor cocktail [18].
2.3. Organotypic slice cultures of rat brain
Rat organotypic slice cultures were prepared from postnatal day
7 (P7) animals and cultured on Millicell inserts (Millipore) as
described previously [18].
2.4. Neuronal viability assay
Hippocampal neurons were seeded at 1.2  105 cells per well in
48-well plates. After treatment, neurons were incubated with MTT
solution (1 mg/ml nal concentration) for 2 h [19].
2.5. Immunouorescence and image analysis
Hippocampal neurons were seeded on coverslips in 24-well
plates at a density of 60,000 cells/coverslip. Phalloidin was used
as a neurite marker. Images of neurons were captured using a Zeiss
confocal microscope with a 63x/1.4 numerical aperture. To quantify
the PSD-95/synapsin-1 images, 5 microscope elds for each condition in 3 independent experiments were measured using Image J
software (NIH, Baltimore, MD) [20].
2.6. Slice preparation and electrophysiology
Hippocampal slices were prepared from 22 to 30 day-old
SpragueeDawley rats, and incubated in cold articial cerebrospinal uid (ACSF) at room temperature [21]. In all experiments,
picrotoxin (10 mm) was added to the ACSF perfusion to suppress
inhibitory GABAA transmission [33]. Extracellular eld potential
recordings were made with an AC amplier (P-5 Series; Grass),
gain  10,000, LP lter (3.0 kHz), and HP lter (0.30 Hz) in the
middle of the stratum radiatum of area CA1 of the hippocampus.
Electric pulses (50 ms, 0.3 Hz, 20e100 mA) were applied to the
Schaeffer collaterals, eliciting compound action potentials from the
presynaptic axons (ber volley) followed by eld excitatory postsynaptic potentials (fEPSPs). The paired pulse facilitation (PPF) index was calculated by (R2-R1)/R1), where R1 and R2 are the peak
amplitudes of the rst and second fEPSPs, respectively [21].
2.7. Statistical analysis
Data are expressed as the mean S.E. of the values from the
number of experiments indicated in the corresponding gures. The
data were evaluated statistically using Student's t test, with p < 0.01
or p < 0.05 indicating a signicant difference between the control
and experimental groups [21].
3. Results
3.1. Abo associate with NL-1 in hippocampal neurons
We prepared a fraction enriched in Abo [11]. Then hippocampal
neurons (14 DIV) were treated with the Abo and co-incubated with
increasing concentrations of sNL-1 to evaluate cell-viability. The
results show that Abo decreased cell viability by 40% with respect to
the control cells, whereas a signicant protective effect was
observed for neurons co-treated with Abo in the presence of
100 nM sNL-1, reaching values close to that of the control at 1 mM

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107

M.C. Dinamarca et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6

sNL-1 (Fig. 1A). The same experimental approach was then conducted in hippocampal slices. Hippocampal organotypic cultures
(14 DIV) were incubated with Abo or Abo plus sNL-1 for 4 days at
37  C; then we evaluated the amount of postsynaptic proteins
remaining after the treatments using a puried fraction enriched in
postsynaptic density proteins (Triton X-100 insoluble fraction).
Both the total and synaptic amount of PSD-95 decreased 75% with
Abo treatment, whereas CaMKII was not affected by the Abo
treatment. However, when the slices were co-incubated with Abo
plus sNL-1, the loss of PSD-95 was signicantly reduced, indicating
that sNL-1 protects against Abo damage at both the total and synaptic levels (Fig. 1B). sNL-1 treatment alone did not affect the stability of PSD-95 or CaMKII. These results suggest that sNL-1 behaves
as a trophic scavenger for Abo and reduces the toxic effects of Abo
on hippocampal neurons. Next, we evaluated whether Abo colocalize with endogenous NL-1 at the synaptic level. Using

immunouorescence analysis of hippocampal neurons, we showed

that after incubation with 1 mM Abo for 1 h, Abo presented a clear
co-localization with NL-1, reaching 6 puncta/10 mm neurite lengths.
Furthermore, from the total amount of Abo bound to dendrites, 60%
was co-localized with NL-1. By contrast, we observed a diffuse and
weak co-localization pattern with NL-2 (20%), which is mainly
located in dendritic shafts (Fig. 1C). These data suggest that in
hippocampal neurons, Abo are mainly distributed according to the
punctuate NL-1 staining pattern that corresponds to excitatory
synapses. To study synapse integrity, we analyzed the number of
synaptic boutons (pair PSD-95/Synapsin-1). Neurons were incubated for 0, 30 and 60 min at 37  C with Abo or Abo plus sNL-1. At
time 0, we observed a normal distribution of synaptic boutons
along the neurite length (Fig. 1D). After 30 min, the Abo treatment
produced a 35% loss of synaptic boutons; this decrease reached 60%
after 60 min of Abo treatment. Under both Abo-treated time points,

Fig. 1. The soluble N-terminal extracellular fragment of NL-1 protects hippocampal neurons from the damage produced by Abo. (A) Cell viability measured using a MTT reduction
assay. Hippocampal neurons (14 DIV) were treated with 5 mM bo alone or with increasing concentrations of sNL-1 (10, 100 and 1000 nM) (n 3). (B) Organotypic hippocampal
slices were treated with 2.5 mM Abo in the absence or the presence of 1 mM sNL-1 over 4 days. PSD-95 and CaMKII were analyzed from total homogenates (H) and Triton X-100
insoluble fractions (enriched in postsynaptic proteins) from each treatment. (C) Immunouorescence of hippocampal neurons treated with 0.5 mM Abo and stained for endogenous
NL-1 or 2 and Abo. The graphs depict the quantication of Abo bound to NL-1 or NL-2 per 10 mm neurites and the quantication of the percent of the total amount of Abo signal that
is co-localized with NL-1 or NL-2 per 10 mm neurites. (D) Representative immunouorescence of PSD-95 (green), synapsin-1 (red) and uorescence labeling of actin by phalloidin
(blue) from hippocampal neurons (21 DIV) treated for 1 h with 1 mM Abo in the absence or presence of 1 mM sNL-1. Quantication of the number of synaptic boutons (PSD-95/
synapsin-1 pairs) per 10 mm neurites (n 3) from neurons treated with Abo alone or with sNL-1. *p  0.01. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.)

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107

M.C. Dinamarca et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6

the loss of synaptic boutons was prevented in the presence of sNL-1

(Fig. 1D), suggesting that sNL-1 scavenges Abo, decreasing the
synaptotoxic effect of Abo in hippocampal neurons.
3.2. Abo interact with NL-1 and GluN2B-containing NMDA
receptors at postsynaptic regions in hippocampal neurons
We evaluated at a biochemical level whether Abo bind to NLs
present in primary cultures of hippocampal neurons (21DIV).
Neurons were incubated with 0.5 mM Abo for 1 h at 37  C and IPP
assays were performed using the anti-Ab antibody 6E10 (Fig. 2A).
Western blots revealed that endogenous NL-1 and NL-2 proteins
are both present in the input of the IPP assay, however, although it
is clear that Abo immunoprecipitated NL-1 (Fig. 2A), only a faint
band was observed for NL-2. To dene whether Abo interact with
synaptically localized NL-1, postsynaptic density (PSD) membranes
were puried from the cerebral cortex of adult rats [18] and incubated with Abo for 2 h at 4  C. Twenty micrograms of the PSD
fraction and 20 mg of the homogenate (H) were used for an IPPassay.
Following IP using an anti-Ab antibody, polypeptides were resolved
by SDS-PAGE and probed by western blotting using anti-NL-1 or
antibodies against the GluN2A or GluN2B subunits of the NMDA
receptor. The IPP assay indicated that endogenous NL-1 is present
in the PSD fraction, corroborating the co-localization previously
observed by immunouorescence. As described previously, Abo
also bound to endogenous GluN2B subunits of NMDA receptors
(Fig. 2B) [10,22]. These results suggest that Abo associate both with
endogenous NL-1 and GluN2B-containing NMDA receptors present
at excitatory synapses at the postsynaptic density of cultured

Fig. 2. Abo interact with NL-1 in cultured hippocampal neurons and in cerebral cortex.
(A) Rat hippocampal neurons (21 DIV) were treated with 0.5 mM Abo for 1 h at 37  C,
then washed, homogenized and immunoprecipitated using an Ab antibody (6E10).
Western blot analysis for NL-1 and NL-2 was then performed. (B) Rat cerebral cortex
was homogenized and the total homogenate (H) and postsynaptic density (PSD) were
puried and incubated with 2.5 mM Abo for 2 h at 4  C and immunoprecipitated with
an anti-Ab antibody. Western blot analysis was performed for NL-1, GluN2A and
GluN2B (NMDA receptor subunits). (n 4).

hippocampal neurons.

3.3. The soluble N-terminal extracellular fragment of NL-1 protects

against the synaptotoxicity of Abo on excitatory synaptic
transmission
Our previous results suggest that sNL-1 can scavenge Abo to
prevent their neurotoxic effects, we used an electrophysiological
approach to evaluate this effect on excitatory synaptic transmission
in adult hippocampal slices. We recorded fEPSPs in the presence of
10 mm picrotoxin to block GABAA-mediated inhibitory synaptic
transmission. Under these conditions, the fEPSP amplitude
decreased (55%) following the addition of 0.5 mM Abo to the
perfusion medium for 30 min (Fig. 3A, black circles). Interestingly,
the slices treated with sNL-1 also showed a decrease in fEPSP
amplitude of approximately 50% after 30 min. We hypothesize that
the presence of sNL-1 in the ACSF could interfere with the endogenous NL-1-neurexin complex, thus altering synaptic transmission.
Regardless we did obtain a protective effect when Abo were coincubated with sNL-1. In these slices, almost no change in fEPSP
amplitude was observed during the rst 15 min; however, there
was a subsequent small reduction of the fEPSP amplitude compared
with controls (Fig. 3A, sNL-1: light gray circles). Fig. 3B shows
normalized EPSCs (20 sweeps) of neurons from control hippocampal slices and after 40 min of treatment with 1.5 mM Abo,
0.25 mM sNL-1 and Abo plus sNL-1 treatments (recorded at
a 80 mV holding potential). Taken together, these results suggest
that NL-1 targets Abo to the synapse whereas the soluble

Fig. 3. The soluble N-terminal extracellular fragment of NL-1 protects against Abo
synaptotoxicity. (A). The effect of Abo or Abo sNL-1 on fEPSP amplitude. Normalized
fEPSCs of rat hippocampal slices. Time course of 0.5 mM Abo, 0.25 mM sNL1 and Abo
plus sNL-1 treatments. (B) EPSCs of control neurons (light gray) and after 40 min of
treatment with 0.5 mM Abo (black), 0.25 mM sNL-1 (dark gray) or Abo plus sNL-1 (gray),
recorded at 80 mV holding potential. The average of 20 sweeps is shown. The mean
of the normalized amplitude of evoked EPSCs in neurons from control, Abo, sNL-1 and
Abo plus sNL-1 treated hippocampal slices (n 5.*p < 0.05, bar represents error
standard).

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107

M.C. Dinamarca et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6

extracellular a/b-hydrolase-fold (ChE-like) domain of NL-1 acts as a

scavenger of Abo, preventing their binding to endogenous NL-1 at
the postsynaptic sites of excitatory synapses.
4. Discussion
To understand the role of Abo species in AD pathology, it is
critical to elucidate the mechanism by which oligomers are targeted to synaptic terminals. Many proteins have been described as
putative receptors for Abo at the postsynaptic region of excitatory
synapses [8,27e29], including glutamatergic receptors (both NMDA
and mGluR5 receptors) [8,22,27,30], Frizzled receptors [28], and
cellular prion protein (PrPc) [29e31], among others. In the case of
cellular PrPc, although it was found to mediate the neurotoxic effects of Abo, only 50% of the oligomers were bound to PrPc, suggesting that other targets are present at the neuronal surface [32].
Another group has indicated that the impairment of LTP induced by
Abo did not depend on cellular PrP protein, also suggesting that
another protein present at the synapse is required for Ab-synaptotoxicity [33]. Increasing evidence suggests that mGluR5 may
contribute to AD pathogenesis by acting as a scaffold for the PrPc/
Abo complex, enabling the propagation of neurotoxic signaling;
this also supports growing evidence that PrPc can act as a coreceptor for Ab [27]. In addition, NMDAR are functionally modulated by both PrPc and PrPSc, wherein PrPc and Ab oligomer
signaling via NMDA receptors may also contribute to AD pathology
[29].
It has been described that the puried extracellular ChE-like
domain of NL-1 binds directly to Ab [11]. In the present work, we

identied that NL-1 targets Abo, via its extracellular ChE-like

domain, to the postsynaptic region of excitatory synapses; this is
the rst time that an interaction between NL-1 and Abo has been
examined at a synaptic level. We further demonstrated a direct
association between NL-1 and Abo by co-immunoprecipitation and
immunouorescence of endogenous NL-1 from hippocampal neurons (Figs. 1 and 2). More specically, we established that the
interaction between Abo and NL-1 occurs in the postsynaptic region. GluN2B-containing NMDAR were co-immunoprecipitated
together with NL-1 and Abo (Fig. 2B). This result supports the
possibility that NL-1 acts as a co-receptor for Abo at the postsynaptic level, a recent study indicating that the extracellular ChElike domain of NL-1 plays an instructive role in the selective
extracellular coupling to NMDAR [34]; a previous study showing
that Abo induce neuronal dysfunction through the activation of
GluN2B-containing NMDAR; the excessive activation of the this
type of NMDAR is one of the proposed mechanisms for Abo synaptotoxicity [22].
In conclusion, we present the rst evidence for a physical
interaction between postsynaptic NL-1 with Abo at excitatory
synapses. Our results suggest that Ab released to the synaptic space
(Fig. 4A) can change its conformation to an amyloidogenic form,
generating Abo; its association with the extracellular a/b-hydrolase
fold (ChE-like) domain of NL-1 increases the local formation of Abo
[11] that eventually induces an impairment in the synaptic connectivity, which involves a decrease in postsynaptic proteins such
as NMDA receptors and PSD-95 at excitatory synapses (Fig. 4B).
Therefore, NL-1 may target Abo to the postsynaptic region of the
synapse. This work provides a new approach for the design of

Fig. 4. The N-terminal extracellular fragment of NL-1 interacts with Abo at excitatory synapses. (A). Depiction of the normal and constitutive release of Ab to the extracellular space
following APP processing in neurons and the adhesion complex of NL-1 and b-neurexin, which plays a key role in the maintenance of synapses [12]. The C-terminal fragment of NL-1
interacts with the PDZ domains of PSD-95 in the postsynaptic region of neurons; NMDA receptors also interact with PSD-95. Ab peptides change their conguration from a native
form (Ab black, circle yellow symbol) to a b-sheet rich conformation that is able to aggregate (Ab red globular symbol). (B). These misfolded Ab peptides interact with the soluble Nterminal extracellular fragment of NL-1 at excitatory synapses, where a local oligomerization of the peptide begins. At this time, the accumulation of Ab oligomers will trigger
synaptic impairment in the postsynaptic region, with a decrease in PSD-95 and NMDA receptors at the neuronal cell surface. (For interpretation of the references to colour in this
gure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107

M.C. Dinamarca et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6

therapeutic drugs that can block the interaction of Abo with the a/
b-hydrolase fold domain of NL-1, decreasing the likelihood of
triggering the postsynaptic neurodegeneration observed in AD.
Acknowledgements
This work was supported by grants from the Basal Center of
Excellence in Aging and Regeneration (CONICYT-PFB 12/ 2007). We
thank the Sociedad Qumica y Minera de Chile (SQM) for grants on
the Role of Potassium in Hypertension and Cognition and The
Effect of Lithium on Human Health to the CARE Biomedical
Research Center. The graphic work was carried out using
Graphique-Science (http://graphiquescience.blogspot.com).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2015.08.107.
References
[1] D. Selkoe, E. Mandelkow, D. Holtzman, Deciphering Alzheimer disease, Cold
Spring Harb. Perspect. Med. 2 (2012) a011460.
[2] A. Serrano-Pozo, M.P. Frosch, E. Masliah, B.T. Hyman, Neuropathological alterations in Alzheimer disease, Cold Spring Harb. Perspect. Med. 1 (2011)
a006189.
[3] M. Buttini, E. Masliah, R. Barbour, et al., Games, b-Amyloid immunotherapy
prevents synaptic degeneration in a mouse model of Alzheimer's disease,
J. Neurosci. 25 (2005) 9096e9101.
[4] L. Alonso-Nanclares, P. Merino-Serrais, S. Gonzalez, J. DeFelipe, Synaptic
changes in the dentate gyrus of APP/PS1 transgenic mice revealed by electron
microscopy, J. Neuropathol. Exp. Neurol. 72 (2013) 386e395.
[5] K. Wilcox, P.N. Lacor, J. Pitt, W.L. Klein, Ab Oligomer-induced synapse
degeneration in Alzheimer's disease, Cell Mol. Neurobiol. 31 (2011) 939e948.
[6] C. Haass, D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessons
from the Alzheimer's amyloid b-peptide, Nat. Rev. Mol. Cell Biol. 8 (2007)
101e112.
[7] W. Wei, L.N. Nguyen, H.W. Kessels, et al., Amyloid b from axons and dendrites
reduces local spine number and plasticity, Nat. Neurosci. 13 (2010) 190e196.
[8] M.C. Dinamarca, J. Rios, N.C. Inestrosa, Postsynaptic receptors for amyloid-b
oligomers as mediators of neuronal damage in Alzheimer's disease, Front.
Physiol. 3 (2012) 1e7.
[9] E.M. Snyder, Y. Nong, C.G. Almeida, et al., Regulation of NMDA receptor trafcking by amyloid-b, Nat. Neurosci. 8 (2005) 1051e1058.
[10] F.G. De Felice, P.T. Velasco, M.P. Lambert, et al., Ab oligomers induce neuronal
oxidative stress through an N-methyl-D-aspartate receptor-dependent
mechanism that is blocked by the Alzheimer drug memantine, J. Biol. Chem.
282 (2007) 11590e11601.
[11] M.C. Dinamarca, D. Weinstein, O. Monasterio, N.C. Inestrosa, The synaptic
protein neuroligin-1 interacts with the amyloid b-peptide. Is there a role in
Alzheimer's disease? Biochemistry 50 (2011) 8127e8137.
[12] D.D. Krueger, L.P. Tuffy, T. Papadopoulus, N. Brose, The role of neurexins and
neuroligins in the formation, maturation, and function of vertebrate synapses,
Curr. Opin. Neurobiol. 22 (2012) 412e422.
[13] F.G. Scholl, P. Scheiffele, Making connections: cholinesterase-domain proteins
in the CNS, Trends Neurosci. 26 (2003) 618e624.

rez, et al., Acetylcholinesterase accelerates

[14] N.C. Inestrosa, A. Alvarez, C.A. Pe
assembly of amyloid-b-peptides into Alzheimer's brils: possible role of the
peripheral site of the enzyme, Neuron 16 (1996) 881e891.
[15] N.C. Inestrosa, M.C. Dinamarca, A. Alvarez, Amyloid-cholinesterase interactions. Implications for Alzheimer's disease, FEBS J. 275 (2008) 625e632.
[16] D. Comoletti, R. Flynn, L.L. Jennings, et al., Characterization of the interaction
of a recombinant soluble neuroligin-1 with neurexin-1b, J. Biol. Chem. 278
(2003) 50497e50505.
[17] R.K. Carlin, D.J. Grab, R.S. Cohen, P.J. Siekevitz, Isolation and characterization of
postsynaptic densities from various brain regions: enrichment of different
types of postsynaptic densities, J. Cell Biol. 86 (1980) 831e843.
[18] F. Gardoni, A. Caputi, M. Cimino, et al., Calcium/calmodulin-dependent protein
kinase II is associated with NR2A/B subunits of NMDA receptor in postsynaptic densities, J. Neurochem. 71 (1998) 1733e1741.
[19] M.C. Dinamarca, W. Cerpa, J. Garrido, J.L. Hancke, N.C. Inestrosa, Hyperforin
prevents b-amyloid neurotoxicity and spatial memory impairments by
disaggregation of Alzheimer's amyloid-b-deposits, Mol. Psychiatry 11 (2006)
1032e1048.
[20] G.G. Faras, I.E. Alfaro, W. Cerpa, et al., Wnt-5a/JNK signaling promotes the
clustering of PSD-95 in hippocampal neurons, J. Biol. Chem. 284 (2009)
15857e15866.
[21] W. Cerpa, G.G. Farias, J.A. Godoy, et al., Wnt- 5a occludes Aboligomer-induced
depression of glutamatergic transmission in hippocampal neurons, Mol.
Neurodegener. 5 (2010) 3.
nicke, M. Mikhaylova, S. Ro
nicke, et al., Early neuronal dysfunction by
[22] R. Ro
amyloid b oligomers depends on activation of NR2B-containing NMDA receptors, Neurobiol. Aging. 32 (2011), 2219e22128.
[23] T.C. Sudhof, Neuroligins and neurexins link synaptic function to cognitive
disease, Nature 455 (2008) 903e911.
[24] J. Koehnke, X. Jin, E.C. Budreck, et al., Crystal structure of the extracellular
cholinesterase-like domain from neuroligin-2, Proc. Natl. Acad. Sci. U. S. A. 105
(2008) 1873e1878.
[25] P. Leone, D. Comoletti, P. Taylor, et al., Structure- function relationships of the
/b-hydrolase fold domain of neuroligin: a comparison with acetylcholinesterase, Chem. Biol. Interact. 187 (2010) 49e55.
[26] G.V. De Ferrari, M.A. Canales, I. Shin, et al., A structural motif of acetylcholinesterase that promotes amyloid b-peptide formation, Biochemistry 40
(2001) 10447e10457.
[27] M. Renner, P.N. Lacor, P.T. Velasco, et al., Deleterious effects of Amyloid b
Oligomers acting as an extracellular scaffold for mGluR5, Neuron 66 (2010)
739e754.
[28] M.H. Magdesian, M.M. CarvalhoM, F.A. Mendes, et al., Amyloid-b binds to the
extracellular cysteine-rich domain of Frizzled and inhibits Wnt/b-catenin
signaling, J. Biol. Chem. 283 (2008) 9359e9368.
[29] A. Hamilton, G.W. Zamponi, S.S. Ferguson, Glutamate receptors function as
scaffolds for the regulation of b-amyloid and cellular prion protein signaling
complexes, Mol. Brain 8 (2015) 18.
[30] J.W. Um, A.C. Kaufman, M. Kostylev, et al., Metabotropic glutamate receptor 5
is a coreceptor for Alzheimer ab oligomer bound to cellular prion protein,
Neuron 79 (2013) 887e902.
[31] A.E. Barry, I. KlyubinI, J.M. Mc Donald, et al., Alzheimer's disease brainderived amyloid-b-mediated inhibition of hLTP in vivo is prevented by
immunotargeting cellular prion protein, J. Neurosci. 31 (2011) 7259e7263.

n, D.A. Gimbel, H.B. Nygaard, et al., Cellular prion protein mediates
[32] J. Laure
impairment of synaptic plasticity by amyloid-b oligomers, Nature 457 (2009)
1128e1132.
[33] C. Balducci, M. Beeg, M. Stravalaci, et al., Synthetic amyloid-b oligomers
impair long-term memory independently of cellular prion protein, Proc. Natl.
Acad. Sci. U. S. A. 107 (2010), 2295e22300.
[34] E.C. Budreck, O.B. Kwon, Jung, et al., Neuroligin-1controls synaptic abundance
of NMDA-type glutamate receptors through extracellular coupling, Proc. Natl.
Acad. Sci. U. S. A. 110 (2013) 725e730.

Please cite this article in press as: M.C. Dinamarca, et al., The soluble extracellular fragment of neuroligin-1 targets Ab oligomers to the
postsynaptic region of excitatory synapses, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/
j.bbrc.2015.08.107