FRMPD4p.Cys618ValfsX8 losses the ability to increase spine density. (A) Cultured hippocampal neurons were transfected with myc–FRMPD4 (WT or FRMPD4p.Cys618ValfsX8) plus EGFP or EGFP alone (control) (DIV 14) and visualized by immunofluorescence staining for EGFP. Images were analyzed by Neurolucida 360 to show different spine shapes. Scale bar, 10 μm. (B) FRMPD4-WT overexpression increased spine density but FRMPD4p.Cys618ValfsX8 did not. Mean ± 95% confidence interval (n = 19 neurons for EGFP, 18 for WT, and 20 for mutant, *P < 0.05, t-test). (C–F) Spines were classified into four categories. FRMPD4-WT overexpression increased mushroom spines but not thin spines, stubby spines and filopodia spines. FRMPD4p.Cys618ValfsX8 did not increase mushroom or other spine categories. Mean ± 95% confidence interval (n = 19 neurons for EGFP, 18 for WT, and 20 for mutant, *P < 0.05, t-test).