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. 2017 May 5;292(18):7327-7337.
doi: 10.1074/jbc.M116.761189. Epub 2017 Mar 10.

Interaction of Amyloid-β (Aβ) Oligomers With Neurexin 2α and Neuroligin 1 Mediates Synapse Damage and Memory Loss in Mice

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Free PMC article

Interaction of Amyloid-β (Aβ) Oligomers With Neurexin 2α and Neuroligin 1 Mediates Synapse Damage and Memory Loss in Mice

Jordano Brito-Moreira et al. J Biol Chem. .
Free PMC article

Abstract

Brain accumulation of the amyloid-β protein (Aβ) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aβ oligomers (AβOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AβOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AβOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AβOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AβO binding to hippocampal neurons and prevented AβO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AβOs in mice. The results indicate that Nrx2α and NL1 are targets of AβOs and that prevention of this interaction reduces the deleterious impact of AβOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AβOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Keywords: Alzheimer disease; amyloid β (Aβ); amyloid β oligomers; brain; memory; neurexin; neuroligin; neuron; synapse.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
AβOs bind Nrxα and NL. A, amino acid sequence alignment reveals significant homology between the IGTVDRS peptide, identified by phage display (see “Results”) and different isoforms of human Nrxs; identical residues are shown in red, and conservative amino acid replacements are shown in blue. B and C, ribbon diagrams of the structures of Nrx1α (B) and the Nrx1β LNS6 domain (C, left) that interacts with NL1 (C, right). The Nrx amino acid sequence motif homologous to the IGTVDRS peptide is highlighted in red (B and C). Nrx residues involved in interaction with NL1 are shown in blue (C), and NL1 residues involved in interaction with Nrx are shown in green (C). The epitope recognized by the anti-Nrx2α antibody used in this study appears in yellow in B. The figures were generated using Visual Molecular Dynamics (University of Illinois), and the atomic coordinates for Nrx1α and the Nrx1β/NL1 complex (PDB codes 3POY and 3BIW, respectively). D, binding assay (see “Experimental Procedures”) between plate-immobilized IG peptide (2.4 or 24 μm, as indicated) or BSA (150 μm, used as a negative control) and AβOs added in soluble form to the wells (0.5, 1 or 2 μm, as indicated). The detected signal was the immunoreactivity toward the anti-Aβ oligomer NU4 antibody (30). E, optical density quantification of the binding assay in D. Error bars represent mean ± S.D. of three wells for each experimental condition. a.u., arbitrary units. F, plate binding assay between immobilized purified recombinant Nrx1α (60 μm) or NL1 (150 μm) and AβOs (0.9 μm) added in soluble form to the wells. The figure shows representative experiments from two to four independent experiments each. G, plate binding assay between immobilized purified recombinant Nrx2α (3 nmol), NL1, NL2, or NL3 (150 μm) and AβOs (0.9 μm) added in soluble form to the wells. Veh, vehicle. H, ligand capture assay (see “Experimental Procedures”) between plate-immobilized AβOs and proteins present in human cortical homogenate. Ligand detection was performed using anti-NL1, anti-Nrx2α, or anti-GABAAR5 antibodies. The figure illustrates a representative experiment from two independent experiments that yielded similar results. I, plate binding assay between immobilized purified recombinant Nrx2α (60 μm) and NL1 (150 μm), with the latter added in soluble form to the wells. Binding of soluble NL1 to immobilized Nrx2α was evaluated, after appropriate washing, by immunoblotting (IB) using anti-NL1 to detect the complex.
Figure 2.
Figure 2.
Anti-Nrx2α, anti-NL1, and the IG peptide attenuate AβO binding to hippocampal neurons. A–C, representative images of AβO binding (NU4 immunoreactivity) in hippocampal cultures exposed to vehicle (A), AβOs (B, 500 nm), or AβOs previously incubated overnight with the IG peptide at a 5:1 Aβ:IG molar ratio (C). Scale bar = 10 μm. D, integrated AβO immunofluorescence levels (NU4 immunoreactivity, vehicle (Veh) and AβOs, n = 5 experiments with independent neuronal cultures and AβO preparations; Aβ:IG complex, n = 4 independent experiments paired with vehicle- and AβO-treated cultures). E, antibodies against Nrx2α and NL1 do not immunoreact with membrane-immobilized AβOs. AβOs (1.5 pmol) were spotted (in triplicate) onto nitrocellulose, and membrane strips were incubated separately with 1 μg/ml polyclonal anti-AβOs (LDN1), 5 μg/ml anti-NL1, 8 μg/ml anti-Nrx2α, or 10 μg/ml anti-cyclophilin B (Ciclo, irrelevant IgG was used as a negative control) and developed by chemiluminescence. F–I, representative images of AβO binding (NU4 immunoreactivity) in hippocampal cultures exposed for 3 h to vehicle (F), AβOs (G, 500 nm), AβOs + anti-Nrx2α (H), or AβOs + anti-NL1 (I). Antibodies were added to the cultures 30 min prior to AβOs. Scale bar = 10 μm. J, integrated AβO immunofluorescence levels (NU4 immunoreactivity). Anti-Nrx2α results are from five experiments with independent neuronal cultures and AβO preparations (8 μg/ml anti-Nrx2α, n = 3; 16 μg/ml anti-Nrx2α, n = 2). Anti-NL1 (5 μg/ml) results are from three experiments with independent neuronal cultures and AβO preparations. K–N, representative images of AβO binding (LDN1 immunoreactivity) in hippocampal cultures exposed for 3 h to vehicle (K), AβOs (L, 500 nm), AβOs + anti-Nrx2α (M), or AβOs + anti-NL1 (N). Antibodies were added to the cultures 30 min prior to AβOs. Scale bar = 10 μm. O, integrated AβO immunofluorescence levels (LDN1 immunoreactivity) in a representative experiment from two experiments that yielded similar results. Error bars correspond to means ± S.D. from three replicates. P–S, representative images of AβO binding (detected by Alexa-streptavidin binding to biotinylated AβOs) in hippocampal cultures exposed for 3 h to vehicle (P), AβOs (Q, 500 nm), AβOs + anti-Nrx2α (R), or AβOs + anti-NL1 (S). Antibodies were added to the cultures 30 min prior to AβOs. Scale bar = 10 μm. T, integrated Alexa-streptavidin fluorescence levels in a representative experiment from two experiments that yielded similar results. Error bars correspond to means ± S.D. from three replicates. In all experiments, 20–30 images (from two to three coverslips) were acquired and analyzed per experimental condition per independent culture. Symbols correspond to mean values from each independent experiment. ***, p < 0.001; ****, p < 0.0001, one-way ANOVA followed by Holm-Sidak post test.
Figure 3.
Figure 3.
Anti-Nrx2α and anti-NL1 prevent AβO-induced neuronal oxidative stress. A–G, representative DCF fluorescence images from hippocampal neurons exposed for 4 h to vehicle (A and E), AβOs (B and F, 500 nm), AβOs + anti-NL1 (C), or AβOs + anti-Nrx2α (G). Scale bars = 60 μm. D and H, integrated DCF fluorescence (normalized by cell number in each image, as determined from bright-field images of the same fields) obtained from five (for anti-NL1) or three (for anti-Nrx2α) independent experiments with different hippocampal cultures and AβO preparations. Fluorescence was quantified using ImageJ. Symbols correspond to mean values from each independent experiment. *, p < 0.05 compared with vehicle, ANOVA followed by Dunnett post test. Veh, vehicle.
Figure 4.
Figure 4.
Anti-Nrx2α and anti-NL1 prevent AβO-induced dendritic spine loss. A–G, representative images of dendritic spines labeled with phalloidin (green, A–C; red, E–G) in cultured hippocampal neurons exposed for 24 h to vehicle (A and E), AβOs (B and F, 500 nm), AβOs + anti-NL1 (C), or AβOs + anti-Nrx2α (G). Scale bars = 10 μm. D and H, spine density (number of spines per 10 μm of dendritic segment), obtained from three (for anti-NL1) or two (for anti-Nrx2α) experiments with independent neuronal cultures and AβO preparations (20 fields/experimental condition; 3 dendritic segments/field). Symbols correspond to mean values from each independent experiment. *, p < 0.05; ***, p < 0.001, one-way ANOVA followed by Holm-Sidak post test. Veh, vehicle.
Figure 5.
Figure 5.
Anti-Nrx2α and anti-NL1 prevent AβO-induced synapse loss. A–C, representative images of merged synaptophysin (SYP, red) and PSD-95 (green) immunofluorescence in hippocampal neurons exposed for 4 h to vehicle (A), AβOs (B, 500 nm), and AβOs + 5 μg/ml anti-NL1 (C). D, quantification of co-localized synaptophysin/PSD-95 puncta. Veh, vehicle. E–G, representative images of merged synaptotagmin (Stg, green) and PSD-95 (red) immunofluorescence in hippocampal neurons exposed for 4 h to vehicle (E), AβOs (F, 500 nm), and AβOs + 8 μg/ml anti-Nrx2α (G). H, quantification of co-localized synaptotagmin/PSD-95 puncta. When used, anti-Nrx2α and anti-NL1 were added to cultures 30 min prior to AβOs. Ten to fifteen fields were imaged and analyzed per experimental condition from four (for anti-NL1) or three (for anti-Nrx2α) experiments with independent cultures and AβO preparations. Symbols correspond to mean values from each independent experiment. *, p < 0.05; **, p < 0.01, one-way ANOVA followed by Holm-Sidak post test. Scale bars in A–C and E–G indicate 10 μm.
Figure 6.
Figure 6.
Anti-Nrx2α and anti-NL1 prevent memory impairment induced by AβOs in mice. A–E, male Swiss mice received one i.c.v. infusion of 1200 ng of anti-Nrx2α, 800 ng of anti-GABAAR subunit 5 (used as a negative control and indicated as IgG), or 750 ng of anti-NL1 30 min prior to i.c.v. infusion of AβOs (10 pmol), as indicated. Mice were tested in the NOR paradigm 24 h after infusion of AβOs (A–C) or in the OL test 8 days after infusion of AβOs (D and E). A and D show schematics illustrating how the NOR and OL tests are performed, respectively. B and C, the percentage of time spent exploring a familiar (old) or novel (new) object used in the test session of the NOR test. E, the percentage of time spent exploring the non-relocated object in the familiar location or the relocated object in the novel location in the OL test. Error bars represent means ± S.E., and symbols represent individual mice in each experimental group. *, p < 0.05, one-sample Student's t test comparing the mean value for the novel object with the fixed value of 50%. Veh, vehicle.

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