Cleavage of Neuroligin-1
Kunimichi Suzuki,1Yukari Hayashi,1Soichiro Nakahara,2Hiroshi Kumazaki,3Johannes Prox,5Keisuke Horiuchi,6
Mingshuo Zeng,3Shun Tanimura,3Yoshitake Nishiyama,3Satoko Osawa,1Atsuko Sehara-Fujisawa,7Paul Saftig,5
Satoshi Yokoshima,3Tohru Fukuyama,3Norio Matsuki,2Ryuta Koyama,2Taisuke Tomita,1,8,* and Takeshi Iwatsubo1,4,8
1Department of Neuropathology and Neuroscience
2Laboratory of Chemical Pharmacology
3Laboratory of Synthetic Natural Products Chemistry, Graduate School of Pharmaceutical Sciences
4Department of Neuropathology, Graduate School of Medicine
The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan
5Institut fu ¨r Biochemie, Christian-Albrechts-Universita ¨t zu Kiel, D-24098 Kiel, Germany
6Department of Orthopedic Surgery, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
7Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
8Core Research for Evolutional Science and Technology, Japan Science and Technology Agency
Neuroligin (NLG), a postsynaptic adhesion molecule,
is involved in the formation of synapses by binding to
a cognate presynaptic ligand, neurexin. Here we
report that neuroligin-1 (NLG1) undergoes ectodo-
main shedding at the juxtamembrane stalk region
to generate a secreted form of NLG1 and a
membrane-tethered C-terminal fragment (CTF) in
adult rat brains in vivo as well as in neuronal cultures.
Pharmacological and genetic studies identified
ADAM10 as the major protease responsible for
NLG1 shedding, the latter being augmented by
synaptic NMDA receptor activation or interaction
with solubleneurexin ligands. NLG1-CTF was subse-
quently cleaved by presenilin/g-secretase. Secretion
of soluble NLG1 was significantly upregulated under
a prolonged epileptic seizure condition, and inhibi-
tion of NLG1 shedding led to an increase in numbers
of dendritic spines in neuronal cultures. Collectively,
neuronal activity-dependent proteolytic processing
of NLG1 may negatively regulate the remodeling of
spines at excitatory synapses.
Formation and maintenance of the synaptic structure is a dy-
namic process that requires bidirectional interactions between
pre- and postsynaptic components. A diverse assortment of
cell adhesion molecules is present at the synapse and organizes
the synaptic specializations of both excitatory and inhibitory
central synapses (Dalva et al., 2007; Siddiqui and Craig, 2011).
Neuroligin (NLG) is one of the potent synaptogenic adhesion
proteins located at the postsynapse, which transsynaptically
binds to a presynaptic ligand, neurexin (NRX) (Ichtchenko
et al., 1995; Irie et al., 1997; Scheiffele et al., 2000; Su ¨dhof,
2008; Bottos et al., 2011). Mammals express four NLG genes
(i.e., NLG1 to NLG4). NLG polypeptides are type 1 transmem-
brane proteins with a large extracellular domain with homology
to acetylcholinesterases but lack critical residues in the active
site and interact with NRXs at the synaptic membrane surface
(Su ¨dhof, 2008). Notably, NLG1 is localized at glutamatergic
postsynapse, and overexpression of NLG1 induces the accumu-
lation of glutamatergic presynapse and postsynapse molecules
in vitro (Song et al., 1999; Scheiffele et al., 2000; Budreck and
Scheiffele, 2007). In contrast, NLG2 triggers the maturation of
GABAergic synapses, implicating specific functions of different
NLGs in theformation and maturation of different chemical types
of synapses in vitro and in vivo (Graf et al., 2004; Varoqueaux
et al., 2004, 2006).
Recent studies revealed that copy number variation or point
mutation in NLG genes are linked to autism spectrum disorder
(ASD), schizophrenia, or mental retardation (reviewed in Su ¨dhof,
2008). Notably, ASD-linked mutations in NLG genes have been
shown to affect the expression, folding, or dimerization of NLG
proteins to compromise their surface expression and binding
to NRXs (Comoletti et al., 2004; Levinson and El-Husseini,
2007; Zhang et al., 2009). Moreover, copy number variations
that are associated with an increased risk of ASD were identified
in NLG1 locus (Glessner et al., 2009). NLG1 knockout (KO) or
transgenic mice showed synaptic dysfunctions and ASD-like
behaviors (Varoqueaux et al., 2006; Chubykin et al., 2007; Blun-
dell et al., 2010; Dahlhaus et al., 2010). Thus, the levels of NLGs
within the synaptic membranes are presumed to directly modu-
late the synaptic functions in vivo. Although several reports indi-
cated that the surface levels of NLG1 are regulated by synaptic
activities through membrane trafficking (Schapitz et al., 2010;
Thyagarajan and Ting, 2010), the regulatory mechanisms to
control protein levels of NLG remains unclear. Here, we show
that NLG1 is sequentially cleaved by ADAM10 and g-secretase
to release its extra- and intracellular domain fragments, respec-
tively. Proteolytic processing of NLG1 resulted in the elimination
of NLG1 on the cell surface, thereby causing a decrease in the
410 Neuron 76, 410–422, October 18, 2012 ª2012 Elsevier Inc.
synaptogenic activity of NLG1. We further show that ADAM10-
mediatedshedding isregulated inanactivity-dependent manner
through NMDA receptor (NMDAR) activation or by binding to
secreted forms of NRXs. Our present results suggest that
neuronal activity and interaction with NRXs regulate the levels
of NLG1 via proteolytic processing to modulate the adhesion
system as well as the functions of synapses.
Proteolytic Processing of NLGs in Brains and Neuronal
NLGs are synaptogenic type 1 transmembrane proteins that
harbor large extracellular domains (Ichtchenko et al., 1995).
Whilethe levels of NLGsarepresumed tobecorrelated with their
physiological and pathological functions (Varoqueaux et al.,
2006; Chubykin et al., 2007; Glessner et al., 2009; Blundell
et al., 2010; Dahlhaus et al., 2010), little information is available
on the proteolytic mechanism of NLGs. Several lines of evidence
have indicated that a subset of type 1 transmembrane proteins
areprocessed by sequential cleavages by ectodomain shedding
and intramembrane cleavage, the latter being executed by
g-secretase (Beel and Sanders, 2008; Bai and Pfaff, 2011). To
test whether the levels of NLGs are regulated by proteolytic
processing, weanalyzed endogenous NLGpolypeptides in adult
rat brains (Figure 1A). Immunoblot analysis using antibodies that
specifically recognize the cytoplasmic region of NLG1 and NLG2
(see Figure S1 available online) revealed immunopositive bands
at ?20–25 kDa, in addition to full-length (FL) protein that
migrated at ?120 kDa. Because the predicted sizes of the cyto-
the ?20–25 kDapolypeptidesrepresent the membrane-tethered
C-terminal fragment (CTF) of endogenous NLGs. Multiple bands
corresponding to CTFs may represent different posttransla-
tional modifications (e.g., glycosylation, see below). To examine
whether these CTFs are processed by the g-secretase activity,
we incubated the membrane fractions of rat brains at 37?C and
detected the appearance of additional bands that migrate faster
than the CTFs with each NLG. Moreover, addition of DAPT,
a specific g-secretase inhibitor, abolished the generation of
the smaller CTFs, in a similar manner to that observed with a
well-known g-secretase substrate, amyloid precursor protein
(APP). These data suggested that NLG-CTFs are cleaved by
the g-secretase activity to release the intracellular domain
(ICD) (Figure 1B). In parallel with the generation of ICDs, we
observed a significant reduction in NLG1-FL upon incubation,
concomitant with the generation of a smaller NLG1 fragment,
which was detected by an antibody against the extracellular
region of NLG1 (Figure 1C). Generation of this extracellular frag-
mentof NLG1was decreasedbytreatmentwith metalloprotease
inhibitors (i.e., EDTA, TAPI2), supporting the notion that the
extracellular domain of NLG1 is processed by ectodomain
shedding. To test whether this processing occurs at synapses
under a physiological condition, we incubated synaptoneuro-
ulation of purified presynaptic boutons attached to postsynaptic
processes (Villasana et al., 2006; Kim et al., 2010) (Figures 1D
and 1E). After ultracentrifugation after incubation, soluble
NLG1 (sNLG1) as well as NLG1-ICD was detected in the soluble
fraction, which was abolished by coincubation with TAPI2 and
DAPT, respectively. To ascertain that these cleavages occur
in situ in neuronal cultures, we analyzed cell lysates and condi-
tioned media (CM) from mouse cortical primary neuronal
cultures obtained from embryonic day (E) 18 pups by immuno-
blotting and detected the secretion of an ?98 kDa single poly-
peptide in the conditioned media, which migrated at an identical
position to that generated upon incubation of the membrane
fractions, by an antibody against the extracellular domain of
NLG1 (Figures 1F and 1G). This band disappeared by treatment
with metalloprotease inhibitors (i.e., GM6001, TAPI2). These
data suggest that the extracellular domain of NLG1 is shed
by the metalloprotease activity to release sNLG1 into the
conditioned media. Furthermore, DAPT treatment caused the
accumulation of CTFs of NLG1 as well as of NLG2. Notably,
simultaneous administration of DAPT and metalloprotease
inhibitors decreased the accumulation of the CTFs. However,
endogenous NLG-ICD, which was observed upon incubation
of microsomes from brain lysates, was hardly detectable in cell
lysates from cultured primary neurons. This suggests that
NLG-ICD is a highly labile endoproteolytic product. These find-
ings led us to speculate thatNLGs areinitially processed bymet-
alloprotease at the extracellular region to generate sNLG and
membrane-tethered NLG-CTF, the latter being further cleaved
by the g-secretase activity (Figure 1H).
ADAM10 and g-Secretase Are Responsible
for the Proteolytic Processing of NLG1
Next we analyzed the metabolism of NLGs in mouse embryonic
fibroblasts from Psen1?/?/Psen2?/?double knockout mice
(DKO cells), which completely lacks the g-secretase activity
(Herreman et al., 2000). Accumulation of NLG-CTFs was
observed upon the overexpression of hemagglutinin (HA)-
tagged NLGs in DKO cells (Figure 2A). However, the levels of
the accumulated NLG-CTFs were significantly reduced by the
coexpression of human PS1, indicating that g-secretase activity
is responsible for the processing of NLG-CTFs. ADAM10 is
known as a responsible enzyme for ectodomain shedding of
a subset of g-secretase substrates (e.g., Notch, APP, cadherin,
and CD44) at the membrane-proximal region of ectodomain
(Saftig and Reiss, 2011). To test whether ADAM10 is involved
in the processing of NLGs, we overexpressed HA-tagged
NLG1 or NLG2 in murine embryonic fibroblasts (MEFs)
obtained from ADAM10 knockout (Adam10?/?) or heterozygous
(Adam10+/?) mice (Figure 2B) (Hartmann et al., 2002). In
Adam10?/?MEF, the generation of sNLG1 was significantly
reduced. In contrast, no change in NLG1 processing was
observed in MEFs obtained from knockout mice of other
ADAMs (i.e., Adam8?/?, Adam17?/?, Adam19?/?, Adam9?/?;
Adam12?/?;Adam15?/?[TKO]) (Zhou et al., 2004; Weskamp
et al., 2006; Kawaguchi et al., 2007; Horiuchi et al., 2007). These
data strongly suggest that ADAM10 is a responsible enzyme for
the shedding of NLG1. Intriguingly, the level of soluble NLG2
secreted fromAdam10?/?MEFwasalmost comparable tothose
from other ADAM knockout MEFs, suggesting that ADAM10
specifically cleaves NLG1 but not NLG2. These data suggest
Proteolytic Regulation of Neuroligin-1
Neuron 76, 410–422, October 18, 2012 ª2012 Elsevier Inc. 411
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