4820? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 121? ? ? Number 12? ? ? December 2011
Valosin-containing protein and neurofibromin
interact to regulate dendritic spine density
Hsiao-Fang Wang,1 Yu-Tzu Shih,1,2 Chiung-Ya Chen,1
Hsu-Wen Chao,1 Ming-Jen Lee,3 and Yi-Ping Hsueh1,2
1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. 2Molecular Cell Biology, Taiwan International Graduate Program,
Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
3Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan.
In the central nervous system of mammals, dendritic spines are
the locations of more than 90% of excitatory synapses (1) and
therefore constitute the functional subcellular structures for
excitatory neurotransmission (2–6). Neurofibromin, a large pro-
tein (2818 aa residues) encoded by the human NF1 gene (7, 8),
is one regulator of dendritic spine formation (9). Mutations of
the gene cause neurofibromatosis type 1 (OMIM 162200), one
of the most common autosomal dominant disorders, affecting
about one in 3,500 individuals. Neurofibromatosis type 1 (NF1) is
characterized by skin pigmentations (café-au-lait spots and freck-
ling) and formations of benign peripheral nerve sheath tumors
(neurofibromas). In addition, many other features are frequently
found in patients with NF1, including cognitive deficits as well as
skeletal lesions and malformations. In children, NF1 is frequently
associated with learning difficulty (10) and greater susceptibility
to autism (11, 12). The function of neurofibromin in synaptogen-
esis (9) and formation of barrel cortex (13) may partially explain
these neurological symptoms. It is also known that neurofibro-
min regulates the functions of osteoclast (14, 15) and skeletal
muscle development (16). Although the Ras/MAPK pathway, the
downstream signaling of neurofibromin, has been implicated
in bone resorption (17), the detailed mechanism underlying the
bony defects in patients with NF1 remains elusive.
The tumor suppressor activity of neurofibromin is largely
dependent on its Ras-specific GTPase-activating protein (RasGap)
activity (reviewed in refs. 18, 19). In addition, neurofibromin also
regulates adenylate cyclase activity through both Gαs-dependent
and -independent pathways, thus controlling the cAMP concen-
tration in cells (20). Our previous study showed that neurofibro-
min is widely distributed in different subcellular compartments
of neurons, including synapses (21). It acts downstream of synde-
can-2, a synaptic heparan sulfate proteoglycan, in the regulation of
dendritic spine formation (9). Neurofibromin interacts with syn-
decan-2 (22) and activates the PKA–Enabled/vasodilator-stimu-
lated phosphoprotein (PKA-Ena/VASP) pathway to promote actin
polymerization and bundle formation (9). Interestingly, although
the PKA pathway is essential for dendritic spine formation, activa-
tion of PKA alone is not sufficient for the process (9), possibly due
to the involvement of multiple downstream pathways of neurofi-
bromin in spinogenesis.
Valosin-containing protein (VCP), also known as p97, is a mul-
tifunctional AAA (ATPases associated with a variety of cellular
activities) protein (reviewed in refs. 23, 24) involved in a variety of
cellular events, including cell cycle control, membrane fusion, ER-
associated protein degradation (ERAD), and autophagy (24–32).
VCP is associated with several neurodegenerative disorders
(reviewed in refs. 33–35). Mutations in the VCP gene result in
inclusion body myopathy with Paget disease of bone and fronto-
temporal dementia (IBMPFD, ref. 36), a dominant inherited dis-
order (OMIM 167320). Expression of mutant VCP in transgenic
mice or introduction of an IBMPFD mutation into mice through
a gene-targeting approach induces degeneration in muscle, bone,
and brain (37, 38), recapturing the phenotypes of the patients with
IBMPFD. Recently, human genetic analysis also indicated that VCP
mutations account for 1%–2% of autosomal dominantly inherited
ALS (39). In addition, VCP interacts with the polyglutamine-con-
taining aggregates that are found in patients with Huntington and
Machado-Joseph diseases (40).
Authorship?note: Hsiao-Fang Wang, Yu-Tzu Shih, and Chiung-Ya Chen contributed
equally to this work.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2011;121(12):4820–4837. doi:10.1172/JCI45677.
Related Commentary, page 4627
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
So far, the mechanism of IBMPFD pathogenesis has not been
elucidated. VCP controls polyubiquitin chain turnover (41) and
contributes to both formation and clearance of the ubiquitylated
inclusion bodies (42). An IBMPFD-associated VCP mutant was
shown to induce aggregation of polyubiquitin-conjugated proteins
in myoblastoma cells (43). VCP mutations have also been shown
to cause the dysfunction of autophagy, which may additionally
contribute to the pathogenesis of IBMPFD (31, 44). In addition to
defects in protein degradation, dystrophic neurites are frequently
found in patients with frontotemporal dementia (FTD) (45–47).
Recently, VCP has been shown to regulate remodeling of neuronal
morphology in Drosophila (48). It is likely that VCP actively contrib-
utes to neuronal morphogenesis and that dysfunction of VCP may
therefore result in neurodegeneration.
VCP forms a homohexameric barrel and hydrolyzes ATP to generate
the mechanical force for its function as a molecular chaperon (23, 27,
49). It possesses two ATPase domains (D1 and D2). The D2 domain
carries the major ATPase activity, while the D1 domain is also the
hexamerization domain of VCP. The N-terminal region (N-domain)
of VCP is involved in the interaction with various adaptors that
direct VCP to various cellular events. The identified IBMPFD muta-
tions are highly clustered in the N- and D1 domains of VCP (36, 47,
50). Interestingly, all mutation hot spots are located at the interface
between the N- and D1 domains (51). Therefore, IBMPFD muta-
tions can change the conformation of the catalytic domains, alter
ATPase activity, and compromise the function of VCP (52).
In this study, we first examined the number of dendritic spines of
CA1 neurons in Nf1+/– mice. Golgi staining showed that the spine
number in Nf1+/– mice is lower than that in the WT littermates,
consistent with our previous finding that neurofibromin plays a
role in controlling spine density of neurons. Using a proteomics
approach, we identified VCP as a neurofibromin-associated pro-
tein. We further provide evidence that the interaction between
neurofibromin and VCP regulates the density of dendritic spines.
Our study found a crosstalk between these two proteins, neurofi-
bromin and VCP, each of which is associated with genetic disease
and regulates neuronal morphogenesis.
Dendritic spine density is lower in Nf1+/– mice. We have previously shown
that neurofibromin regulates dendritic spine formation, particu-
larly the initiation step of spinogenesis, namely dendritic filopodia
The density of dendritic spines is reduced in Nf1+/– heterozygous mice. (A) First branches, (B) second branches, and (C) the remaining higher-
order branches of apical dendrites of CA1 pyramidal cells. For each group, 2 representative images are shown. The right panels indicate the
quantitative results of the spine density. Data represent mean plus SEM. The numbers of analyzed dendrites were (A) WT, 51; Nf1+/–, 55; (B)
WT, 21; Nf1+/–, 12; (C) WT, 51; Nf1+/–, 30. Scale bars: 2 μm. ***P < 0.001.
4822? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
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, 13), 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 1). These results support a role for the Nf1 gene in the regu-
lation of dendritic spine density in mouse brain.
Neurofibromin interacts with VCP. To further explore the func-
tion 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 arti-
cle; doi:10.1172/JCI45677DS1) 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 interac-
tion between neurofibromin and VCP was specific, because control
IgG did not precipitate VCP (Supplemental Figure 1A). In addi-
tion to VCP, p47, a cofactor of VCP, was also present in the pre-
cipitate 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 identi-
fied 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 2A). The recip-
rocal immunoprecipitation also showed the precipitation of neu-
rofibromin by VCP antibody from rat brain extracts (Figure 2A).
Non-immune control IgG was used again as negative control to
ensure the specificity of co-immunoprecipitation (Figure 2A).
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.
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 pres-
ence 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 2B), supporting the associa-
tion 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 2C)
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 endog-
enous neurofibromin (Figure 2C), 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 2D). These frag-
ments 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 experi-
ment. 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 2D), 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 effec-
tively removed the CSRD fragment from the immunoprecipitates
(Figure 2E). By contrast, the LRD still associated with the D1D2
fragment (Figure 2E). These data also support the specific interac-
tion 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 2F).
Note that in addition to the expected full-length protein at
approximately 100 kDa, we frequently detected Myc-tag–immu-
noreactive protein species with an apparent molecular weight less
than 95 kDa in precipitates as well as inputs (Figure 2, B, C, and F).
Smaller protein fragments were also found for the Myc-tagged D2
and D1D2 truncated mutants (Figure 2, C and D). These faster-
migrating protein species are likely C-terminal proteolytic prod-
ucts, 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 cul-
tured cells, we performed a GST fusion pull-down assay to validate
the direct binding of neurofibromin and VCP. As shown in Figure 2G,
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 hypoth-
esized 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). Bio-
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
chemical 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
VCP interacts with neurofibromin. (A) Two-directional immunoprecipitations of neurofibromin and VCP. Adult rat brain extracts were used
for immunoprecipitation using the indicated antibodies. Immunoblotting was then performed. (B) Co-immunoprecipitation of p47, VCP, and
neurofibromin. HEK293T cells were transfected with Myc-tagged VCP and Myc-tagged p47 or vector control as indicated. One day later, cell
extracts were harvested for immunoprecipitation using neurofibromin antibody. Immunoblotting using Myc tag antibody revealed the pres-
ence of both VCP and p47. (C–F) Interaction between neurofibromin and VCP in transfected HEK293T cells. Variant Myc-tagged VCP con-
structs and HA-tagged NF1 constructs were cotransfected into HEK293T cells as indicated, and cell lysates were harvested 24 hours later and
immunoprecipitated using antibodies as indicated. (D) Myc-tagged D1D2 construct was cotransfected with variant neurofibromin fragments.
GRD1DN, dominant negative mutant of GRD1. (E) 2 mM DTT was added in lysates to reduce oxidation. (G) GST pull-down assay. His-tagged
D1D2 of VCP was mixed with LRD or GRD1 GST fusion proteins. The protein complex was then pulled down using glutathione agarose and
analyzed by immunoblotting.
4824? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
Reduction of VCP expression impairs dendritic spine formation. (A) Knockdown effect of the VCP miRNA construct in HEK293T cells. Myc-
tagged WT VCP (VCPWT) or a silent mutant of VCP (VCPmt) and non-silencing control or VCP miRNA construct were cotransfected into cells. One
day later, cells were harvested for immunoblotting analysis using Myc tag and GFP antibodies. (B) Knockdown of VCP in cultured hippocampal
neurons. Transfection of VCP miRNA and non-silencing control was carried out at 12 DIV. Immunostaining using VCP antibody was performed at
18 DIV. The relative VCP protein levels revealed by immunostaining were quantified. Values are presented as means plus SEM. Non-silencing,
n = 51; VCP miRNA, n = 35. Scale bar: 10 μm. (C) Cell morphology of VCP-knockdown neurons. Cultured hippocampal neurons were transfected
with various plasmids (Control: non-silencing control plus GW1 vector control; miRNA: VCP miRNA plus GW1 vector control; Rescue: VCP
miRNA plus miRNA-resistant VCP silent mutant) at 12 DIV and harvested for immunostaining using a GFP antibody at 18 DIV. The lower panels
show enlarged images (×5.3) of the boxed regions marked in the upper panels. Scale bar: 20 μm. (D–F) Cumulative probability distributions and
means plus SEM of protrusion density (D), protrusion width (E), and protrusion length (F). (D–F) More than 48 neurons, 193 dendrites, and 794
spines for each group from 2 independent experiments were analyzed. In D, P < 0.01, rescue versus control; P < 0.001, miRNA versus control
and versus rescue. **P < 0.01, ***P < 0.001.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
mouse VCP expression in HEK293T cells (Figure 3A). In cultured rat
hippocampal neurons, the VCP miRNA construct also reduced the
expression of endogenous VCP (Figure 3B). 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 den-
dritic spines measured at 18 DIV (Figure 3, 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 com-
pared with the non-silencing control (Figure 3, E and F). To rule out
the possibility of an off-target effect of VCP miRNA, a rescue experi-
ment was performed by cotransfection of a VCP mutant resistant to
miRNA. Expression of this VCP silent mutant efficiently increased
the spine number (Figure 3, 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.
To further confirm the significance of VCP in controlling den-
dritic 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 4A). Compared with the vector con-
trol, the presence of the D1D2 fragment reduced the density of
dendritic spines (Figure 4, B–D; KS test in Figure 4C, P = 0.013;
t test in Figure 4D, P = 0.0014). By contrast, the N-terminal region
of VCP, which does not interact with neurofibromin, did not obvi-
ously influence the density of dendritic spines (Figure 4, E–G), sup-
porting 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 activ-
ity of VCP contributes to the effect of the D1D2 fragment on
spine density. However, the construct carrying the K524A muta-
tion 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.
In conclusion, the above analyses using VCP miRNA and the
D1D2 construct suggested a role of VCP in the regulation of den-
dritic spine density.
Overexpression of the neurofibromin-binding domain of VCP inhibits spine formation. (A) Immunostaining using Myc-tag antibody reveals the
similar expression and distribution of Myc-tagged VCP, D1D2, and N-domain in cultured hippocampal neurons. (B) Myc-tagged VCP D1D2
construct and (E) Myc-tagged N-domain and vector control were cotransfected with GFP-actin, as indicated, into cultured hippocampal neurons
at 12 DIV. Six days later, the neuronal morphology was monitored by detection of GFP immunoreactivity. In B, the lower panels show enlarged
images (×5.3) of the boxed regions marked in the upper panels. (C and F) Cumulative probability distributions and (D and G) graph of protrusion
densities obtained from B and E, respectively. More than 22 neurons and 86 dendrites for each group of experiments were analyzed. P < 0.05,
DID2 versus control; **P < 0.01. Scale bars: 10 μm (A); 20 μm (B); 5 μm (E). Values are presented as mean plus SEM.
4826? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
Disruption of the interaction between neurofibromin and VCP results
in reduced dendritic spine density. To confirm the role of the interac-
tion between neurofibromin and VCP in spinogenesis, the LRD
fragment (Figure 5A), 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 5, B–D; KS test in Figure 5C, control vs. WT LRD, P < 0.001;
t test in Figure 5D, control vs. LRD, P < 0.001), supporting the
role of the interaction between neurofibromin and VCP in the
regulation of dendritic spine density.
We then asked whether mutations identified in the NF1 gene of
patients with NF1 would affect the interaction between neurofi-
bromin and VCP and whether these mutations lead to the reduc-
tion 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 1), of which only 3 have
been identified in previous studies (54–56). Examination of the
familial segregation and screening of the control group (>300 Tai-
wanese participants) suggested that these mutations are patho-
genic. Of these 18 mutations, 14 (77.8%) result in a truncated pro-
tein, 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 1).
In addition to a part of the LRD, the truncated mutant proteins
lack more than one-third of the C-terminal residues of neurofibro-
min. 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 5A). 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 5, E and F). By contrast, deletion of the residue
Y1587 almost completely abolished this interaction (Figure 5, E
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.
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and F). These data suggest a crit-
ical role of this tyrosine residue
in the interaction between VCP
and neurofibromin LRD.
We then investigated the
biological significance of the
Y1587 deletion. Because the
overexpression of WT LRD
(Figure 5, B–D) likely disrupted
the interaction between endog-
enous 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 5F).
Indeed, expression of the LRD
Y1587Δ mutant did not reduce
spine number (Figure 5, B–D;
KS test in Figure 5C, control
vs. Y1587Δ, P = 0.83; WT LRD
vs. Y1587Δ, P < 0.001; t test in
Figure 5D, control vs. Y1587Δ,
P = 0.85; WT LRD vs. Y1587Δ,
P < 0.001). By contrast, the LRD
C1909R mutant, which is capa-
ble of interacting with VCP, was
able to reduce the spine den-
sity (Figure 5, B–D; KS test in
Figure 5C, control vs. C1909R,
P = 0.002; t test in Figure 5D,
control vs. C1909R, P < 0.001),
though the inhibitory effect
was significantly weaker than
that of WT LRD (Figure 5, B–D;
KS test in Figure 5C, P < 0.001;
t test in Figure 5D, 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
To further confirm the impor-
tance 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 exam-
ined whether transfection of
full-length rNf1 increases the
density of dendritic spines in
Nf1+/– neurons and whether the
full-length Y1587Δ mutant loses
Characteristics of patients with NF1 carrying mutations in the LRD regionA
Plexiform NF on the neck
No dementia in
Poor school performance;
UBO in thalamus
and basal ganglia
Tibia bony defect
Tibia bony defect,
Plexiform NF at
the right knee
NF at the right leg
c.5839 C→T (CGA→TGA)
AA total of 18 sequence variants were identified from 250 patients. The mutations consist of 5 frameshift, 9 missense, 1 deletion, and 3 aberrant splicing mutations. Fourteen mutations led to a premature
stop codon, resulting in truncation of the LRD region. BThe Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff (http://www.hgmd.cf.ac.uk/ac/index.php) was searched to determine
whether the mutations have been reported previously by other laboratories. CAll patients had both café-au-lait spots and neurofibroma. In addition to these 2 phenotypes, other characteristics of individual
patients are listed. NF, neurofibroma; UBO, unidentified bright object comprising a nonspecific bright signal in the T2-weighted image of brain MRI with unknown biological significance; MPNST, malignant peripheral nerve sheath tumor. DInformation about the patient’s school performance is lacking. F.Hx., family history; poor school performance: the lowest 10% in a cohort or a class; ADHD, attention deficit
4828? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
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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 6, A–C; Nf1+/– control vs.
Nf1+/– rNf1, t test, P < 0.001; KS test, P = 0.007) as well as at 18 DIV
(Figure 6, D–F; Nf1+/– control vs. Nf1+/– rNf1, t test, P < 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 6, A–C; Nf1+/– rNf1 vs. Nf1+/– Y1587Δ, t test,
P < 0.001; KS test, P < 0.001) or at 18 DIV (Figure 6, D–F; Nf1+/–
rNf1 vs. Nf1+/– Y1587Δ, t test, P < 0.001; KS test, P < 0.001). These
results support the importance of the residue Y1587 for the activ-
ity of neurofibromin in controlling spine density.
Clinically, the proband with the mutation Y1587Δ had mul-
tiple 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 1).
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, 52).
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 7A) or endogenous full-length neurofibromin (Figure 7B),
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.
Because previous observations showed that IBMPFD mutant
VCP can form aggregates in myoblastoma cells (43), we then won-
dered 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 simi-
lar to that of WT VCP; each protein was widely distributed, with a
tendency to concentrate at the cell cortex (Figure 7C). 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 pro-
teasome and to regulate degradation of ubiquitinated proteins
(58, 59), 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 7D). 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 expres-
sion of VCP K524A mutant or knockdown of VCP (Figure 7, E
and G). In these experiments, MG132, a potent proteasome inhibi-
tor, was either added to the cultures 4 hours before harvesting cell
extracts or omitted. Although MG132 increased the signals of pro-
tein ubiquitination, it did not increase the total neurofibromin
protein levels in either VCP-knockdown cells or K524A mutant–
expressing cells (Figure 7, 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 7, 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 7H). Besides, we did not
find protein aggregates of IBMPFD mutants in neurons (Figure 7H).
Like WT VCP, the IBMPFD mutant proteins also entered dendritic
spines (Figure 7H). Thus, IBMPFD mutations did not noticeably
alter expression levels or the subcellular distribution of VCP. How-
ever, the R95G mutant significantly reduced the density of den-
dritic spines (Figure 7, I–K; KS test in Figure 7K, control vs. R95G,
P < 0.001; WT vs. R95G, P = 0.006; t test in Figure 7J, control vs.
R95G, P < 0.001; WT vs. R95G, P < 0.001). Although the effect
Overexpression of full-length rNf1, but not Y1587Δ mutant, rescues
the dendritic spine phenotype of Nf1+/– neurons in culture. Mouse
cultured cortical neurons were transfected at DIV7 (A) or DIV12 (D)
and harvested for staining using neurofibromin and GFP antibodies at
DIV10 (A) or DIV18 (D). For WT neurons, GFP-actin and vector con-
trol were transfected. For Nf1+/– neurons, GFP-actin was cotransfected
with full-length rNf1, full-length Y1587Δ mutant, or vector control, as
indicated. B and C, and E and F are quantitative analyses of A and
D, respectively. Values in B and E represent the mean plus SEM. The
numbers of analyzed dendrites for each group were: (B and C) WT, 79;
Nf1+/– control, 106; Nf1+/– rNf1, 37; Nf1+/– Y1587Δ, 59; (E and F) WT,
94; Nf1+/– control, 110; Nf1+/– rNf1, 49; Nf1+/– Y1587Δ, 44. Scale bars:
10 μm in the whole images; 2 μm in the enlarged images (A); 2 μm (D).
In C, P < 0.01, Nf1+/– rNf1 versus Nf1+/– control; P < 0.001, Nf1+/– con-
trol versus WT and Nf1+/– rNf1 versus Nf1+/– Y1587Δ. In F, P < 0.001,
Nf1+/– control versus WT and versus Nf1+/– rNf1, and Nf1+/– Y1587Δ
versus Nf1+/– rNf1. **P < 0.01, ***P < 0.001.
4830? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
exhibited by the R155H mutant was weaker, it still reduced the
density of dendritic spines as compared with the vector control
(Figure 7, I–K; KS test in Figure 7K, P = 0.012; t test in Figure 7J,
P = 0.028). By contrast, WT VCP did not appear to impair the spine
density (Figure 7, 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+/– neu-
rons, expression of WT VCP increased the spine density to a level
comparable to that in WT neurons (Figure 8, A and B). By con-
trast, the VCP R95G mutant did not rescue the density of den-
dritic spines. In Nf1+/– neurons, the R95G mutant further reduced
the spine density compared with vector control (Figure 8, A and
B). These data support the conclusion that VCP is located down-
stream of neurofibromin in controlling 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 8C). No obvious difference in the VCP
protein levels in total homogenates between Nf1+/– mice and WT
littermates was detected (Figure 8C). In several subcellular frac-
tions, including light membrane (P3) and lysed synaptosomal
membrane (LP1) fractions, the distribution of VCP protein was
also comparable in WT and Nf1+/– brains (Figure 8C). However, in
Nf1+/– brains, the level of VCP protein was lower in the crude syn-
aptic vesicle (LP2) and synaptic cytosol (LS2) fractions, which con-
tain vesicles and cytosolic fractions at both pre- and post-synaptic
sites (Figure 8C). By contrast, VCP protein was more abundant in
the total soluble cytosolic fraction (S3) of Nf1+/– mice as compared
with WT littermates (Figure 8C). 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 for-
mation. 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 learn-
ing disability (60). We therefore asked whether lovastatin treat-
ment 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 res-
cue the effect of VCP knockdown on spine density. However, at a
higher concentration (5 μM), the spine density of lovastatin-treat-
ed VCP-knockdown neurons was comparable to that of neurons
treated with lovastatin alone (Figure 9, A and B). In VCP-knock-
down neurons, 5 μM lovastatin treatment increased the density
of dendritic spines compared with vehicle control (Figure 9, 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 9C). Similar results were obtained with simvas-
tatin, with regard to both rescuing the effect of VCP knockdown
on spine density (Figure 9D) and absence of interference with VCP
knockdown (Figure 9E). Taken together, these data suggest that a
statin-sensitive pathway is also involved in VCP-mediated spino-
genesis. It is consistent with our hypothesis that VCP and neurofi-
bromin work together to regulate neural function.
VCP and dendritic spines. IBMPFD is characterized by three clinical
features: myopathy, osteolytic bone lesions, and FTD (44). Using
a knockdown approach and IBMPFD mutants, we provide several
lines of evidence that VCP plays a role in dendritic spine forma-
tion. Expression of the IBMPFD mutants reduces the number of
dendritic spines in cultured hippocampal neurons by 10%–18%.
Since synapse loss has been suggested in various neurological dis-
orders associated with dementia (61–67), the ability of VCP in the
regulation of spinogenesis may be relevant to clinical features of
dementia in patients. VCP does not represent the only example
where a gene responsible for FTD is involved in neuronal morpho-
genesis. Chromatin-modifying protein 2B (CHMP2B), the caus-
ative gene for FTD3 (68), also regulates dendritic morphology and
the number of dendritic spines (69–71). These studies imply that
FTD may be caused, at least partially, by cumulative alterations in
neuronal morphology, including dendritic arborization and den-
dritic spinogenesis. To confirm this hypothesis, characterization
of genetic mouse models is required in the future.
VCP is a multifunctional protein involved in ERAD, autophagy,
and membrane fusion. It is still unclear how IBMPFD mutants
reduce the density of dendritic spines. Protein degradation
IBMPFD mutations reduce the interaction of neurofibromin and VCP
and the density of dendritic spines. (A) LRD and (B) neurofibromin
interact weakly with IBMPFD mutants. Variant Myc-tagged VCP con-
structs 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. Immu-
noblotting 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) cul-
tured 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 con-
trol. 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 immu-
noblotting analysis. For F and G, immunoprecipitation was performed
using neurofibromin antibody to precipitate endogenous neurofibro-
min. 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 inde-
pendent 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.
4832? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
through ERAD and autophagy pathways is certainly a possibil-
ity. Furthermore, membrane fusion is likely involved in the pro-
cess, because vesicle trafficking is actively involved in the regula-
tion of synapse formation and activity. In agreement with this
assumption, we found that p47, a cofactor of VCP playing an
important role in membrane fusion, was co-immunoprecipitated
with neurofibromin and VCP. p47 interacts with the N-terminal
region of VCP; the C-terminal D1 and D2 domains of VCP are
involved in the interaction with neurofibromin. Neurofibro-
min, VCP, and p47 likely form a protein complex to regulate
neuronal morphology. However, it is possible that all of these
events, including ERAD, autophagy, and membrane trafficking,
contribute to dendritic spine formation. More investigations
need to be carried out to further elucidate the molecular role of
VCP in synapse formation.
VCP forms a hexameric barrel. ATP hydrolysis provides the
energy to change the conformation of the hexameric barrel
and achieve its function as a molecular chaperon (49). IBMPFD
mutations cluster at the interface of the N- and D1 domains and
modulate ATPase activity and D2 ring conformation of VCP (52).
These mutations do not hit the hexamerization site of the D1
region. Therefore, these mutant proteins still form hexameric bar-
rels in solution (52). Mutant VCP may form heterohexamers with
WT VCP, thus impairing the function of WT VCP. This would
explain, at least partially, why the IBMPFD mutations have a
dominant effect of impairing the function of VCP in cells. It may
also explain why the IBMPFD mutants that interact weakly with
neurofibromin have a dominant-negative effect on the control of
dendritic spine density.
Although IBMPFD mutations induce protein aggregates of
VCP in C2C12 myoblastoma cells (43), we did not observe such
aggregates in transiently transfected HEK293T cells or in cul-
tured neurons. These data suggest that muscle cells are more
susceptible to IBMPFD mutations, which may be relevant to
the observation that, in patients with IBMPFD, the mean age of
presentation for inclusion body myopathy was 42 years, whereas
VCP acts downstream of neurofibromin in the regulation of dendritic spine density. (A and B) Cultured cortical neurons isolated from Nf1+/– mice
and WT littermates were cotransfected with GFP-actin and Myc-tagged WT VCP or R95G mutant or vector control at 12 DIV. The density of den-
dritic spines was determined at 18 DIV by staining using GFP and Myc antibodies. (A) Mean values plus SEM of protrusion density. **P < 0.01,
***P < 0.001. (B) Cumulative probability of protrusion density. In total, 19–26 neurons and 31–82 dendrites were analyzed. P < 0.01, WT
R95G versus WT WT and Nf1+/– control versus WT control; P < 0.001, Nf1+/– WT versus Nf1+/– control and versus Nf1+/– R95G. (C) Subcellular
distribution of VCP in Nf1+/– brain. Subcellular fractions of WT and Nf1+/– brains were isolated by a series of centrifugations (95) and analyzed
by immunoblotting. Synaptophysin (SVP38) was used as a loading control of LP2. H, total homogenate; P1, crude nuclei, unbroken cells, and
debris; S1, supernatant of P1; P3, light membrane fraction (including ER and Golgi body); S3, soluble cytosolic fraction; LP1, lysed synaptosomal
membrane; LP2, crude synaptic vesicles; LS2, soluble synaptic cytosol. The signals were quantified using Gel-Pro Analyzer (Media Cybernetics).
The intensity ratios of WT to Nf1+/– of each fraction are shown.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
that for FTD was 53 years (36). In addition, since the IBMPFD
mutants reduced the spine density without obviously causing
VCP aggregation in neurons, our study also suggests that pro-
tein aggregation is unlikely a cause of neuronal morphological
defects induced by IBMPFD mutations.
The molecular basis of NF1. NF1 is also an autosomal dominant dis-
order. Although there are four major features of NF1 (i.e., café-au-
lait spots, peripheral neurofibromas, skinfold freckling, and Lisch
nodules), many minor features and medical complications are pres-
ent in a substantial number of patients with NF1 (72), increasing
Statin treatment abates the effect of VCP knockdown on dendritic spine density. Cultured hippocampal neurons were cotransfected with GFP-actin
and vector control or VCP miRNA at 12 DIV and treated with lovastatin (Lova.) at 15 DIV for 3 days or simvastatin (Simva.) at 14 DIV for 4 days.
Quantitative measurements of spine density are shown as (A) cumulative probability distributions and (B and D) graph of protrusion densities.
For A and B, more than 53 neurons and 213 dendrites for each group were analyzed. For D, more than 30 dendrites for each group were ana-
lyzed. In the presence of statins, knockdown efficiency of VCP miRNA assayed by immunostaining using VCP antibody was also evaluated in C
and E. For VCP-knockdown neurons, somas were outlined based on the GFP signal. In C, sample sizes of each group were as follows: vehicle
plus control, 51; vehicle plus miRNA, 35; lovastatin plus control, 59; lovastatin plus miRNA, 26. In A, P < 0.001, miRNA vehicle versus control
vehicle and miRNA lovastatin 2 μM versus control lovastatin 2 μM. *P < 0.05, ***P < 0.001. Scale bars: 10 μm (C and E).
4834? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
the difficulty of effective therapeutic treatment. So far, it is unclear
why clinical features of NF1 patients vary markedly. One reason may
be the lack of a mutational hot spot in the NF1 gene. Mutations
identified in patients with NF1 are widely spread over the entire
gene. Different domains of neurofibromin are expected to carry out
specific biological functions; however, this issue has not yet been
fully addressed. With the exception of the GRD and LRD regions,
the remaining parts of neurofibromin lack recognizable protein
domains. Therefore, numerous efforts have been directed to iden-
tify neurofibromin-interacting proteins (22, 73–78). Identification
of candidate proteins that interact with neurofibromin may provide
better and more effective treatments for individual NF1 patients
who carry a mutation in a specific domain. In the present study,
we identified and characterized the interaction of VCP and neu-
rofibromin. We also found that the reduced spinogenesis in VCP-
knockdown neurons was rescued by lovastatin and simvastatin. The
results support the notion that neurofibromin and VCP contribute
to the same pathway to control spinogenesis and also imply that,
for patients carrying a mutation in the LRD region of the NF1 gene,
statins may be good candidates as therapeutic agents. In fact, a pre-
vious study has shown that lovastatin treatment reverses the learn-
ing deficits of Nf1+/– mice (60, 79). Furthermore, lovastatin improves
osteoblast activity and rescues tibial dysplasia and the bone healing
defect caused by loss of Nf1 (79, 80). However, a randomized con-
trolled trial showed that simvastatin treatment does not improve
cognitive function in patients with NF1 (81). A possible explanation
for this conflict might be that statin may be only effective in a frac-
tion of NF1 patients, such as patients carrying mutations that alter
the interaction between neurofibromin and VCP. More investiga-
tions have to be carried out to evaluate this possibility.
The effects of statins. Statins are well known to inhibit HMG-CoA
reductase, the rate-limiting enzyme of the mevalonate pathway. The
mevalonate pathway produces cholesterol and other isoprenoids.
Therefore, statins are able to reduce the cholesterol level and lipid
modification of proteins in cells. It has been shown that lipid modifi-
cation is critical for the activity of Ras and that lovastatin inhibits Ras
isoprenylation and activities in controlling cell growth and neuronal
differentiation (82, 83). Since neurofibromin also inhibits Ras activ-
ity through its GAP domain, Li and colleagues used statins to res-
cue the learning defects of Nf1+/– mice (60). In addition to protein
isoprenylation, statins may also influence the density of dendritic
spines through regulation of the cholesterol level, since cholesterol
has been shown to be involved in spinogenesis (84). Although cho-
lesterol is required for spinogenesis, the cholesterol/glycerophospho-
lipid molar ratio gradually decreased during neuronal differentiation
(85). Therefore, it is likely that in mature neurons, even though the
total cholesterol level is lower, cholesterol is enriched at dendritic
spines to maintain spine morphology. Taken together, these studies
also suggest that cholesterol homeostasis is important for neuronal
development. Interestingly, VCP has been shown to be involved in
sterol-induced dislocation of HMG-CoA reductase from the ER,
which leads to ubiquitination and degradation of HMG-CoA reduc-
tase (86–88). Therefore, treatment with statins may rescue an imbal-
ance of cholesterol synthesis and thus reverse the spine phenotype in
VCP-deficient neurons. It will be interesting to further confirm the
role of statins in the VCP-regulated pathway.
The crosstalk between neurofibromin and VCP. Mutations of NF1 and
VCP result in different genetic disorders. However, when the phe-
notypes of NF1 and IBMPFD are analyzed in detail, some similar,
though not identical, features can be found. NF1 patients are prone
to learning disability, whereas some IBMPFD patients display the
features of dementia. The function of neurofibromin and VCP in the
regulation of dendritic spine density may account, at least in part, for
these phenotypes. In our 250 NF1 patients, 18 mutations occurred
in the LRD region (Table 1). Among these patients, 5 of 18 (27.8%)
developed mental subnormality. The percentage of NF1 patients
with mental subnormality, including poor school performance and
attention deficit hyperactivity disorder, was as high as 60% in previ-
ous studies (89), while in a study focusing on a Taiwanese cohort, the
total was less than 5% (3 of 68). The evidence provided in this report
suggests that the learning disability in patients with an NF1 muta-
tion in the LRD region is relatively common. This clinical feature
supports a genetic interaction between VCP and neurofibromin.
In addition to learning disability, 9 of 18 patients (50%) with muta-
tions in the LRD region of NF1 had problems in the skeletal system,
including short stature, tibia bone defects with pseudoarthrosis,
and scoliosis. Abnormal skeletal development occurs in 10%–20% of
NF1 patients (90). Multiple forms of scoliosis occur in at least 10%
of NF1 patients (91); our experience suggests that the frequency of
bone defects in NF1 patients might be as high as 25% (17 of 68) (57).
Thus, patients with a mutation in the LRD region of the NF1 gene
are also susceptible to bone dysplasia and defects. This correlation
also favors a genetic interaction between neurofibromin and VCP,
since IBMPFD mutations also have an impact on bone metabolism.
So far, it is not clear why the phenotype of a mutation in the NF1
gene occurs at an early stage, while that of a mutation in the VCP
gene occurs at a later stage of life. This discrepancy may indicate
that another regulation, such as other reacting protein or posttrans-
lational modification, is involved in regulation of the role of neuro-
fibromin and VCP in spinogenesis. It is also likely that neurofibro-
min and VCP have other functions in neuronal morphogenesis and
therefore display differential phenotypes. For instance, the C1909R
mutation of neurofibromin did not obviously affect the interaction
between the LRD fragment and VCP. However, overexpression of the
LRD C1909R mutant still had a weaker impact on spinogenesis in
cultured hippocampal neurons, suggesting that there is an alterna-
tive pathway downstream of neurofibromin to control spinogenesis
that does not require an interaction with VCP.
In conclusion, our study provides the first evidence to our
knowledge that VCP regulates dendritic spine formation, which
may correlate with the dementia phenotype in patients with
IBMPFD. Moreover, VCP interacts with neurofibromin to regu-
late dendritic spine formation, implying an interconnection
between NF1 and IBMPFD.
Human research subjects
Patients with NF1 were clinically assessed by clinical geneticists and/or
neurologists. All the clinical information pertinent to the diagnosis ful-
filled the NIH diagnostic criteria of NF1, and, if applicable, laboratory test
results from patients with a NF1 mutation were interpreted by a member of
our research team as well as referral physicians. The mental subnormalities
and school performance were reported by either the patients themselves or
their accompanying family members.
Mutation analysis of the NF1 gene
Two hundred and forty-six Taiwanese patients with NF1 were recruited.
Genomic DNA from peripheral blood samples of patients was extracted for
screening. Sequencing reactions of the region containing 6 exons (exons 27a,
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
27b, 28, 29, 30, and 31), which encompasses the LRD of neurofibromin, were
performed. In total, 18 sequence variants were identified, comprising 5 frame-
shift, 9 missense, 1 deletion, and 3 splicing aberrant mutations (Table 1).
Fourteen mutations (77.8%) led to a premature stop codon truncating the
LRD region. The genetics study was approved by Institutional Review Board
of the National Taiwan University Hospital, Taipei, Taiwan.
Antibodies and chemicals
The following antibodies were used in this report: neurofibromin NF1(N)
and GST (Santa Cruz Biotechnology Inc.), VCP (for immunoblotting, BD
Biosciences; for staining, Bethyl), GFP (Invitrogen), ubiquitin FK2 (Assay
Designs), HA (both mouse monoclonal 12CA5 and rat monoclonal, Roche),
and Myc 9B10 (Cell Signaling Technology). Simvastatin and lovastatin were
obtained from Sigma-Aldrich. MG132 was purchased from Calbiochem.
Rat brain or cells were homogenized in lysis buffer (1× PBS buffer contain-
ing 1% Triton X-100, 1–2 mM DTT, 2 μg/ml leupeptin, 2 μg/ml aprotinin,
10 μg/ml pepstatin, and 1 mM N-tosyl-l-phenylalanylchloromethyl ketone)
and centrifuged at 100,000 g at 4°C to remove cell debris. The solubilized
extract was subjected to immunoprecipitation using specific antibodies
and control non-immune IgG. After mixing by rotation for 4–5 hours at
4°C, the precipitates were sequentially washed 5 times with PBS contain-
ing 0.2% Triton X-100, twice with PBS, and once in 10 mM Tris-HCl buffer
(pH 7.4). The proteins in the washed precipitates were then analyzed by 2D
gel electrophoresis or immunoblotting.
NF1. Human neurofibromin fragments were amplified by PCR using the
following primers: (a) for CSRD (cysteine/serine-rich domain, aa 543–909
of type I protein), 5′-GAAGATCTCAGGAAGCAATGGAGGCTCTG and
5′-GAAGATCTTTACACTTTCTCATGGTTACACACCAT; (b) for GRD1
and GRD2 (GAP-related domain type I, aa 1168–1530 of type I protein,
and GAP-related domain type II, aa 1168–1551 of type II protein), 5′-GAA-
GATCTGGTTACCACAAGGATCTCCAGA and 5′-GAAGATCTTTAGT-
GCTCTGGAGGACCCAGGTAT; (c) for LRD (leucine repeat domain, aa
1545–1950 of type I protein), 5′-GAAGATCTAGTTCAAAGTTTGAG-
GAATTTATGACT and 5′-GAAGATCTTTAAACTCTTTGTCGTTTG-
GCATCATC; (d) for CTD (C-terminal domain, aa 2260–2818 of type I
protein), 5′-GAAGATCTGGACCTGACACTTACAACAGTCA and 5′-GAA-
GATCTTCACACGATCTTCTTAATGCTATT, where underlining denotes
the recognition sequence of the restriction endonuclease BglII. The PCR
products were then subcloned into the BglII site of the vectors HA-GW1 or
pGEX4T1. The full-length rat Nf1 expression construct was a gift of Shige-
ki Shibahara (Department of Molecular Biology and Applied Physiology,
Tohoku University School of Medicine, Tohoku, Japan). Site-directed muta-
genesis was carried out using the primer pair 5′-GGGAATCCTATTTTC-
TATGTTGCACGGAGGTTCAAA-3′ and 5′-TTTGAACCTCCGTGCAA-
CATAGAAAATAGGATTCCC-3′ to introduce the Y1587 deletion mutation
into the full-length rNf1 gene.
VCP. A set of primers — A: 5′-GGGGTACCGCCTCTGGAGCC-
GATTCAA; B: 5′-GAAGATCTTTAGCCATACAGGTCATCGTCATT; C:
5′-GAAGATCTTTAGCCTACTTCATTCAAGGATTCCTC; D: 5′-GAA-
GATCTTA TGATGACATCGGTGGTTGCA; E: 5′-GAAGATCTTTAATC-
CATAGTAACTGC CAGGGAATTC; F: 5′-GAAGATCTGACTTCCGGT-
GGGCTTTGAG — was used to amplify VCP fragments from EST clone
BC043053. The combinations used were as follows: (a) for full-length
VCP, primers A and B; (b) for fragment N, primers A and C; (c) for frag-
ment D1, primers D and E; (d) for fragment D2, primers F and B; (e) for
fragment ND1, primers A and E; and (f) for fragment D1D2, primers D
and B. The PCR products were then subcloned into vectors Myc-GW1 or
pET28a. For vector-based miRNA constructs, the target sequence No. 1,
5′-TAGGATAGCAGGATCAATGAT-3′; for mouse and rat Vcp genes and
the sequence No. 2, 5′-ATCCATTTCTGTCAGGATCTG-3′; for all human,
mouse, and rat VCP genes were determined by using the BLOCK-iT RNAi
Designer tool (Invitrogen). The corresponding paired oligonucleotides
were then inserted into the vector pcDNA 6.2-GW/EmGFP-miRNA
using the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP
(Invitrogen). The plasmid pcDNA 6.2-GW/EmGFP-miR-neg control was
employed as a negative control for knockdown experiments; it contained
the sequence 5′-GAAATGTACTGCGCGTGGAGACGTTTTGGCCACT-
GACTGACGTCTCCACGCAGTACATTT-3′ that was predicted not to
target any known vertebrate gene as an insert. The QuikChange XL Site-
Directed Mutagenesis Kit (Agilent) was applied to generate silent mutants
resistant to No. 1 miRNA constructs using primers 5′-CATTGATCCT-
GCTATTTTGAGACCTGGCCGTCTA-3′ and 5′-TAGACGGCCAGGTCT-
CAAAATAGCAGGATCAATG-3′ (mutated sites are underlined). Plasmid
EGFP-actin was purchased from Clontech.
Hippocampal neuronal culture and transfection
Culture and transfection of rat embryonic hippocampal and mouse embry-
onic cortical neurons were performed as described previously (9, 92).
Analyses of immunofluorescence staining and dendritic spine
To study dendritic spine formation, we performed transfection at 12 DIV,
and immunofluorescence staining at 18 DIV. Cultured hippocampal neu-
rons were washed twice in PBS and fixed in PBS containing 4% PFA and 4%
sucrose for 10 minutes at room temperature. After washing, cells were per-
meabilized with 10% methanol and 0.2% Triton X-100 in PBS for 5 minutes
and then blocked with 3% BSA/0.1% NHS in PBS for 1 hour. The primary
antibody was diluted in PBS containing 3% BSA and 0.1% normal horse
serum and added to cells for a further overnight incubation at 4°C. After
washing, secondary antibody conjugated with Alexa Fluor 488, 555, or 594
(Invitrogen) was added. Following incubation for 1 hour at room tempera-
ture and washing to remove unbound antibodies, samples were analyzed
with a confocal microscope (LSM510 or LSM700; Zeiss) equipped with a
Plan-Apochromat 63× NA 1.4 oil objective lens (Zeiss). Images were cap-
tured with LSM or Zen acquisition and analysis software (Zeiss) at 20–22°C
and acquired as Z-series of 5–12 sections spaced 0.6–0.8 μm apart. The Z-series
were then projected into single images. For publication, the images were
processed with Photoshop (Adobe), with minimal adjustment of bright-
ness or contrast applied to the whole images.
The intensities of VCP immunoreactivities were measured using
ImageJ software (http://rsbweb.nih.gov/ij/). The signals of GFP were
employed to outline the soma of transfected neurons. The intensities of
VCP protein were then compared in control and VCP miRNA–transfect-
ed neurons. Morphometric measurement was performed using ImageJ.
The number of spines present on the dendrites (along 20 μm of each
dendrite starting from a point 20 μm away from the soma) was manually
counted and traced. The maximum length from the tip of the spine to
the dendritic shaft and the head width of each spine were manually mea-
sured by ImageJ region measurement tools. The data were then exported
to Excel (Microsoft) for further analysis. The cumulative probability dis-
tributions were determined and statistically analyzed by KS test based
on the D and corresponding P values. In addition, the mean and SEM
of each group were determined and analyzed using a 2-tailed Student’s t
test. The experiments were repeated using at least 2–3 independent cul-
ture preparations. Because dendritic spine formation is highly sensitive
to culture conditions, such as the quality of B27 supplement (93), each
4836? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 12 December 2011
experiment was repeated using the same lot of B27 supplement. The
data from repeated experiments were pooled for statistical analysis only
when the variation of the control group was not significantly different
between repeated experiments.
Two-month-old Nf1+/– mice and WT littermates (94) were obtained from
the Jackson Laboratory and sacrificed by cervical dislocation. Whole brains
were taken, immediately placed into cold PBS, and then processed for Golgi
staining (Rapid GolgiStain Kit, FD NeuroTechnologies). Coronal sections
of 150-μm thickness were cut on a vibratome and plated on gelatin-coat-
ed microscopy slides. Images were collected using a Zeiss AxioImager-Z1
microscope. Pyramidal cells at the CA1 region of the hippocampus were
selected for determination of the number of spines.
We are grateful to the study participants. We also thank Sue-Ping
Lee and Yi-Ling Lin at the Institute of Molecular Biology, Aca-
demia Sinica, and the Proteomic Core Facility at the Institute of
Biological Chemistry, Academia Sinica, for technical assistance.
We further thank Gunnar Johansson for suggestions on the manu-
script and Heiko Kuhn for manuscript editing. H.-F. Wang is sup-
ported by a postdoctoral fellowship from Academia Sinica. This
work was supported by Academia Sinica (to Y.P. Hsueh) and grants
from the National Science Council (NSC 98-2321-B-001-002, NSC
99-2321-B-001-032, NSC-100-2321-B-001-032, and NSC 98-2311-
B-001-012-MY3 to Y.-P. Hsueh).
Received for publication November 5, 2010, and accepted in
revised form September 21, 2011.
Address correspondence to: Yi-Ping Hsueh, Academia Sinica, Insti-
tute of Molecular Biology 128, Academia Rd. Sec. 2, Taipei 115,
Taiwan. Phone: 886.2.27899311; Fax: 886.2.27826085; E-mail:
email@example.com. Or to: Ming-Jen Lee, Department of Neu-
rology, National Taiwan University Hospital, No. 7, Chung-Shan
South Road, Taipei 100, Taiwan; Phone: 886.2.23123456 ext.
65342; Fax: 886.2.23418395; E-mail: firstname.lastname@example.org.
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