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

• 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 neuroligin-2, accumulation of synapsin were observed(D-I; G-I)

Neuroligin-expressing cells induce the formation of presynaptic structures

• 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.

• 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-transfectedHEK293 cells

• 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

• 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

• 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

• 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)

• 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.

• 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 neuroligin-1 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 AChE-homologous 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 neuroligin-GPI-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 GPI-neuroligin, immunostained with anti-HA antibodies.(D-f)control beads coated with lipid bilayers containing Texas Red-conjugated 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

• 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:• 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.

• 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.