Fig 2: Levels of NCAM2 are reduced in synaptosomes of cultured hippocampal neurons treated with Aβ1-42 oligomers.(a) Western blot analysis of homogenates and synaptosomes isolated from cultured hippocampal or cortical neurons either mock treated or incubated with Aβ1-42 oligomers for 24 h. Blots were probed with antibodies against NCAM2 and synaptophysin used as a loading control. Note that NCAM2 is enriched in synaptosomes versus homogenates in hippocampal neurons and this enrichment is reduced in hippocampal neurons treated with Aβ1-42. Levels of NCAM2 in synaptosomes from cortical neurons are not reduced in response to Aβ1-42. NCAM2 levels are increased in homogenates of neurons treated with Aβ1-42 versus mock-treated neurons. Graphs show mean+s.e.m. fold change in NCAM2 levels in cell culture homogenates with levels in mock-treated neurons set to 1 (left panels), and mean+s.e.m. of the synaptic enrichment of NCAM2 defined as a ratio of NCAM2 levels in synaptosomes to homogenates (right panels) from n=5 independent experiments. *P<0.05 paired t-test. (b) Western blot analysis of the cell culture media collected from the hippocampal and cortical neurons either mock-treated or incubated with Aβ1-42 oligomers for 24 h. Blots were probed with antibodies against the extracellular domain of NCAM2. Note that levels of the soluble ∼100 kDa extracellular fragments of NCAM2 are increased in the culture media from cultured hippocampal neurons treated with Aβ1-42. Graphs show quantification of the blots (mean+s.e.m., n=3 independent experiments for hippocampal neurons, n=6 independent experiments for cortical neurons) with levels in mock-treated cultures set to 1. *P<0.05 paired t-test. For a,b, full-length versions of the blots are shown in Supplementary Fig. 10.

Fig 3: Cleavage of the membrane-adjacent extracellular fragment of NCAM2 is increased in AD brains.(a) Diagram showing the structure of NCAM2. Ig, immunoglobulin-like domain; Fn, fibronectin type III domain. Peptides used in the cleavage assay and corresponding to aa682-701 (NCAM2aa682-701) and aa666-685 (NCAM2aa666-685) of human NCAM2 are shown below. Asparagine 689 and aspartic acid 693 exchanged to alanine in the cleavage assay shown in c are highlighted in bold. (b) Scheme of the peptide cleavage assay. Peptides labelled with FITC and biotin at the N- and C-terminus, respectively, were incubated either with the total brain lysate or lysate of synaptosomes. Non-cleaved peptides and biotin-containing fragments of the cleaved peptides were removed using streptavidin-coated beads. The remaining FITC fluorescence was used as an estimate of peptide cleavage. (c) Graphs show the efficiency of the peptide cleavage (mean+s.e.m.) with the fluorescence signals for NCAM2aa682-701 in controls set to 100%. Lysates from eight controls and eight AD patients were analysed. Note that the efficiency of NCAM2aa682-701 cleavage is higher in AD cases and particularly in synaptosomes. NCAM2aa682-701 cleavage is reduced by mutating aspartic acid 693. *P<0.05 (compared as indicated), ^P<0.05 (compared with NCAM2aa682-701 cleavage in AD) analysis of variance with Tukey's multiple comparisons test.

Fig 4: NCAM2 accumulates in excitatory synapses of cultured hippocampal neurons.(a) Low-magnification image of a cultured hippocampal neuron labelled by indirect immunofluorescence with antibodies against NCAM2, synaptophysin and MAP2. Note expression of NCAM2 along MAP2 positive dendrites. NCAM2 is also expressed in astrocytes (marked a) which are present in these cultures. Scale bar, 20 μm. (b) High-magnification image of dendrites of neurons co-labelled with antibodies against NCAM2, synaptophysin and MAP2. Arrows show clusters of NCAM2 partially overlapping with synaptophysin accumulations. NCAM2-negative synapses are also observed (arrowheads). Scale bar, 10 μm. (c) High-magnification image of a dendrite of a cultured hippocampal neuron labelled with antibodies against NCAM2, synaptophysin and PSD95. NCAM2 clusters partially overlap with accumulations of PSD95 and synaptophysin (arrows). Scale bar, 10 μm. Three-dimensional analysis of the co-localization within the outlined area is on the right. Z-stack has been acquired with 0.15 μm steps. The xz and yz sections along the dashed lines on the xy image are shown. Note co-localization of the NCAM2 cluster with synaptic markers. (d) Negative control, that is, labelling performed without primary antibodies, is shown. Scale bar, 10 μm.

Fig 5: Disruption of NCAM2 functions at the neuronal cell surface promotes glutamatergic synapse disassembly.(a–e) Cultured hippocampal neurons were either mock-treated or incubated with the recombinant soluble extracellular domains of NCAM2 (NCAM2-ED), antibodies against the extracellular domain of NCAM2 (NCAM2mAb), or Aβ1-42 oligomers. In a,b, neurons were labelled with antibodies against the extracellular domain of GluR1 before permeabilization of membranes with detergent, and co-labelled with antibodies against synaptophysin after permeabilization of membranes with detergent. Representative images of dendrites are shown (a). Note co-localization of cell surface GluR1 accumulations with synaptophysin clusters in mock-treated neurons, and increased levels of non-synaptic cell surface GluR1 accumulations in neurons treated with NCAM2-ED, NCAM2mAb or Aβ1-42. Graphs (b) show the percentage of synaptic and non-synaptic GluR1 clusters relative to total number of GluR1 clusters along dendrites and numbers of synaptophysin accumulations per dendrite length (mean+s.e.m.). *P<0.0001 (analysis of variance with Dunnett's multiple comparison test, n>80 dendrites from 20 neurons were analysed in each group). In c, neurons were labelled with antibodies against the extracellular domain of NR1 before permeabilization of membranes with detergent, and co-labelled with antibodies against synaptophysin after permeabilization of membranes with detergent. Graphs show the percentage of synaptic and non-synaptic NR1 clusters relative to total number of NR1 clusters along dendrites (mean+s.e.m.). *P<0.0001 (analysis of variance with Dunnett's multiple comparison test, n>85 dendrites from 20 neurons were analysed). In d,e, neurons were co-labelled with fluorescent phalloidin and synaptophysin antibodies. Representative images of dendrites are shown in d. Note higher labelling intensity and co-localization with synaptophysin of the phalloidin-labelled polymerized actin accumulations in control neurons versus neurons treated with Aβ1-42, NCAM2-ED or NCAM2mAb. Note increased numbers of filopodia and lamellipodia in neurons treated with Aβ1-42, NCAM2-ED or NCAM2 mAb. Graphs (e) show ratio of the dendrite area-to-length and phalloidin labelling intensity of dendrites of neurons. Mean values+s.e.m. are shown. *P<0.0001 (analysis of variance with Dunnett's multiple comparison test, n=50 dendrites from 20 neurons were analysed in each group). Scale bar, 10 μm (in a,d).