Control of Axonal Growth and Regeneration of Sensory
Neurons by the p110d PI 3-Kinase
Britta J. Eickholt1*, Aminul I. Ahmed1, Meirion Davies2, Evangelia A. Papakonstanti3, Wayne Pearce3, Michelle L. Starkey2, Antonio Bilancio3,
Anna C. Need4, Andrew J. H. Smith5, Susan M. Hall2, Frank P. Hamers6, Karl P. Giese4, Elizabeth J. Bradbury2, Bart Vanhaesebroeck3,7
1Medical Research Council Centre for Developmental Neurobiology, King’s College London, London, United Kingdom, 2Neurorestoration Group,
Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom, 3Ludwig Institute for Cancer Research, London, United
Kingdom, 4Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King’s College London, London, United Kingdom, 5Gene Targeting
Laboratory, The Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom, 6Rudolf Magnus Institute of Neuroscience,
University Medical Centre Utrecht, Utrecht, The Netherlands, 7Department of Biochemistry and Molecular Biology, University College London,
London, United Kingdom
The expression and function of the 8 distinct catalytic isoforms of PI 3-kinase (PI3K) in the nervous system are unknown.
Whereas most PI3Ks have a broad tissue distribution, the tyrosine kinase-linked p110d isoform has previously been
shown to be enriched in leukocytes. Here we report that p110d is also highly expressed in the nervous system.
Inactivation of p110d in mice did not affect gross neuronal development but led to an increased vulnerability of dorsal
root ganglia neurons to exhibit growth cone collapse and decreases in axonal extension. Loss of p110d activity also
dampened axonal regeneration following peripheral nerve injury in adult mice and impaired functional recovery of
locomotion. p110d inactivation resulted in reduced neuronal signaling through the Akt protein kinase, and increased
activity of the small GTPase RhoA. Pharmacological inhibition of ROCK, a downstream effector of RhoA, restored axonal
extension defects in neurons with inactive p110d, suggesting a key role of RhoA in p110d signaling in neurons. Our data
identify p110d as an important signaling component for efficient axonal elongation in the developing and regenerating
Citation: Eickholt BJ, Ahmed AI, Davies M, Papakonstanti EA, Pearce W, et al (2007) Control of Axonal Growth and Regeneration of Sensory Neurons
by the p110d PI 3-Kinase. PLoS ONE 2(9): e869. doi:10.1371/journal.pone.0000869
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases
which regulate a wide variety of biological responses in different
cell types . In the nervous system, PI3K activity contributes
to the establishment of appropriate connectivity by regulating
various cellular processes, including neuronal differentiation,
survival, migration, extension and guidance [2–5]. The 8
isoforms of mammalian PI3Ks have been grouped into three
classes (I, II, and III) . The class IA subset of PI3Ks signal
downstream of Tyr kinases and Ras, and are heterodimers
composed of one of three p110 catalytic subunits-p110a, p110b
or p110d-in complex with one of the three regulatory subunit
(collectively called ‘p85s’). Detailed information on the tissue
distribution of the p110a and p110b isoforms is not available,
although evidence for a broad expression of both isoforms has
been presented [6–9]. On the other hand, p110d is known to be
highly enriched in leukocytes [7,10,11]. Gene-targeting studies
in the mouse have uncovered non-redundant roles of specific
p110 PI3K isoforms in immunity, metabolism and cardiac
function [12,13]. In contrast, the expression and function of the
distinct PI3K isoforms in the nervous system have not been
Here, we report that expression of p110d PI3K is highly
enriched in the embryonic nervous system in the mouse at stages
concomitant with the extension and guidance of neuronal
processes. Genetic or pharmacological inactivation of p110d in
sensory neurons led to a reduction in PI3K signaling, increased
sensitivity to growth cone collapse and deficient axonal elongation
under limiting growth conditions. In addition, mice with inactive
p110d show impaired axonal regeneration and functional recovery
following a sciatic nerve crush injury. These results identify p110d-
mediated PI3K signaling as a crucial component for efficient
RESULTS AND DISCUSSION
p110d expression is highly enriched in the nervous
To date, no detailed information on the distribution of the distinct
PI3K isoforms in neuronal tissue has been reported. In order to
analyze the expression of p110a and p110d, reporter mice were
generated in which a b-Gal/LacZ reporter gene was inserted into
the endogenous p110a or p110d gene locus by homologous
recombination [14,15]. An internal ribosome entry site (IRES)-
LacZ sequence was targeted into the last exon of the p110 gene,
immediately after the stop codon (schematically shown for p110d
in Figure 1A). This allowed independent production of the p110
Academic Editor: Brian McCabe, Columbia University, United States of America
Received June 5, 2007; Accepted August 16, 2007; Published September 12,
Copyright: ? 2007 Eickholt et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work was funded by the Wellcome Trust grant (GR067121) to BJE,
the King’s College Trustees (AA) and the MRC (EJB). Personal support has been in
part by EU FP5 QLG1-2001-02171 (to AB), EU Marie Curie (E.P), and EU FP6-502935
(to WP). Additional grant support was provided by the Biotechnology and
Biological Science Research Council (BB/C505659/1) and the Ludwig Institute for
Cancer Research (to BV). The ISCR Gene Targeting Laboratory (AJS) was supported
by the BV is a consultant for Piramed, Slough, UK. Biotechnology and Biological
Science Research Council
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: Britta.J.Eickholt@kcl.
PLoS ONE | www.plosone.org1 September 2007 | Issue 9 | e869
protein and the b-Gal enzyme from the same bicistronic mRNA,
encoded by the p110 gene promoter(s). These mice are hereafter
referred to as p110alzand p110dlzmice.
As expected, the p110a isoform of PI3K was found to be widely
expressed (Figure 1B), and high expression of p110d in the
embryonic liver, the principal location of hemopoiesis, supports
previous reports on the expression of p110d in adult mice
leukocytes [7,11]. Unexpectedly strong signals of p110d were also
observed in the nervous system, especially in the spinal cord,
dorsal root ganglia (DRG), cranial sensory ganglia and peripheral
nerves (Figure 1C). Stainings at various time points showed that
this pattern of LacZ expression generally reflects the appearance of
differentiated neurons in both the central and peripheral nervous
system. At E12.5, for example, the p110d/LacZ signal followed the
wave of retinal ganglion cell differentiation in the central retina
(Figure 1D), and was not detected in the neuroblast layer at any
developmental stage analyzed (E12–E18; Figure 1D and data not
shown). In addition, p110d/LacZ expression was enriched during
neuronal migration, for example at E12.5 in the facial motor
nuclei within the hindbrain during movement to the final
rhombomere location (Figure 1E). Axonal processes and cell
bodies within the vomeronasal epithelium also expressed p110d/
LacZ, shown in Figure 1F. Cross sections through the spinal cord
at lumbar levels revealed highly enriched p110d/LacZ staining in
the DRG, interneurons of the spinal cord and the spinal motor
neuron pool (Figure 1G).
In adult mice, high p110d/LacZ expression was also present in
neurons, for example in specific brain regions, including the
hippocampus, cortex and thalamus (Figure S1A–C). Immunoblot-
ting of brain extracts confirmed the enrichment of p110d protein
in distinct brain regions (Figure S1D). This contrasts with the
uniform distribution of p110a and p110b, as well as various forms
of the p85 regulatory subunits (Figure S1D).
Inactivation of p110d increases the vulnerability of
sensory neurons to growth cone collapse and
decreases axonal extension
To determine the contribution of p110d to neuronal development
and function, we analyzed mice in which p110d was inactivated as
a result of the introduction of a germline point mutation which
renders the kinase inactive (p110dD910A; . Homozygous
p110dD910A/D910Amice, hereafter referred to as p110d kinase-
inactive (KI) mice, are viable and fertile . In these mice,
expression of the mutated p110d protein and the other PI3K
subunits was equivalent to that of the wild-type (WT) proteins in
brain homogenate (Figure S2), demonstrating the absence of
compensatory PI3K expression. Gross morphology of the nervous
system (data not shown) and hippocampus-dependent learning
behavior (Figure S3) were unaffected in p110d KI mice, suggesting
that the establishment and functioning of the neuronal circuitry
required for complex behavioral tasks does not depend on p110d
We next assessed the responsiveness of neurons to PI3K
inhibition using the pan-PI3K inhibitor LY294002 , which
induces growth cone collapse in sensory neurons [16,17]. Our
analyses showed that this response was significantly greater in
p110d KI than in WT DRG neurons (Figure 2A). This indicates
that DRG neurons with inactive p110d are more sensitive to
global PI3K inhibition, and also provides evidence that the
remaining PI3K isoforms could not compensate for the loss of
p110d activity. IC87114, a p110d-selective small molecule
inhibitor , also induced growth cone collapse in WT DRG
neurons, but had little effect on p110d KI DRG neurons
(Figure 2A), indirectly confirming the selectivity of this compound.
Responsiveness of DRG neurons to physiological stimuli that
utilize PI3K/Akt signaling was also assessed. Growth cone collapse
induced by the axon guidance molecule Sema3A, known to
decrease PI3K signaling [17,19], was 50% higher in p110d KI
DRG neurons at low concentration of Sema3A (Figure 2B, C).
Figure 1. Expression pattern of p110a and p110d as assessed by X-
gal staining of lacZ (b-Gal) reporter mice. (A) p110d gene locus of
p110dlzmice. (B) Broad expression of p110a/LacZ in p110alzmice at
E10.5. Scale bar, 500 mm (C) Side view of an E13.5 p110dlzembryo
reveals high X-gal staining in the developing central and peripheral
nervous system, and the liver (LV). Scale bar, 1 mm. (D) p110d/LacZ
expression in retinal ganglion cells (RGC) in the central retina at E12.5.
Scale bar, 100 mm (E) p110d/LacZ expression in the facial motor nuclei
(FMN) within the hindbrain as they migrate from rhombomere (r) 4 to
their final position in r6 at E12.5. Scale bar, 150 mm. (F) p110d/LacZ
expression in axonal processes and cell bodies within the vomeronasal
epithelium (shown here at E15.5). Scale bar, 100 mm. (G) p110d/LacZ
expression in the DRG, motor neuron pool (MN) and interneurons (IN) in
cross-sections through the spinal cord at E13.5. Scale bar, 100 mm.
PLoS ONE | www.plosone.org2 September 2007 | Issue 9 | e869
Integrin activation through the substrate laminin is known to
activate PI3K signaling in the growth cone . Laminin-
mediated axonal elongation in p110d KI DRG neurons was
reduced by almost 30% at lower (10 mg/ml) but not at higher
(20 mg/ml) concentrations of laminin (Figure 3). Taken together,
these observations uncover a p110d PI3K signaling pathway,
important for the maintenance of optimal axonal outgrowth in an
inhibitory environment and under lower substratum availability.
Genetic or pharmacological inhibition of p110d in cultured DRG
neurons did not alter apoptosis (as measured by apoptotic nuclei;
data not shown), indicating that this pathway is independent of
PI3K-mediated survival responses.
Reduced axonal regeneration in the injured sciatic
nerve of mice with inactive p110d
Given that the absence of p110d activity limits axonal outgrowth
in embryonic neurons, we assessed the axonal growth potential of
adult p110d KI neurons. Adult peripheral nerve axons are capable
of functional regeneration in mammals, with a crush injury of the
sciatic nerve being a well-established injury model to study axonal
growth . On day 3, extension of regenerating neuronal fibers
2 mm distal from the injury site was reduced in p110d KI mice
(Figure 4A,B), without any differences in the presence of
Figure 2. Increased sensitivity of p110d KI DRG growth cones to collapse in response to PI3K inhibition or Sema3A treatment. (A) Growth cone
collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110d-selective inhibitor). Both drugs were used at 10 mM for
10 min (n$6 independent experiments). *p,0.01. (B) E13.5 DRG explants were cultured for 24 h on 20 mg/ml laminin and treated with Sema3A
(0.3 mg/ml) for 30 min. Increased growth cone collapse in response to Sema3A (0.3 mg/ml) in p110d KI DRGs (right). Each data point represents the
%6SEM (n$3 independent experiments). In each experiment at least 80 growth cones were counted. *p,0.02. (C) Example of DRG explants in each
group, which have been stained for bIII-tubulin (red) and phalloidin (green). Scale bar, 20 mm.
Figure 3. Reduced outgrowth of p110d KI DRG neuron under limiting
substrate conditions. Left panel, example of E13.5 DRG neurons derived
from WT and p110d KI mice, cultured on 10 mg/ml laminin for 24 h.
Right panel, relative axonal length in p110d KI and WT DRG neurons at
1, 5, 10 and 20 mg/ml laminin (right). Length was expressed relative to
that of WT DRG neurons cultured in the presence of 10 mg/ml laminin.
Each point represents the mean of at least 3 experiments6SEM, each
experiment was carried out in duplicate. n=60 neurons in each
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cytoskeletal breakdown products 4 mm distally from the site of
alter normal axonal degeneration. p110d activity has been shown to
be essential for CSF-1-driven in vitro chemotaxis of macrophages
[22,23], which might affect axonal regeneration due to impaired
inflammatory response at the injury site in p110d KI mice. No
changes in recruitment of macrophages (stained by F4/80) into the
injured nerve were detected (Figure 4B). Next, we compared the
capacity of DRG soma to upregulate growth-associated SPRR1A
(small proline-rich repeat protein 1A) during regeneration. SPRR1A
has been shown to peak 1–2 weeks following sciatic nerve injury in
adult mice and its depletion reduces axonal outgrowth in vitro .
We co-stained for ATF3 (Activating Transcription Factor 3), which
is produced de novo in sensory neurons following sciatic nerve injury
and is widely used as a marker for nerve injury . No significant
difference was observed in the percent of ATF3-positive neurons in
injured DRGs obtained from WT and p110d KI mice in L4 DRGs
7 days after nerve injury, demonstrating that the sciatic nerve injury
was equivalent in the two groups (data not shown). However, the
regeneration marker SPRR1A was significantly reduced in L4 DRG
soma of p110d KI mice (Figure 4C), indicating that loss of p110d
function impairs regenerative capacity of adult sensory neurons.
Inhibition of axonal regeneration correlates with
impaired functional recovery
We next assessed whether the anatomical evidence for p110d-
dependency for optimal axonal regeneration correlated with
functional recovery using an automated quantitative gait analysis
system, the CatWalk, to assess recovery of locomotion following
nerve injury on days 1, 3, 7, 10, 14 and 21 . Prior to injury,
gait analysis assessed by the CatWalk on a number of locomotor
parameters revealed no differences between WT and p110d KI
mice (Figure 5A, B, S4). In contrast, following unilateral injury to
the sciatic nerve, p110d KI mice showed a significant decrease in
the recovery of the ability to bear weight on the injured paw
(Figure 5A, B). Whilst both WT and p110d KI mice display
functional recovery during the first 10 days post-lesion, the relative
Figure 4. Reduced axonal regeneration in the injured sciatic nerve of
p110d KI mice. 3 days post-injury, the sciatic nerves of WT and p110d KI
mice were fixed and cryo-sectioned. (A) Average relative fluorescence
intensity profile of anti-bIII-tubulin labeling across a one-pixel line along
the entire nerve segment, following cropping of the micrographs to
a fixed pixel segment. (B) High-power micrographs of the sciatic nerve
segments 2 mm distal to the injury, labeled with anti-bIII-tubulin (left
panels and green in right panels), anti-F4/80 (red) and Hoechst (blue).
Scale bar, 50 mm. (C) At 7 days post injury, L4 DRGs of WT and p110d KI
mice were fixed, vibratome-sectioned, and co-labeled with ATF3 (red)
and SPRR1A (green). Percentage of SPRR1A and ATF3 co-labeling over
ATF3-only positive DRG neurons in WT and p110d KI mice. Data are
from 3 WT and 4 p110d KI mice, and are presented as mean6SEM.
*p,0.05. Scale bar, 50 mm.
Figure 5. Impaired functional recovery in p110d KI mice following
sciatic nerve crush injury. Paw pressure intensity during continuous
locomotion was assessed using the CatWalk quantitative gait analysis
system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and
21 post-injury. (A) Intensity profiles of 5 steps during a single run from
each paw, 7 days post-injury. Each intensity profile shows two traces,
a higher trace of the maximal relative intensity and a lower trace of the
average relative intensity. (B) Reduced relative paw pressure intensity
for each paw during recovery in p110d KI mice compared to WT mice.
Data presented is the mean6SEM of $6 animals. p,0.001, 2-way
repeated measures ANOVA.
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paw pressure intensity in p110d KI mice was significantly lower in
comparison to WT mice (Figure 5B; p,0,001, 2-way repeated
measured ANOVA). These differences were most apparent at later
time-points (day 10 and day 14 post-lesion; Figure 5B; p,0.05,
Tukey test). These results indicate that following sciatic nerve
injury, the lack of functional p110d led to a decreased ability of the
axons to undergo regenerative growth, which in turn led to
decreased functional recovery.
Reduced Akt and increased RhoA/PTEN signaling in
neurons with inactive p110d
We next investigated the effect of p110d inactivation on signaling
in neurons. PI3K signaling drives many aspects of neuronal
morphology through the coordinated phosphorylation of proteins
that regulate cytoskeletal dynamics, protein synthesis, and
transcriptional activity. Akt is an important effector through
which PI3K controls axon elongation and morphological
responses induced by neurotrophins [27,28]. A substantial
decrease in activatory Akt phosphorylation was observed in
p110d KI DRG neurons cultured in the presence of NGF
(Figure 6A). In contrast, phosphorylation of GSK-3b, an effector
of the PI3K/Akt pathway in many cell types  and a crucial
determinant of axonal growth and guidance [27,30,31], was not
affected (Figure 6A). This lack of inactivation of GSK-3b may
allow the observed normal neurite outgrowth under non-limiting
conditions in p110d KI mice (Figure 3) and suggests the presence
of alternative, p110d-independent regulatory pathways for GSK-
Figure 6. Reduced Akt and increased RhoA/PTEN signaling in DRG neurons with inactive p110d. (A) E13.5 DRG neurons of WT and p110d KI mice
were cultured for 24 h before processing for Western blot analysis using the indicated antibodies. (B) Inactivation of p110d in p110d KI mice leads to
an increase in lipid phosphatase activity in PTEN immunoprecipitates from homogenates of brain (left panel) or DRGs (right panel) from WT and p110d
KI mice. Graphs show the lipid phosphatase activity in 3 independent experiments, measured in triplicates. p,0.001. (C) Inactivation of p110d does
not affect Rac, but increases RhoA activity. Left panel, brain lysate from WT and p110d KI mice was subjected to pull-down with GST-PBD or GST-RBD,
followed by SDS-PAGE and immunoblotting using antibodies to Rac1 or RhoA. Right panel, graphs represent the mean6SEM of Rac1-GTP (left) or
RhoA-GTP loading of 2 experiments, each performed in triplicate. (D) Inhibition of ROCK rescues the effects of p110d inactivation on axonal
elongation at 10 mg/ml laminin. Left panels, example of an E13.5 DRG explant (e) of p110d KI mice cultured for 24 h in the absence (control) or
presence of Y27632 (10 mM). Scale bar, 20 mm. Right panels, length of axons extending from WT and p110d KI DRG explants, in the absence (2) or
presence (+) of Y27632 (10 mM). Each data point represents the mean6SEM of 3 independent experiments.
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3b. Another member of the signaling pathway downstream of
PI3K activation is mTor and its effector p70S6K, which controls
the initiation of protein synthesis . DRG neurons from p110d
KI mice showed a substantial decrease in p70S6K phosphoryla-
tion, whilst levels of MAPK phosphorylation and Bcl-xl protein
were not affected (Figure 6A). These results indicate that p110d
PI3K activity exerts control over the Akt/p70S6K pathway, which
is not compensated for by p110a or p110b.
p110d has recently been demonstrated to inhibit the activity of
the tumor suppressor PTEN through a pathway involving RhoA
. Similary, in p110d KI DRGs and brain homogenates, PTEN
lipid phosphatase activity was constitutively elevated (Figure 6B).
In addition, GTP-loading of RhoA, but not Rac, was significantly
increased in p110d KI brain extracts (Figure 6C). These observa-
tion is in line with the idea that p110d can suppress cellular RhoA
but not Rac activity under basal conditions . RhoA is a critical
mediator of the inhibitory effect of several axon guidance
molecules and myelin associated inhibitors [32,33]. Inhibition of
RhoA, or its downstream effector ROCK, is able to restore out-
growth in an inhibitory environment provided by myelin or myelin
associated inhibitors [32,33]. In order to test the significance of
increased RhoA function for p110d signaling during axonal
elongation, we inhibited ROCK using the small molecule inhibitor
Y27632 [34–36]. As expected, axons extending from p110d KI
DRG explants were significantly shorter than WT axons
(Figure 6D). However, treatment with Y27632 restored axonal
length in p110d KI neurons to the lengths seen in WT neurons
(Figure 6D). These data are consistent with a model whereby
inactivation of p110d leads to increases in ROCK activity as
a consequence of higher levels of active RhoA. Previous work has
indicated RhoA/ROCK signaling in the control of PTEN ,
thus raising the possibility that PTEN activity in neurons functions
downstream of RhoA. Such deregulation of signaling by p110d is
consistent with the phenotypes observed in this study .
The major finding of this study is the identification of a function of
the p110d PI3K in controlling effective axonal elongation under
less favorable conditions and during insult to the nervous system.
This is consistent with the idea that although p110d is similar to
p110a and p110b in terms of structure and substrate specificity, it
is restricted in its expression and function (Figure 1; [7,11,38]). It
remains to be determinded if the p110a and p110b isoforms of
PI3K play similar roles in neurons. Homozygous inactivation of
p110a or p110b leads to embryonic lethality [8,9,15], precluding
investigation in this area until conditional p110a/p110b mutant
mice will become available. p110d is considered to be an
interesting therapeutic target in inflammation and auto-immunity
[12,14,39], and the development of small molecule inhibitors
against p110d is in progress . The data reported here suggest
that p110d inhibitors may not have adverse effects on the steady-
state functioning or the development of the nervous system.
Nonetheless, caution should be exercised given that these com-
pounds may have undesirable effects under conditions of nerve
injury or ongoing neurological degeneration.
MATERIALS AND METHODS
Gene targeting to create the p110alzmice and p110dlzmice has
been described elsewhere [14,15]. All mice were littermates and
backcrossed for 10 generations onto the C57B16/J strain. Animals
were maintained in individually-ventilated cages on a 12 h light-
dark cycle, with free access to food and water. All experiments
were undertaken in accordance with the UK Animals (Scientific
Procedures) Act 1986.
Embryo and tissue preparation
p110alzand p110dlzmice were transferred into ice-cold PBS, and
fixed for 1 h on ice in fixing buffer (4% paraformaldehyde/0.2%
glutaraldehyde/2 mM MgCl2/5 mM EGTA/0.02% NP40). Em-
bryos were then washed 3 times in washing buffer (PBS/2 mM
MgCl2/0.01% sodium deoxycholate/0.02% NP40/5 mM EGTA)
and post-fixed in 4% paraformaldehyde for 1 h. b-galactosidase
expression was visualized by incubation in X-gal developing buffer
(PBS/5 mM K3Fe(CN)6/5 mM K4(CN)6/2 mM MgCl2/0.01%
sodium deoxycholate/0.02% NP40, 1 mg/ml X-gal) overnight at
room temperature. Embryos were then washed in PBS, post-fixed in
4% paraformaldehyde for 1 h, and washed twice in distilled water
before dehydration though a series of 70% ethanol, 95% ethanol,
and 100% ethanol. Following incubation in methylsalicylate for 1 h,
embryos were rehydrated in 100% ethanol, 95% ethanol, 70%
ethanol, and 30% ethanol and finally washed in PBS before being
embedded in 10% gelatin and vibratome-sectioned at 50 mm.
Sciatic nerve crush
Forinvivoregenerationstudies,6to8 week-old malemicewereused.
The mice were assessed before surgery to establish baseline-walking
(0.5 mg/kg) and ketamine (75 mg/kg), and the left sciatic nerve was
tendon using forceps compression (10 sec) and the crush site labeled
with lamp black. The muscle and skin layers were sutured and
animals were allowed to recover in the cage post-operatively.
The CatWalk gait analysis system was used to assess functional
recovery of locomotion following sciatic nerve crush injury .
The animal traversed a meter long walkway with a glass floor and
2 perspex walls spaced 8 cm apart, housed in a darkened room.
Light from 2 encased white fluorescent tubes entered the glass
floor through the distal edge of the glass, and was totally internally
reflected. Light scatters only where a paw contacts the glass,
illuminating the area of paw contact. This reflected light was
captured using videocamera (Sentech 705, 8.5 mm, f=1.4, variable
focus and variable iris) equipped with a wide-angle objective and
a frame grabber (Matrix Vision SG-board) connected to a PC
running the CatWalk 500 software for capture and analysis .
Each mouse ran across the CatWalk one day before surgery to
establish baseline locomotor parameters. Following surgery, animals
ran the Catwalk on days 1, 3, 7, 10, 14 and 21. The program was set
to capture the paw prints from the middle section of the run. At least
2 runs per animal were performed on each day. Data was analyzed
by labeling all areas containing one or more pixels above a certain
analysis threshold. In a second interactive pass, these areas were
assigned to each of the paws (left and right fore and hind paws: LF,
RF, LH, RH). Data generated from the program was exported to
Excel, yielding several parameters including average area and
intensity for each paw, the regularity index, and duration of swing
and stance phase. Statistical significance was evaluated using two-
way repeated measures ANOVA and Tukey post-hoc comparisons
(Sigma Stat 3.0.1, SPSS Inc.).
Morris water maze
Mice for behavioral testing were housed in groups of 2 to 4 in
individually-ventilated cages, with food and water ad libitum and
maintained on a 12 h light-dark cycle. Mice tested were males
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between the ages of 2 and 6 months. The Morris water maze
protocol has been described previously . Briefly, the mice were
trained in a 1.5 m pool with a 10 cm platform. The water was
maintained at 24–27uC, and made opaque with non-toxic white
paint. The animals received 12 training trials per day, in blocks of
4, with 1 h in between each block, and a 90 sec maximum swim
time. On days 3 and 5, a probe trial was given, in which the
platform was removed and the animal was allowed to swim for
90 sec before being removed from the pool. The movement of the
animals whilst in the pool was videotaped and recorded by
a computer tracking system (HVS Image, Hampton, UK). The
behavioral data were analyzed with the ‘HVS water program’ and
Sigmastat (SYSTAT Software, SSPS Science Inc).
For stainings of DRG neurons in situ, E13.5 WT and p110d KI
mice were paraformaldehyde fixed, embedded in 20% gelatin and
vibratome-sectioned at 80 mm. Anti-P-p70S6K (Cell Signaling
Technology) was applied at 1:300 overnight at 4uC. Following
extensive washes, bound antibodies were detected using Alexa
conjugated secondary antibodies (1:1000). Sections of WT and
p110d KI mice were analyzed by confocal microscopy at the same
scanning settings for direct intensity comparison (optical slice size
2 mm, Zeiss LSM 5 META confocal laser scanning microscope).
For assessment of axon regeneration in the peripheral nerve, mice
were deeply anaesthetized with pentobarbitone (80 mg/kg, i.p.)
3 days after nerve crush and transcardially perfused with 10 ml
saline followed by 50 ml paraformaldehyde (4% in 0.1 M
phosphate buffer). The entire left and right sciatic nerves were
removed, post-fixed in 4% paraformaldehyde for 2 h, transferred
to 20% sucrose overnight, mounted in OCT (BDH, UK), and
cryosectioned longitudinally for subsequent immunohistochemis-
try using anti-bIII-tubulin antibody (1:800, Covance), anti-F4/80
(Serotec) and the nuclear dye Hoechst (Sigma). For assessment of
regeneration-associated markers in the cell bodies, 7 days after the
sciatic nerve crush, mice were sacrificed (as above). DRGs from
lumbar segments 4 and 5 were dissected, stored in 4% para-
formaldehyde at 4uC for 2 h, washed once in PBS and embedded
in 10% gelatin, followed by cutting of 20 mm sections on ice using
a vibratome (Leica, Speed 3-Frequency 7). Sections were trans-
ferred to PBS+1% sodium azide, and anti-SPRR1A antibody
(kindly provided by S. Strittmatter, Yale University) was applied at
1:7000 in PBS/0.2% Triton-X100 and incubated overnight at
room temperature, under mild agitation. Sections were washed
extensively, and bound antibody was detected using biotinylated
horse anti-mouse secondary antibody (Jackson ImmunoResearch;
1:400, 90 min), ABC reagent (Vector Labs; 1:250, 30 min),
biotinyl tyramide (PerkinElmer Life Sciences; 1:75, 10 min) and
extra-avidin FITC (Sigma; 1:500, 2 h). Sections were then
incubated with rabbit anti-ATF3 (1:400, Santa Cruz), overnight
at 4uC. After extensive washes, sections were incubated with
donkey anti-rabbit TRITC (1:200, Jackson). All sections were
mounted on slides in Vectashield fluorescent mounting medium
(Vector Laboratories Ltd., UK).
Age-matched WT and p110d KI embryos were isolated at the
appropriate age, and transferred into ice-cold DMEM. DRGs
were dissected from E13.5 mouse embryos and extramesenchymal
tissue was removed using a sharpened 0.2 mm tungsten wire.
DRGs were then plated on glass coverslips previously coated with
poly-L-lysine and laminin (both at 20 mg/ml; Sigma). For primary
neuronal cultures, DRGs were incubated in trypsin (1 mg/ml,
diluted in HBSS) for 10 min at 37uC, and dissociated using a fire-
polished Pasteur pipette. Neurons were either cultured at low
density (50 cells/mm2) for neurite outgrowth assays on glass
coverslips coated as described above, or in laminin-coated 6-well
dishes (150 cells/mm2) for biochemical analysis. Explants and
primary DRG neurons were incubated at 37uC/5% CO2for 24 h
in DMEM/10% FCS/Pen/Strep supplemented with 20 ng/ml
NGF (Promega). Pharmacological inhibitors were used as de-
scribed; LY294002 was purchased from Calbiochem. The collapse
assay using purified Sema3A-Fc or PI3K inhibitors was performed
as previously described .
Neuronal cultures were treated as indicated and paraformalde-
hyde-fixed (4% paraformaldehyde/PBS/10% sucrose) for 30 min
before permeabilization for 5 min with PBS/1% Triton 6100.
Neurons were then labeled with Phalloidin-Alexa488 (1:50 in
PBST) and anti-bIII-tubulin antibody (1:800; Covance). For each
experiment, the collapsed growth cones were counted and repre-
sented as a % ratio. Each experiment was performed a minimum
of 4 times, and the average % collapsed was determined. Standard
errors of the mean were determined as the (standard deviation/
square root (number of experiments)). For determination of neurite
length, neurons were labeled with the anti-bIII-tubulin antibody,
and the KS300 program (Zeiss) was used to measure neurite
length in each treatment.
SDS-PAGE and Western blotting
Dissociated neurons were washed twice with ice-cold PBS lysed for
30 min in ice-cold lysis buffer (10 mM Tris.HCl pH 7.4, 250 mM
sucrose, 10 mM MgCl2, 0.5% NP40, complete protease inhibitor
cocktail (Roche), 2 mM sodium orthovanadate, 0.1 mM DTT,
25 mM NaF). All cell lysates was adjusted to equal concentrations,
and 20 mg protein was separated by SDS-PAGE and blotted onto
nitrocellulose (Hybond ECL, Amersham Biosciences). Primary
antibodies were applied for 1 h followed by 3 washes in TBST.
MAPK, anti-Bclxl and anti-P-GSK-3b (Cell Signaling Technology),
anti-actin (Roche), anti-p110b (sc-602; Santa Cruz). Antibodies to
p110a or p110d were generated in-house . Bound antibody was
detected using HRP-conjugated secondary antibody (Vector Labs)
diluted in blocking milk, which was applied for 1 h. After extensive
washes in TBST, blots were developed on MXB film (Kodak) using
an ECL (Amersham Biosciences) detection system.
GTPase and PTEN lipid phosphatase activity assays
The Rac or RhoA activation assays were performed using GST-
PBD (p21-binding domain of PAK) or GST-RBD (Rho binding
domain of Rhotekin), respectively, as described . In brief,
brain tissue was lysed in Mg2+lysis buffer (Upstate) and mixed with
GST-PBD or with GST-RBD bound to glutathione-agarose and
incubated for 1 h at 4uC. Bound protein was washed, and
suspended in sample buffer. Proteins were then separated by SDS-
PAGE, transferred to PVDF membranes and blotted with the
indicated antibodies. PTEN lipid phosphatase activity was
measured as previously described using malachite green reagent
for the detection of phosphate release . Similar results were
also obtained using a PTEN activity ELISA kit (Echelon).
in the brain. Coronal sections of the brain of (A) p110d lz and (B)
Expression of p110d and other class IA PI3K isoforms
PLoS ONE | www.plosone.org7 September 2007 | Issue 9 | e869
WT adult mice reveal restricted expression of p110d/LacZ in
several brain regions, including the cortex (Cx), hippocampus (H)
and thalamus (Th). Sections were counterstained with nuclear fast
red. Scale bar, 1 mm. (C) p110d expression in different brain areas
as assessed by X-gal staining of adult lacZ (b-Gal) reporter mice.
(D) Expression of PI3K isoforms and the CD45 pan-leukocyte
marker in lysates of different brain regions and thymus of adult
WT mice. CD45 was found to be expressed in thymus and not in
the brain, indicating that X-gal signals do not derive from resident
leukocytes in the brain.
Found at: doi:10.1371/journal.pone.0000869.s001 (1.37 MB TIF)
campus of p110d KI mice. Tissue extracts from the hippocampus
from adult WT and p110d KI mice were immunoblotted with PI3K
isoform-specific antibodies as indicated. Anti-b-actin staining was
used as internal control for equal protein loading.
Found at: doi:10.1371/journal.pone.0000869.s002 (0.15 MB TIF)
Expression of class IA PI3K proteins in the hippo-
mice in the Morris water maze. (A) WT and p110d KI mice were
trained with 12 trials per day in blocks of 4 trials. The time to
reach the hidden platform is shown; there was no difference
between the genotypes. (B) After training, day 3 and 5 probe trials
were performed to assess selective searching in the quadrant where
the platform used to be (TQ). Both genotypes searched selectively
indicating normal spatial memory in p110d KI mice. (C) The
‘platform crossings’ during the probe trials showed the same
accuracy in WT and p110d KI mice. Each data point represents
the mean+SEM (n=11 mice/group). During the probe trials the
swim speeds did not differ between the genotypes (data not shown).
Found at: doi:10.1371/journal.pone.0000869.s003 (1.70 MB TIF)
Normal spatial memory development of p110d KI
prior to injury. WT and p110d KI mice were assessed for 6
locomotor parameters using the CatWalk quantitative gait analysis
system to obtain baseline values. (A) The Regularity Index, an
index that quantifies the % of steps assigned to one of 6 normal
step sequences , is equivalent between WT and p110d KI
mice. (B) The base of support (measured in arbitrary units)
represents the width between the two hind paws and indication of
the stability of posture during locomotion. The base of support
does not differ between WT and p110d KI mice. (C, D) For each
hind paw, the average area of contact and the average intensity of
light reflected at each point of contact (which is indicative of the
pressure applied by the paw upon the glass surface) are equivalent
between WT and p110d KI mice. Both average area and average
intensity are measured in arbitrary units. (E, F) The stance phase is
timed while the paw is placed upon the glass and the swing phase
is timed between paw placements. The duration of swing and
stance phases (in sec) between the two groups do not differ. In each
evaluation, every data point represents the mean+SEM (n=6
mice/group). p.0.1 in all parameters.
Found at: doi:10.1371/journal.pone.0000869.s004 (0.47 MB TIF)
p110d KI mice display normal locomotor parameters
We thank Mark Holt for the fluorescence intensity measurements, and
Siobhan Jordan, Klaus Okkenhaug and Sara Sancho for their contribu-
tions to the early phase of this work.
Conceived and designed the experiments: BV BE MD EB EP KG.
Performed the experiments: BE AA MD MS EB EP WP AB AN. Analyzed
the data: BE AA MD EB SH EP KG AN. Contributed reagents/
materials/analysis tools: BV AS FH EB WP. Wrote the paper: BV BE.
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