university of pisa department of reproductive medicine and

UNIVERSITY OF PISA

Department of Reproductive Medicine and Child Development

Ph.D. THESIS

IN

Pathophysiology of Reproduction and Sexology

RAPID SIGNALING OF ESTROGEN TO WAVE1 AND MOESIN

CONTROLS NEURONAL SPINE FORMATION VIA THE ACTIN

CYTOSKELETON

DEFENDER:

Dr. Angel Matias Sanchez

Chief:

Prof. Andrea Riccardo Genazzani

Tutor:

Dr. Tommaso Simoncini

A mis ángeles, Delfina y Valentino, por iluminar mi vida con tanto amor.

A mi padres, Tito y Mirta, por darme las fuerzas, apoyo y sabiduría de creer que con esfuerzo todo se puede….

Al amor de mi vida, Marina, por su incondicionalidad, amor y fuerzas que me brindo desde el primer día.

INDEX

SUMMARY.......................................................................................................................... 6

RIASSUNTO................................................................................................................. ....... 7

INTRODUCTION............................................................................................................... 8

OVERVIEW ON ESTROGEN........................................................................................... 8

ESTROGEN RECEPTORS............................................................................................... 12

ER ACTION MECHANISMS.......................................................................................... 14

Membrane initiated signals (MIS) of estrogens

Nuclear signals (NS) of estrogens

ROLE OF ESTROGEN IN THE BRAIN......................................................................... 20

Estrogen and cell movement

Estrogen and the cytoskeleton

Moesin and actin remodeling

WAVE1 and spine dendrite formation

Role of estrogen receptor alpha (ERα) in WAVE1 and moesin regulation

AIMS.................................................................................................................................. 31

RESULTS........................................................................................................................... 32

Estrogen induces rapid cytoskeletal and cell membrane remodeling and dendritic spine formation in cortical neurons

Estrogen rapidly induces WAVE1 and moesin phosphorylation

Estrogen regulates WAVE1 via estrogen receptor α (ERα)

ERα signals to WAVE1 via a G protein/c-Src initiated cascade

Interaction of ERα with Gαi and Gβ is required to E2 signaling to c-Src

Estrogen signals to WAVE1 via cyclin-dependent kinase 5 (Cdk5)

Differential signaling of ER to moesin and WAVE1 in neurons

Estrogen signaling to WAVE1 results in Arp-2/3 membrane translocation

Estrogen regulates the formation of dendritic spines via WAVE1 and moesin in cortical neurons

DISCUSSION................................................................................................................... 39

MATERIALS AND METHODS................................................................................... 43

Neuronal cell cultures

Immunoblottings

Immunoprecipitations

Quantitative analysis of cell membrane morphology and thickness and of actin fibres remodeling

Cell immunofluorescence

Kinase assays

Gene silencing with RNA interference

Transfection experiments

Statistical analysis

REFERENCES................................................................................................................... 49

FIGURE LEGENDS.......................................................................................................... 60

SUPPLEMENTAL DATA............................................................................................... 66

FIGURES............................................................................................................................ 68

SUPPLEMENTAL DATA............................................................................................... 83

ACKNOWLEDGEMENT................................................................................................ 88

DECLARATION............................................................................................................... 89

6

SUMMARY

Estrogens are important regulators of neuronal cell morphology, and this is thought to be

critical for gender-specific differences in brain function and dysfunction. Dendritic spine

formation is dependent on actin remodeling by the WASP-family verprolin homologous

(WAVE1) protein, which controls actin polymerization through the actin-related protein

(Arp)-2/3 complex. Emerging evidence indicates that estrogens are effective regulators of

the actin cytoskeleton in various cell types via rapid, extranuclear signaling mechanisms.

We here show that 17-estradiol (E2) administration to rat cortical neurons leads to

phosphorylation of WAVE1 on the serine residues 310, 397, and 441 and to WAVE1

redistribution toward the cell membrane at sites of dendritic spine formation. WAVE1

phosphorylation is found to be triggered by a Gαi/Gβ�protein-dependent, rapid

extranuclear signaling of estrogen receptor α to c-Src and to the small GTPase Rac1. Rac1

recruits the cyclin-dependent kinase (Cdk5) that directly phosphorylates WAVE1 on the

three serine residues. After WAVE1 phosphorylation by E2, the Arp-2/3 complex

concentrates at sites of spine formation, where it triggers the local reorganization of actin

fibers. In parallel, E2 recruits a Gα13-dependent pathway to RhoA and ROCK-2, leading to

activation of actin remodeling via the actin-binding protein, moesin. Silencing of WAVE1

or of moesin abrogates the increase in dendritic spines induced by E2 in cortical neurons.

In conclusion, our findings indicate that the control of actin polymerization and branching

via moesin or WAVE1 is a key function of estrogen receptor α in neurons, which may be

particularly relevant for the regulation of dendritic spines. (Molecular Endocrinology 23:

1193–1202, 2009)

7

RIASSUNTO

Gli estrogeni sono importanti regolatori della morfologia delle cellule neuronali e questo

sembra essere responsabile delle differenze genere-correlate nelle funzioni e disfunzioni

cerebrali. La formazione delle spine dendritiche dipende dal rimodellamento actinico da

parte di WAVE-1 (WASP-family verprolin homologus), che controlla la polimerizzazione

actinica attraverso Arp2/3 (actin-related protein 2/3). Studi recenti evidenziano che gli

estrogeni sono i regolatori effettivi del citoscheletro actinico in vari tipi cellulari attraverso

segnali rapidi extranucleari. In questo studio dimostriamo che il trattamento dei neuroni

corticali di ratto con 17β-estradiolo (E2) porta alla fosforilazione di WAVE-1 a livello di

ser-310, 397 e 441 e alla ridistribuzione di WAVE-1 sulla membrana cellulare, nei siti di

formazione delle spine dendritiche. La fosforilazione di WAVE-1 sembra essere innescata

da un segnale extranucleare rapido dell’ERα (dipendente da una proteina Gαi/Gβ) che

attiva c-Src e la GTPasi Rac1. Rac1 recluta la chinasi ciclina-dipendente (Cdk5) che fosforila

direttamente WAVE-1 sui tre residui serinici. Dopo la fosforilazione di WAVE-1, il

complesso Arp2/3 si concentra nei siti di formazione delle spine dendritiche, dove è

innescata la riorganizzazione locale delle fibre actiniche. In parallelo, E2 recluta una via

Gα13-dipendente che attiva RhoA e ROCK-2, portando all’attivazione del rimodellamento

actinico attraverso la proteina legante l’actina, moesin. Il silenziamento di WAVE-1 o di

moesin annulla l’incremento delle spine dendritiche indotto dall’E2 nei neuroni corticali.

In conclusione le nostre scoperte indicano che il controllo della polimerizzazione actinica e

la ramificazione attraverso WAVE1 è una funzione chiave dell’ ERα nei neuroni,

particolarmente rilevante per la regolazione delle spine dendritiche. (Mol. Endoc., 2009).

A.M. Sanchez Rapid Signaling of Estrogen in the Brain

8

INTRODUCTION

OVERVIEW ON ESTROGEN

Estrogens, like other steroid hormones, are naturally occurring

cyclopentanophenanthrene compounds whose synthesis begins with cholesterol.

Estrogens play key roles in development and maintenance of normal sexual and

reproductive function in both men and women [1]. Although, their levels are significantly

higher in women of reproductive age. They are mainly produced by the ovary and in part

by the adrenal cortex, and three estrogens occur naturally in the female [2-3]. In

premenopausal women, 17β-estradiol (E2), produced by the ovary granulosa, is the

estrogen produced in the largest quantity and is the most potent as it has the highest

affinity for its receptors. In pre-menopausal women, circulating estradiol levels fluctuate

from 40 to 200-400 pg/mL during the menstrual cycle [3]. After menopause, estradiol

levels drop to less than 20 pg/mL. The second estrogen is estrone (E1), a less potent

metabolite of estradiol. Estrone is produced from androstenedione, the immediate

precursor of estrone, in the liver and adipose tissue. In post-menopausal women, the

ovary ceases to produce estradiol while the adrenal gland continues to produce

androstenedione. As the result, the level of estrone remains unchanged while the plasma

level of estradiol falls significantly. The third endogenous estrogen is estriol (E3), also a

metabolite of estradiol (Fig. A). Estriol is the principal estrogen produced by the placenta

during pregnancy, and is found in smaller quantities than estradiol and estrone in non-

pregnant women [2-4].


9

Fig. A. The chemical structures of estrone, 17β-estradiol, and estriol.

In the target tissues such as reproductive organs, estrogens exhibit important biological

functions. Among these, the ovarian cycle, the development of both sexual primary and

secondary characters are dependent on estrogen [5]. Besides regulation of physiological

functions in reproductive tissues, estrogens exert a vast range of biological effects and

influence many other physiological processes in mammals including cardiovascular health,

bone integrity, cognition, and behaviour [1].

Estrogens play important roles in bone homeostasis, being involved in modeling of bone

during adolescence. They initiate pubertal growth and later limit longitudinal bone growth

by inducing closure of the epiphysial growth plate. Even in adult life, sex steroids appear

to influence the remodeling of bone. E2, in particular, is crucial for the maintenance of

bone mass in females [6] as is evident from the rapid loss of trabecular bone and

development of osteoporosis that occurs after ovariectomy or at menopause [7-8].

Several factors, known to be important in regulating differentiation and function of


10

osteoblasts and osteoclasts, are regulated by E2. In osteoblasts, E2 stimulates synthesis

and secretion of the anabolic growth factor IGF-I and inhibits that of thecytokines, IL-1,

tumor necrosis factor (TNF), and IL-6 [9] that are involved in bone resorption [10].

Estrogen appears to be protective in the cardiovascular system, in part due to favourable

modulation of serum lipid profiles [11-12]. The 30% of these effects are due to a reduction

of total cholesterol by increasing the LDL receptor (LDLr) expression in the liver [13-14]

and inhibiting the LDLr oxidation at vascular level [15]. Recent evidence indicates that the

70% of estrogen effect is also due to its direct action on the vasculature, as demonstrated

by gender differences in smooth muscle contractility, with a greater response to

noradrenaline or phenylephrine in aortas isolated from male than female rats [16]. E2

rapidly induces the activity of endothelial nitric oxide synthase (eNOS), which produces

the vasodilator nitric oxide (NO) [17]. Furthermore, estrogens are able to prevent the

proliferation and the migration of vascular smooth muscular cells (VSMC) both directly

[18] and indirectly blocking the mitogenic action of growth factors [19-20]. Prior to

menopause, women have a lower incidence of coronary heart disease compared with age-

matched men [21-22]. Moreover, estrogen replacement therapy reduces mortality due to

coronary heart disease in post-menopausal women ([11]. Deprivation of estrogens by

ovariectomy results in enhanced vascular contraction in aortas of female rats [16]. In

ovariectomized rats, chronic estrogen replacement suppresses endothelium-dependent

contractions mediated by the cyclo-oxygenase/prostaglandin H synthase pathway [23].

Studies in humans also demonstrate a beneficial vascular action of estrogen, with


11

endothelium-dependent vasodilatation being enhanced by the administration of 17β-

estradiol at physiological levels [24-26].

In central nervous system, estrogen has been found to contribute to promote synapse

formation and plasticity and to limit neuronal damage and death, besides its capability to

protect from neurodegenerative process. Furthermore, the 17β-estradiol plays an

important role as antioxidant reducing the oxygen reactive species (ROS) by increasing the

protein level of tioredoxin, an important protein that protect from lipid peroxidation after

oxidative stress [27].

Postmenopausal women are in an estrogen-deprived state and are at risk for stroke and

other neurodegenerative diseases [28]. Epidemiological evidence suggests that

postmenopausal estrogen therapy (ET) reduces the risk or delays the onset of Alzheimer

disease [29]. Estrogen loss from natural or surgical menopause has been associated with a

decline in cognitive function [30] that is reversed by ET. ET has been shown to affect

cognitive function during brain aging as well [31-32].

Given this widespread role for estrogen in human physiology, it is not surprising that

estrogen is also implicated in the development or progression of numerous diseases,

which include but are not limited to osteoporosis, neurodegenerative diseases,

cardiovascular disease, insulin resistance, lupus erythematosus, endometriosis, obesity

and various types of cancer (breast, ovarian, colorectal, prostate, and endometrial).

Indeed, in addition to proliferative effects on normal cells, estrogen is considered as a

stimulant for the initiation and promotion of tumors in these organs [5]. This expanded


12

view of estrogen action reflects the findings of a large number of clinical and

epidemiological studies which gathered on the effects of exogenous estrogen

administration on the post-menopausal women health. Based on data from both clinical

and animal studies, estrogen is implicated in the development of breast cancer stimulating

proliferation of mammary cells by increasing cell division and DNA synthesis [33]. Risk

factors associated with breast cancer reflect cumulative exposure of the breast epithelium

to E2 [2, 34]. There is also evidence that estrogens, together with gonadotropins,

contribute to the etiology of ovarian cancer in humans [33, 35]. Whereas, clinical studies

indicate that the incidence of colon cancer is lower in women than in men [36], and data

from the Women’s Health Initiative indicate a significantly reduced incidence of colon

cancer in postmenopausal women receiving combined ‘Hormone Replacement

Therapy’(HRT; estrogen plus progestin) [2, 37].

ESTROGEN RECEPTORS

The biological actions of estrogens are traditionally mediated by binding to one of two

specific estrogen receptors (ERs), ERα or ERβ, that are encoded by different genes located

on different chromosomes (locus 6q25.1 and locus 14q23-24.1, respectively) [38-40]. ERα

and ERβ, like all the members of the nuclear receptor super-family, are modular proteins

sharing common regions, named A/B, C, D, and E/F, as well as a high sequence homology

(Fig. B). These regions participate in the formation of independent but interacting

functional domains. The N-terminal domain (A/B region) is involved in both inter-

molecular and intra-molecular interactions as well as in the activation of gene


13

transcription. The DNA binding domain (DBD, C region) allows ER to dimerize and to bind

to the specific ERE sequence on DNA through its two “zinc finger” structures. The hinge

domain (D region) has a role in receptor dimerization and in binding to chaperone heat-

shock proteins (Hsp). The ligand binding domain (LBD, E/F region, C-terminal) comprises

the E2-binding domain and works, synergistically with the N-terminal domain in the

regulation of gene transcription [10, 41-42].

Fig. B. A schematic structural comparison of human ERα and ERβ functional domains. Receptor

domains are illustrated with different colored boxes, and the approximate size of each domain is

indicated. The A/B domain contains the ligand-independent transcriptional-activation function AF-

1, the C domain represents the DNA-binding-domain (DBD), the D domain corresponds to the

hinge region, and the E domain contains the hormone-binding domain (LBD) and the hormone-

dependent transcriptional-activation function AF-2. The number inside each box of ERβ refers to

the percentage of amino acid identity.

ERs contain two regions called activation functions (AFs) important for ligand-dependent

transcriptional activity (Fig. 1.1) [10, 41-42]. AF-1 and AF-2 regions of ERs, interacting with

a number of transcription co-activators, can activate transcription independently but in


14

most cases, they synergize with one another in a promoter- and cell-context specific

manner [43]. AF-1 could be activated even in a ligand independent manner, depending on

the phosphorylation status of ER. In particular, the Ser118 residue in the AF-1 region of

ERα, as well as residues Ser106 and Ser124 in the AF-1 region of ERβ, are the

phosphorylation sites essential for the ligand-independent activation of ERs through the

Ras mitogen activated protein kinase (MAPK) signaling cascade [44].

Recent progress in studies on genomic and cDNA sequences has accelerated the

identification of gene splice variants in the NR super-family. Numerous mRNA splice

variants exist for both ERs and the best characterized splice variants are ERα46 and ERβcx,

which are frequently co-expressed with their wild-type counterparts. The exact function

and potential role of these and other ERs splice variants in physiology and human disease

remain to be elucidated [45-46].

ER ACTION MECHANISMS

The mechanisms of ERα and ERβ action are complex pathways that involve two distinct

types of signaling which lead to protein kinase activation mediate of rapid or

“nongenomic” effects (rapid membrane-initiated mechanism) and direct or indirect

transcription of target genes (nuclear mechanism) (Fig. C). All these pathways synergize

each other to determine the overall effects of E2.


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Fig. C. Models of estrogen action. In the “classical” pathway of estrogen action (i), estrogen or

other selective estrogen receptor modulators (SERMs) bind to the estrogen receptor (ER), a ligand-

activated transcription factor that regulates transcription of target genes in the nucleus by binding

to estrogen response element (ERE) regulatory sequences in target genes and recruiting

coregulatory proteins (CoRegs) such as coactivators. Rapid or “nongenomic” effects of estrogen

may also occur through the ER located in or adjacent to the plasma membrane (ii), which may

require the presence of “adaptor” proteins, which target the ER to the membrane. Activation of

the membrane ER leads to a rapid change in cellular signaling molecules and stimulation of kinase

activity, which in turn may affect transcription. Lastly, other non-ER membrane-associated

estrogen-binding proteins (EBPs) may also trigger an intracellular response (iii).


16

Membrane initiated signals (MIS) of estrogens

The nuclear initiated signals of steroid hormones occurs after a time-lag of at least 2 hours

after E2 stimulation and explains some of hormone functions in physiological and

pathological situations [47-48]. This picture was challenged when a physiological dose of

E2 was reported to increase the uterine cAMP level in ovariectomized rats within 15

seconds, an effect too rapid to be accounted for genomic action(s). This event was not

abrogated by transcriptional inhibitors and was termed “rapid or nongenomic”. Actually

the term “non-genomic” is not adequate when referring to rapid changes that may also

initiate new gene transcription [47, 49]. Various signaling pathways are activated upon E2

binding to ERs. These rapid events may be classified into four main signaling cascade:

phospholipase C (PLC)/protein kinase C (PKCs) [13, 20, 50-51], Ras/Raf/MAPK [52-54],

phosphatidyl inositol 3 kinase (PI3K)/AKT [48, 55-56], and cAMP/protein kinase A (PKA)

[57-58]. These pathways present numerous interactions with several other pathways

(Figure D). The ERα:E2 complex interacts with the IGF-1 receptor, leading to IGF-1

receptor activation and hence to MAPK signaling pathway activation [59]. In addition, the

ERα:E2 complex activates the EGF receptor by a mechanism that involves activation of

guanine nucleotide exchange proteins (G-proteins), Src, and matrix metalloproteinases,

leading to an increase in extracellular regulated kinases (ERK) and PI3K/AKT activities [52,

59-61]. In endothelial cells the Src/PI3K/AKT pathway mediates rapid E2-dependent

activation of eNOS and the release of nitric oxide. AKT and PKC could also modulate the

MAPK pathway through Raf phosphorylation [17, 48, 62]. It is important to note that

activation of signaling pathways by E2 is cell type-specific. Indeed, the effect of E2 on PKC


17

Figure D. Schematic model illustrating the relationship between the mechanisms of action of E2.

Palmitoylation (PA) allows the estrogen receptor (ER) localization at the plasma membrane. E2

binding induces ER association to signaling proteins, and triggers the activation of signaling

cascades. The kinase activations phosphorylate ER, modulate transcriptional coactivators

recruitment, and enhance AP-1 and Sp-1 activation. After dimerization ERs directly interact with

ERE on DNA. ERs-DNA indirect association occurs through protein-protein interactions with the Sp-

1 and AP-1 transcription factors.

activity has been observed in the preoptic area of female rat brain slices, but not in the

hypothalamus or cortex [63]. The activation of G-protein/Src/PI3K/MAPK pathway by E2


18

was evident in late, but not early, differentiated rat pre-adipocytes ([52]. The differential

requirement of Src/PI3K or intracellular calcium for MAPK activation is also observed in

diverse cell types [52, 60]. Different PKC isoforms are rapidly activated by E2 in HepG2 and

MCF7 cells [50]. As a whole, these studies indicate that the rapid actions of E2 depend on

a number of conditions such as the set of signal transduction molecules and downstream

targets present in the target cell, thus the responses are likely to be diverse. All these

results point to the concept that ERα is the primary endogenous mediator of rapid E2

actions.

Nuclear signals (NS) of estrogens

In the “classical” mechanism of action, estrogens diffuse into the cell membrane and bind

to ERs causing ERs to dissociate from heat shock proteins, to dimerize and to traslocate

into the nucleus. The nuclear ERα- or ERβ-E2 complex directly binds DNA through the ERE

(estrogen responsive element) sequences or indirectly through protein-protein interaction

with activator protein-1 (AP-1) or stimulating protein (Sp-1), resulting in recruitment of

coregulatory proteins (coactivators or corepressors) to the promoter, increased or

decreased mRNA levels, protein synthesis, and physiological responses ([2, 33] (Figure C-

E). A large subset of coregulatory proteins (e.g., steroid receptor coactivator-1, 2, and 3)

helps the hormone-receptor complex to recruit histone acetyltransferases and

methyltransferases which, in turn, possess chromatin remodeling ability and tether

activated receptors to the basal transcriptional machinery [64]. Both ERα and ERβ are

capable of regulating gene transcription through this classical mechanism involving ERE.


19

ERβ seems to be a weaker transactivator [65]. AF-1 activity of ERβ is weak compared with

that of ERα on ERE, whereas their AF-2 activities are similar [65]. In turn, when both AF-1

and AF-2 functions are active in a particular cell and/or on a particular promoter, the

activity of ERα greatly exceeds that of ERβ, whereas ERα and ERβ activities are similar

when only AF-2 is required [2, 66]. It has been postulated that differences in the ERα and

ERβ activities are due to differences in the ability of the receptors to interact with

coregulatory proteins, because of the low amino acid identity in A/B domain of ERs (Figure

B) [2, 64].

Another category of gene promoters, lacking any ERE-like sequences, requires a second

DNA-binding transcription factor (e.g., Sp-1 and AP-1) to mediate ER association with the

DNA [67] (Figure E). Although ERα and ERβ have similar effects on ERE-mediated gene

transcription, the receptors show opposite effects on promoters containing AP-1 [68]. E2

activates AP-1-mediated gene transcription when bound to ERα but inhibits promoter

activity when bound to ERβ [68]. The converse is true for anti-estrogens, such as

tamoxifen, raloxifene, and ICI 164384, which are AP-1 transcriptional suppressors via ERα

and activators via ERβ [68-69]. Similar to AP-1, E2 binding to ERα induces transcriptional

activation when associated with Sp-1 in GC-rich regions. However, E2 binding to ERβ does

not result in the formation of a transcriptionally active complex at a promoter containing

Sp-1 elements [70]. Consequently, these differences in transcriptional activity between

the ERα and ERβ may account for the major differences in their tissue specific biological

actions.


20

Figure E. Schematic model illustrating the action mechanisms of E2. In the first panel (a) is

depicted the classical interaction of the activated receptor with ERE on DNA. In panels b and c are

representations of the indirect effects of ERs on transcription interactions. This occurs through

protein-protein interactions with the Sp1 (b), AP-1 (c). The panel d represents the E2-non-genomic

mechanism. AP-1, activating factor-1; Sp-1, stimulating factor-1.

ROLE OF ESTROGEN IN THE BRAIN

Estrogen and cell movement

Sex steroid hormones are fundamental regulators of cell growth, proliferation, and

migration. Indeed, estrogens direct the development of several tissues and organs [71]

and orchestrate cell growth and proliferation during adult life [72]. Cells respond to the

environment through the expression of transmembrane receptors that sense extracellular

stimuli and activate an elaborate network of intracellular signaling molecules. Given the

large number of different signaling molecules and the complex relationships between

them, a major challenge is to understand how specific signals generate unique responses.


21

The architecture of signaling networks plays an important role in achieving signaling

specificity [73].

The control of cell morphology and of the interaction of the cell with the extracellular

environment requires a number of regulators orchestrating the different cytoskeletal

components and their interactions with the cell membrane and with anchorage proteins

[74]. Recent findings indicate that extra-nuclear signaling of estrogen is a fundamental

regulator of cell morphology in diverse cellular lines, including neuronal cells [75-76], and

that many of these actions are played via rapid signaling to the actin cytoskeleton

achieved via actin recruitment [77-78].

The brain is an important target of sex steroids, like estrogen, that plays multiple

regulatory roles. These hormones are powerful mediators of dynamic brain differentiation

into a male or female phenotype. This phenomenon has been originally described in

studies on neuronal cell migration in the developing pre-optic area/anterior hypothalamus

[79]. The effects of sex hormones turn into differences in neuronal cell number, glial

complexity, neurochemical expression and synaptic connectivity [80] and are related to

estrogens in the brain. Estrogen acts as neurotrophin regulating cell death, neurogenesis,

neurotransmitter plasticity, neuronal migration and synaptogenesis [81] and dynamically

modulates brain ontogeny by influencing neuronal cell movement [82-83].

In the recent years, the knowledge on how estrogen interferes with mammalian brain

functions and development has broadened substantially. In the adult brain, estrogen is

not only involved in the neuroendocrine feedback regulation at the hypothalamic and


22

pituitary level but also in the control of motor and cognitive functions. After the

demonstration that the estrogen-synthesizing enzyme aromatase and both estrogen

receptor α and β isoforms are expressed in many brain areas during ontogeny, it was soon

realized that estrogen modulates neuronal differentiation, notably by influencing cell

migration, survival and death, and synaptic plasticity of neurons. These effects were

initially seen in the classical target area for estrogen, the hypothalamus, but later studies

revealed actions of estrogen also in other regions. Estrogens do not act only on the early

stages of neuronal development but also on mature neurons for the maintenance and

reorganization of dendritic structures. Dendrites are the principal cellular sites where

neurons receive, process, and integrate inputs from their multiple pre-synaptic partners.

Both the shape of dendritic trees and the density and shape of their spines can undergo

significant changes during the development and life of a neuron. Dendritic spines are

composed from actin microfilaments that undergo to dynamic remodeling responsible for

brain development of neuronal circuits under the regulation of several extracellular

stimuli [84]. Estrogen is involved in the formation of dendritic spines during development

and of their structural plasticity at mature synapses, increasing dendritic spine density

through the dynamic control of actin filaments [85] (Fig. F).

One of the most intriguing actions that these hormones perform is the control of brain

plasticity, that is thought to be critical for key functions of this organ, such as memory,

learning and making cognition possible via neuronal/glial remodeling [86-87]. At the basis

of brain plasticity is the ability of neurons and glial cells to remodel their mutual

connections which requires major changes in cell morphology [88-89].


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Figure F. Complete neuron cell diagram. Neurons (also known as neurones and nerve cells) are

electrically excitable cells in the nervous system that process and transmit information. In

vertebrate animals, neurons are the core components of the brain, spinal cord and peripheral

nerves.

These morphological modifications are tightly dependent on the generation of dynamic

structural changes of the actin cytoskeleton, via actin polymerization and de-

polymerization that also represent the starting platform for other important cellular

processes such as cytokinesis, adhesion and motility. When a cell remodels its shape, the

actin cytoskeleton is reorganized, and protrusive membrane structures, such as

lamellipodia and filopodia, are formed at the leading edge. These structures generate the


24

locomotive force in migrating cells and are critical in the generation of cell–cell

interconnections [74].

Clinical studies also suggest that the lack of estrogens, such as in women after the

menopause, may be related to the progression of brain degenerative diseases, like

Alzheimer’s Dementia or Parkinson’s disease [90-91], and the hypothesis that estrogen

administration to postmenopausal women might decrease the progression of these

conditions is lingering [92].

Estrogen and the cytoskeleton

The cytoskeleton is an organized structure formed by the connection of three different

types of filaments: actin, microtubules and intermediate filaments. These cytoskeletal

complexes interact together as a dynamic network contributing to cell functions such as

structural integrity, shape, division and cell motility. Indeed, in most animal cells, the

cytoskeleton is the primary actor in creating the driving forces through which cells move

[93-94]. Actually, although other non-actin cytoskeletal complexes aid in coordinating cell

movement and in empowering translocation, the actin cytoskeleton through highly

dynamic remodeling and continuous creation of new actin networks is considered

essential for pushing the cell forward in the surrounding environment [74], especially at

front membrane cell protrusions that allow the very first steps of cell motility. This

restructuring of cell shape is achieved with the help of a number of accessory proteins and

signaling pathways [95] that mediate signals of growth factor and cytokines.


25

Estrogens are fundamental modulators of cell morphology and motility in different cellular

lines. Binding their receptors sex hormones may modulate different rapid intracellular

cascades such as G protein, tyrosine kinase, c-Src, small GTPases, and protein kinases

pathways finally leading to the activation of actin remodeling and cell movement. In this

context, we have recently described how the treatment of diverse cell lines with

physiological doses of estradiol led to a rapid remodeling of actin cytoskeleton with a loss

of stress fibers [77-78]. This cytoskeletal rearrangement, associated with the development

of specialized cell membrane structures such as ruffles and pseudopodia, is obtained

through the activation of the actin-regulatory protein, moesin [77-78]. A number of

studies underlined the fundamental contribution of the ezrin–radixin–moesin (ERM)

family of proteins to cytoskeletal processes responsible for many vital cellular functions

such as cell motility [96].

Moesin and actin remodeling

Moesin, a member of the ezrin/radixin/moesin (ERM) family, is an actin-binding protein

that plays an important role in cell motility by linking the actin cytoskeleton to a variety of

membrane anchoring proteins [97-98]. In quiescent conditions moesin exists in an auto-

inhibited conformation and phosphorylation of Thr558 within the C-terminal actin-binding

domain by the Rho-associated kinase (ROCK), results in a conformational change and in

the association with the scaffold protein, ezrin/radixin/moesin-binding protein 50 (EBP50)

on moesin’s NH2-terminal end and with F-actin on moesin’s COOH-terminal end to

mediate the linkage of microfilaments to membranes in cell surface microvilli [99-100].


26

Estrogen activates moesin in breast cancer and endothelial cells through a rapid, extra-

nuclear signaling cascade originated by the interaction of ERα with the G protein Gα13.

This process leads to the recruitment of RhoA and of the Rho-associated kinase, ROCK-2

and to moesin activation. This pathway leads to the formation of membrane ruffles and

pseudopodia which interact with the extracellular matrix and with nearby cells, thus

promoting cell migration [77-78]. Estrogen exposure leads to rapid modifications of the

cytoskeleton’s organization and to modified interaction with the surrounding environment

and nearby cells [77-78, 101]. The control of cell morphology may well also be relevant for

the actions of estrogen in the central nervous system, where morphological changes in

neuron/neuron interconnections and dendritic spine density ensue related to the cyclical

changes in estrogen levels [102-103].

WAVE1 and spine dendrite formation

A large amount of literature supports a key role of the Rho family of GTPases (Rho, Rac1

and cdc42) in coordinately regulating the polymerization of the actin cytoskeleton and

turnover during cell migration [104] through the activation of different downstream

effectors such as c-Src kinase, PI3K (phosphoinositide-3 kinase), PTEN phosphatase (which

converts phosphoinositides PIP3 into PIP2) and the WASP family of proteins. The discovery

of this group of cytoskeletal-regulatory proteins has contributed to clarify the molecular

basis of cell movement. Indeed, signaling from Rho small GTPases to the Arp2/3 complex,

the WASP family members WASP, N-WASP and WAVE promote the formation and

branching of actin filaments [105-107] (Figure G). In particular, WAVE1 protein that is


27

Figure G. Model of actin nucleation by WASP-ARP complex

regulated by Rac1 plays a key role also in the induction of actin branching and dendrite

spine formation [108].

The WAVE1 complex seems to be transported by kinesin-1, and this transport has been

suggested to be important for axon elongation [109]. Several reports have also indicated

the involvement of N-WASP in spine formation [106, 110-111]. Phosphorylation and de-


28

phosphorylation of WAVE1 are also involved in spine formation. Kim et al. recently

reported that the WAVE1 complex was active during spine formation, and that cyclin

dependent kinase-5 (CDK5) phosphorylates the proline-rich region of WAVE1 in the

WAVE1 complex. The phosphorylation sites in the proline rich region of WAVE1 are not

conserved in WAVE2 and WAVE3 [112]. Loss of WAVE1 function in vivo or in cultured

neurons results in a decrease in mature dendritic spines [112], suggesting that

phosphorylation/de-phosphorylation of WAVE1 in neurons has an important role in the

formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic

spine morphology, critical for the development and maintenance of these structures.

Indeed, WAVE1 knockout mice exhibit deficits in learning and memory [113], suggesting

the intriguing hypothesis that the increased prevalence of cognitive impairment and of

some degenerative disorders observed in conditions of estrogen withdrawal might to

some extent be due to the lack of WAVE1 control by these steroids in brain cells. Thus, the

finding that estrogen controls neuronal cell morphology through the rapid modulation of

WAVE1 heralds promises from both the biological and medical perspective, like

Alzheimer’s Dementia or Parkinson’s disease.

Although this is a relatively recent area of investigation, it significantly expands our

perception of the scope of estrogens non-genomic signaling and sheds new insight into

their ability on the control of cell movement, which may be critically relevant for a various

of physiological or pathophysiological processes. Given the critical action of WASP family

proteins on actin remodeling and cell motility, an interesting open question remains the

role played by sex hormones, as suggested by recent observations [114].


29

Role of estrogen receptor alpha (ERα) in WAVE1 and moesin regulation

Estrogen plays an important role in changes taking place in the brain through the regulation of

growth and differentiation of axons and dendrites, influence on plasticity, support of survival as

well cognitive and behavioral functions. The classical mode of estrogen action is through the

activation of its receptors. Estrogen receptor (ER) presents two isoforms, ER alpha (ERα) and ER

beta (ERβ) [115].

The interaction of ERα with Gαi and Gβ has been recently reported in endothelial cells [116].

Consistent with our previous results in endothelial and breast cancer cells [78, 117], ERα is also

able to interact with Gα13 in the presence of E2. Through the activation of Gα13 at the cell

membrane, ERα has a privileged access to the pathways that signal to the actin cytoskeleton. In

fact, Gα13 is responsible for the activation of the RhoA/ROCK-2/moesin cascade pathway [118],

and may be this pathway is also functional in estrogen-induced neuronal movement.

On the other hand, WAVE1 signaling cascade is recruited via Cdk5. This leads to intracellular

localization of WAVE1 at the cell membrane where it drives the accumulation of actin filaments

and increases the mechanical force necessary for neurite extension and growth-cone

translocation. Increasing evidence indicates that a number of dynamic functional modifications

induced by estrogens in neurons are plaid via rapid extra-nuclear signaling pathways recruited by

estrogen receptors at the cell membrane or within the cytoplasm [101, 119], in various cell types,

including neurons [120-121]. These rapid signaling actions of estrogen receptors render estrogens

flexible modulators of neuronal function. It is intriguing to find that the modifications of the

neuronal cytoskeleton and cell membrane are achieved through this category of action of

estrogen receptors. Whether such rapid modifications of the structure of neuronal membrane and

of the number of dendritic spines can in any way be related to changes in neuronal function that


30

could lead to improved or preserved cognition or memory is difficult to say. However, it is well

established that brain function undergoes rapid modifications in the presence of changing

estrogen concentrations, as shown by animal models and by the brain originated disturbances

(e.g. vasomotor symptoms) seen after delivery or menopause, supporting the idea that sex

steroids, including estradiol might have a role as fast regulators of central nervous system

function.


31

AIMS

The aim of the present study was to explore the molecular basis of the action of 17β-

estradiol on cortical neuronal cells morphology. In particular, we wished to identify

whether these actions may require the regulation of the actin cytoskeleton via WAVE1 or

moesin and to characterize the intracellular cascades that may be recruited during this

signaling.


32

RESULTS

Estrogen induces rapid cytoskeletal and cell membrane remodeling and dendritic spine

formation in cortical neurons

As a first step in identifying the effects of estrogen on neuronal morphology, we studied

the actions of a rapid estrogen exposure on the actin cytoskeleton. Treatment with 17-

estradiol (E2, 10 nM) resulted in a rapid change in actin organization, with a remodeling of

the actin cytoskeleton toward the cell membrane resulting in a thickening of the

membrane (Fig. 1, A–C). This phenomenon was time dependent and transient, being

maximal after 15–20 min and reversing to baseline after 60 min (Fig. 1, A–C). In parallel,

an increased number of dendritic spines was found that followed the same time course

(Fig. 1, A and D).

Estrogen rapidly induces WAVE1 and moesin phosphorylation

WAVE1 is regulated by phosphorylation on three serine residues in the proline-rich

domain (Ser310, Ser397, and Ser441) [112]. Treatment with E2 (10 nM) rapidly increased

the phosphorylation of WAVE1 on all three serine residues along a temporal pattern that

was consistent with that of actin remodeling (Fig. 2, A and B). In parallel, E2 induced a

dynamic phosphorylation on Thr558 of the actin-binding protein moesin (Fig. 2C), which is

responsible for triggering actin remodeling toward the membrane in endothelial and

breast cancer cells [78, 117]. Upon exposure to E2, WAVE1 was redistributed toward the


33

plasma membrane, in particular along the dendritic protrusions (Fig. 2D), in parallel with

actin and cell membrane remodeling (Fig. 2, E and F).

To test whether actin remodeling by WAVE1 or moesin is required for the estrogen-

dependent membrane rearrangement in neurons, we silenced WAVE1 with small

interfering RNAs (siRNAs) and moesin with antisense phosphorothioate oligonucleotides

(PONs). WAVE1 expression was significantly reduced when neurons were transfected for

48 h with WAVE1 siRNAs (supplemental Fig. A, published as supplemental data). In

WAVE1-silenced neurons, E2 failed to induce actin reorganization (Fig. 2, G and H). Moesin

silencing resulted in lack of actin remodeling in response to E2, in the absence of any

modification of WAVE1 expression or localization (Fig. 2, G and H). As control, transfection

of a sense (inactive) oligonucleotide for moesin had no effect on cytoskeletal changes or

WAVE1 expression during E2 administration (Fig. 2, G and H).

Estrogen regulates WAVE1 via estrogen receptor α (ERα)

Phosphorylation of WAVE1 induced by E2 identified as a shift in the gel migration was

prevented by the pure ER antagonist ICI 182,780 (100 nM) (Fig. 3A). To identify which ER

isoform mediates the signaling of E2 to WAVE1, we used the preferential ERα agonist

4,49,40-(4 propyl-[1H]- yrazole-1,3,5-triyl) trisphenol (PPT, 10 nM) or the ERβ agonist 2,3-

bis(4-hydroxyphenil)-propionitrile (DPN, 10 nM). WAVE1 phosphorylation was detected

only in the presence of E2 or PPT, suggesting that ERα supports the signaling to WAVE1,

whereas ERβ is not required (Fig. 3A). To substantiate this observation, we silenced ERα in


34

cortical neurons with siRNAs. This resulted in a reduction of ERα expression, along with a

dramatic decrease in WAVE1 phosphorylation on all three serine sites during exposure to

estrogen (Fig. 3, B and C). This happened in the absence of modifications of the expression

of WAVE1 or ERβ (Fig. 3B). In agreement, remodeling of actin and cell membrane

localization of WAVE1 induced by E2 were prevented by ICI 182,780 and supported by the

ERα agonist PPT but not by the ERβ-selective agonist DPN (supplemental Fig. B).

ERα signals to WAVE1 via a G protein/c-Src initiated cascade

To look for the cascades involved in the signaling of ERα to WAVE1 in neurons, we used a

variety of approaches to interfere with E2 signaling to WAVE1. PD98059 (5 mM), which

inhibits the ERK1/2 MAPK, and wortmannin (30 nM), an inhibitor of phosphatidylinositol-3

kinase, were unable to reduce the phosphorylation of WAVE1 by E2 (supplemental Fig. C).

WAVE1 phosphorylation was instead prevented by the G protein inhibitor pertussis toxin

(PTX, 100 ng/ml) and by the inhibitor of c-Src, PP2 (0.2 µM) (supplemental Fig. C),

indicating that ERα recruits a G protein/c-Src cascade to WAVE1.

Interaction of ERα with Gαi and Gβ is required to E2 signaling to c-Src

Consistent with the previous results, E2 administration resulted in c-Src phosphorylation

in neurons (Fig. 4A). c-Src phosphorylation was prevented by PTX and by inactivating the G

proteins Gαi and Gβ with either a dominant-negative construct or with specific siRNAs but

not by inactivating Gα13 with a dominant-negative construct (Fig. 4A). The interaction of


35

ERα with Gαi and Gβγ has been recently reported in endothelial cells [116]. With co-

immunoprecipitation experiments, we also find a ligand-induced interaction of ERα with

Gαi1 and Gβ1 (Fig. 4, B and C). During E2 induced binding of ERα with Gβ1, the interaction

of Gαi1 and Gβ1 is decreased (Fig. 4C). Consistent with our previous results in endothelial

and breast cancer cells [78, 117], ERα is also able to interact with Gα13 in the presence of

E2 (Fig. 4D).

c-Src is a transducer of signals from G proteins to small GTPases [122], and WAVE is

involved in actin reorganization and in the formation of membrane ruffles induced by the

small GTPase Rac1 [123]. We thus studied whether Rac1 is recruited during the signaling

of ERα to WAVE1. Indeed, during treatment with E2, cortical neurons transfected with a

Rac1 dominant-negative construct did not show an enhanced WAVE1 phosphorylation but

actually displayed a slight decrease vs. baseline (Fig. 4E). Rac1 is recruited downstream of

c-Src, because the Rac1 dominant-negative construct does not impair activation of c-Src

by E2 (supplemental Fig. D).

Blockade of G proteins (with PTX), c-Src (with PP2), or Rac1 (with the dominant-negative

construct) all resulted in a visible inhibition of WAVE1 membrane translocation and of

actin remodeling induced by E2 (supplemental Fig. E), indicating that a signaling cascade

involving G proteins, c-Src, and Rac1 mediates the regulatory effects of E2 on WAVE1 in

cortical neurons.


36

Estrogen signals to WAVE1 via cyclin-dependent kinase 5 (Cdk5)

Cdk5 phosphorylates WAVE1 on the serine residues Ser310, Ser397, and Ser441 (12). To test

the requirement of Cdk5 for estrogen signaling in neuronal cells, we immunoprecipitated

Cdk5 and used the immunoprecipitates (IPs) to perform kinase assays using

dephosphorylated histone H1 as the target for serine phosphorylation. E2 administration

resulted in a rapid increase in Cdk5 activity, which was sensitive to roscovitine (50 µM), a

selective inhibitor of Cdk5 (Fig. 5A). Cdk5 activation in the presence of E2 was also

prevented by the c-Src inhibitor (PP2) as well as by transfection of a Rac1 dominant-

negative construct (Fig. 5B), overall suggesting that ERα recruitment of c-Src and Rac1

leads to Cdk5 activation.

Consistent with this hypothesis, WAVE1 phosphorylation on the three Cdk5 target sites

induced by E2 was inhibited by roscovitine (Fig. 5C). In addition, transfection of neurons

with three constructs encoding for WAVE1 with serine to alanine mutations [112] deleting

the Cdk5 serine phosphorylation sites (Fig. 5D) resulted in decreased dendritic spine

density, particularly with the mutated Ser310 construct (S310A-WAVE1) (Fig. 5, E and F),

confirming the hypothesis that E2 regulates WAVE1 and dendritic spine formation via

Cdk5.

Differential signaling of ER to moesin and WAVE1 in neurons

To confirm that ERα recruits different signaling avenues to regulate moesin and WAVE1,

we exposed neurons to E2 (10 nM) during the blockade of Gαi, Gβ, c-Src, Rac1, and Cdk5


37

(involved in the signaling to WAVE1) or of Gα13, RhoA, or Rho-associated kinase (involved

in signaling to moesin). Western analysis of WAVE1 and moesin phosphorylation showed

that the blockade at any level of the c-Src/Rac1/Cdk5 cascade results in impaired

phosphorylation of WAVE1 but not of moesin (Fig. 5G). Conversely, blockade of the

Gα13/RhoA/Rho associated kinase impairs moesin phosphorylation by E2 but not WAVE1

phosphorylation (Fig. 5G). Blockade of Gαi or Gβ resulted in inhibition of both moesin and

WAVE1 phosphorylation, suggesting that these two G proteins may be involved in

downstream signaling to these actin regulators via multiple pathways.

Estrogen signaling to WAVE1 results in Arp-2/3 membrane translocation

Actin nucleation by the Arp-2/3 complex is critical for the rapid formation of an actin

network at the leading edge of cells [124]. In response to Rac1, the Arp-2/3 complex

initiates the formation of lamellipodia. WAVE1 acts as a scaffolding complex relaying

signals from small GTPases to the Arp-2/3 complex [125].

Cortical neurons treated with E2 displayed a membrane translocation of Arp-2 that was

time consistent with the phosphorylation of WAVE1 (Fig. 6, A and B). Blockade of c-Src

with PP2 or of Rac1 by transfection of a dominant-negative construct or of Cdk5 with

roscovitine blocked the estrogen-induced membrane translocation of Arp-2, supporting

the concept that estrogen signals to WAVE1 and Arp-2 through a c-Src/Rac1/Cdk5

cascade.


38

Estrogen regulates the formation of dendritic spines via WAVE1 and moesin in cortical

neurons

The formation of excitatory synaptic connections in neurite extensions is dependent on

the development of actin-rich dendritic spines [108]. Actin staining in cortical neurons

demonstrated a net enrichment in dendritic spine density after treatment with E2 (Fig. 7,

A and B). Silencing of WAVE1 with siRNAs significantly decreased the number of spines

induced by E2 (Fig. 7, A and B). As control, the transfection of a siRNA-resistant WAVE1

construct reversed the effect of the siRNA (Fig. 7, A and B). Moesin silencing with specific

PONs also prevented the increase in dendritic spines by E2 (Fig. 7, A and B).


39

DISCUSSION

The control of cell morphology and of the interaction of the cell with the extracellular

environment requires a number of regulators orchestrating the different cytoskeletal

components and their interactions with the cell membrane and with anchorage proteins

[74]. These phenomena are key determinants of brain plasticity, making cognition,

memory, or learning possible via neuronal/glial remodeling [89]. Dendrites are the sites

where neurons receive, process, and integrate inputs from their multiple presynaptic

partners. Both the shape of the dendritic trees and the density and shape of their spines

can undergo significant changes during the development and life of a neuron. Dendritic

spines are composed mainly of actin microfilaments that undergo a continuous

remodeling that is important for the development of neuronal circuits [84].

Estrogens stimulate the formation of dendritic spines as early as during embryonic

neuronal development and continue to act on mature neurons regulating their structural

plasticity at mature synapses and increasing dendritic spine density through the dynamic

control of actin filaments [85].

The key finding of this work is that ERα uses the actin controllers WAVE1 and moesin in

neurons to induce a rapid cytoskeletal remodeling that supports the formation of

dendritic spines. In the presence of its ligand, ERα recruits a Gαi-Gβ/c-Src/

Rac1/Cdk5/WAVE1 cascade (Fig. 8). This turns into WAVE1 phosphorylation and

translocation to the sites where the actin cytoskeleton and the cell membrane are actively

remodeled and in the parallel membrane localization of the Arp-2/3 protein complex,


40

which is responsible for the extension and branching of actin filaments and thus for cell

membrane remodeling. The parallel recruitment of the Ezrin/Radixin/Moesin (ERM) actin-

binding protein, moesin, via a Gα13/RhoA/ROCK-2 cascade (Fig. 8) contributes to the

remodeling of the cytoskeleton and is required for the estrogen-induced dendritic spine

formation.

Recent evidence on cultured neurons and in intact mice shows that loss of WAVE1 results

in a dramatic decrease in mature dendritic spines [112], suggesting that in neurons, the

regulation of the actin cytoskeleton is critical for the development and maintenance of

these structures. Thus, the finding that estrogen controls neuronal cell morphology

through the rapid modulation of WAVE1 heralds promises from both the biological and

medical perspective. Indeed, WAVE1 knockout mice exhibit deficits in learning and

memory [113], suggesting the intriguing hypothesis that the increased prevalence of

cognitive impairment and of some degenerative disorders observed in conditions of

estrogen withdrawal might to some extent be due to lack of WAVE1 control by these

steroids in brain cells.

When E2 hits ERα, a signaling cascade to Cdk5 is recruited. This leads to intracellular

localization of WAVE1 at the cell membrane where it drives the accumulation of actin

filaments and increases the mechanical force necessary for neurite extension and growth-

cone translocation. Although all the three identified WAVE proteins are known to localize

at growth cones, their respective roles are unclear [126]. The WAVE1 complex seems to be

transported by kinesin-1, which has been suggested to be particularly important for axon

elongation [108], whereas other reports indicate an involvement of neural WASP in spine


41

formation [111]. Phosphorylation and dephosphorylation of WAVE1 seem to be necessary

for spine formation, as well. During spine formation, the WAVE1 complex is

hyperphosphorylated on the proline-rich region by Cdk5, resulting in a later functional

regulation of the Arp-2/3 complex [126]. Thus, Cdk5-dependent phosphorylation of

WAVE1 and the subsequent Arp-2/3-mediated branching of actin is a newly identified

mechanism by which dendritic spine formation is regulated by estrogen. It is likely that

this phenomenon results from the synergic regulation of multiple converging pathways, as

suggested by the seemingly paradoxical reduction of WAVE1 phosphorylation induced by

E2 during Rac1 blockade. This suggests that E2 also recruits a yet unidentified inhibitory

pathway that reduces Cdk5 phosphorylation of WAVE1 that is independent of Rac1 (Fig.

8). In addition, the requirement of moesin for optimal cytoskeletal remodeling and spine

formation also stands for the existence of an integrated signaling network enacted by ERs

in neurons to control the architecture of the cytoskeleton.

Increasing evidence indicates that a number of dynamic functional modifications induced

by estrogens in neurons are made via rapid extranuclear signaling pathways recruited by

ERs at the cell membrane or within the cytoplasm [101, 119], in various cell types,

including neurons [120-121]. These rapid signaling actions of ERs render estrogens flexible

modulators of neuronal function. It is intriguing to find that the modifications of the

neuronal cytoskeleton and cell membrane are achieved through this category of action of

ERs. Whether such rapid modifications of the structure of neuronal membrane and of the

number of dendritic spines can in any way be related to changes in neuronal function that

could lead to improved or preserved cognition or memory is difficult to say. However, it is


42

well established that brain function undergoes rapid modifications in the presence of

changing estrogen concentrations, as shown by animal models and by the brain originated

disturbances (e.g. vasomotor symptoms) seen after delivery or menopause, supporting

the idea that sex steroids might have a role as fast regulators of central nervous system

function.

In conclusion, we here show that estrogens are implicated in neurite extension in

neuronal cells acting through rapid, extranuclear signal transduction pathways recruited

by ERα. Estradiol acts through the activation of G proteins via ERα, leading to the

recruitment of a c-Src/Rac1/Cdk5/WAVE1/Arp-2/3 cascade. Through this cascade, E2

supports the rearrangement of the actin cytoskeleton at sites of ongoing spine formation

in cortical rat neurons. The parallel G protein-dependent activation of the RhoA/ROCK-

2/moesin cascade contributes to this phenomenon. The identification of these original

rapid actions of estrogen in brain cells increases our understanding of the complex actions

of sex steroids in the central nervous system and may provide new tools to interfere with

some of the gender-related degenerative changes observed in the brain throughout aging.


43

MATERIALS AND METHODS

Neuronal cell cultures

Primary cultures of cortical neurons were obtained from embryonic day 18 rat fetuses as

described [75]. Cortical cells were dissected with 0.02% trypsin (Invitrogen) for 5 min at

37°C and then dissociated. Cells were plated on poly-D-lysine-coated 60 mm Petri dishes

at a density of 0.5–1 x 105 cells/cm2. Neurons were grown in Neurobasal medium

(Invitrogen) supplemented with 10 U/ml penicillin, 10 µg/ml streptomycin, 0.5mM

glutamine, 25 µM glutamate, and 2% B27 (Invitrogen). These culture conditions generate

cultures that are > 95% neuronal and ≤ 5% glial [127]. 17β-estradiol, PTX, PD98059 and

wortmannin were from Sigma-Aldrich (Saint-Louis, MO); roscovitine and PP2 were from

Calbiochem; ICI 182,780, PPT and DPN were obtained by Tocris Cookson (Avonmouth, UK).

Whenever an inhibitor was used, the compound was added 30-45 minutes before starting

the treatments.

Immunoblottings

Cell lysates were separated by SDS-PAGE (8-12%). Immunoblottings were performed with

antibodies against: Histone H1, Phospho-Histone H1 (P-Ser27), WAVE1 (Sigma-Aldrich

Laboratories); Actin (C-11), Arp2 (H-84), ERα (H-184), ERβ (N-19), c-Src (H-12), Rac1 (C-14)

(Santa Cruz Biotechnology, Santa Cruz, CA); Cdk5 (268-283) (Calbiochem); Phospho-Src

(Tyr416) (Cell Signaling Technology); WAVE1 (c-term), Phospho-Ser310-WAVE1, Phospho-

Ser397-WAVE1, Phospho-Ser441-WAVE1. Primary and secondary Abs were incubated with


44

the membranes with standard technique. Immunodetection was accomplished using

enhanced chemiluminescence. Chemiluminescence was acquired with a quantitative

digital imaging system (Quantity One, BioRad, Hercules, CA) allowing to check for

saturation. Overall emitted photons were quantified for each band, particularly for loading

controls, which were homogeneously loaded.

Immunoprecipitations

Primary cortical neurons were washed with ice-cold PBS and lysed with: 20mM Tris-HCl,

pH 7.4, 10 mM EDTA, 100 mM NaCl, 1% Igepal, 1 mM Na3VO4, 50 mM NaF, 0.1 mg/L

PMSF, 0.3 mg/L aprotinin, and 0.01% protease inhibitor mixture (Sigma-Aldrich

Laboratories). The immunoprecipitating antibody Arp-2 (2 µg) was added to equal

amounts of cortical neuron lysates (0.5–1 mg) in 500 µl of lysis buffer for 1 h at 4°C with

gentle rocking. Subsequently, 40 µl of 1:1 Protein-A-agarose was added and gently rocked

for 2 additional hours at 4°C. The mixture was then centrifuged at 12,000 x g for 5 min at

4°C. The supernatant was removed, and the immunoprecipitates were washed with 500 µl

of: 20 mM Tris-HCl, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1% IGEPAL, 1 mM Na3VO4, 50

mM NaF, 0.1 mg/L PMSF, 0.3 mg/L aprotinin, and 0.01% protease inhibitor mixture

(Sigma-Aldrich laboratories). Immunoprecipitated proteins were separated under

reducing and denaturing conditions by 10% SDS-PAGE and transferred to a polyvinylidene

difluoride membrane (Millipore). Nonspecific binding was blocked with 5% skim milk in

PBS–Tween. Membranes were incubated with anti-WAVE1 (1:1000, Sigma-Aldrich


45

Laboratories) or anti-Arp2 (1:900; H-84, Santa Cruz Biotechnology, Santa Cruz, CA)

antibody.

Quantitative analysis of cell membrane morphology and thickness and of actin fibres

remodeling

The remodeling of actin fibres and the morphological changes of the membrane were

quantified by assessing the intensity of actin fluorescence after conversion of the coloured

pixels to greyscale using the Leica QWin image analysis and processing software (Leica

Microsystems, Wetzlar, Germany). This analysis was performed selecting random boxes

including the extra- and intra-cellular space across the membrane, and the linear intensity

of the signal was spatially recorded. We sampled five areas per each cell, and we repeated

this on 40 different cells per experimental condition. Number of spine protrusions per 10

μM of dendrite length were measured in 5 different dendrites per cell and repeated in 40

different cells.

Cell immunofluorescence

Neuronal cells were grown on coverslips. Cells were fixed with 4% paraformaldehyde for

30 min and permeabilized with 0.1% Triton for 5 min. Blocking was performed with PBS

containing 3% bovine serum albumin for 30 min. Cells were incubated with anti-WAVE1

(1:750; Sigma-Aldrich Laboratories) and anti-Arp2 (H-84) (1:300; Santa Cruz

Biotechnology, Santa Cruz, CA) overnight at 4º C followed by incubation with a


46

fluorescein-conjugated goat anti-rabbit secondary antibody (1:200; Vector Laboratories).

Then cells were incubated with Texas Red-phalloidin (Sigma) for 30 min. After washing the

nuclei were counterstained with or 4'-6-diamidino-2-phenylindole (DAPI) (Sigma) and

mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA).

Immunofluorescence was visualized using an Olympus BX41 microscope and recorded

with a high-resolution DP70 Olympus digital camera.

Kinase assays

Neuronal cells were harvested in 20 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 0.5%

IGEPAL, 1 mM Na3VO4, 50 mM NaF, 0.1 mg/L PMSF, 0.3 mg/L aprotinin, and 0.01%

protease inhibitor mixture (Sigma-Aldrich Laboratories). Equal amounts of cell lysates

were immunoprecipitated with anti-Cdk5 (2 µg) antibody. Subsequently, 40 µl of 1:1

Protein-A-agarose was added and the IPs were washed three times with 20 mM Tris-

HCl,10 mM EDTA, 150 mM NaCl 0.1% IGEPAL, 0.1 mg/ml PMSF. Two additional washes

were performed in kinase assay buffer (20 mM MOPS, 25 mM -glycerophosphate, 5 mM

EGTA, 1 mM DTT) and the samples were resuspended in this buffer: 5 g of Histone H1

de-phosphorylated (H4524, Sigma-Aldrich Laboratories) together with 500 M ATP and 75

mM MgCl2 were added to each sample and the reaction was started putting the samples

at 30°C for 20 min. The reaction was stopped on ice and by resuspending the samples in

Laemmli Buffer. The samples were separated with SDS-PAGE and western analysis was

performed using Abs recognizing anti-Cdk5 (268-283, Calbiochem) and anti-phospho-

Histone H1.4 (pSer27) (H7664, Sigma-Aldrich Laboratories).


47

Gene silencing with RNA interference

Two synthetic small interfering RNAs targeting ERα (siRNA SMARTpool ESR1, Dharmacon,

USA) and WAVE1 (siRNA SMARTpool WAVE1, Dharmacon, USA) were used at the final

concentration of 50-75 nM. Cortical neurons were treated 48 hours after siRNA

transfection. The efficacy of gene silencing was checked with western analysis and found

to be optimal at 48 hours.

Transfection experiments

The dominant-negative Rac1 T17N 2XMYC-tagged (N-terminus) (Rac1 DN) was from the

Guthrie cDNA Resource Center (www.cdna.org). The insert was cloned in pcDNA3.1+. The

plasmid (10 μg) was transfected into cortical neurons using Lipofectamine (Invitrogen,

Carlsbad, CA). Parallel cells were transfected with empty pcDNA3.1+ plasmid. Cells (60-

70% confluent) were treated 24 h after transfection.

Statistical analysis

All values are expressed as mean ± SD. Statistical analyses and graphics were done using

InStat from GraphPad Prism 5 Software. Statistical differences between mean values were

determined by ANOVA, followed by the Fisher’s protected least significance difference

(PLSD).


48

ACKNOWLEDGEMENTS

This work has been supported by the PRIN grant 2004057090_007 by the Italian University

and Scientific Research Ministry (MIUR) to T.S.

We are grateful to Dr. Yong Kim and Dr. Paul Greengard (Laboratory of Molecular and

Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, New York

10021, USA), for providing the Abs vs. P-Ser310, P-Ser397 and P-Ser441 WAVE1 isoform.


49

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60

FIGURE LEGENDS

Fig. 1. E2 induces dendritic spine formation and dynamic actin remodeling in cortical

neurons. A, Neuronal cells were treated for different times with E2 (10 nM). Actin fibers

were stained with phalloidin linked to Texas Red (red labeling), and nuclei were

counterstained with 4�,6-diamidino-2-phenylindole (blue labeling). The insets show

dendritic spines at higher magnification from the white boxes. The yellow boxes on the

cells indicate a sample cellular area that is analyzed in the colored graphs. In these graphs,

the longitudinal axis represents the gray level, and the horizontal axis indicates pixels.

Blue, red, and yellow areas indicate the extracellular, plasma membrane, and cytoplasmic

fractions, respectively. B and C, Mean intensity ratio of actin staining in the

membrane/cytoplasm in the same experiment. The results are derived from the sampling

of five areas of the cell membrane of 40 different random cells. Areas of minimum and

maximum cell membrane thickness were always included. The results are expressed as

the mean ± SD of the measurements. *, P < 0.05 vs. 0 min. D, Mean number (±SD) of

dendritic protrusions per 10 µm dendrite length. *, P < 0.05 vs. 0 min. CON, Control.

Fig. 2. Estrogen activates actin remodeling in neurons via WAVE1 and moesin. A, Protein

extracts from neuronal cells treated with 10 nM E2 for 0–120 min were assayed with

Western analysis for their overall content of WAVE1, actin, or P-Ser310-, P-Ser397-, and P-

Ser441-WAVE1. B, Densitometric analysis of the P-WAVE1 bands in A. C, Western analysis

for wild-type and Thr558-P-moesin of neuronal protein extracts treated with E2 (10 nM)


61

for 0–60 min. D, Neuronal cells were treated for different times with E2 (10 nM). Actin

fibers were stained with phalloidin linked to Texas Red (red labeling), WAVE1 was stained

with fluorescein isothiocyanate (green labeling), and nuclei were counterstained with 4,6-

diamidino-2-phenylindole (blue labeling). E, Mean ± SD thickness of the cell membrane

and mean number ± SD of membrane-localized WAVE1, estimated as WAVE1 spots at the

cell membrane per cell, indicated as the mean of the measurement of 40 different cells

per condition. F, Mean intensity ratio of actin staining in the membrane/cytoplasm in the

same experiment. The results are derived from the sampling of five areas of the cell

membrane of 40 different random cells. Areas of minimum and maximum cell membrane

thickness were always included. *, P < 0.05 vs. 0 min. G, Neuronal cells were transfected

with siRNA vs. WAVE1 or with antisense or sense PONs toward moesin for 48 h. WAVE1

expression and cellular localization status was checked by staining for WAVE1 (fluorescein

isothiocyanate). Actin fibers were stained with phalloidin-Texas Red, and nuclei were

counterstained with 4�,6-diamidino-2-phenylindole. H, Mean ± SD thickness of the cell

membrane as well as the intensity of actin staining in the cytoplasm and membrane in the

same experiment. Technical details are as in E and F. D and G, The white boxes on the cells

indicate a sample cellular area that is analyzed in the colored graphs. In these graphs, the

longitudinal axis represents the gray level, and the horizontal axis indicates pixels. Blue,

red, and yellow areas indicate the extracellular, plasma membrane, and cytoplasmic

fractions, respectively. CON, Control.


62

Fig. 3. Estrogen signals to WAVE1 via ERα. A, Cortical neurons were exposed for 20 min to

10 nM E2 in the presence or absence of the pure ER antagonist ICI 182,780 (ICI; 100 nM)

or with the ERα-selective ligand PPT (10 nM) or the ERβ-selective agonist DPN (10 nM).

Total phosphorylation of WAVE1 was assayed with Western analysis as a gel migration

shift in the band. B, Cortical neurons were transfected with siRNA vs. ERα (siRNA ERα) or

with vehicle, and protein analysis for ERα, ERβ, actin, or wild-type (WAVE1) or P-Ser310-,

P-Ser397-, and P-Ser441-WAVE1 was performed on cell lysates after treatment for 20 min

with 10 nM E2. C, The graph displays the quantitative analysis of the intensity of the bands

in B, obtained as number of photons measured by the ChemiDoc digital imaging system

and evaluated with the Quantity One Software (Bio-Rad, Hercules, CA). CON, Control.

Fig. 4. ERα signals to WAVE1 via Gαi, Gβ, c-Src, and Rac1. A, Cortical neuronal cells were

exposed for 20 min to 10 nM E2, in the presence or absence of the G protein inhibitor PTX

(100 ng/ml) or the c-Src inhibitor PP2 (0.2 µM) or after transfection with dominant-

negative Gα13 or Gαi constructs or siRNAs vs. G�1. Wild-type c-Src or P-Tyr416-c-Src were

assayed in cell extracts. B–D, Neuronal cell protein extracts were immunoprecipitated

with antibodies toward the indicated G proteins and co-immunoprecipitation of ERα, ERβ,

and Gαi (C) was tested by Western analysis. E, Cortical neurons were treated for 20 min

with 10 nM E2, with or without transfection of a dominant-negative Rac1 for 48 h. Rac1,

wild-type WAVE1, or P-Ser310-, P-Ser397-, and P-Ser441-WAVE1 were assayed in cell

extracts. CON, Control.


63

Fig. 5. Estrogen signals to WAVE1 via a Gαi-G�/c-Src/Rac1/Cdk5 cascade, whereas

signaling to moesin requires a Gα13/RhoA/ROCK2 pathway. A–C, Cortical neurons were

treated with E2 in the presence or absence of the specific Cdk5 inhibitor roscovitine (Rosc;

50 µM) or the c-Src inhibitor PP2 (0.2 µM) or after transfection with a dominant-negative

Rac1 construct. A and B, Whole protein extracts were immunoprecipitated with an

antibody against Cdk5. The IPs were used in a kinase assay to phosphorylate histone H1

(substrate of Cdk5). Phosphorylated histone H1 was detected after Western analysis with

a phosphospecific antibody. C, Neuronal cell content of Cdk5, WAVE1, and P-Ser310-, P-

Ser397-, and P-Ser441-WAVE1 were assayed with Western analysis. D–F, Cortical neurons

were exposed to 10 nM E2 for 20 min with or without transfection of WAVE1 constructs

with inactivating mutations of the Ser310, Ser397, and Ser441 Cdk5 phosphorylation sites.

D, Western analysis of wild-type or P-Ser310-, P-Ser397-, or P-Ser441-WAVE1 was

performed. E, Dendritic spine morphology and number were measured with

immunofluorescence after staining actin fibers with phalloidin/Texas Red. Scale bar, 5 µm.

F, The graph shows the quantitative analysis of spine density calculated as the number of

spines per 10 µm dendrite length, normalized vs. nontransfected, vehicle-treated neurons

and expressed as a percentage. The results are expressed as the mean ± SD. *, P < 0.05 vs.

E2 in nontransfected cells. G, Cortical neurons were treated with E2 in the presence or

absence of the G protein inhibitor PTX (100 ng/ml), the c-Src inhibitor PP2 (0.2 µM), the

Cdk5 inhibitor roscovitine (Rosc; 50 µM), or the Rho-associated kinase inhibitor (ROCK-2)

Y-27632 (Y, 10 µM) or after transfection with dominant-negative Gα13, Gαi, Rac1, or RhoA


64

constructs or with siRNAs vs. G�1. Western analysis for actin, wild-type, and Thr558-P-

moesin, wild-type, and P-Ser310-, or P-Ser397-, or PSer441-WAVE1 was performed.

Fig. 6. Estrogen signaling to WAVE1 turns into membrane localization of the Arp-2/3

complex. Cortical neurons were treated for 20 min with 10 nM E2 in the presence or

absence of the Cdk5 inhibitor roscovitine (Rosc; 20 µM) or the c-Src inhibitor PP2 (0.2 µM)

or after transfection with a dominant-negative Rac1 construct. A, Cells were stained with

an antibody against Arp-2 (fluorescein isothiocyanate), actin fibers were stained with

phalloidin linked to Texas Red, and nuclei were counterstained with 4,6-diamidino-2-

phenylindole. Yellow arrows indicate membrane-localized Arp-2. B, Quantification of the

membrane-localized Arp-2 complexes in the different conditions. Results are expressed as

percent vs. control cells (mean ± SD). *, P < 0.05 vs. control. Membrane-localized Arp-2

complexes were counted in 40 different cells. CON, Control.

Fig. 7. Estrogen increases dendritic spine density via WAVE1 and moesin. Cortical

neurons were incubated in the presence of 10 nM E2 for 20 min in baseline conditions or

after silencing of WAVE1 with specific siRNAs or of moesin with antisense PONs Sense

PONs for moesin served as control. A, Dendritic spine morphology and number were

measured with immunofluorescence after staining actin fibers with phalloidin/Texas Red.

Scale bar, 5 µm. B, The graph shows the quantitative analysis of spine density expressed as

the number of spines per 10 µm dendrite length. The results are expressed as the mean ±

SD. *, P < 0.05 vs. control. CON, Control.


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Fig. 8. Signaling cascades of ERα to WAVE1 and moesin and dendritic spine formation in

cortical neurons. Binding of 17-β-estradiol (E2) to the specific membrane receptors (ERα)

induces phosphorylation of the moesin and WAVE1 cascade. ERα uses the actin

controllers WAVE1 and moesin to induce a rapid cytoskeletal remodeling that supports

the formation of dendritic spines. In the presence of E2, ERα recruits a Gαi–Gβ/c–

Src/Rac1/Cdk5/WAVE1 cascade. This turns into WAVE1 phosphorylation and translocation

to the sites where the actin cytoskeleton and the cell membrane are actively remodeled

and in the parallel membrane localization of the Arp2/3 protein complex, which is

responsible for the extension and branching of actin filaments and thus for spine dendrite

formation. The parallel recruitment of the ERM actin-binding protein, moesin, via a

Gα13/RhoA/ROCK-2 pathway contributes to the remodeling of the cytoskeleton and is

required for the estrogen-induced dendritic spine formation. Moesin activation is finely

modulated by Gαi protein-dependent intracellular pathways.


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SUPPLEMENTAL DATA

Fig. A. Cortical neurons were transfected with siRNA vs. WAVE1 (siRNAs WAVE1) or with

vehicle, and protein analysis for WAVE1 or actin was performed on cell lysates after

treatment for 20 min with estradiol. The graph shows the intensity of the band quantified

as number of photons measured by the ChemiDoc digital imaging system and evaluated

with the Quantity One Software (Bio-Rad).

Fig. B. Cortical neurons were exposed for 20 min to 10 nM E2 in the presence or absence

of the pure ER antagonist ICI 182,780 (ICI; 100 nM), or with the ERα-selective ligand PPT

(10 nM) or the ERβ-selective agonist DPN (10 nM). Actin was stained with phalloidin/Texas

Red, WAVE1 was stained with FITC and the nuclei were counterstained with DAPI. Yellow

arrows indicate membrane-localized WAVE1.

Fig. C. Cortical neurons were exposed for 20 min to 10 nM E2 in the presence or absence

of the pure ER antagonist ICI 182,780 (ICI; 100 nM), or with the ERα-selective ligand PPT

(10 nM) or the ERβ-selective agonist DPN (10 nM). The bar graph shows the quantification

of the membrane-localized WAVE1 complexes in the different conditions. * = p < 0.05 vs.

control. Membrane-localized WAVE1 complexes were counted in 40 different cells. The

table shows the mean ± SD thickness of the cell membrane as well as the intensity of actin

staining in the cytoplasm and membrane in the same experiment. The results are derived

from the sampling of five areas of the cell membrane of 40 different random cells. Areas

of minimum and maximum cell membrane thickness were always included.


67

Fig. D. The graphs show the intensity of the bands in Fig. 4A in the text. Band intensity was

quantified as number of photons measured by the ChemiDoc digital imaging system and

evaluated with the Quantity One Software (Bio-Rad).

Fig. E-F. Cortical neuronal cells were exposed for 20 min to 10 nM E2, in the presence or

absence of the c-Src inhibitor, PP2 (0,2 µM) or of the G protein inhibitor PTX (100 ng/ml).

Other cells were treated after transfection with a dominant-negative Rac1 (RAC1TN17DN)

for 48 h. WAVE1 intracellular localization was checked by staining with FITC, actin fibres

were stained with phalloidin-Texas Red and nuclei were counterstained with DAPI. Yellow

arrows indicate membrane-localized WAVE1 complexes. The bar graph shows the

quantification of the membrane-localized WAVE1 complexes in the different conditions. *

= p < 0.05 vs. control. Membrane-localized WAVE1 complexes were counted in 40

different cells. The table shows the mean ± SD thickness of the cell membrane as well as

the intensity of actin staining in the cytoplasm and membrane in the same experiment.

The results are derived from the sampling of five areas of the cell membrane of 40

different random cells. Areas of minimum and maximum cell membrane thickness were

always included.


68

FIGURES

Fig. 1A


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Fig. 2


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Fig. 3


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Fig. 4


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Fig. 5


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Fig. 6


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Fig. 7


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Fig. 8


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SUPPLEMENTAL DATA


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85

Fig. C

Fig. D


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87

Fig. F


88

ACKNOWLEDGEMENT

I would like to express my deep appreciation to the tutor Dr. Tommaso Simoncini and to Professor Andrea Riccardo Genazzani. They are guiding me into the exploration of scientific projects with novel ideas and top skills. They have been always next to me, encouraging my every tiny advancement, solving the problems and difficulties, which I will remember in my heart forever.

In the laboratory MCGEL directed by Dr. Tommaso Simoncini, I met good colleagues and great friends. I will never forget, Maria Silvia, Paolo, Filippo, Silvia,Chiara, Xiao –Dong, Lorenzo, Veronica, Antonella, Sara,Eleonora and Santhosh. Thanks for their help.

Finally I want to remember my family and in special my wife Marina Flamini and my angels Delfina and Valentino. In support of my dreams, they have always encouraged me. Your love makes the fountain of force in my heart to drive me make progress. Thanks.

Angel Matias Sanchez


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DECLARATION

I, the undersigned, hereby declare that the work contained in this thesis is my own original work. The results present in this thesis were published in:

1. Sanchez AM, Flamini MI, Fu XD, Mannella P, Giretti MS, Goglia L, Genazzani AR, Simoncini T. Rapid signaling of estrogen to WAVE1 controls neuronal spine formation via the actin cytoskeleton. Molecular Endocrinology. 2009 Aug;23(8):1193-202. Epub 2009 May 21. Impact Factor: 5,389.

2. Sanchez AM, Simoncini T. Extra-nuclear signaling of ERα to the actin cytoskeleton in the central nervous system. Steroids. 2009 Dec 14. Impact Factor: 2,588. Review.

Regarding the remainder of the results, I hereby declare that I have not previously submitted any part of it at any university for a degree.

Signature............................................... Date...................................................

university of pisa department of reproductive medicine and

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