Fig 2: CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 µm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with >). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 µm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 µm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with >). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 µm.

Fig 3: The effect on plasma membrane expression of AMPA receptor subunits in hippocampal culture after presynaptic silencing with Bafilomycin (BAF).(A) Control synapses immunolabeled with anti-GluA1 in non-permeabilized culture. (B) As in (A), but treated with Bafilomycin (BAF) before fixation. (C) Control synapses immunolabeled with anti-GluA2 in non-permeabilized culture. (D) As in (C), but treated with Bafilomycin (BAF) before fixation. (E) Quantification of difference in maximal punctate GluA1 labeling intensities between control and Bafilomycin-treated cultures. There is no significant difference. (F) Quantification of difference in maximal punctate GluA2 labeling intensities between control and Bafilomycin-treated cultures. There is a slight, but significant decrease in labeling intensities after Bafilomycin treatment. Asterisk denotes statistical significance (p < 0.001). Scale bar: 10 µm.

Fig 4: 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 Aß1-42 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 Aß1-42. 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 Aß1-42, NCAM2-ED or NCAM2mAb. Note increased numbers of filopodia and lamellipodia in neurons treated with Aß1-42, 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).

Fig 5: a2d-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors(A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), a2d-1, a2d-2, or a2d-3 (n = 4 per group). ***p < 0.001 versus the control vector group.(B) Original blots and quantification show that a2d-1 reduces cell membrane expression of heteromeric GluA1/GluA2 receptors in HEK293 cells. Surface membrane proteins were isolated by biotinylation and were precipitated with an anti-GFP antibody (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the P3 group.(C) Original blots and quantification show that nerve injury increases a2d-1 but reduces GluA2 in spinal cord synaptosomes (n = 5 rats per group). *p < 0.05, ***p < 0.001 versus the sham group.(D and E) Original blots and quantification show that nerve injury reduces heteromeric GluA1/GluA2 receptors in spinal cord synaptosomes (n = 5 rats per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group.Mann-Whitney U test was conducted in (A)–(E).