Valosin-containing protein and neurofibromin interact to regulate dendritic spine density
Hsiao-Fang Wang, … , Ming-Jen Lee, Yi-Ping Hsueh
Hsiao-Fang Wang, … , Ming-Jen Lee, Yi-Ping Hsueh
Published November 21, 2011
Citation Information: J Clin Invest. 2011;121(12):4820-4837. https://doi.org/10.1172/JCI45677.
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Research Article

Valosin-containing protein and neurofibromin interact to regulate dendritic spine density

Abstract

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder characterized by progressive myopathy that is often accompanied by bone weakening and/or frontotemporal dementia. Although it is known to be caused by mutations in the gene encoding valosin-containing protein (VCP), the underlying disease mechanism remains elusive. Like IBMPFD, neurofibromatosis type 1 (NF1) is an autosomal dominant disorder. Neurofibromin, the protein encoded by the NF1 gene, has been shown to regulate synaptogenesis. Here, we show that neurofibromin and VCP interact and work together to control the density of dendritic spines. Certain mutations identified in IBMPFD and NF1 patients reduced the interaction between VCP and neurofibromin and impaired spinogenesis. The functions of neurofibromin and VCP in spinogenesis were shown to correlate with the learning disability and dementia phenotypes seen in patients with IBMPFD. Consistent with the previous finding that treatment with a statin rescues behavioral defects in Nf1+/– mice and providing further support for our hypothesis that there is crosstalk between neurofibromin and VCP, statin exposure neutralized the effect of VCP knockdown on spinogenesis in cultured hippocampal neurons. The data presented here demonstrate that there is a link between IBMPFD and NF1 and indicate a role for VCP in synapse formation.

Authors

Hsiao-Fang Wang, Yu-Tzu Shih, Chiung-Ya Chen, Hsu-Wen Chao, Ming-Jen Lee, Yi-Ping Hsueh

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Figure 7

IBMPFD mutations reduce the interaction of neurofibromin and VCP and the density of dendritic spines.

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IBMPFD mutations reduce the interaction of neurofibromin and VCP and the...
(A) LRD and (B) neurofibromin interact weakly with IBMPFD mutants. Variant Myc-tagged VCP constructs and vector control were cotransfected with HA-tagged LRD or singly transfected into HEK293T cells. One day later, (A) Myc and (B) neurofibromin antibodies were used for immunoprecipitation. Immunoblotting analysis was performed using the antibodies as indicated. The relative intensities to WT lane are shown. Distribution of Myc-tagged VCP and IBMPFD mutants in (C) HEK293T cells and (H) cultured hippocampal neurons was examined by immunostaining using Myc and GFP antibodies. Cotransfection with GFP was performed to outline cell morphology. Expression of (D) IBMPFD mutants, (E and F) VCP K524A mutant, and (G) VCP miRNA construct did not obviously influence the total protein level of neurofibromin. HEK293T cells were transfected with variant VCP constructs, VCP miRNA, or vector control. Immunoblotting was then performed to examine the protein levels of neurofibromin. CASK and/or actin were used as internal controls. In E, F, and G, MG132 was added to cultures 4 hours before harvest or omitted. Ubiquitin antibody FK2 (αUb) was also included in immunoblotting analysis. For F and G, immunoprecipitation was performed using neurofibromin antibody to precipitate endogenous neurofibromin. Immunoblotting using FK2 antibody did not reveal obvious signals in the neurofibromin precipitates. (I) IBMPFD mutants decrease spine density. Cultured hippocampal neurons were cotransfected with GFP-actin and vector control, WT VCP, or IBMPFD mutants (R95G and R155H) at 12 DIV. Cells were fixed at 18 DIV and stained with Myc and GFP antibodies. The morphology of spines was then analyzed based on the GFP signals. (J) Protrusion density. Values are presented as mean plus SEM. (K) Cumulative probability distributions. More than 34 neurons and 146 dendrites for each group collected from 2 independent experiments were analyzed. Scale bars: (C) 10 μm; (H) upper panels, 10 μm; lower panels, 5 μm; (I) 2 μm. In K, P < 0.05, R155H versus control; P < 0.01, R95G versus WT; P < 0.001, R95G versus control. *P < 0.05, ***P < 0.001.