Transcript Nonneurolnal cells engineered to express neuroligins

Neurexin mediates the assembly of
presynaptic terminals
Camin Dean1, 3, Francisco G Scholl2, 3, Jenny Choih1, Shannon
DeMaria1, James Berger1, Ehud Isacoff1 & Peter Scheiffele2
07/2003
At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic
membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic
membranes into synaptic junctions are unknown.
Neurexins are neuronal cell surface proteins that exhibit a high degree of diversity. 3 genes for neurexins
have been described, each of which is transcribed from two promoters. This results in six principal
neurexin transcripts that are subject to extensive alternative splicing, generating a family of thousands
of differentially expressed proteins.
Neuroligin-1 was originally isolated as a splice-specific ligand of beta-neurexins by affinity
chromotography. Neuroligins and neurexins bind to each other and form asymmetric intercellular
junctions.
Nonneurolnal cells engineered to express neuroligins induce accumulation of
synapsin in contacting axons
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Model system: by culturing explants of
neonatal pontine tissue, “purified”
presynaptic axons can be prepared and then
paired in vitro with a purified preparation of
their in vivo targets, cerebellar granule cells.
Upon contact with granule cells, clusteing of
synaptic vesicles is observed within pontine
axons(J-L). No such synapsin concentation is
observed when pontine axons contact
nonneuronal cells such as GFP-expressing
HEK293 cells (A-C).
In contrast, when HEK293 cells were
transfected with a construct encoding
epitope-tagged neuroligin- 1ab or neuroligin2, accumulation of synapsin were
observed(D-I; G-I)
Neuroligin-expressing cells induce the formation of presynaptic structures
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A: contact between pontine axons and a
HEK293 cell transfected with an HA-tagged
neuroligin-1ab expression construct. On the
HEK293 cell surface, 15nm gold
particles(used to label transfected cells) are
marked by arrowheads.Two junctional areas
with electron dense material(*), synaptic
vesicles profiles (sv), a mitohondrion in the
axon (M), and the nucleus of the HEK293
cells (N) are labeled.
B: similar view of a contact between pontine
axons and mock-transfected HEK293 cells.
C-G:serial sections of the contact in A.
H: several axonal processes growing over a
single neuroligin-expressing HEK293 cell.
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In an initial screen (see
Supplementary Fig. 1 online), the
authors generated chimeric proteins
between the neuroligin ectodomain
and its structural homologue
acetylcholinesterase (AChE). Using
this approach, they identified three
sequence elements that are
essential for neuroligin function:
one located in the center of the
domain (mutated in NLG/AChE-2)
and two within the carboxy (C)terminal portion of the domain
(NLG/AChE-4 and NLG/AChE-6)
Synaptic vesicles undergo turnover at contacts formed with neuroligin-transfected
HEK293 cells
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One salient fature of synaptic function is the regulated exocytosis of vesicles in response to depolarization of the presynaptic
membrane, followed by a reinternalization of synaptic vesicle membrane.
Co-cultures of transfected HEK293 cells and pontine explants were briefly (5min) incubated in depolarizing solution with an antibody
raised against luminal domain of synaprotagmin(A-E). Captured synaptotagmin antibodies, representing synaptic vesicles that had
undergone exocytosis, were visualized with secondary antibodies(B)
Large synaptotagmin-positive axonal structures not in contact with neuroligin-expressing cells showed only little vesicle turnover(D,
arrows). Vesicle turnover was quantified: confocal scans were performed and for recognizable spots on target, the average pixel
intensity in a rectangle of 5x5mkm was calculated for the Cy3 and Cy5 channels, representing the staining for internalized
synaptotagmin luminal domain antibodies and total synaptotagmin detected by cytoplasmic domain antibodies.
CASK-Lin-2, member of MAGUK family of proteins, localizes to synapses and interacts with neurexin. Using triple staining and
serial z-sectioning in a confocal microscope, CASK/Lin-2 was found colocalized with the presynapric vesicle clusters in pontine
axons, contacing with neuroligin-expressing cells.
Neuroligin activity is inhibited by soluble beta-neurexin
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Soluble beta-neurexin was added to a
coculture of pontine explants and their in
vivo target - cerebellar granule cells. Addition
of nrx_Fc at a concentration of 50 mkg/ml
reduced neuroligin-induced vesicle-clustering
in axons by 45%(D-E), whereas a control IgG
had no significant effect (A-C).
Endogenous neurexins are concentrated at synapses
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Pan-neurexin antibodies:
Antibodies against the cytoplasmic tail of
neurexin-1. Due to sequence similarities
between neurexin family members, this
antiserum is likely to recognize the alpha and
beta isoforms ofneurexin-1, neurexin-2 and
neurexin-, but not proteins in the
neurexiniV/CASPR/paranoidin family.
In immunostained dissociated cultures of
cerebellar granule cells and hippocampal
neurons, the the antiserum revealed a
punctate distribution of the protein that
showed extensive overlap with the synaptic
vesicle marker synaptobrevin, demonstrating
that endogenous neurexins are indeed
concentrated at synapses (e–g) .In isolated
axons emerging from pontine explants
neurexins were strongly enriched in growth
cones, appropriately localized to mediate
initial interactions with neuroligins in the
postsynaptic target cell ( b–d). In vivo,
pontine mossy fiber axons form synapses
with cerebellar granule cells in the inner
granular layer (IGL) of the cerebellar cortex.
Neurexin was detected in the same regions of
the IGL as the synaptic vesicle marker
synaptobrevin (h–j), consistent with an
enrichment at mossy fiber–granule cell
synapses.
Recrutement of neurexin to neuroligin-induced synapses
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To observe neurexin recruitment to newly forming synapses, the authors transfected
dissociated cerebellar granule cells with a neuroligin-1 expression vector before
synapses were formed in culture (at the day of plating). Neuroligin overexpression
increased the number of synaptic vesicle clusters formed fivefold, and there was a strong
accumulation of endogenous neurexin in neuronal processes at sites of contact with the
neuroligin-expressing cells (Fig. 5). Confocal imaging revealed that neuroligin-1 was
concentrated at these immature synaptic contacts and that synaptic vesicles and neurexin
colocalized at points of contact with neuroligin-expressing cells (Fig. 5e–h)
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In hippocampal cultures that had been
maintained in vitro for 12 d,
endogenous neuroligin-1 was strongly
concentrated at synapses
(Supplementary Fig. 3).
(a-c) Hippocampal neurons were
immunostained with antibodies against
neuroligin-1 (a, green in overlay) and
antibodies against the synaptic vesicle
marker synaptophysin (b, red in
overlay). (d-f)
Hippocampal neurons
expressing EGFP. Cells were
transfected after 12 days in vitro,
maintained for two days and
immunostained with antibodies against
synapsin (e, red in overlay). EGFP
fluorescence is shown in (d, green in
overlay). (g-i)
Hippocampal neurons
expressing HA-tagged neuroligin-1.
Cells were transfected after 12 days in
vitro, maintained for two days and
immunostained with antibodies against
the HA-tag to detect neuroligin (green)
and antibodies against PSD-95 (red).
Conclusion: neuroligin-1 stimulates pre- and
postsynaptic differentiation and can recruit
neurexins to newly forming synapses.
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To test whether neuroligin–neurexin
interactions are sufficient to induce synaptic
differentiation in the absence of other
postsynaptic factors, the authors purified the
AChE-homologous ectodomain of neuroligin1 attached to a glycosylphosphatidylinositol
(GPI) anchor. The recombinant protein was
reconstituted into liposomes that were coated
onto 5-m silica beads. This approach allows
presentation of the functional domain of
neuroligin-1 on a surface with dimensions
approximating a neuronal dendrite, in a freely
diffusible form (Supplementary Fig. 4 online;
When these neuroligin-coated beads were
applied to hippocampal neurons in culture,
we observed clustering of neurexin and
synaptic vesicle proteins at the bead contact
sites (Fig. 6a–d). Synaptic vesicles that
accumulated at these sites showed
depolarization-induced turnover, as indicated
by uptake of an antibody directed against a
luminal epitope of synaptotagmin (Fig. 6e–g).
These findings show that the AChEhomologous domain of neuroligin-1, as a
purified protein, is sufficient to induce the
formation of functional synaptic vesicle
release sites in axons.
Lower panel:Characterization of neuroliginGPI-coated silica beads. (a) DIC image of
uncoated silica beads.(b) lipid-coated silica
beads labeled with Texas Red-conjugated
dipalmitol-glycerol that was incorporated into
the lipid bilayers visualised by fluorescence
microscopy.
© silica beads coated with bilayers containing HA-tagged GPIneuroligin, immunostained with anti-HA antibodies.
(D-f)control beads coated with lipid bilayers containing Texas Redconjugated dipalmitol-glycerol (d, red in overlay) were added to 10
DIV hippocampal cultures for 24 hours and were immunostained for
the synaptic marker synapsin (e, green in overlay)
Hypothetical model for induction of presynaptic differentiation by
lateral clustering of neuroligin/neurexin
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a) Oligomers of neuroligin-1 (red) in the
postsynaptic membrane recruit multiple
neurexins (blue) in the presynaptic
membrane, resulting in lateral clustering
of neurexins. The cytoplasmic tails of the
clustered neurexin proteins recruit
scaffolding and signaling molecules,
possibly via the PDZ-binding motif at the
C-terminus. This scaffold may then signal
the assembly of the exocytotic machinery
and recruit additional neurexins and
thereby neuroligins to form an expanding
contact zone. Postsynaptic neuroligin-1
oligomers may contribute to the assembly
of the postsynaptic specializations by
interaction with signaling and/or
scaffolding proteins such as PSD-95, which
also binds to glutamate receptors
The most important findings:
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overexpression of neuroligin stimulates pre- and postsynaptic differentiation in cultured
hippocampal neurons, suggesting that neuroligin is a limiting component of the
postsynaptic machinery involved in synapse formation.
Neuroligin activity depends on its interaction with neurexins.
Endogenous neurexins are concentrated in synaptic terminals,
Postsynaptic multimers of neuroligin-1 are sufficient to trigger the recruitment of
neurexin to newly forming synaptic sites and
Clustering of neurexin induces recruitment of synaptic vesicles.
Recently, it has been found that novel neuroligin, neuroligin - 4, is expressed in human
heart. Neuroligin 4 has 63-73% amino acid identity with the other members of the
human neuroligin family, and the same predicted domain structure.
Mouse monoclonal antibodies against rat neuroligins 1 and recognaze neuroligin- 4, and
they were used to detect neuroligin - 4 overexpressed in COS-7 cells.
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Confocal immunofluorescence analysis of
erbB receptor distributions at adult rat
gastrocnemius NMJ. En face (A1, D1, G1)
and side view (B1, E1, H1) images of NMJs
stained with Bodipy-Btx. The NMJs were
double-labeled with antibodies against erbB2
(A2, B2), erbB3 (D2, E2), and erbB4 (G2,
H2) and visualized using a Cy3-conjugated
secondary antibody. Color overlays are
shown with -Btx in green and the respective
antibody in red (A3-H3). Schematics of these
distributions are shown in C, F, and I. ErbB2
is enriched in the secondary folds. ErbB3 is
enriched in the Schwann cell. A gap between
the primary gutter and the erbB3
immunoreactivity can be seen in E3 (arrow).
ErbB4 is enriched in the secondary folds.
Scale bar, 10 µm.