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Mol. Cell. Neurosci. 32 (2006) 37 – 48
Local functions for FMRP in axon growth cone motility and
activity-dependent regulation of filopodia and spine synapses
Laura N. Antar,a Chanxia Li,b Honglai Zhang,a Reed C. Carroll,a and Gary J. Bassell b,*
a
Department of Neuroscience, Rose F. Kennedy Center for Mental Retardation, Albert Einstein College of Medicine, 1410 Pelham Parkway, Bronx,
NY 10461, USA
b
Departments of Cell Biology and Neurology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322, USA
Received 27 July 2005; revised 31 January 2006; accepted 14 February 2006
Available online 2 May 2006
Genetic deficiency of the mRNA binding protein FMRP results in the
most common inherited form of mental retardation, Fragile X
syndrome. We investigated the localization and function of FMRP
during development of hippocampal neurons in culture. FMRP was
distributed within granules that extended into developing axons and
growth cones, detectable at distances over 300 Am from the cell body.
In mature cultures, FMRP granules were present in both axons and
dendrites, with pockets of higher concentrations appearing intermittently, along distal axon segments and near synapses. MAP1b mRNA, a
known FMRP target, was also localized to axon growth cones.
Morphometric analysis of growth cones from the FMR1 KO revealed
both excess filopodia and reduced motility. At later stages during
synapse formation, FMR1 KO neurons exhibited excessive filopodia
and long spines along dendrites, yet there was a marked decrease in
the density of spine-like protrusions juxtaposed to presynaptic
terminals. In contrast, there was no difference in the density of shaft
synapses between FMR1 KO and WT. Brief depolarization of WT
neurons resulted in increased numbers of filopodia and spine synapses,
whereas no additional morphologic changes were observable in
dendrites of FMR1 KO neurons that already had increased density
of filopodia – spines. These findings suggest that alterations in the
regulation of axonal growth and innervation in FMR1 KO neurons
may contribute to the dendritic and spine pathology in Fragile X
syndrome. This work has broader implications for understanding the
role of mRNA binding proteins in developmental and proteinsynthesis-dependent plasticity.
D 2006 Elsevier Inc. All rights reserved.
Introduction
Fragile X syndrome (FXS) is caused by the lack of expression
of the Fragile X Mental Retardation Protein (FMRP), an mRNA
binding protein that localizes to several neuronal compartments,
including dendritic spines, where it may play a role in regulation of
protein-synthesis-dependent synaptic plasticity (Feng et al., 1997;
* Corresponding author. Fax: +1 404 727 0570.
E-mail address: [email protected] (G.J. Bassell).
Available online on ScienceDirect (www.sciencedirect.com).
1044-7431/$ - see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.mcn.2006.02.001
Antar et al., 2004). FMRP associates with over a hundred different
mRNAs (Brown et al., 2001; Miyashiro et al., 2003). The absence
of FMRP leads to translational dysregulation which can compromise neuronal and synaptic morphology (reviewed in Broadie and
Pan, 2005).
FMRP has been shown to associate with mRNAs that encode
proteins regulating the cytoskeleton and synapses. One such
FMRP target is the microtubule associated protein (MAP1b)
mRNA, which is inappropriately translated in both Drosophila
dFmr1 (dfxr) null (Zhang et al., 2001a,b; Pan et al., 2004) and
mouse FMR1 knockout models (Brown et al., 2001; Lu et al.,
2004). Furthermore, in Drosophila, genetic and biochemical
interactions have been observed between dFMRP and the small
GTPase Rac1 which regulate the actin cytoskeleton (Schenck et
al., 2003). Furthermore, in Drosophila, dFMRP was shown to
repress translation of profilin mRNA, encoding a regulatory actin
binding protein (Reeve et al., 2005). In addition, dFmr1 mRNA
interacts with the mRNA encoding the small GTPase Rac1 (Lee
et al., 2003). It remains unknown whether these dFMRP targets
are also bound by mammalian FMRP. In murine fibroblasts,
FMRP binds to the 5VUTR of mRNA encoding protein
phosphatase 2A catalytic subunit (PP2Ac), a cofilin phosphatase
(Castets et al., 2005). In mouse brain, FMRP is associated with
mRNA encoding calcium calmodulin-dependent protein kinase
IIa mRNA (Zalfa et al., 2003), which is known to be translated at
synapses (Scheetz et al., 2000). In cultured neurons from FMR1
knockout mice, there is deficient activity-induced synthesis of
postsynaptic density protein PSD-95 (Todd et al., 2003).
Collectively, these studies suggest a role for FMRP in the local
translation of proteins important for regulation of cytoskeletal and
synaptic structure.
Dendritic spines are the major postsynaptic locus of excitatory
synaptic innervation in the brain (Yuste and Bonhoeffer, 2004).
Within these specialized compartments, local protein synthesis
may drive synapse-specific plasticity underlying brain development, learning and memory (Steward and Worley, 2002). The loss
of the mRNA binding protein FMRP gives rise to an abnormal
dendritic spine phenotype that has been characterized by long, thin
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L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
immature looking spines in both Fragile X patients and FMR1
knockout mice (Irwin et al., 2000, 2001; Greenough et al., 2001).
In Golgi-stained sections from FMR1 KO mice, there also appears
to be an increase in total spine density in vivo (Irwin et al., 2002);
however, this phenotype was only transient during postnatal
development in a subsequent study (Nimchinsky et al., 2001).
The spine phenotype in FXS may be due to altered translational
regulation needed for spine development and maturation of
morphologic form during a critical period.
FMRP may be regulated by neuronal activity, specifically glutamatergic signals known to affect dendritic spines and synaptic
plasticity. Activation of metabotropic glutamate receptors
(mGluR) can stimulate the local synthesis of FMRP (Weiler et
al., 1997) and also promote its trafficking from the soma into
dendrites (Antar et al., 2004, 2005). mGluR activation is also
associated with a protein-synthesis-dependent lengthening of
dendritic spines (Vanderklish and Edelman, 2002) and long-term
depression LTD at hippocampal synapses (Huber et al., 2001).
Interestingly, in FMR1 knockout mice, there is an exaggerated
mGluR-dependent LTD (Huber et al., 2002). This suggests that
mGluR signaling affects learning and memory in a pathway that
involves FMRP.
In this study, we have investigated the localization and
function of FMRP during hippocampal neuronal development in
culture. Here, we report the presence of FMRP in growth cones
of developing axons, as well as within distal segments of
mature axons that innervate dendrites. We also show that
FMRP-associated MAP1b mRNA is localized to axon growth
cones. Analysis of FMR1 KO neurons revealed altered growth
cone morphology and motility and loss of activity-dependent
regulation during spine synapse formation. We show that FMRP
also controls morphologic changes of filopodia and spines
during activity-dependent synaptogenesis. This research is
significant for understanding the role of FMRP in regulation
of neuronal morphology and developmental plasticity and the
impairments in these processes that contribute to Fragile X
syndrome.
Results
High resolution localization of FMRP in dendrites, axons and
spine synapses
We have previously shown that FMRP is distributed in granules
within dendrites and spines of cultured rat hippocampal neurons
(Antar et al., 2004). Quantitative analysis indicated that over 70%
of spine synapses, defined as F-actin-rich protrusions colocalized
with synapsin, contained FMRP. Here, we have cultured hippocampal neurons from newborn mice isolated from WT and FMR1
KO. Fluorescence imaging depicted phalloidin staining of F-actin
(green) that labeled dendritic surface protrusions (Fig. 1A). FMRP
was distributed in granules throughout the dendrites, including
distal branches (Fig. 1B). Higher magnification analysis of
deconvolved and 3D-reconstructed dendritic subregions depicted
FMRP signal within phalloidin-labeled spines (Fig. 1E, arrows).
FMRP granules (red) were often colocalized to sites of synaptic
contacts (Figs. 1E, F), as identified with anti-synapsin (blue). Of
interest, FMRP was also detected in the thinner axonal processes;
one such distal axon branch is shown depicting fifteen FMRP
granules (Fig. 1F, yellow arrowheads). As a control, we did not
observe FMRP immunostaining in dendrites or axons from FMR1
KO neurons (Fig. 1H).
High resolution analysis of dendrite and spine morphology in WT
and FMR1 KO
The morphologic appearance of dendritic surface protrusions,
in wild-type and FMR1 knockout neurons, was visualized using
high resolution immunofluorescence and 3D reconstruction. In
one approach, WT and FMR1 KO neurons were triple stained
with phalloidin, synapsin and FMRP (Fig. 1). In the second
scheme, neurons were triple stained with phalloidin, MAP2 and
synapsin (Fig. 2). Fluorescence images were acquired through a
Z-series, deconvolved and 3D-reconstructed. Visual analysis of
3D reconstructions frequently depicted an excess of long, thin
dendritic protrusions in FMR1 KO, compared to WT, and these
protrusions were often not associated with synapsin puncta (Figs.
1K, L—black arrowheads; Figs. 2K, L—black arrowheads).
Spine synapses, as defined as stubby or long protrusions,
colocalized with synapsin, were more frequent in WT dendrites
than KO.
In our quantitative analysis of protrusion density, those having a
long and thin morphology were designated as filopodia – spines
(Fig. 3A), as done by others (Prange and Murphy, 2001), since it is
difficult during this stage of development in culture (2 weeks) to
unequivocally distinguish spines from filopodium based solely on
morphologic criteria. This is because filopodia are abundant and
many spines have an ambiguous morphology (long, thin filopodiallike morphology, often lacking an obvious terminal swelling or
bulbous head). FMR1 KO neurons had 99.44% more dendritic
protrusions (filopodial – spines) than their WT counterparts (WT:
2.88 T 0.25 vs. KO: 4.88 T 0.07 per 10 Am, P < 0.0004, Student’s t
test). In contrast, quantitative analysis of those protrusions which
colocalized with synapsin, i.e. spine synapses, showed that KO had
a 60.16% decrease in their overall number (Fig. 3B) (WT: 4.02 T
0.38 vs. KO: 2.51 T 0.22, P < 0.0010, Student’s t test). In order to
determine if the density of protrusion associated with presynaptic
terminals differed by morphology, they were further divided into
two morphologic categories: ‘‘short’’ or ‘‘long’’. In both cases,
there was a significant diminution of dendritic protrusions
associated with synapsin in the KO (Figs. 3C, D) (long
protrusions/synapsin: 1.66 T 0.186 WT vs. 0.8561 T 0.137, P <
0.0009; short protrusions/synapsin: 2.37 T 0.25 WT vs. 1.66 T
0.17, P < 0.0243, Student’s t test). Decreased protrusion/synapsin
density in the FMR1 KO was not due to an overall decrease in total
synapse density (WT 9.99 T 0.98 vs. KO 8.42 T 0.52, P = 0.1562,
Student’s t test). Note that the vast majority of synapses are shaft
synapses, and there was no statistically significant difference in the
total density of synapsin puncta (n = 400) following analyses of
over fifteen neurons (two dendrites per neuron).
FMR1 KO neurons have loss of activity-dependent regulation of
filopodia – spine and spine synapse number
We examined whether KCl stimulation affects protrusion
number differently in FMR1 KO neurons compared to WT. We
quantified both the total density of protrusions (filopodial –
spines) (Fig. 3A) and protrusions that did colocalize with
synapsin (Figs. 3B, C, D). In WT cultures, there was a
significant increase (56.94%) in protrusion density after KClinduced depolarization (2.88 T 0.25 vs. 4.52 T 0.51, P < 0.014,
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
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Fig. 1. High resolution localization of FMRP within dendrites, axons and synapses of cultured mouse hippocampal neurons (16 DIV). (A – F) Wild-type (WT)
neuron showing triple label detection of F-actin using FITC-phalloidin (A, green), anti-FMRP (B, red) and anti-synapsin (C, blue). Optical sections were
deconvolved and 3D-reconstructed (see Experimental methods). Overlay of all three (D) depicts two regions of interest (insets e, f) enlarged in panels E and F.
(E) FMRP granules were distributed throughout the dendrite and were detectable in bulbous spines (white arrows) and long spines (yellow arrow). FMRP
granules (red) often colocalized with synaptic contacts (blue). (F) FMRP granules were present in both the thicker dendrite, often clustered beneath synapses
(white arrowheads), as well as within the thinner distal axonal branches (yellow arrowheads) that made contacts with the dendrite. (G – L) FMR1 KO neuron
showing triple label detection of F-actin (G, green), anti-FMRP (H, red) and anti-synapsin (I, blue). (H) FMRP immunostaining was not detectable in KO
neurons. (J – L) Dendrites from FMR1 KO neurons displayed excess of long protrusions (black arrowheads) that did not appear to have synaptic contacts,
despite the abundance of synaptic contacts (blue) along the dendritic shaft.
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L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
Fig. 2. High resolution analysis of dendritic and spine morphology in WT and FMR1 KO neurons (16 DIV). (A – F) Wild-type (WT) neuron showing
triple label detection of F-actin using FITC-phalloidin (A, green), anti-synapsin (B, red) and anti-MAP2 (C, blue). Optical sections were deconvolved
and 3D-reconstructed (see Experimental methods). Overlay of all three (D) depicts two regions of interest (insets e, f) enlarged in panels E and F. (E)
Detection of MAP2 filled the dendritic shaft (blue), whereas F-actin marked spine-like protrusions (green) that were short and stubby (white arrows) or
long and thin (yellow arrowhead); both types juxtaposed to synaptic contacts (red). Long, thin spines were occasionally noted that did not colocalize
with anti-synapsin (black arrowhead). (G – L) In contrast, FMR1 KO neurons displayed an excess of long protrusions, having either filopodial form or
spine-like terminal swellings, which were not colocalized with synaptic contacts (black arrowheads in panels K and L). FMR1 KO neurons less
frequently displayed long and thin protrusions (yellow arrowhead) or short and stubby spines (white arrowhead) that were contacted on their surface by
anti-synapsin puncta (red).
Student’s t test), but in KO cultures, the already high numbers of
protrusions occluded any further significant increase by KCl
(Fig. 3A). Similarly, the density of protrusions/synapsin in WT
neurons increased upon KCl stimulation by 52% (Fig. 3B, no
drug: 4.02 T 0.37 vs. stimulus: 6.12 T 0.399, P < 0.0004,
Student’s t test), whereas the density of protrusions/synapsin
from KO neurons did not respond to KCl-induced depolarization
(no drug: 2.52 T 0.22 vs. stimulus: 2.36 T 0.25, P < 0.0006,
Student’s t test). In the KO, the absence of KCl-induced
increases in protrusion/synapsin density was apparent from both
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
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Fig. 3. FMR1 KO neurons show impaired activity-dependent regulation of total dendritic protrusions: filopodia and spines. (A) Quantitative analysis of total
long, thin protrusions (designated filopodia – spines) indicated that KO neurons have increased density compared to WT. In response to KCl depolarization, WT
dendrites extrude increased numbers of filopodial – spine protrusions, whereas further KCl-induced increases were occluded in FMR1 KO neurons by already
high number of protrusions. This analysis excluded short and stubby protrusions. (B) The density of protrusions in contact with a synapsin punctum was
reduced in the KO compared to WT. KCl depolarization increased protrusion (synapsin positive) density in WT, but not FMR1 KO neurons. (C, D) Similarly,
decreased protrusion (synapsin positive) density and absence of KCl-induced increases were also observed when protrusions were subclassified as long and
thin (C) or short and stubby that lacked a stem (D); both colocalized with synapsin puncta.
types of classification: long, short spine synapses (Figs. 3C, D).
These results indicate that neuronal activity can stimulate the
growth of dendritic protrusions, likely both filopodia and spines
(including spine synapses), yet FMR1 KO neurons are deficient
in these morphologic responses. Instead, the KO phenotype is
characterized by an excess of apparently uninnervated protrusions and a reduction in spine synapses.
Localization and function of FMRP at the axonal growth cone
In younger neurons (4 DIV), FMRP granules localized to
both developing dendrites and axons and were detectable at
distal sites within growth cones. Examination of a polarized
neuron is shown in Fig. 4 that depicts the presence of FMRP
granules in an axon that is 340 Am in length. FMRP granules
distributed to the distal-most axon and growth cone where it
extended into filopodial tips (Fig. 4E). We also performed
fluorescence in situ hybridization (FISH) using oligonucleotide
probes to MAP1b mRNA, a known FMRP target mRNA
(Darnell et al., 2001; Lu et al., 2004; Antar et al., 2005), and
similarly demonstrated that it, too, is detectable in growth cones
of developing axons (Fig. 5).
Next, we analyzed the density of filopodia on growth cones of
developing neurites at early stages in culture (4 DIV), in neurons
cultured from WT or FMR1 KO mice. There was a hyperabundance of filopodia emerging from growth cone heads of
FMR1 KO neurons compared to their WT counterparts (Fig. 6).
This increase in filopodial number was also observed during timelapse video microscopy (see Supplemental data). Five images were
taken from corresponding timepoints in each 15-min movie and
averaged to represent the mean number of filopodia in that growth
cone. Quantification revealed that FMR1 KO growth cones contain
significantly more filopodia than their WT counterparts in live
neurons (KO 13.11 T 1.51; WT 8.50 T 1.09; P = 0.0001, Student’s t
test). This dynamic analysis of filopodial excess in live KO
neurons compliments the data done on fixed cells. We note that the
increased density of growth cone filopodia was not attributed to
changes in the overall area of the growth cone (WT 109.04 T
16.34, KO 112.31 T 19.87, Student’s t test) expressed as variance
over mean (Fig. 6D).
To determine if the filopodial phenotype from FMR1 KO
neurons was reversible, EGFP – FMRP or EGFP alone was
transfected and allowed to express overnight (Fig. 7). To visualize
F-actin containing filopodia, transfected neurons were stained with
rhodamine-phalloidin (Figs. 7A, B). We observed that FMR1 KO
neurons expressing EGFP – FMRP had reduced number of growth
cone filopodia (6.38 T 0.45) compared to cells express EGFP alone
(13.22 T 1.05) (Fig. 7C). Filopodial density in KO expressing
EGFP – FMRP was even less than non-transfected WT neurons
(8.50 T 1.09; see Fig. 6C), perhaps because overexpressed EGFP –
FMRP, in rescued KO neurons, is present at higher levels than
endogenous FMRP in WT neurons.
Functional differences in FMR1 KO and WT neurons were
shown to include significant differences in growth cone motility as
measured by total growth cone distance (absolute value) traveled
in microns. Movement of growth cone centroids of both FMR1
KO and WT mice were tracked on their x and y axes. Histories
shown for WT and FMR1 KO growth cones reveal tighter
clustering of the centroid in the FMR1 KO growth cone when
compared to the more dynamic behavior of the WT growth cone
(Fig. 8A). Then, all growth cone x, y coordinates were averaged
together to examine total distance traveled, as a measure of growth
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Discussion
Local functions for FMRP in regulation of neuronal morphology
during development
In cultured hippocampal neurons, FMRP was localized to both
growth cones of developing axons and spine synapses within
dendrites and axons. In that neuronal activity and specific signals
can stimulate FMRP trafficking within dendrites and synapses
(Antar et al., 2004, 2005), the localization of FMRP may be
essential for its function in control of neuronal morphology. In this
study, we observe that FMR1 KO neurons display excessive
growth cone filopodia and dendritic filopodia – spines. In addition,
there was loss of growth cone dynamics (in young neurons) and
inability of dendrites to respond to activity and sculpt new
protrusions, including spine synapses.
Our findings of excess filopodia and reduced growth cone
dynamics in neurons from the FMR1 knockout mouse have some
parallels to morphologic impairments observed in the dFmr1 (dfxr)
null fly. In the fly, the dfxr phenotype includes axonal overgrowth
of muscle, enlarged presynaptic terminals (Zhang et al., 2001a,b),
excessive axonal branching and increased architectural complexity
(Pan et al., 2004). Collectively, these studies indicate a broad role
of FMRP as regulator of neuronal morphology during development. These observations have broader implications for understanding the pathogenesis of Fragile X syndrome.
A possible role for FMRP in spine development and spine synapse
stabilization
Fig. 4. Localization of FMRP to distal axon and growth cone. Cultured WT
neurons were fixed (4 DIV) and processed for immunofluorescence
detection of FMRP (red) and F-actin (green). (A) Three overlapping DIC
images of a polarized neuron were acquired at low magnification and
merged to show entire neuron and its processes. This axon is 340 Am in
length. The axon growth cone (inset) was imaged at higher magnification to
depict F-actin (B, green) and presence of FMRP granules (C, red). (D, E)
Image reconstruction of deconvolved Z-series shows merge of FMRP
granules (yellow arrowheads) and F-actin (green) in growth cone filopodia
(inset from panel D is enlarged in panel E).
cone motility, which demonstrated that FMR1 KO growth cones
had diminished rate of movements (protrusive and retractive) over
time (Fig. 8B).
The spine phenotype exhibited in FXS and in FMR1 KO mice
has been extensively studied (Irwin et al., 2000, 2001, 2002).
Through Golgi impregnation of neurons, it has been shown, both in
humans and FMR1 KO mice, that there is an abnormal elevation in
protrusion number and an accompanying morphologic anomaly:
overly long, thin protrusions (Greenough et al., 2001). Additionally, EGFP expression of hippocampal neurons in slices has shown
that this abnormal phenotype exists, only transiently in the first
postnatal week (Nimchinsky et al., 2001). However, these studies
did not examine whether these excessive spines were actually
innervated. This information is important in order to understand the
disease pathogenesis.
Our data suggest that FMR1 KO dendrites have an excess in
the total number of dendritic surface protrusions (referred to as
filopodia – spines). These long protrusions include both thin spines
and dendritic filopodia, which have been shown in some studies to
be spine precursors (Jontes and Smith, 2000), yet other evidence
indicates that they are separate structures important for formation
of shaft synapses (Fiala et al., 1998). An excess of dendritic
filopodia has been described in various forms of mental
retardation (Purpura, 1974). Dendritic filopodia densely populate
dendrites during early dendritic development, where they initiate
contact with local axons, ultimately recruiting functional presynaptic boutons (Ziv and Smith, 1996). While filopodial density
normally decreases following synaptogenesis (Jontes and Smith,
2000), filopodia may remain in excess in disease states, perhaps
due to delayed synaptic contacts. Alternatively, an excess in
filopodia in FXS may be a consequence of the loss of mature
spines (Fiala et al., 1998).
In contrast to the excess of long protrusions (filopodia – spines),
we observed a significant reduction in the density of protrusions
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
43
Fig. 5. Localization of MAP1b mRNA granules in axonal growth cones. Cultured neurons were fixed (4 DIV) and processed for fluorescence in situ
hybridization (FISH) using digoxigenin-labeled oligonucleotide probes which were detected by immunofluorescence. (A) DIC image of neuron and axonal
growth cone (inset) is enlarged at the right showing (B) DIC, (C) FISH and (D) merge. MAP1b mRNA granules are detected in the axon shaft and growth cone.
(E – H) Another axon growth cone is shown that has navigated upward to contact a cell body from another neuron. (G) MAP1b mRNA granules are present in
the growth cone. (I – L) Control neuron that was hybridized with sense probe revealing only sparse background signal (K, L).
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L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
Fig. 6. Hyperabundance of filopodia emerges from growth cones of cultured hippocampal neurons (3 DIV) from FMR1 KO. Selected phase images from timelapse video-microscopy show growth cones from (A) WT and (B) FMR1 KO neurons (micron bars, 5 Am). (C) Quantitative analysis shows that growth cones
from FMR1 KO neurons contained significantly more filopodia than their WT counterparts (P < 0.0001). (D) There was no difference in growth cone area
between FMR1 KO and WT neurons. See Supplementary data.
that contain synapsin immunoreactive presynaptic terminals,
suggesting a reduction in spine synapse number in the FMR1
KO. Our observations of spine synapse loss are consistent with
morphologic and physiological findings of a delay in the formation
of functional spines in hippocampal neurons from FMR1 KO
(Braun and Segall, 2000). During the first week in culture, KO
neurons do not form synaptic contacts as quickly as WT, as
determined through measurement of spontaneous synaptic activity.
Furthermore, KO cultures have fewer mono- and poly-synaptic
connections at 2 – 3 weeks in culture, as determined by a longer
duration to afferent stimulation in WT than KO and by a smaller
total number of excitatory events per response in KO (Braun and
Segall, 2000).
It has been hypothesized that the structural and functional
defects in FXS may result from impaired metabotropic glutamatereceptor-dependent plasticity (Huber et al., 2002; Bear et al.,
2004). The potential spine loss associated with exaggerated LTD
may be followed by excess growth of immature and poorly
innervated spines. The finding of diminished spines with mature
bulbous morphology (Irwin et al., 2000) may also account for the
low numbers of AMPARs found in synaptosomes from cerebral
cortex of FMR1 KO animals (Li et al., 2002). There may be fewer
AMPARs because there are excessive filopodia or immature spines
that are devoid of AMPARs and fewer mature spines that do house
AMPARs.
While a number of studies indicate that filopodial and spine
outgrowth are driven by neuronal activity and glutamatergic
signals (Jontes and Smith, 2000), our findings suggest that the
inter-relationships between these structures can be modulated by
FMRP. We show here that KCl depolarization can promote
increases in both the density of filopodia and spines. However,
in the FMR1 KO, the already high levels of these protrusions
occluded any further addition in response to KCl. One speculation
is that FMRP may act to limit or repress morphologic changes at
basal states and permit activity-dependent changes in synaptic
connections.
Possible FMRP-mediated translation involved in spine
development and stabilization
Future analysis of the FMR1 KO phenotype may reveal
impaired activity-dependent translation of proteins that are needed
for spine synapse maturation or stabilization, perhaps including
those proteins involved in the assembly of the postsynaptic density
and/or regulation of glutamate receptor insertion and cycling. It has
been noted that there appears to be an excess of protein synthesis in
synaptosomes isolated from FMR1 knockout mice (Zalfa et al.,
2003). CaMKIIa and Arc mRNAs were both abnormally elevated
in polyribosomes fractions from FMR1 KO brain (Zalfa et al.,
2003). It will be interesting to discern if the excess in protein
synthesis under basal states occludes any further stimulus-induced
translation needed for filopodial conversion or spine maturation. In
support of this idea, FMR1 KO neurons have reduced mGluRdependent translation of PSD-95 (Todd et al., 2003), which could
impair the ability to enrich PSD-95 at developing synapses and
impair spine development.
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
45
Fig. 7. Transfection of human FMRP (EGFP fusion) into FMR1 KO neurons rescued the growth cone filopodial phenotype. The images show phalloidin
staining of F-actin in FMR1 KO neurons transfected with the marker EGFP (not shown). (A) FMRI KO neurons transfected with EGFP show an abnormally
increased number of growth cone filopodia like untransfected neurons. (B) KO neurons transfected with EGFP – FMRP show a normal number of growth cone
filopodia, comparable to the number seen in WT cells. (C) Quantification of filopodial numbers depicted in panels A and B.
The filopodial phenotype of FMR1 KO neurons is recapitulated in
the growth cone
Here, we report the localization of FMRP granules to the
leading edge of axonal growth cones. Analysis of growth cones
from the FMR1 KO at 4 DIV using time-lapse microscopy of live
neurons revealed an excess of filopodia, analogous to the dendritic
phenotype of more mature neurons. In addition, we observed that
growth cones from FMR1 KO neurons were much less dynamic.
These exhibited a net decrease in the total distance traveled, as
measured by summating forward and rearward displacements
(absolute values) of the growth cone center and dividing by time.
While it is clear that local protein synthesis can affect chemotropic
responses of growth cones to axon guidance molecules (Campbell
and Holt, 2001), the identity of the mRNAs involved in this
response has not been well characterized. One example is the
localization of h-actin mRNA to growth cones that is mediated by
binding of the zipcode by the mRNA binding protein ZBP1 (Zhang
et al., 2001b). Disruption of this interaction results in growth cones
that cannot protrude and often show retractive behavior (Zhang et
al., 2001b). However, this phenotype is very different from that
described here for FMR1 KO neurons, where net extension is
unaffected, but there is failure to display dynamic cycles of
protrusion and retraction during net neuritic growth.
The growth cone phenotype of FMR1 KO neurons likely results
from impaired translation of many FMRP-target mRNAs. One
attractive candidate mRNA is microtubule associated protein
MAP1B mRNA (Darnell et al., 2001). In cultured neurons from
FMR1 knockout mice, there was excessive translation of MAP1b
mRNA and delayed down-regulation of MAP1b protein during
development (Lu et al., 2004). The aberrantly elevated MAP1b
protein was correlated with abnormally increased microtubule
stability in FMR1 knockout neurons. Here, we also show that
MAP1b mRNA is localized to axon growth cones. It will be
interesting to test whether altered local regulation of MAP1b mRNA
in growth cones contributes to their impaired motility in FMR1 KO.
Loss of FMRP may result in impaired regulation of Rho
GTPases that control the actin cytoskeleton (Schenck et al., 2003).
It is known that FMRP and dFMRP also bind and regulate
translation of mRNAs encoding proteins affecting F-actin assembly, including Rac1, cofilin phosphatase and profilin (Lee et al.,
2003; Castets et al., 2005; Reeve et al., 2005). It is of interest that
mutations of PAK3 (p21-activated kinase 3), which is in the same
pathway as Rac1 and cofilin, also lead to mental retardation, and its
genetic loss in cultured neurons results in excess of filopodial
protrusions and decreased density of mature spine synapses (Boda
et al., 2004). These and other studies provide interesting links
between Rho proteins, spine development and mental retardation
(van Galen and Ramakers, 2005) and suggest prolonged retention
of dendritic filopodia and delayed spine synapse development as a
common feature of mental retardation. It will be interesting to
know whether dysregulation of these actin pathways also leads to
46
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
decapitated and hippocampi removed and dissociated. Cells from KO and
WT cultures were determined to have comparable survival rates and were
plated at 50K cells/60-mm dish. Neurons were plated on poly-l-lysinecoated coverslips (1.0 mg/ml), either on 15-mm glass coverslips for
immunofluorescence and in situ hybridization or onto Bioptechs (Bioptechs, Inc., Butler, PA) coverslips for live cell imaging. Neurons were
attached to the substrate in minimal essential medium (MEM) with FBS
(10%) for 2 h, inverted onto dishes containing astroglia and grown in
defined Neurobasal Medium (Gibco, Grand Island, NY) with Glutamax
(Gibco) and B-27 supplements (Gibco) (Goslin et al., 1998), except as
noted. Cells, control or drug-treated, were fixed with 4% paraformaldehyde
in 1 PBS at RT for 18 min or processed for live cell imaging.
Drug treatments
Bath application of 10 mM KCl was done for 30 min directly in the
culture dishes containing coverslips of hippocampal neurons.
Immunofluorescence
Immunofluorescence (IF) was performed as described previously
(Antar et al., 2004) on 16 DIV cultures for filopodial and spine analysis
and 4 DIV for growth cone analysis. For primary antibody incubations (1
h, RT), FMRP was detected with mouse antibody (1C3; 1:500, Chemicon,
Temecula, CA) and synapsin with a polyclonal antibody (1:500, Sigma).
For secondary antibody incubations (1 h, RT), a Cy3 or Cy5 flourochromeconjugated anti-mouse or anti-rabbit antibody was used (1:750) (Jackson
ImmunoResearch, West Grove, PA). Alexa 488-conjugated phalloidin (or
TRITC phalloidin for EGFP-transfected cells) was used to stain F-actin
(1:500; Molecular Probes, Eugene, Oregon).
Fluorescence in situ hybridization (FISH)
Fig. 8. Growth cones from FMR1 knockout neurons have impaired
dynamics and speed. (A) x – y coordinates were plotted every 15 s for
each growth cone centroid over 15 min. Lines connect the centroid pathway
over the time course. Histories are shown for WT and FMR1 KO growth
cones. Note the tight clustering of the centroid in the FMR1 KO growth
cone when compared to the more dynamic behavior of the WT growth
cone. This difference appears to be facilitated by an increase in motility. (B)
Total growth cone movement (protrusion and regression) was tracked every
15 s for 15 min from live KO and WT neurons at 3 DIV. Each point
represents the average motility of several growth cones by measuring
movement of centroids (geometric centers) of each growth cone over 1 min.
FMR1 KO growth cones had significantly diminished growth cone motility
( P < 0.05). The total distance traveled with unweighted averages is
depicted in the inset ( P < 0.05).
impaired axon growth cone and presynaptic innervation in FXS.
Thus, the phenotype of FXS may be broader in scope than
previously thought, having both dendritic and axonal phenotypes
that converge at the developing synapse.
Experimental methods
Five antisense oligonucleotide probes (50 nts) were designed to mouse
microtubule associated protein MAP1b mRNA (see Supplementary data).
Sense oligonucleotides were synthesized as negative controls. Oligonucleotides were synthesized on a DNA synthesizer incorporating four
amino-modified thymidines (C6dT), purified and chemically labeled using
digoxigenin succinamide ester (Roche Molecular Biochemicals) as
described previously (Antar et al., 2004). In situ hybridization was
completed as previously described (Antar et al., 2004, 2005). The
digoxigenin-labeled oligonucleotide probes were detected by immunofluorescence using Cy3-conjugated monoclonal antibody to digoxigenin and
Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch) as
described previously (Antar et al., 2004). Coverslips were mounted with
gelvatol with n-propyl gallate (6 mg/ml) as an anti-bleaching agent.
Image acquisition and analysis
Fluorescence was visualized using 60 or 40 Plan-Neofluar
objectives, 100 W mercury arc lamp and HiQ bandpass filters (Chroma
Tech) on a Nikon Eclipse inverted microscope. Images were captured with a
cooled CCD camera (Quantix, Photometrics) from eight cultures using IP
Lab software (Scanalytics). Neurons labeled by triple label fluorescence
were imaged in each channel along the z axis and deconvolved using a 3D
blind algorithm (Autoquant X, Bitplane). Images were thresholded,
superimposed and registered using fiduciary beads present in the mounting
medium. Volume rendering and 3D reconstruction were performed using
Imaris software (Bitplane, Inc. USA).
Hippocampal culture
Morphologic definitions
Hippocampi were dissected and cultured from hemizygous male and
homozygous female FMR1 KO mice ( P 0 – P 1) and their wild-type
counterparts of the same genetic background, birthed from separate litters
on the same day (Jackson Lab, Bar Harbor, ME). Paired wild-type and KO
experiments were performed on the same day at the same time using cells
grown on the same glia and medium in a co-culture system. P0 mice were
Protrusions having a long, thin morphology were designated as
filopodia – spines (Fig. 3A), as done by others (Prange and Murphy, 2001),
since it is difficult during this stage of development in culture (2 weeks) to
unequivocally distinguish spines from filopodium based solely on morphologic criteria. This is because filopodia are abundant and spines often have
L.N. Antar et al. / Mol. Cell. Neurosci. 32 (2006) 37 – 48
47
an ambiguous morphology (long, thin filopodial-like morphology, often
lacking an obvious terminal swelling or bulbous head). Dendritic protrusions
that were juxtaposed to synapsin-positive punctum were designated
‘‘protrusions/synapsin’’ (Fig. 3B). These were further subclassified as ‘‘long
protrusions/synapsin’’ (Fig. 3C) or ‘‘short protrusions/synapsin’’ (Fig. 3D),
this later category likely representing spine synapses having a bulbous or
stubby morphology with a short neck or lacking a visible neck.
Data for protrusion density measurements were collected from a single
primary and two secondary dendrites, the former measured at least 10 Am
from the cell soma (for statistical analysis, n = protrusions per 10 Am of
dendrite). WT neurons and KO neurons were examined from 6 separate
cultures, ten to fifteen neurons per experimental condition per culture. For
each condition, this analysis involved the scoring of over 300 protrusions.
Xing for assistance with figure preparation. We also thank Steve
Warren for FMR1 knockout mice. This work was supported by
FRAXA fellowship to L. Antar and NIH NS051127 and Dana
Foundation awards to G. Bassell.
Live cell imaging
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The human EGFP – FMRP behind the human FMR1 promoter was
generated as follows: a 2.8-kb EcoRI/NheI fragment of the human FMR1
promoter from plasmid pE5.1 (a generous gift of Dr. David Nelson) was
cloned into the EcoRI site of pEGFP-1 (BD Biosciences Clontech) to
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pEGFP-C1 (BD Biosciences Clontech) to put FMRP in frame with EGFP.
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Acknowledgments
We thank Jennifer Darnell for the EGFP – FMRP construct
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.mcn.2006.02.001.
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