Fig. 5

Regulation of dendritic spine formation by DGCR2-NRXN1 interaction. A DGCR2 promotes NRXN1β–NLGN1 interaction through its ECD. HEK 293T cells were co-transfected with NRXN1β-myc/CFP, NLGN1-YFP and FLAG-hDGCR2, ∆ECD or ∆ICD. Lysates were immunoprecipitated with anti-myc antibody and Protein A/G agarose, and resulting complexes were blotted with anti-NLGN1, anti-myc and anti-FLAG antibodies. Inputs were blotted for perspective proteins as control. Actin served as a loading control. IgG-HC, IgG heavy chain; IgG-LC, IgG light chain; NS, non-specific band. B HEK 293T cells were co-transfected with NRXN1β-myc/CFP, NLGN1-YFP, and an increasing amount of FLAG-hDGCR2. Lysates were immunoprecipitated with anti-myc antibody and Protein A/G agarose, and resulting complexes were blotted with anti-NLGN1, anti-myc and anti-FLAG antibodies. Inputs were blotted for perspective proteins as control. Actin served as a loading control. C-D ΔECD mutant of DGCR2 cannot rescue reduced spine density caused by DGCR2 knockdown. Primary hippocampal neurons were transfected at DIV 9 with indicated constructs and fixed for staining at DIV 16. Representative images of neuronal morphology (upper panel) and dendrite spines (lower panel) of cultured hippocampal neurons. Scale bars as indicated. Spine density (per 10 μm) quantitative analysis of data in C (D). n = 18 neurons for each condition. ** p < 0.005, *** p < 0.001. n. s., p > 0.05, One-way ANOVA. E-F DGCR2-ECD treatment impairs spine development. HEK 293T cells were transfected with secreted FLAG-hDGCR2-ECD or its mock construct. The conditional medium (CM) of transfected HEK 293T cells was collected and added to the primary hippocampal neurons at DIV 9 ~ 10 for 3 ~ 4 days. Representative images of neuronal morphology (upper panel) and dendrite spines (lower panel) of cultured hippocampal neurons (E). Scale bars as indicated. Spine density (per 10 μm) quantitative analysis of data in E (F). n = 10 neurons for each condition. *** p < 0.001