Figure 5. Overexpression of the VCP-binding domain of neurofibromin reduces the number of dendritic spines.

(A) Mutations in the LRD region of the NF1 gene identified from NF1 patients. Locations of mutation sites are indicated by asterisks. (B) Cotransfection of GFP-actin and vector control, HA-tagged WT LRD, or LRD mutants (Y1587Δ and C1909R) into cultured hippocampal neurons at 12 DIV. Six days later, immunostaining was performed using GFP and HA tag antibodies. Only GFP signals are shown. Scale bar: 5 μm. (C) Cumulative probability distributions and (D) graph of protrusion densities. More than 52 neurons and 224 dendrites collected from 2 independent experiments were analyzed. In C, P < 0.01, C1909R versus control and versus Y1587Δ; P < 0.001, C1909R, Y1587Δ, and control versus WT. ***P < 0.001. Values are presented as mean plus SEM. (E) Myc-tagged D1D2 and (F) Myc-tagged VCP were cotransfected with HA-tagged WT and mutant LRD into HEK293T cells as indicated. One day later, total cell extracts were harvested and immunoprecipitated with a Myc antibody. Immunoblotting analysis was then performed using sequentially HA and Myc tag antibodies.