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www.elsevier.com/locate/molbrainresMolecular Brain Research 128 (2004) 8–19
Research report
Correlation between synaptogenesis and the PTEN phosphatase
expression in dendrites during postnatal brain development
Claudia Perandonesa,b,1, Roxana Veronica Costanzoa,b,1, Valeria Kowaljowc,Omar Hilario Pivettab, Hector Carminattia, Martın Radrizzania,b,*
a1 Fundacion Instituto Leloir, (IIBBA-CONICET, IIB-FCEN-UBA), Avenue Patricias Argentinas 435, Buenos Aires 1405, ArgentinabCentro Nacional de Genetica Medica, ANLIS-Dr. Carlos G. Malbran, Avenue Las Heras 2670, 4j Piso, (1425), Buenos Aires, Argentina
c Instituto de Investigacion Medica Mercedes y Martın Ferreyra, Casilla de Correo 389, 5000 Cordoba, Argentina
Accepted 30 May 2004
Available online 28 July 2004
Abstract
The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene codifies a lipid inositol 3V-phosphatasethat negatively regulates cell survival mediated by the phosphatidyl inositol 3V kinase (PIP3-kinase)–protein kinase B/Akt signaling pathway.Recently, PIP3-kinase was involved in axon polarization, but PTEN functions in dendrites are uncertain. Using amino-terminal antibodies
against the catalytic domain, we found a 34 kDa fragment of PTEN protein detected only in mouse brain tissue, present in neuron dendrites
and spines of cerebral cortex, cerebellum, hippocampus and olfactory bulb. The PTEN-fragment reaches the synaptic fraction with a positive
temporal correlation with synaptic stabilization in postnatal cerebellum and brain. In the weaver mutant mice, PTEN was absent only in the
Purkinje cells dendrites that cannot receive the granule cells synaptic input. Furthermore, the activated p-Akt/PKB was present in axons but
not in dendrites of mature neuron cells. P-Akt was also altered by the weaver mutation maintaining the inverse correlation with the PTEN-
fragment in Purkinje cell dendrites. In contrast, the expression of this fragment was not affected by the staggerer mutation.
Together, these results suggest that synaptogenesis is a necessary process for polarization in PIP3 pathway mediated by the PTEN
catalytic-fragment into dendrites of CNS neurons.
D 2004 Elsevier B.V. All rights reserved.
Theme: Cellular and molecular biology
Topic: Staining, tracing, and imaging techniques
Keywords: Phosphatidylinositol phosphatase; Weaver; Cerebellum; PTEN; Dendrites; Synaptogenesis
1. Introduction dolfi group demonstrated that PTEN haploinsufficiency was
PTEN was initially identified as a tumor suppressor
mutated in glioblastomas, breast, prostate and kidney can-
cers. Germline mutations of PTEN result in Cowden,
Bannayan–Ruvalcaba and Lhermitte –Duclos diseases
(LDD), in which disorganized benign tumors and malig-
nances appear in multiple organs [10]. Recently, the Pan-
0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.molbrainres.2004.05.021
* Corresponding author. Laboratory of Neurosciences, Fundacion
Instituto Leloir, (IIBBA-CONICET, IIB-FCEN-UBA), Avenue Patricias
Argentinas 435, Buenos Aires 1405, Argentina. Tel.: +54-11-4863-4011 to
4019; fax: +54-11-4865-2246.
E-mail address: mradrizzani@leloir.org.ar (M. Radrizzani).1 C.P. and R.C. contributed equally to this work.
necessary and sufficient to produce this type of pathology in
mice and that its absence was lethal for embryonic devel-
opment [9]. Furthermore, the Cre-loxP system was used to
inactivate PTEN in the mouse brain, resulting in deletion of
PTEN in granule neurons of the cerebellum and the dentate
gyrus. Loss of PTEN in these cells resulted in seizures,
ataxia and premature death. Also, a low proliferative index
and elevated PKB/Akt phosphorylation can be seen, resem-
bling LDD [3,13,19].
PTEN encodes a phosphatase with a tensin-like domain
at the N terminal and a novel domain of unknown function
at the C terminal. The PTEN enzyme is a dual-specificity
protein phosphatase and a phosphatidylinositol phosphate
(PIP) phosphatase. The PIP phosphatase activity is specific
C. Perandones et al. / Molecular Br
for the 3-position of the inositol ring [18]. PTEN can
dephosphorylate the 3V position of PIP3 to generate PIP2, a
biochemical function that antagonizes the activity of PI3K,
which converts PIP2 to PIP3. These opposing effects are
also observed on cell proliferation and survival. The PI3K/
PTEN intracellular signaling cascade has been intensely
reviewed [5]. Briefly, growth factor stimulation of cells
causes activation of PI3K and an increase in cellular levels
of the membrane phospholipid phosphatidylinositol (3,4,5)
triphosphate (PIP3), a key mediator in cell survival.
Accumulation of PIP-3 at the membrane allows recruit-
ment of proteins containing the pleckstrin homology (PH)
domain. One of these proteins is the proto-oncogene
serine/threonine kinase Akt. Upon membrane recruitment,
Akt is activated by phosphorylation. Activated Akt is a
well established survival factor, exerting anti-apoptotic
activity by preventing the release of cytochrome C from
mitochondria and inactivating Forkhead transcription fac-
tors (FKHR), which are known to induce the expression of
genes that are critical for apoptosis. Despite homology to
protein phosphatases, PTEN dephosphorylates the D3
position of the inositol ring of PIP3 and negatively
regulates PKB/Akt activities [36,37].
The mutation of PTEN in gliomas, the prevalence of
neurological defects in patients with mutated PTEN, and the
growing recognition of PIP3s as neuronal regulators have
led us to evaluate the role of PTEN and the PI3K-protein
kinase B/Akt signaling pathway in dendrite development
[1]. Using amino-terminal antibodies against the catalytic
domain of PTEN, we found a 34 kDa fragment of the
protein present in mouse only in brain tissue, mainly in
neuron dendrites of cerebral cortex, cerebellum, hippocam-
pus and olfactory bulb. We observed a positive correlation
between the synaptic stabilization process and detection of
the PTEN fragment in the synaptic fraction. In the weaver
mutant mice, the PTEN fragment was absent only in the
dendrites of Purkinje cells, the ones that showed impaired
synaptogenesis, while the p-Akt levels were significantly
increased. In contrast, the expression of this fragment was
not affected by the staggerer mutation.
Together, these results suggest a prominent role of the
synaptogenesis process in the regulation of the PTEN/PI3-
kinase signaling pathway.
2. Materials and methods
2.1. Experimental animals
Mice were from the inbred weaver strain (Kcnj6wv
B6CBACa Aw-J/A-Kcnj6wv), the staggerer mutant strain
(C57BL/6J-Rorasg/+) and control C57BL/6J (Rora+/
Rora+), all derived from Jackson Laboratories (Bar Har-
bor, ME, USA) and raised in the Research Department—
Centro Nacional de Genetica Medica-ANLIS Dr. Carlos G.
Malbran.
2.2. Anti-PTEN antisera
PTEN synthetic peptide sequence between aminoacids
33 to 47 (mouse IAMGFPAERLEGVYR, antigen grade)
was commercially obtained (Alpha Diagnostic International,
San Antonio, TX, USA) and used as immunogen. The
carboxy-terminal of synthetic peptide (3 mg/ml) was
cross-linked to Bovine Serum Albumin (0.5 ml of 4 mg/
ml BSA) using 1-ethyl-3-(3-dimethylamino-propyl) carbo-
diimide (EDAC, 15 mg/ml) (Sigma-Aldrich, Boston).
Rabbits were immunized to produce polyclonal antibodies
using Freund’s adjuvant by intradermic injection in the neck
[7].
The antiserum was diluted with PBS–5% milk, 1:50 for
immunohistochemistry and 1:400 for Western blot. Preim-
mune serum was used as negative control and albumin
cross-linked peptide was employed for specific competition
(at dilution 100:1). Further controls were made using
adenocarcinomas of endometrium and colon. As was
expected, PTEN was absent in both negative control adeno-
carcinomas in contrast to the positive control tissues (data
not shown).
2.3. Secondary antibodies
For detection in tissue slices, a goat anti-rabbit antibody
coupled to peroxidase was used as a secondary antibody
(Promega, Madison, WI) (incubated for 1 h at room tem-
perature (RT), at dilution 1:1000). An anti-rabbit, affinity-
purified antibody, coupled to Cy3 (1 h at RT, dilution 1:200)
(Sigma, St. Louis, MO) was used as a secondary antibody
for fluorescence [31].
2.4. Western blot
Tissues and brain regions were dissected and homoge-
nized 1:10 w/v in RIPA buffer (Tris–HCl 50 mM, pH: 7.5,
NaCl 150 mM, Nonidet P40: 1%, Deoxycholate: 0,5% and
SDS 0,1%) with a protease inhibitor cocktail at a final
concentration of 500 AM AEBSF, HCl 150 nM Aprotinin, 1
AM E� 64, 0.5 mM EDTA, Disodium Salt and 1 AMLeupeptin Hemisulfate (Set 1: Cat. No. 539131, Calbio-
chem-Novabiochem, La Jolla, CA). Fifty micrograms of
proteins measured with the Lowry method were loaded in
each lane of 10% SDS-PAGE. Standard molecular weight
markers: Albumin Bovine Serum 66 kDa, Ovalbumin 45
kDa, Glyceraldehyde-3-Phosphate 36 kDa, Carbonic anhy-
drase 29 kDa, Trypsinogen 24 kDa, Trypsin inhibitor 20
kDa, alpha-lactalbumin 14.2 kDa were used (Sigma). PTEN
amino terminal rabbit polyclonal antibodies were diluted
with PBS–5% milk 1:400 for Western blot. A monoclonal
anti-rabbit gamma chain specific antibody coupled to alka-
line phosphatase (Clone RG-96, purified antibody, Sigma)
was used as a secondary antibody on Western blots (1 h at
RT, dilution 1:10,000) and developed with BCIP/NBT color
development substrate (5-bromo-4-chloro-3-indolyl-phos-
ain Research 128 (2004) 8–19 9
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–1910
phate/nitro blue tetrazolium; Promega). A commercial goat
antibody against the PTEN amino-terminal was used as
control (dil. 1:500, PTEN N-19 sc-6818, Santa Cruz Bio-
tech., CA) and developed with anti-goat IgG coupled to
alkaline phosphatase provided by Santa-Cruz Biotech (dil.
1:1000, anti-goat IgG-AP, sc-2022, Santa Cruz Biotech.).
2.5. Subcellular fractionation
Sucrose differential centrifugation was used to obtain
crude enriched subcellular fractions [28]. Brains or cerebella
were dissected, chopped with a surgical blade, and homog-
enized in presence of Hepes 5 mM pH 7.4, Sucrose 0.32 M
and protease inhibitor cocktail (Set 1, Calbiochem-Nova-
biochem). All procedures were made in an ice bath. Con-
tamination was controlled using Anti-Synaptophysin (a gift
from Dr. A. Caceres, Instituto de Investigacion Medica
Mercedes y Martın Ferreyra) and Anti-Histone1 Monoclo-
nal antibodies (sc-8030, Santa Cruz Biotech.) in each
fraction.
2.6. Immunohistochemistry
Wild type (C57BL/6J) or weaver mutant mice cerebella
were dissected at different postnatal ages, fixed in alcohol/
acid (95% ethanol, 5% acetic acid) for 3 h at 4 jC,dehydrated, and embedded in paraffin. Tissue slices (5
Am) were mounted on silanized (Silane, Sigma) glass cover
slips, deparaffinized, rehydrated, and blocked using 5%
BSA in PBS for 1 h. The slices were incubated with rabbit
polyclonal antibody overnight at 4 jC, rinsed twice with
PBS, incubated with secondary antibody (goat anti-rabbit
peroxidase, 1:1000) for 1 h, and developed with 3.3V-diaminobenzidine (DAB) (Gibco BRL, Gaithersburg,
MD). Fluorescence detection was made with Cy3 secondary
antibody (goat anti-rabbit, 1:200; Sigma) and commercial
antibody was detected with donkey antigoat antibody cou-
pled to fluorescein–isothiocyanate chloride (Santa Cruz
Biotech.). Nuclear contrast marker was performed incubat-
ing slices with bis-benzamide as DNA for 1 h (1:1000)
(Hoechst reactive no. 33342, 0.5 Ag/ml; Sigma), rinsed three
times and mounted with an aqueous medium (FluorSave
reagent; Calbiochem-Novabiochem).
Images were obtained using a BX-60 Olympus micro-
scope with an Olympus-UTVO.5xC camera (Olympus,
Japan) and merged with CoolSNAP-Pro color program
(Media Cybernetics, MD). Confocal images were obtained
in a Carl Zeiss LSM510 laser microscope and merged using
the Adobe PhotoShop program.
2.7. Electron microscopy
Adult male mice were perfused with saline, followed by
4% paraformaldehyde/in 0.1 M of Caccodilate. The brains
were taken out and 40 Am thick vibratome sections were
collected and stained for PTEN as indicated above with a
Peroxidase coupled secondary antibody, followed by a DAB
labelling reaction. Sections were extensively washed, osmi-
cated for 1 h (1% OsO4 in PB), dehydrated through a
graded series of ethanol and propylene oxide, and embedded
in Spurr’s resin by a 48 h polymerisation at 60 jC. Ultrathinsections were obtained with a Sorvall ‘‘Porter Blum’’ MT2-
B ultramicrotome, contrasted with uranyl acetate and Rey-
nold’s lead citrate, and observed in a JEOL 1200 EX2
transmission electron microscope at 85 kV.
3. Results
3.1. PTEN expression in the CNS
The great majority of existent antibodies against the
PTEN protein are directed to its carboxy terminal domain
due to the low immunogenic properties of the amino
terminal domain of the protein. However, because the N-
terminal domain contains the enzymatic side of PTEN
(phosphatase domain) and the majority of mutations occur
within it, we decided to develop a new antibody to assess
the role of the catalytic domain in neurite differentiation.
Our antibody was directed against residues located in the
ph2-a 1 loop of the phosphatase domain, the insertion that
forms the side wall of the active site pocket [16].
The rabbit antiserum obtained against this amino-termi-
nal peptide of PTEN shows two bands of 60 and 34 kDa in a
comparative Western blot revealed with commercial antise-
rum (Fig. 1, (i)). Both bands and peptide dot blots disap-
peared when antiserum was blocked with the synthetic
peptide in a competitive assay (compare the first and second
lanes). The band of 60 kDa is consistent with findings of
other laboratories [17,36] however, the 34 kDa one, has
only been previously reported once [14].
The 60 kDa band was sharper and slimmer with our
antiserum than its counterpart detected by goat antiserum,
suggesting that our antibody recognizes a more specific
PTEN isoform [8]. By contrast, the low molecular weight
band can be detected with the same intensity by both antisera.
Then, the cellular distribution of PTEN was compared
using both antibodies in cerebellar slices (Fig. 1, (ii)). Both
antisera detected perikarya and nuclear labels in Purkinje
and granule cell layers in a double labeling assay. Although
these findings were consistent with previous reports [14], a
novel label was observed in the dendritic shafts of the
molecular layer (A, B, and C). The localization of PTEN
in dendrites was confirmed using both antibodies. Specific-
ity of the immunostaining was confirmed by a number of
controls. Staining was not evident when primary antibody
was omitted or a pre-immune antiserum was used (D).
3.2. Tissues and brain expression of PTEN fragment
We assessed PTEN fragment expression in different
tissues and brain regions using Western blot technique
Fig. 1. (i) PTEN Antiserum specificity. Rabbit antiserum pre-incubated with blocking peptide used as control. The antiserum directed against the PTEN peptide
was able to recognize two bands of 60 and 34 kDa, respectively, which have been indicated by black arrows. Commercial goat antiserum against amino-
terminal domain also showed two spots of the same electrophoretic mobility. Competition of the rabbit antiserum with the synthetic peptide is shown in the first
lane (Blocking peptide). The gray arrow indicates the peptide dot position in the membrane. (ii) Colocalization of PTEN amino-terminal antibodies in mouse
cerebellum. Brain sections were incubated with our rabbit antibody and a commercial PTEN goat antiserum, developed with Cy3 labeled anti-rabbit secondary
antibody (Red, A) and anti-goat antibody labeled with secondary antibody coupled to FITC, respectively (green, B). The merged image shows PTEN
antibodies colocalizations in yellow (C). Strong signals were detected in cell bodies and Purkinje dendritic shafts of the molecular layer (ML). Colocalization
was observed also in the cytoplasm of the Purkinje cell layer (PcL) and, with low intensity, in neurons of the internal granule layer (IGL). Although, we have
observed no labeling with our rabbit antibody in the mossy fibers, they are intensely stained with the goat-antibody. Also, in the granule cell axons, an intense
labeling with antibodies against the phosphatase domain can be detected in the molecular layer. Controls were made using normal goat and rabbit serums and
the previously mentioned secondary antibodies (D). Magnification was � 400.
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–19 11
(Fig. 2). Our antibody against PTEN displayed a band of 60
kDa, mostly in brain and thyroid gland lanes. This band was
faint in lung and other tissues. Interestingly enough, the 34
Fig. 2. (A) PTEN expression pattern in adult mice. Western blot detection with
stained with Coomasie blue is shown. Molecular weights are indicated on the le
mobility of the entire PTEN and the amino terminal PTEN fragment. PTEN expre
detected in brain. (B) PTEN expression pattern in brain. Different molecular weigh
Ag of protein from different brain regions indicated at the top. Molecular weight ma
different for each brain specific region.
kDa PTEN isoform could only be detected in brain. Fur-
thermore, in brain, the PTEN fragment was widely
expressed in variable amounts in almost every region,
the rabbit antibody against PTEN peptide. At the bottom, a duplicated gel
ft side of the gel. The black arrows highlight the apparent electrophoretic
ssion has been evaluated in different tissues. PTEN fragment has only been
ts of PTEN isoforms are indicated using black arrows. Each lane contains 50
rker positions are indicated at the left. The ratio between the two isoforms is
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–1912
showing the lowest intensity in the medulla and the highest
in the cerebral cortex (Fig. 2B). The PTEN fragment
reached the synaptosomal subcellular fraction a finding in
concordance with its novel localization (Fig. 3A). The 60
kDa PTEN was absent in the synaptic fraction of all ages
tested.
The PTEN label was also detected in dendrites of
Purkinje cells. Western blot analysis of the PTEN-fragment
expression during cerebellar postnatal development showed
its peak during the third week of development (Fig. 3B),
when the synapsis between granule cells and Purkinje spines
of dendrites are selected and stabilized [2].
3.3. PTEN fragment and synaptogenesis
Taking into account the positive correlation observed
between the synaptogenesis process and the PTEN frag-
ment, we decided to evaluate the expression pattern of
PTEN in the weaver mutant mouse model, where synapto-
genesis is severely impaired. In these ataxic mice, the
granule cells die in the pre-migratory external granule cell
layer and synaptogenesis between granule- and Purkinje
Fig. 3. (A) Subcellular localization of PTEN. Homogenate, nuclear and synapt
Synaptophysin antibody (arrow at 38 kDa) or PTEN amino-terminal antibodies. C
(P#) were analyzed using the PTEN amino-terminal antibody. The PTEN spot has
nuclear fraction and the 34 kDa one in the synaptosomal fraction (arrows). (B) PT
from homogenates or synaptosomes of cerebellum were compared in Western blo
fractions and homogenates, showing an increase at P20. The 60 kDa isoform was u
in normal and weaver mice during postnatal development. Nuclear and synaptic c
anti-PTEN peptide rabbit antiserum. Black arrows indicate the molecular weight of
spot only in the nuclear fraction having its maximum level at P17. This difference
present in all synaptic brain fractions of the weaver and control mice at all ages. In
weaver mutant cerebellum, the synaptic-fractions were devoid of PTEN fragment
are indicated at the right of the gel. Ad: adult; WvP17: weaver mutant mouse at
cells does not progress [35]. Wild type and mutant brains
and cerebellar subcellular fractions of adult and P10–P17
(synaptic stabilization period) mice were compared by
Western blot technique (Fig. 3C). Although nuclear frac-
tions showed both bands, only the complete isoform had an
increase at P17 in normal mice. By contrast, the weaver
mutant mice showed no differences between P17 and adult
cerebella. According to previous reports, the 60 kDa protein
has not been detected in the cerebellum and brain synaptic
fractions. We detected an increase of the 34-kDa PTEN
fragment during development in both, cerebellar and brain
synaptic fractions. PTEN fragment was undetectable in P17
and adult cerebellum of weaver mutant mice, contrasting to
the brain where differences in PTEN bands intensity be-
tween genotypes could not be detected. In summary, the
impaired synaptogenesis in the mutant weaver mice selec-
tively affects the expression of the 34 kDa PTEN fragment.
3.4. Localization of PTEN in brain slices
The presence of the 34 kDa fragment in the synaptic
fraction and the detection of this small band specifically in
ic crude fractions obtained from adult mouse brains were incubated with
erebellar and synaptic homogenates from mice of different postnatal ages
been split in the subcellular fraction. The band of 60 kDa is enriched in the
EN fragment expression during cerebellar development. Proteins obtained
ts. The minor band of PTEN was detected with goat antiserum in synaptic
ndetectable in this subcellular fraction. (C) Subcellular localization of PTEN
erebellar fractions of different postnatal ages (P#) were analyzed using the
the spots that have been detected by this assay. Western blots show a 60 kDa
has not been observed in the weaver mutant. PTEN fragment of 34 kDa was
cerebellum, this isoform shows an increase with during development. In the
label at P17 and in adult mice. Molecular weight marker (MWM) positions
postnatal day 17; WvAd: adult weaver mutant mice.
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–19 13
the brain lead us to identify PTEN-expressing cells using
our antiserum in sagittal sections of mouse brain. Strong
dendritic immunostaining was observed in cerebellum,
olfactory bulb, hippocampus, frontal and posterior cortex
(Fig. 4), as well as in striatum, and basal ganglia (not
shown). Cytoplasmic staining could be observed in the
perikarya of major neurons which also showed nuclear
PTEN labeling. In order to assess the nuclear PTEN
labeling, we performed Hoescht counterstaining. PTEN
and the DNA fluorescence labeling were co-localized in a
large population of cells but not in all nuclei. The double
labeling was observed in cells of the internal granular layer
Fig. 4. PTEN Immunostaining of mouse adult brain. Immunofluorescence of n
incubated with rabbit antibody against the peptide, developed with Cy3 (red)
Colocalizations can be observed in violet. Scale bars, 50 Am. Magnification at
antibodies. The highest intensity of the label was observed in nuclei during granule
(ML). In the Purkinje cell layer (PcL), the label was detected in the cytoplasm and
Layer (IGL), nuclei have a homogeneous labeling. (B) Posterior cortex. In thi
Cytoplasmatic label has been detected around the nuclei forming speckles in large
(Mi) dendrites that projected to the External Plexiform Layer (EPL). Nuclear label
(D) Frontal cortex. PTEN labeling looked like the one observed for the apical dendr
absent in pial surface cells. (E) Hippocampus PTEN staining can be observed in
different in the CA3 area. (F) Under magnification, almost all extrapyramidal cel
of cerebellum and olfactory bulb (Fig. 4A and C). In
occipital and frontal cortex, the proportion of PTEN stained
nuclei is lesser than that observed in other regions like
cerebellum (Fig. 4B and D).
3.5. Ultrastructural localization of PTEN in dendrites of
Purkinje cells
In order to provide a more accurate localization of PTEN
in dendrites, we performed an electron microscopic analysis.
The evaluation of PTEN immunostained profiles in the
normal cerebellar cortex revealed the abundancy of labeled
uclear, cytoplasmatic and dendritic PTEN localization. All sections were
and nuclei were counterstained with Hoechst DNA fluorescence (blue).
200� . (A) Cerebellar cortex. Not all the nuclei were labeled with rabbit
cells migration and in the dendrites of Purkinje cells in the molecular layer
in nuclei of Purkinje cells (speckled pattern). In the Internal Granular Cell
s region, PTEN staining can be observed in dendrites and many nuclei.
neurons. (C) Olfactory bulb. The stain was clearly observed in Mitral cell
can be detected in Mitral cells and in the internal granule cells layer (IGL).
ite extensions of the Pyramidal neurons [38]. Nuclear labeling of PTEN was
dendrites and many nuclei. Pattern of PTEN nuclear expression was highly
ls of hippocampus showed nuclear and dendritic labeling.
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–1914
dendrites dispersed through all the molecular layer. PTEN
labeled dendritic shafts (Fig. 5A and B) and spines (Fig. 5C
and D) were readily identified by the presence of electron-
dense labeled substance. We also observed the asymmetric
synapsis on the dendritic spines of the Purkinje spiny
branchlets, characterized by a thick postsynaptic density
(PSD), a widened cleft with dense staining material, and a
presynaptic axonal varicosity filled with round clear vesicles
(Fig. 5D).
3.6. PTEN expression during postnatal development
We detected an increase in the expression of the PTEN 34
kDa fragment during the second postnatal week of brain
development. In order to confirm the correlation between age
and dendritic expression of the PTEN fragment during
definite steps of synaptogenesis, we analyzed brain slices
from different selected ages. Cerebral cortex and hippocam-
pal neurons displayed intense dendritic staining in concor-
dance with Western blot findings (Fig. 6A, B and D). PTEN
labeling showed mostly nuclear localization in all layers. A
faint staining was observed in the proximal portion of
Fig. 5. Electron photomicrographs illustrating PTEN-immunostained dendrites o
PTEN labeling can be easily recognized by its electron dense speckled pattern. T
arrowheads point the mitochondria. (B) High magnification of the panel A. (C) P
density (PSD) shows an intense PTEN staining (arrowhead). The asterisk marks
magnification of the panel C. Scale bar 500 nm (15.000�).
dendrites in the cerebral cortex at P10. In pyramidal neuronal
dendrites, the staining showed an increase at postnatal day 17,
reaching it maximum expression in the adult mouse.
In hippocampus, dendrites of CA1 and Gyrus dentate
neurons, showed an increase in the expression of PTEN
fragment during development, as we have observed in
cerebellum and cortex (Fig. 6H and J). Neurons of CA3
region showed a lack of dendritic PTEN expression (Figs.
4F and 6I). However, the nuclei showed intense and
homogeneous labeling at P10. PTEN nuclear stain was
increased during development, but its nuclear pattern
changed in the CA3 area. Nuclear stain was homogeneous
in CA3 neurons at P10 (Fig. 6F), increasing with different
intensity at P17 (Fig. 6G) and finally only a subset of
neurons was labeled in adult mice (Figs. 4E and 6H).
PTEN dendritic label was clearly seen in Purkinje cells
during development of the cerebellum. At P10, granule cells
of the external layer still proliferate; the neurons start to
differentiate and the deeper neurons start their migration
inward into the molecular layer. It was at this time that
PTEN was highly expressed in the nuclei and stayed during
granule cell migration. When granule cells pass across the
f Purkinje cells. (A) PTEN-immunoreactive Purkinje cell dendritic shaft.
he asterisks mark the cisternae of the smooth endoplasmic reticulum. The
TEN-immunoreactive Purkinje cell dendritic spine. The thick postsynaptic
the presynaptic axonal varicosity filled with round clear vesicles. (D) High
Fig. 6. PTEN expression in frontal cortex during postnatal development: PTEN can be observed in nuclei of all layers at postnatal day 10 (A), 17 (B) and in
adult mice (D), showing similar levels of expression at all ages. Dendritic labels can be seen as dashes in the deeper layers at P10, increasing in the superficial
layers at P17 reaching its higher expression in adult mice. The Antibody label was efficiently blocked by PTEN synthetic peptide coupled to albumin (C).
Magnification shows Peroxidase staining in the pyramidal neurons of the V layer (black arrow, E). Scale bars, 50 Am. PTEN expression in hippocampus during
postnatal development. Hippocampus areas CA1, CA3 and Gyrus dentate are indicated in the figures (F–J). Magnification of the CA3 area was performed.
PTEN label can be detected in the nuclei of almost all neurons at postnatal day 10 (F). At P17, different patterns of staining were observed, coexisting cells with
a low and an intense labeling in the same area (G). In the adult mice, the difference between both subpopulations, in terms of labeling intensity, became higher
(I). The nuclear stain of the same hippocampal CA3 region was compared using Hoechst dye (H). Nuclear labeling of PTEN in CA1 and Gyrus Dentate was
homogeneous in almost neurons and its intensity increased with age. In dendrites, PTEN expression increased with age. Under magnification extrapyramidal
cells showed nuclear and dendritic labeling (J). Scale bars, 100 Am. Scale bars, 20 Am (for E and J).
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–19 15
Purkinje cell layer, PTEN labeling was notoriously dimin-
ished and became higher in the internal granule layer (Fig.
7A and D). Nuclear stain of PTEN was present in Purkinje
cells at P10. In adults, the PTEN nuclear labeling showed
a weak speckled pattern (Fig. 7C and F). PTEN label has a
bulk increase of intensity from P10 to P17 in the internal
granule cell layer nuclei, decreasing in adults (compare
Fig. 7A, B and C).
3.7. PTEN and p-Akt in normal, weaver, and staggerer
mutant mice brain
Cerebellar granule cell axons make contact with Pur-
kinje cells secondary branch dendrites during synapto-
genesis. These primary contacts change the physical
place of the shaft to a dendritic spine. Half of the total
synapsis was found in this subcellular compartment at P14
[15]. P-Akt antiserum in brain slices showed an intense
labeling in axons and also in perikarya of Purkinje cells. In
contrast, there was no staining of the dendritic shafts
during development (Fig. 8). Rabbit antibodies against
the PTEN peptide revealed changes in nuclear localization
and cytoplasmic enrichment of PTEN between P14 and
adult mice. An inverse correlation between the presence of
p-Akt and PTEN in axons and dendrites has been ob-
served. In the adult weaver mutant mouse, granule cells
are absent in the internal granule layer and Purkinje cells
are organized in multiple layers. Purkinje cells showed
Fig. 7. PTEN in cerebellum during postnatal development: Comparison of PTEN expression at different postnatal ages: P10 (A and D), P17 (B and E) and adult
(C and F) cerebella. Magnification showed PTEN label in D, E and F (630�). The histology showed the nuclear distribution of PTEN at P10, with increasing
levels of staining at P17 and a severe decay in adult mice (A, B, C, 100�). By contrast, Purkinje cells dendrites increased their label during all ages of
development, having their highest expression in the adult. A very weak stain can be observed in the granule cells in the external granule layer (EGL), showing
an increase in its intensity during the migration process inward to molecular layer (ML). PTEN expression declined when the cells reached the Internal
Granular Layer (IGL). Scale bar 100 Am in A–C or 50 Am in D–F. Purkinje cells layer (PcL).
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–1916
axon, perikarya and dendritic p-Akt labeling in the weaver
mutant cerebellum. Contrasting to p-Akt, only a weak
staining of PTEN was detected in the nucleus of the
neurons of weaver’s cerebellum, without label in dendrites
at P14 persisting to adult. Therefore, an inverse correlation
between p-Akt and PTEN labeling in dendrites is observed
in the weaver mouse cerebella. Weaver mutants do not
have any p-Akt or PTEN staining differences comparing to
the wild type mice in other regions like olfactory bulb,
striatum, hippocampus and cerebral cortex (data not
shown).
In the staggerer mutant mouse, the Purkinje cell’s differ-
entiation is blocked causing a congenital ataxia and a
cerebellar hypoplasia. Purkinje cell somata and dendrites
are smaller than normal at all stages [33]. In this model, the
granule cells form only primitive junctions with the Purkinje
cells dendritic shafts. These specialized junctions are not
superseded by the normal parallel fiber:Purkinje spine
synapsis and disappear by the third week. The staggerer
Purkinje cells showed an intense PTEN staining of the
dendritic spines and shafts (Fig. 8C).
4. Discussion
The PI3-kinase/p-Akt signaling pathway has been im-
plicated at different levels in neurite function. It has been
demonstrated that PI3 kinase inhibition suppressed neurite
outgrowth of PC12 cells [12]. Also, the relevant roles of
this pathway in initial axon polarization [32], growth [20]
and regeneration in cell culture [23] were previously
described. However, up to now, there have been no reports
showing the localization of PTEN, the downregulator of
this pathway, in neurites. The data presented in this work
support the existence of a new isoform of the PTEN
phosphatase protein. Although this isoform can be detected
in many different cell lines like colon carcinoma T84 cells
(data not shown), it seems to be specific to brain neuron
cells ‘‘in vivo’’. Taking into account not only that anti-
bodies directed against the carboxy-terminal domain can-
not detect this isoform but also its molecular weight, we
assume that the regulatory domain has been excluded from
this PTEN fragment. This finding allows us to suggest a
different mechanism of regulation for the PTEN neurite
activity.
We have observed a strict correlation between the
PTEN spatio-temporal pattern of expression and its func-
tion in central nervous system development. We have
detected the highest nuclear expression during granule
cell migration through the cerebellar molecular layer, an
observation that is in concordance with the relevant role
described for this protein in cerebellar architecture
[3,13,19]. Nuclear PTEN expression seems to be a very
sharply regulated process since it shows variable levels
Fig. 8. PTEN-fragment/p-Akt expression in normal and ataxic mutant mice cerebella. Comparative confocal PTEN localization in normal (A), weaver (B) and
staggerer (C) P17 cerebellar cortex. Panels (D) and (E) showed PTEN labeling in normal and weaver adult cerebellar cortex, respectively. A magnification of
the PTEN molecular layer’s stain in the normal adult mice is shown in panel (F). Panels (G) and (H) show the p-Akt labeling in normal and weaver mutant
mice, respectively. The control was performed using normal rabbit antiserum (I). Asterisks indicate the nuclear staining in granule cells during migration.
External granule layer (EGL), Molecular layer (ML); Purkinje cells layer (PcL); Internal Granular Layer (IGL). Scale bars 20 Am in A, B, C, E, G, H and I.
Scale bar 10 Am in D and 2 Am in F.
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–19 17
during neuronal differentiation. This variability of PTEN
nuclear levels seemed to be highest in the CA3 area of
the hippocampus, where the distribution of the nuclear
labeling showed a very heterogeneous pattern. Surpris-
ingly, in this area, many neurons expressed high levels of
PTEN, while others showed low or undetectable expres-
sion levels. It is worth highlighting that this labeling
pattern has only been observed using our antibody, which
is directed against the active site of the protein. Being
that the CA3 hippocampus area has been assigned as
responsible for episodic memory [22], we suggest that
PTEN activity can be directly or indirectly involved in
this complex process.
During the synaptic stabilization process, the dendritic
shafts showed a progressive enrichment of the 34 kDa
isoform of PTEN. This observation is in concordance
with the role of PTEN in the downregulation of the
protein synthesis mediated by the PI3K and TOR path-
ways [21,24,27,29,38]. Using a conditional gene disrup-
tion approach to inactivate PTEN during postnatal
development in a cell specific manner, Marino et al.
[19] found that Purkinje cells had a noticeable increase
in cell size, while the dendritic processes showed severe
thickening. This observation supports our finding of the
local role of the PTEN fragment in the arrest of the
dendritic growth or synaptic plasticity.
Many works confirmed that local dendrite protein
synthesis is involved in neuronal communication effi-
ciency in the adult brain [11,25]. Being the fact that
PTEN has been confirmed as a downregulator of cell
growth in neurons and that its inactivation causes dys-
regulation of cell growth in LDD [4], PTEN could be
considered as a local protein synthesis regulator upon
dendritic requests.
The mouse neurological mutant weaver has a point
mutation in girk2, a gene encoding of a G-protein-
coupled inwardly, rectifying the potassium channel [26].
The most obvious effect of the mutation is the cell loss
that occurs before the granule cells complete migration to
the internal granule cell layer [34]. Ultrastructural studies
C. Perandones et al. / Molecular Brain Research 128 (2004) 8–1918
of the agranular cortex in the weaver mutant mice
revealed that despite the presence of innumerable free
postsynaptic differentiations (mainly Purkinje cell dendrit-
ic spines), the number of synaptic junctions was severely
reduced. Thus, the Purkinje cells are unable to receive
their correspondent synaptic inputs [30]. In this model,
the 34 kDa PTEN fragment was absent only in the
dendrites of Purkinje cells, supporting the hypothesis that
the synthesis of this fragment is regulated by the synapto-
genesis process.
Staggerer is a classical mutation of RORa that blocks
Purkinje cell differentiation, resulting in congenital ataxia
and cerebellar hypoplasia. Developmental studies in stagger-
er mice indicated that the immature synaptic arrangements
and cell morphology are intrinsic to mutant Purkinje cells,
while subsequent loss of granule cells is a secondary conse-
quence. The granule cells form only primitive junctions with
Purkinje cell dendritic shafts. These specialized junctions are
not superseded by the normal parallel fiber:Purkinje spine
synapses and disappear by the third week. In the staggerer
model, the 34 kDa PTEN fragment was present in dendrites of
Purkinje cells, suggesting that the expression of this fragment
can be triggered by the initial contact between parallel fibers
and dendrites of Purkinje cells during the synaptogenesis
process.
Loss of PTEN leads to activation of the PI3K pathway and
thereby to phosphorylation and activation of Akt [5,6].
Because our antibody was directed to the active site of the
PTEN phosphatase, we expected to find no p-Akt levels in the
regions, which showed intense PTEN labeling. We found an
inverse correlation between the presence of PTEN fragment
and activated pAkt in normal mice. To investigate whether
and to what extent activation of Akt is implicated in neuronal
abnormalities, we set out to characterize the expression
pattern of activated Akt in the weaver mice using a p-Akt-
specific antibody. In the dendrites of Purkinje cells of the
weaver mice, the inverse correlation between PTEN and Akt
has also been observed.
Both previous published reports and the present data
support the role of PTEN as a local downregulator of the
PI3 kinase pathway in dendrites. Our findings showed that
the PTEN 34 kDa isoform, regulated by the synaptogenesis
process, plays a key role in the polarization of the PI3kinase
pathway in neurons. This paper provides further insight into
the regulation and function of local protein synthesis in
neuronal dendrites.
Acknowledgements
We thank Dr. Carina Ferrari for electron microscopic
analysis assistance. This work was supported by ‘‘Programa
Nacional de Ataxias y Huntington Argentina-ANLIS-Dr.
Carlos G. Malbran’’, the ‘‘Fundacion Alberto J. Roemmers’’
and the School of Natural Sciences, University of the
Buenos Aires (UBACyT: X-124).
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