research report correlation between synaptogenesis and...

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: [email protected] (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


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.


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.


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).


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).


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.


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


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).

References

[1] K.T. Akama, B.S. McEwen, Estrogen stimulates postsynaptic density-

95 rapid protein synthesis via the Akt/protein kinase B pathway,

J. Neurosci. 23 (6) (2003) 2333–2339.

[2] J. Altman, Postnatal development of the cerebellar cortex in the rat: II.

Phases in the maturation of Purkinje cells and of the molecular layer,

J. Comp. Neurol. 145 (4) (1972) 399–463.

[3] S.A. Backman, V. Stambolic, A. Suzuki, J. Haight, A. Elia, J.

Pretorius, M.S. Tsao, P. Shannon, B. Bolon, G.O. Ivy, T.W. Mak,

Deletion of PTEN in mouse brain causes seizures, ataxia and defects

in soma size resembling Lhermitte–Duclos disease, Nat. Genet. 29 (4)

(2001) 396–403.

[4] S. Backman, V. Stambolic, T.W. Mak, PTEN function in mammalian

cell size regulation, (Review)Curr. Opin. Neurobiol. 12 (5) (2002)

516–522.

[5] L.C. Cantley, B.G. Neel, New insights into tumor suppression: PTEN

suppresses tumor formation by restraining the phosphoinositide 3-

kinase/AKT pathway, Proc. Natl. Acad. Sci. U. S. A. 96 (8) (1999)

4240–4245.

[6] D.A. Cantrell, Phosphoinositide 3-kinase signaling pathways, Re-

view, J. Cell. Sci. 114 (8) (2001) 1439–1445.

[7] J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.

Strober (Eds.), Current Protocols in Immunology, vol. 1, John Wiley

& Sons, New York, 1995, Section II Unit 2.4.

[8] T.E. Creighton (Ed.), Proteins. Structures and Molecular Properties,

W.H. Freeman and Company, New York, 1996, p. 353.

[9] A. Di Cristofano, B. Pesce, C. Cordon-Cardo, P.P. Pandolfi, Pten is

essential for embryonic development and tumour suppression, Nat.

Genet. 19 (4) (1998) 348–355.

[10] R.J. Gorlin, M.M. Cohen, L.M. Condon, B.A. Burke, Bannayan–

Ruvalcaba syndrome, Am. J. Med. Genet. 44 (1992) 307–314.

[11] C. Jiang, E.M. Schuman, Regulation and function of local protein

synthesis in neuronal dendrites, Trends Biochem. Sci. 27 (10)

(2002) 506–513.

[12] K. Kimura, S. Hattori, Y. Kabuyama, Y. Shizawa, J. Takayanagi,

S. Nakamura, S. Toki, Y. Matsuda, K. Onodera, Y. Fukui, Neurite

outgrowth of PC12 cells is suppressed by Wortmannin, a specific

inhibitor of phosphatidylinositol 3-kinase, J. Biol. Chem. 272

(1994) 16089–16092.

[13] C.H. Kwon, X. Zhu, J. Zhang, L.L. Knoop, R. Tharp, R.J. Smeyne,

C.G. Eberhart, P.C. Burger, S.J. Baker, Pten regulates neuronal soma

size: a mouse model of Lhermitte–Duclos disease, Nat. Genet. 29 (4)

(2001) 404–411.

[14] M.B. Lachyankar, N. Sultana, C.M. Schonhoff, P. Mitra, W. Poluha,

S. Lambert, P.J. Quesenberry, N.S. Litofsky, L.D. Recht, R. Nabi, S.J.

Miller, S. Ohta, B.G. Neel, A.H. Ross, A role for nuclear PTEN in

neuronal differentiation, J. Neurosci. 20 (4) (2000) 1404–1413.

[15] D.M. Landis, R.L. Sidman, Electron microscopic analysis of postnatal

histogenesis in the cerebellar cortex of staggerer mutant mice,

J. Comp. Neurol. 179 (4) (1978) 831–863.

[16] J.O. Lee, H. Yang, M.M. Georgescu, A. Di Cristofano, T. Maehama,

Y. Shi, J.E. Dixon, P. Pandolfi, N.P. Pavletich, Crystal structure of

the PTEN tumor suppressor: implications for its phosphoinositide

phosphatase activity and membrane association, Cell 99 (3) (1999)

323–334.

[17] D.M. Li, H. Sun, TEP1, encoded by a candidate tumor suppressor

locus, is a novel protein tyrosine phosphatase regulated by transform-

ing growth factor beta, Cancer Res. 57 (11) (1997) 2124–2149.

[18] T. Maheama, J.E. Dixon, The tumor suppressor, PTEN/MMAC1,

dephosphorylates the lipid second messenger, phosphatidylinositol

3,4,5-trisphosphate, J. Biol. Chem. 273 (1998) 13375–13378.

[19] S. Marino, P. Krimpenfort, C. Leung, H.A. van der Korput, J.

Trapman, I. Camenisch, A. Berns, S. Brandner, PTEN is essential

for cell migration but not for fate determination and tumorigenesis

in the cerebellum, Development 129 (14) (2002) 3513–3522.

[20] A. Markus, J. Zhong, W.D. Snider, Raf and akt mediate dis-


tinct aspects of sensory axon growth, Neuron 35 (1) (2002)

65–76.

[21] G.B. Mills, Y. Lu, E.C. Kohn, Linking molecular therapeutics to

molecular diagnostics: inhibition of the FRAP/RAFT/TOR compo-

nent of the PI3K pathway preferentially blocks PTEN mutant cells

in vitro and in vivo, Proc. Natl. Acad. Sci. U. S. A. 98 (18) (2001)

10031–10033 (Comment on: Proc. Natl. Acad. Sci. U. S. A. 2001

98 (18) 10314-9 and 10320-5).

[22] S.J. Mizumori, K.E. Ragozzino, B.G. Cooper, S. Leutgeb, Hippocam-

pal representational organization and spatial context, Hippocampus 9

(4) (1999) 444–451.

[23] K. Namikawa, M. Honma, K. Abe, M. Takeda, K. Mansur, T. Obata,

A. Miwa, H. Okado, H. Kiyama, Akt/protein kinase B prevents injury-

induced motoneuron death and accelerates axonal regeneration,

J. Neurosci. 20 (8) (2000) 2875–2886.

[24] M.S. Neshat, I.K. Mellinghoff, C. Tran, B. Stiles, G. Thomas, R.

Petersen, P. Frost, J.J. Gibbons, H. Wu, C.L. Sawyers, Enhanced sen-

sitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR, Proc.

Natl. Acad. Sci. U. S. A. 98 (18) (2001) 10314–10319.

[25] L.E. Ostroff, J.C. Fiala, B. Allwardt, K.M. Harris, Polyribosomes

redistribute from dendritic shafts into spines with enlarged synapses

during LTP in developing rat hippocampal slices, Neuron 35 (3)

(2002) 535–545.

[26] N. Patil, D.R. Cox, D. Bhat, M. Faham, R.M. Myers, A.S. Peterson, A

potassium channel mutation in weaver mice implicates membrane

excitability in granule cell differentiation, Nat. Genet. 11 (2) (1995)

126–129.

[27] K. Podsypanina, R.T. Lee, C. Politis, I. Hennessy, A. Crane, J. Puc,

M. Neshat, H. Wang, L. Yang, J. Gibbons, P. Frost, V. Dreisbach, J.

Blenis, Z. Gaciong, P. Fisher, C. Sawyers, L. Hedrick-Ellenson, R.

Parsons, An inhibitor of mTOR reduces neoplasia and normalizes

p70/S6 kinase activity in Pten+/� mice, Proc. Natl. Acad. Sci.

U. S. A. 98 (18) (2001) 10320–10325.

[28] M. Radrizzani, H. Carminatti, O.H. Pivetta, V.P. Idoyaga-Vargas,

Developmental regulation of Thy 1.2 rate of synthesis in the mouse

cerebellum, J. Neurosci. Res. 42 (1995) 220–227.

[29] B. Raught, A.C. Gingras, N. Sonenberg, The target of rapamycin

(TOR) proteins, ColloquiumProc. Natl. Acad. Sci. U. S. A. 98 (13)

(2001) 7037–7044.

[30] P. Rossi, G. De Filippi, S. Armano, V. Taglietti, E. D’Angelo, The

weaver mutation causes a loss of inward rectifier current regulation in

premigratory granule cells of the mouse cerebellum, J. Neurosci. 18

(10) (1998) 3537–3547.

[31] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), , 2nd ed., Molecular

Cloning: A Laboratory Manual, vol. 3, Cold Spring Harbor Labora-

tory Press, Cold Spring Harbor, NY, 1989, Chap. 18.

[32] S.H. Shi, L.Y. Jan, Y.N. Jan, Hippocampal neuronal polarity specified

by spatially localized mPar3/mPar6 and PI 3-kinase activity, Cell 112

(1) (2003) 63–75.

[33] R.L. Sidman, P.W. Lane, M.M. Dickie, Staggerer, a new mutation in

the mouse affecting the cerebellum, Science 137 (1962) 610–612.

[34] R.J. Smeyne, D. Goldowitz, Development and death of external gran-

ular layer cells in the weaver mouse cerebellum: a quantitative study,

J. Neurosci. 9 (5) (1989) 1608–1620.

[35] C. Sotelo, Anatomical, physiological and biochemical studies of the

cerebellum from mutant mice. II. Morphological study of cerebellar

cortical neurons and circuits in the weaver mouse, Brain Res. 94 (1)

(1975) 19–44.

[36] V. Stambolic, A. Suzuki, J.L. de la Pompa, G.M. Brothers, C. Mirtsos,

T. Sasaki, J. Ruland, J.M. Penninger, D.P. Siderovski, T.W. Mak,

Negative regulation of PKB/Akt-dependent cell survival by the tumor

suppressor PTEN, Cell 95 (1) (1998) 29–39.

[37] H. Stocker, M. Andjelkovic, S. Oldham, M. Laffargue, M.P. Wymann,

B.A. Hemmings, E. Hafen, Living with lethal PIP3 levels: viability of

flies lacking PTEN restored by a PH domain mutation in Akt/PKB,

Science 295 (5562) (2002) 2088–2091.

[38] K.L. Whitford, P. Dijkhuizen, F. Polleux, A. Ghosh, Molecular con-

trol of cortical dendrite development, Annu. Rev. Neurosci. 25 (2002)

127–149.

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