Dendritic spine density is lower in Nf1+/– mice.
We have previously shown that neurofibromin regulates dendritic spine formation, particularly the initiation step of spinogenesis, namely dendritic filopodia formation, in cultured hippocampal neurons. Because it was shown earlier that the loss of an Nf1
allele results in cognitive deficits and impaired formation of barrel cortex (10
), we examined spine density in Nf1+/–
mice using Golgi staining to evaluate the effect of Nf1
haploinsufficiency on neuronal morphology in vivo. Compared with WT littermates, the spine densities of CA1 pyramidal neurons in Nf1+/–
mice were significantly lower, irrespective of whether first-, second-, or higher-order branches of apical dendrites were analyzed (Figure ). These results support a role for the Nf1
gene in the regulation of dendritic spine density in mouse brain.
The density of dendritic spines is reduced in Nf1+/– heterozygous mice.
Neurofibromin interacts with VCP.
To further explore the function of neurofibromin, a neurofibromin antibody was employed to identify the neurofibromin-associated proteins by co-immunoprecipitation from rat brain extracts. The precipitates were then separated by 2D gel electrophoresis and analyzed by mass spectrometry. In comparison with the control IgG, the most robust protein precipitated by neurofibromin antibody appeared as a spot at approximately 90 kDa on the 2D gel (Supplemental Figure 1A; supplemental material available online with this article; doi:
) and was identified as VCP using both MALDI-TOF (Supplemental Figure 1B) and MS/MS analyses (Supplemental Figure 1C). In the MALDI-TOF analysis, a total of 36 peptides matched rat VCP (coverage of 36%). In the MS/MS analysis, 7 peptides were identical to rat VCP protein. The interaction between neurofibromin and VCP was specific, because control IgG did not precipitate VCP (Supplemental Figure 1A). In addition to VCP, p47, a cofactor of VCP, was also present in the precipitate of neurofibromin antibodies (Supplemental Figure 1, A and B), suggesting that neurofibromin forms a complex with VCP and p47. Other protein spots on the 2D gel were also analyzed by MALDI-TOF. However, none of the promising protein candidates were identified. Since VCP was the most promising protein identified from the 2D gel, we focused on VCP in the current study.
To confirm the interaction between neurofibromin and VCP, we performed co-immunoprecipitation-immunoblotting assays using adult rat brain extracts. The result showed that VCP was coprecipitated by neurofibromin antibody (Figure A). The reciprocal immunoprecipitation also showed the precipitation of neurofibromin by VCP antibody from rat brain extracts (Figure A). Non-immune control IgG was used again as negative control to ensure the specificity of co-immunoprecipitation (Figure A). Because neurofibromin and VCP are not neuron-specific proteins, we also examined the interaction between neurofibromin and VCP in non-neuronal cells. Similar to the results obtained with rat brain extracts, neurofibromin antibody precipitated endogenous neurofibromin as well as endogenous VCP from HEK293T cell extract (see below). These immunoblot analyses were consistent with the results obtained by mass spectrometric analyses showing that neurofibromin associates with VCP.
VCP interacts with neurofibromin.
The presence of p47 in the neurofibromin protein complex was also confirmed by co-immunoprecipitation. Myc-tagged VCP and Myc-tagged p47 were cotransfected into HEK293T cells. The presence of VCP and p47 in the immunocomplex of neurofibromin can then be examined simultaneously by using a Myc tag antibody for immunoblotting. Indeed, both VCP and p47 were precipitated by neurofibromin antibody (Figure B), supporting the association of p47 with the neurofibromin protein complex.
The C-terminal D1D2 region of VCP is required for the interaction with the LRD region of neurofibromin.
To delineate the binding domains of VCP involved in the association with neurofibromin, a series of Myc-tagged constructs containing different domains of VCP (Figure C) was expressed in HEK293T cells and immunoprecipitated using neurofibromin antibody. Only full-length VCP and the construct encompassing the D1 and D2 domains of VCP interacted with endogenous neurofibromin (Figure C), suggesting that the C-terminal D1 and D2 regions but not the N-terminal region of VCP are involved in the interaction with neurofibromin.
To identify the VCP-interacting domain of neurofibromin, we divided neurofibromin into 7 fragments. Among them, only 4 fragments expressed soluble proteins (Figure D). These fragments contained the cysteine/serine-rich domain (CSRD), the GAP-related domain (GRD), the leucine-rich repeat domain (LRD), or the C-terminal domain (CTD) (53
). In addition to type I neurofibromin carrying the type I isoform of GRD (GRD1), the alternative splice variant GRD2 was also included in our experiment. These constructs were then tagged with an HA cassette and cotransfected with a Myc-tagged D1D2 construct of VCP. The co-immunoprecipitation experiment conducted with a Myc antibody showed that the LRD of neurofibromin is highly enriched in the precipitates (Figure D), suggesting that the LRD is the interaction site for the D1D2 fragment. We noticed that the CSRD fragment, perhaps due to the high content of cysteine residues in the CSRD, had a low solubility and tended to aggregate. Therefore, the trace amount of this protein detected in the Myc antibody precipitate may correspond to aggregates of oxidized CSRD protein generated during antibody and protein A binding. To check this possibility, we reduced the time of binding with antibody and protein A (from 4 to 3 hours) and increased the concentration of DTT (from 1 mM to 2 mM) to reduce oxidation. Indeed, these modifications effectively removed the CSRD fragment from the immunoprecipitates (Figure E). By contrast, the LRD still associated with the D1D2 fragment (Figure E). These data also support the specific interaction between the LRD and D1D2 constructs.
To further confirm the interaction between the LRD with full-length VCP, we cotransfected HEK293T cells with Myc-tagged VCP and HA-tagged LRD constructs. Similar to the result obtained with the D1D2 fragment, the LRD fragment was coprecipitated with full-length VCP protein using Myc tag antibody (Figure F). Note that in addition to the expected full-length protein at approximately 100 kDa, we frequently detected Myc-tag–immunoreactive protein species with an apparent molecular weight less than 95 kDa in precipitates as well as inputs (Figure , B, C, and F). Smaller protein fragments were also found for the Myc-tagged D2 and D1D2 truncated mutants (Figure , C and D). These faster-migrating protein species are likely C-terminal proteolytic products, because the C-terminal region of VCP has been shown to be sensitive to proteolytic degradation (52
In addition to co-immunoprecipitation from rodent brain and cultured cells, we performed a GST fusion pull-down assay to validate the direct binding of neurofibromin and VCP. As shown in Figure G, purified His-tagged D1D2 of VCP was precipitated by the fusion protein GST-LRD but not GST-GRD1. These data suggest a direct protein-protein interaction between neurofibromin and VCP.
VCP regulates the spine density in cultured hippocampal neurons.
Because neurofibromin regulates dendritic spine formation, we hypothesized that VCP may also be involved in this process. Subcellular distribution of VCP in neurons was first examined. Using GFP to outline cell morphology of neurons, we found that VCP was widely distributed in different compartments of neurons, including soma, dendrites, and dendritic spines (Supplemental Figure 2A). Biochemical fractionation indicated that VCP protein is present in the light membrane (P3), crude synaptosomal (P2), and crude synaptic vesicle (LP2) fractions (Supplemental Figure 2B). The presence of VCP protein in the synaptic fractions supports the possibility that VCP locally regulates synapse morphology or density.
The microRNA (miRNA) knockdown approach was then employed to explore the role of VCP in dendritic spine morphology and density. We generated an artificial miRNA construct coexpressing Emerald GFP (EmGFP) to concurrently label transfected cells and outline the cell morphology. The artificial miRNA was designed to target a site within the VCP gene that is identical in rat and mouse. Therefore, the miRNA construct is expected to reduce the expression of both rat and mouse VCP genes. In addition, a non-silencing control expressing an miRNA sharing no significant homology with mammalian genomes (see Methods for details) was used as a negative control. As expected, the VCP miRNA knockdown clones effectively silenced Myc-tagged mouse VCP expression in HEK293T cells (Figure A). In cultured rat hippocampal neurons, the VCP miRNA construct also reduced the expression of endogenous VCP (Figure B). In our culture system, it took at least 2 weeks for neurons to be fully differentiated, i.e., to form mature dendritic spines, the location of excitatory synapses. Therefore, in this study, we routinely performed transfection at 12 days in vitro (DIV) and examined neuronal morphology at 18 DIV. Indeed, knockdown of endogenous VCP reduced the density of dendritic spines measured at 18 DIV (Figure , C and D; Kolmogorov-Smirnov [KS] test, P < 0.001; t test, P < 0.001). Notably, neither the spine length nor the width of the spine heads was affected as compared with the non-silencing control (Figure , E and F). To rule out the possibility of an off-target effect of VCP miRNA, a rescue experiment was performed by cotransfection of a VCP mutant resistant to miRNA. Expression of this VCP silent mutant efficiently increased the spine number (Figure , C and D; miRNA vs. rescue, KS test, P < 0.001; t test, P < 0.001), supporting the role of VCP in regulating the density of the dendritic spines.
Reduction of VCP expression impairs dendritic spine formation.
To further confirm the significance of VCP in controlling dendritic spine density, we investigated the effects of the different fragments of VCP in cultured hippocampal neurons. Similar to full-length VCP, the D1D2 and N-domain constructs were widely distributed in neurons (Figure A). Compared with the vector control, the presence of the D1D2 fragment reduced the density of dendritic spines (Figure , B–D; KS test in Figure C, P = 0.013; t test in Figure D, P = 0.0014). By contrast, the N-terminal region of VCP, which does not interact with neurofibromin, did not obviously influence the density of dendritic spines (Figure , E–G), supporting the specific effect of the D1D2 region on downregulation of the spine density. Moreover, since the D1 and D2 regions possess an ATPase activity, we then investigated whether the ATPase activity of VCP contributes to the effect of the D1D2 fragment on spine density. However, the construct carrying the K524A mutation resulting in ATPase inactivation seemed to have a very strong cytotoxicity to cultured neurons (data not shown). We therefore could not evaluate whether the ATPase activity of VCP is involved in the regulation of dendritic spine density.
Overexpression of the neurofibromin-binding domain of VCP inhibits spine formation.
In conclusion, the above analyses using VCP miRNA and the D1D2 construct suggested a role of VCP in the regulation of dendritic spine density.
Disruption of the interaction between neurofibromin and VCP results in reduced dendritic spine density.
To confirm the role of the interaction between neurofibromin and VCP in spinogenesis, the LRD fragment (Figure A), a region of neurofibromin interacting with VCP, was overexpressed in cultured hippocampal neurons. Compared with vector control, expression of the LRD fragment reduced the spine density of cultured hippocampal neurons (Figure , B–D; KS test in Figure C, control vs. WT LRD, P < 0.001; t test in Figure D, control vs. LRD, P < 0.001), supporting the role of the interaction between neurofibromin and VCP in the regulation of dendritic spine density.
Overexpression of the VCP-binding domain of neurofibromin reduces the number of dendritic spines.
We then asked whether mutations identified in the NF1
gene of patients with NF1 would affect the interaction between neurofibromin and VCP and whether these mutations lead to the reduction in spine density. Mutation screening of 250 Taiwanese NF1 patients fulfilling NIH diagnostic criteria led to the identification of 18 mutations in the LRD region (Table ), of which only 3 have been identified in previous studies (54
). Examination of the familial segregation and screening of the control group (>300 Taiwanese participants) suggested that these mutations are pathogenic. Of these 18 mutations, 14 (77.8%) result in a truncated protein, 3 lead to a change of a single amino acid (mutants p.A1655T, p.T1787M, and p.C1909R), and 1 results in the deletion of a single amino acid (residue Y1587, c.4759_4761delTAT; Table ). In addition to a part of the LRD, the truncated mutant proteins lack more than one-third of the C-terminal residues of neurofibromin. This fact makes it difficult to evaluate the specific role of the interaction between these truncated NF1 mutants with VCP. We therefore focused on the missense and single amino acid deletion mutants (Figure A). The interactions between these LRD mutants and the D1D2 fragment or full-length VCP were examined by co-immunoprecipitation. When compared with WT LRD, T1787M, C1909R, and A1655T mutations did not noticeably affect the interaction between the LRD domain and the D1D2 region or full-length VCP (Figure , E and F). By contrast, deletion of the residue Y1587 almost completely abolished this interaction (Figure , E and F). These data suggest a critical role of this tyrosine residue in the interaction between VCP and neurofibromin LRD.
Characteristics of patients with NF1 carrying mutations in the LRD regionA
We then investigated the biological significance of the Y1587 deletion. Because the overexpression of WT LRD (Figure , B–D) likely disrupted the interaction between endogenous VCP and neurofibromin and thus reduced spine density, we expected that overexpression of the LRD Y1587Δ mutant would not affect spine density, given that this mutant cannot interact with VCP (Figure F). Indeed, expression of the LRD Y1587Δ mutant did not reduce spine number (Figure , B–D; KS test in Figure C, control vs. Y1587Δ, P = 0.83; WT LRD vs. Y1587Δ, P < 0.001; t test in Figure D, control vs. Y1587Δ, P = 0.85; WT LRD vs. Y1587Δ, P < 0.001). By contrast, the LRD C1909R mutant, which is capable of interacting with VCP, was able to reduce the spine density (Figure , B–D; KS test in Figure C, control vs. C1909R, P = 0.002; t test in Figure D, control vs. C1909R, P < 0.001), though the inhibitory effect was significantly weaker than that of WT LRD (Figure , B–D; KS test in Figure C, P < 0.001; t test in Figure D, P < 0.001). In conclusion, the results of these analyses suggest that the Y1587Δ mutation in the NF1 gene disrupts the interaction between neurofibromin and VCP and, as a result, affects dendritic spinogenesis.
To further confirm the importance of residue Y1587 in the function of neurofibromin, we used cultured cortical neurons prepared from Nf1+/–
mice. The aforementioned data suggested that deletion of one copy of the Nf1
gene reduced the spine density of pyramidal neurons in mouse brain. We then examined whether transfection of full-length rNf1
increases the density of dendritic spines in Nf1+/–
neurons and whether the full-length Y1587Δ mutant loses this ability. Two sets of experiments with different time points for transfection and immunostaining were performed. In the first set, conditions identical to those in the experiments described above were chosen, i.e., transfection and staining were carried out at 12 DIV and 18 DIV, respectively. In the second set, transfection was done at 7 DIV and staining was carried out at 10 DIV. Our previous study indicated that neurofibromin regulates the initial step of dendritic spinogenesis, namely dendritic filopodia formation (9
). We therefore expected that exogenous NF1 should also increase the number of dendritic filopodia in relatively young neurons, such as at 10 DIV. Indeed, compared with vector control, expression of full-length rNf1 increased the density of dendritic protrusion in Nf1+/–
cortical neurons at 10 DIV (Figure , A–C; Nf1+/–
control vs. Nf1+/–
< 0.001; KS test, P
= 0.007) as well as at 18 DIV (Figure , D–F; Nf1+/–
control vs. Nf1+/–
< 0.001; KS test, P
< 0.001). By contrast, expression of the full-length Y1587Δ mutant did not increase the protrusion density in Nf1+/–
neurons at 10 DIV (Figure , A–C; Nf1+/–
rNf1 vs. Nf1+/–
< 0.001; KS test, P
< 0.001) or at 18 DIV (Figure , D–F; Nf1+/–
rNf1 vs. Nf1+/–
< 0.001; KS test, P
< 0.001). These results support the importance of the residue Y1587 for the activity of neurofibromin in controlling spine density.
Overexpression of full-length rNf1, but not Y1587Δ mutant, rescues the dendritic spine phenotype of Nf1+/– neurons in culture.
Clinically, the proband with the mutation Y1587Δ had multiple café-au-lait spots with numerous cutaneous neurofibroma, which are the typical phenotypes of patients with NF1. This patient was diagnosed with mental subnormality in 2004 at the age of 63 and has been experiencing dementia for the past 3 years. Among the 18 patients with an NF1
mutation in the LRD region, 5 were diagnosed as having mental subnormality characterized by either poor school performance or dementia (27.8%, Table ). In a previous study, the frequency of mental subnormality of NF1 patients was less than 5% in a Taiwanese cohort (3 of 68, ref. 57
). The higher frequency of mental subnormality in NF1 patients carrying mutations in the LRD region may be related to the interaction between neurofibromin and VCP and the role of LRD in the regulation of dendritic spine density.
VCP IBMPFD mutants interact weakly with neurofibromin and impair spinogenesis.
Because the identified IBMPFD mutations are clustered at the interface of the N-domain and the D1 region, it is believed that they can alter the conformation of the hexameric barrel formed by the D1 domain during ATP binding and ATP hydrolysis (51
). Because both D1 and D2 domains are required for the interaction with neurofibromin, we speculated that neurofibromin recognizes a special conformation of VCP that may be sensitive to IBMPFD mutations. To investigate this possibility, we conducted co-immunoprecipitation experiments to determine whether IBMPFD mutations would result in a decrease in the interaction of VCP and neurofibromin in HEK293T cells. Compared with WT VCP, both VCP R155H and R95G mutants, which occur most frequently in IBMPFD, interacted weakly with the cotransfected LRD fragment (Figure A) or endogenous full-length neurofibromin (Figure B), suggesting that IBMPFD mutations in the N-terminal region of VCP reduce the protein-protein interaction mediated through the C-terminal D1 and D2 regions of VCP.
IBMPFD mutations reduce the interaction of neurofibromin and VCP and the density of dendritic spines.
Because previous observations showed that IBMPFD mutant VCP can form aggregates in myoblastoma cells (43
), we then wondered whether the reduction of the interaction between an IBMPFD mutant and neurofibromin is caused by a change in the subcellular distribution of mutant VCP protein. However, in HEK293T cells, the subcellular distribution of R155H and R95G mutants was similar to that of WT VCP; each protein was widely distributed, with a tendency to concentrate at the cell cortex (Figure C). We did not find any obvious VCP protein aggregates in HEK293T cells. This result indicates that protein aggregation of IBMPFD mutants is cell type–specific and also suggests that the reduced interaction of IBMPFD mutants and neurofibromin is unlikely to be due to an altered subcellular distribution of IBMPFD mutants.
Although VCP has been shown to associate with the 26S proteasome and to regulate degradation of ubiquitinated proteins (58
), the interaction between VCP and neurofibromin did not appear to regulate the protein stability of neurofibromin, as the protein levels of neurofibromin were comparable in HEK293T cells expressing WT VCP and those expressing the IBMPFD mutant (Figure D). In addition to IBMPFD mutants, the effects of VCP knockdown and K524A mutation were also examined. Similarly, the levels of neurofibromin protein were not altered by expression of VCP K524A mutant or knockdown of VCP (Figure , E and G). In these experiments, MG132, a potent proteasome inhibitor, was either added to the cultures 4 hours before harvesting cell extracts or omitted. Although MG132 increased the signals of protein ubiquitination, it did not increase the total neurofibromin protein levels in either VCP-knockdown cells or K524A mutant–expressing cells (Figure , E and G). Moreover, after enrichment by immunoprecipitation, we still could not detect ubiquitinated neurofibromin after alteration of VCP protein level or expression of K524A mutant (Figure , F and G). Taken together, all of these analyses suggest that VCP does not influence the protein level or ubiquitination of neurofibromin.
Cultured hippocampal neurons were then used to assess the effect of IBMPFD mutations on spinogenesis. Similar to the results in HEK293T cells, expression of the R95G and R155H mutants was comparable to that of WT VCP in cultured hippocampal neurons. The intensity of immunoreactivity of WT VCP was comparable to that of R95G and R155H mutants (Figure H). Besides, we did not find protein aggregates of IBMPFD mutants in neurons (Figure H). Like WT VCP, the IBMPFD mutant proteins also entered dendritic spines (Figure H). Thus, IBMPFD mutations did not noticeably alter expression levels or the subcellular distribution of VCP. However, the R95G mutant significantly reduced the density of dendritic spines (Figure , I–K; KS test in Figure K, control vs. R95G, P < 0.001; WT vs. R95G, P = 0.006; t test in Figure J, control vs. R95G, P < 0.001; WT vs. R95G, P < 0.001). Although the effect exhibited by the R155H mutant was weaker, it still reduced the density of dendritic spines as compared with the vector control (Figure , I–K; KS test in Figure K, P = 0.012; t test in Figure J, P = 0.028). By contrast, WT VCP did not appear to impair the spine density (Figure , I–K). These data suggest that the presence of IBMPFD mutants impairs dendritic spine formation.
VCP acts downstream of neurofibromin in the regulation of dendritic spine density.
We then used Nf1+/– cortical neurons to investigate whether VCP acts downstream of neurofibromin. In Nf1+/– neurons, expression of WT VCP increased the spine density to a level comparable to that in WT neurons (Figure , A and B). By contrast, the VCP R95G mutant did not rescue the density of dendritic spines. In Nf1+/– neurons, the R95G mutant further reduced the spine density compared with vector control (Figure , A and B). These data support the conclusion that VCP is located downstream of neurofibromin in controlling spine density.
VCP acts downstream of neurofibromin in the regulation of dendritic spine density.
Our previous study had shown that neurofibromin was widely distributed in the various membrane fractions purified from rat brains, including the light membrane fraction, lysed synaptosomal membrane fractions, and crude synaptic vesicle fraction (22
). Here we found that the neurofibromin protein levels in Nf1+/–
brains were lower than those in WT brains regardless of the subcellular fraction examined (Figure C). No obvious difference in the VCP protein levels in total homogenates between Nf1+/–
mice and WT littermates was detected (Figure C). In several subcellular fractions, including light membrane (P3) and lysed synaptosomal membrane (LP1) fractions, the distribution of VCP protein was also comparable in WT and Nf1+/–
brains (Figure C). However, in Nf1+/–
brains, the level of VCP protein was lower in the crude synaptic vesicle (LP2) and synaptic cytosol (LS2) fractions, which contain vesicles and cytosolic fractions at both pre- and post-synaptic sites (Figure C). By contrast, VCP protein was more abundant in the total soluble cytosolic fraction (S3) of Nf1+/–
mice as compared with WT littermates (Figure C). These results suggest that the subcellular distribution of VCP is influenced by neurofibromin.
Taken together, our results suggests that loss of one copy of the Nf1 gene reduces the level of VCP in crude synaptic vesicles and synaptosomal cytosol and may thus impair dendritic spine formation. Overexpression of VCP in Nf1+/– neurons may ectopically increase the VCP level at synapses and rescue the defects in the density of dendritic spines.
Statin treatment rescues the effect of VCP knockdown on spine density.
A previous study showed that lovastatin, an inhibitor of the HMG-CoA reductase, rescues the defects of Nf1+/–
mice in learning disability (60
). We therefore asked whether lovastatin treatment rescues the defect in spinogenesis resulting from silencing expression of VCP. Treatment of cultured hippocampal neurons with lovastatin at a concentration of 2 μM for 3 days did not rescue the effect of VCP knockdown on spine density. However, at a higher concentration (5 μM), the spine density of lovastatin-treated VCP-knockdown neurons was comparable to that of neurons treated with lovastatin alone (Figure , A and B). In VCP-knockdown neurons, 5 μM lovastatin treatment increased the density of dendritic spines compared with vehicle control (Figure , A and B). The effect of lovastatin was unlikely due to interference with Vcp
miRNA knockdown, because the Vcp
miRNA construct still effectively knocked down the expression of VCP in the presence of this statin (Figure C). Similar results were obtained with simvastatin, with regard to both rescuing the effect of VCP knockdown on spine density (Figure D) and absence of interference with VCP knockdown (Figure E). Taken together, these data suggest that a statin-sensitive pathway is also involved in VCP-mediated spinogenesis. It is consistent with our hypothesis that VCP and neurofibromin work together to regulate neural function.
Statin treatment abates the effect of VCP knockdown on dendritic spine density.