Journal: Nature Communications

Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease

doi: 10.1038/ncomms9836

Figure Lengend Snippet: ( 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 Aβ 1-42 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 Aβ 1-42 . 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 Aβ 1-42 , NCAM2-ED or NCAM2mAb. Note increased numbers of filopodia and lamellipodia in neurons treated with Aβ 1-42 , 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 ).

Article Snippet: To confirm that Aβ 1-42 associates with NCAM2, NCAM2 immunoprecipitates were also analysed with the rabbit monoclonal antibody (D3E10, Cell Signaling) recognizing Aβ 1-42 , but not full-length APP or other Aβ species.

Techniques: Cell Culture, Incubation, Recombinant, Comparison, Control